LIBRO DE RESUMENES - Congreso SEBBM Salamanca 2016
Transcription
LIBRO DE RESUMENES - Congreso SEBBM Salamanca 2016
Entidades colaboradoras: Expositores: SEBBM Salamanca 2016 Organiza: SEBBM Salamanca 2016 XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular Del 5 al 8 de septiembre Colaboradores: Patrocinadores de Reuniones de Grupos y Cursos: Patrocinador general del Congreso: Socios Protectores: XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular Patrocinadores de Conferencias y Premios: LIBRO DE RESÚMENES www.sebbm.es/xxxixcongreso XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM Salamanca 2016 Del 5 al 8 de septiembre Primera edición: Septiembre 2016 © los autores, 2016 XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular Publica: Sociedad Española de Bioquímica y Biología Molecular (SEBBM) Producción editorial: Rubes Editorial, S.L. Sicilia, 253 – 6º 4ª. 08025 Barcelona Índice Invitación. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Agradecimientos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Comité organizador . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Junta directiva de la SEBBM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Socios protectores de la SEBBM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Calendario del Congreso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Conferencias plenarios / Plenary Lectures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Simposios / Symposia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 S1. S2. S3. Biomedicina molecular / Molecular biomedicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulación génica y comunicación celular / Gene regulation and cellular communication . . . . . . . . . . . . . . . . . Estructura y función de biomoléculas / Structure and function of biomolecules . . . . . . . . . . . . . . . . . . . . . . . . 17 23 29 Pósters / Posters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 P01. P02. P03. P04. P05. P06. P07. P08. P09. P10. P11. P12. P13. P14. P15. P16. Apoptosis / Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bases moleculares de la patología / Molecular bases of pathology . . . . . . . . . . . . . . . . . . . Biología del desarrollo / Developmental biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biología molecular computacional / Computational molecular biology . . . . . . . . . . . . . . . . Biomembranas y bioenergética / Biomembranes and bioenergetics . . . . . . . . . . . . . . . . . . Bioquímica de la nutrición / Nutritional biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . Bioquímica perinatal / Perinatal biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bioquímica y biología molecular de plantas / Biochemistry and molecular biology of plants . Biotecnología molecular / Molecular biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Estructura y función de proteínas / Protein structure and function . . . . . . . . . . . . . . . . . . Genómica y proteómica / Genomics and proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolismo del nitrógeno / Nitrogen metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurobiología molecular / Molecular neurobiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Parasitología molecular / Molecular parasitology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Radicales libres y estrés oxidativo / Free radicals and oxidative stress . . . . . . . . . . . . . . . . Regulación de la expresión génica y dinámica del genoma / Regulation of gene expression and genome dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P17. Regulación metabólica / Metabolic regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P18. Señalización celular / Cellular signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P19. Transgénesis en mamíferos / Transgenesis in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . P20. Transportadores de membrana / Membrane transporters . . . . . . . . . . . . . . . . . . . . . . . . . . PW1. Enseñanza de la bioquímica / Teaching biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . PW2. Workshop: Investigador emergente / Workshop emergent investigator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 42 59 63 64 68 72 73 75 80 92 96 97 104 111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 127 140 152 154 159 162 Índice de autores / Authors index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Invitación Querid@ amig@, En nombre del comité organizador te doy la bienvenida al XXXIX Congreso de la SEBBM. La sede, el Palacio de Congresos de Castilla y León, está situada en el mismo centro de Salamanca, a pocos metros del Edificio Histórico de la Universidad o de la plaza Mayor. El acto inaugural (5 de septiembre a las 19:00 h) contará con la presencia del Prof. Sir Paul Nurse, premio Nobel de Fisiología y Medicina en 2001, quien impartirá una conferencia. Seguidamente, iremos paseando al Colegio Arzobispo Fonseca para disfrutar del cóctel de bienvenida en su bello claustro. Durante los dos días y medio siguientes ofreceremos el marco que nos permita disfrutar de las discusiones científicas. Para facilitarlo, hemos dotado de tiempo suficiente entre las sesiones de simposios y las conferencias plenarias con la pretensión de que ningún póster quede sin ser visitado ni discutido. Además, hemos recuperado el Poster Party de la tarde, con el cual pretendemos catalizar el intercambio de ideas. Animo también a los congresistas a interesarse por los nuevos y excelentes productos que las empresas patrocinadoras nos muestran desde sus stands, colocados junto a los pósters. Desde aquí me gustaría agradecer a las empresas patrocinadoras su constante apoyo, sin el cual este Congreso no podría celebrarse. Me gustaría destacar dos novedades en este Congreso. La primera, facilitar que sean los propios socios de la SEBBM los que tomen la iniciativa de elaborar propuestas de simposios mediante concurrencia competitiva. Y, la segunda, destacar la figura del investigador emergente en un simposio plenario que nos permita conocer mejor nuestro potencial investigador. Naturalmente, con respecto a los congresos anteriores mantendremos las ya clásicas conferencias plenarias y premios (Alberto Sols-Fundación BBVA, Fundación Ramón Areces, L’Oréal-UNESCO, Leloir, Niemeyer, PABMB, Fischer-Scientific, Joven Investigador-Biotools, Margarita Lorenzo-Fundación Lilly), así como las reuniones de grupo, esencia del Congreso de la SEBBM. Por otro lado, continuaremos con las actividades satélite, tales como el Curso de Iniciación a la Investigación en Bioquímica y Biología Molecular, el Foro del Emprendedor, la Reunión de Coordinadores de Másteres del Área de Bioquímica, la Bioquímica en la Ciudad, y los Talleres, de enorme interés entre los más jóvenes, y entre los no tan jóvenes. Con la nueva conexión ferroviaria de vía rápida, la ciudad de Salamanca está a tan solo una hora y media de Madrid. La coincidencia del Congreso con las fiestas patronales de la ciudad nos permitirán además disfrutar de los conciertos en la plaza Mayor y otras distracciones de índole gastronómica. Con sus universidades, claustros y palacios, Salamanca es de incuestionable interés cultural. Por todos estos motivos, en nombre del Comité Organizador es un placer darte la bienvenida y animarte a participar activamente en el XXXIX Congreso de la SEBBM. JUAN PEDRO BOLAÑOS Presidente del Comité Organizador 4 Agradecimientos Nuestro agradecimiento a las entidades públicas y privadas que han colaborado económicamente en la realización del XXXIX Congreso de la SEBBM. Ministerio de Economía y Competitividad Consejo Superior de investigaciones científicas (CSIC) Junta de Castilla y León Ayuntamiento de Salamanca Universidad de Salamanca Instituto de Biología Fundacional y Genómica (IBFG) Instituto de Investigación Biomédica de Salamanca (IBSAL) Instituto de Neurociencias Castilla y León Centro de Investigación del Cáncer (CIC) Fundación BBVA Fundación Ramón Areces L’Oréal – For Women in Science Fundación lilly FEBS PABMB Eppendorf Bruker Fisher Scientific Biotools Roche Geirli Bio-Rad Biogen Conda – Pronadisa Controltécnica Cultek Ecogen Frontexbiomed GE Healthcare Gilson Marie Curie Alumni Association Merck Nucleus Panreac AppliChem Stab Vida Vitro VWR International Werfen 5 Comité organizador COMITÉ HONORÍFICO Excmo. Sr. Juan Vicente Herrera, Presidente de la Comunidad Autónoma de Castilla y León Crisanto Gutiérrez (Tesorero de la SEBBM) Joaquim Ros (Coordinador de la Comisión Asesora de Congresos de la SEBBM) Ilma. Sra. Dra. Carmen Vela, Secretaria de Estado de Universidades e Investigación Excmo. Sr. Daniel Hernández Ruipérez, Rector Magnífico de la Universidad de Salamanca COMITÉ CIENTÍFICO Ángeles Almeida (Instituto de Investigación Biomédica de Salamanca, Hospital Universitario de Salamanca-Universidad de Salamanca) COMITÉ EJECUTIVO Juan Pedro Bolaños (Presidente, Instituto de Biología Funcional y Genómica, Universidad de Salamanca) Isabel Fariñas (Universidad de Valencia) Ángeles Almeida (Secretaría Científica, Instituto de Investigación Biomédica de Salamanca, Hospital Universitario de Salamanca-Universidad de Salamanca) Arantxa Tabernero (Coordinación Actividades Satélite, Instituto de Neurociencias de Castilla y León, Universidad de Salamanca) Emilio Fernández (Tesorero, Instituto de Biología Funcional y Genómica) Carlos López-Otín (Universidad de Oviedo) Salvador Moncada (Universidad de Manchester, Reino Unido) OTROS COMITÉS Curso de Iniciación a la Investigación Arantxa Tabernero (Instituto de Neurociencias de Castilla y León, Universidad de Salamanca) Reunión de coordinadores de grados y posgrados Carmen Guerrero (Coordinación Logística, Centro de Investigación del Cáncer) Isabel Muñoz-Barroso (Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca) Ángel Hernández (Coordinación Logística, Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca) Foro del emprendedor César Roncero (Coordinación Logística, Instituto de Biología Funcional y Genómica) Juan Carlos Arévalo (Instituto de Neurociencias de Castilla y León, Universidad de Salamanca) Actividades Bioquímica en la Ciudad Federico Mayor (Presidente de la SEBBM) Concepción Lillo (Instituto de Neurociencias de Castilla y León, Universidad de Salamanca) María Ángeles Ros (Vocal de Congresos de la Junta Directiva de la SEBBM) Ana Velasco (Instituto de Neurociencias de Castilla y León, Universidad de Salamanca) Itziar Alkorta (Vocal de Empresas y Cónsules de la Junta Directiva de la SEBBM) David Díaz (Instituto de Neurociencias de Castilla y León, Universidad de Salamanca) 6 Junta directiva de la SEBBM PRESIDENTE Federico Mayor Menéndez VICEPRESIDENTE Manuel Palacín Prieto VOCALES Juan Pedro Bolaños Hernández Carmen Caelles Franch Irene Díaz Moreno Iztiar Alkorta Calvo SECRETARIA Almudena Porras Gallo Fernado Moreno Herrero TESORERO Crisanto Gutiérrez Armenta PRESIDENTE ELECTO Felix María Goñi Urcelay Marian Ros Lasierra Socios protectores de la SEBBM Los socios protectores de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM) contribuyen al progreso de la ciencia. 7 Calendario del Congreso * " " *-'( $$.* " %& !"# $ !"# $ )3 "4 )56)5!-) 76)56 )5-) 6) 56) 86) 96 ) -58 % % ( )3 "4 )5 6)58-) 6)59-)56)576 ) 6) !6) 6) % ( ) ($ 8 "(6 & " " !! ! ! ! +( ) +( * " /0-1%#"0 2 +< ,2= ,# )2 +, *3 0&#> " +' $ %&' #;<$:4 )2 )),? #(&( ( $ %&' )("3&+$:46$4& $ '# "(($:4&$4 ! $ %&' " #$$ #+ "" '()(* Conferencias plenarios Plenary Lectures 9 10 Salamanca 2016 Conferencia de apertura / Opening Lecture Alberto Sols - Fundación BBVA CP01-1 Controlling the Cell Cycle Matthew Swaffer, Andrew Jones, Helen Flynn, Bram Snijders, Paul Nurse The Francis Crick Institute, Londres, UK Like other eukaryotes wild type fission yeast cells control their cell cycle through multiple cyclin dependent kinases (CDKs) with roles at different cell cycle stages. However, all these CDKs can be replaced by a single CDK, suggesting that the order of cell cycle events can be controlled by quantitative changes in CDK activity rather than by qualitatively different CDKs. Using phosphoproteomics combined with a novel in vivo protein kinase assay, we have shown that activity increases as cells proceed through the cell cycle and that different substrates have differing sensitivities to absolute kinase activity. Substrates required early in the cell cycle for S-phase become phosphorylated at low CDK activity whilst substrates require later for mitosis need higher CDK activity to become phosphorylated. We propose that this is the core organising principle ordering events in the cell cycle which helps explain the plasticity and redundancy observed between CDKs in Metazoan eukaryotes. Conferencia Premio Fisher Scientific: mejor artículo científico de joven científico/a / Fisher Scientific Award Lecture: best scientific paper of a young scientist CP02-1 AMPK and PFKFB3 mediate glycolysis and survival in response to mitophagy during mitotic arrest Elena Doménech Cruz1, Carolina Maestre1, Lorena Esteban2, David Partida1, Gonzalo Fernández-Miranda3, Ramón Campos-Olivas1, Manuel Pérez1, Diego Megías1, Katherine Allen4, Miguel López5, Asish Saha6, Guillermo Velasco7, Eduardo Rial2, Raúl Méndez3, Patricia Boya2, María Salazar-Roa1, Marcos Malumbres1 1 Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, ES, 2 CIB, Madrid, ES, 3IRB Barcelona, Barcelona, ES, 4Boston Universisty School of Medicine, Massachusetts, US, 5CIMUS, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de Compostela, ES, 6Division of Endocrinology, Diabetes & Nutrition, Boston University School of Medicine, Massachusetts, US, 7 Universidad Complutense de Madrid, Madrid, ES Blocking mitotic progression has been proposed as an attractive therapeutic strategy to impair proliferation of tumour cells. Inhibition of mitotic kinases such as Aurora A or Plk1, or treatment of cells with microtubule poisons that disrupt the proper dynamics of the spindle, results in mitotic arrest, a feature that may be used to prevent tumour cell proliferation. However, how cells survive during prolonged mitotic arrest is not well understood. At this stage, transcription is strongly suppressed, translation is limited to a subset of messenger RNAs, and the absence of the nuclear envelope temporally maintains on hold multiple regulatory mechanisms that are based on the separation of the nuclear and cytoplasmic compartments. Although death in mitosis is at least partially mediated by apoptosis, the molecular pathways that control survival or death in mitosis are not well understood. Here we show that survival during mitotic arrest is affected by the special energetic requirements of mitotic cells. Prolonged mitotic Conferencias plenarios / Plenary Lectures arrest results in mitophagy-dependent loss of mitochondria, accompanied by reduced ATP levels and the activation of AMPK. Oxidative respiration is replaced by glycolysis owing to AMPK-dependent phosphorylation of PFKFB3 and increased production of this protein as a consequence of mitotic-specific translational activation of its mRNA. Induction of autophagy or inhibition of AMPK or PFKFB3 results in enhanced cell death in mitosis and improves the anti-tumoral efficiency of microtubule poisons in breast cancer cells. Thus, survival of mitotic-arrested cells is limited by their metabolic requirements, a feature with potential implications in cancer therapies aimed to impair mitosis or metabolism in tumour cells. Conferencia plenaria Leloir / Leloir Plenary Lecture CP03-1 Más que un inhibidor de quinasas dependientes de ciclinas: una nueva función de p21 en el replisoma Sabrina Mansilla1, Agustina P Bertolin1, Valerie Bergoglio2, Marina Gonzalez Besteiro1, María Belén de la Vega1, Marie-Jeanne Pillaire2, Nicolás Calzetta1, Natalia Paviolo1, Paola Campodónico1, María Belén Federico1, Jean-Sebastien Hoffmann2, Vanesa Gottifredi1 1 Fundación Instituto Leloir-Instituto de Investigaciones Bioquímicas de Buenos Aires. Consejo de Investigaciones Científicas y Técnicas, C.A.B.A, AR, 2Cancer Research Center of Toulouse, Inserm U1037, CNRS ERL5294 ; University Paul Sabatier, Toulouse, FR Los niveles del inhibidor p21 quinasa dependiente de ciclina (CDK) son bajos en la fase S e insuficiente para inhibir CDK. Hemos demostrado que la p21 endógena, en lugar de ser residual es funcional y necesaria para preservar la estabilidad genómica de las células no estresadas. La pérdida de expresión basal de p21 retrasa la elongación del ADN naciente, provocando defectos permanentes en la finalización de la replicación y promoviendo la inestabilidad de regiones genómicas que son difíciles de replicar, es decir, sitios frágiles comunes (SFC). Encontramos que la región de p21 necesaria para promover la replicación es su dominio de interacción con PCNA (PIR ) y no su dominio de unión CDK. Por otro lado, identificamos a la polimerasa especializada kappa como la responsable de los defectos replicativos que aumentan cuando los niveles de p21 se eliminan, ya que no se detectaron alteraciones en el proceso de replicación ni aumento de la inestabilidad genómica en células donde p21 y la polimerasa kappa fueron eliminadas. Por lo tanto, con un mecanismo que es completamente independiente de su capacidad de regular CDK, p21 endógeno impide un tipo de inestabilidad genómica que no se desencadena por las lesiones del ADN endógenas sino por una desregulación en la elección de la polimerasa de ADN durante la síntesis de ADN genómico. Conferencia plenaria / Plenary Lecture L’Oréal-UNESCO For Women in Science CP04-1 Modulation of neural plasticity in Alzheimer’s disease Nancy Ip Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong-Kong, CN Proper brain function depends on the intricate interplay of well co-ordinated signaling pathways among different types of brain cells, deregulation of which can lead to neural dysfunctions that contribute 11 Conferencias plenarios / Plenary Lectures to various brain disorders. Our research interest is focused on unravelling the molecular and cellular mechanisms underlying these pathways to enhance our understanding of brain functions as well as to identify specific molecular targets associated with brain disorders. In this seminar, I will talk about our recent work, which has led to the identification of new targets for Alzheimer’s disease (AD). Increasing evidence suggests that synaptic dysfunction plays a significant role in the onset and progression of AD. We have recently identified distinct receptor-mediated signaling pathways, including receptor tyrosine kinase EphA4, which can regulate hippocampal synaptic strength and plasticity. Specifically, we have demonstrated the role of EphA4 as a negative regulator of synaptic plasticity and its importance in mediating synaptic dysfunctions during progression of AD, thus identifying this receptor as a promising target for drug development. We have also identified a small molecule EphA4 inhibitor from an in-house traditional Chinese medicine database and validated its ability to alleviate impaired synaptic plasticity in an AD transgenic mouse model. In addition to synaptic plasticity, innate immunity has emerged to be an important mediator of the pathogenesis of AD. Our recent work has suggested that impairment of interleukin-33 (IL-33) signaling is associated with early progression of the disease. Importantly, injection of IL-33 rescues synaptic dysfunctions and contextual memory deficits in an AD transgenic mouse model. The beneficial action of IL-33 is mediated by regulating the activation state of microglia: enhancing their β-amyloid phagocytic capacity and reducing proinflammatory response in the brain. Together, our work highlights the identification of novel molecular targets for neurodegenerative diseases, and opens up new opportunities in AD drug discovery and development Conferencia Premio Joven Investigador SEBBM-BIOTOOLS: a la actividad científica en España / SEBBM-BIOTOOLS Young Award Lecture: for scientific work in Spain CP05-1 The emerging role of stress kinases in tissue homeostasis and pathophysiology Guadalupe Sabio Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, ES Obesity is currently a major health problems that has reached epidemic proportions. Obesity is associated with other disorders collectively known as “metabolic syndrome”, characterized by impaired glucose tolerance, dyslipidaemia and hypertension. Moreover, obesity may be a predisposing factor for the development of some types of cancer, such as hepatocellular carcinoma. Inflammation plays an important role in obesity and its complications. Stress activated protein kinases (SAPKs), an important family of kinases implicated in the transduction of stress signals into the cell, could be key regulators of the development of diabetes and cancer. While the role of JNK in metabolic diseases has been studied for a long time, less is known about the role of the other stress kinase family, p38MAPK. We will discuss about the role of p38MAPK pathway in the pathophysiology of obesity and the development of related complications, such as cancer, diabetes and cardiovascular diseases. 12 XXXIX Congreso SEBBM Conferencia Premio Científico Margarita Lorenzo (Fundación Lilly) / Award Lecture Margarita Lorenzo (Fundación Lilly) CP06-1 La disminución de la expresión de s-resistina en el hipotálamo mejora la respuesta central y periférica a la insulina en ratas Wistar María Rodríguez Pérez Área de Bioquímica, Facultad de Ciencias Ambientales y Bioquímica; Centro Regional de Investigaciones Biomédicas, UCLM, Toledo La s-resistina es una variante no secretable de resistina, generada por splicing alternativo, que se expresa principalmente en tejido adiposo blanco e hipotálamo de ratas Wistar. Resultados previos confirmaron que células 3T3-L1 que expresaban s-resistina mostraron un incremento en la respuesta inflamatoria, así como un descenso en el transporte de glucosa estimulado por insulina. Además, la s-resistina, al igual que la resistina, bloqueó la vía de señalización de la insulina inhibiendo la fosforilación de IR, IRS-1 y Akt, e incrementó los niveles de SOCS-3. Por tanto, s-resistina podría actuar como un factor no secretado que regula la sensibilidad del adipocito a la insulina. En este trabajo se analiza el papel de s-resistina en el hipotalámo. Para ello se ha disminuido la expresión central de esta isoforma corta de resistina, inyectando, de forma i.c.v, un lentivirus que contiene un RNAi frente a dicha isoforma (LV-RNAi-s-res). La diminución central de s-resistina incrementa los niveles basales de fosforilación en Tyr, tanto en IR como en IRS-1, mientras que disminuye los niveles de fosforilación en Ser307 en IRS-1 en el hipotálamo. También se observa un incremento de los niveles de mRNA y de fosforilación en STAT-3, mientras que éstos disminuyen en el caso de PTP1b. Además, en los animales tratados con el LV-RNAi-s-res se promueven los efectos anorexigénicos, reduciendo la expresión de NPY e incrementando la de POMC, lo que conlleva un descenso de la ingesta. Estos datos muestran, por tanto, una mejora en la vía de señalización de insulina en aquellos animales que presentan disminuidos los niveles de expresión de s-resistina en el hipotálamo. Asimismo, se observa un incremento en la sensibilidad periférica a la insulina, como lo demuestran las curvas de tolerancia a la glucosa obtenidas en los animales tratados. Todos estos resultados sugieren que s-resistina podría actuar como un sensor neuronal intracrino, que actuaría regulando la sensibilidad a la insulina en todo el organismo. Conferencia plenaria Niemeyer / Niemeyer Plenary Lecture CP07-1 Mitochondrial non-coding RNAs: novel targets for cancer therapy? Luis Burzio Andes Biotechnologies Sp.A, Fundación Ciencia & Vida, and Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago de Chile, CL A family of mitochondrial lncRNAs (ncmtRNAs) arising from the mitochondrial 16S rRNA gene will be described. They comprise a Sense (SncmtRNA) and Antisense (ASncmtRNA) members, containing long inverted repeats (IR) and stem. These transcripts are differentially expressed according to proliferative status; both transcript types are expressed in normal proliferating cells, but not in resting cells. Tumor cells (human and mouse) express the SncmtRNA but down regulate the Salamanca 2016 ASncmtRNAs. We proposed that the ASncmtRNAs is tumor suppressor and a generalized hallmark of cancer. Knock-down of ASncmtRNA with antisense oligonucleotides (ASK for short) induces selective death of tumor cells. Tumor cell death displays characteristics of apoptosis and is mediated by down-regulation of Survivin and XIAP. Cell death is preceded by a drastic reduction in proliferative index, mediated through a marked decrease only of Cyclin D1 and B1. In addition, ASK induces drastic inhibition of sphere formation together with down regulation of N-cadherin and β-caveolin, pro-metastasic factors.The reduction in survivin, both cyclins, N-cadherin and β-caveolin seems to be mediated by microRNAs arising upon ASncmtRNA knock down. Indeed, ASK induces several microRNAs derived from the ASncmtRNAs. Among them, we found strong increase of hsa-miR-1973 and hsa-miR-4485 derived from the stem of the ASncmtRNAs. Mimics assay demonstrate that hsa-miR-4485 expression inhibition of cyclin D1 and B1 plus N-cadherin. Translation studies demonstrate that the ASncmtRNAs are potent target for cancer therapy in vivo. This results will include examples of syngeneic (melanoma cells and renal carcinoma cells) as well as xenograph assays with human prostate carcinoma (PC3 cells) and breast carcinoma (MDA-MB-231 cells). Conferencia plenaria SEBBM / SEBBM Plenary Lecture CP08-1 Epithelial plasticity in health and disease Ángela Nieto Instituto de Neurociencias, CSIC-UMH, Alicante, ES Epithelial homeostasis is crucial to maintain tissue architecture, and therefore, it needs to be tightly regulated in the adult. By contrast, embryonic cells show a high degree of epithelial plasticity required for proper morphogenesis and, in particular, for the implementation of massive cell movements that occur at gastrulation and neural crest delamination among other processes. We have been interested in the analysis of cell movements, plasticity and epithelial to mesenchymal transitions (EMT) for many years and found that the reactivation of developmental EMT-like programs in adult cells leads to several pathologies including tumor progression and organ degeneration. While the epithelial and mesenchymal cells are usually considered as extreme phenotypes, intermediate EMT states also exist. Under those circumstances cells depict an intermediate phenotype expressing both epithelial and mesenchymal markers and from which they can reverse to the original state or move towards a more mesenchymal phenotype. This hybrid transitory state can favor coordinated cell migration or wound healing and it can also enable the formation of clusters of migratory cancer cells than can better survive in the blood-stream and have increased tumor initiating potential. In contrast to the hybrid transitory state, there are particular contexts in which an intermediate phenotype Conferencias plenarios / Plenary Lectures can be defined as a final state, as we have recently shown to occur during renal fibrosis. I will present new data to suggest that evolution has favored the maintenance of the epithelial phenotype by attenuating the function of some EMT inducers. I will discuss the implications that this mechanism might have in embryonic development and in tumor progression toward the metastatic state. Conferencia de clausura / Closing Lecture Fundación Ramón Areces CP09-1 Angiogenesis revisited: role and (therapeutic) implications of endothelial metabolism Peter Carmeliet Laboratory of Angiogenesis and Vascular Metabolism, Vesalius Research Centre (VRC), Department of Oncology, KU Leuven, Leuven, BE The past 40 years of research in the angiogenesis field have focused on identifying genetic signals such as VEGF and Notch, which determine vessel sprouting. However, the role and therapeutic potential of targeting endothelial cell (EC) metabolism have been largely overlooked. We have recently reported that ECs are glycolysis addicted and that glycolysis importantly co-determine vessel sprouting downstream of VEGF and other pro-angiogenic signals. In addition, we documented that ECs are rather unique in utilizing fatty acid-derived carbons for the de novo synthesis of deoxyribonucleotides for DNA synthesis during EC proliferation when vessels sprout. Moreover, targeting (blocking) glycolysis and fatty acid oxidation inhibit pathological angiogenesis and induce tumor vessel normalization (thereby reducing metastasis and improving chemotherapy), suggesting that these metabolic pathways are new targets for anti-angiogenic drug development without evoking systemic side effects. Furthermore, lymphatic ECs differ from other EC subtypes in their metabolic requirements for lymphangiogenesis. Since many of these metabolic targets are pharmacologically druggable, these metabolic pathways represent a new promising target for therapeutic anti-angiogenesis. References [1] S. Schoors, et al. & P. CARMELIET. Fatty acid carbon is essential for dNTP synthesis in endothelial cells. Nature 520, 192-197 (2015). [2] S. Schoors, et al. & P. CARMELIET. Partial and transient reduction of glycolysis by PFKFB3-blockade reduces pathological ocular angiogenesis. Cell Metab 2014; 19(1):37-48. [3] K. De Bock, et al. & P. CARMELIET. Role of PFKFB3-driven glycolysis in vessel sprouting.Cell 154, 651-663 (2013). 13 14 Simposios Symposia 15 16 Simposio 1. Biomedicina molecular / Molecular biomedicine 17 18 Salamanca 2016 S1.1. Señalizacion por quinasas de estrés en la salud y en la enfermedad / Stress kinase signaling in heart and disease Simposios / Symposia to the effects of feeding a high fat diet, including protection against insulin resistance and failure of obesity development. We have used tissue-specific JNK-deficient mice to probe the mechanism of JNK regulation of insulin resistance and obesity. We show that JNK plays different roles in multiple tissues and that the phenotype of whole body JNK-deficient mice reflects the interactions between these different JNK-dependent processes. The molecular mechanisms of JNK function will be discussed. S1.1-1 News from alternative p38 in inflammation Ana Cuenda Centro Nacional de Biotecnología, CSIC, Madrid, ES p38MAPK pathways are central to inflammatory processes. The p38MAPK group has four members encoded by different genes, p38α, p38β, p38γ and p38δ. While the roles of the p38 isoform have been widely studied in the context of inflammation and tumour development, the knowledge of the in vivo role of p38γ and p38δ in these processes is still very limited. Our laboratory is studying essential functions of these two less studied alternative p38MAPKs, in vivo and in cultured cells. This strategy has lead to the discovery of unexpected cross talk between p38γ/p38δ and ERK1/2 pathway controlling cytokine production in cells during inflammation. The regulation of inflammatory processes by p38γ/p38δ has an impact in the development of different diseases such as colon and skin inflammation, arthritis, or colon cancer associated to colitis. We will discuss how, in specific settings, the p38MAPK components, p38γ and p38δ, operate in inflammatory processes, and the mechanisms underlying these unexpected functions. S1.1-2 Regulation of tumorigenesis by p38 MAPK signaling Ángel R. Nebreda Institute for Research in Biomedicine (IRB Barcelona) and ICREA, Barcelona, ES The p38a MAPK pathway plays key roles in the cellular responses to stress but can also integrate signals that affect many other cellular processes in a cell context- and cell type-specific manner. Studies using genetically modified mice have elucidated in vivo functions for p38a, and provided insights into how this pathway can suppress tumor initiation. There is evidence that normal cells rely on p38a signaling to engage various tumor suppressor mechanisms. However, p38a does not seem to be usually mutated in human tumors, and it appears that thissignaling pathway sometimes can acquire new roles facilitating tumor development. Using mouse models of cancer, we have obtained genetic evidence that transformed epithelial cells rely on the p38a pathway for survival and proliferation. Experiments using chemical inhibitors support a role for p38a signaling in tumor development in vivo. We are also investigating how the p38a pathway in stromal cells contributes to tumorigenesis. I will present results showing that myeloid cells rely on p38a signaling to support tumor progression by several mechanisms. S1.1-3 CONFERENCIA PABMB Metabolic stress signaling by the JNK signaling pathway S1.2. Versatilidad de las células mieloides. Aspectos básicos en polarización y reprogramación funcional de las mismas y su potencial terapéutico / Versatility of myeloid cells: basic aspects aspects on polarization and functional reprogramming and therapeutic potential S1.2-1 Polarización de macrófagos: aspectos básicos y nuevos reguladores Sonsoles Hortelano Unidad de Terapias Farmacológicas, ISCIII, Madrid, ES Las células mieloides constituyen un grupo heterogéneo esencial en inmunidad. En los últimos años, los macrófagos han adquirido especial relevancia, no solo porque desempeñan un papel clave en la defensa contra patógenos y contribuyen a la homeostasis del tejido y la reparación; sino porque además se encuentran implicados en un amplio espectro de patologías como el cáncer, la obesidad, enfermedades respiratorias o en enfermedades inflamatorias crónicas como la enfermedad de Crohn. Esta diversidad en sus funciones viene determinada por la capacidad que presentan estas células para adaptarse al medio ambiente que les rodea, pudiendo adoptar distintos estados de activación. Simplificando la gran heterogeneidad de estas células, podemos distinguir dos tipos de macrófagos, identificados como M1 (macrófagos clásicos o pro-inflamatorios) y M2 (macrófagos alternativos o anti-inflamatorios), que difieren en la expresión de receptores, funciones y producción de citoquinas y quimioquinas. El delicado balance entre ambas poblaciones viene marcado por un conjunto de señales, incluyendo factores de transcripción, cambios epigenéticos, reguladores post-transcripcionales,…, que modifican sus estados de activación. A pesar de los recientes avances en el estudio de la biología de los macrófagos, todavía queda mucho camino por recorrer. Sin duda, una mejor comprensión de las bases moleculares y los mecanismos implicados en la plasticidad de estas células permitirá el diseño de estrategias terapéuticas más eficaces. S1.2-2 Los receptores Notch en el control de la activación proinflamatoria de los macrófagos Roger J. Davis Howard Hughes Medical Institute (HHMI), University of Massachusetts, Chevy Chase, US María José Martínez Díaz-Guerra Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Medicina, Universidad de Castilla-La Mancha, Albacete, ES The cJun NH2-terminal kinase (JNK) signaling pathway is implicated in the pathogenesis of diabetes.High fat diet-induced obesity causes activation of JNK in insulin target tissues. JNK-deficient mice are resistant Los receptores Notch constituyen una ruta de señalización muy conservada que es esencial en procesos de desarrollo y diferenciación celular. En el sistema inmune está bien documentada su participación en el desarrollo 19 Simposios / Symposia y la activación de las células T y B. Múltiples trabajos han asociado la activación de la ruta de Notch con procesos inflamatorios, sin embargo los mecanismos por los que la infección y la inflamación modulan la señal de Notch, y cómo esta ruta puede condicionar la respuesta de las células mieloides, no están completamente establecidos. Nuestro grupo estudia la función que los receptores Notch desarrollan en la respuesta de los macrófagos a la activación por los receptores Toll. Estos inducen la expresión de todos los receptores Notch, especialmente Notch1, y de ligandos de las familias Jagged y Delta, así como la de genes diana clásicos de la vía como Hes1 y Hey1 y 2. La activación de los receptores Toll incrementa también la expresión y/o activación de diferentes proteasas implicadas en el procesamiento de los receptores Notch como la furina, ADAM10 y ADAM17, y la presenilina1 que forma parte del complejo γ-secretasa, así como el de otras moléculas que favorecen la activación de estas proteasas como la tetraspanina TSPAN33. La señalización a través de los receptores Notch a su vez favorece la expresión de diversas citoquinas proinflamatorias tras la activación de los receptores Toll como IL-6, IL-12 o TNFα, o enzimas como la Óxido Nítrico Sintasa inducible, o COX-2. Este efecto está mediado por la unión directa de Notch/RBP-J a los promotores de algunos de estos genes, o de forma indirecta por un incremento en la actividad de factores de transcripción como NFκB e IRF8. Nuestros resultados y los de otros grupos muestran que los receptores Notch actúan favoreciendo la activación de los macrófagos en la respuesta inmune e inflamatoria. Este proceso parece condicionar el desarrollo de algunas patologías inflamatorias, lo que sitúa a estos receptores, sus moduladores y sus dianas como posibles puntos de acción farmacológica. S1.2-3 Control de la polarización antiinflamatoria de macrófagos humanos y sus implicaciones patológicas Ángel L. Corbí Grupo de Biología de las Células mieloides, Centro de Investigaciones Biológicas (CIB), CSIC, Madrid, ES Macrophages display a huge functional heterogeneity and acquire proor anti-inflammatory functions depending on the surrounding tissue microenvironment. In response to Th1 cytokines, macrophages acquire pro-inflammatory, bactericidal, tumoricidal and immunogenic activities (“M1 polarization”), whose hallmark is the production of TNFalpha and IL-12/IL-23. Conversely, other factors (IL-4, TGFbeta) promote “M2 polarization”, characterized by enhanced anti-inflammatory, scavenging, tumor-promoting, tissue repair and pro-angiogenic functions. The pathophysiological relevance of the balance between macrophage polarization states is best exemplified during inflammation. M1 macrophages predominate at the initial stages of an inflammatory response, whereas M2 macrophages drive resolution of inflammation, and their sequential occurrence is required for termination of inflammatory responses, and for tissue repair after injury. In fact, deregulation of the macrophage polarization balance leads to chronic inflammatory pathologies (e.g., rheumatoid arthritis, fibrosis, obesity), and its subversion is used by tumors to escape from immune-surveillance and to foster their own proliferation and dissemination. Thus, determination of the mechanisms underlying the differences between polarization states should lead to the identification of molecules whose targeting might allow modulating macrophage effector functions under homeostatic or pathological conditions. We have previously defined the transcriptomic profiles of macrophages generated in the presence of either GM-CSF (pro-inflammatory GM-MØ) or M-CSF (anti-inflammatory M-MØ), and identified genes/proteins whose expression distinguishes pro-inflammatory macrophages or homeostatic tissue-resident macrophages in vivo. With this background, the presentation will focus on the existence of an anti-inflammatory gene signature in human macrophages, whose acquisition is dependent on the MAFB transcription factor. The importance of MAFB in the anti-inflammatory polarization of human macrophages will be illustrated with data generated on samples from 20 XXXIX Congreso SEBBM a rare osteolytic disease (MCTO). Besides, the presentation will address the molecular basis of the serotonin ability to inhibit pro-inflammatory cytokine production, describing the receptors involved and identifying the serotonin-regulated genes in human macrophages. S1.3. Hipoxia: de los mecanismos moleculares a las aplicaciones clínicas/ Hypoxia: from molecular mechanisms to clinical applications S1.3-1 Acute oxygen sensing mechanisms José López-Barneo Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, Sevilla, ES Acute O2-sensing is essential for adaptation of mammals to changing environments and pathophysiological conditions. Upon exposure to hypoxia, arterial chemoreceptors, in particular the carotid body (CB), mediate fast (seconds) cardiorespiratory responses (hyperventilation and sympathetic activation) that compensate the lack of O2. These life-saving reflexes are necessary for survival at high altitude and at sea level contribute to the eupneic drive to breathe as well as to increase ventilation in hypoxemic patients with chronic obstructive pulmonary diseases. In addition to the CB, other organs, such as the adrenal medulla (AM) and the pulmonary arteries, are also responsive to acute hypoxia. Therefore, the understanding of the acute O2 sensing mechanisms has a major biomedical interest. Responsiveness to hypoxia depends on specialized chemoreceptors cells, which contain O2-sensitive K+ channels. In CB cells, closure of these channels during hypoxia leads to cell depolarization and the release of transmitters that activate excitatory fibers terminating at the brain stem respiratory center. We have shown that ablation of the Ndufs2 gene, which encodes a 49 kD protein at the ubiquinone binding site of mitochondrial complex Iresults in mice with selective abolition of the hypoxic ventilatory response. Ndufs2-deficient chemoreceptor cells from the CB and the AM are insensitive to hypoxia although show normal responses to other stimuli such as hypercapnia or hypoglycemia. Our current model of acute O2 sensing proposes that the slow down of mitochondrial electron transport during hypoxia causes a succinate-dependent accumulation of reduced quinone (QH2), which results in a burst of NADH and ROS. These, in turn, modulate ion channels in O2-sensing membrane microdomains. S1.3-2 Hypoxic regulation of the innate immune response Sarah Walmsley MRC Centre for Inflammation Research, University of Edinburg, Edinburg, UK The treatment of both sepsis and the adult respiratory distress syndrome represent huge clinical challenges and areas of unmet therapeutic need. In acute settings, hypoxemia is associated with increased morbidity and mortality of bacterial infections, particularly in the context of development of ARDS. We questioned whether more prolonged hypoxic preconditioning could modify the clinical outcome to infection. In both a local subcutaneous S. aureus skin infection model and a systemic S. pneumoniae model of bacteraemic pneumonia pathophysiological responses to infection are pre-determined by prior exposure to hypoxia. We observe these outcomes to be a consequence of the transcriptional regulation of leukocyte HIF-1a pathways, with myeloid cell responses to altered oxygen and nutrient availability a critical determinant of overall morbidity and mortality. This work provides evidence of imprinted changes in the functional memory of Salamanca 2016 myeloid cell populations, and supports the concept that strategies to target the host response in addition to direct anti-microbial targeting will be required to improve outcomes where HIF-1 hypoxia and infection co-exist. S1.3-3 Identification of non-coding genetic variants in samples from respiratory disease patients that affect the transcriptional response to hypoxia Luis del Peso Departamento de Bioquímica, Universidad Autónoma de Madrid, Madrid, ES A wide range of diseases course with an unbalance between the consumption of oxygen by tissues and its supply. This situation triggers a transcriptional response, mediated by the hypoxia inducible factors Simposios / Symposia (HIFs), that aims to restore oxygen homeostasis. Little is known about the inter-individual variation in this response and its role in the progression of disease. Herein, we sought to identify common genetic variants affecting the hypoxia response elements (HREs) and characterize their effect on transcription. To this end, we constructed a list of genome-wide HIF-binding regions from publicly available experimental datasets and studied the genetic variability in these regions by targeted re-sequencing of genomic samples from 96 chronic obstructive pulmonary disease patients and 144 obstructive sleep apnea patients. This study identified 13 frequent variants in potential HREs, which is significantly lower than expected by chance. Analysis of the loci containing these variants by means of reporter assays showed that these regions drove the transcription of reporter genes under low oxygen that the alternate alleles blunted this response. Finally, using genome editing we confirmed the functional role of these variants in a physiological context. Altogether our results suggest that common polymorphisms contribute to determine the efficiency of transcriptional activation of target genes by hypoxia. 21 22 Simposio 2. Regulación génica y comunicación celular / Gene regulation and cellular communication 23 24 Salamanca 2016 S2.1. Fronteras en biología de plantas: de las moléculas al organismo / Frontiers in plant biology: from molecules to the organism (Simposio Hispano-Mexicano) Simposios / Symposia demostrado que la señal responsable de los fenotipos de interés es un apocarotenoide generado por el rompimiento de carotenos lineales por la digoxigenasa CCD4. Estudios recientes han identificado genes alterados por esta señal con funciones en el desarrollo de la hoja, así como secuencias en cis importantes para esta regulación. S2.1-3 La reparación por escisión de bases como mecanismo de control epigenético S2.1-1 Abordajes sistemáticos para el estudio de la división de las células madre en plantas Ana I. Caño Delgado Centre de Recerca en Agrigenòmica (CRAG), Bellaterra (Cerdanyola del Vallés), ES El mantenimiento de los nichos de células madre en una pregunta central de la biología fundamental. Nuestro laboratorio ha identificado recientemente una vía de señalización necesaria para la quiescencia celular del nicho de células madre en la raíz primaria de la planta modelo Arabidopsis. BRAVO (BRASSINOSTEROIDS AT VASCULAR AND ORGANIZING CENTRE) es un factor de transcripción de tipo R2R3-MYB que funciona como un represor específico de la división del centro quiescente en el nicho de células madre de la raíz. El análisis molecular de la vía BRAVO ha revelado un mecanismo que controla las divisiones de células específicas de esteroides mediada por Brasinoesteroides. Estos resultados abren una nueva línea para el estudio de las células madre de la planta. Actualmente, estamos llevando a cabo la identificación de las redes de señalización que operan durante la división de las células madre basándonos en la integración de los datos genómicos con resolución celular, en combinación con imágenes de alta resolución espaciotemporal y modelado matemático. En Salamanca, presentaremos una visión actualizada de la señalización en el Centro Quiescente (QC) y en las células madre vasculares adyacentes, así como nuestro avances hacia la identificación de las firmas transcripciones de células madre basadas en el análisis genómico de una sola célula. S2.1-2 La participación de carotenos lineales en el desarrollo de la hoja Patricia León, Julio Sierra, Elizabeth Cordoba, Ernesto Llamas Instituto de Biotecnología UNAM MX, Cuernavaca, MX Los cloroplastos son esenciales para la fotosíntesis, sin embargo, estos organelos también funcionan como fabricas en la síntesis de múltiples compuestos esenciales de las plantas. La diferenciación del cloroplasto depende de señales nucleares coordinadas a la diferenciación del tejido fotosintético. Sin embargo, los plastidos también generan señales retrógradas con que transmiten su estado metabólico y de diferenciación alterando la expresión de nuclear y regulando el desarrollo y las respuestas de la planta. Se reconocen múltiples vías de señalización retrógrada, pero sólo pocas de las señales responsables. Estudios recientes han mostrado que los carotenoides en adición a su función capatadora de luz y fotoprotectiva, también sirven como señales regulatorias. La caracterización de la mutante clb5 de Arabidopsis ha proporcionado evidencias que intermediarios lineales de carotenoides son capaces de modular la expresión nuclear y plastídica, así como el desarrollo de la hoja. Nuestro trabajo demuestra que los fenotipos asociados asociados a clb5 son una consecuencia de la mutación en la enzima zeta coroteno desaturasa (ZDS), que provoca la acumulación de los intermediarios fitoflueno y -carotenos, y no se observan en otras mutantes deficientes de carotenos. El patrón de expresión de ZDS en la planta apoya a que esta enzima juega un papel importante durante el desarrollo de la planta. Análisis genéticos y moleculares han Dolores Córdoba-Cañero, María Isabel Martínez-Macías, Jara Teresa Parrilla-Doblas, María Isabel Ponferrada-Marín, Teresa Morales-Ruiz, María Victoria García-Ortiz, Casimiro Barbado, Iván Devesa, Raphael Ricon, Rafael R. Ariza,Teresa Roldán-Arjona Departamento de Genética, Universidad de Córdoba (UCO)/Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC)/ Hospital Universitario Reina Sofía, Córdoba, ES La ruta de Reparación por Escisión de Bases (Base Excision Repair, BER) es esencial para eliminar lesiones frecuentes del ADN y se ha estudiado con detalle en modelos animales y microbianos, pero no en plantas. Este mecanismo de reparación consta de varias etapas y se inicia por la acción de enzimas denominadas ADN glicosilasas, que eliminan bases dañadas. El trabajo llevado a cabo en Arabidopsis por nuestro grupo y por otros laboratorios ha permitido identificar una familia de ADN glicosilasas específicas de plantas (familia ROS1/DME) que eliminan 5-meticitosina (5-meC). La 5-meC no es una lesión, sino una marca epigenética que suprime la expresión génica y desempeña un papel clave en el desarrollo y la diferenciación celular. Las enzimas de la familia ROS1/DME inician una ruta de escisión para borrar la metilación del ADN y no tienen equivalentes en células animales. Son ADN glicosilasas atípicas, anormalmente voluminosas y con un dominio catalítico discontinuo con residuos que intervienen en el reconocimiento de la base metilada y su escisión. Tras liberar la 5-meC estas enzimas rompen el esqueleto azúcar-fosfato, generando un hueco mono-nucleotídico con un extremo 3´ bloqueado por un fosfato o por un aldehído insaturado. Una ADN fosfatasa (ZDP) o una endonucleasa (APE1L) convierten estos extremos en un grupo 3´-OH, permitiendo así que una ADN polimerasa aún por identificar inserte una citosina no metilada y una ADN ligasa (LIG1) selle la cadena procesada. En esta ruta de desmetilación también intervienen proteínas adicionales. En conjunto, estos resultados demuestran que las plantas usan la ruta BER no solo para proteger su genoma, sino también para reprogramar su epigenoma. S2.2. Más allá de los genes: análisis funcional del resto del genoma / Beyond the genes functional analysis of the rest of the genome S2.2-1 Regulation of genome function: genes versus structure Miguel Manzanares Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, ES Recent studies have demonstrated an unexpected link between chromatin structure and gene activity. The generation of high-resolution chromatin interaction maps has revealed that the genome folds into distinct megabase-scale modules termed topologically associated domains (TADs) that show a direct relationship to gene expression and regulation within them. CTCF is a ubiquitously expressed and highly conserved chromatin architectural protein enriched at the boundaries 25 Pósters / Posters of these domains. Most strikingly, TAD organization is mostly stable among different cell-types and developmental stages independent of the gene activity status within them, and also shows a surprising evolutionary conservation in structure. Furthermore, depletion of CTCF or other chromatin structural proteins does not cause a dramatic disassembling of the organization of the genome. Altogether, these observations pose the conundrum of why is the genome organized in such way, and how does this occur? We are exploring these issues in different developmental systems to explore how the structuring of the genome can regulate gene expression. S2.2-2 Transcriptional regulation of CD69 gene through the enhancer located in the conserved non-coding sequence (CNS)2 Pilar Lauzurica, Laura Notario, Teresa Laguna, Miguel G. Fontela, Isabel Cervera, Jennifer Redondo, Berta Vázquez Instituto de Salud Carlos III, Majadahonda, Madrid, ES The CD69 type II C-type lectin is one of the earliest indicators of leukocyte activation acting in lymphocyte migration and cytokine secretion. CD69 expression in hematopoietic lineage undergoes rapid changes depending on the cell-lineage, the activation state or the localization of the cell where it is expressed, suggesting a complex and tightly controlled regulation. We have examined the molecular mechanism underlying mouse CD69 gene transcription in silico, in vitro and in vivo. Analysis of the CD69 gene revealed evolutionary conservation at the promoter and at four noncoding sequences (CNS) that were called CNS1, CNS2, CNS3, and CNS4. These regions were found to be hypersensitive sites in DNase I digestion experiments, and chromatin immunoprecipitation assays showed specific epigenetic modifications. Through in silico studies, we analyzed several regulatory features of the 4 CNSs, confirming a major function of CNS2 in the transcriptional regulation of CD69. In addition, multiple transcription binding sites are identified in the CNS2 region by DNA cross-species conservation analysis. CNS2 displayed constitutive and inducible enhancer activity in transient transfection assays in T cells. By functional approaches we defined a core region of 226bp located within CNS2 as the main enhancer element of CD69 transcription in the hematopoietic cells analyzed. However, using a transgenic approach to test CNS function, we found that the CD69 promoter conferred developmentally regulated expression during positive selection of thymocytes but could not support regulated expression in mature lymphocytes. Inclusion of CNS1 and CNS2 caused suppression of CD69 expression, whereas further addition of CNS3 and CNS4 supported developmental-stage and lineage-specific regulation in T cells but not in B cells. S2.2-3 Using CRISPR tools to functionally analyze the non-coding genome Davide Seruggia1, Santiago Josa1, Almudena Fernández López2, Lluís Montoliu3 1 CNB-CSIC, Madrid, ES, 2CIBERER-ISCIII y CNB-CSIC, Madrid, ES, 3 Centro Nacional de Biotecnología (CNB-CSIC), Madrid, ES Most part of mammalian genomes is non-coding and contains DNA regulatory elements that are essential for proper gene expression. However, these key genetic elements are surrounded by many repetitive elements, such as retrotransposons, which interfere with homologous recombination-mediated approaches and, hence, usually 26 XXXIX Congreso SEBBM prevent any direct functional assessment. The recent development and implementation of genome-editing technologies, notably CRISPR-Cas, has enabled, for the first time, targeting these small regulatory elements and allowed their functional evaluation to assess their relevance in overall gene expression regulation. For many years we were traditionally using large-genomic size transgenes (i.e. yeast artificial chromosomes) to assess the function of regulatory elements of certain experimental gene models. However, this approach had many limitations, mostly related with the random nature of the genetic modification, prone to chromosomal position effects. We have applied CRISPR-Cas9 tools to functionally analyze several key DNA regulatory elements that we have been characterizing in the mouse tyrosinase locus. We tested the gene-editing tools in cells first and we next produced the corresponding genome-edited animals. In this presentation we will discuss the variety of mutated alleles generated, the mosaicism detected in these experiments and how we have been able to segregate most of these alleles in order to analyze the phenotype of any of them, independently. S2.3. Papel de los exosomas y carga exosómica en sinapsis inmune, cáncer y células neuronales / Role of exosomes and exosomic cargo in the immune synapse, cáncer and neural cells S2.3-1 Immune Cell-to-Cell communication: Mechanisms of microRNA and proteins sorting into exosomes Francisco Sánchez-Madrid Intercellular Communication Laboratory, National Center Cardiovascular Research and Instituto Investigación Sanitaria Princesa, Universidad Autónoma de Madrid, Madrid, ES Exosomes are released by most cells to the extracellular environment and are involved in cell-to-cell communication, mediating transfer of proteins and RNAs through structures as the Immunological synapse. Exosomes arising from different cell types show enrichment for a shared set of specific proteins, including tetraspanins, adhesion molecules, endosomal proteins, and heat-shock proteins, among others. They are also very much enriched in RNAs, including miRNAs and other non-coding RNAs. Exosome composition is not a mere copy of cytosolic content; rather, specific proteins and nucleic acids are selectively sorted into exosomes. The amount and content of exosomes can moreover change in response to stimuli such as cell activation, hypoxia or cell stress. Such changes in exosome composition determine the final outcome of exosome-mediated communication. However, the mechanisms that control these processes are not well understood. First, we will discuss on the mechanisms of miRNA and protein sorting into EVs. In this regard, we have identified sequence motifs present in miRNAs that control their localization into exosomes. The protein heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) specifically binds exosomal miRNAs through the recognition of these motifs and controls their loading into exosomes. Moreover, hnRNPA2B1 in exosomes is sumoylated, and sumoylation controls the binding of hnRNPA2B1 to miRNAs. Second, we will discuss post-translational modifications of proteins that control exosomal sorting. Our recent data demonstrate that ISGylation, an ubiquitin-like modification, controls exosome secretion. Salamanca 2016 Simposios / Symposia S2.3-2 S2.3-3 Exosomes and metastasis: the dangerous alliance Extracellular vesicles as messengers between neural cells and beyond Héctor Peinado Microenvironment and Metastasis Group, Molecular Oncology Program, Spanish National Cancer Research Centre (CNIO), Madrid, ES Cancer is a systemic disease, while most of the research effort has been focused on analyzing the primary tumor, there is a lack of information on how the tumor microenvironment influences metastasis. Prominent roles for stromal cells, such as fibroblasts, endothelial cells, lymphatic endothelial cells, bone marrow-derived cells, soluble factors and secreted vesicles have been established. Exosomes are secreted vesicles carrying lipids, proteins, RNA and DNA molecules. By carrying these molecules, and facilitating their cell-to-cell transfer, exosomes can modulate the behavior of resident cells that can impact disease progression. Exosomes can serve as vehicles for horizontal transfer of proteins, RNA and DNA to the surrounding cells, thus promoting additional modifications in the tumor and metastatic microenvironments. Our studies in demonstrated that tumor-secreted exosomes are a major tumor-derived factor that acts systemically to promote metástasis in a process that we have termed “education”. Tumor exosomes fuse specifically to stromal cells within the metastatic microenvironments favoring organotropic metastasis. Our most recent data demonstrate that exosomes secreted by metastatic models promote vascular leakiness in the sentinel lymph node favoring metastatic spread. We found that lymphatic endothelial cells and subscapular sinus macrophages are the main cell types uptaking exosomes in the lymph nodes promoting cellular and molecular alterations in the lymph node microenvironment fostering metastasis. The analysis of human lymphatic fluid suggest that proteomic cargo and particles are increased in melanoma patients opening the possibility of the use of circulating vesicles in lymphatic fluid as biomarkers and as a source of novel markers in pre-metastatic sentinel lymph nodes that predict relapse and metastatic potential. Eva-Maria Krämer-Albers Department of Molecular Cell Biology, Johannes Gutenberg University (JGU), Mainz, DE Exosomes are universally secreted vesicles that mediate cell communication in multiple tissues, including the CNS. Exosomes deliver a specific set of biomolecules including proteins, lipids and small non-coding RNAs modulating the phenotypic behavior of recipient cells.Our recent work revealed that exosomes, are involved in bidirectional interaction between CNS glia cells and neurons. Myelinating oligodendrocytes release exosomes in response to neurotransmitter signalling and activation of glial ionotropic glutamate receptors. These exosomes are internalized by neurons via endocytosis and the exosome cargo is functionally recovered by the recipient neurons. Neurons appear to benefit from exosome internalization by increased resistance to stress conditions such as oxidative stress, starvation and ischemia indicating a role of glia to neuron exosome transfer in glial support and neuroprotection. Oligodendroglial exosomes have the ability to modulate a broad spectrum of neuronal functions. Treatment of cultured neurons with isolated glial exosomes affects action potential firing and axonal transport, activates signal transduction pathways, and regulates neuronal gene expression. We further studied exosome release from oligodendrocytes derived from PLP- and CNP-deficient mice, which are characterized by secondary axonal degeneration, and found that exosome secretion in these mouse models is impaired. Intriguingly, exosomes derived from knock-out oligodendrocytes lack neuroprotective activity and the ability to promote axonal transport. In summary, we propose that oligodendroglial exosomes function as vehicles for the transfer of biomolecules from oligodendrocytes to neurons and are implicated in neuroprotection and glial maintenance of axonal integrity. Supported by DFG. 27 28 Simposio 3. Estructura y función de biomoléculas / Structure and function of biomolecules 29 30 Salamanca 2016 S3.1. Dinámica estructural del genoma/ Genome structural dynamics S3.1-1 Mammalian sperm exhibit priming of embryonic and adult regulatory landscapes Víctor Corcés Department of Biology, O. Wayne Rollings Research Center Emory University, Atlanta, US Environmental factors increase the frequency of metabolic disorders. The mechanisms by which these epiphenotypes are transmitted between the exposed and subsequent generations through the paternal germline remains poorly understood. The nuclei of mammalian sperm are highly condensed, the DNA is mostly covered by protamines with only a few retained nucleosomes, and epigenetic information stored in the form of DNA methylation is quickly erased from paternal chromosomes shortly after fertilization. Experiments carried out in our lab suggest a more complex picture of mouse sperm, suggesting the presence of multiple histone modifications, nucleosomes positioned around transcription start sites and transcription factor binding sites, and the presence of multiple transcription factors. Most promoters in mouse sperm contain the elongating form of RNA polymerase II, and are flanked by several positioned nucleosomes marked by a variety of active histone modifications. The sperm genome is bound by several transcription factors, including Nanog, Oct 4, Mediator, condensin, and the pioneer factor FoxA1. These proteins are found at promoters, enhancers, and super-enhancers, many of which are active in mESCs or adult tissues. CTCF and cohesin are also present in sperm DNA, where they mediate interactions that organize the sperm genome into domains and compartments that overlap extensively with those found in mESCs. This information suggests that mammalian sperm contain a rich and complex palette of epigenetic information that could be altered by environmental factors to paint novel phenotypic outcomes in the next generation. Simposios / Symposia S3.1-3 Structural dynamics of the breast cancer genome in response to hormones Miguel Beato Centro de Regulación Genómica (CRG), Barcelona Science and Technology Institute (BIST), Barcelona, ES Eukaryotic cells decode environmental information via receptors and signalling networks that converge in the cell nucleus to adjust an integrated gene expression response. We study the response of breast cancer cells to progestins (Pg) acting via the progesterone receptor (PR) to decipher the underlying molecular mechanisms. The organization in nucleosomes of the DNA sequences recognized by PR is a requisite for the initiation of chromatin remodelling leading to displacement of histones H1 and H2A/H2B. Remodelling depends on PR-associated enzymes, including histone modifying enzymes and ATP-dependent remodelers, as well as activated PARP1 that leads to accumulation of Poly-ADP-Ribose (PAR) in he cell nucleus. These epigenetic processes are required for the rapid regulation of thousand of genes and ultimately for cell proliferation in response to hormone. In addition to nucleosomes, higher levels of genome organization also participates in hormone action. The conserved partition of the genome in consecutive topological associating domains (TADs) contributes to coordination of the hormonal response. Hormone regulated genes tend to segregate into TADs that respond as a whole with either activation or repression of transcription. Thus, TADs behave as “regulons” in the response of cells to external signals. High-resolution Hi-C data reveal the role of dominant enhancers in organizing the differential hormonal response of the TADs. The extensive chromatin remodelling in response to hormones requires large amounts of ATP, which is mainly generated in the cell nucleus by NUDIX5 that uses ADP-Ribose (ADPR) derived from PARG-mediated hydrolysis of PAR. NUDIX5 is an homodimer that catalyses hydrolysis of ADPR to AMP and R-5-P. In response to hormone NUDIX5 is dephosphorylated at T45, leading to a conformational change that enables the reaction of ADPR with PPi to generate ATP and R-5-P. NUDIX5 is overexpressed in breast cancers and is a marker for poor prognosis. Thus, it represents a novel target for breast cancer management. S3.1-2 Genome architecture mapping of multi-enhancer contacts in embryonic stem cells Ana Pombo Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine (MDC), Berlín, DE S3.2. Formación y funcionamiento de los ribosomas / The synthesis and function of ribosomes S3.2-1 The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption causes disease. Technologies based on chromosome conformation capture (3C) have profoundly expanded our understanding of the role of genome architecture in gene regulation. We introduce Genome Architecture Mapping (GAM), a novel genome-wide method for measuring three-dimensional chromatin topology. Exploration of the most prominent chromatin contacts detected in mouse ES cells using GAM identifies most specific chromatin contacts between active genes and enhancers across very large genomic distances. GAM also reveals abundant three-way contacts genome-wide, especially between the enhancers most highly occupied by pluripotency transcription factors and highly transcribed genomic regions.Our results highlight a previously inaccessible complexity in genome architecture and a major role for contacts related with gene expression in the structural organisation of the genome in mammalian nuclei. Ribosome assembly and functions of ribosomal proteins Jesús de la Cruz Instituto de Biomedicina de Sevilla, Universidad de Sevilla, Sevilla, ES Ribosomes are highly complex ubiquitous macromolecular assemblies that are responsible for the synthesis of all cellular proteins from mRNA templates. In all cells, ribosomes are composed of two ribosomal subunits, the large one about twice the size of the small one. In eukaryotes, biogenesis of cytoplasmic ribosomes is a complex process that takes successively place in the nucleolus, nucleoplasm and cytoplasm. The speed, directionality and accuracy of this process depend on a myriad of small nucleolar RNAs and protein trans-acting factors. Functional analyses have also demonstrated that many ribosomal proteins directly contribute to the maturation and nucleo-cytoplasmic transport of pre-ribosomal particles. The principles governing the assembly of ribosomal proteins and how these molecules 31 Pósters / Posters assemble together remain challenging questions, especially in eukaryotes. Our laboratory is interested in understanding the role of ribosomal proteins, mainly from the large subunit, in ribosome biogenesis and function. In this presentation, I provide details on how selected ribosomal proteins contribute to these processes. Moreover, many evolutionarily conserved ribosomal proteins possess extra eukaryote-specific aminoor carboxy-terminal extensions and/or internal loops. I will also discuss on an emerging interesting question in our field, the functional significance of these extensions for the proper assembly and function of ribosomes. S3.2-2 The activity of the ribosome synthesis pathway during proliferation. Causes and consequences of nucleolar stress Mercedes Dosil Departamento de Bioquímica y Biología Molecular y Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, ES Ribosome synthesis takes place within the most prominent nuclear substructure, the nucleolus. It is a multistep process that entails pre-rRNA transcription, pre-rRNA processing, and assembly of ribosomal proteins imported from the cytoplasm. These activities require the participation of more than 200 trans-acting factors. The majority of studies on ribosome formation have been performed in yeast, and it has long been assumed that most features are similar in humans. However, although the basic aspects are conserved, there are many maturation events, and links to other processes, that are unique of higher eukaryotes. One such link is the nucleolar stress response, which is mediated by p53, and is triggered by alterations of ribosome synthesis. This response is receiving intense attention because it is the cause of various human diseases known as ribosomopathies. Despite this growing interest, progress in understanding ribosome synthesis in humans is greatly hampered by technical limitations. Powerful approaches used in yeast, such as genetic screens to identify trans-acting factors or purification of pre-ribosomes by tandem-affinity chromatography, are not possible in human cells. In our laboratory we have optimized a series of techniques to monitor the formation of different pre-ribosomal complexes in human cells. We are studying normally-growing cells and cells blocked at specific steps of ribosome maturation. Data will be shown on the defects in ribosome synthesis that occur in Diamond-Blackfan anemia and on the mechanisms underlying p53 activation in this ribosomopathy. S3.2-3 The function of ribosomes at the structural level. Cryo-electron microscopy analysis of translating ribosomes Mikel Valle Unidad de Homeostasis de Proteínas, CIC BioGUNE, Derio, Bizkaia, ES The ribosome is the large macromolecular complex where translation takes place. This bio-synthesis of proteins involves numerous steps and the functioning of ribosomes is modulated by the interaction with several translation factors. The design of ribosomes into two subunits is related to their specialized work in reading the mRNA (small ribosomal subunit) and writing the coded polypeptide (large ribosomal subunit). The interplay between subunits takes care of the selection of cognate aminoacyl-tRNAs, and the maintenance of the reading frame in the mRNA. The presence of structured mRNAs, the nature of the nascent polypeptide chain, and the involvement of cellular factors during stress conditions, are some of the elements 32 XXXIX Congreso SEBBM that regulate translation by ribosomes. The highly dynamic ribosomal structure has been a subject of deep investigation. Cryo-electron microscopy (cryoEM) has contributed to unveil the structure/function relationship of the translating ribosome. The latest developments in cryoEM technique have improved the resolution of the attainable 3D maps, and there is a large number of ribosomal cryoEM structures at atomic detail. These advances will be discussed in the context of ribosomal functioning. S3.3. Dinámica de la cromatina y estabilidad genómica / Chromatin dynamics and genome stability S3.3-1 Nucleosome positioning in vivo driven by the DNA sequence Francisco Antequera Instituto de Biología Funcional y Genómica, CSIC, Universidad de Salamanca, Salamanca, ES Nucleosomes facilitate the packaging of the genome inside the nucleus and modulate the access of regulators to the DNA molecule. Most of the yeast genome is organized in a pattern of positioned nucleosomes that is stably maintained under different genetic backgrounds and physiological conditions. While the contribution of chromatin remodelers and DNA binding proteins to maintain this organization is well established, the relevance of the DNA sequence to nucleosome positioning in the genomic context remains controversial. We have searched for sequence determinants associated with positioned nucleosomes in several species of fission and budding yeasts. We have found that the distribution of the four mononucleotides along mononucleosomal DNA follows a well-defined pattern. Despite the high degree of histone conservation, these nucleosomal signatures are species-specific and are present in coding and non-coding regions. In the case of open reading frames, they correctly predict the relative distribution of codons on mononucleosomal DNA and establish a direct connection between the position of each codon around the histone octamer and protein composition. High-resolution mutagenenesis of mononucleosomal DNA reveals that sequence changes alter the nucleosomal pattern at the level of individual nucleosomes suggesting the existence of sequence elements contributing to positioning. Functional analyses of these elements show that it is possible to target nucleosomes to specific positions on natural or synthetic DNA molecules in the genomic context. S3.3-2 The importance of the mitotic spindle integrity in DNA repair Facundo Ramos, María Teresa Villoria, Encarnación Dueñas, Andrés Clemente-Blanco Instituto de Biología Funcional y Genómica, CSIC, Universidad de Salamanca, Salamanca, ES Maintenance of genome integrity is a vital aspect of our cellular physiology. Our hereditary information encoded in the DNA is constantly threatened by both endogenous and environmental genotoxic stresses that could alter our genomic material. To combat this threat, eukaryotic cells have evolved a series of mechanisms, collectively known as DDR, to survey several features of the cellular response, including the detection of the lesion, a transient cell cycle Salamanca 2016 arrest and the repair of the broken DNA. During the last years it has becoming clearer the biochemical mechanisms operating at each stage of the DDR. However, little is known about the spatial regulation of their components during the DNA damage response and how the relocation of the DNA lesion itself can influence in the repair process. Interestingly, previous studies have indicated that DNA breaks are re-localized from the nucleoplasm to the nuclear periphery, suggesting that nuclear compartmentalization of DNA lesions could comprise another layer in the regulation of the DNA repair pathway. Nevertheless, whether the nuclear periphery harbours an environment that is permissive for DNA repair and its implications in maintaining genome integrity is a subject that still under debate. Remarkably, new data coming from our group indicate that the cell cycle phosphatase Cdc14 is involved in DNA repair by controlling the tethering of a DNA lesion into the spindle pole body (SPB) region of the nuclear envelope. This function is attained by preserving the integrity of the metaphase spindle, a vital requirement to stimulate DSB-SPB interaction and thus DNA repair. Accordingly, disruption of spindle stability impairs both DSB-SPB interaction and DNA repair by homologous recombination. These observations directly connect spindle integrity with DNA repair and reveal that DSBs are preferentially tethered to the SPBs to be restored. Importantly, this new function of Cdc14 could provide a physiological mechanism that spatially regulates the DNA damage response and therefore the fate of the repair process. Simposios / Symposia S3.3-3 Cohesin functions in chromosome architecture and inheritance Ana Losada Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, ES Cohesin is a ring-shaped complex that entraps the sister chromatids from the time they emerge from the replication fork until their separation in anaphase, thereby ensuring both faithful chromosome segregation and accurate DNA repair by homologous recombination. Cohesin plays also an important role in genome compartmentalization. It promotes long-range DNA looping which contributes to transcriptional regulation, organization of DNA replication factories and locus rearrangement by recombination. Recently, mutations in cohesin genes have been identified in a number of human cancers. Understanding the contribution of cohesin mutations to tumourigenesis requires a deep knowledge of the multiple functions of the complex. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21 and either SA1 or SA2. Cohesion factors Pds5, Wapl and Sororin bind cohesin and modulate its interaction with chromatin. There are also two versions of Pds5 in vertebrates, Pds5A and Pds5B, and both interact with cohesin-SA1 and cohesin-SA2. In order to investigate the functional specificity of all these different cohesin complexes both at the cellular level and in the context of an organism, we have generated mouse models carrying knock out alleles for the variant subunits SA1, SA2, Pds5A and Pds5B. I will discuss what we have learnt from the characterization of these mice. 33 34 Pósters Posters 35 36 Salamanca 2016 Pósters / Posters Las comunicaciones libres aceptadas al XXXIX Congreso SEBBM se muestran por grupos científicos. El doble código R indica que el póster ha sido seleccionado como comunicación oral en una reunión de grupo científico. Las letras “r” y “m” tras el código de la comunicación indican, respectivamente, que optan al Premio Roche al mejor panel y al Premio Lilly–Margarita Lorenzo. El Libro de resúmenes on-line, en formato buscador, está disponible en la red y para smartphone y Tablet en: http://www.sebbm.com/xxxixcongreso/ Además de este listado, existe el Libro de resúmenes impreso que se entrega a los congresistas que lo solicitaron en el momento de formalizar su inscripción. P01. Apoptosis / Apoptosis P01-1 Lipid raft platforms as major scaffolds in regulating apoptotic signaling Faustino Mollinedo, Consuelo Gajate Laboratory of Cell Death and Cancer Therapy, Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, ES Membrane lipid rafts are highly ordered membrane domains enriched in cholesterol, sphingolipids and gangliosides, which form specialized domains with distinct composition and physical properties. Rafts provide an additional level of compartmentalization, serving as sorting platforms and hubs for recruitment of distinct signaling processes. We found that the ether phospholipid edelfosine, considered as the prototype of a family of compounds collectively known as alkylphospholipid analogs with interesting biomedical activities, showed affinity for cholesterol and accumulated in lipid rafts in a number of malignant hematological cells. This led to an efficient in vitro and in vivo antitumor activity by inducing translocation of Fas/CD95 death receptor as well as downstream signaling molecules to rafts, as assessed by different approaches, including microscopy techniques. Then, additional antitumor drugs were shown to act, at least in part, by recruiting death receptors in lipid rafts. The partition of death receptors together with downstream apoptotic signaling molecules in membrane rafts led us to postulate the concept of a special liquid-ordered membrane platform coined as “cluster of apoptotic signaling molecule-enriched rafts” (CASMER). CASMERs act as scaffolds for apoptosis signaling compartmentalization, facilitating and stabilizing protein-protein interactions by local assembly of cross-interacting molecules, thus leading to apoptosis. This process also occurs under physiological death receptor activation. The fact that raft/ death receptor-mediated apoptotic activation can be pharmacologically promoted, exacerbating a physiological process, opens up a new way to modulate apoptosis with interesting biomedical applications. P01-2 Killing cancer cells safely. The new antitumoral drug ABTL0812 induces autophagy-mediated cancer cell death by a novel and drugable mechanism of action José Miguel Lizcano de Vega Protein Kinases & Signal Transduction Lab Unitat de Medicina, Dept. Bioquímica i Biologia Molecular & Institut de Neurociències. Universitat Autònoma de Barcelona, Bellaterra, ES ABTL0812 is a novel first-in-class, small molecule which showed antiproliferative effects on tumor cells in phenotypic assays. Here we described the mechanism of action of this antitumoral drug, which is currently in clinical development. We used two human cancer paradigms, lung and pancreatic adenocarcinoma (A549 and MiaPaCa-2 cells, respectively). ABTL0812 impairs cancer cell proliferation and tumor growth in animal models (tumor xenografts), resulting in autophagy-mediated cancer cell death, without affecting non-tumoral cells. No signs of apoptosis were observed, and autophagy-deficient cells (Atg5-/-) were resistant to ABTL cytotoxicity. Importantly, ABTL0812 synergizes with standard fist-line chemotherapy (docetaxel and gemcitabine) to induce cancer cell death and tumor growth reduction in animal models. We identified the primary target of ABTL0812 by in silico high-throughput screening, comparing ABTL0812 structure against ChEMBL15 database. ABTL0812 binds and activates Peroxisome Proliferator-Activators Receptors (PPAR) alpha and gamma, resulting in upregulation of Tribbles-3 pseudokinase (TRIB3) gene expression. Upregulated TRIB3 binds cellular Akt, which induces suppression of the Akt/mTORC1 axis. Pharmacological inhibition of PPARs or TRIB3 silencing prevents ABTL0812 cytotoxicity. ABTL0812 induces Akt inhibition in cancer cells, human tumor xenografts, and peripheral blood mononuclear cells from patients enrolled in phase I/Ib fist-in-human clinical trial. Thus, ABTL0812 has a unique and novel mechanism of action, that induces autophagic cancer cell death and defines a new and drugable cellular route: PPARs-TRIB3-Akt-mTORC1. Furthermore, the low toxicity and high tolerability support further clinical development of ABTL0812. P01r-3 Temozolomide does not improve γ-irradiation-triggered caspase-dependent cell death in glioblastoma-derived human cells Laura Martínez-Escardó1, María Sánchez-Osuna1, Pilar Baldominos1, Jordi Bruna2, Victor J. Yuste1 1 Cell Death, Senescence & Survival Research Group, Dept. Bioquímica y Biología Molecular - Instituto de Neurociencias, Facultad de Medicina, Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès, ES, 2Neuro-Oncology Unit, Hospital Universitari de Bellvitge - Institut Català d´Oncologia (ICO), Duran i Reynals, L’Hospitalet de Llobregat, ES Despite treatment with the most rigorous surgical interventions, along with radio-chemotherapy regimens, glioblastoma (GBM) continues to rank amongthe most aggressive human cancers. Although radio-chemotherapy has improved the overall survival of GBM-affected patients up to ~14 months, almost all of them suffer recurrences. Previous results from our group demonstrated that after a broad panel of cytotoxic stimuli, GBM cells undergo caspase-dependent cell death without showing both oligonucleosomal DNA degradation and apoptotic nuclear morphology. Therefore we hypothesize that the refractoriness of this kind of tumors towards the current treatment could be due to an impairment of GBM cells to undergo canonical apoptotic cell death. With this aim we decided to characterize the biochemical and morphological changes that GBM cells suffer when exposed to the standard combination employed in the clinics, which combines γ-irradiation and the alkylating agent temozolomide. In 37 Pósters / Posters this sense, γ-irradiation triggered caspase-dependent cell death and partially provoked the appearance of apoptotic nuclear morphologies. On the other hand, GBM-derived cells cultured in the presence of high concentrations of temozolomide were unable to undergo cell death. Unexpectedly, the cytotoxic effect by γ-irradiation was not enhanced when temozolomide was added to the culture media. Although preliminary, our results suggest that the cytotoxic effect of the current standard treatment could be mainly due to radiotherapy instead of temozolomide or their combination. L.M.-E. holds a predoctoral contract (Personal Investigador en Formació) from Universitat Autònoma de Barcelona. P.B. is a M.Sc. student in Advanced Immunology from Universitat de Barcelona and Universitat Autònoma de Barcelona. V.J.Y. is a Doctor Distinguished Researcher under a “Retention of Research Talent” contract from “Programa Banco de Santander”. This work has been supported by MINECO/FEDER (SAF2012-31485), Generalitat de Catalunya, AGAUR, Grups de Recerca Consolidats (2014SGR-1609) and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED). P01-4 Cytochrome P450 induction in SH-SY5Y cells and its effect on neurotoxicity promoted by xenobiotics Jesús Fernández Abascal, Massimo Valoti University of Siena, Siena, IT Background: Cytochrome P-450 (CYP) is involved in the oxidative metabolism of both endogenous and exogenous compounds such as drugs, pesticides, or hormones. Some CYP isoforms are inducible by β-naphthoflavone (β-N), phenobarbital and ethanol in the rat (Raza and Avadhani 1988); and they can be found in brain mitochondria, leading to a potential toxicity generated by several apoptotic mechanisms (Boopathi, Anandatheerthavarada et al. 2000). An important question is whether their induction would promote neurotoxicity due to activation of endogenous substrates, or whether induction would lead to a more sensitive system to exogenous toxins. This metabolic activity may be related with promotion of Parkinson Disease (PD). In fact, some in vitro studies indicate that CYP is able to protect cells against MPP+ toxicity (Mann and Tyndale 2010). This is an important regard of the neurodegenerative process as the mechanisms involved in PD are thought to be multifactorial in nature. Hence, the study of how CYP-induction may predispose the brain to subsequent damage caused by exogenous toxins is significant. In addition, knowledge of CYP metabolism of neuroprotective compounds in the dopaminergic neurons is important for treatment of PD patients. Aim: The main goal of this project is to elucidate any involvement of CYP in mitochondrial impairment and any subsequent damage. The induction of CYP isoforms in SH-SY5Y cells and their potential effect upon neurotoxicity of several xenobiotics has been studied. Methods: Undifferentiated neuroblastoma cells has been treated with various CYP inducers: β-N, cyclophosphamide (CPA), ethanol and acetaminophen. Western blot and qRT-PCR were used to identify the expression of different CYP isoforms. Cell viability was recorded by colorimetric assay: pre-incubation for 24 hours with each inducer alone, then either co-incubation of the same inducer and a toxin (Rotenone, Paraquat or MPP+), or incubation just with the toxin alone. A study of apoptotic population was carried out by fluorescent-activated cell sorting (FACS); and the mitochondrial membrane potential was measured to identify the early apoptotic cells. Results: Cyclophosphamide (CPA) showed to induce the isoforms 2D6, 2E1, and 1A1. β-Naphtoflavone (β-N) increased the expression of CYP 2E1 and 1A1. Ethanol increased the expression of CYP 2E1. Acetaminophen only increased the 2E1 isoform. The MTT assay showed β-N to partially neuroprotect cells against MPP+ toxicity only when the induction of 38 XXXIX Congreso SEBBM CYP was maintained during the treatment with the toxin. The analysis of apoptotic population by FACS supported this results, showing a decrease of late apoptotic cells. However, preliminary results from mitochondrial membrane potential measurement indicate that early apoptotic cell populations remain equal. Conclusions: CYP isozymes are inducible by different compounds in the SH-SY5Y cells, and are involved in cell survival against toxicity of inhibitors of Complex I of electron transport chain. These results could provide a new insights in the development of PD and the metabolism of dopaminergic cells. Other experiments will be done in order to study the relationship between neurotoxins and CYP, either in undifferentiated or RA-differentiated SH-SY5Y cells. Acknowledgment: The presented research is granted by TINTIN - a Marie Curie ITN funded project (2013-17). GA: 608381. References [1] Boopathi, E., H. K. Anandatheerthavarada, S. V. Bhagwat, G. Biswas, J. K. Fang and N. G. Avadhani (2000). “Accumulation of mitochondrial P450MT2, NH(2)-terminal truncated cytochrome P4501A1 in rat brain during chronic treatment with beta-naphthoflavone. A role in the metabolism of neuroactive drugs.” J Biol Chem 275(44): 34415-34423. [2] Mann, A. and R. F. Tyndale (2010). “Cytochrome P450 2D6 enzyme neuroprotects against 1-methyl-4-phenylpyridinium toxicity in SH-SY5Y neuronal cells.” Eur J Neurosci 31(7): 1185-1193. [3] Raza, H. and N. G. Avadhani (1988). “Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver.” J Biol Chem 263(19): 9533-9541. P01-5 Estudio de la correlación entre la expresión de la enzima α(1,6)fucosiltransferasa y la proteína DR4 de la vía de proapoptosis celular TRAIL/DR4 en las líneas de cáncer colorrectal SW480/SW620 y validación en una cohorte de pacientes Rubén López-Cortés1, Elsa Núñez Rodríguez1, Laura Muinelo Romay2, Emilio Gil Martín1, Almudena Fernández Briera1 1 Grupo BB1, Dpto. Bioquímica, Genética e Inmunología, Universidad de Vigo, Vigo, ES, 2Grupo de Oncología Médica Traslacional, CHUS, Santiago de Compostela, ES DR4 (Death Receptor 4) es una glicoproteína proapoptótica de la que se sospecha que está core-fucosilada. Estudios previos de nuestro grupo investigador han encontrado indicios de correlación inversa entre los niveles de expresión de la alfa(1,6)fucosiltransferasa (FucT-8), enzima responsable de la core-fucosilación, y la expresión de DR4. Nuestro trabajo actual pretende profundizar en esta observación inicial. Usando como modelo celular las líneas isogénicas SW480 y SW620, junto con sus respectivos clones shRNA atenuados para la expresión de FucT-8, hemos logrado demostrar la up-regulation de DR4 en los clones knocked-down de ambas líneas celulares. La inducción de apoptosis con TRAIL (Tumor Necrosis Factor-Related Apoptosis Inducing Ligand), por otra parte, condujo a una mayor expresión de FucT-8, mientras, sin embargo, la expresión de DR4 se mantuvo inalterada. En SW480 todos los clones (silvestre, control y knocked-down) mostraron fragmentación de las caspasas iniciadoras Casp8 y Casp9, pero la mayor transducción de la señal apoptótica se halló a través de las caspasas efectoras Casp3, Casp6 y Casp7 en los clones atenuados. El perfil de expresión de las caspasas en SW620 reflejó el bloqueo de la vía mitocondrial descrito en esta línea. Otro foco de nuestra atención ha sido la vía de supervivencia celular MAPK/ERK, que se activa por fosforilación del transductor ERK1/2 (pERK1/2). Los clones knocked-down de SW480 mostraron un nivel de expresión de pERK1/2 mayor que los control pero Salamanca 2016 insensible a la estimulación con TRAIL. Esta diferencia no se observó en SW620. Para validar la correlación entre FucT-8 y DR4 se recurrió al análisis de fracciones de membrana procedentes de una cohorte de pacientes con cáncer colorrectal. Los especímenes de tejido tumoral mostraron mayores niveles de expresión de FucT-8, y menores de DR4, que los de tejido sano, lo que refuerza los indicios de partida sobre la evolución inversa de la expresión de ambas proteínas en el cáncer colorrectal. Estudio financiado mediante el Contrato-Programa CN2011/024, Xunta de Galicia. RLC es contratado FPU12/03662 del MECD. P01-6 Las caspasas 3 y 6 participan en la proteólisis de Tau y en la apoptosis inducidas por estrés hiperosmótico en células de neuroblastoma humano SH-SY5Y Marta Olivera Santa-Catalina1, Montaña Caballero Bermejo1, Ricardo Argent1, Juan Carlos Alonso1, Francisco Centeno2, María Jesús Lorenzo1 1 Departamento de Bioquímica y Biología Molecular y Genética. Facultad de Veterinaria. Universidad de Extremadura, Cáceres, ES, 2 Departamento de Bioquímica y Biología Molecular y Genética. Facultad de Ciencias. Universidad de Extremadura, Badajoz, ES La proteína asociada a microtúbulos Tau tiene como función principal la regulación de la dinámica de los microtúbulos. Este proceso está a su vez regulado por las modificaciones postraduccionales que sufre la proteína Tau, como la proteólisis. Esta modificación aberrante promueve la formación de filamentos y agregados de la proteína (NFT, del inglés, neurofibrilary tangles), que llevan a la muerte de las neuronas. Entre las proteasas responsables de la proteólisis de Tau destacan las caspasas, que también juegan un papel fundamental en la apoptosis celular. El objetivo del presente trabajo fue estudiar el efecto del estrés hiperosmótico inducido por sorbitol sobre la proteólisis de Tau. Además, se analizó la posible relación entre la activación de caspasas y apoptosis observada bajo estas condiciones. Células de neuroblastoma humano SH-SY5Y se incubaron con sorbitol durante diferentes periodos de tiempo y la fragmentación de Tau y la activación de las caspasas 3 y 6 fueron analizadas mediante Western blot. El estrés hiperosmótico promovió la proteólisis de Tau y la activación de ambas caspasas. Usando inhibidores farmacológicos específicos, se detectó que la proteólisis de Tau tenía lugar en el extremo amino-terminal y que las caspasas 3 y 6 participaron en dicha fragmentación. De forma paralela, el sorbitol indujo apoptosis en las células tratadas, proceso en el que también están involucradas las caspasas. Nuestros resultados sugieren una posible relación entre proteólisis de Tau y apoptosis celular en condiciones de estrés hiperosmótico. Trabajo financiado por los proyectos: BFU2007-67577-C02-02/BMC, PRIS10018, GRU10046 y GR15164 FEDER-Junta de Extremadura. P01-7 Células madre amnióticas epiteliales: cambios en su proliferación y apoptosis durante su diferenciación hepática Rodrigo Riedel1, Antonio Pérez Pérez2, Bernardo Maskin3, Mariana Jaime3, Ornella Parolini4, Víctor Sánchez Margalet2, Cecilia Varone1, Julieta Maymó1 1 IQUIBICEN-CONICET-Depto. de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, AR, 2Depto. de Bioquímica Médica y Biología Molecular, Universidad de Sevilla, Sevilla, ES, 3Hospital Nacional Alejandro Posadas, Buenos Aires, AR, 4Centro di Ricerca E. Menni -Fondazione Poliambulanza- Istituto Ospedaliero, Brescia, IT Pósters / Posters Las células madre aisladas de la placenta y sus membranas fetales, representan una atractiva e importante alternativa para la medicina regenerativa, dadas las ventajas que poseen respecto a otras células madre: el fácil acceso a los tejidos, un uso que no genera debate ético y un potencial ilimitado para proveer células. Las células madre amnióticas epiteliales (hAEC), aisladas del amnios de la placenta humana a término, son pluripotentes, expresan marcadores embrionarios, no son tumorigénicas y poseen propiedades inmunomoduladoras. Estas características posicionan a las hAEC como ideales candidatas para su utilización en la medicina regenerativa. La falla hepática es una de las mayores causas de morbilidad y mortalidad en el mundo; y si bien el trasplante representa la mejor manera de tratar la enfermedad hepática crónica y aguda, existen varios obstáculos al respecto. Las hAEC han sido propuestas como fuente alternativa de hepatocitos, dado su capacidad de diferenciación hepática. El objetivo del presente trabajo fue estudiar algunos aspectos de la proliferación y apoptosis de las hAEC, durante su diferenciación hepática temprana y tardía. La diferenciación se llevo a cabo durante 30 días con factores específicos (EGF + dexametasona) o con medio condicionado de la línea celular hepática HepG2 (MC). Previamente hemos determinado que las hAEC adquieren morfología hepática y expresan marcadores específicos hepáticos (α-fetoproteína, α1-AT, albúmina, CYP3A4, CYP7A1), luego del tratamiento con factores. Determinamos que el EGF incrementó significativamente la proliferación y la viabilidad de las hAEC medidas por incorporación de 3H-T y ensayo de MTT, respectivamente. Se produjo un aumento en la expresión de Ki-67 y una disminución de Caspasa-3 clivada y PARP clivada, determinadas por IF y WB. El tratamiento con MC produjo el efecto contrario. Los resultados obtenidos contribuyen a la comprensión de los mecanismos celulares y moleculares que ocurren durante la diferenciación hepática de las hAEC. P01r-8 Impaired function of the WD40 domain of ATG16L1 caused by the T300A Crohn’s disease risk polymorphism Inmaculada Serramito Gómez, Felipe Xosé Pimentel Muiños Instituto de Biología Molecular y Celular del Cáncer (IBMCC), Salamanca, ES A coding allele of human ATG16L1 (rs2241880; T300A) increases the risk of Crohn’s disease, but the underlying molecular defects introduced by this mutation are not fully understood. We show that T300A alters the ability of the C-terminal WD40-repeat domain of ATG16L1 to interact with an amino acid motif that recognizes this region. This alteration impairs the unconventional autophagic activity of TMEM59, a transmembrane protein that contains the WD40 domain-binding motif, and disrupts its normal intracellular trafficking and ability to engage ATG16L1 in response to bacterial infection. Notably, these defects are independent of ATG16L1 T300A caspase cleavage. In addition, TMEM59-induced autophagy is blunted in cells expressing the fragments generated by caspase 3 cleavage of the risk allele, whereas canonical autophagy remains unaffected. These results suggest that the T300A polymorphism alters the function of motif-containing molecules that engage ATG16L1 through the WD40 domain, either by influencing this interaction under non-stressful conditions or by inhibiting their downstream autophagic signalling after caspase-mediated cleavage. P01r-9 Efecto protector de la leptina en explantos placentarios sometidos a un pH ácido Antonio Pérez Pérez1, Ayelen Rayen Toro2, Teresa Vilariño-García3, Julieta Maymó2, Cecilia Varone2, Víctor Sánchez-Margalet3 1 Hospital Universitario Virgen Macarena, Sevilla, ES, 2Departamento de Química Biológica, FCEN-UBA. IQUIBICEN, CONICET, Buenos Aires, AR, 3Departamento de Bioquímica Médica, Biología Molecular e Inmunología, Universidad de Sevilla, Hospital Universitario Virgen Macarena, Sevilla, ES 39 Pósters / Posters La disminución del intercambio gaseoso a nivel de la placenta es una complicación grave durante la gestación. La perturbación del pH del medio interno puede alterar funciones fetales, maternas y también placentarias. En particular, la disminución del pH puede alterar la homeostasis fetal provocando daños tisulares irreparables o hasta la muerte fetal por acidosis metabólica. En este contexto decidimos estudiar el efecto de la disminución del pH sobre el proceso de apoptosis en explantos placentarios y la acción de la leptina bajo dichas condiciones. Basándonos en antecedentes previos de nuestro grupo de investigación y en el rol central de p53 en la regulación de la apoptosis, hipotetizamos que los cambios de pH en las células placentarias aumentan la apoptosis alterando la expresión de p53 así como también la de sus genes diana. En este contexto nos planteamos estudiar un posible efecto antiapoptótico de la leptina en células trofoblásticas sometidas a diferentes pH. Explantos de placenta humana a término fueron incubados en medio DMEM F-12 a diferentes pH (7,4, 7 y 6,8) en presencia o ausencia de leptina 10 nM. Se analizaron por Western blot y qRT-PCR los niveles de p53 y sus efectores p21, BAX y BCL-2, así como también el fragmento derivado por rotura de PARP-1 y la forma activa de caspasa-3. Encontramos un aumento en la fosforilación (residuo Ser 46) de p53, así como de la expresión de p21, PARP-1 y la activación de caspasa-3 en los explantos incubados a pH 7 y 6,8. Por el contrario, estos efectos fueron atenuados en presencia de leptina 10 nM tanto a pH 7 como a pH 6,8. La relación BCL-2/BAX disminuyó a pH 7 y 6,8 en comparación con explantos incubados a pH 7,4, efecto que se contrarrestó por el tratamiento con leptina. Estos datos demuestran un potencial efecto antiapoptótico de la leptina que involucra el eje de regulación de p53 en explantos placentarios cultivados a pH ácidos, sugiriendo que la leptina placentaria posee un efecto protector contra el daño generado por el pH acido en células trofoblásticas. P01-10 Characterization of the release of histones in glioblastoma-derived cells after cytotoxic insult and its relationship with the apoptotic endonuclease DFF40/CAD Pilar Baldominos, Laura Martínez-Escardó, Víctor J. Yuste Cell Death, Senescence & Survival Research Group, Dept. Bioquímica y Biología Molecular - Instituto de Neurociencias, Facultad de Medicina, Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès, Bellaterra, ES Among primary brain tumours, glioblastoma (GBM) is the most lethal and the one with highest prevalence. Conventional therapy involves a combinatorial strategy of surgical resection, chemotherapy with temozolomide and irradiation. However, the benefits of the current approach are negligible and the overall survival of patients is prolonged, at beast, for no more than 16 months. Our group has previously described the resistance of glioblastoma-derived cells to chemotherapy and their inability to complete the apoptotic cell programme. Histones are DNA binding proteins that have been described to be released to the extracellular milieu during apoptosis. In these conditions, histones are recognized by receptors of the toll-like receptor family from innate immune system cells, so they are characterized as DAMPs (damage associated molecular patterns) molecules. Here, we have studied the ability of glioblastoma-derived cells to liberate histones after challenged with different cytotoxic stimuli, with the immunological implications it might have. Moreover we have characterized the relationship between the release of histones and the endonuclease DFF40/CAD. Previous studies of our group showed DFF40/CAD is essential for finishing the apoptotic programme and it is down-regulated in glioblastoma-derived cells. 40 XXXIX Congreso SEBBM Work supported by “Ministerio de Economía y Competitividad (MINECO)/ Fondo Europeo de Desarrollo Regional (FEDER)” Grant SAF2012-31485, and by “Generalitat de Catalunya” Grant SGR2009-346. P.B. performed this work under the Final Master Thesis Work of the interuniversitary MSc in Advance Immunology from “Universitat de Barcelona” and “Universidad Autónoma de Barcelona”. P01r-11 The new antitumor drug ABTL0812 induces autophagy-mediated cell death in human glioblastoma cell lines Pau Muñoz-Guardiola1, Valeria Rizzuto2, Tatiana Erazo2, Nora Diéguez2, Rubén Melgarejo2, Elisabet Megías2, Carles Domènech3, José Miguel Lizcano2 1 Protein Kinases & Signal Transduction Lab. Unitat de Medicina, Dept. Bioquímica i Biologia Molecular & Institut de Neurociències. Universitat Autònoma de Barcelona (UAB); Ability Pharmaceuticals SL. Campus UAB, Tiana, ES, 2Protein Kinases & Signal Transduction Lab. Unitat de Medicina, Dept. Bioquímica i Biologia Molecular & Institut de Neurociències. Universitat Autonoma de Barcelona (UAB), Bellaterra, ES, 3Ability Pharmaceuticals SL. Campus UAB, Bellaterra, ES ABTL0812 is a first-in-class molecule with antitumor activity which has successfully passed phase I/Ib clinical trials in patients with advanced solid tumors. ABTL0812 is a polyunsaturated fatty acid derivativewhich induces cell death in a broad panel of cancer cell lines, without affecting cell viability of non-tumoral cells. ABTL0812 inhibits Akt by upregulating expression of the pseudokinase TRIB3, leading to suppression of the Akt-mTORC1 axis and autophagy-mediated cancer cell death. ABTL0812 do not activate apoptosis in any of the cancer cell lines tested. ABTL0812 also inhibits tumor growth of lung and pancreatic xenograft models, with a similar efficacy to that of gold-standards-of-care docetaxel and gemcitabine. Here, we present preliminary results on the effect of ABTL0812 treatment in two human glioblastoma cell lines, LN18 and U87-MG. Glioblastoma multiforme is the most common and lethal primary malignant brain tumor, and the therapeutic strategies are few and poor. Aggressiveness of glioblastoma is mostly due to diffuse infiltration, angiogenesis and resistance to apoptosis. To overcome apoptotic resistance, autophagy has emerged as an alternative to kill cancer cells. Here we describe by first time that ABTL0812 induces autophagy-mediated cell death of human glioblastoma cell lines LN18 and U87-MG, with an IC50 value similar to that showed in other human cancer cell lines (pancreas, lung, prostatic, neuroblastoma, breast or endometrial cancers). We also characterize the precise mechanism of action by which ABTL0812 exerts a cytotoxic effect in glioblastoma. Acknowledgements: ABTL0812 is owned by Ability Pharmaceuticals SL, a Biotech Company of the UAB Parc de Recerca. Work supported by MINECO, Grant INNPACTO Ref. IPT-2012-0614-010000, and Fondo Europeo de Desarrollo Regional (FEDER). P01-12 Desarrollo de ensayos citómicos de alto contenido para la predicción y clasificación del riesgo tóxico de compuestos químicos José Enrique O`Connor1, Susana Balaguer1, Guadalupe Herrera2 Universidad de Valencia, Valencia, ES, 2Fundación Incliva, Valencia, ES 1 El desarrollo de métodos in vitro de análisis de la toxicidad bioquímica de compuestos químicos es una prioridad y ha conducido a la disponibilidad Salamanca 2016 de técnicas relativamente sencillas con aceptable capacidad de detección de efectos in vitro y de predicción de toxicidad aguda in vivo. La citómica se está convirtiendo en una herramienta esencial para el estudio de toxicidad celular in vitro. En nuestro laboratorio, los ensayos citómicos de toxicidad sobre líneas celulares humanas clasifican razonablemente los compuestos según su riesgo de toxicidad en una escala de toxicidad in vivo en humanos. Sin embargo, la comparación de los datos con el sistema internacional de clasificación (Global Harmonizing System, GHS), basado en datos de toxicidad in vivo en rata, resultaba en una escasa capacidad de clasificación correcta.Hemos puesto a punto y validado una plataforma de ensayos citómicos miniaturizados y de alto contenido (integridad de membrana, toxicidad mitocondrial y estrés oxidativo/nitrosativo) para la determinación de parámetros generales de citotoxicidad y específicos de estrés oxidativo en tres líneas celulares de rata. Aplicando técnicas estadísticas y bioinformáticas hemos determinado la capacidad la plataforma de ensayos para predicción de toxicidad in vivo y clasificación de compuestos según el GHS. Los resultados muestran que los ensayos citómicos miniaturizados y de alto contenido clasifican correctamente la toxicidad de un gran número de compuestos químicos de acuerdo al criterio GHS. La capacidad de clasificación mejora respecto al método in vitro de referencia, basado en los valores de IC50 en el test de captación de Rojo Neutro por 3T3. Financiado por Universitat de València, Convocatòria Accions Especials 2015 (UV-INV-AE15-349700). P01r-13 El bloqueo farmacológico de Mcl-1 sensibiliza a las células de cáncer de vejiga al tratamiento con Paclitaxel Rocío Jiménez Guerrero1, Jessica Gasca Bellido1, Mª Luz Flores de Mera1, María Tortolero García2, Francisco Romero Portillo2, Miguel Ángel Japón Rodríguez3, Carmen Sáez Torres3 1 Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocío/CSIC, Universidad de Sevilla, Jerez de la Frontera, ES, 2Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Sevilla, ES,3Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocío/CSIC, Universidad de Sevilla. Departamento de Anatomía Patológica, Hospital Universitario Virgen del Rocío, Sevilla, ES Los taxanos como Paclitaxel y Docetaxel son agentes antimitóticos que se utilizan en diferentes tipos de cáncer incluyendo el de vejiga, y constituyen una alternativa terapéutica para el tratamiento en segunda línea del cáncer avanzado de vejiga músculo-invasivo. Sin embargo, las células tumorales acaban desarrollando mecanismos de resistencia que hacen necesaria la búsqueda de nuevas alternativas y/o de terapias sensibilizadoras. En este sentido aparece Obatoclax (GX15-070) como inhibidor específico del dominio BH3 de Mcl-1, una proteína clave en la muerte celular por apoptosis tras parada en mitosis, y que está muy relacionada con la resistencia al tratamiento con taxanos en diversos tipos tumorales. Actualmente están en marcha varios ensayos clínicos en fase I y II con Obatoclax en algunas enfermedades hematológicas, pero aún se desconoce su papel en tumores sólidos y su mecanismo de acción sigue siendo motivo de controversia. Nosotros hemos observado que en células de cáncer de vejiga Obatoclax induce muerte celular y que en células resistentes a Paclitaxel es capaz de sensibilizar a dicho tratamiento. Además, hemos estudiado la influencia de Obatoclax en la progresión del ciclo celular.Hemos observado también que este fármaco induce autofagia en líneas celulares de cáncer de vejiga y que dicha autofagia puede ser protectora o inductora de muerte celular. En este trabajo discutimos los posibles mecanismos por los que Obatoclax sensibiliza al tratamiento con taxanos relacionados con el papel de la autofagia citoprotectora o citotóxica dependiendo del tipo celular. Pósters / Posters P01r-14 Histone H2AZ is involved in transmissible cytotoxicity mechanisms leading to multiple myeloma cell death Beatriz Martín-Antonio1, Guillermo Suñe1, Lorena Pérez1, Amer Najjar2, Álvaro Urbano-Ispizua1 1 IDIBAPS-Hospital Clinic Josep Carreras Leukaemia Research Institute, Barcelona, ES, 2The University of Texas M.D. Anderson Cancer Center. Department of Cancer Systems Imaging, Houston, Texas, US Cord blood derived natural killer cells (CB-NK) are a feasible source of NK cells for immune cell therapy in hematological malignancies. We have shown that CB-NK exert a transmissible cytotoxicity towards multiple myeloma (MM) cells which involves protein vesicle transfer between MM cells exposed to CB-NK (1ºMM) and MM cells exposed to 1ºMM cells (2ºMM cells). However, this transmission between MM cells could have a diluent effect of the CB-NK cytotoxicity which could be a tumor cell survival mechanism. To gain insights into this mechanism, we performed TRANS-SILAC proteomics to identify which proteins were transferred between CB-NK and MM cells. 1ºMM cells acquired 9.5% of CB-NK proteins, these proteins were transferred to 2ºMM cells. As a consequence, 1ºMM cells diluted their CB-NK protein content. Furthermore, while in resting conditions, the proteome transfer between MM cells was minimal, under the presence of CB-NK, 1ºMM cells transferred a higher amount of their proteome to CB-NK and to 2ºMM cells. Among the transferred proteins, we focused on the Histone H2AZ variant 1. In vivo confocalmicroscopy showed direct and secondary transfer of H2AZ between MM cells associated to cell death. H2AZ overexpression in CB-NK increased CB-NK cytotoxicity vs MM cells. H2AZ overexpression in MM cells caused Caspase independent MM cell death with features of DNA damage. H2AZ-mediated cytotoxicity was transmissible from H2AZ-overexpressing MM cells to neighboring MM cells causing DNA damage in the 2ºMM population. While the transfer of H2AZ from CB-NK to MM cells occurred through vesicles, between MM cells it was through intercellular nanotubes connecting MM cells. These findings show the relevance of cell-cell communication in mechanisms leading to MM cell death. P01m-15 Apoptosis in adventitial layer of hydatid cysts Edgar Mauricio Jiménez Vargas, Rodolfo Paredes, Felipe Correa, Carlos González, Ivan Contreras, Caroll Stoore Escuela de Medicina Veterinaria, Facultad de Ecología y Recursos Naturales, Universidad Andrés Bello, Santiago, CL Cystic Echinococcosis (CE) is a zoonotic infection with high prevalence in part of Eurasia, Africa, Australia, and South America that represents a major public health and economic burden in many countries. Fertile cysts are capable to generate protoscolex, while for unknown reason, some cysts are unable to produce (infertile hydatid cysts). Previous reports showed that apoptosis could be involved in a negative regulation of protoscoleces generation, leading to hydatid cyst infertility but no report are available about cell apoptosis in adventitial layer and the relationship with other parasitic disease like fascioliasis. Materials and Methods: Animals were evaluated to characterize the presence of CE and fasioliasis (FS) in cattle slaughtered at abattoir in Santiago, Chile. Samples were processed for routine histology and stained with heamatoxylin/eosin. Samples from animal with both fertile and infertile cyst from CE and with or without DS was included in this study, using 5 samples per condition. Histological samples were evaluated by immunofluorescence and TUNEL Assay; digital images were obtained 41 Pósters / Posters XXXIX Congreso SEBBM using an Olympus BX 41 Microscope and analyzed with software for morphometric analysis (Image Pro-Plus, Media Cybernetics, USA). The adventitial layer apoptotic index was calculated as follows: apoptotic nuclear area in adventitial layer x 100/total nuclear area in adventitial layer. Kruskal-Wallis test was performed using IBM SPSS Statistics 22 (IBM Corporation) software, p <0.05. Results: The apoptotic index in infertile cysts (0.18 ± 0.35%) was significantly higher (p = 0.032) compared with fertile cysts (0.045 ± 0.068%), as the presence of co-infection with liver fluke, both fertile cysts as infertile has a lower apoptotic index without significant difference (p > 0.05). Conclusion: The highest index of apoptosis cells in the adventitia layer of hydatid cysts is related to infertility cysts. The presence of distomatosis decreases cell apoptosis. Acknoledgments: Fondecyt /Chile No. 1161475, Proyecto interno UNAB DI-1388-16/I. P02. Bases moleculares de la patología / Molecular bases of pathology P02-1 Sensibilización de las células de cáncer de mama a la radioterapia mediante modulación epigenética José Neptuno Rodríguez López1, Rebeca González Guerrero1, Luis Sánchez del Campo1, Ana Moreno1, Juan Cabezas Herrera2, María Fernanda Montenegro1 1 Departamento de Bioquímica y Biología Molecular A, Universidad de Murcia, Murcia, ES, 2Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, ES El cáncer, además de considerarse una enfermedad genética, puede también considerarse una enfermedad epigenética. Las modificaciones epigenéticas además de ser causantes de los cambios en la expresión de los genes, participan activamente en la respuesta de las células al daño del ADN. Como los factores epigenéticos están regulados por múltiples elementos, las terapias dirigidas a componentes específicos de la maquinaria epigenética pueden no ser eficaces. A diferencia de estas, las terapias dirigidas a la inhibición del ciclo de la metionina pueden inhibir indirectamente la metilación tanto de proteínas como del ADN afectando a una gran cantidad de genes y vías de señalización. Siguiendo esta misma línea, hemos desarrollado una terapia adyuvante dirigida a la epigenética de la respuesta al daño del ADN en las células de cáncer de mama que consigue aumentar la apoptosis y reducir, in vivo, la metástasis a distancia. En nuestro laboratorio hemos observado que una terapia combinada dirigida a desacoplar el metabolismo de la adenosina usando dipiridamol en presencia de un nuevo antifolato sintético, 3-O-(3,4,5-trimetoxibenzoil)-(-)–catequina, bloquea eficiente y simultáneamente el ciclo del ácido fólico y de la metionina en células de cáncer de mama sensibilizándolas a la radioterapia. Esta combinación impide, entre otros, la movilización de 53BP1 y BRCA1 a las zonas de la cromatina que flanquean las roturas de doble cadena del ADN evitando así la reparación del daño del ADN en las células de cáncer de mama expuestas a la radiación ionizante. Así, la terapia combinada que proponemos puede definirse como una estrategia global dirigida a la maquinaria epigenética de las células tumorales. Financiado por un proyecto del (SAF2013-48375-C2-1R) y la FAECC. 42 MINECO-Fondos FEDER P02-2 Extracellular heat shock protein 90 binding to TGFβ receptor I participates in TGFβ-mediated collagen production in myocardial fibroblasts Jenny Milagros Gómez1, Francisco Javier Ruano Calvo2, Antonio Aires Trapote3, Iñigo Casafont Parra4, Ana María Palanca Cuñado4, Aitziber López Cortajarena3, Ana Victoria Villar Ramos4 1 Hospital de Laredo, Cantabria, ES, 2Hospital Universitario Marqués de Valdecilla, Cantabria, ES, 3CIC-BiomaGUNE, Gipuzcoa, ES, 4 Universidad de Cantabria, Cantabria, ES The pathological remodeling heart shows an increase in left ventricular mass and an excess of extracellular matrix deposition that can over time cause heart failure. Transforming growth factor β (TGFβ) is the main cytokine controlling this process. The molecular chaperone heat shock protein 90 (Hsp90) has been shown to play a critical role in TGFβ signaling by stabilizing the TGFβ signaling cascade. We detected extracellular Hsp90 in complex with TGFβ receptor I (TGFβRI) in fibroblasts and determined a close proximity between both proteins suggesting a potential physical interaction between the two at the plasma membrane. It was supported by in silico studies predicting Hsp90 and TGFβRI extracellular domain interaction. Extracellular Hsp90 inhibition lessened the yield of collagen production as well as the canonical TGFβ signaling cascade. These observations together with the significant increased activity of Hsp90 at the plasma membrane of TGFβ-treated myocardial fibroblasts pointed to a functional cooperative partnership between the extracellular Hsp90 and TGFβRI in the fibrotic process. We propose that an extracellular population of Hsp90 bound to TGFβRI behaves as an active participant in collagen production in TGFβ-activated fibroblasts. We also offer an in vivo insight into the role of Hsp90 during cardiac remodeling in murine aortic banding model suffering from pathological cardiac remodeling and detect circulating Hsp90 overexpressed in remodeling mice. P02r-3 Identification of MicroRNAs involved in the metabolic basis of renal fibrogenesis Verónica Miguel Herranz1, Marta Fierro Fernández1, Ricardo Ramos2, Santiago Lamas1 1 Centro de Biología Molecular Severo Ochoa CSIC-UAM, Madrid, ES, 2Genomic Facility, Parque Científico de Madrid, Madrid, ES Excessive accumulation of extracellular matrix (ECM) proteins is the hallmark of fibrotic diseases. In the kidney, it is the final common pathway of prevalent clinical conditions as type 2 diabetes mellitus and arterial hypertension, leading to chronic renal failure. This process is dependent on the activation of ECM synthesis in phenotypically transformed fibroblasts known as myofibroblasts. While archetypal cytokine mediators such as TGF-β play a fundamental role in this transformation, recent work has shown that lipid metabolic alterations, specifically a reduction in fatty acid oxidation (FAO) are important pathogenetic coadjuvators. MicroRNAs are small non-coding RNAs that regulate gene expression at the post-transcriptional level and controlling multiple biological processes, including fibrogenesis. To identify miRNAs involved in the metabolic regulation of renal fibrosis, we performed studies in in the renal fibrosis mouse models of unilateral ureteral obstruction (UUO) and folic acid nephropathy (FAN). From a total of 175 array-analyzed microRNAs in the UUO model 74 showed altered expression in the fibrotic samples. In silico analysis of the FAO-related enzyme carnitin-palmitoyl transferase 1 (CPT1A) 3´UTR identified miR-33-5p and miR-124-3p as potential candidates. We found downregulation of miR-33-5p in the UUO and FAN models by 40 and 50%, respectively, while miR-124-3p levels were unchanged. TGF-β1-induced fibrogenesis in the human renal cell line HKC-8 as well as in primary human renal epithelial cells (RECs) and primary human tubular renal epithelial cells (HTECs). We found 60% downregulation of miR-124-3p levels in TGF-β1-treated HTEC. Salamanca 2016 A 60% decrease in the luciferase reporter activity of the CPT1A 3´UTR after transfection with miR-124-3p confirmed its target specificity. Consistently, miR-124 overexpression downregulated CPT1A mRNA and protein levels by 60% in HTECs and resulted in a 3-fold increase of TGF-β1-induced α-SMA expression. These results support the role of miRNAS in a novel and potentially complex metabolic regulatory pathway related to renal fibrosis. P02m-4 La disminución de la expresión de s-resistina en el hipotálamo mejora la respuesta central y periférica a la insulina en ratas Wistar María Rodríguez Pérez1, C. Pintado1, V. López-Gómez Carreño2, N. Gallardo2, E. Moltó1, C. Arribas1, A. Andrés2 1 Área de Bioquímica, Facultad de Ciencias Ambientales y Bioquímica; Centro Regional de Investigaciones Biomédicas, UCLM, Toledo, ES, 2Facultad de Ciencias y Tecnologías Químicas, Ciudad Real, ES; Centro Regional de Investigaciones Biomédicas, UCLM, Toledo, ES La s-resistina es una variante no secretable de resistina, generada por splicing alternativo, que se expresa principalmente en tejido adiposo blanco e hipotálamo de ratas Wistar. Resultados previos confirmaron que células 3T3-L1 que expresaban s-resistina mostraron un incremento en la respuesta inflamatoria, así como un descenso en el transporte de glucosa estimulado por insulina. Además, la s-resistina, al igual que la resistina, bloqueó la vía de señalización de la insulina inhibiendo la fosforilación de IR, IRS-1 y Akt, e incrementó los niveles de SOCS-3. Por tanto, s-resistina podría actuar como un factor no secretado que regula la sensibilidad del adipocito a la insulina. En este trabajo se analiza el papel de s-resistina en el hipotalámo. Para ello se ha disminuido la expresión central de esta isoforma corta de resistina, inyectando, de forma i.c.v, un lentivirus que contiene un RNAi frente a dicha isoforma (LV-RNAi-s-res). La diminución central de s-resistina incrementa los niveles basales de fosforilación en Tyr, tanto en IR como en IRS-1, mientras que disminuye los niveles de fosforilación en Ser307 en IRS-1 en el hipotálamo. También se observa un incremento de los niveles de mRNA y de fosforilación en STAT-3, mientras que éstos disminuyen en el caso de PTP1b. Además, en los animales tratados con el LV-RNAi-s-res se promueven los efectos anorexigénicos, reduciendo la expresión de NPY e incrementando la de POMC, lo que conlleva un descenso de la ingesta. Estos datos muestran, por tanto, una mejora en la vía de señalización de insulina en aquellos animales que presentan disminuidos los niveles de expresión de s-resistina en el hipotálamo. Asimismo, se observa un incremento en la sensibilidad periférica a la insulina, como lo demuestran las curvas de tolerancia a la glucosa obtenidas en los animales tratados. Todos estos resultados sugieren que s-resistina podría actuar como un sensor neuronal intracrino, que actuaría regulando la sensibilidad a la insulina en todo el organismo. Financiación: PI-2007/60 (JCCM) FISCAM y BFU2012-39705-C03-01 Mineco, España. P02-5 Identification of new mechanisms involved in the postnatal regeneration of endocrine pancreas: role of the autophagy in beta-cells and its relationship with IGF-2 Elisa Fernández-Millán1, David Álvarez-Cilleros2, Juan de Toro-Martín3, Esther Lizárraga-Mollinedo4, Fernando Escrivá2, Carmen Álvarez Escolá2 1 CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, ISCIII), Madrid, ES, 2Dpto. Bioquímica y Biología Molecular II, Facultad de Farmacia, UCM, Madrid, ES, 3Institute of Nutrition and Functional Foods (INAF), Laval University, Pósters / Posters Quebec, CA, 44ULB Center for Diabetes Research, Medical Faculty, Université Libre de Bruxelles (ULB), Bruxelles, BE Aim: The developing endocrine pancreas undergoes substantial remodeling during the postnatal period, which triggers a transformation from fetal to adult phenotype of beta-cells. This beta-cell turnover is achieved by a transient wave of apoptosis that is temporally associated with a lack of expression of IGF-2 within islets. Because IGF-1R pathway is upstream of mTORC1, a negative regulator of autophagy, we expected autophagy to be activated in beta-cells during neonatal period. Thus, our study aimed to characterize the basal autophagic activity during endocrine pancreas remodeling and its contribution to neonatal beta-cell apoptosis. Methods: Autophagy markers were measured by Western blot in the pancreas of rats on postnatal (PN) day 4, 14 and 23. Autophagosome formation was analyzed by TEM. Neonates were treated with rapamycin (3 mg/kg, 5 days) and beta-cell apoptosis was quantified by TUNEL assay. In order to determine the effect of IGF-2 on autophagy, in vitro studies were performed in INS-1E cells and neonatal islets. Results: Under basal conditions, a significant decrease of LC3II levels was observed in the pancreas of neonates on PN14 and PN23 compared with PN4. This decrease was accompanied by an accumulation of p62 (>2-fold vs. PN4). Similarly, TEM revealed a reduction in the number of autophagic vacuoles per beta cell in PN14 (49.7%, p<0.05) and PN23 rats (64.24%, p<0.05) compared with PN4 rats. Sub-chronic treatment with rapamycin, induced a significant reduction (p<0.01) in the beta-cell apoptosis observed on PN14. Finally, kinetic experiments showed that long- but not short-term supplementation of beta-cells with IGF-2 resulted in a significant increase in LC3-II formation. Conclusion: The blockade of autophagic activity in the endocrine pancreas seems to be required during postnatal remodeling in order to ensure the correct beta-cell turnover by apoptosis. This event is probably associated to disappearance of islet IGF-2 expression. Acknowledgments: MINECO (BFU2011/25420), CAM (S2010/BMD-2423) and CIBERDEM (ISCIII), Spain. P02-6 Development of new cell genetic tracing tools for the study of intratumour heterogeneity Laura Quevedo1, Laura González-Silva1, Thaidy Moreno1, Carlos Revilla1, Dieter Saur2, Roland Rad2, Ignacio Varela1 1 Instituto de Biomedicina y Biotecnología de Cantabria (CSIC-UC-Sodercan), Departamento de Biología Molecular, Universidad de Cantabria, Santander, ES, 2Department of Internal Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich, DE; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, DE Intratumour heterogeneity has been observed in multiple cancers and has been postulated as a critical aspect for tumour metastasis and treatment resistance. Therefore, a further characterization of its role in cancer progression and metastasis has become essential to increase our understanding of cancer biology and to improve the treatment of cancer patients1. The use of cell lineage tracing systems, combined with the use of genetically modified mouse models, could be applied in this context. Several genetic tracing systems, based in a random CRE-mediated recombination event in an allele with multiple fluorescent markers surrounded by incompatible lox sites have been created2. Once this recombination occurs, the cell and its genetic descendants are permanently labelled with the same fluorescent marker. Nevertheless, these systems present several limitations like a reduced number of potential colour combinations or problems in the unique identification of the markers. Here we have identified new incompatible lox sites that, together with an efficient selection of fluorescent markers, have allowed us to design a new system that will be able to produce up to 15 different colour combinations 43 Pósters / Posters that can be uniquely identified by confocal microscopy and FACS. This system will be combined with cancer mouse models to study the role and dynamics of intratumour heterogeneity in cancer progression. References [1] Gerlinger, M. et al. “Intratumor heterogeneity and branched evolution revealed by multiregion sequencing”. N. Engl. J. Med. 366, 883–892 (2012). [2] Cai, D., Cohen, K. B., Luo, T., Lichtman, J. W. & Sanes, J. R. “Improved tools for the Brainbow toolbox”. Nat. Methods 10, 540–547 (2013). P02r-7 Obesity and type 2 diabetes alters the immune properties of human adipose derived stem cells Noelia Keiran, Carolina Serena, Victoria Ceperuelo-Mallafre, Miriam Ejarque, Kelly Roche, Catalina Núñez-Roa, Joan Vendrell, Sonia Fernández-Veledo Hospital Universitari de Tarragona Joan XXIII. Institut d´Investigació Sanitària Pere Virgili, Universitat Rovira i Virgili, Tarragona, ES; CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, ES Adipose tissue-derived stem cells (ASCs) are proposed as an alternative stem cell source to bone marrow-derived cells for immune cell therapy. However, microenvironmental factors may impact the functionality of this population in human adipose tissue (AT). We hypothesized that the fat depot in addition to the donor phenotype controls the immunomodulatory capacity of ASCs. Focusing on obesity and type 2 diabetes (T2D) as metabolic disorders that might affect the immune response of ASCs, we compared the inflammatory response of ASCs from subcutaneous and visceral AT of age-matched donors (lean n=4, body mass index [BMI] 21.981.9; obese n=4 BMI 33.12.1 and T2D n=4 BMI 35.31.5). Obese and particularly T2D-derived ASCs showed increased expression of inflammatory markers, activation of NLRP3 inflammasome and higher migration, invasion and phagocytosis capacities than those derived from lean donors. Remarkably, ASCs derived from obese and T2D subjects exhibited a reduction in typical immunosuppressive activities attributed to stem cells. Accordingly, obese and T2D-ASCs were less effective in suppressing lymphocyte proliferation, activating the M2 macrophage phenotype, and in increasing TGF-b1 secretion, than lean-derived ASCs. Treatment of lean hASCs with IL-1b mimicked thedysfunctional immune behaviour of obese and T2D hASCs. Conversely, combined treatment with IL1RA and TGF-b1 reverted the phenotype of obese- and T2D-ASCs. These data indicate that the donor metabolic phenotype compromises the immunomodulatory properties of ASCs. These results are relevant not only for understanding the physiology of ASCs in terms of cell-based therapies but also for their role as key regulators of the immune response. P02-8 Papel de la Kleisina NCAPH en el desarrollo y evolución del cáncer de mama HER2 positivo Sonia Castillo-Luva1, Adrián Blanco Gómez2, Jesús Pérez-Losada2 Universidad Complutense de Madrid, Madrid, ES, 2Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC). Universidad de Salamanca/CSIC. Salamanca, España, Salamanca, ES 1 El cáncer de mama es la neoplasia más frecuente en mujeres y causa de gran morbimortalidad siendo un problema importante de salud pública. Es una enfermedad compleja y heterogénea, con un rango de manifestaciones variable tanto a nivel clínico, como histopatológico, molecular y de la respuesta al tratamiento. (1) Los estudios de expresión génica en carcinoma ductal invasivo (IDC), obtenidos mediante análisis de microarrays en bases de datos, nos han permitido identificar a un miembro del complejo Condensina I como un transcrito sobreexpresado en IDC versus tejido de 44 XXXIX Congreso SEBBM mama normal. El complejo Condensina I participa en la correcta segregación cromosómica y su alteración podría dar lugar a aneuploidías. (2) Hemos encontrado asociación significativa entre el pronóstico de la enfermedad y la expresión de este complejo en la base de datos pública TCGA que contiene información molecular y clínica de más de 825 pacientes con cáncer de mama. (3) Mujeres con niveles altos de expresión del complejo en el tumor presentaron una menor supervivencia que aquellas que presentaron niveles bajos. Es más, niveles elevados de expresión de Condensina I se asociaron con una reducción de supervivencia libre de metástasis. Para estudiar el posible papel que desempeña Condensina I en la evolución del cáncer de mama se ha generado un modelo de ratón transgénico que sobre-expresa el gen del componente Kleisina I del complejo Condensina I y el proto-oncogén Erbb2/Neu. (4) Nuestros datos preliminares indican que niveles altos de expresión de KLEISINA I modifican las características de desarrollo tumoral como son el número de tumores y la capacidad de diseminación en animales expuestos a un estímulo hormonal, como es el embarazo. Sin embargo, no afecta a características temporales de la enfermedad como son la latencia o la duración de la enfermedad. Financiación: Este proyecto está financiado por el Ministerio de Ciencia e Innovación (MINECO/FEDER) SAF2015-64499-R Referencias [1] Polyak K. Heterogeneity in breast cancer. J Clin Invest. 2011 Oct;121(10):3786-8. [2] Wood AJ, Severson AF, Meyer BJ. Condensin and cohesin complexity: the expanding repertoire of functions. Nat Rev Genet. 2010 Jun;11(6):391-404. [3] Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature, 2012 Oct 4;490(7418):61-70. [4] Guy CT, Webster MA, Schaller M, Parsons TJ, CardiffRD, Muller WJ. Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10578-82. P02-9 Tumor suppressor ARF regulates tissue microenvironment and tumor growth through modulation of macrophage polarization Lidia Jiménez, Sandra Herranz, María Ángeles Higueras, Alfonso Luque, Sonsoles Hortelano Unidad de Terapias Farmacológicas. Instituto de Investigaciones de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Majadahonda, ES Tumor microenvironment has been described to play a key role in tumor growth, progression, and metastasis. Macrophages are a major cellular constituent of the tumor stroma, and particularly tumor associated macrophages (TAMs or M2-like macrophages) exert important immunosuppressive activity and a pro-tumoral role within the tumor microenvironment. Alternatively-reading frame (ARF) gene is widely inactivated in human cancer. We have previously demonstrated that ARF deficiency severely impairs inflammatory response establishing a new role for ARF in the regulation of innate immunity. On the basis of these observations, we hypothesized that ARF may also regulates tumor growth through recruitment and modulation of the macrophage phenotype in the tumor microenvironment. Xenograft assays of B16F10 melanoma cells into ARF-deficient mice resulted in increased tumor growth compared to those implanted in WT control mice. Tumors from ARF-deficient mice exhibited significantly increased number of TAMs as well as microvascular density. Transwell assays showed crosstalk between tumor cells and macrophages. On the one hand, ARF-deficient macrophages modulate migratory ability of the tumor cells. And on the other, tumor cells promote the skewing of ARF-/- macrophages toward a M2-type polarization. In conclusion, these results demonstrate that ARF deficiency facilitates the infiltration of Salamanca 2016 macrophages into the tumor mass and favors their polarization towards a M2 phenotype, thus promoting tumor angiogenesis and tumor growth. This work provides novel information about the critical role of ARF in the modulation of tumor microenvironment. P02-10 8,9-dehydrohispanolone-15,16-lactol diterpene prevents LPS-triggered inflammatory responses by inhibiting endothelial activation Lidia Jiménez-García1, Paqui G. Través2, Raquel López-Fontal3, Sandra Herranz1, Sandra Amarilla-Quintana1, Beatriz de las Heras4, Sonsoles Hortelano1, Alfonso Luque1 1 ISCIII, Majadahonda, ES, 2The Salk Institute, La Jolla, California, US, 3Universidad Europea de Madrid, Villaviciosa de Odón, ES, 4 Universidad Complutense de Madrid, Madrid, ES Endothelial activation contributes to lung inflammatory disorders by inducing leukocyte recruitment to pulmonary parenchyma. Consequently, vascular-targeted therapies constitute promising strategies for the treatment of inflammatory pathologies. Here we evaluated the effect of 8,9-dehydrohispanolone-15,16-lactol diterpene (DT), previously described as a regulator of LPS-treated macrophages, on lung endothelium during inflammation. Pulmonary lung endothelial cells pre-treated with DT and activated with LPS or TNF- exhibited reduced expression of the pro-inflammatory cytokines Cxcl10, Ccl5 and Cxcl1 compared with non-treated cells, whereas the anti-inflammatory molecules IL1r2 and IL-10 were induced. Consistent with this result, DT pre-treatment inhibited NF-B nuclear translocation in endothelial cells activated by LPS or TNF-. Besides, conditioned medium from these cells failed to stimulate leukocytes as measured by a reduction in adhesive ability of the leukocyte cell line J774 to fibronectin. Additionally, DT reduced the expression of the endothelial adhesion molecules E-selectin, VCAM-1 and ICAM-1 after activation. Similarly, expression of VCAM-1 and ICAM-1 molecules on the lung endothelial layer of C57/BL6 mice pretreated with DT and challenged with LPS were unchanged. Finally, inhibition of vascular adhesion molecule expression by DT decreased the interaction of J774 cells with lung endothelial cells in an inflammatory environment. Thus, DT exhibits an efficient anti-inflammatory activity on lung vasculature by inhibiting the expression of endothelial adhesion molecules and chemotactic factors. Our findings establish DT as a novel endothelial inhibitor for the treatment of inflammatory-related diseases triggered by gram-negative bacteria or by the associated cytokine TNF-. P02-11 Telomere status and telomerase activity: clinical usefulness in non-small cell lung and colorectal cancer Tamara Fernández-Marcelo1, Carmen De Juan Chocano1, Ana Gómez2, Andrés Sánchez Pernaute3, Florentino Hernando2, José-Ramón Jarabo2, Marina Zorita4, Antonio-José Torres García3, Pilar Iniesta Serrano1 1 Departamento de Bioquímica y Biología Molecular II. Facultad de Farmacia. Universidad Complutense; Instituto de Investigación Sanitaria Hospital Clínico San Carlos (IdISSC), Madrid, ES, 2 Servicio de Cirugía Torácica. Hospital Clínico San Carlos. Instituto de Investigación Sanitaria Hospital Clínico San Carlos (IdISSC), Madrid, ES, 3Servicio de Cirugía General y del Aparato Digestivo. Hospital Clínico San Carlos. Instituto de Investigación Sanitaria Hospital Clínico San Carlos (IdISSC), Madrid, ES, 4Departamento de Bioquímica y Biología Molecular II. Facultad de Farmacia. Universidad Complutense, Madrid, ES Pósters / Posters Lung cancer remains the most common cancer in men and colorectal cancer (CRC) is the 3rd most common in men and the 2nd in women1. For both, the identification of new biomarkers is still needed to predict the course of the disease. Telomere dysfunction and increase in telomerase activity (TA) are frequent events in cancer2,3; however, their role as prognostic factors remains unconfirmed. We evaluated TA and telomere length (TL) in 142 non-small cell lung cancers (NSCLCs) and 132 CRCs, and their corresponding control tissues, from patients submitted to potentially curative surgery at S. Carlos Hospital, Madrid. Group oriented curves for disease-free survival were calculated according to the Kaplan-Meier method considering TA, TL and T/N ratio (TL in tumour vs. control) and differences were evaluated using the Log-Rank test (only patients submitted to curative surgery & median follow-up period: 5 years). Software: SPSS 19 & Cutoff Finder4. Positive TA was found in 86.6% of NSCLCs. The mean TL (MTL) was 6.56±0.26 Kb in NSCLCs and 7±0.19 Kb for controls (P=0.027). The mean T/N ratio was 0.93±0.03. Positive TA was detected in 82.5% of CRCs. The MTL was 5.49±0.23 Kb in CRCs and 7.32±0.33 Kb in controls (P<0.001). The mean T/N ratio was 0.79±0.02. In NSCLCs, the patients whose tumours showed a TL<7.29 Kb or T/N ratio<0.97 had a significantly worse clinical evolution (P=0.034 & 0.040, respectively; and both were independent prognostic markers of the stage). The absence of TA in the tumour conferred a better clinical evolution (P=0.039). In CRCs, the most favorable prognosis was found in the patients with a MTL in tumours <6.35 Kb or T/N ratio<0.665 (P<0.001 & P=0.043, respectively). The MTL in CRCs was shown to be an independent prognostic parameter. No recurrences were detected in the group of patients who had TA negative tumours (P=0.184)5,6. Telomere attrition seems to confer a different prognosis according to the tumour type: negative prognosis in NSCLCs and positive in CRCs. Exploring the molecular basis which underlies telomere shortening, as we previously hypothesized7, could be useful to establish personalized therapies. References [1] http://globocan.iarc.fr/ [2] J Cell Mol Med. 2011;15(6):1227-38. [3] Cancer Treat Rev. 2013;39(5):444-56. [4] PLoS One. 2012;7(12):e51862. [5] J Exp Clin Cancer Res. 2015;34:78. [6] PLoS One. 2016;11(2):e0149626. [7] Oncology. 2012;82(3):153-64. P02r-12 Relevance of BMP9-Mediated signaling in oval cell function during chronic liver injury. Crosstalk with the HGF/c-MET pathway Annalisa Addante1, María García-Álvaro2, César Roncero1, Margarita Fernández1, Laura Almalé1, Nerea Lazcanoiturburu1, Julian Sanz3, Patricia Saperas3, Se-Jin Lee4, Isabel Fabregat5, Steven Dooley6, Peter ten Dijke2, Blanca Herrera1, Aránzazu Sánchez1 1 Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Complutense University of Madrid (UCM), Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, ES, 2Department of Molecular Cell Biology and Cancer Genomics Centre Netherlands, Leiden University Medical Centre, RC Leiden, Leiden, NL, 3Department of Pathology. Hospital Clínico San Carlos, Madrid, ES, 4Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, US, 5Bellvitge Biomedical Research Institute - IDIBELL, L’Hospitalet de Llobregat, Barcelona, ES, 6Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, DE Oval cells (OCs) constitute a bi-potential progenitor cell population from adult liver. Under chronic liver disease (CLD), they become activated and proliferate and differentiate into cholangiocytes and/or hepatocytes to compensate for the cellular loss contributing to maintain liver homeostasis. It is well recognized the critical role of the TGF-β in CLD, but the role 45 Pósters / Posters of other members of the TGF-β superfamily, the BMPs, is only partly understood. We focused our attention in BMP9, a member of the BMP family that has recently emerged as a critical regulator of liver pathology; and explored its potential role in the regulation of OC. For this, WT and BMP9 KO mice were submitted to 0.1% DDC-supplemented diet for 2, 4 or 6 weeks, as a model of liver damage associated with OC expansion. We found a greater expansion of the oval cell population in BMP9 KO mice respect to WT in response to a DDC diet, which suggests that BMP9 plays a negative role in OC-mediated liver regeneration. BMP9 suppressor effects were confirmed in vitro, as BMP9 decreases oval cell number and induces apoptosis. Using a model of OC lines harboring a genetically inactivated HGF receptor (c-Met) tyrosine kinase (Met-/- cells) and its control (Metflx/flx cells) we found that HGF/c-Met signaling inhibits BMP9 suppressor effects. Furthermore, our results indicate that BMP9 activates the Smad pathway in OCs, being this activation diminished in Met-/- OC. Consistently, Smad activation by BMP9 in Metflx/flx oval cells is increased after co-treatment with HGF. Searching for the mechanisms mediating the BMP9/HGF crosstalk, we found that the expression of ALK1, the BMP9 high affinity type I receptor, is up-regulated when OC are treated with BMP9 and HGF. More importantly, loss of ALK1 expression abolishes both the HGF-mediated amplification of BMP9-triggered Smad signaling in OC and HGF protective effects. In conclusion, BMP9 emerges as a novel regulator of OC population exerting suppressor effects. We also provide novel evidence of an interesting signaling crosstalk between BMP9 and HGF/c-Met pathways in OC regulation during CLD. P02-13 Ifg1r media en la regeneración del epitelio bronquiolar murino regulando la cinética de recuperación tras la ablación selectiva de las células club Icíar Paula López García1, Sergio Piñeiro-Hermida1, Rosete Sofia Pais1, Raquel Torrens1, Andreas Hoeflich2, José G. Pichel1 1 Centro de Investigación Biomédica de La Rioja (CIBIR) / Fundación Rioja Salud, Logroño, ES, 2Institute for Genome Biology, Leibniz-Institute for Farm Animal Biology (FBN), Dummerstorf, DE La regeneración y reparación del epitelio pulmonar son vitales para mantener la función e integridad de las vías aéreas. El desequilibrio entre el daño epitelial y la recuperación de éste es la causa de numerosas enfermedades crónicas pulmonares como asma, EPOC, fibrosis pulmonar y cáncer de pulmón. La señalización mediada por IGFs (Insulin-like Growth Factors) se ha relacionado con estas patologías, pero sus mecanismos de acción en este órgano son poco conocidos. La inmuno-localización de su receptor IGF1R revela altos niveles de expresión en las células del epitelio pulmonar. Para determinar la función de IGF1R en el epitelio del pulmón murino se generaron mutantes condicionales Nkx2.1-Cre; Igf1r fl/fl que expresan la recombinasa Cre en el epitelio respiratorio. La falta de IGF1R altera la diferenciación del epitelio bronquiolar en ratones adultos ocasionando un incremento de la proliferación y cambios en la morfología de las células club en los bronquiolos distales, pero sin ocasionar impacto morfológico aparente en el parénquima alveolar. Durante la recuperación del daño bronquiolar inducido por la ablación selectiva de las células club con naftaleno, la deficiencia de IGF1R mantiene los defectos histológicos, aumenta la proliferación y retrasa la diferenciación de las células club y ciliadas. Además, los pulmones de los ratones mutantes también revelan un aumento de expresión de Igf1, Insr, Igfbp3 y de marcadores de precursores epiteliales, un descenso en la proteína Scgb1a1, y alteraciones de los mediadores de la señalización de IGFs. Estos resultados demuestran que IGF1R está implicado en el control de la proliferación y diferenciación celular del epitelio bronquiolar durante su regeneración, y fundamentan su posible participación en patologías respiratorias. 46 XXXIX Congreso SEBBM P02m-14 Effects of a novel synthethic microneurotrophin DHEA-derivative BNN27 in the neurodegenerative and inflammatory component of diabetic retinopathy Silvia Lisa Ferrer1, Ruth Iban-Arias2, Niki Mastrodimou2, Despina Kokona3, Panagiota Iordanidou2, Mara Boumpouli2, Achilleas Gravanis2, Ioannis Charalampopoulos2, Kyriaki Thermos2 1 Instituto de Neurociencias de Castilla y Leon, Salamanca, ES, 2 University of Crete, Heraklion, GR, 3Inselspital, Universitätsspital Bern, Bern, CH Diabetes may induce diabetic retinopathy (DR), a chronic eye disease that lead to retinal cell death and blindness. DR is caused by three components: neurodegeneration, inflammation and neovascularization, but available treatments target only the neovascularization component. Therefore, new therapeutics must be developed to treat the neurodegeneration and inflammatory components. DHEA, a neurosteroid hormone that binds to TrkA receptor with high specificity (Lazaridis, 2011), has neuroprotective and anti-inflammatory effects in the retina (Kokona, 2012; Straub, 1998). Novel DHEA derivatives, such as the spiro-epoxy derivative BNN27, have been synthesizedthat lacking the endocrine side effects of DHEA (Calogeropoulou, 2009). The main objective of this study was to investigate the neuroprotective and anti-inflammatory effect of BNN27 in the rat streptozotocin (STZ) model of DR. We have shown that 4 weeks after STZ injection, the number of ganglion and amacrine cells decreased (Mastrodimou, 2015). Treatment of the diabetic rat with BNN27 (10 mg/kg, ip) for one week, protected amacrine cells that express nitric oxide synthetase and tyrosine hydroxylase and ganglion cell axons. This neuroprotection was shown to be due to BNN27 activation/ phosphorylation of the TrkA receptor and its prosurvival signaling pathway (ERK1/2 kinases) as well as the reduction of the phosphorylation of pro-apoptotic kinases SAPK/JNK. In addition, the transcription factor related to the activation of the inflammatory response, NFκΒ, increased in diabetic retina. An increase in the phosphorylation of Akt, the kinase implicated in the upstream signaling of pro-inflammatory cytokines, was observed in DR. BNN27 reversed Akt activation and the expresion of the pro-inflammatory cytokines TNFα and IL-1β, and increased the expression of the anti-inflammatory cytokines, IL-10 and IL-4 in DR.In conclusion, our results suggest that the DHEA-derivative BNN27 has neuroprotective and anti-inflammatory effects in the diabetic retina and is a putative therapeutic candidate for the treatment of DR. P02-15 Inflammatory up-regulation in the pathogenesis of calcific aortic valve stenosis Iván Parra Izquierdo1, Irene Castaños-Mollor1, Javier López2, Cristina Gómez1, Sergio Ayuso1, Alberto San Román2, Mariano Sánchez Crespo1, Carmen García-Rodríguez1 1 Instituto de Biología y Genética Molecular, CSIC-Universidad de Valladolid, Valladolid, ES, 2Instituto de Ciencias del Corazón, Hospital Clínico Universitario, Valladolid, ES Background and aim: Calcific aortic valve stenosis (CAVS), the most prevalent valvulopathy in Western countries, is characterized by valve thickening. Many evidences point to inflammation as a key event for the development of fibrosis and valve calcification. Since interferons (IFN) coordinate a diverse array of cellular programs through transcriptional regulation of immunologically relevant genes, the aim of this study was to elucidate their role in the inflammatory process in human valve cells. Materials and methods: Endothelial and interstitial valve cells were isolated from stenotic aortic valve (obtained from valve replacement), and non-stenotic aortic and pulmonary valves (obtained from heart transplant recipients). After exposure to recombinant type I and II IFN, the expression Salamanca 2016 of pro-inflammatory molecules was addressed by Western blot and flow cytometry, and monocyte adhesion assayed. Results: Experiments showed a blatant array of cell-specific responses to IFN, including the expression of COX-2, ICAM-1 and E-selectin in endothelial valve cells. Notably, the response was higher in aortic than in pulmonary valve cells. IFN also promoted monocyte adhesion to endothelial monolayers. In control/stenotic interstitial cells, IFNγstrongly induced ICAM-1; in addition, stenotic cells were responsive to IFNα. Strikingly, IFN strongly cooperated with LPS (TLR4 ligand) and Pam2CSK4 (TLR2/6 ligand) to induce COX-2 and ICAM-1 expression, being the synergistic effect higher in aortic than in pulmonary valve cells. Conclusions: IFN are more potent inducers of inflammation in aortic than pulmonary valve cells, thus correlating with clinical differences since pulmonary valve rarely suffer CAVS. Data open the way to explore IFN as a potential therapeutic target for CAVS. P02m-16 La expresión de la ciclooxigenasa-2 en hepatocitos atenúa la esteatohepatitis no alcohólica y la fibrosis hepática en ratones Omar Motiño García-Miguel1, Noelia Agra Andrieu1, Rocio Brea Contreras1, Carmelo García-Monzón2, Javier Vargas-Castrillón2, Lisardo Boscá Gomar3, Marta Casado Pinna4, Rafael Mayoral Moñibas5, Ángela María Martínez Valverde6, Daniel Frances7, Paloma Martín-Sanz3 1 Instituto de Investigaciones Biomédicas Alberto Sols-UAM-CSIC, Madrid, ES, 2Instituto de Investigación Sanitaria Princesa-Hospital Universitario Santa Cristina, Madrid, ES, 3Instituto de Investigaciones Biomédicas Alberto Sols-UAM-CSIC y Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERrhd), Madrid, ES, 4Instituto de Biomedicina de Valencia-CSIC, Valencia, ES, 5Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERrhd) y Universidad de California-La Jolla, San Diego, US, 6Instituto de Investigaciones Biomédicas Alberto Sols-UAM-CSIC y Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas asociadas (CIBERdem), Madrid, ES, 7Instituto de Fisiología Experimental-CONICET, Rosario, AR La ciclooxigenasa 2 (COX-2) participa en diferentes enfermedades hepáticas, pero se conoce muy poco sobre la importancia de COX-2 en el desarrollo y progresión de la esteatohepatitis no alcohólica. Este estudio se diseñó con el objetivo de esclarecer el papel de la expresión de COX-2 en hepatocitos en la patogénesis de la esteatohepatitis y la fibrosis hepática. En el presente trabajo, ratones transgénicos para COX-2 específica del hepatocito (hCOX-2-Tg) y sus correspondientes controles wild-type (Wt) fueron alimentados con una dieta deficiente en metionina y colina (MCD) para establecer un modelo experimental de esteatohepatitis no alcohólica, o inyectados con tetracloruro de carbono (CCl4) para inducirles fibrosis hepática. En nuestro modelo animal, los ratones hCOX-2-Tg alimentados con la dieta MCD presentaron menor grado de esteatosis, “ballooning” e inflamación que los ratones Wt, en parte debido a una disminución del reclutamiento e infiltración de los macrófagos hepáticos, con la correspondiente disminución en los niveles de citoquinas pro-inflamatorias en suero. Además, los ratones hCOX-2-Tg mostraron una atenuación significativa del incremento del estrés oxidativo y la apoptosis hepática inducida por la dieta MCD observada en los ratones Wt. Aún más, los ratones hCOX-2-Tg tratados con CCl4 presentaban, de forma significativa, estadíos menores de fibrosis y un menor contenido hepático de colágeno, hidroxiprolina y marcadores pro-fibrogénicos que los controles Wt. De forma conjunta, nuestros datos indican que la expresión constitutiva de COX-2 en el hepatocito atenúa el desarrollo de la esteatohepatitis no alcohólica y la fibrosis hepática en ratones, mediante una reducción de la inflamación, el estrés oxidativo y la apoptosis, y mediante una modulación de la activación de las células estrelladas hepáticas, respectivamente, Pósters / Posters sugiriendo un posible papel protector de la inducción de COX-2 en la progresión de la enfermedad del hígado graso no alcohólico, EHGNA. P02-17 Study of genetic factors determining the heterogeneous activation of signaling pathways associated with cardiac pathophysiology and their contribution to the individual susceptibility to cardiotoxicity caused by chemotherapy Aurora Gómez Vecino1, Roberto Corchado Cobos2, Susana Fraile Martín3, Carmen García Macías3, María Isidoro García4 1 Instituto de Biología Molecular y Celular del Cáncer (IBMCC)Centro de Investigación del Cáncer (CIC), Zamora, ES, 2Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC). Universidad de Salamanca/CSIC, Salamanca, ES, 3Servicio de Patología Molecular Comparada. Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC). Universidad de Salamanca, Salamanca, ES, 4Instituto de Investigación Biosanitaria de Salamanca (IBSAL); Servicio de Bioquímica Clínica. Hospital Universitario de Salamanca, Salamanca, ES The cardiotoxicity of anthracyclines is a complex trait. The degree of cardiac damage is in part mediated by an imbalance between different intracellular signaling pathways such as p38MAPK or PI3K / AKT which at the same time, have been described as being important in other processes of cardiac tissue. Working hypothesis: (i) Interindividual differences in cardiac levels of signaling pathways may contribute to the different susceptibility among patients to cardiac damage produced by anthracyclines (ii) Identification of genomic regions associated with different levels of these signaling proteins is a strategy to identify part of the genetic component of cardiotoxicity. Material and methods: We studied a cohort of mice with ERBB2 + breast cancer generated by a backcross between two syngeneic strains, C56BL/6 and FVB (carrier of the transgene MMTV-ErbB2 / Neu). The animals were treated with doxorubicin alone or in combination with docetaxel. Histopathological parameters of cardiac damage were quantified using the Ariol automated system. Levels of the following proteins were quantified by Luminex: pCREB (Ser133), pAKT (Ser473), pSTAT5A / B (Tyr694 / Tyr699), pSTAT3 (Ser707), p70S6K (Thr412), p38 MAPK (Thr180 / Tyr182), pJNK (Thr183 / Tyr185), NFKB (Ser536) and pERK1 / 2 (Thr185 / Tyr187). Results: (i) The genetic background influences the activation of heart intracellular signaling pathways. (ii) The levels of activation of these pathways are correlated with histopathological parameters of heart damage. (iii) There are specific and common genetic regions of susceptibility of complex trait (QTL) associated with both processes. Conclusion: We used the levels of different intramyocardial signaling pathways as intermediate phenotypes for the identification of part of the genetic component of susceptibility to heart damage caused by chemotherapy. These results will require further validation to be later transferred to the human population. P02-18 Effects of Tributyltin Chloride on Cybrids with or without an ATP Synthase Pathologic Mutation Ester López Gallardo1, Laura Llobet Sese2, Sonia Emperador Ortiz1, Julio Montoya2, Eduardo Ruiz Pesini3 1 Universidad de Zaragoza- CIBER de Enfermedades Raras, Zaragoza, ES, 2Universidad de Zaragoza, Zaragoza, ES, 3Universidad de Zaragoza-Fundación ARAID, Zaragoza, ES Background: The oxidative phosphorylation system (OXPHOS) includes nuclear chromosome (nDNA)- and mitochondrial DNA (mtDNA)-encoded 47 Pósters / Posters polypeptides. Many rare OXPHOS disorders, such as striatal necrosis syndromes, are due to genetic mutations. Despite important advances in sequencing procedures, causative mutations remain undetected in some patients. It is possible that etiologic factors, such as environmental toxins, are the cause of these cases. Indeed, the inhibition of a particular enzyme by a poison could imitate the biochemical effects of pathological mutations in that enzyme. Moreover, environmental factors can modify the penetrance or expressivity of pathological mutations. Objectives: To study the interaction between p.MT-ATP6 and an environmental exposure that may contribute phenotypic differences between healthy individuals and patients suffering from striatal necrosis syndromes or other mitochondriopathies. Methods: We analyzed the effects of the ATP synthase inhibitor tributyltin chloride (TBTC), a widely distributed environmental factor that contaminates human food and water, on transmitochondrial cell lines with or without an ATP synthase mutation that causes striatal necrosis syndrome. Doses were selected based on TBTC concentrations previously reported in human whole blood samples. Results: TBTC modifies the phenotypic effects caused by a pathological mtDNA mutation. Interestingly, wild-type cells treated with this xenobiotic show similar bioenergetics when compared with the untreated mutated cells. Conclusions: In addition to the known genetic causes, our findings suggest that environmental exposure to TBTC might contribute to the etiology of striatal necrosis syndromes. P02-19 Puesta a punto de un modelo celular para el estudio de la función OXPHOS en la enfermedad de Alzheimer Alba Pesini, Eldris Iglesias, Pilar Bayona-Bafaluy, Nuria Garrido, Carmen Hernández-Ainsa, Ester López-Gallardo, Julio Montoya, Eduardo Ruiz-Pesini Universidad de Zaragoza, Zaragoza, ES La enfermedad de Alzheimer (AD) es un proceso neurodegenerativo del sistema nervioso central. Esta patología se caracteriza por pérdida de memoria, cambios de comportamiento y demencia. Estos procesos mentales dependen del correcto funcionamiento de las redes neuronales, que a su vez dependen de las sinapsis entre las neuritas. La generación de neuritas requiere producción de membrana plasmática. Nosotros proponemos que el sistema OXPHOS, por su implicación en la síntesis de novo de nucleótidos de pirimidina que participan en la síntesis de fosfolípidos, juega un papel muy importante en la elaboración de estas neuritas. Para probar nuestra hipótesis, usamos una línea celular, neuroblastoma SH-SY5Y, con capacidad de diferenciación a neurona. Dado que las neuronas colinérgicas son una de las células más afectadas en la AD, vamos a poner a punto un modelo de neurona colinérgica para el estudio de la función OXPHOS. Lo primero que llevamos a cabo es la caracterización genética y celular. Posteriormente estudiamos mediante citometría de flujo diferentes parámetros de diferenciación neuronal y de neurona colinérgica. También analizamos por ELISA los niveles de acetilcolina celular. Finalmente, hemos estudiado los cambios que operan en el sistema OXPHOS (actividad y cantidad del complejo respiratorio IV y consumo de oxígeno) durante este tipo de diferenciación celular y los efectos que provocan diferentes compuestos que actúan sobre el sistema OXPHOS en la diferenciación. Esta caracterización basal nos va a permitir analizar la función OXPHOS en la síntesis de novo de nucleótidos de pirimidina, en la generación de membrana y, finalmente, en la diferenciación neuronal. 48 XXXIX Congreso SEBBM P02-20 Analysis of genetic and phenotypic interactions between DNA damage / genotoxicity pathways in heart tissue and heart damage caused by anthracyclines and taxanes Roberto Corchado Cobos(#)1, Aurora Gomez Vecino(#)2, Carmen García Macías3, Telmo Rodrigues Teixeira3, María Isidoro García4, María Asunción García Sánchez5, Julie Milena Galvis Jiménez6, Isabel Ramos Fernández1, Adrián Blanco Gómez(*)7, Pedro Luis Sánchez Fernández(*)8, Jesús Pérez Losada(*)7 1 Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC). Universidad de Salamanca/CSIC, Plasencia, ES, 2 Instituto de Investigación Biosanitaria de Salamanca (IBSAL), Salamanca, ES, 3Servicio de Patología Molecular Comparada. Instituto de Biología Molecular y Celular del Cáncer (IBMCCCIC). Universidad de Salamanca, Salamanca, ES, 4Instituto de Investigación Biosanitaria de Salamanca (IBSAL); Servicio de Bioquímica Clínica. Hospital Universitario de Salamanca, Salamanca, ES, 5Departamento de Ciencias Biomédicas y del Diagnóstico. Facultad de Medicina. Universidad de Salamanca, Salamanca, ES, 6Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC). Universidad de Salamanca/CSIC, Salamanca. Instituto de Investigación Biosanitaria de Salamanca (IBSAL) Salamanca. Instituto Nacional de Cancerología de Colombia, Bogotá, CO, 7Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC). Universidad de Salamanca/CSIC. Instituto de Investigación Biosanitaria de Salamanca (IBSAL), Salamanca, ES, 8 Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC). Universidad de Salamanca/CSIC. Servicio de Cardiología Hospital Universitario de Salamanca, Salamanca, ES Introduction: Anthracyclines are among the most widely used chemotherapeutic agents in the treatment of a variety of tumors. The identification of genetic and molecular factors responsible for the increased risk of CDA (cardiotoxicity due to anthracyclines) will contribute to a better understanding of their pathophysiology, which could lead to new approaches to predict, prevent and treat this serious complication of chemotherapy. Working hypothesis: Based on two premises: (i) anthracyclines have a pro-genotoxicity effect. Differences in anti-genotoxicity pathways and genetic variants could contribute to different susceptibility to CDA. (ii) The usefulness of a simplified model system to identify genetic determinants involved in the quantitative inheritance of complex traits. Materials and methods: We treated a cohort of mice carrying ERBB2 breast cancer with doxorubicin alone (N = 85) or in combination with docetaxel (N = 77). The cohort was generated by a backcross between two genetically homogeneous strains, C57BL/6 and FVB, with the latter carrying the MMTV- ErbB2 / Neu transgene. Histopathologic heart damage was assessed by quantification of histologic parameters using Ariol automated system. Cardiac level of some key anti-genotoxicity proteins: ATR total, pp53 (Ser15), P21 Total, Total MDM2, pHistone H2AX (Ser139), pCHK1 (Ser345) and pCHK2 (Thr68) were quantified. Results: We identified: (i) differences dependent on the genetic background in both cardiotoxicity and the levels of proteins implicated in the pathways protecting against genotoxicity; (ii) activation of anti-genotoxicity pathways were associated with chemotherapy cardiotoxicity; (iii) quantitative trait loci (QTLs) specific and common to cardiotoxicity and the levels of the pathways studied. Conclusion: We identified genetic determinants associated with anthracycline cardiotoxicity using components of the anti-genotoxic pathways as subphenotypes. Crosses of syngeneic mouse strains are useful in these studies, but require further validation in the human population. (#) Igual contribución como primeros autores. (*) Igual contribución como autores senior. Salamanca 2016 P02r-21 Xenobiotics that affect oxidative phosphorylation alter differentiation of human adipose-derived stem cells at concentrations that are found in human blood Laura Llobet, Janne M. Toivonen, Julio Montoya, Eduardo Ruiz-Pesini, Ester López-Gallardo Universidad de Zaragoza, Zaragoza, ES Adipogenesis is accompanied by differentiation of adipose tissue-derived stem cells to adipocytes. As part of this differentiation, biogenesis of the oxidative phosphorylation system occurs. Many chemical compounds used in medicine, agriculture or other human activities affect oxidative phosphorylation function. Therefore, these xenobiotics could alter adipogenesis. We have analyzed the effects on adipocyte differentiation of some xenobiotics that act on the oxidative phosphorylation system. The tested concentrations have been previously reported in human blood. Our results show that pharmaceutical drugs that decrease mitochondrial DNA replication, such as nucleoside reverse transcriptase inhibitors, or inhibitors of mitochondrial protein synthesis, such as ribosomal antibiotics, diminish adipocyte differentiation and leptin secretion. By contrast, the environmental chemical pollutant tributyltin chloride, which inhibits the ATP synthase of the oxidative phosphorylation system, can promote adipocyte differentiation and leptin secretion, leading to obesity and metabolic syndrome as postulated by the obesogen hypothesis. P02r-22 Una mutación en TSFM causa ataxia de inicio infantil y cardiomiopatía no obstructiva Sonia Emperador Ortiz1, M. Pilar Bayona-Bafaluy1, Ana Fernández-Marmiesse2, Carmen Hernández-Ainsa1, Mercedes Pineda3, Blanca Felgueroso4, Ester López-Gallardo1, Rafael Artuch3, Iria Roca2, Eduardo Ruiz-Pesini1, María Luz Couce2, Julio Montoya1 1 Universidad de Zaragoza, Zaragoza, ES, 2Hospital Clínico Universitario de Santiago de Compostela. Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Santiago de Compostela, ES, 3Hospital Sant Joan de Deu. Institut de Recerca Pediàtrica (IRP-HSJD), Barcelona, ES, 4Hospital Materno Infantil Teresa Herrera, A Coruña, ES El sistema de fosforilación oxidativa (OXPHOS) es una ruta bioquímica implicada en muchos procesos celulares clave. Este sistema está constituido por la cadena de transporte de electrones (ETC) formada por los complejos I al IV (CI-CIV) y la ATP sintasa (CIV). Estos complejos que forman el sistema OXPHOS incluyen 13 polipéptidos codificados en el DNA mitocondrial (mtDNA), pero este mtDNA también codifica para 22 tRNA y 2 rRNA necesarios para la expresión de dichos polipéptidos. El sistema de traducción mitocondrial depende también de muchas proteínas codificadas en el DNA nuclear (nDNA) de manera que mutaciones en cualquiera de los componentes del sistema de traducción mitocondrial pueden ser responsables de diferentes patologías implicadas en la disfunción mitocondrial. En este trabajo describimos un paciente que sufre ataxia progresiva de inicio infantil y cardiomiopatía hipertrófica, portador de una mutación en homocigosis en el gen que codifica para el factor de elongación de la traducción mitocondrial Ts (TSFM). Mediante estudios bioquímicos, moleculares y celulares confirmamos la patogenicidad de esta mutación, al rescatar el fenotipo normal en fibroblastos del paciente tras la transfección de la proteína wild-type. Recientemente se han descrito varios casos de diferentes mutaciones en este mismo gen, en pacientes con fenotipos similares, y hay un número creciente de publicaciones que describen mutaciones en genes nucleares relacionados con la maquinaria de traducción mitocondrial. Pósters / Posters Por ello consideramos necesario el análisis de estos genes en pacientes con una clínica de enfermedad mitocondrial tras descartar alteraciones en el mtDNA. Financiación: ISCIII; FIS (PI14/00005); CIBERER. P02-23 The Fibronectin Synergy Site is Essential for Platelet Function María Benito-Jardón1, Mercedes Costell Rosselló2 Universitat de València ERI en Biotecnología y Biomedicina Campus de Burjassot, Burjassot, ES, 2Universitat de València, Burjassot, ES 1 The large extracellular matrix (ECM) protein Fibronectin (FN) is a structural component of tissues but it is also found as a soluble plasma protein. FN regulates cell activity by binding cellular receptors, like integrins. The major binding site for integrins in FN is the RGD motif located in the 10 th FN type III repeat. Additionally, the α5β1 and αIIb3 integrins can also bind the synergy site (DRVPPSRN) in the FN type III9. The synergy site has been described as important to mediate either the initial cell adhesion or the reinforcement of the integrin-FN bond. The arginines within the synergy sequence were mutated to generate a mouse with impaired synergy site (FNsyn). The homozygous synergy mutant mice were viable and fertile, but tail bleeding times were 1.6-fold increased compared to wild-type littermates. Study of the thrombus formation after inducing an injury in the microvasculature by intravital microscopy showed that the occlusion of arterioles in FNsyn mice occurred significantly slower. Platelets mediate this process and contain the FN ligands α5β1 and αIIb3 integrins. Platelets of the FNsyn mice accumulated less FN, pointing that their ability of binding FN is reduced. Since αIIb3 can also bind fibrinogen (Fg), to abolish any compensation by Fg we generated the double mutant mice FNsynβ3-/-. Contrary to the β3 -/-, the FN synβ3-/-mice died at E16.5 and from E12.5 display multiple skin bleedings and oedema due to a defect in the separation of the lymphatic system from the blood vessels. This process is platelet-dependent. To deepen in the platelet function on a FNsyn background we performed platelet spreading and flow chamber adhesion assays on FNsyn and FN wt, which showed that β3-/- platelets are able to spread on FNwt but not on FN syn and both β3 -/- and β3 +/+ were unable to adhere to FN syn under physiological flow conditions while they could adhere to the FN wt. These results confirmed the in vivo observations that the FN synergy site plays an important role in haemostasis and in absence of β3 integrin becomes essential for platelet function. P02-24 Role of interferons and pro-inflammatory mediators on macrophages in mechanical stress in asthma María Ester Quesada del Bosque, Hitasha Rupani, Arjana A. Sivaloganathan, Peter H. Howarth, Tilman Sanchez-Elsner Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK Asthma is a chronic inflammatory disease that is characterised by airway narrowing due to inflammation and remodeling. This pathology makes airways hyperresponsive to a variety of environmental factors allergens, viruses, among others. Our group has studied the role of the alveolar macrophages (AMs) from asthmatics in response to stress produced by respiratory viruses, mainly rhinovirus (RV), which have shown a reduction in RV-induced interferons (INFs) in severe asthmatics compared to healthy. Airways 49 Pósters / Posters epithelium also plays an active role in immunity and inflammation and has been shown to be impaired in asthmatics, being it more permeable to environmental factors and overexpresses proinflammatory cytokines. Part of our team (Grainge et al., N Engl J Med 2011;364:2006-15) showed how bronchoconstriction affects epithelium remodeling stimulating collagen synthesis and an increase in the number of glandular cells. Numerous environmental factors interact to influence susceptibility and trigger bronchoconstriction. Our goal is to evaluate the effect of stress suffered from asthmatics when exposed to triggers environments on the immune system, alveolar macrophages, on their response to virus. We have recruited 24 mild asthmatics that underwent methacholine challenge (bronchoconstriction) or saline challenge (control group, no bronchoconstriction). Our preliminary data show significant differences in the immune response after the asthma attack, with macrophages expressing less inflammatory cytokines after bronchoconstriction than before, when exposed to RV. These results suggest that macrophages may become less pro-inflammatory after bronchoconstriction, thus enhancing inflammation in the lungs. P02-25 PSA-NCAM en el cáncer colorrectal: expresión en pacientes, líneas tumorales y estabilidad de la cola de PSA Almudena Fernández Briera1, Lucía Patiño Álvarez1, Almudena Fernández Briera2, Carlos Villaverde Taboada3, Emilio Gil Martín1 1 Dpto. Bioquímica, Genética e Inmunología. Universidad de Vigo, Vigo, ES, 2Universidad de Vigo, Vigo, ES, 3Dpto. Estadística e Investigación Operativa. Universidad de Vigo, Vigo, ES La pérdida de la adhesión entre células tumorales es uno de los factores que fomenta su desagregación y posterior diseminación y metástasis. Nuestro grupo viene trabajando en una de las moléculas de adhesión, la NCAM (o molécula de adhesión de células neurales) en el cáncer colorrectal (CCR). La NCAM es una glicoproteína de membrana involucrada en la adhesión célula-célula y célula-matriz. Presenta tres isoformas principales, conocidas por sus respectivas Mr, NCAM-120, 140 y 180. Estas isoformas pueden unir un homopolímero dealfa(2,8) siálico (ácido polisiálico o PSA) en 2 de sus 6 sitios de N-glicosilación. La retención de agua y fuerte repulsión electrostática del PSA se cree que favorecen la diseminación de algunos tipos tumorales. La puesta a punto de un protocolo para la inmunodetección de NCAM y PSA en una misma muestra (patente Ref. ES2571302 A1) nos ha permitido valorar la expresión de la proteína y de su grado de polisialilación en la fracción citosólica y de membrana de 21 especímenes pareados de tejido sano y tumoral de 21 pacientes con CCR, así como en 7 líneas tumorales de colon y en cerebro de ratón. Los resultados demuestran la expresión de NCAM-140 en 20 de los 21 pacientes analizados y una aparente tendencia a sobreexpresarse en el tejido tumoral. Por su parte, el PSA rinde una señal intensa y continuada desde Mr ~180 hasta >260, que sugiere la presencia también de NCAM-180 fuertemente polisialilada. Esta polisialilación (en su mayoría presumiblemente NCAM-180) disminuye en gran parte en el tejido tumoral (p=0,008, de acuerdo con el test de Wilcoxon). El estudio de la estabilidad térmica y frente al pH del PSA, tanto en el tejido sano y tumoral de CCR como en cerebro postnatal de ratón, confirman la presencia de NCAM-140 y 180 en ambos tejidos. Asimismo, se ha comprobado la expresión de NCAM-140 en las líneas tumorales de colon HT-29, Caco-2, SW480 (no así en las SW620), DLD-1, HTC116 y Colo-205 en sus estados adhesivo y en suspensión. 50 XXXIX Congreso SEBBM P02r-26 Mitochondrial dynamics and mitochondrial DNA Aida Rodríguez Nuevo, Eduard Noguera, Àngels Díaz Ramos, Antonio Zorzano Institute for Research in Biomedicine (IRB Barcelona), Barcelona, ES; CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, ES; Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, ES Mitochondria are dynamic organelles, which undergo fission and fusion events. Fission is the process by which mitochondria are divided. The opposite process is fusion: Mitofusin 1 and 2 are responsible for the outer membrane fusion, whereas Opa1 is for the inner membrane fusion. The flexibility entailed by these events is crucial to ensure mitochondrial quality and functionality. Moreover, mitochondrial DNA (mtDNA) integrity is tightly related to these proteins. Several authors purpose Opa1 as determining for mtDNA stability. The absence of diffusion of photo-activatable mitochondrial GFP protein in Opa1 deficient cell showed complete fragmentation of the mitochondrial network. Using a C2C12 muscle cells we observed that ablation of Opa1 induced mtDNA instability. Confocal microscopy analysis revealed a reduction of mtDNA labelling and a misplacement of the mtDNA extra-mitochondrially. We plan to investigate the effects of these observations, which are expected to be crucial to understand the importance of both Opa1 and mtDNA regulation in the skeletal muscle cell context. P02-27 Non-invasive monitoring of hypoxia-inducible factor activation by optical imaging during antiangiogenic treatment in a xenograft model of ovarian carcinoma Beatriz Martínez-Poveda1, Valentí Gómez2, Marisa Alcaide-Germán2, Sara Perruca2, Silvia Vázquez2, Emilio Alba3, Oriol Casanovas4, María Laura García-Bermejo5, Luis del Peso2, Benilde Jiménez2 1 Universidad de Málaga, Andalucía Tech, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Málaga, ES, 2Departamento de Bioquímica, Instituto de Investigaciones Biomédicas (CSIC-UAM), Madrid, ES,3Servicio de Oncología Médica, Hospital Virgen de la Victoria, Málaga, ES, 4Laboratorio de Angiogénesis Tumoral, IDIBELL, L’Hospitalet de Llobregat, ES, 5 Departamento de Patología, Hospital Ramón y Cajal, Madrid, ES Targeting the hypoxia response pathway and angiogenesis are two promising therapeutic strategies for cancer treatment. Their use as single strategies has important limitations. Thus, development of combined regimens has become an important step toward improving therapeutic efficacy. Also, non-invasive monitoring of the response to targeted biological therapies, as well as determination of the optimal schedule for combination regimens has become an active field of research over the last five years, with relevance for both preclinical and clinical settings. Here, we used an optical imaging method to non-invasively monitor the functional changes in HIF activity in response to antiangiogenic treatment in a xenograft model of human ovarian carcinoma. A bioluminescent reporter construct containing nine copies of the hypoxia response element upstream of the luciferase gene (9xHRE-luciferase) was characterized in vitro in a panel of tumor cell lines and in vivo in a subcutaneous xenograft model of ovarian carcinoma by means of optical imaging. We showed that in OVCAR-3 subcutaneous xenografts, the most abrupt change in the HIF functional reporter occurs before the onset of massive tumor growth. However, this system failed to detect hypoxia induced upon antiangiogenic treatment due to the compensating effects of increased hypoxia and decreased tumor cell viability caused by imbalanced neovascularization vs. tumor expansion. Therefore, the readout based on HIF functional reporter Salamanca 2016 could be conditioned by the dynamics of tumor growth and angiogenesis, which is highly variable depending on the tumor type, tumor model and stage of progression P02r-28 Unraveling new roles for macrophages in cardiac repair upon myocardial infarction Laura Alonso Herranz1, Pilar Gonzalo1, Marta Cedenilla1, Vanessa Nuñez1, Luis Jesús Jiménez-Borreguero1, Carlos López-Otín2, Alicia G. Arroyo1, Mercedes Ricote1 1 Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, ES, 2Universidad de Oviedo, Oviedo, ES After myocardial infarction (MI), monocyte infiltration and subsequent differentiation into macrophages is essential for cardiac recovery, but mechanisms remain not well defined. To understand the role of macrophages, we performed two different models of injury in C57BL/6 mice: permanent left anterior coronary artery ligation (LAD-ligation) and cryoinjury. LAD-ligation resulted in more pronounced worsening of the LV function and higher fibrotic deposition compared to cryoinjury. In order to analyze the possible role of macrophages in the different functional outcome, we compared the genetic signature of isolated cardiac macrophages upon injury and found mainly differences in matrix-remodeling genes. Interestingly persistent elevated levels of MT1-MMP were found in post-LAD versus post-CRYO macrophages. This result prompted us to investigate the particular functions of this protease in macrophages after cardiac injury. Consequently, LAD-ligation was performed in a transgenic mouse line with macrophage specific deletion of the MT1-MMP(MT1-MMPDLysM mice). Histological and echocardiographic analysis in MT1-MMPDLysM mice upon MI showed a decrease in LV dilatation and fibrosis, better preserved healthy myocardium and higher density of arterioles that finally lead to amelioration of cardiac dysfunction compared to their control littermates. This study unravel that macrophage MT1-MMP absence attenuates cardiac dysfunction by increasing arteriogenesis, and suggest new treatment options for cardiac ischaemic disease based on metalloprotease modulation in cardiac macrophages. Funding: TV3 Marató Foundation (MTV/PIC1203) and “Obra Social La Caixa” (International PhD “la Caixa” - Severo Ochoa 2014). P02-29 Lack of LOXL2 (lysil oxidase-like 2) reduces mouse renal fibrosis after surgical unilateral ureteral obstruction Pósters / Posters roles in organ development, cicatrization, and fibrosis. Irrespective of its catalytic activity, LOXL2 has been shown to be an inductor of epithelial-mesenchymal transition (EMT). In this work, unilateral ureteral obstruction (UUO), an experimental model of obstructive nephropathy characterized by EMT and renal fibrosis (Grande et al., Nat Med. 2015; 21:989-97) was performed in LOXL2 gain and loss of function mice (Martin et al., EMBO J. 2015; 34:1090-109), and its effects in ECM proteins expression and renal fibrosis were analyzed as previously described (Muñoz-Felix et al.; KidneyInt. 2014; 85:319-32). Lack of expression of LOXL2 resulted in lowered ECM proteins (collagen-I and alfa-SMA) expression and reduced interstitial renal fibrosis compared with WT mice after 15 days of UUO. These results uncover a role of LOXL2 in kidney fibrosis, thus being a potential target to pharmacological intervention in human chronic kidney disease. P02m-30 Vochysia rufa stem bark extract protects endothelial cells against high glucose damage Neire Moura de Gouveia1, Sonia Ramos2, M. Ángeles Martín2, Luis Goya Suárez2, Olga Palomino3 1 Department of Biofunctional Science, Faculty Mineirense, Mineiros, Goias, Brasil, BR, 2Department of Metabolism and Nutrition, Institute of Science and Food Technology and Nutrition (ICTAN – CSIC), Madrid, ES, 3Department of Pharmacology, Faculty of Pharmacy, Universidad Complutense de Madrid, Madrid, ES Increased oxidative stress by persistent hyperglycemia is a widely accepted factor in vascular damage responsible for type 2 diabetes complications. The plant Vochysia rufa (Vr) has been used in folk medicine in Brazil for the treatment of type 1 and 2 diabetes. In this study, the protective effect of a Vr stem bark extract against a challenge by a high glucose concentration on EA.hy926 (EA) endothelial cells was evaluated. The Vr stem bark was extracted with water by maceration and then evaporated until dryness under vacuum. Treatment with 0.5-100 μg/mL of Vr extract for 24h did not affect cell viability. The extract was diluted at concentrations of 5, 10 and 25 μg/mL and maintained for 24 hours along with 30 mM of glucose to evaluate the protective effect of the extract on the EA cells. The treatment of EA cells with 30 mM of glucose for 24h significantly increased the cell damage. Furthermore, EA cells treated with 30 mM of glucose showed a decrease of reduced glutathione (GSH) concentration and increased protein carbonyl levels and activity of antioxidant enzymes, compared to control. Vr concentrations significantly reduced cell damage evoked by glucose. While 5 and 10 μg/mL Vr evoked a partial protection against the glucose insult, 25 μg/mL Vr fully recovered GSH, antioxidant enzymes and carbonyls to baseline levels. This study demonstrates that phytochemicals from V. rufa stem bark may help to protect endothelial cells against oxidative damage by modulating GSH concentration, antioxidant enzyme activity and protein carbonyl levels. Carmen Arizmendi López1, María Teresa Grande2, María Auxiliadora Aparicio2, Alberto Martín Martín3, Annette G. Düvet2, Luis Gamello-Pozuelo2, Miguel Ángel Arévalo4, Amparo Cano3, José Miguel López Novoa2 1 Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, Salamanca, ES, 2Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, ES, 3 Departamento de Bioquímica, UAM, Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Madrid, ES, 4Departamento de Histología y Anatomía Humana, Universidad de Salamanca, Salamanca, ES M. Rodríguez Quiroga1, L. Vázquez Iglesias1, L. Barcia Castro1, M. Páez de la Cadena1, F. J. Rodríguez Berrocal1, O. J. Cordero2 1 Departamento de Bioquímica, Genética e Inmunología. Universidad de Vigo, Vigo, ES, 2Departamento de Bioquímica y Biología Molecular. Universidad de Santiago de Compostela, Santiago de Compostela, ES LOXL2 is the member of the lysil oxidase family most abundant in the kidney. The family classical catalytic activity is the formation of aldehydes from lysine residues in collagen and elastin precursors. These aldehydes undergo spontaneous chemical reactions with other lysyl oxidase-derived residues that results in cross-linking of proteins of the extracellular matrix (ECM) resulting in its stabilization. Lox enzymes have shown important Cancer Stem Cells (CSCs) include a subpopulation of cells responsible for tumour survival, resistance to chemotherapy, recurrence and metastases, called Metastatic Stem Cells (MetSCs). Recently, DPP-IV/CD26 was proposed as a new CSC and MetSCs biomarker in colorectal cancer (CRC). In a panel of CRC cell lines from patients at different stage: SW1116 (stage A), HT-29, Caco-2, SW480 (stage B), SW620 (lymph node P02-31 CD26: a colorectal cancer stem cell marker 51 Pósters / Posters metastasis), DLD -1 (stage C), COLO205 (peritoneal metastasis) and T84 (lung metastasis), we had evaluated by flow cytometry the presence of the most favourite biomarkers candidates for CRC CSCs and MetSCs: CD133, CD44, EpCAM, LGR5, riboflavin-dependent autofluorescence, and CD26. Here we studied them in the spheroids originated from those lines in vitro. All cell lines could form spheres in the first generation, confirming the presence of CSCs in all cell lines. SW1116 showed the smallest spheres and the lower number of cells disaggregated from the spheres. All cell lines, except SW1116, did form subspheres in serial passages. Efficiency was essentially maintained through passages in these cell lines with self-renewal, except SW620 (loss of efficiency) and T84 (enhanced efficiency). E-cadherin expression among sphere-forming cells showed quite equivalent levels and higher than in the cell lines, as expected for proliferating cells in epithelial state, although not all (particularly Colo205 cells) are E-cadherin+. Sphere-forming cells also showed enhanced levels of LGR5 expression (2-3-fold in general). Still, the heterogeneity remains, as DLD-1 and Colo205 showed similar frequencies (11%) in comparison to the HT-29 (52%) and Caco-2 (88%) cell lines with much higher expression. To note, the cell lines with higher frequencies of LGR5+ also show a high frequency of the LGR5+ EpCAMhigh subset. The other biomarkers showed an important heterogeneity where CD26 and CD133 mark different CSCs subsets. Financiación: Plan Nacional I+D+I (PI15/02007), Fundación Científica AECC (GCB13131592CAST) y Xunta de Galicia (GRC2014/019 y R2014/039). P02-32 Relación entre arginina, poliaminas, obesidad y resistencia insulínica: estudio en ratones ob/ob, un modelo de obesidad y riesgo cardiovascular Sandra Tavárez Alonso, Giovanna Pulido Núñez, Encarna Morant Peiró, Lidia López López, Eulalia Alonso Iglesias Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia, ES Numerosas evidencias experimentales sustentan beneficios de la arginina en las patologías de riesgo cardiovascular y, concretamente, en la obesidad y la resistencia insulínica (RI). Los mecanismos moleculares propuestos para la acción de la arginina se han relacionado con su papel como sustrato precursor del óxido nítrico. Sin embargo la implicación en estos efectos de las poliaminas, otros metabolitos derivados de la arginina relacionados tanto con la obesidad como con la liberación y acción de la insulina, permanece sin ser evaluada. Con este objetivo hemos analizado en nuestro estudio los niveles de poliaminas (putrescina, espermidina y espermina; HPLC) en páncreas y músculo de ratones obesos (ob/ob) y control, los efectos sobre ellos de la suplementación de arginina (1% en el agua de bebida), así como su relación con la RI (índice HOMA), grado de adiposidad y otros parámetros de deterioro metabólico. En nuestro modelo, la suplementación de arginina redujo significativamente la ganancia ponderal, el peso de los paquetes grasos, el estrés oxidativo (MDA), la inflamación (IL-6) y, particularmente, la RI (disminución de glucemia e insulinemia). Estos efectos se relacionan con cambios en los niveles de poliaminas que incluyen disminución de espermina en páncreas y elevación muscular de putrescina. De acuerdo con estos resultados, los efectos metabólicos de la arginina parecen implicar diferentes procesos y vías, algunas de las cuales podrían suponer cambios sustanciales en los niveles de poliaminas. Estos policationes, según se ha propuesto, podrían ejercer papeles selectivos estimuladores o permisivos en relación a la síntesis y liberación de insulina y en la sensibilidad a la misma. Financiado con las ayudas CONSOLIDER-INGENIO CSD2007-00063 y AGL2014-58205-REDC. 52 XXXIX Congreso SEBBM P02-33 La citoquina proinflamatoria TNF-α promueve la fosforilación y la deslocalización de la proteína asociada a microtúbulos Tau en células de neuroblastoma humano SH-SY5Y diferenciadas Montaña Caballero Bermejo, Marta Olivera Santa-Catalina, Juan Carlos Alonso, Ricardo Argent, Francisco Centeno, Mª Jesús Lorenzo Benayas Universidad de Extremadura, Cáceres, ES La enfermedad de Alzheimer (EA) es un desorden neurodegenerativo asociado a la edad. Uno de los marcadores neuropatológicos de esta enfermedad es la presencia de ovillos neurofibrilares (NFT) compuestos por la proteína Tau hiperfosforilada y proteolizada. Se ha descrito que TNF-α colocaliza con los NFT en las zonas del cerebro afectadas por la EA, lo que sugiere que esta citoquina puede regular la formación, la agregación y la deposición de los NFT. Además, la fosforilación de Tau aumenta en un entorno inflamatorio, aunque de momento se desconoce la importancia de las citoquinas sobre la fosforilación de residuos específicos de Tau. El objetivo de este trabajo fue estudiar el efecto de la citoquina TNF-α sobre la fosforilación residuo específica y la localización intracelular de la proteína Tau en células SH-SY5Y diferenciadas. El tratamiento de las células con TNF-α produjo un aumento de los niveles y de la fosforilación de Tau en los residuos Thr-50 y Thr-69 de manera dependiente a la concentración utilizada. El aumento en los niveles de Tau inducidos por TNF-a se previno en presencia de actinomicina D y de cicloheximida lo que sugiere que esta citoquina induce la expresión genética de Tau en nuestras condiciones experimentales. Además, TNF-a promovió cambios en la localización de las formas de Tau fosforiladas, que pasaron de encontrarse principalmente en las neuritas a concentrarse en el soma celular y en núcleo. Nuestros resultados sugieren que la inflamación puede contribuir a la patología neurofibrilar de la proteína Tau. Trabajo financiado por los proyectos: GRU10046, GRU15164 (FEDERJunta de Extremadura) y FIS PS/1330. P02-34 Different expression levels of DLK1 inversely modulate proliferation and oncogenic potential of human MDA-MB-231 breast cancer cells through inhibition of NOTCH signaling María Julia González Gómez1, Ana Isabel Naranjo Pastor2, Victoriano Baladrón García2, Jorge Laborda Fernández1, María Luisa Nueda Sanz1 1 Biochemistry and Molecular Biology Branch, Department of Inorganic, Organic Chemistry and Biochemistry. School of Pharmacy. University of Castilla-La Mancha/CSIC, Albacete, ES, 2 Biochemistry and Molecular Biology Branch, Department of Inorganic, Organic Chemistry and Biochemistry. Medical School; CRIB/Biomedicine Unit. University of Castilla-La Mancha/CSIC, Albacete, ES NOTCH receptors participate in cell proliferation and survival of several types of cancer cells. Evidence accumulated indicates that these receptors can function as oncogenes or as tumor suppressors depending on the cellular context. The EGF-like protein DLK1 acts as a NOTCH signaling inhibitor and it is also involved in the regulation of cell growth and cancer. In this work, we focused on the role of the protein DLK1 in the control of breast cancer cell growth, where NOTCH receptors have been shown to play both antagonist roles. We found that human DLK1 inhibit NOTCH1 signaling in MDA-MB-231 breast cancer cells. The proliferation rate and migration capabilities of these cells depended upon the level of NOTCH1 activation and signaling as regulated by DLK1. In particular, high levels of DLK1 expression leaded to a great decrease in NOTCH1 signaling, which associated Salamanca 2016 to a decrease of breast cancer cell proliferation and migration. On the contrary, lower levels of NOTCH1 signaling inhibition caused by lower levels of DLK1 overexpression led to an increase of in vitro MDA-MB-231 cell migration, and both in vitro and in vivo cell proliferation. The data presented in this work suggest that a fine regulation of NOTCH signaling plays an important role in the control of breast cancer cell proliferation and migration. Financial Support: This work was supported by funds from the Spanish Cancer Association (AECC) and the Health Council of the Regional Government of Castilla-La Mancha, Spain (PI-2008/20), and from the Ministry of Economy and Competitiveness (BFU2010-16433). P02-35 Relación entre la calpaína-2 y la actividad nucleolar en células humanas de cáncer de colon Marcelino Telechea-Fernández1, Rosa Zaragozá2, Concha García3, Juan R. Viña3, Andrés Cervantes1, Elena R. García-Trevijano3 1 Dpto. Hematología y Oncología Médica. Facultad Medicina. Univ. Valencia. IIS INCLIVA, Valencia, ES, 2Dpto. Anatomía y Embriología Humana. Facultad Medicina. Univ. Valencia. IIS INCLIVA, Valencia, ES, 3Dpto. Bioquímica y Biología Molecular. Facultad Medicina. Univ. Valencia. IIS INCLIVA, Valencia, ES La desregulación de la calpaína-2 (CAPN2), una Cys-proteasa que participa en el procesamiento limitado de múltiples proteínas, se han asociado a una menor supervivencia de pacientes con cáncer colorectal (CCR). Sin embargo, aún no se ha establecido el papel de la CAPN2 en la progresión tumoral y su posible valor pronóstico. El tamaño/número de nucléolos son factores de mal pronóstico en la progresión tumoral. Se analizó la posible relación entre la CAPN2 y la actividad nucleolar en células humanas de CCR, DLD-1. No se observó una correlación entre la expresión de CAPN2 y el tamaño/número de nucléolos como indicador de malignidad tumoral. Sin embargo, mediante inmunotinción, se identificó a la CAPN2 como una CAPN de localización preferentemente nucleolar. Se ha sugerido la autoproteólisis como forma de limitar la actividad CAPN sobre sus sustratos. Se estudiaron la autoproteólisis de la CAPN2 y su papel en el nucléolo. Mientras que la CAPN2 se encontraba en su forma completa en el citosol, en el nucleolo estaba proteolizada. Es más, observamos una correlación entre el procesamiento de la CAPN2 nucleolar y la malignidad tumoral en células de CCR con mutaciones KRASG13D frente a KRASWT. Las medidas de actividad enzimática en el nucléolo confirman que la CAPN2 nucleolar se encontraba plenamente activa. El extremo Nt de la histona H3 aparecía proteolizado en nucleolos donde se observaba una colocalización de CAPN2 y el extremo Ct de la histona H3. El tratamiento de las células con calpeptina bloquea dicha proteólisis, lo que sugiere que la CAPN2 puede ser un mediador del procesamiento de la histona H3 nucleolar. Nuestros datos sugieren que el papel de la CAPN2 nucleolar en células tumorales podría ser el de estimular la biogénesis ribosomal a través del procesamiento de la histona H3 y consecuentemente, la estimulación de la expresión de genes ribosomales. Financiado: BFU-2013-46434-P a J.R.V y R.Z; PI12/02394 a E.R.GT ambos proyectos cofinanciados por FEDER; GVPROMETEOII/2014-055 a J.R.V. y GVPROMETEO/2013-005 a AC. P02-36 Regulators of metal homeostasis normalize the FRDA phenotypes in a Drosophila melanogaster model of the disease Pablo Calap Quintana1, Sirena Soriano2, José Vicente LLorens1, María José Martínez-Sebastián1, María Dolores Moltó3 1 University of Valencia, Burjassot, ES, 2University of Valencia, Burjassot, ES; Baylor College of Medicine, Houston, US, 3University of Valencia, Burjassot, ES, CIBERSAM, INCLIVA, Valencia, ES Pósters / Posters The neurodegenerative disease Friedreich’s ataxia (FRDA) is caused by a reduction in the synthesis of the mitochondrial protein frataxin. Frataxin deficiency results in several biochemical disturbances including impaired iron-sulphur cluster biogenesis and accumulation of mitochondrial iron coupled to cytosolic iron depletion. Besides the alteration in the iron homeostasis, it has been recently shown that other metals such as Cu or Zn could play a role in the pathogenesis of the disease. Using an RNAi-based model of FRDA in Drosophila, we previously found that the levels of iron, zinc, copper, manganese and aluminum were also increased in the FRDA flies. We therefore set out to test whether genetic modification of key pathways regulating metal content and distribution would improve FRDA phenotypes by restoring metal homeostasis in fly models of the disease. For this purpose, two frataxin knockdown lines were used showing a neurodegenerative phenotype (rough eye) or a decreased motor performance phenotype, respectively. Changes in the expression of different homeostasis regulators, transporters or chaperones of Fe, Zn and Cu were able of suppressing the rough eye and the motor performance phenotype of the FRDA model flies. Overexpression of the Metal-responsive Transcription Factor-1 and knockdown of the metal sequestering metallothioneins (MT) also had a beneficial effect on both phenotypes. According to our results, the Fe related modifiers, some of the zinc transporters and the overexpression of MTF-1 could be exerting their beneficial effect by restoring the normal iron level. The knockdown of the MT as well as the MTF-1 overexpression also restored the increased level of lipid peroxidation found in the FRDA flies, highlighting the critical role of metal dysregulation in the oxidative stress alterations of FRDA. Different chelators for Cu and Zn were able of improving the motor impairment of frataxin depleted flies, showing that the imbalance of other metals besides Fe must be taken into account to fully understand the pathology of FRDA. P02-37 Análisis de la metilación del gen DPPIV en ADN sérico de pacientes con cáncer colorrectal como posible mecanismo regulador Loretta De Chiara1, Mar Ferreira Carrera1, Olalla Otero Estévez1, Oscar Cordero2, María Páez de la Cadena1, José Ignacio Rodríguez Prada3, Pamela Estévez Boullosa3, Lucía Cid Gómez3, David Remedios Espino4, Francisco Javier Rodríguez Berrocal1, Vicenta Soledad Martínez Zorzano1 1 Dpto. de Bioquímica, Genética e Inmunología. Universidade de Vigo, Vigo, ES, 2Dpto. Bioquímica y Biología Molecular, Universidade de Santiago de Compostela, Santiago de Compostela, ES, 3Servicio de Aparato Digestivo. Hospital Álvaro Cunqueiro EOXI Vigo, Vigo, ES, 4Servicio de Aparato Digestivo. Complexo Hospitalario Universitario de Ourense, Ourense, ES Previamente hemos descrito el descenso de los niveles séricos de la proteína CD26 soluble (CD26s) en pacientes con cáncer colorrectal (CCR) y adenomas avanzados (AA). En este estudio se analiza la metilación del promotor del gen codificante de CD26, gen DPPIV, en ADN sérico de pacientes con neoplasia avanzada (CCR o AA) y de individuos sanos, para determinar si la disminución de CD26s en el suero de los pacientes se debe al silenciamiento de DPPIV por hipermetilación de la isla CpG analizada. Se cuantificó el porcentaje de metilación en las muestras (21 con neoplasia avanzada y 27 sanos) mediante qPCR específica de metilación con sondas TaqMan. Para facilitar el estudio, la isla CpG de 317 pb descrita en el promotor de DPPIV, se subdividió en 3 subislas CpG (1, 2 y 3). No se encontraron diferencias estadísticamente significativas en el porcentaje medio de metilación para ninguna de las subislas analizadas entre los individuos sanos y los pacientes con neoplasia avanzada. Se observó una correlación positiva y estadísticamente significativa en la metilación entre las 3 subislas en los individuos sanos. Sin embargo, en los pacientes no encontramos correlación entre las subislas 1-2 y 1-3, pero sí entre las subislas 2-3. Tampoco se observó correlación entre el porcentaje de metilación y los niveles séricos de CD26s. Estos resultados sugieren que 53 Pósters / Posters la metilación del promotor de DPPIV, al menos en la isla CpG estudiada, no está relacionada con la reducción de CD26s en suero de pacientes con neoplasia avanzada. Financiación: Plan Nacional I+D+I 2014-2020 (AES) ISCIII-FEDER (PI15/02007), Fundación Científica AECC (GCB13131592CAST), “Axudas consolidación e estructuración de unidades de investigación competitiva” (GRC2014/019) y REGICC (R2014/039) de Xunta de Galicia. P02-38 Effects of Cyclin D-CDK4/6 axis inhibition on PTEN-deficient neoplasias Maria Alba Dosil1, Cristina Mirantes1, Nuria Eritja1, Isidre Felip1, Raul Navaridas1, Sonia Gatius1, Maria Santacana1, Eloi Garí2, Xavier Matias-Guiu1, Xavier Dolcet1 1 Oncologic Pathology Group. Dept. Ciències Mèdiques Bàsiques, Universitat de Lleida. Hospital Universitari Arnau de Vilanova. Institut de Recerca Biomèdica de Lleida, IRBLleida, Lleida, ES, 2 Cell Cycle Group. Dept. Ciències Mèdiques Bàsiques, Universitat de Lleida. Hospital Universitari Arnau de Vilanova. Institut de Recerca Biomèdica de Lleida, IRBLleida, Lleida, ES PTEN is one of the most frequently mutated genes in human cancers. A significant proportion of human malignancies present PTEN deficiencies. Among all of them, the frequency of alterations in PTEN is particularly high in endometrial carcinomas. Absence of PTEN leads to an abnormal cell division, which is one of the most important hallmarks in cancer. CDKs, their cognate Cyclins and CDKs inhibitors have been found mutated in a significant fraction of human neoplasias and are presented as key targets in the treatment of the pathogenesis of cancer. For that reason, several CDK inhibitors have been developed as a strategy to generate new anticancer drugs. Palbociclib or PD-332991 inhibits specifically CDK4/CDK6 and it has been approved by the US Food and Drug Administration for use in metastatic breast cancer. First of all, we have studied PTEN-driven tumorigenesis in the context of Cyclin D1 deficiency in the endometrium, thyroid and prostate. Histological examination by pathologists revealed that Cyclin D1 deficiency did not impairs PTEN tumorogenesis neither in thyroid nor in prostate. Interestingly, Cyclin D1 absence displayed a slight but not statistically significant reduction in the incidence and progression of endometrial lesions. Next, we have used a Tamoxifen-inducible PTEN knock-out mouse model to assess the anti-tumoral effects of Palbociclib on endometrial and prostate tumors, and thyroid hyperplasias. Our results demonstrate that Palbociclib treatment triggers shrinkage of endometrial lesions, but show poors beneficial effect on thyroid hyperplasias and prostate carcinomas. To date, this is the first pre-clinical study evaluating the response to Palbociclib in endometrial, thyroid and prostate malignancies driven by PTEN deficiency. P02-39 Estudios de preferencia metabólica en células del “microambiente angiogénico” Mª Carmen Ocaña1, Beatriz Martínez-Poveda1, Ana R. Quesada2, Miguel Ángel Medina2 1 Universidad de Málaga, Andalucía Tech, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias e IBIMA (Instituto de Biomedicina de Málaga), Málaga, ES, 2Universidad de Málaga, Andalucía Tech, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias e IBIMA (Instituto de Biomedicina de Málaga); Unidad 741, CIBER de Enfermedades Raras (CIBERER), Málaga, ES El “redescubrimiento” del efecto Warburg a principios de este siglo XXI y de la elevada glutaminolisis tumoral han contribuido a un renovado interés por el metabolismo en oncología. Además, estudios recientes han dado 54 XXXIX Congreso SEBBM importancia al metabolismo de células endoteliales. Resulta interesante indagar más en el metabolismo de este tipo celular así como de otras células de su microentorno (células tumorales e inflamatorias, entre otras). Mediante distintas aproximaciones metodológicas y utilizando distintos tipos celulares del “microambiente angiogénico” se pretende conocer más sobre el papel de la glucosa y la glutamina, así como la posible utilización de ácidos grasos como el palmitato, en estos tipos celulares. La adquisición de estos conocimientos podría suponer el comienzo de una posible vía para “atacar” a la angiogénesis patológica no solo con compuestos anti-angiogénicos, sino utilizando simultáneamente como diana el metabolismo endotelial y/o tumoral. Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The “CIBER de Enfermedades Raras” is an initiative from the ISCIII (Spain)]. This communicaction has the support of a travel grant “Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech”. P02-40 Generación de una proteína quimera de endoglina soluble y proteína fluorescente verde (GFP). Posibles aplicaciones en el estudio de la función de endoglina soluble Lidia Ruiz Llorente, Carmen Langa, Carmelo Bernabeu Centro de Investigaciones Biológicas (CIB) y Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, ES Endoglina (Eng; CD105) es una glicoproteína de membrana de tipo I que funciona en células endoteliales como receptor auxiliar de TGF-βs y BMPs, y que modula la fisiopatología vascular. Además de las dos isoformas de membrana, L-endoglina (long endoglin) y S-endoglina (short endoglin), existe una forma de endoglina soluble (sol-Eng) que se genera por la acción de la metaloproteasa-14 (MMP-14) sobre la región extracelular de la proteína de membrana (López-Novoa y Bernabeu, 2010). Se han descrito niveles elevados de esta forma soluble en pacientes con preeclampsia, hipercolesterolemia, aterosclerosis y cáncer (Valbuena-Díez et al., 2012). Además, sol-Eng es un marcador de daño cardiovascular en pacientes con hipertensión y diabetes y tiene una acción patogénica en preeclampsia. Endoglina soluble inhibe la angiogénesis, así como la proliferación, migración e invasión celular (del Castillo et al., 2015 y Gallardo-Vara et al., 2016). Sin embargo, los mecanismos de acción de sol-Eng no han sido elucidados todavía y son necesarias nuevas herramientas y aproximaciones experimentales en este campo. En nuestro laboratorio hemos clonado el dominio extracelular de endoglina en el plásmido pEGFP-N1 para generar una proteína de fusión con GFP dando lugar al vector pEGFP-N1/ENG.EC. Este vector ha sido secuenciado y la proteína de fusión ha sido caracterizada mediante transfecciones transitorias y estables en células CHO-K1 por técnicas de citometría, SDS-PAGE, inmunodetección, ELISA y microscopio de fluorescencia. La construcción aquí descrita podría utilizarse como herramienta en el estudio de la funcionalidad de sol-Eng tanto en situaciones control como patológicas. P02r-41 Diseño y caracterización de modelos celulares para el estudio del síndrome de Pearson Carmen Hernández Ainsa, Pilar Bayona-Bafaluy, Julia Barrios, Eduardo Ruiz Pesini, Julio Montoya, Sonia Emperador Universidad de Zaragoza, Zaragoza, ES El síndrome de Pearson (PS, OMIM-557000) es una enfermedad rara, caracterizada principalmente por anemia sideroblástica y disfunción pancreática exocrina, aunque se pueden ver implicados otros sistemas como el renal. Se debe a la presencia de una única deleción grande en el mtDNA, Salamanca 2016 Pósters / Posters o SLSMD (single large-scale mtDNA deletion), en un alto porcentaje de heteroplasmia, que suele ser más abundante en sangre que en otros tejidos. El tratamiento es sintomático y se basa en transfusiones y reemplazamiento de enzimas pancreáticos, pero a pesar de ello los pacientes suelen morir en la infancia. Los individuos que sobreviven desarrollan síndrome de Kearns-Sayre (KSS), por lo que se intenta retrasar la aparición de los síntomas neurológicos mediante el tratamiento con ácido ascórbico y ácido folínico. Para estudiar si la dinámica de propagación de las SLSMD se podría ver afectada por factores como su tamaño y localización, porcentaje de heteroplasmia, fondo genético nuclear o mitocondrial, estado celular proliferativo o no proliferativo, el tipo celular maduro o las condiciones de cultivo; se están generando y estudiando distintos modelos celulares. En primer lugar, se están realizando estudios genéticos y bioquímicos con fibroblastos en cultivo obtenidos a partir de biopsias de piel de dos pacientes PS, con deleciones de distinto tamaño, y de un paciente control. También se han obtenido híbridos transmitocondriales o cíbridos mediante la fusión fibroblastos enucleados y plaquetas de pacientes, con la línea celular de osteosarcoma 143b rho0. En todos los casos se han conseguido diferentes porcentajes de heteroplasmia, lo que permitirá hacer estudios comparativos. Actualmente, también se está trabajando en el desarrollo de células pluripotentes inducidas (iPSC) a partir de fibroblastos de pacientes PS. Estos modelos celulares permitirán aumentar el conocimiento sobre esta enfermedad. (ALP) and generated by hydrolysis of ATP via eNPP1. For the first time, the present study investigated extracellular pyrophosphate metabolism during hemodialysis sessions (including its synthesis via eNPP1 and its degradation via ALP) in physiological conditions. Methods and Findings: 45 patients in hemodialysis were studied. Physiological ALP activity represents only 4–6% of clinical activity. ALP activity increased post-hemodialysis by 2% under ideal conditions (87.4 ± 3.3 IU/L vs. 89.3 ± 3.6 IU/L) and 48% under physiological conditions (3.5 ± 0.2 IU/L vs. 5.2 ± 0.2 IU/L). Pyrophosphate synthesis by ATP hydrolysis remained unaltered post-hemodialysis. Post-hemodialysis plasma pH (7.45 ± 0.02) significantly increased compared with the pre-dialysis pH (7.26 ± 0.02). The slight variation in pH (~0.2 units) induced a significant increase in ALP activity (9%). Addition of phosphate in post-hemodialysis plasma significantly decreased ALP activity, although this effect was not observed with the addition of urea. Reduction in phosphate levels and increment in pH were significantly associated with an increase in physiological ALP activity post-hemodialysis. A decrease in plasma pyrophosphate levels (3.3 ± 0.3 μmol/L vs. 1.9 ± 0.1 μmol/L) and pyrophosphate/ATP ratio (1.9 ± 0.2 vs. 1.4 ± 0.1) post-hemodialysis was also observed. Conclusion. Extraction of uremic toxins, primarily phosphate and hydrogen ions, dramatically increases the ALP activity under physiological conditions. This hitherto unknown consequence of hemodialysis suggests a reinterpretation of the clinical value of this parameter P02-42 Evaluation of the kinase inhibitory and anti-angiogenic effects of damnacanthal 1 2 P02-44 2 Javier A. García-Vilas , Ana R. Quesada , Miguel Ángel Medina Torres Universidad de Málaga, Andalucía Tech, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias e IBIMA (Instituto de Biomedicina de Málaga), Málaga, ES, 2Universidad de Málaga, Andalucía Tech, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias e IBIMA (Instituto de Biomedicina de Málaga), Unidad 741, CIBER de Enfermedades Raras (CIBERER), Malaga, ES 1 Damnacanthal is a natural anthraquinone initially isolated from roots of Morinda citrifolia L., a small evergreen tropical tree usually known as noni. Damanacanthal is the most potent known inhibitor of p56Ick tyrosine kinase. Herein we show that damnacanthal is a broad spectrum kinase inhibitor. We use docking analysis and molecular dynamics simulations to get further insight on the inhibition of FAK, VEGFR-2 and c-Met by damnacanthal. Since these three and other kinases inhibited by damnacanthal are involved in angiogenesis, we suspected that this natural compound could also have anti-angiogenic effects. This was confirmed by a combination of in vitro, ex vivo and in vivo assays. [Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The “CIBER de Enfermedades Raras” is an initiative from the ISCIII (Spain)]. This communication has the support of a travel grant “Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech”. P02-43 Alkalosis and dialytic clearance of phosphate increases phosphatase activity: a hidden consequence of hemodialysis El análogo de las estrigolactonas GR-24 inhibe la angiogénesis in vitro e in vivo Paloma Carrillo1, Beatriz Martínez-Poveda1, Miguel Ángel Medina2, Ana R. Quesada2 1 Universidad de Málaga, Andalucía Tech, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias e IBIMA (Instituto de Biomedicina de Málaga), Málaga, ES, 2Universidad de Málaga, Andalucía Tech, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias e IBIMA (Instituto de Biomedicina de Málaga); Unidad 741, CIBER de Enfermedades Raras (CIBERER), Málaga, ES Muchas fitohormonas han mostrado un gran potencial en la prevención y terapia contra el cáncer. Las estrigolactonas son hormonas vegetales derivadas de los caroteinoides que están implicadas en la inhibición de la ramificación de la raíz y el brote, promover la germinación de plantas parásitas e intervenir en el establecimiento de simbiosis con micorrizas arbusculares. Se ha descrito la capacidad antitumoral de diferentes análogos de estrigolactonas, entre ellos GR-24, frente a diferentes líneas celulares tumorales in vitro y en modelos xenográficos. En este estudio se ha evaluado la capacidad citotóxica y anti-angiogénica de GR-24, tanto in vitro como in vivo. In vitro, GR-24 presenta una IC50 entre 50 y 90 μM en diversas líneas celulares tumorales y endoteliales. Además, afecta a pasos clave del proceso angiogénico, como son la proliferación, diferenciación, migración y capacidad de degradación de la matriz extracelular de células endoteliales, a concentraciones menores que su IC50. En los ensayos in vivo, GR-24 muestra un gran efecto inhibidor sobre la formación de vasos sanguíneos en la membrana corioalantoidea de pollo y sobre la formación de vasos intersegmentales en embriones de Danio rerio. En conjunto, estos resultados sugieren que GR-24 puede ser un nuevo compuesto prometedor en la terapia anti-angiogénica y otras enfermedades dependientes de angiogénesis. Ricardo Villa Bellosta1, Emilio González Parra2, Jesús Egido2 1 Instituto de Investigacion Sanitaria, Fundación Jiménez Díaz, Madrid, ES, 2Fundación Jiménez Díaz, Madrid, ES [Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The “CIBER de Enfermedades Raras” is an initiative from the ISCIII (Spain)]. Background: Extracellular pyrophosphate is a potent endogenous inhibitor of vascular calcification, which is degraded by alkaline phosphatase This communicaction has the support of a travel grant “Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech”. 55 Pósters / Posters P02-45 Statins upregulate microRNA-122 and microRNA-27n in HepG2 cells A. Benito-Vicente, A. Etxebarria, X. Moreno-Monreal, U. Galicia-García, S. Jebari, E. Aizquibel, A Larrea-Sebal, A. Etxaniz, D. González-Bullón, K. B.Uribe, R. Alonso-Estrada, H. Ostolaza, C. Martín Instituto Biofísika (UPV/EHU, CSIC) and Dpt. Biochemistry and Molecular Biology, UPV/EHU, Portugalete, ES Statin administration decreases cardiovascular risk and, cholesterol lowering with statins has become a cornerstone of cardiovascular disease prevention for a wide range of patients. Despite this, muscle problems, increased incidence of diabetes, increased body weight, reduced exercise capacity and other adverse symptoms are associated with statin use. Besides of their major positive health benefits by inhibiting hydroxymethylglutarylCoAReductase (HMGCR), the rate-limiting enzyme of cholesterol synthesis, it has been proposed that statinsinteract directly with the epigenome. The epigenetic mechanisms postulated to be responsible for these changes included DNA methylation and histone modification. To date, there is limited evidence available on the effects of statins on a third, recently described arm ofepigenetics: microRNA (miRNA) expression. It has been established that miRNAs coordinate metabolism by directly affecting the transcriptome, modulate multiple target transcripts and thus heavily influence gene expression patterns. miR-33, may be key to control HDL levels via repression of sterol transporters in the liver. In addition, miRNAs are involved in molecular mechanisms that regulate the lipid homeostasis and can contribute importantly to cardiovascular disease development. Generally, miRNAs act intracellularly, but it has been described that they can be released to the extracellular medium and be detected circulating in plasma where they are specially stable and resistant to RNAse degradation. miRNA profiles have been detected extracellularly in inflammation processes, cardiovascular disease, atherosclerosis, diabetes, obesity and aging. Different studies show that miRNAs in the medium can be protected in lipid vesicles or by conjugation onto proteins. It has been demonstrated that HDL can bind and transport endogenous miRNAs, particularly miR-223 to hepatocytes and that the inflammatory properties of HDL are due in part to the transference of miR-223 to the endothelial cells where the expression of adhesion molecules is suppressed. Very recently, it has been proposed that atorvastatin induces energy depletion and inhibition of creatine synthesis in liver cells. Understanding how the statin treatment cause adverse effects on triglyceride and fatty acid synthesis, lipid infiltration in the liver and peripheral tissues, or the mechanism that leads to enhanced risk of statin- induced type 2 diabetes mellitus would help the management of these diseases. In this work we examined the effects of four statins (atorvastatin, pravastatin, rosuvastatin and simvastatin; 5 μM for 24 and 48 h) on miR-122, mir-33a and mir-27b levels in HepG2 cells and in the culture medium. XXXIX Congreso SEBBM these circumstances, we did not observe a direct correlation between ROS production and cell death caused by the Aß because both Aß 40 and Aß 42 increased neuronal death while Aß 40 did not change ROS production. Conversely, we observed that those Aß that are able to cross neuron membrane (Aß 25-35 and Aß 42) increased ROS production but those remained outside (Aß 40) not changed ROS generation. No significant differences were found between Aß 40 and Aß 42 regarding the extent of the deleterious effects of both peptides on neuronal viability or synaptophysin expression. However, Aß 40 elicited a clear delocalization of PSD-95 and synaptotagmin from prospective synapsis to the neuronal soma suggesting the occurrence of an immediate effect of Aß 40 on synaptic disassembling. The formation of Aß 40- or Aß 42-serum albumin complexes avoided the effects of these peptides on neuronal viability, synaptophysin expression and PSD-95/synaptotagmin disarrangement suggesting that sequestration of Aß by albumin prevents deleterious effects of these peptides in neurons. This suggests that Aß borne by albumin can be safely transported through body fluids. P02r-47 DeUbiquitinating enzymes (DUBs) as new regulators of the Hypoxia Pathway Teresa Martín-Mateos, Encarnación Pérez-Andrés, Onintza Carlevaris, Sara Pozo, Edurne Berra CIC bioGUNE, Derio, ES Oxygen homeostasis is crucial for aerobic organisms as oxygen is the final electron acceptor to generate ATP through the oxidative phosphorilation. Accordingly, low-oxygen availability (or hypoxia) can produce irreversible damages, even when transient. Adaptation to reduced oxygen availability is indeed a major physiologic challenge but it is also associated with pathologies such as ischemic diseases, inflammatory and metabolic disorders, Alzheimer and cancer. The adaptive response is triggered by the hypoxia-signalling pathway, which is under exquisite control through the ubiquitin-proteasome system (UPS). The family of DeUbiquitinating enzymes (DUBs) specifically deconjugates ubiquitin from targeted proteins, playing major roles in the UPS control. Because of the reversible ubiquitination’s crucial role to fine tune the hypoxia pathway and in the light of DUBs being druggable enzymes, we carried out an unbiased loss-of function screen. Of the nearly 79 DUBs encoded by the Human genome and predicted to be active, we individually inhibit the expression of 66 DUBs using pools of small hairpin RNAs (shRNAs) and analyzed for their effect in hypoxia-driven luciferase reporter activity. Here, we will discuss the results of our screen, the validation of the selected HITs measuring the effect of DUBs silencing on endogenous hypoxia-inducible target gene expression as well as our more recent data regarding the study of the new hypoxia specific DUBs we have identified. P02-48 P02r-46 Aberrant co-localization of synapsis proteins promoted by Alzheimer amyloid-ß peptides. Protective effect of human serum albumin Marta Domínguez-Prieto, Ana Velasco, Arantxa Tabernero, José María Medina Instituto de Neurociencias de Castilla y León (INCyL), Universidad de Salamanca, Salamanca, ES Amyloid-ß (Aß) 25-35, Aß 40 and Aß 42 significantly decreased neuronal viability although the higher effect was showed by Aß 35-25. This may be due to a more penetrating ability performed by Aß 25-35 since under our experimental circumstances Aß 25-35 was internalized by the neuron and it reached mitochondria. However, Aß 40 did not entry neuron cytosol and Aß 42 was internalized by neuron but not reached mitochondria. Under 56 Validation of pharmacological chaperones targeting the oncoprotein nucleophosmin Andoni Cuevas, Marián Alonso-Mariño, Sonia Bañuelos, María Ángeles Urbaneja Biofisika Institute (UPV/EHU, CSIC), Dpt Biochemistry & Molecular Biology, University of the Basque Country, Leioa, ES Nucleophosmin (NPM), a highly and ubiquitously expressed protein, mainly localized in nucleoli but able to shuttle between nucleus and cytoplasm, plays crucial roles in ribosome maturation and export, centrosome duplication, cell cycle progression, and response to a variety of stress stimuli. Much interest in this protein has arisen since the discovery that heterozygous mutations in the terminal exon of the NPM1 gene are responsible for the unfolding of NPM C-terminal domain and consequently its nucleolar release, which leads to massive and aberrant cytoplasm Salamanca 2016 accumulation of the protein. Given that those genetic and cellular alterations are the most frequent in acute myeloid leukemia (AML), NPM is considered as a promising target for this disease. We have validated the effects of two HTS-based selected hits on HeLa cells transfected with AML-related NPM mutants. Both candidates interfere with unfolding-related protein aggregation and prevent aberrant cytoplasm localization of NPM mutants, prompting them as promising pharmacological chaperones for AML therapy. Pósters / Posters rápida de oligómeros sin periodo de latencia, con una amplia distribución de tamaños moleculares. Las especies oligoméricas formadas se han separado mediante cromatografía de exclusión molecular y ultrafiltración y se ha caracterizado su morfología y su estructura mediante AFM y FT-IR. La toxicidad de los oligómeros sobre cultivos de células SH-SY5Y se ha determinado utilizando el ensayo WST-1. Las diferencias estructurales observadas entre los oligómeros tempranos de Abeta40 y Abeta42 pueden ser responsables de sus diferentes propiedades citotóxicas. P02-49 P02r-51 p38γ/p38δ and TPL2 are new components of the Dectin-1 signalling pathway regulating Candida albicans infection Identification and functional characterization of C14ORF39, a novel synaptonemal complex protein essential for meiotic recombination and mouse fertility Alejandra Escós López1, Miguel Ángel Martín-Serrano1, Dayanira Alsina-Beauchamp2, Ana Risco1, Ana Cuenda1 1 Centro Nacional de Biotecnología, Madrid, ES, 2Mount Sinai Hospital, Nueva York, US Candida albicans is a frequent etiologic agent in sepsis associated with a high mortality in immune compromised patients. p38γ/p38TM modulate bacterial lipopolysaccharide-induced sepsis by maintaining the steady-state levels of TPL2, the MKK kinase that mediates ERK1/2 activation after TLR stimulation. However, their contribution in fungal sepsis remains unknown. Here we show that p38γ/TM deficiency partially protected against Candida albicans infection, decreased macrophage and neutrophil recruitment to infected kidneys, and reduced the production of cytokines and chemokines, such as CCL2 and KC. Moreover, p38γ/p38TM deletion impaired the innate immune response to Candida albicans and to the Dectin-1 ligand Curdlan by blocking ERK1/2 activation and reducing IL-1βproduction in macrophages. We found that TPL2 is essential for Dectin-1 signalling in mouse macrophages and human monocytes. We proposethe existence of a new TAK1-TPL2-MKK1-ERK1/2 pathway activated by Dectin-1 engagement, and regulated byp38γ/p38TM, whose targetingmay offer novel strategies for antifungal agent design. P02-50 Caracterización de la estructura y la citotoxicidad de oligómeros tempranos de Abeta40 y Abeta42 Bertrand Morel1, María Paz Carrasco-Jiménez2, Xiomara Gálvez-Escolano2, Carmen Marco2, Antonio Andrés García-Valdivia1, Esperanza Padín1, David Ruzafa1, Francisco Conejero-Lara1 1 Departamento de Química Física e Instituto de Biotecnología, Universidad de Granada, Granada, ES, 2Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias, Universidad de Granada, Granada, ES La formación de agregados fibrilares del péptido beta-amiloide (Abeta) en el cerebro es una de las señas de identidad de la enfermedad de Alzheimer (EA). Abeta es el componente principal de las placas seniles, que se observan en los espacios extracelulares del cerebro de enfermos de EA. Sin embargo, es cada vez más evidente que son las formas oligoméricas solubles de Abeta las principales responsables de los efectos citotóxicos sobre las neuronas y en última instancia de la pérdida de la función cognitiva asociada a la EA. De las dos formas más abundantes de Abeta, Abeta40 y Abeta42, es este último el que más contribuye a la toxicidad celular. Desafortunadamente, debido a su pequeño tamaño, su heterogeneidad e inestabilidad, se sabe aún muy poco sobre las características estructurales de estos oligómeros, los mecanismos de su formación o las razones fisicoquímicas de su citotoxicidad. En este trabajo hemos estudiado la formación de oligómeros de Abeta40 y Abeta42 durante las fases tempranas del proceso de agregación, partiendo de las formas monoméricas de ambos péptidos. Las cinéticas de agregación a 37ºC seguidas mediante dispersión dinámica de luz y fluorescencia de tioflavina T y bis-ANS indican una acumulación Natalia Felipe-Medina1, Laura Gómez-H.1, Manuel Sánchez-Martín1, Owen R. Davies2, Isabel Ramos1, Ignacio García-Tuñón1, Dirk G. de Rooij3, José Luis Barbero4, Ricardo Benavente5, Elena Llano6, Alberto M. Pendás1 1 Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, ES, 2Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK, 3Reproductive Biology Group, Division of Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, NL, 4Centro de Investigaciones Biológicas (CSIC), Madrid, ES, 5Department of Cell and Developmental Biology, Biocenter, University of Würzburg, Würzburg, DE, 6Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca). Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, ES Meiotic recombination generates crossing over between homologous chromosomes which are essential for genome haploidization. Humans differ largely in the number of crossover across the genome. The synaptonemal complex (SC) provides the structural framework for synapsis and crossing over processing. Recently, one anonymous gene variant (C14ORF39) influencing human recombination rate was identified. Here, we show that C14ORF39 is a novel component of the central element of the synaptonemal complex that interacts with the well-known synaptonemal complex central element 1 (SYCE1). Structurally, by homology modeling C14ORF39 shows a repeated linear repeats of triple helical bundle and suggests to act as a structural linker of the SC. Mice lacking C14ORF39 are defective in synapsis of homologous chromosomes at meiotic prophase I which provokes an arrest at pachytene-like stage and consequently infertility. In agreement to be a modifier of recombination rate in humans, C14ORF39 is essential for the appropriate processing of intermediate recombination nodules just before reciprocal recombination and crossover take place. P02-52 Genetics of obesity: can an old dog teach us new tricks? Giles SH. Yeo University of Cambridge, Metabolic Research Labs, MRC Metabolic Diseases Unit, Addenbrooke’s Hospital, Cambridge, UK It is clear that the cause of obesity is a result of eating more than you burn.What is more complex to answer is why some people eat more than others? Over the past 20 years, insights from human and mouse genetics have illuminated multiple pathways within the brain that play a key role in the control of food intake. We now know that the brain leptin-melanocortin pathway is central to mammalian food intake control, with genetic disruption resulting in extreme obesity. These, however, remain rare, with the major burden of disease carried by those of us with ‘common obesity’. In recent years, genome-wide association studies have revealed more 57 Pósters / Posters than 100 different candidate genes linked to BMI, with most, including many components of the melanocortin pathway, acting in the CNS and influencing food intake. So while severe disruption of the melanocortin pathway results in severe obesity, subtle variations in these same genes influence where you might sit in the normal distribution of BMI. As we now enter this ‘post-genomics’ world, can this new information influence our treatment and management of obese patients? P02-53 Modelo murino de las alteraciones metabólicas encontradas en pacientes con mutaciones en IGF1R Carmen Patricia Pérez Matute1, Iciar P. López1, Emma Recio-Fernández1, Raquel Torrens1, Sergio Piñeiro-Hermida1, Elisa Wirthgen2, Andreas Hoeflich2, José A. Oteo3, José G. Pichel1 1 Fundación Rioja Salud-CIBIR, Logroño, ES, 2Institute for Genome Biology, Leibniz-Institute for Farm Animal Biology (FBN), Dummerstorf, DE, 3Hospital San Pedro-CIBIR, Logroño, ES IGF1R (Insulin-like Growth Factor type 1 Receptor) es un receptor transmembrana de expresión ubicua con una gran similitud con el receptor de la insulina y con funciones biológicas esenciales. Pacientes con mutaciones alélicas en el gen IGF1R presentan alteraciones metabólicas y endocrinas. El objetivo del presente trabajo fue analizar metabólicamente un modelo animal de deficiencia de IGF1R. Se han empleado ratones mutantes UBC-CreERT2; Igf1rfl/fl con deleción de Igf1r inducida postnatalmente con tamoxifeno. Los ratones fueron sacrificados 9 semanas después de la inducción de la deleción y la sangre y los diferentes tejidos fueron extraídos para su análisis. Los ratones hembra mutantes presentaron mayor peso corporal y grasa periovárica que sus controles, sin observarse diferencias en los machos. El peso del hígado se vio incrementado en los ratones mutantes de ambos géneros. Además, el contenido hepático de triglicéridos fue significativamente superior en los ratones macho mutantes. A nivel sistémico, los mutantes presentaron elevados niveles plasmáticos de insulina y del índice de resistencia a la insulina HOMA. Los niveles plasmáticos de IGF-1 también se vieron incrementados en los mutantes, al igual que IGFBP3. Los niveles de IGFBP2 se vieron disminuidos en los mutantes. Este estudio demuestra que los ratones mutantes son insulino-resistentes, con elevados niveles de IGF-1 y de triglicéridos en hígado, aunque algunos de estos efectos son dependientes del género. Nuestros resultados sugieren que este modelo de deficiencia en IGF1R refleja alteraciones metabólicas observadas en pacientes con deleción en este gen, y, por tanto, puede ser una herramienta útil para el conocimiento en profundidad de los mecanismos que subyacen a la fisiopatología de esta mutación. P02-54 Estudio de la hemostasia en modelos murinos de Telangiectasia Hemorrágica Hereditaria Cristina Egido Turrión, Claudia Ollauri Ibáñez, Laura Ruiz Remolina, Alicia Rodríguez Barbero, José Miguel López Novoa, Miguel Pericacho Bustos Departamento de Fisiología y Farmacología. Universidad de Salamanca. IBSAL, Salamanca, ES La Telangiectasia Hemorrágica Hereditaria (HHT) es una enfermedad rara causada, en un 90% de los casos, por mutaciones en los genes de endoglina o de ALK1. Clínicamente se caracteriza por la presencia de telangiectasias y malformaciones arteriovenosas cuya rotura provoca graves hemorragias muy difíciles de detener que comprometen la vida del paciente. La frecuencia e intensidad de estas hemorragias hace que los pacientes necesiten transfusiones periódicas, lo que merma su calidad de vida e incrementa el gasto sanitario. Tradicionalmente se ha considerado 58 XXXIX Congreso SEBBM que la HHT es una enfermedad provocada por una angiogénesis deficiente y que los sangrados son consecuencia de la rotura de unos vasos sanguíneos frágiles. Sin embargo, nuestra hipótesis es que esa mala angiogénesis puede ser responsable de la elevada frecuencia de los sangrados, pero que su gravedad es consecuencia de alguna alteración de la hemostasia en estos pacientes. Resultados previos de nuestro grupo han demostrado que el dominio RGD de endoglina interacciona con integrinas leucocitarias y regula el proceso de transmigración. Por ello nos planteamos que también pueda interaccionar con las integrinas plaquetarias y regular así la formación o estabilización del trombo. Para analizar esta hipótesis hemos estudiado la hemostasia y la estabilización del trombo en 2 modelos murinos de HHT: ratones heterocigotos en endoglina (ENG+/-) y en ALK1 (ALK1+/-). Nuestros resultados demuestran que en ambos casos hay un aumento del tiempo de sangrado que se acompaña de un retraso en la estabilización del trombo, siendo las diferencias más claras en el caso de los ratones deficientes en endoglina. Estos resultados permitirían la búsqueda de nuevos enfoques terapéuticos para el control de las hemorragias de los pacientes de HHT. P02r-55 Differential DNA damage response through alternative splicing of the DDR factor RAP80 Erika Zodda1, Francesca Mateo1, Mònica Pons1, Bruna Oriol1, Inés Izquierdo2, Marta Cascante2, Timothy M. Thomson1 1 IBMB-CSIC, Barcelona, ES, 2Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Barcelona, Barcelona, ES Alternative splicing governs cell-type, lineage-specific or environmentally regulated expression of variant forms of mRNAs and their encoded proteins that exert differential functions specifically and exquisitely adapted to a particular cellular context. By employing a cell model in which distinct tumor cell subpopulations display clearly differentiated epithelial or mesenchymal phenotypes and gene programs, we have identified alternatively spliced mRNAs with potential impact on the self-renewal capacities of these cell subpopulations. For this, we have applied RNAseq followed by analysis of mRNA isoforms differentially expressed between the two subpopulations and shRNA-mediated transcript knockdowns. This has led us to discover that mRNAs for RAP80, an adaptor protein that tethers BRCA1 to sites of DNA damage through an ubiquitin-interacting motif (UIM), are expressed as either predominantly epithelial or predominantly mesenchymal isoforms, and that these isoforms switch their expression in epithelial or mesenchymal cells as a function of expression of the alternative splicing regulators ERSP1/2. We have found that the predominantly epithelial isoform of RAP80 mRNA encodes a functional protein that provides more efficient repair in response to DNA damage. We have also found that the expression of RAP80 is essential for the maintenance of self-renewal phenotypes of the epithelial tumor cell subpopulation of our dual-cell model, which displays traits of cancer stem cells (CSCs). In summary, we have discovered that alternative splicing regulates efficient DNA damage repair mediated by RAP80, associated with an epithelial gene program, and that RAP80 is necessary for the maintenance of CSC properties. These findings represent the first description of differential DDR linked to epithelial CSC characteristics regulated through alternative splicing. P02-56 Efecto de la Restrición Calórica (RC) en el metabolismo de cerámidas en el hipotálamo. Relación con la lipotoxicidad y el desarrollo de resistencia a insulina con la edad Cristina Pintado Losa1, María Rodríguez Pérez1, Virginia Gómez-López Carreño2, Carmen Arribas Mocoroa1, Eduardo Moltó Pérez1, Antonio Andrés Hueva2, Nilda Gallardo Alpizar2 Salamanca 2016 1 Facultad de Ciencias Ambientales y Bioquímica. UCLM, Toledo, ES, 2Facultad de Ciencias y Tecnologías Químicas. UCLM, Ciudad Real, ES El hipotálamo vincula las señales de hormonas y nutrientes al control de la ingesta, el metabolismo energético y la sensibilidad a insulina. El desarrollo de resistencia a insulina con la edad es resultado de una serie compleja de alteraciones, incluyendo un desequilibrio en el flujo lipídico que conduce a la síntesis de ceramidas en neuronas hipotalámicas que afectaría al mantenimiento de la homeostasis energética a través de la activación de vías inflamatorias y/o la inducción de estrés de retículo endoplásmico (RE) y lipotoxicidad. Un mejor conocimiento de la regulación del metabolismo lipídico en el hipotálamo podría conducir a la identificación de nuevas estrategias para la prevención y tratamiento de la obesidad y la diabetes. Se estudió mediante RT-PCR a tiempo real la variación con el envejecimiento de la expresión de genes implicados en la síntesis, degradación y metabolismo de ceramidas, así como de marcadores de estrés de RE, respuesta a proteínas mal plegadas (UPR) e inflamación en el hipotálamo de ratas alimentadas ad libitum o en restricción calórica (RC). La RC produjo un aumento en la expresión de genes de degradación de ceramidas y descenso en los de síntesis. Por otro lado la RC provocó la disminución de la expresión de CHOP, mientras que no se observaron variaciones ni con el envejecimiento o la RC en la expresión de Grp78 y PDI. El patrón de expresión en marcadores y citoquinas pro-inflamatorias se altera con la RC, disminuyó la expresión de iNOS, IL-1β y TNFα en ratas de 8 meses, pero se obtuvieron efectos opuestos en las de 24 meses. Los resultados sugieren que la RC en edades tempranas provocaría una disminución en la generación de ceramidas y en las vías inflamatorias en el hipotálamo, siendo ésta una posible intervención terapéutica que mejorase la sensibilidad central a la insulina. Pósters / Posters P02-58 p75NTR antagonists attenuate photoreceptor cell loss in murine models of retinitis pigmentosa María Platón-Corchado1, Pablo F. Barcelona2, Miguel Marchena1, Alberto M. Hernández-Pinto1, Sean Jmaeff2, Catalina Hernández-Sánchez1, H. Uri Saragovi2, Enrique J. de la Rosa1 1 Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, ES, 2 Lady Davis Institute-Jewish General Hospital, McGill University, Montreal, Quebec, CA Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies that courses with photoreceptor cell degeneration and death. ProNGF signaling through p75NTR has been associated to neurodegenerative conditions. Thus, we have explored the possible role of p75NTR in the course of RP, as well as its value as pharmacological target for RP treatment. Quantitative RT-PCR analysis showed no differences in the expression levels of the proNGF/p75NTR system components between the RP model rd10 and wild type mouse retinas. However, unprocessed proNGF levels, measured by ELISA, increased in the rd10 retina at early degenerative stages prior to the peak of photoreceptor cell death, and remained elevated for the period of major of photoreceptor cell loss. Reduced proNGF processing concurs with increased α2-macroglobulin expression, an inhibitor of that processing, as well as with increased levels of pro-inflammatory cytokines and GFAP expression. Retinal explants treated with p75NTR antagonists for 24 h showed significantly reduced levels of photoreceptor cell death, as determined by TUNEL assay. This effect was accompanied by a decrease in retinal reactive gliosis. A single intravitreal or subconjuntival injection of the p75NTR antagonist THX-B in rd10 and RhoP mouse RP models elicited a neuroprotective effect on photoreceptor cells, observable five days later as an increase in the thickness of the outer nuclear layer, where photoreceptor nuclei are located. p75NTR-antagonists supported photoreceptor cell survival both on retinal explants and in vivo, in two different RP models, thus providing an initial proof of concept on a possible therapy for RP. P02-57 RasGRF2 controls nuclear trafficking in photoreceptor cells David Jimeno García1, Camela Gómez1, Nuria Calzada1, Pedro de la Villa2, Concepción Lillo3, Eugenio Santos1 1 Centro de Investigación del Cáncer-Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, ES, 2Departamento de Fisiología, Universidad de Alcalá, Alcalá de Henares, ES, 3INCYL, IBSAL (Universidad de Salamanca), Salamanca, ES Analyses of RasGRF1 knockout (GRF1-KO), GRF2-KO and the GRF1/2 double-KO mouse retinas, revealed specific alterations in cone photoreceptors in the GRF2-KO and GRF1/2-DKO. These alterations consist in the specific accumulation of misplaced “ectopic” nuclei in the photoreceptors segments area. The pathological displacement of cone nuclei occurred postnatally and peaked between postnatal day 11 (P11) and P15 coinciding with the so called “late migratory phase” of cone nuclei. The late migratory phase is a physiological process whereby cone nuclei move towards the outer limiting membrane (OLM) where they reach their final position. In the GRF2-KO and GRF1/2-DKO retinas this movement is deregulated and cone nuclei are located closer to the OLM and in some regions of the retina even trespass it. Using detailed inmunocytochemical analyses we have identified disruption of the OLM and accumulation of activated, phosphorylated forms of PAK, MLC2 and VASP, molecules known to participate in nuclear migration and cytoskeletal reorganization, in cone photoreceptors of GRF2-KO and GRF1/2-DKO retinas. Electroretinographic recordings show specific impairment of cone photoreceptor cells in GRF1-KO and GRF1/2-DKO retinas. These data support a critical role of GRF2 in cone nuclear migration and proper retinal development and functioning. P03. Biología del desarrollo / Developmental biology P03. Biología del desarrollo / Development biology P03-1 A novel function of aPKC controlling apical cell trafficking Sol Sotillos Martín Centro Andaluz de Biología del Desarrollo/CSIC/UPO, Dos Hermanas, ES Cell polarity is a basic characteristic of any cell, required for their proper physiological function. To establish cell polarity a number of determinants are required (reviewed in (1)) to restrict the different functional domains in the cell. When establish and for maintenance of cell polarity a proper trafficking to and recycling from the membrane is required. And, vice versa, once established cell polarity is necessary to define the cell domains for trafficking delivery. Thus, an intermingled between cell polarity and cell trafficking is essential to maintain each other and for the proper cell physiology. Recently we have shown a new interacting point between cell polarity and trafficking at the level of the polarity determinant aPKC and the Rab11-adaptor protein Nuclear fallout (Nuf, (2)). aPKC is a Ser/Thr kinase of the Par complex essential in most of the polarity processes. Nuf belongs to the Rab11-family interacting proteins (Rab11-FIP (3)) that function as an adaptor of Rab11 to the microtubule motor protein kinesin and the dynein complex (2, 4). We have demonstrated that active aPKC interacts directly with and phosphorylates Nuf, modifying its 59 Pósters / Posters XXXIX Congreso SEBBM cellular distribution. Furthermore, aPKC is a cargo of the Nuf-Rab11 recycling endosomes and it seems to be regulating its own recycling in the cell by phosphorylation and displacement of Nuf from the apico-lateral cortex. References [1] E. Rodriguez-Boulan, I. G. Macara: Organization and execution of the epithelial polarity programme. Nat Rev Mol Cell Biol 15, 225-242 (2014). [2] F. J. Calero-Cuenca et al.: Nuclear fallout provides a new link between aPKC and polarized cell trafficking. BMC Biol 14, 32 (2016). [3] C. P. Horgan, M. W. McCaffrey: The dynamic Rab11-FIPs. Biochem Soc Trans 37, 1032-1036 (2009). [4] B. Riggs et al.: The concentration of Nuf, a Rab11 effector, at the microtubule-organizing center is cell cycle regulated, dynein-dependent, and coincides with furrow formation. Mol Biol Cell 18, 3313-3322 (2007). GTPases, are widely deemed as potential therapeutic targets owing to their protumorigenic functions. However, the sparse use of animal models has precluded the full understanding of their in vivo pathophysiological roles. Here, we report that the hematopoietic-specific Vav1 GEF unexpectedly acts as a tumor suppressor by buffering Notch1 signaling in lymphocytes. This noncanonical function entails the nucleation of cytoplasmic complexes between Cbl-b and the active Notch1 intracellular domain (ICN1) that favor the ubiquitinylation-mediated degradation of ICN1. Genetic ablation of Vav1 upregulates ICN1 signaling in immature T cells and, in collaboration with ancillary mutations, triggers the rapid development of T cell acute lymphoblastic leukemia (T-ALL). This pathway is downregulated at the transcriptional level in human T-ALL of the TLX+ subtype, further underscoring its potential tumor suppressing roles. These results call for an overall reevaluation of Rho GEF function in cancer. P03-2 P03-4 From buds to paws, a temporal transcriptome analysis When to decide to divide: a Soxist point of view 1 1 2 Marc Fernández Guerrero , Rocío Pérez Gómez , Fabrice Darbellay , Lucille Delisle2, Denis Duboule2, Marian Ros1 1 Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Consejo Superior de Investigaciones Científicas, Universidad de Cantabria (CSIC), Santander, ES, 2School of Life Sciences, Federal Institute of Technology, Lausanne, Lausanne, CH Limbs develop from limb buds composed of a core of limb progenitor cells covered by the surface ectoderm. A variety of experiments in chick and mouse embryos indicate that the fate of the distal limb progenitors is progressively restricted: early progenitors can form the whole proximo-distal elements of the limb while late progenitors can only form the digits. Here we have used RNA-seq to reveal the transcriptional features associated with this developmental transition. The analysis was performed in mouse forelimbs of 4 different stages (E9.5, E10.5, E11.5 and E12.5) and restricted to the distal 150μm with ectodermal and mesodermal components analyzed separately. Two biological replicates were used for each condition. Of 46,480 Ensembl-annotated genes, 3,416 were differentially expressed between ectoderm and mesoderm. In each tissue, the time serie analysis revealed that major changes occur between E9.5 and E10.5. The accuracy of the expression patterns was confirmed by in situ hybridization and by the truthful coincidence with published expression patterns. According to their temporal expression, six different dynamics of gene expression were identified. In general, the average expression of transcription factors decreased with age, whereas the average expression of extracellular molecules increased with age. The differentially expressed genes were further subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and showed diverse functional categories and signaling pathways. We are exploring their relevance in orchestrating age-specific developmental capacities of each tissue. P03-3 An unexpected tumor suppresor role for the Rac1 exchange factor Vav1 in T cell acute lymphoblastic leukemia Javier Robles Valero1, Luis Francisco Lorenzo-Martín1, Mauricio Menacho Márquez1, Antonio Abad1, Lluis Espinosa2, Anna Bigas2, Xosé R. Bustelo1 1 Centro de Investigación del Cáncer (CIC-CSIC), Universidad de Salamanca, Salamanca, ES, 2Institut Hospital del Mar d’Investigacions Mèdiques, Barcelona, ES Rho GDP/GTP exchange factors (GEFs), the enzymes that stimulate Rho 60 Aixa V. Morales1, Mar Muñiz1, Elena Calleja1, Alejandra C. Quiroga1, Marco A. Cañizares1, Lingling Li1, Silvia Nicolis2, Véronique Lefebvre3 1 Instituto Cajal, CSIC, Madrid, ES, 2University of Milano-Bicocca, Milano, IT, 3Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, US During the development of the nervous system, the generation of hundreds of subtypes of neurons and glial cells relies upon the relatively fast production, amplification, specification and differentiation of a pool of neural progenitors and neural stem cells (NSCs). Surprisingly, this strategy is retained to some extent in niches in the adult nervous system throughout lifetime under physiological conditions. Although the molecular mechanisms involved in both embryonic and adult neurogenesis are conserved, it is not clear how the differences in the cell production rates and in the temporal extent of neurogenesis can be attained. Genes of the Sox family of transcription factors are essential during neurogenesis. In the developing spinal cord, we have determined that Sox5 controls cell cycle exit of neural progenitors and the specification of subtypes of dorsal interneurons, counteracting the Wnt signalling pathway (Martínez-Morales et al., 2010; Quiroga et al., 2014). More recently, we have characterized that both Sox5 and Sox6 are expressed in the majority of NSCs in one of the longer lasting neurogenic niches, the subgranular zone (SGZ) of the dentate gyrus of the adult mouse hippocampus.Using inducible targeted deletions: Sox5fl+/fl+ and Sox6fl+/fl+ mice crossed to a transgenic Sox2-cre-ERT2 line inducible by tamoxifen, we have determined that Sox6 is required for the self-renewal ability of the NSCs of the DG, while Sox5 is required for restricting the proliferation of the NSCs. These results suggest that Sox5 and Sox6 control the activation, proliferation and/or stemness of NSCs during adult hippocampal neurogenesis. P03-5 Generación de mutantes específicos para distintas isoformas de polychaetoid mediante la tecnología CRISPR/Cas9 en Drosophila Marta Carrasco-Rando, Mar Ruiz-Gómez CBMSO, Madrid, ES Uno de los principales componentes de la barrera de filtración glomerular implicada en el ultrafiltrado de la sangre en vertebrados es el diafragma de filtración (DF), un complejo multiproteico que actúa como filtro molecular y plataforma de señalización intracelular. En Drosophila las células encargadas de la filtración de la hemolinfa (nefrocitos) presentan una estructura análoga, tanto molecular como funcionalmente, al DF de vertebrados. El objetivo principal de nuestro laboratorio es el empleo de Drosophila como modelo para caracterizar nuevos componentes y Salamanca 2016 reguladores del DF. Así estamos llevando a cabo el análisis funcional de polychateiod(pyd), el ortólogo de ZO-1, en la formación y mantenimiento de los DF en los nefrocitos. Este gen da lugar, por splicing alternativo, a varios transcritosque codifican para proteínas que presentan los dominios comunes PDZ, SH3 y Guk, conservados en ZO-1, pero difieren en otros dominios alternativos. En la deficiencia para pyd los nefrocitos aparecen aglutinados y presentan una expresión y localización aberrante de los principales constituyentes del DF. El análisis ultraestructural de estos mutantes confirma que carecen de diafragmas de filtración. Para analizar la contribución de los distintos dominios a la función de Pyd en nefrocitos hemos empleado la tecnología CRISPR/cas9 para generar nuevas líneas mutantes específicas para diferentes isoformas. Pósters / Posters E2, lo cual sugiere que el cAMP ejerce un efecto sobre la inducción de leptina por E2. Por otro lado se demostró que la sobreexpresión de CBP (proteína de unión a CREB con función de acetiltransferasa) produce un aumento en el efecto de E2 sobre la expresión de leptina. También se observó, en transfecciones transitorias, que la sobreexpresión de HDAC-1, una histona deacetilasa, disminuyó la inducción de E2 sobre leptina sugiriendo que las modificaciones en acetilaciones de histonas podrían estar involucradas en su acción, y el tratamiento con TSA (Triscjostatin A, inhibidor de deacetilasas) contrarresta este efecto, sugiriendo que las modificaciones de las histonas en el DNA podrían estar influyendo en su acción. Estos resultados proveen nuevas evidencias acerca de los mecanismos por los cuales el E2 regula la expresión de la leptina placentaria. Futuros ensayos permitirán estudiar en detalle los complejos proteicos formados entre en el promotor de leptina en respuesta a E2. P03r-6 Sprouty1 and Sprouty2 cooperate to pattern internal genitalia independently of Ret signaling Marta Vaquero Susagna1, Carlos Anerillas Aljama1, Sara Cuesta Sancho2, Joan Ribera Calvet1, Mario Encinas Martín1 1 Universitat de Lleida, Lleida, ES, 2IRB Lleida, Lleida, ES Sprouty proteins are feedback inhibitors of receptor tyrosine kinase signaling pathway. Deletion of Spry1 in mice leads to defects in the development of the genito-urinary system due to hyperactivation of Ret signaling. On the other hand, targeted deletion of Spry2 causes craniofacial and enteric innervation defects owing to excessive FGFR and Ret signaling, respectively. Besides these distinct roles in mouse organogenesis, recent data indicate that the different Spry genes play overlapping roles during development. In the present work we describe that Spry1; Spry2 double heterozygous mice exhibit internal genitalia abnormalities that are not caused by dysregulated Ret signaling. Mutant females exhibit imperforate vagina and hydrometrocolpos, whereas males show seminal vesicle duplication. These observations are consistent with abnormal Wolffian duct development, which shows strong Ret expression on its caudal end from E14.5 to E16.5, embryonic ages between which Müllerian duct fusion and seminal vesicle formation occur. However, Ret deletion does not affect vagina or seminal vesicle development. Moreover, unlike kidney defects found in Spry1 knockout mice, removal of both alleles of Ret in the context of Spry1; Spry2 heterozygosity do not rescue vaginal defects or seminal vesicle duplication. Taken together, these observations indicate that Spry1 and Spry2 cooperate during the development of the caudal Wolffian duct in a Ret-independent manner. P03-8 Tyrosine 53 is essential for Sprouty1 function during genitourinary development Sara Cuesta Sancho1, Marta Vaquero Susagna2, Carlos Anerillas Aljama2, Mario Encinas Martín2 1 IRB Lleida, Lleida, ES, 2IRB Lleida/Universidad de Lleida, Lleida, ES Sprouty family genes are feedback inhibitors of receptor tyrosine kinase (RTK) signalling. Mice lacking Sprouty 1 have defects in urogenital development reminiscent of human CAKUT (congenital abnormalities of the kidney and lower urinary tract). The mechanisms of action of Sprouty proteins are controversial but several in vitro studies point to a conserved tyrosine in the N-terminus of Spry family members (Tyr53 in Spry1) as a master regulator of their activity. To address the role of Tyr 53 in vivo, our group has generated knockin mice bearing a Tyrosine to Alanine mutation in residue 53 of Spry1. Despite expressing normal levels of the protein, these mice present genitourinary abnormalities indistinguishable from those found in Spry1 knockout mice such as fused supernumerary kidneys, and hydroureter/megaureter. However, unlike Spry1 knockout mice, Spry1Y53A mice show reduced weight and viability at weaning. Summing up, our results indicate that Tyr53 is required for Spry1 function in genitourinary development of mice displaying the same effect characterized in Spry1 knockout mice. The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement n° 609396. P03-7 Efectos de la vía del AMPc sobre la acción del estradiol en la expresión de leptina placentaria Malena Schanton1, Antonio Peréz Peréz2, José Luis Dueñas3, Victor Sanchez Margalet2, Cecilia Laura Varone1 1 Departamento de Química Biológica, FCEN, UBA, IQUIBICEN, CONICET, Buenos Aires, AR, 2Departamento de Bioquímica Médica y Biología Molecular, Universidad de Sevilla, Sevilla, AR, 3Hospital Universitario Virgen Macarena, Sevilla, ES La leptina es una proteína expresada en placenta que durante la gestación regula la implantación y el desarrollo embrionario. Resultados previos de nuestro laboratorio han demostrado que el estradiol (E2) regula la expresión de leptina placentaria a nivel transcripcional involucrando efectos genómicos y no genómicos. Previamente demostramos que CREB regularía la expresión basal de leptina, el objetivo del presente trabajo es estudiar el efecto de vía AMPc sobre la acción del E2 en la expresión de la leptina placentaria. Se utilizaron células BeWo y explantos de placenta humana a término como modelos experimentales. Al utilizar los inhibidores de la vía de PKA, H89 y SQ22536 se observó por Western blot y por ensayos de genes reporteros, una disminución de la expresión de leptina mediada por P03-9 Efecto de la inhibición de la quinasa activada por AMP, AMPK, en la función del espermatozoide humano Violeta Calle Guisado1, Ana Hurtado de llera1, David Martín-Hidalgo1, Lauro González Fernández2, José Mijares Gordún3, Mari Cruz Gil Anaya1, Ignacio Santiago Álvarez Miguel4, Luis Jesús García Marín1, María Julia Bragado González1 1 Facultad de Veterinaria, Universidad de Extremadura, Cáceres, ES, 2Centro de Estudos de Ciência Animal/Instituto de Ciências, Tecnologias e Agroambiente da Universidade do Porto (CECA/ICETA), Oporto, PT, 3Centro de Cirugía de Mínima Invasión Jesús Usón, Clínica Norba, Cáceres, ES, 4Centro de Cirugía de Mínima Invasión Jesús Usón, Instituto Extremeño de Reproducción Asistida, Cáceres, ES La proteína quinasa activada por AMP, AMPK regula el metabolismo celular dependiendo de la carga energética. La forma activa de la AMPK está fosforilada en la treonina 172. Nuestro grupo ha identificado previamente esta proteína y su función en el espermatozoide porcino. 61 Pósters / Posters Nuestro objetivo es estudiar la presencia, localización intracelular y la función de la AMPK en el espermatozoide humano. Eyaculados procedentes de donantes sanos se separaron mediante gradiente de densidad en dos poblaciones de alta y baja calidad espermática. La identificación y localización de la proteína se llevó a cabo mediante Western blotting e inmunohistoquímica. La motilidad y viabilidad espermática se evaluaron en presencia o ausencia del compuesto C (CC), inhibidor específico de la AMPK, mediante el sistema ISAS de análisis espermático o citometría de flujo, respectivamente. La AMPK se localiza en los diferentes compartimentos de espermatozoide humano con una intensidad variable. La forma activa, p-Thr172-AMPK, presenta una mayor intensidad en la población espermática de alta motilidad. La inhibición de la actividad de AMPK con CC reduce significativamente todos los parámetros evaluados: porcentaje de espermatozoides mótiles, velocidades (VCL, VSL o VAP), progresividad y otros coeficientes de motilidad, todo ello sin afectar la viabilidad espermática. Este trabajo identifica por primera vez la AMPK y su forma activa en el espermatozoide humano y muestra su implicación en la motilidad espermática. Nuestros resultados nos permiten seguir trabajando en las posibles implicaciones de la AMPK en la función del espermatozoide y en su uso en la mejora de las técnicas de reproducción asistida. P03-10 Inhibition of APAF-1 with LPT99 prevents cisplatin-induced apoptosis in HEI-OC1 auditory cells Blanca Cervantes1, Isabel Sánchez-Pérez2, Carmen Herrero3, Isabel Varela-Nieto4 1 Centro de Investigación Biomédica en Red de Enfermedades Raras. Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Madrid, ES, 2Dpto. Bioquímica. Fac. Medicina. Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Madrid, ES, 3Spiral Therapeutics, San Francisco, US, 4Centro de Investigación Biomédica en Red de Enfermedades Raras. Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM. IdiPAZ, Instituto de Investigación Hospital Universitario La Paz , Madrid, ES HEI-OC1 (House Ear Institute – organ of Corti 1) is an epithelial otic cell line that was derived by Kalinec et al. (2003) from the cochlea of a transgenic mouse called Immortomouse™, which harbors a temperature-sensitive mutant of the SV40 large T antigen. The modulation of culture conditions allows the progression of the progenitor cells to a differentiated hair cell-like with a phenotype similar to that of the adult organ of Corti. One the most interesting characteristic of these cells is their sensitivity to ototoxic drugs such as aminoglycoside antibiotics or antineoplastic agents as cisplatin, which can cause sensorineural hearing loss. Cisplatin is a highly effective chemotherapeutic agent, but it has significant ototoxic side effects. Apoptosis is an important mechanism of cochlear hair cell loss following exposure to cisplatin. The present study examined the effects of LPT99, a second generation of APAF-1 inhibitors on cisplatin-induced apoptosis. Viability and growth rate of HEI-OC1 cells were determined using a crystal violet based staining method and the activation of Caspase-3, a biochemical marker for apoptosis cell death was evaluated with immunocytochemistry. We observed a dose-dependent decrease in cell viability after challenge with cisplatin (0-5 μg/mL); however survival rate increased in the presence of 1 μM LPT99. According, cells treated with cisplatin showed an IC50 of 4.47±1.94 μg/mL, which increased dramatically to 10.51 ± 3 μg/mL in the presence of LPT99. In addition, immunofluorescence studies suggest that the activity of Caspase-3 after cisplatin treatment decreased in the presence of LPT99. These results suggest that LPT99 protected the cells HEI-OC1 against cisplatin-induced apoptosis by inhibiting of APAF-1. Supported by the FP7-PEOPLE-2013-IAPP-TARGEAR. 62 XXXIX Congreso SEBBM P03r-11 The early epaxial enhancer of Myf5 has a dual role during embryonic development Macarena López-Mayorga1, Natalia Moncaut2, Cristina Vicente-García1, Lydia Teboul3, Peter W. J. Rigby2, Jaime Carvajal García-Valdecasas1 1 Centro Andaluz de Biología del Desarrollo, Sevilla, ES, 2 The Institute of Cancer Research, London, UK, 3MRC Harwell, Oxfordshire, UK The determination and specification of skeletal muscle in vertebrates is orchestrated by the myogenic regulatory factors (MRFs): Myf5, Mrf4, MyoD and Myogenin. Myf5 is the first to be expressed in the embryo, initiating and co-ordinating the myogenic cascade. In absence of Myf5, progenitors fail to be specified; activation of MyoD rescues the phenotype and myogenesis progresses. In Myf5/MyoD KO animals rescue does not take place and no skeletal muscle is formed. Transcription of the Mrf4/Myf5 locus is controlled by over 25 elements. The Early Epaxial Enhancer (EEE) operates in the dermomyotomal dorsomedial lip (DML) and is the first to activate Myf5 at E8.5 dpc. We have generated a mouse allele in which the EEE has been selectively deleted, abrogating early Myf5 expression in the DML. Homozygous animals do not show an overtly phenotype, as is the case for the full Myf5 KO lines. Transcriptome comparison between WT and EEE-KO animals reveals a series of genes downregulated in the KO, providing – for the first time – an insight into the Gene Regulatory Networks downstream of Myf5. Two major pathways are affected: one involved in the myogenic process; the second related to chondriogenesis. To unravel the full phenotype of the EEE-KO, we have crossed this line with the MyoD-KO, thus abolishing the rescue by the second MRF. No muscle groups are absent in these animals, indicating that not a single muscle derives exclusively from the DML, but bost of the body muscles show marked atrophy. The EEE element contains two peaks of sequence conservation; transgenic analyses show only one to be essential for DML expression. Our results suggest that the EEE has thus a dual function: to drive the early expression of Myf5 in the DML, and to maintain expression levels at later stages. P03-12 DLK2 favorece la diferenciación y maduración condrocítica en la línea celular ATDC5 A. Ruiz-García1, Laura Alguacil Camacho1, S. Rivero2, J. Laborda1, J.J. García-Ramírez1 1 Departamento de Química Inorgánica, Orgánica y Bioquímica. Facultad de Medicina/CRIB, Universidad de Castilla-La Mancha, Albacete, ES, 2The Chidren’s Research Institute Department of Clinical Neurology, NW Washinton DC, US La proteína de membrana DLK2, y su homóloga DLK1, pertenecen al grupo de ligandos no canónicos de los receptores NOTCH. Ambas proteínas DLK presentan una región extracelular con seis dominios EGF-like, una región transmembrana y una corta región intracelular. Las proteínas DLK desempeñan un importante papel en la proliferación y la diferenciación celular, en procesos como la adipogénesis, hematopoiesis, miogénesis, condrogénesis y osteogénesis. Desde hace tiempo se conoce que DLK1 promueve la maduración condrogénica de las células mesenquimáticas, aunque curiosamente impide su maduración final a condrocitos. Además, DLK1 ha sido descrito como un inhibidor de la diferenciación condrocítica de la línea celular de ratón ATDC5 sometida a estímulos diferenciadores. Estudios previos realizados en nuestro laboratorio revelan que los genes Dlk1 y Dlk2 se expresan de forma complementaria durante la diferenciación condrocítica en embriones de ratón. Así, Dlk1 se expresa en las células menos maduras, como los precondrocitos y los condrocitos columnares, mientras que Dlk2 se expresa en fases más diferenciadas, como los condrocitos hipertróficos y los condrocitos terminales. En este Salamanca 2016 trabajo analizamos el papel de la proteína DLK2 durante la diferenciación condrogénica de la línea ATDC5 en respuesta a insulina. Para ello, hemos obtenido transfectantes estables en esta línea celular, con mayor y menor expresión de DLK2, y hemos analizado varios parámetros específicos de la diferenciación condrocítica. Nuestros resultados muestran que DLK2 es capaz de estimular tanto la expresión de marcadores condrocíticos tempranos, como Col2a1 y AGC, como de marcadores tardíos, como Col10a y Runx2. Además, DLK2 potencia la formación de condensaciones celulares y la producción de matriz extracelular. Nuestros resultados muestran que, al contrario que DLK1, DLK2 es un potente activador de la diferenciación y de la maduración de condrocitos. Estos resultados servirán de base para investigar en profundidad los mecanismos mediante los cuales esta proteína regula la diferenciación condrocítica. P03-13 Functional analysis of transcriptional regulators during auditory development Thomas Schimmang Instituto de Biología y Genética Molecular, CSIC-Universidad de Valladolid, Valladolid, ES Transcriptional regulatory networks are essential during the formation and differentiation of organs. The transcription factor N-myc is required for proper morphogenesis of the auditory organ and to control correct patterning of its sensory epithelium, the organ of Corti. We have recently shown that the Otx2 gene, a mammalian ortholog of the Drosophila orthodenticle homeobox gene, is a crucial target of N-myc during inner ear development. Otx2 expression is lost in N-myc mouse mutants, and N-myc misexpression in the chick inner ear leads to ectopic expression of Otx2. Furthermore, Otx2 enhancer activity is increased by N-myc misexpression, indicating that N-myc may directly regulate Otx2. Inactivation of Otx2 in the mouse inner ear leads to ectopic expression of prosensory markers in non-sensory regions of the cochlear duct. Upon further differentiation, these domains give rise to an ectopic organ of Corti, together with the re-specification of non-sensory areas into sensory epithelia. Therefore, the Otx2-positive domain of the cochlear duct shows a striking competence to develop into a mirror-image copy of the organ of Corti. Taken together, these data show that Otx2 acts downstream of N-myc and is essential for patterning and spatial restriction of the sensory domain of the mammalian auditory organ. P04. Biología molecular computacional / Computational molecular biology P04-1 Formation and maintenance of nitrogen-fixing cell patterns in filamentous cyanobacteria Javier Muñoz-García, Saúl Ares Grupo Interdisciplinar de Sistemas Complejos (GISC) y Departamento de Matemáticas, Universidad Carlos III de Madrid, Leganés, ES Cyanobacteria forming one-dimensional filaments are paradigmatic model organisms of the transition between unicellular and multicellular living forms. Under nitrogen-limiting conditions, in filaments of the genus Anabaena, some cells differentiate into heterocysts, which lose the possibility to divide but are able to fix environmental nitrogen for the colony. These heterocysts form a quasiregular pattern in the filament, representing a prototype of patterning and morphogenesis in prokaryotes. Pósters / Posters Recent years have seen advances in the identification of the molecular mechanism regulating this pattern. We use these data to build a theory on heterocyst pattern formation, for which both genetic regulation and the effects of cell division and filament growth are key components. The theory is based on the interplay of three generic mechanisms: local autoactivation, early long-range inhibition, and late long-range inhibition. These mechanisms can be identified with the dynamics of hetR, patS, and hetN expression. Our theory reproduces quantitatively the experimental dynamics of pattern formation and maintenance for wild type and mutants. We find that hetN alone is not enough to play the role as the late inhibitory mechanism: a second mechanism, hypothetically the products of nitrogen fixation supplied by heterocysts, must also play a role in late long-range inhibition. The preponderance of even intervals between heterocysts arises naturally as a result of the interplay between the timescales of genetic regulation and cell division. We also find that a purely stochastic initiation of the pattern, without a two-stage process, is enough to reproduce experimental observations. P04r-2 Optimizing T cell epitope vaccine formulation for maximum population coverage Pedro A. Reche, Magdalena Molero-Abraham, Mario Solis-Lopez, María E. Lafuente Departamento de Microbiología I, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, ES T cells recognize small peptide antigens when presented in the cell surface of target cells bound to human leukocyte antigens (HLA) molecules. These peptides are known as T cell epitopes, and there is potential for using such epitopes in vaccine formulations. A T cell epitope vaccine will only be effective on those individuals presenting the HLA molecules capable of binding and presenting the epitopes included in the vaccine. Thereby, HLA polymorphism pose a major handicap to the development of T cell epitope-based vaccines covering the entire population. HLA peptide-biding specificity is determined by the polymorphisms and HLA polymorphic variants are expressed at vastly variable frequencies in different ethnic groups. In response to this problem, some authors, included us have defined HLA supermotifs to identify promiscuous epitopes binding to groups of HLA molecules known as supertypes. These HLA supermotifs are however unspecific and can result in discarding relevant epitopes. In contrast, we describe here a new algorithm that, by taking peptide binding specificities of the HLA molecules into consideration, minimizes the potential epitopes required to develop a broadly protective vaccine. P04-3 Learning the genome. Artificial Neural Networks applied to genomic data Davide Bau CNAG-CRG, Valencia, ES The three-dimensional organization of chromatin drives gene expression by bringing together genes and their interacting partners. Different genes in different cell types are expressed differently, despite sharing the same DNA sequence. Epigenetic modifications defines cell specificity; specific patterns of covalent modifications of the histone tails impact gene expression by altering chromatin structure or recruiting histone modifiers in a cell specific way. Learning how the epigenomic landscape contributes to the cellular outcome could help understanding how the genome is regulated. Using machine learning techniques, I have integrated histone modifications, chromatin accessibility and gene expression data to study the impact of post-translational modifications on genome architecture. My results show that learning from a subset of all the chromosomes is sufficient to 63 Pósters / Posters accurately predict a set of features on the remaining chromosomes. My predictions provide a useful tool to help understanding how the genome is organized and to complement incomplete genomic data. Keywords: Genome architecture, Structural biology, Epigenetics, Genomics, Integrative modeling, Bioinformatics, Machine Learning. XXXIX Congreso SEBBM splicing patterns that recapitulate those of undifferentiated cells, are controlled by MBNL1 and involve multiple cancer drivers, including the mitotic gene NUMA1. We show that NUMA1 alternative splicing induces enhanced cell proliferation and centrosome amplification in non-tumorigenic mammary epithelial cells. P04-4 Decomposing variation in heterogeneous clinical omic data Francisco José Campos-Laborie, José Manuel Sánchez-Santos, Javier de las Rivas Sanz Centro de Investigación del Cáncer (CiC-IBMCC, CSIC/USAL/IBSAL), Salamanca, ES Individual diversity is one of the most complex issues to deal with in omic studies of large populations. Most of the current approaches to detect differences using new generation omic-wide data are based on the analyses of significant mean or median changes that reflect average alterations in the whole population studied versus their controls or references. In this situation the biomarkers that are only related to a subset of samples are difficult to detect and often wrongly assigned, but in many occasions such sample subsets are quite relevant for the biological study performed. Considering that technical and batch-associated variations are mostly treated and corrected by robust normalization methods developed in recent years (RUV, SVA) (PMIDs: 25150836, 22257669), we explore different methodologies to understand the frequent heterogeneity in disease sample sets that come from specific clinical or biological features only associated to a subset of the population. Moreover, the classical normalization approaches often apply ways to reduce or remove unwanted variation (PMID: 26286812), but our scope is not to decrease or alter the sample signals but to identify omic biomarkers that are modified only in a sub-population of the clinical cohorts studied. The first method proposing the identification of disease outliers using genomic data was COPA (PMID: 16895932), and several other more recent approaches have tried to tackle this problem. One efficient approach to identify features associated to subsets of a population can be to explore in a recursive way the existing dependent relationships between features and samples provided by large-scale omic datasets. This type of comprehensive omic data (genomic, transcriptomic, etc) can facilitate the finding of new significant biomarkers related to hidden or not clear phenotypic conditions. The stratification of patients according to their omic profiles achieved using recursive heuristic methods is a way that we propose to assign new biomarker features between closely related states and to subset samples depending on their omic patterns. P05. Biomembranas y bioenergética / Biomembranes and bioenergetics P05-1 Molecular dynamics study of the interaction of the HIV gp41 LLP2 segment with model membranes Emmanuel Fajardo-Sánchez1, David Carrillo-Trevinyo1, Vicente Galiano2, José Villalaín1 1 IBMC-UMH, Elche, ES, 2Dpto. Física y Arq. Computadores-UMH, Elche, ES The construction of new antivirals to prevent the infection of the human immunodeficiency virus (HIV) is one of the biggest biomedical challenges nowadays, and the complexity of the interactions between the virus and the host cell is one of the main problems to achieve that objective. The basic knowledge of those interactions is one of the most important backgrounds which can describe this process properly. The present study aims to understand the molecular interaction between the membrane and one of the endodomain segments of HIV protein gp41 known as LLP2. LLP2 plays a key role in the infection process, but little is known about the molecular interactions between this segment and the membrane. We have designed a model membrane specifically composed of POPC/ POPE/POPI14/POPS/OSM/CHL1 to study the membrane interaction of the LLP2 segment by molecular dynamics (MD). The peptide is initially located at a distance of 5Å of membrane surface without touching any of the membrane lipids. At the end of the MD simulation, LLP2 seems to interact stronger with POPI14 than with the other lipids in the membrane. These data would imply that this gp41 segment interacts specifically with negatively-charged phospholipids and therefore gp41 function could depend on specific lipid interaction. These data should help us in our understanding of the molecular mechanism of HIV life cycle as well as making possible the future development of potent inhibitor molecules, which might target gp41 segments involved in membrane binding. P05r-2 P04-5 The role of RNA processing alterations in cancer biology Eduardo Eyras Universitat Pompeu Fabra, Barcelona, ES Alternative splicing of mRNAs profoundly influences almost all biological processes during neoplastic transformation. However, the specific alternative splicing mechanisms that are disrupted in tumors are not yet exhaustively characterized. We systematically analyzed mutation, copy number and gene expression patterns of 1348 RNA-binding protein (RBP) genes in 11 solid tumor types, together with alternative splicing changes and the enrichment of binding motifs in the alternatively spliced sequences. Our comprehensive study reveals widespread alterations in the expression of RBP genes, as well as novel mutations and copy number variations in association with multiple alternative splicing changes in cancer drivers and oncogenic pathways. In particular, we found altered 64 CD98hc at the crossroad of oxidative stress and amino acid availability Sara Cano Crespo, Laura Rodríguez de la Ballina, Manuel Palacín Institute for Research in Biomedicine (IRB Barcelona); Spanish Biomedical Research Center in Rare Diseases (CIBERER); Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, ES CD98hc, the only ubiquitously expressed heavy subunit of Heteromeric Amino acid Transporters (HATs) is overexpressed in highly proliferative cells, in both physiological and pathological conditions. CD98hc associated transporters (i.e. xCT, LAT1 and y+LAT2 in wild type fibroblasts) support cell survival in vitro and cell proliferation in vitro and in vivo. Cell survival depends on the capacity of the cell to counterbalance the oxidative stress via CD98hc-xCT. Indeed, the deficiency of CD98hc-xCT can not be compensated, leading to cell death by ferroptosis. Supplementation of culture media with beta mercaptoethanol (β-ME) rescues CD98hc deficient Salamanca 2016 cell survival. In such conditions, CD98hc-null cells suffer modulation of other CD98hc-independent amino acid (AA) transporters (for instance induced expression of peptide transporter 1 (PEPT1)), which triggers intracellular AA imbalance with reduced levels of branched chain AA (BCAA) and aromatic (ARO) AAs. Moreover, CD98hc ablation have an impact in mitochondria at both metabolic and morphology levels and ablated cells show oxidative stress. Along with all of the aforementioned adaptations CD98hc-null cells present limited proliferation which interestingly, is rescued by external supply of dipeptides containing BCAAs and ARO AAs which can enter via PEPT1. This supplementation also recovers part of the mitochondrial phenotype, suggesting a strong dependency on BCAAs and ARO AAs availability for cell proliferation and energy metabolism and therefore a strict requirement of CD98hc-LAT1 and CD98hc-y+LAT2in our cell model. P05-3 The role of HIV-1 gp41’s lentivirus lytic peptides in the interaction of the envelope protein with viral membrane cholesterol Jon Ander Nieto Garai1, Eneritz Bilbao Eraña1, Francesc Xabier Contreras Gómez2, Maier Lorizate Nogales1 1 Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco and Instituto Biofisika (CSIC, UPV/EHU), Leioa, ES, 2Instituto Biofisika (CSIC, UPV/EHU) and Ikerbasque Basque Foundation for Science, Leioa, ES The HIV-1 envelope protein (env) is involved in the fusion between the virus and the host cell, a key process in virus infectivity. Env’s recruitment mechanism to the nascent virion’s lipid bilayer is yet unknown. The elucidation of env’s recruitment and its lipid environment would point out new therapeutic targets to hamper virus entry and therefore the spread of the infection, and would aid in the generation of immunogens against different envelope epitopes. It has been suggested that the targeting of env to budding regions requires lipid raft like membrane domains, the gag polyprotein and the three Lentivirus Lytic Peptide(LLP) domains in the cytoplasmic tail (CT) of the protein. In this regard, LLPs they have been described to play a role in the expression of env to plasma membrane, its fusogenicity and in the incorporation of env to the viral particles. Therefore, the aim of this work is to study the env-cholesterol interaction in viral context, more precisely the role of the LLP domains in such interaction. HEK 293T cells were transfected with proviral plasmids including two envdeletion mutants: ΔLLP1 and ΔLLP1-3. Transfected cells were incubated with radioactively labeled photoactivatable [3H]-photo-cholesterol. This lipid allows the study of specific in vivo interactions as it covalently binds to any molecule closer than 3Å. The cell culture supernatant was then collected and ultra-pure viral particles were purified. Our results suggest that env is associated to cholesterol in the viral membrane. Nevertheless, in the ΔLLP1-3 mutant the env-cholesterol interaction is significatively reduced. Therefore, env seems to be associated to raft-like lipids in the viral membrane and the LLP domains could play a key role in this interaction, changing protein structure or affecting the direct interaction of the CT with cholesterol. We plan to study the biological significance of the loss of env-cholesterol interaction. We also plan to study the interaction of lipids with other viral proteins and even cellular accessory proteins present in the budding site that may play a role in env recruitment. P05-4 Mechanism of binding of C1B domain of PKCε to lipid membranes Juan Carmelo Gómez-Fernández, Antonio L. Egea-Jiménez, Senena Corbalán García Pósters / Posters Departamento de Bioquímica y Biología Molecular A, Universidad de Murcia, Murcia, ES C1 domains are members of the Cys-rich domains superfamily, formed by 50-51 amino acids residues present in many types of proteins, as it is the case of the classical and novel Protein Kinases C (PKCs). Both types of PKCs, classical and novel isoenzymes, possess two C1 subdomains, C1A and C1B, although it is not totally clear why two modules are needed. C1 domains are known to interact with diacylglycerol and exogenous agents like phorbol esters. In a previous report, we demonstrated the importance of not only diacylglycerol but also of anionic phospholipids, specifically 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), for the interaction of the C1B domain with lipid membranes, among which the C1Bε domain had the highest binding affinity than any other C1B domains of novel isoenzymes. In this work we have studied the role of positively charged amino acids residues located on top of the C1Bε domain and their involvement in the interaction membrane-protein. Some anionic residues were replaced by alanine and we characterized the effect of single, double and triple mutations. Results show that binding is decreased by increasing of the number of residues mutated in the domain, and it be even abolished in the presence of diacylglycerol. In conclusion, the electrostatic interactions derived of these positively amino acids residues is important to give place to membrane docking which is further stabilized by interaction with the diacylglycerol. This work was supported by grants from Ministerio de Economía y Competitividad-FEDER (BFU2014-52269-P) and from Fundación Séneca (Comunidad Autónoma de Murcia) (19409/PI/14). P05-5 Intercellular communication in the adult carotid body germinal niche Valentina Annese, Elena Navarro-Guerrero, Aida Platero-Luengo, Verónica Sobrino, Ricardo Pardal Instituto de Biomedicina de Sevilla (IBiS). Hospital Universitario Virgen del Rocío/CSIC, Universidad de Sevilla. Dpto. de Fisiología Médica y Biofísica, Sevilla, ES Neural stem cells rise high interest among scientists due to their possible use against neurodegenerative disease. Nervous tissue-specific stem cells reside in specialized niches that allow them to respond to physiological demands. A profound knowledge of the functioning of these niches would allow us to improve the use of stem cells for cell therapy. We have studied the biology of a subpopulation of neural crest-derived stem cells that persist in an adult peripheral chemoreceptor organ: the carotid body. This organ has the capacity to adapt to a hypoxemic situation by increasing in size and cell number. The carotid body parenchyma contains a subpopulation of neural stem cells able to give rise to neurogenesis in response to hypoxia, activating proliferation and completing differentiation into new neuronal cells. Recent data in our group show that these stem cells are even more versatile, being able to participate also in the angiogenic process that takes place in the organ in parallel to neurogenesis. Carotid body stem cells are able to convert into new vascular cells in response to the hypoxic stimulus. We are studying in detail the biology of this population of adult carotid body neural stem cells. We have identified restricted progenitors within both neuronal and mesenchymal differentiation lineages. We are particularly interested in the membrane mechanisms by which carotid body germinal niche cells interact to each other, since these communication abilities determine the functioning of the niche and therefore the behavior of stem cells. Altogether, our results on the characterization of the carotid body germinal niche might have high impact on the use of these stem cells for cell therapy. 65 Pósters / Posters P05-6 Mitochondrial permeabilization by Bax at the single molecule level Ana J. García Saez University of Tuebingen, Tuebingen, DE Bax is a key regulator of apoptosis that mediates mitochondrial fragmentation and permeabilization of its outer membrane through a poorly understood mechanism. We have combined electron paramagnetic resonance with single molecule microscopy and superresolution microscopy to determine the structural organization, oligomeric state and nanoscale assembly of active, full-length Bax in the membrane at different spatial scales. We found that Bax organizes into multiple oligomeric species based on dimer units. Each Bax molecule in the dimer is characterized by a stable dimerization domain and a more flexible piercing domain. The most important structural change during Bax activation is the opening of the hairpin formed by helices 5 and 6 into a clamp-like structure. Moreover, in the mitochondria of apoptotic cells, active Bax clusters into a broad distribution of distinct architectures, including full rings, as well as linear and arc-shaped oligomeric assemblies. Remarkably, both rings and arc assemblies of Bax can perforate the membrane, as revealed by atomic force microscopy. Our data support a new molecular mechanism in which Bax dimers assemble into oligomers with even number of molecules that fully or partially delineate pores of different sizes to permeabilize the mitochondrial outer membrane. P05-7 Sea anemone actinoporins: Protein molecules dancing at the water-membrane edge Álvaro Martínez del Pozo Departamento de Bioquímica y Biología Molecular, Universidad Complutense de Madrid, Madrid, ES Sea anemone actinoporins constitute a fascinating family of multigene proteins because of their dual behavior at the water-membrane interface. In water they are mostly monomeric and remain stably folded as soluble globular proteins. However, upon interaction with lipid membranes of specific composition they become oligomeric integral membrane structures resulting in lytic pores that are lethal for their target cells. Actinoporins pore formation is efficient in sphingomyelin (SM) containing membranes. However, it is also known that cholesterol (Chol) is required for effective pore formation as a consequence of the biophysical changes and intermolecular interactions established within the lipidic membrane. In spite of the intensive work developed for more than 20 years in this direction, it is not yet fully understood how the bilayer lipid composition or the physical state of the membrane affect pore formation. Within this idea, we have studied how esterols, interfacial hydrogen bonding, membrane width and the saturation degree of the SM species employed affect actinoporins pore forming ability. Finally, we have recently shown how the appearance of actinoporins as multigene families that give rise to many protein isoforms in the same individual species can be explained in terms of a synergistic interaction to produce heteropores with highly different pore-forming activity. P05-8 Glycosylation-dependent partitioning of IFN-γ receptor in lipid and actin nanodomains is critical for JAK/STAT signaling Ornella Morana1, Cédric Blouin2, Christophe Lamaze2, Xabier Contreras3 1 Instituto Biofisika (UPV/EHU-CSIC); Departamento de Bioquímica, Universidad del País Vasco, Leioa, ES, 2Institut Curie - Centre de Recherche, PSL Research University, Membrane Dynamics and 66 XXXIX Congreso SEBBM Mechanics of Intracellular Signaling Laboratory; Centre National de la Recherche Scientifique (CNRS), París, FR, 3Instituto Biofisika (UPV/EHU-CSIC); Departamento de Bioquímica, Universidad del País Vasco; IIKERBASQUE, Basque Foundation for Science, Leioa, ES Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied IFN-γ receptor signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. This mutation by adding a neo-N-glycan on IFN-γR2 subunit blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for JAK/STAT activation by IFN-γ could not occur. Removing IFN-γR2 T168N bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. P05r-9 Characterization of the calcium binding mode of Rabphilin-3A Jesús Baltanás Copado1, María Dolores Pérez-Sánchez2, Juan C. Gómez-Fernández2, Senena Corbalán-García2 1 Universidad de Murcia, Úbeda, ES, 2Dpt. of Biochemistry and Molecular Biology A, University of Murcia, Murcia, ES C2 domains are ubiquitous conserved Ca+2 activated membranedocking modules present in a wide range of Ca+2-regulated proteins. They consist of 130 residues andshare a common fold composed of two four-stranded β-sheets arranged in a compact β-sandwich connected by surface loops and helices. Many of these C2 domains have been demonstrated to function in a Ca2+-dependent membrane-binding manner and hence actas cellular Ca2+ sensors. Calcium ions bind in a cupshaped invagination formed by threeloops at one tip of the β-sandwich where the coordination spheres for the Ca2+ ions are incomplete. This incomplete coordination sphere can be occupied by neutral and anionic phospholipids, enabling the C2 domain to dock at the membrane. The binding to Ca+2of the C2A, C2B and C2AB domain of Rabphilin 3A and different membrane model systems composed of PtdIns(4,5)P2 and PtdSer have been characterized by using isothermal titration calorimetry. The C2A domain shows a very low affinity to bind Ca+2. This interaction relies on a three sequential binding mode with dissociation constants of 117, 524 and 710 uM. The C2B domain fitted to a one set ofsites model with a dissociation constant of 2 uM, indicating that the C2B domain has ahigher affinity for Ca+2 than the C2A domain. Previous work in our laboratory hasenabled us to capture an intermediate state of the Ca+2-bound state of the C2A domain. This suggests a different mechanism of ions interaction with a large rearrangement inthe calcium binding regions (CBRs) of rabphilin C2A domain that might explain thelow Ca+2 affinity. Strikingly, when the calorimetric study was performed with the C2AB domain, the results fitted into a two set of sites, being the dissociation constants 1.4 uM and 3.8 uM for the first and second components, respectively. Site-directed mutagenesis of the residues involved in calcium binding has confirmed that the C2B domain confers Rabphilin 3A with a quick response to Ca2+ and the C2A with a slow response due to its low affinity. These results indicate that Rabphilin 3A might play different roles at several states of the vesicle fusion cycle, depending on the Ca2+ and target lipids available at each moment at the plasma membrane and transport vesicles. Salamanca 2016 Acknowledgements: This work has been sponsored by grants BFU2014-52269-P (MINECO, Spain-FEDER) and 19409/PI/14 (Fundación Seneca, Region de Murcia). Pósters / Posters no improvements in GABARAP lipidation or GABARAP-PE induced fusion were observed, perhaps due to structural differences between yeast and human proteins. The data support the notion that these two ubiquitin-like systems are involved in driving the biogenesis of the autophagosomal membrane. P05r-10 Multifuncional C2 domains David López-Martínez, Teresa Coronado-Parra, Juan C. Gómez-Fernández, Senena Corbalán-García Dpt. of Biochemistry and Molecular Biology A, University of Murcia, Murcia, ES The C2 domains are protein modules frequently involved in targeting proteins to cell membranes. They share a common fold of two four-stranded-β-sheets arranged in a compact β-sandwich and surrounded by variable loops and helices, whose variations in the aminoacidic composition give them the capacity to bind phospholipids in a calcium-dependent manner. We have shown for numerous C2 domains that they interact differently with Ca2+, phosphatidylserine and phosphoinositides, conferring different functional abilities to dock at the plasma membrane with different target messenger affinity mechanism. Moreover, we have shown that the C2A domain of synaptotagmin 1 is a non- PI(4,5)P2 responder because of a glutamic residue instead of one critical lysine. Different affinities to bind different phosphoinositides in membrane models are controlled by other amino acidic residues adjacent to the key interacting lysines. Our discovers shed light on how these domains develop structural changes to modulate their sensitivity and specificity to various cellular signals, and provide structural and functional description about in what manner, C2 domains are controlled by a dual-target mechanism. P05-11 Autophagosomal elongation: Interplay between Atg12-Atg5/Atg16 complex and LC3/GABARAP systems Marina N. Iriondo, Anton Zuriñe, Ainara M. López-Pardo, Javier H. Hervás, Ane Landajuela, Alicia Alonso, Félix M. Goñi Instituto Biofisika (UPV/EHU, CSIC); Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Bilbao, ES Macroautophagy mediates the degradation of long-lived proteins and organelles through the de novo formation of double-membrane autophagosomes (APs) that sequester cytoplasmic material and deliver it to the vacuole/lysosome. How APs form, especially how the membrane expands and eventually closes upon itself is an area of intense research. This process requires the concerted actions of a distinctive set of the so-called Atg proteins that include two ubiquitin-like proteins, Atg12 and Atg8. Membrane association of the human Atg8 homologues (LC3/ GABARAP) is mediated by the covalent attachment to phosphatidyl ethanolamine (PE) and has been related to both membrane expansion and membrane fusion, but the underlying molecular mechanisms are poorly understood. Furthermore, it has been shown that the Atg12–Atg5/Atg16 complex in yeast acts as an E3 enzyme for the conjugation reaction of Atg8, enhancing the E2 activity of Atg3 and specifying the site of Atg8–PE production. In the present study the two ubiquitin-like conjugation systems (yeast Atg12 and human Atg8) were used to analyse the molecular mechanisms by which the presence of Atg12-Atg5/Atg16 would improve human LC3/GABARAP lipidation and its ability to induce membrane tethering and fusion, leading to autophagosome elongation. With this purpose, we have performed experiments of protein-protein and protein-lipid interaction using different model membranes and a variety of biophysical approaches i.e. ultracentrifugation, fluorescence spectroscopy, or confocal microscopy. Atg12–Atg5/Atg16 improved GABARAP recruitment to lipid bilayers, in the form of giant unilamellar vesicles (GUVs), however Acknowledgements: This work has been sponsoredby grants from the Basque Government (IT838-13) and FEDER/Mineco (BFU2015-66306-P). P05-12 Alteraciones de la homeostasis de colesterol por éteres lipídicos antitumorales Pablo Ríos-Marco1, Marisol Fernández-Ortiz2, Xiomara Gálvez-Escolano2, José Manuel Jiménez-López2, Antonio Ríos3, Carmen Marco2, María Paz Carrasco Jiménez2 1 Instituto de Parasitología y Biomedicina López - Neyra, CSIC, Granada, ES, 2Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias, Universidad de Granada, Granada, ES, 3 Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, ES Según la Organización Mundial de la Salud, el cáncer es una de las principales causas de morbilidad y mortalidad en el mundo. Los éteres lipídicos se presentan como una alternativa terapéutica a utilizar en quimioterapia contra el cáncer. Se destacan los alquilfosfolípidos (APL), siendo la edelfosina (1-O-octadecil-2-O-metil-rac-glicero-3fosfocolina) representante de la primera generación de APL, así como los éteres lipídicos glicosilados (GAEL), en los que se sustituye el grupo fosfocolina de edelfosina por un grupo monosacárido. Nuestro grupo de investigación ha demostrado que ambos análogos lipídicos alteran la homeostasis del colesterol al interrumpir el tráfico intracelular de este lípido hasta el retículo endoplasmático (RE) en células tumorales humanas, provocando así una respuesta colesterogénica, que se traduce en una síntesis de colesterol incrementada. En estos experimentos hemos utilizado como control positivo el agente U18666A, que bloquea el transporte de colesterol desde los compartimentos endosomales/ lisosomales al RE. Hemos comprobado que el tratamiento con β-hidroxipropil-ciclodextrina moviliza el colesterol retenido por U18666A en los compartimentos endosomales/lisosomales aumentando el transporte de colesterol a RE. Sin embargo, este efecto no es observable cuando las células son tratadas con los diferentes éteres lipídicos, concluyendo que estos agentes no retienen el colesterol en estos compartimentos. Un efecto diferencial entre APL y GAEL consiste en que solo edelfosina inhibe significativamente la síntesis del fosfolípido fosfatidilcolina. Nuestros resultados concluyen que ambos grupos de éteres lipídicos presentan mecanismos diferentes responsables de su actividad antitumoral. Esta investigación ha sido financiada por la Junta de Andalucía (P11-CVI-7859). P05-13 Yeast-based system for expression and purification of functional native human plasma membrane Ca2+-ATPase isoform PMCA4b Isaac Corbacho, Francisco F. García-Prieto, Ara E. Hinojosa, María Berrocal, Ana M. Mata Depto. de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, Badajoz, ES Calcium (Ca2+) plays an important role in cell signaling, and its homeostasis is implicated in several diseases and neurodegenerative disorders. Among the different transporters and channels regulating 67 Pósters / Posters Ca2+ levels, human plasma membrane calcium ATPases (PMCAs) are responsible for the extrusion of calcium out of the cell. Thus, understanding PMCAs underlying mechanisms is crucial. However, there is a limited availability of these proteins for functional and structural analysis, since traditionally they have been obtained from either human or pig brain preparations, yielding a heterogeneous mixture of PMCA isoforms. In this work, using the yeast Saccharomyces cerevisiae system, we show a new and enhanced method for the expression of the full-length human PMCA isoform 4b (hPMCA4b). We have also improved a method for the purification of this native isoform by calmodulin-agarose affinity chromatography. One of the most relevant features of this work is that, when compared to PMCAs purification from pig brain, our method provides a pure single isoform instead of a mixture of isoforms, essential for fine-tuning the activity of PMCA4b. Another relevant feature is that the method described in this work has a superior yield of protein than previously established methods used to purify PMCA proteins expressed in yeasts. This work was supported by Ministerio de Economía y Competitividad (BFU2011-23313 and BFU2014-53641-P); Junta de Extremadura (GR10108 and GR15139), Fundación Caja de Extremadura and FEDER. P05r-14 Polyamine-RNA-membrane interactions: from the past to the future of biology Carlos Acosta Andrade1, Ibai Artetxe2, Marta G. Lete2, Kepa Ruiz Mirazo3, Felix M. Goñi2, Francisca Sánchez Jiménez1 1 Departamento de Biología Molecular y Bioquímica, Universidad de Málaga; and Unit 741 of CIBER de Enfermedades Raras, Málaga, ES, 2Biofisika Institute (CSIC, UPV/EHU); and Department of Biochemistry, University of the Basque Country, Leioa, ES, 3Biofisika Institute (CSIC, UPV/EHU); and Department of Biochemistry, University of the Basque Country. Department of Logic and Philosophy of Science, University of the Basque Country, Leioa, ES Biogenic polyamines (PA), e.g. spermine, spermidine, putrescine, are widely spread amino acid derivatives, occurring in living cells across the whole evolutionary scale. Their amino groups confer them a markedly basic character at the cellular pH. We have tested the interaction of PA with negatively-charged phospholipids (e.g. PIP2, abundant in the nuclear membrane), and with nucleic acids (mainly tRNA was used for practical reasons). PA induced aggregation of lipid vesicles containing acidic phospholipids, and of nucleic acids. Aggregation was detected using both spectroscopic and fluorescence microscopy methods (the latter with giant unilamellar vesicles). PA-liposome complexes were partially disaggregated when nucleic acids were added to the mixture, indicating a competition of lipids and nucleic acids for PA, in a multiple equilibrium phenomenon. Equivalent observations could be made when vesicles composed of oleic acid and 1-decanol (1:1 mol ratio) were used instead of phospholipid liposomes. The data could evoke putative primitive processes of proto-biotic evolution. At the other end of the time scale, the system may constitute an interesting tool in the development of nanoscale drug delivery. Key words: polyamines, lipids, phosphoinositides, membrane dynamics, LUCA, nanoparticles. P05-15 Role of PKC alpha in breast cancer progression Senena Corbalán García, Consuelo Marín-Vicente, Sonia Reverte-Ródenas, Juan Carmelo Gómez-Fernández Dpto. Bioquímica y Biología Molecular A, Facultad de Veterinaria, Regional Campus of International Excellence Campus Mare Nostrum, Universidad de Murcia, IMIB-Arrixaca, Murcia, ES 68 XXXIX Congreso SEBBM Cancer cells show a great versatility in signal transduction pathways. One of the groups of proteins involved in these cellular processes is the family of Protein Kinase C. These enzymes phosphorylate their targets in residues of serine or threonine and have been implicated in a myriad of processes important to tumour progression such as proliferation, migration or apoptosis. They are ubiquitous and regulated by phosphorylation, interaction with other proteins and different cofactors such as Ca2+ and lipids. Depending on the isoenzyme and the cellular type, their involvement can be pro-oncogenic or anti-oncogenic. So far, no efficient therapy based on the use of these proteins has been developed for impairing tumorigenesis. In the present work, we use transcriptomics to study the effect of supressing the expression of classical PKC alpha in breast cancer model cells. The Estrogen Receptor+ and Progesterone Receptor+ MCF7 cells were knocked down in PKC alpha by using siRNA technology. A total of 13461 unique mRNA transcripts were analyzed in an Affymetrix microarray. Among them, 168 probes were significantly up-regulated and 79 probes were down-regulated. Gene Ontology analyses in several databases identified down-regulated genes of the MAPK and ErbB families, while the up-regulated genes rendered inositol phosphate metabolism and phosphatidylinositol signalling as the most important groups. These results indicate that PKC alpha is directly involved in the oncogenic process by controlling key regulatory pathways. Furthermore, its down-regulation promotes the over-expression of many other proteins indicating that PKC alpha might act as a break to control their activity. This work was supported by Ministerio de Economía y Competitividad (Madrid), Grant [BFU2011-22828] and co-financed by FEDER. P06. Bioquímica de la nutrición / Nutritional biochemistry P06-1 Disminución en parámetros de estrés oxidativo e inflamación en tejido adiposo visceral y hepático mediante la suplementación con aceite de rosa mosqueta, a ratones alimentados con dieta alta en grasa Gladys Tapia Opazo, Daniel González-Mañán, Camila Dossi Muñoz, Amanda D’Espessailles Tapia Facultad de Medicina, Universidad de Chile, Santiago, CL Introducción: El aceite de Rosa Mosqueta (RM, Rosa rubiginosa) contiene ácido alfa-linolénico, precursor de los ácidos grasos omega-3, el eicosapentaenoico (EPA) y el docosahexaenoico (DHA). EPA y DHA tienen propiedades antiinflamatorias, antioxidantes y sensibilizadoras de la insulina, además estimulan la beta oxidación e inhiben la lipogénesis de novo, por lo cual son agentes protectores del daño inducido por la obesidad, la resistencia a la insulina, el estrés oxidativo y la inflamación; alteraciones que se relacionan con la esteatosis hepática y síndrome metabólico. Objetivo: Evaluar si la suplementación oral con aceite de RM disminuye los parámetros de estrés oxidativo e inflamación inducidos por una dieta alta en grasa, en tejido hepático y adiposo visceral. Metodología: Se alimentaron ratones (n=9, por grupo) machos C57BL/6J durante 12 semanas, dividiéndose en 4 grupos: (i) dieta control (20% proteínas, 70% carbohidratos, 10% lípidos); (ii) dieta control y RM (1,94 mg ALA/ g peso corporal/día, American Bioprocess Ltda); (iii) dieta alta en grasas (20% proteínas, 20% carbohidratos, 60% lípidos); (iv) dieta alta en grasas y RM. Se evaluaron parámetros generales (peso corporal, grasa Salamanca 2016 visceral, histología), estrés oxidativo [niveles séricos de malondialdehido (ELISA); niveles hepáticos de Nrf2 (IHQ), Hemooxigenasa-1 (Western blot) y proteínas carboniladas] e inflamación [niveles hepáticos de PPAR-α (IHQ); niveles séricos (ELISA) y expresión (qPCR) de TNF-α e IL-1β en hígado y tejido adiposo visceral]. Resultados: Se observó en el grupo con dieta alta en grasa un aumento significativo (ANOVA unifactorial, Test de Newman Keuls, P<0,05) en parámetros de estrés oxidativo e inflamación, junto con niveles de Nrf2, Hemo-oxigenasa1 y PPAR-α disminuidos en comparación con el grupo alimentado con dieta alta en grasa y suplementado con RM. Conclusión: La suplementación dietaria con aceite de RM disminuye el estrés oxidativo y la inflamación hepática y visceral asociado a la prevención de la esteatosis hepática. FONDECYT 1140547. P06r-2 La alimentación con dieta de cafetería durante la lactancia en ratas afecta los niveles de mir-222 en leche Catalina Amadora Pomar Oliver1, Heriberto Castro García2, Catalina Picó Segura1, Juana Sánchez Roig1, Andreu Palou Oliver1 1 Laboratorio de Biología Molecular, Nutrición y Biotecnología (Nutrigenómica y Obesidad), Universidad de las Islas Baleares (UIB); y CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Palma de Mallorca, ES, 2Facultad de Nutrición y Salud Pública, Universidad Autónoma de Nuevo León, Nuevo León, MX El objetivo de este estudio ha sido analizar en ratas el efecto de la alimentación con dieta de cafetería durante la lactancia sobre los niveles en leche de mir-222, seleccionado por su potencial relación con la obesidad. Para distinguir los efectos debidos a la obesidad per se, se estudió también un grupo de madres obesas alimentadas con dieta de cafetería hasta un mes antes de la gestación (madres postcafetería). Se recogieron muestras de leche a día 5, 10 y 15 de lactancia y se determinaron los niveles de mir-222. Tras el destete se sacrificaron crías de los distintos grupos, en condiciones de alimentación y de ayuno de 12 h, para analizar la expresión de genes relacionados con el metabolismo glucídico y lipídico en el tejido adiposo blanco (TAB) e hígado. Los niveles de mir-222 en leche aumentaron durante la lactancia en todos los grupos, si bien a día 15 los niveles de mir-222 fueron superiores en las madres del grupo de cafetería, pero no en las de postcafetería, en comparación con las controles. Tras el destete, las crías control presentaron niveles de expresión de Prkaa1 (AMPKa1) (una de las posibles dianas del mir-222) incrementados en respuesta al ayuno, tanto en hígado como en TAB. Este aumento no se observó en las crías de madres del grupo cafetería. Las crías de madres del grupo cafetería mostraron también una alterada respuesta a los ciclos de alimentación/ ayuno en la expresión de genes implicados en vías metabólicas reguladas por la AMPK: Srebf1, Fasn, Scd1 y Gck en hígado, y Pparg, Srebf1 y Scd1 en TAB. Estas alteraciones no se observaron en crías de madres postcafetería. En conclusión, la alimentación con dieta de cafetería en ratas durante la lactancia resulta en un incremento de los niveles de mir-222 en leche. Aunque no se conocen los posibles efectos, el mayor suministro de mir-222 a las crías a través de la leche podría relacionarse tentativamente con la alteración en la expresión de Prkaa1 y de genes implicados en la lipogénesis y metabolismo de la glucosa en respuesta a los ciclos de alimentación/ayuno. Si bien hacen falta más estudios para demostrar la existencia de una relación causa/efecto. P06-3 Diet and sex hormones regulate hepatic Synaptotagmin 1 mRNA in mice Sara Sancho-Knapik1, Clara Gabas-Rivera1, Sonia Gascón1, Luis Herrera Marcos1, Roberto Martínez-Beamonte2, María A. Navarro1, Pósters / Posters Joaquin C. Surra3, Carmen Arnal4, Jesús de la Osada García5 Departamento de Bioquímica y Biología Molecular y Celular, Zaragoza, ES, 2Departamento de Bioquímica y Biología Molecular, Zaragoza, ES, 3Departamento de Producción Animal, Zaragoza, ES, 4 Departamento de Patología Animal, Zaragoza, ES, 5Universidad de Zaragoza. Dpto de Bioquímica y Biología Molecular y Celular, Zaragoza, ES 1 The expression of Synaptotagmin 1 (Syt1) has been found to be associated with the lipid droplets in liver. Here, we studied the expression of Syt1in Apoe-deficient mice receiving cholesterol, Western diet, squalene, and oleanolic acid. We also studied the influence of sex and impact of surgical castration. Dietary cholesterol increased hepatic Syt1 expression, an effect that was enhanced when cholesterol was combined with saturated fat present in a Western diet. This potentiation was modified by the administration of 10 mg/kg oleanolic acid or 1 g/kg squalene. Females fed chow or Western diet showed higher levels of hepatic Syt1 expression as compared to male mice on the same diet. Surgical castration of males did not modify the Syt1 expression; however, ovariectomy led to decreased levels. The data show thathepatic Syt1 expression is influenced by diet and hormonal milieu. P06-4 Aplicación de la metabolómica en la búsqueda de biomarcadores útiles para evaluar la efectividad de los alimentos funcionales Manuel Suárez, Susana Suárez-García, Anna Arola-Arnal, Gerard Aragonès, Francisca Isabel Bravo, Maria Josepa Salvadó, Begoña Muguerza, Cinta Bladé, Lluís Arola Grupo de Nutrigenómica, Departamento de Bioquímica y Biotecnología, Universidad Rovira i Virgili, Tarragona, ES En los últimos años, el gran avance experimentado por las técnicas analíticas ha provocado un salto cualitativo en el campo de la nutrición y salud. En concreto, la aplicación de la metabolómica, la cual nos permite conocer de forma precisa las moléculas que componen el metaboloma de un individuo, es de gran interés en la búsqueda de biomarcadores que confirmen la efectividad de los alimentos funcionales. De este modo, siguiendo una aproximación no dirigida, se pueden determinar aquellas moléculas que se ven alteradas en una situación de enfermedad. Una vez seleccionadas las que pueden servir como biomarcadores, mediante el uso de técnicas de análisis dirigidas será posible evaluar de forma directa si la ingesta de un alimento funcional es capaz de revertir esta situación, retornando el metabolito a sus valores adecuados. Esta estrategia implica que se inicia la búsqueda del biomarcador sin tener conocimiento previo de cuáles son las moléculas que pueden verse modificadas y requiere el uso de equipos altamente precisos y bases de datos que permitan obtener una lista de todos los metabolitos existentes para, a continuación, determinar cuáles son los que pueden utilizarse como biomarcadores mediante el uso de técnicas de análisis estadístico de tipo multivariante. Así pues, siguiendo este tipo de aproximación, hemos determinado las moléculas que se modifican tras la ingesta crónica de una dieta alta en grasas en el proceso de desarrollo de dislipidemia y que se podrán utilizar como marcadores para evaluar la efectividad de alimentos funcionales dirigidos a revertir el progreso de esta enfermedad. Estudio financiado por el proyecto AGL2013-40707-R del Ministerio de Educación y Ciencia del Gobierno de España y fondos FEDER. 69 Pósters / Posters P06-5 Altered levels of circulating lysoglycerophospholipids are indicative of early steatosis in hamsters Susana Suárez-García, Manuel Suárez, Aïda Pascual-Serrano, Anna Arola-Arnal, Begoña Muguerza Marquínez, Cinta Bladé, Lluís Arola Grupo de Nutrigenómica, Departamento de Bioquímica y Biotecnología, Universidad Rovira i Virgili, Tarragona, ES The identification of early biomarkers of pathology can enhance clinical diagnosis and contribute to its prevention. This is especially important in lifestyle related diseases because early diagnosis allows treatments based on changes in unhealthy habits, without resort to drugs. In previous studies, we applied non-targeted metabolomics to identify differential circulating patterns of lysoglycerophospholipids (LysoPLs) after chronic intake of high-fat diet (HFD). The aim of the present work was to determine the LysoPL content of plasma and liver of HFD-fed animals to investigate if they could be circulating biomarkers of liver damage. Hamsters were distributed into 2 groups (n=8) and fed either normal-fat diet (NFD) or HFD. Interestingly, the liver of HFD-fed animals showed a mild grade of steatosis after 30 days of feeding without alterations in plasma transaminases. Targeted metabolomics (UHPLC-MS/MS) was used to quantify LysoPLs. The results showed significant changes in lysophosphatidylcholines (LysoPCs) and lysophosphatidylethanolamines (LysoPEs) among groups. Multivariate analysis showed a clear clusterization among groups and in both tissues the predictive value of the model was adequate. A strong correlation across liver and plasma LysoPL content was obtained. Circulating LysoPCs 16:1, 17:1, 20:5 and 22:5 were decreased in steatotic hamsters and had the highest number of significant correlations across liver. Our results suggest that altered levels of plasma LysoPLs, in particular LysoPCs, indicate an early degree of steatosis in hamsters. LysoPL evaluation could be a useful tool for the non-invasive diagnosis of steatosis. Work funded by AGL2013-40707-R from Spanish Ministerio de Economía y Competitividad, Fondos FEDER and 2016 FI_B2 00070 and 00107 fellowships. P06r-6 Caracterización de la actividad glutenásica de las heces humanas Sergio Gutiérrez Getino1, Jenifer Pérez-Andrés2, Honorina Marinez-Blanco1, Miguel Ángel Ferrero1, Javier Casqueiro2, Leandro B. Rodríguez-Aparicio1 1 Departamento de Biología Molecular, Área de Bioquímica y Biología Molecular, Universidad de León. Facultad de Veterinaria, León, ES, 2Departamento de Biología Molecular, Área de Microbiología, Universidad de León. Facultad de Ciencias Biológicas y Ambientales, León, ES La enfermedad celíaca es una enteropatía inflamatoria crónica con manifestaciones sistémicas que afecta hasta el 3% de la población. Aunque su etiología es probablemente multifactorial, su desarrollo depende por completo de la exposición de los individuos, genéticamente predispuestos, al gluten del trigo y proteínas similares del centeno y la cebada ingeridos habitualmente con la dieta. Estas proteinas son resistentes a la degradación completa por las enzimas digestivas humanas, lo que da lugar a la aparición en el lumen intestinal de péptidos de alto peso molecular ricos en prolina y glutamina. En los pacientes celiacos, algunos de estos péptidos, como el 13-mer o el 19-mer, son responsables de la activación de la respuesta inmune innata y otros, como el 33-mer, son responsables de la activación de la respuesta 70 XXXIX Congreso SEBBM inmune adaptativa. Por bioensayo y mediante técnicas electroforéticas y cromatográficas hemos podido aislar, identificar y caracterizar varias proteinas presentes en las heces humanas con actividad glutenásica. Todas ellas son capaces de degradar los péptidos 13-mer, 19- mer y 33-mer que son tóxicos para los individuos celiacos. Estos resultados permiten establecer las posibles diferencias enzimaticas existentes entre individuos sanos y celiacos y abren una nueva via en el desarrollo de estrategias enzimáticas encaminadas tanto al diagnóstico como al tratamiento de esta enfermedad. Investigación subvencionada por la Direción general de Investigacion (SAF2015-64306-R) y por la Junta de Castilla y León (LE283U14). P06-7 Hipoxia y señalización de insulina en los adipocitos Maider Varela1, Fermín Milagro Yoldi2, Alfredo Martínez Hernández2, Carlos De Miguel Vázquez3 1 Universidad de Navarra, Pamplona, ES, 2Centro de Investigación en Nutrición, Universidad de Navarra; Departamento de Ciencias de Alimentación y Fisiología, Universidad de Navarra; CIBER Fisiopatología Obesidad y Nutrición (CIBERObn), Instituto de Salud Carlos III, Pamplona, ES, 3Departamento de Bioquímica y Genética, Universidad de Navarra; Centro de Investigación en Nutrición, Universidad de Navarra; CIBER Fisiopatología Obesidad y Nutrición (CIBERObn), Instituto de Salud Carlos III, Pamplona, ES La hipoxia asociada a la hipertrofia e hiperplasia del tejido adiposo se ha relacionado con el desarrollo de resistencia a la insulina en los adipocitos. Estudios previos indican que el receptor de insulina (IR) funcional se encuentra ubicado principalmente en unas invaginaciones de la membrana plasmática denominadas caveolas. La caveolina-1 (Cav-1) es la principal proteína estructural de las caveolas y se sabe que es importante para la correcta activación de la señalización de la insulina en el adipocito. Sin embargo, se desconoce si la hipoxia es capaz de alterar la expresión y función de Cav-1 impidiendo la transmisión de la señal de la insulina. En este trabajo se investigó la regulación de Cav-1 y el estado de la vía de la insulina inducidos por la hipoxia en los adipocitos de ratón 3T3-L1. Hemos observado que la hipoxia altera la expresión de proteínas relacionadas con la señalización de insulina y reduce significativamente la acumulación de triglicéridos en el adipocito. Por otra parte, la captación de glucosa inducida por insulina también disminuyó cuando se incubaron las células en hipoxia (1%O2) durante 48 horas, mientras que la captación de glucosa basal aumentó significativamente en comparación con las células control. Este resultado fue consistente con el aumento de la expresión del transportador de glucosa independiente de insulina, Glut1, y con la disminución de la expresión del transportador sensible a insulina, Glut4. Por otro lado, la hipoxia disminuyó significativamente la expresión y fosforilación de Cav-1. Con el fin de determinar si Cav-1 está regulada por HIF-1, células 3T3-L1 diferenciadas fueron tratadas con equinomicina, un potente inhibidor de HIF-1α. Tras 24 horas de hipoxia, la equinomicina revirtió el nivel de expresión de Cav-1 al observado en las células control. Un ensayo de inmunoprecipitación de cromatina demostró que HIF-1 se une directamente a los elementos de respuesta de hipoxia (HRE) en el promotor de la Cav-1, probablemente regulando su expresión. En conjunto, estos resultados indican que Cav-1 puede ser un gen diana de HIF-1 en la hipoxia, contribuyendo al desarrollo de resistencia a la insulina en adipocitos. Salamanca 2016 P06-8 Alteraciones metabólicas y marcadores moleculares asociados a la ingesta de dietas desequilibradas en macronutrientes Paula Oliver, Andreu Palou Universidad de las Islas Baleares (UIB) y CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Palma de Mallorca, ES El consumo de dietas desequilibradas ha aumentado debido a la mayor ingesta generalizada de grasas y proteínas, y a la tendencia de reducir la cantidad de carbohidratos para controlar el peso. Estos desequilibrios dietéticos podrían estar incrementando el riesgo de sufrir problemas de salud a largo plazo. Un mayor consumo de grasa se relaciona con obesidad y síndrome metabólico. Además, se ha descrito un fenotipo denominado “falso delgado” o “metabolically obese normal-weight” (MONW), caracterizado por presentar complicaciones metabólicas del estado obeso pero en ausencia de obesidad. Por otra parte, las dietas hiperproteicas se han mostrado efectivas a corto plazo para perder peso y normalizar parámetros metabólicos, pero existen controversias sobre su inocuidad a largo plazo. Nuestro grupo ha analizado el impacto sobre la salud de la ingesta crónica de dietas ricas en grasas o proteínas. Administramos ambas dietas a ratas en cantidad isocalórica a una dieta control, para evitar el sobrepeso y mimetizar el fenotipo MONW en el caso de los animales alimentados con dietas hiperlipídicas. Las ratas MONW, sin ser obesas, presentaron mayor adiposidad y alteraciones relacionadas con el síndrome metabólico (resistencia a insulina e inflamación). Por otra parte, la ingesta crónica de dietas hiperproteicas produjo un descenso en el peso corporal que podría relacionarse con un menor contenido hídrico pero no con menor adiposidad. Además, estos animales mostraron signos de riesgo patológico, como incremento de marcadores de inflamación y mayor tamaño renal. Ambas dietas desequilibradas incrementaron la deposición de triglicéridos en el hígado, incrementando así el riesgo de padecer enfermedades metabólicas. Nuestros resultados muestran también que el desequilibrio en macronutrientes afectó a la expresión génica de células sanguíneas, y que este material celular podría usarse para la identificación de biomarcadores tempranos de alteraciones metabólicas relacionadas con la ingesta de dietas desequilibradas, como el hígado graso. Debido al efecto nocivo de la ingesta continuada de dietas desequilibradas es clave disponer de biomarcadores tempranos para poder prevenir futuros problemas de salud. P06-9 Regulación de la expresión de calpaína 1 en el hígado de rata: importancia del mantenimiento de los niveles de Vitamina A Lucía Rodríguez-Fernández1, Rosa Zaragozá Colom2, Concha García3, Luís Torres3, Joaquin Timoneda1, Juan Viña Ribes3, Teresa Barber3 1 Dpto. Bioquímica y Biología Molecular, Universidad de Valencia, Valencia, ES, 2Dpto. Anatomía y Embriología Humana, Universidad de Valencia; IIS INCLIVA, Valencia, ES, 3Dpto. Bioquímica y Biología Molecular, Universidad de Valencia; IIS INCLIVA, Valencia, ES La vitamina A es necesaria para el correcto desarrollo embrionario y la organogénesis, ejerciendo además importantes efectos sobre la visión, reproducción, función inmune, proliferación, diferenciación celular y apoptosis. Muchas de estas funciones son realizadas por el derivado de la vitamina A, el ácido retinoico, que regula la expresión de una gran variedad de proteínas, a través de mecanismos transcripcionales y no transcripcionales. Las calpaínas son un conjunto de cisteín-proteasas dependientes de calcio implicadas en la regulación de la ruta mitocondrial y lisosomal de la apoptosis. Las isoformas CAPN1 y 2 son las mejor caracterizadas y difieren en la concentración de calcio Pósters / Posters necesaria para su activación. Las calpaínas se encuentra activas en variados procesos fisiológicos como la diferenciación celular o patológicos como el daño tisular y el cáncer. Para elucidar el papel que juega la vitamina A y su derivado el ácido retinoico en la expresión de las calpaínas, se ha estudiado su expresión en el hígado de ratas deficientes en vitamina A y en hígado de ratas control tratadas con ácido retinoico. Tanto en deficiencia de vitamina A y administración de ácido retinoico a ratas control, se observa un aumento significativo de la expresión del mRNA de la CAPN1. En paralelo con el aumento de expresión se observa claramente, en ambos modelos, la autoproteolisis del extremo Nt, lo que indica que se ha producido la activación de la CAPN1. Dicha activación podría participar en el daño tisular observado en la deficiencia en vitamina A y en la administración farmacológica de ácido retinoico. Financiación: BFU2013-46434-P; GVPROMETEOII/2014-055. P06-10 El aceite de rosa mosqueta (Rosa rubiginosa) preacondiciona al hígado frente al daño por isquemia reperfusión hepática Camila Giacinta Dossi Muñoz, Gladys Tapia Opazo Universidad de Chile, Santiago, Chile, CL En el tejido hepático la interrupción parcial o total del flujo sanguíneo (Isquemia=I) seguido de su posterior recuperación (Reperfusión=R) por largos periodos de tiempo genera daño hepático (evidenciado por aumento de las transaminasas séricas) y estrés oxidativo. Se han descrito algunas estrategias que preacondicionan al tejido, disminuyendo o evitando el daño por IR, con importante aplicabilidad clínica. El aceite de rosa mosqueta (RM), dado su alto contenido de ácidos grasos esenciales y antioxidantes, posee propiedades antiinflamatorias y antioxidantes, las cuales podrían ayudar a disminuir el daño por IR. Objetivo: Determinar si la administración dietaria de aceite de rosa mosqueta (Rosa rubiginosa) preacondiciona al hígado, disminuyendo el daño hepático y el estrés oxidativo inducido por isquemia seguida de reperfusión. Metodología: Ratas machos, cepa Sprague Dawley (n=9 por grupo) se dividieron en 4 grupos experimentales: (a) Sham, (b) IR, (c) RM-sham y (d) RM-IR. Los animales fueron suplementados con aceite de rosa mosqueta (0,4 mLd/PO) o dosis isovolumétricas de solución salina (NaCl) durante 21 días. La cirugía IR (1 hora de isquemia y 20 horas de reperfusión) y laparotomía media sin isquemia (Sham) fueron realizadas 21 días después del tratamiento. Se evalúo: i) peso corporal, peso hepático y de tejido adiposo visceral, ii) actividad de transaminasas séricas AST y ALT, iii) histología hepática y iv) estrés oxidativo hepático (proteínas oxidadas y TBARS). Resultados: El grupo (b) presentó aumento significativo (ANOVA unifactorial, Test Newman Keuls p<0,05) en la actividad de transaminasas séricas, daño histológico (ambos parámetros de daño hepático) y en los marcadores de estrés oxidativo respecto al grupo (d). Las histologías hepáticas del grupo (b), presentaron alteración de la histo-arquitectura hepática, focos de necrosis e inflamación, alteraciones no evidenciadas en los otros grupos experimentales. No se observaron diferencias significativas en incremento del peso corporal, peso hepático y de tejido adiposo visceral. Conclusión: La suplementación con aceite de RM preacondiciona frente al daño por IR, asociado a una disminución de parámetros de daño y de estrés oxidativo. FONDECYT 1140547 71 Pósters / Posters P07. Bioquímica perinatal / Perinatal biochemistry P07m-1 Calpain-specific proteolytic processing of adhesion proteins in mammary tissue and breast cancer cell lines Rosa Zaragozá1, Lucía Rodríguez-Fernández2, Iván Ferrer-Vicens2, Concha García2, Sara Oltra3, Elena R. García-Trevijano2, Juan R. Viña2 1 Dpto. Anatomía y Embriología Humana. Facultad Medicina. Univ. Valencia. IIS INCLIVA, Valencia, ES, 2Dpto. Bioquímica y Biología Molecular. Facultad Medicina. Univ. Valencia. IIS INCLIVA, Valencia, ES, 3Servicio Oncología. Fund. Investigación Hosp. Clínico Universitario. IIS INCLIVA, Valencia, ES Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in breast cancer. Calpains (CAPNs) are known to cleave adhesion proteins. The isoform-specific function of CAPN1 and -2 was explored in two models of cell-adhesion disruption: mice mammary gland during involution and different subtypes of breast cancer cell lines. In both models, E-cadherin, b-catenin, p-120 and talin-1 were cleaved. Both CAPNs were able to cleave adhesion proteins from lactating mammary gland in vitro. Nevertheless, CAPN2 was the only isoform found to co-localize with E-cadherin in cell-junctions at the peak of lactation. CAPN2/E-cadherin in vivo interaction analyzed by proximity ligation assay (PLA), was dramatically increased during involution. Calpain inhibitor administration prevented the cytosolic accumulation of truncated E-cadherin cleaved by CAPN2. Conversely, in breast cancer cells, CAPN2 was restricted to the nuclear compartment. The isoform-specific expression of CAPNs and CAPN activity was dependent on the breast cancer subtype. However, CAPN1 and CAPN2 knock-down cells showed that cleavage of adhesion proteins and cell migration was mediated by CAPN1 independently of the breast-cancer subtype. Data presented here suggest that the subcellular distribution of CAPN1 and -2 is a major issue in target-substrate recognition and therefore, it determines the isoform-specific role of CAPNs during disruption of cell adhesion in either a physiological or a pathological context. Grants: BFU-2013-46434-P to J.R.V & R.Z; PI12/02394 to E.R.GT both of the projects including FEDER grants; and GVPROMETEOII/2014-055 to J.R.V. P07-2 Neonatal overfeeding alters hepatic insulin sensitivity during lactation and leads to long-term insulin resistance and fatty liver in mice: Key role of Mogat1 Marta Ramon-Krauel1, Thais Pentinat2, Maria Vilà2, Ricky Pérez-Wienese2, Sílvia Ribó2, Judith Cebrià2, Susana Kalko3, Rubén Díaz1, Uwe Tietge4, Torsten Plosch4, Josep Jiménez-Chillarón2 1 Hospital Sant Joan de Deu, Esplugues de Llobregat, ES, 2Institut de Recerca Pediàtrica, Hospital Sant Joan de Deu, Esplugues de Llobregat, ES, 3IDIBAPS, Universitat de Barcelona, Barcelona, ES, 4 Groningen University, Groningen, NL Background: Excessive energy intake and rapid weight gain early in life are associated with obesity, type 2 diabetes, hepatic steatosis and other features of the metabolic syndrome. The monoacylglycerol acyltransferase (MGAT) is an enzyme involved in an alternative pathway 72 XXXIX Congreso SEBBM for triglyceride (TAG) synthesis and storage. It has been recently proposed to have potential implications in the pathogenesis of hepatic insulin resistance (IR). Objective: To understand the mechanisms that contribute to (1) the development of early IR and (2)the long-term programming of metabolic disease in a mouse model of childhood obesity. Method: Here we used a mouse model of neonatal overnutrition (ON) and accelerated growth that develops obesity, IR, glucose intolerance and hepatic steatosis with aging. To gain insight about the mechanisms that lead to IR and steatosis, we performed gene expression profiling (Affymetrix) in livers from Control and ON Mice. Results: The Ontology with highest significance was the Lipid Biosynthetic Pathway. Strikingly, Mogat1 ranked with highest significance in this list. We confirmed Mogat1 up-regulation in liver samples from 4-6 month old ON mice. Importantly, Mogat1 was already elevated in livers of lactating 15-day-old ON mice. Furthermore we found no changes on other pathways that could contribute to lipid synthesis and storage in the liver (synthesis de novo, oxidation or VLDL transport). Mogat1 catalyzes the conversion of Monoacylglycerol to DAG, which activates PKCε that in turn contributes to IR. In agreement, we found that Mogat1 over expression resulted in DAG hepatic accumulation and higher PKCε activation in ON mice. Conclusion: Mogat1 might be a key player in the development of IR and hepatic steatosis. Therefore, targeting MGAT activity in the liver might be a novel potential strategy to improve hepatic insulin sensitivity. P07-3 Estudio de los mecanismos de regulación epigenética y su relación con la presencia de diabetes mellitus gestacional en mujeres europeas Eva de la Fuente Luelmo1, María Haro García1, Danuta Dudzik2, Coral Barbas2, Mª Pilar Ramos Álvarez1 1 Universidad CEU San Pablo, Madrid, ES, 2CEMBIO Facultad de Farmacia, Universidad CEU San Pablo, Madrid, ES La diabetes mellitus gestacional (DMG) es una enfermedad multifactorial en la que participan distintos factores ambientales, genéticos y epigenéticos y cuya prevalencia sigue aumentando. Asimismo, el ambiente intrauterino en el que se desarrolla un embrión, puede afectar a la programación fetal condicionando las enfermedades del recién nacido en su vida adulta. La enzima metionina adenosiltransferasa (MAT1A) participa en el ciclo de la homocisteína y es la encargada de crear la S-adenosilmetionina (SAM), principal donante de grupos metilo en los procesos de metilación de numerosas moléculas. De dicho ciclo forma parte también la metionina sintasa (MTR) enzima catalizadora de la última etapa de la síntesis de metionina. La correcta función de ambas afectará a la regulación epigenética de la expresión génica. Son numerosos los polimorfismos descritos sobre ambos genes. El objetivo de este estudio ha sido obtener las frecuencias alélicas de las variantes rs7087728 de MAT1A y rs1805087de MTR en una población de mujeres gestantes con y sin DMG, y observar la existencia de una asociación entre el fenotipo y el genotipo, mediante el estudio de la metilación total del DNA. Los resultados muestran que en el grupo de mujeres con DMG son significativamente más frecuentes el alelo C en MAT1A y el G en MTR. Por otra parte, el estudio de la cuantificación total del nivel de metilación del DNA del tejido placentario reveló un mayor nivel de metilación en las placentas de las mujeres con DMG respecto a las controles. Estos resultados sugieren una posible relación entre ciertos mecanismos de regulación epigenética y la presencia de DMG, pudiendo contribuir al desarrollo de patologías asociadas al síndrome metabólico en los recién nacidos en etapas posteriores de su vida. Salamanca 2016 P07-4 Peroxisome proliferator-activated receptor gamma modulates late pregnancy energy homeostasis and metabolic adaptations Patricia Corrales-Cordón1, Yurena Vivas1, Mónica Díez–Hochleitner2, Adriana Izquierdo-Lahuerta1, Daniel Horrillo1, Ismael Velasco1, Cristina Martínez-García1, Julio Sevillano2, Mercedes Ricote3, Manuel Ros1, María Pilar Ramos2, Gema Medina-Gómez1 1 Universidad Rey Juan Carlos, Alcorcón, ES, 2Universidad CEU San Pablo, Madrid, ES, 3Centro Nacional de Investigaciones Cardiovasculares, Madrid, ES Pregnancy requires a progressive adaptation of maternal energy metabolism, which includes adipose tissue expansion and pancreatic β-cells adaptation, in the function and structure of these tissues. Insulin resistance develops predominantly during late gestation, as part of the metabolic adaptations that support fetus development and growth. Peroxisome proliferator-activated receptor γ (PPARγ) is involved in adipogenesis, glucose and lipid metabolism and modulation of insulin sensitivity. Moreover, PPARγ plays an important role in β-cell proliferation in other pathologic situations like obesity. Our aim is to study the role of PPARγ in β-cell adaptation during gestation with different study conditions. We have created two models: transgenic PPARγ2 knockout (PPγ2KO-C) mice and specific PPARγ knockout mice in pancreatic β-cell (βγKO). At D15-D16 of gestation GTT or ITT were performed, and animals were sacrificed at D18.βγKO females were also fed with high fat diet 3 weeks before pregnancy. Lack of PPARγ2 induced higher insulin resistance associated with lower serum adiponectin levels during late pregnancy. Indeed, ablation of PPARγ2 induced changes in fat mass distribution, which specially affected the perigonadal deposit. Moreover, there was an altered expression of genes involved in lipid metabolism and inflammation in adipose tissue. In the other hand, results in βγKO mice have shown decreased pancreatic β-cell mass despite high serum levels of insulin during pregnancy. Their pancreatic weight was lower compared with the WT animals. There were also differences in placenta metabolites between βγKO and WT pregnant mice. These data indicated that an appropriate expression of PPARγ is necessary to ensure a normal adipose tissue and pancreas metabolism during gestation, particularly within the late phase of pregnancy when a state of insulin resistance is established. Acknowledgements:BFU2012-33594,BFU2013-47384-R,BFU2015-70454-REDT, BSAF2014-56671-R and 30VCPIGI02. P08. Bioquímica y biología molecular de plantas / Biochemistry and molecular biology of plants P08-1 Dengue virus capsid peptide binds specifically a HMMM model membrane Emmanuel Fajardo-Sánchez1, Vicente Galiano2, José Villalaín1 Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche-Alicante, ES, 2Dpto. Física y Arq. Computadores, Universidad Miguel Hernández, Elche-Alicante, ES 1 Pósters / Posters Dengue virus (DENV) C protein is crucial for viral assembly in order to ensure specific encapsidation of its genome. DENV C protein associates with intracellular membranes through a conserved hydrophobic domain and accumulates in endoplasmic reticulum-derived lipid droplets which could provide a platform for capsid formation during assembly. In a previous work (Nemesio et al., 2013), we described a region in DENV C protein which induced a nearly complete membrane rupture of several membrane model systems, which was coincident with the theoretically predicted highly hydrophobic region of the protein (peptide DENV2C6). In this work we have performed a molecular dynamics (MD) simulation of the interaction of a peptide corresponding to this DENV C region with different membrane model systems. Unrestrained all-atom MD was carried out using NAMD by using the CHARMM36 force field for the lipid molecules and the CHARMM general force field for the peptide. We have used a Highly Mobile Membrane-Mimetic (HMMM) lipid bilayer model for the MD. We show that DENV2C6 is capable of binding specifically to membranes and that this binding is modulated by lipid composition through specific lipid-peptide interactions (Figure 1). These data corroborate our previous results and open a new avenue for the development and identification of peptides, which might be used as new drugs against the DENV life cycle, through the modulation of membrane structure. The research conducted in this work was funded by grant BFU2013-43198-P (Ministerio de Economía y Competitividad, Spain) to J.V. P08-2 Development of a DNA-based system suitable for olive oil authentication Alba Alonso-Rebollo, Natividad Ortega, Sonia Ramos-Gómez, María D. Busto, David Palacios, María C. Pilar-Izquierdo, Manuel Pérez-Mateos Universidad de Burgos, Burgos, ES Olive oil is recognized worldwide not only for its taste, nutritional and health benefits, but also for being the most valuable of the agro-food Mediterranean industry. Mechanical processes to which the olives are subjected result in the production of olive oil considered as extra virgin (EVOO) or virgin (VOO). DNA-based methods are actually considered as one of the most suitable methods for olive oil traceability and authentication. Currently there are several reports of molecular markers applied for olive cultivar fingerprinting. This work presents the design of a DNA-based system suitable for extra virgin olive oil (EVOO) traceability and authentication, independently of the cultivar, which allows not only the olive detection, but also its quantification, by using SYBR-Green qPCR technology. Chloroplast DNA is more abundant and more resistant than nuclear DNA. Moreover, the lower mutation rate of this DNA leads on less intra-cultivar variability and makes it suitable for species identification. The trnL gene has been applied in species identification, including olive and, based on this gene, four new olive-specific amplification systems were designed and analyzed on basis their sensitivity, specificity and efficiency by qPCR, after their optimization. One of the systems proved specificity, great sensitivity and high efficiency, resulting suitable for olive DNA detection in EVOO. The qPCR analysis of six different commercial EVOOs, tested by triplicate, showed that system trnL-E allowed not only the detection of DNA but also its quantification in any of the triplicates from each EVOO. Thus, we obtained a trnL-based qPCR system suitable for its application in extra-virgin olive oil authentication and traceability. 73 Pósters / Posters P08-3 El silenciamiento del gen Fanpr3.1 de fresa (Fragaria x ananassa) genera resistencia a la infección por Colletotrichum acutatum y sugiere una función en defensa semejante a la de los genes Atnpr3/Atnpr4 en Arabidopsis José J. Higuera-Sobrino1, Francisco Amil-Ruiz1, José Garrido-Gala1, Ayman Lekhbou1, Enriqueta Moyano1, Isabel Arjona-Girona2, Juan M. Arjona-López2, José A. Mercado3, Fernando Pliego-Alfaro3, Juan Muñoz-Blanco1, Carlos J. López-Herrera2, José L. Caballero1 1 Departamento de Bioquímica y Biología Molecular, Edif. Severo Ochoa-C6, Universidad de Córdoba, Córdoba, ES, 2Departamento de Protección de Cultivos, Instituto de Agricultura Sostenible, CSIC, Córdoba, ES, 3Departamento de Biología Vegetal, Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora, IHSM-UMA-CSIC, Universidad de Málaga, Málaga, ES Nuestro grupo está interesado en la interacción fresa/Colletotrichum, como sistema modelo, y en dilucidar y caracterizar genes y mecanismos moleculares relacionados con la defensa de la planta de fresa a éste y otros patógenos. Entre los genes que hemos identificado, por su marcado interés aplicado, está el gen Fanpr3.1, semejante a miembros de la familia multigénica de factores de transcripción NPR de Arabidopsis. AtNPR1 es regulador positivo de la ruta de señalización, mediada por SA, que conduce a la expresión de proteínas PR de defensa y a la inducción de SAR (Systemic Adquired Resistance). AtNPR3 y AtNPR4 comparten entre sí el 73% de identidad y tan solo un 34,5% y 36% de identidad, respectivamente, con AtNPR1. AtNPR3 y AtNPR4 son sensores moleculares de SA y se unen a AtNPR1 condicionando su actividad de una manera compleja, actuando como adaptadores para su reclutamiento por Cullin 3-based E3 ubiquitin ligasas y su degradación posterior en el proteasoma. Análisis filogenéticos indican que FaNPR3.1 está más cercana a AtNPR3 y AtNPR4 que a AtNPR1 y AtNPR2. FaNPR3.1 presenta identidad del 61,6% y del 61,5% con AtNPR3 y AtNPR4, respectivamente, y del 36,8% y de 37,6% con AtNPR1 y AtNPR2. Por tanto, no podemos predecir la función de FaNPR3.1 en fresa mediante comparación de secuencias, ya que FaNPR3.1 es parcialmente semejante a ambos subgrupos de proteínas de Arabidopsis. Así, hemos obtenido construcciones plasmídicas, mediante sistema Gateway, para sobreexpresión/silenciamiento del gen Fanpr3.1 en la fresa y hemos desarrollado un sistema de ensayo fitopatológico tras su expresión transitoria en fruto. Los análisis preliminares indican que el silenciamiento del gen Fanpr3.1 mediante RNAi provoca resistencia del fruto a la infección por C. acutatum y la sobreexpresión del mismo en fruto aumenta sensiblemente la susceptibilidad a dicho patógeno. Estos datos sugieren que FaNPR3.1 podría actuar en fresa de manera semejante a como lo hace su ortólogo FaNPR4 en Arabidopsis, regulando negativamente la expresión de proteínas PR de defensa y SAR. P08-4 Farmacognosia como estrategia terapéutica para el tratamiento del cáncer de pulmón Wilber Romero-Fernández1, Israel Manjarres2, Cristian Carvajal2, Andrés Tayo2, Mileidys Pérez-Alea3 1 Facultad de Ciencias de la Salud. Universidad Técnica de Ambato, Ambato, EC, 2Facultad de Ciencias e Ingeniería en Alimentos. Universidad Técnica de Ambato, Ambato, EC, 3Instituto de Investigación Vall d´Hebron, Barcelona, ES Las enzimas aldehído deshidrogenasas (ALDH) son una familia de enzimas que oxidan los aldehídos endógenos y exógenos tóxicos a sus correspondientes ácidos carboxílicos débiles, jugando un papel clave en el mantenimiento de la homeostasis celular. Por otra parte, se ha 74 XXXIX Congreso SEBBM demostrado que las células madre tumorales (CMT) son la fuerza motriz de la tumorigénesis y la principal responsable de resistencia a terapia, progresión, recidiva y metástasis. Las CMT se caracterizan por su acelerada actividad metabólica, dando lugar a una elevada producción de aldehídos reactivos y radicales libres. Evidencias experimentales demuestran que las CMT con elevada actividad ALDH son altamente proliferativas, tumorigénicas, resistentes a la muerte celular y responsables de la reaparición del tumor y de metástasis en enfermedades hematológicas malígnas y en tumores de pulmón, mama, próstata, colon y cerebro. A todo esto se suma que en la actualidad seguimos utilizando principios activos derivados de plantas como prototipos para el desarrollo de fármacos más específicos, eficaces y menos tóxicos. En vista del papel de las ALDH y concretamente de la isoforma ALDH1A1 en la fisiopatología del cáncer de pulmón, consideramos que la ALDH1A1 representa una importante diana terapéutica para el desarrollo de fármacos basados en la etnofarmacología. Eliminar eficazmente la población de CMT podría representar una eficiente estrategia para el tratamiento del cáncer de pulmón. En este trabajo se muestra el uso potencial de la farmacognosia en la inhibición de la ALDH1A1 que promueve la inducción de apoptosis e incrementa la eficacia de los tratamientos convencionales del cáncer de pulmón. P08-5 Variaciones en el metabolismo oxidativo de semillas de soja (Glycine max L. Merr.) en respuesta al ataque de Nezara viridula (L.) Ivana Sabljic, Karina Balestrasse, Jésica Barneto, Romina Giacometti, Jorge A. Zavala, Eduardo A. Pagano Facultad de Agronomía, Universidad de Buenos Aires, INBA (UBA-CONICET), Buenos Aires, AR Nezara viridula (chinche verde – southern stink bug) (Hemiptera: Pentatomidae) es una de las principales plagas del cultivo de soja en muchas regiones productivas. Su ataque causa una disminución en el rendimiento, la calidad de la semilla, disminuyendo la viabilidad y el vigor, y el valor comercial de los granos. Existe información de que el ataque de insectos provoca la estimulación de la defensa antioxidante modificando la actividad de varias enzimas. Los principales mecanismos involucrados comprenden las rutas de señalización celular, la síntesis de compuestos de defensa y la expresión de enzimas consumidoras de especies reactivas de oxígeno. El objetivo de este trabajo fue comparar la respuesta antioxidante de dos genotipos contrastantes: IAC-100 (resistente) y Davis (susceptible) cuando son atacados por adultos de Nezara viridula (L.). Las actividades de las enzimas Guaiacol peroxidasa (GPOX), catalasa (CAT) y superóxido dismutasa (SOD) fueron cuantificadas espectrofotométricamente en extractos de semillas obtenidas de plantas de soja crecidas en invernadero, en el estadio R6, después de 72 de iniciado el ataque de los insectos y comparadas con las de semillas no dañadas. Para analizar las generación de H2O2 las semillas fueron aisladas y sumergidas en una solución 1% de 3,3-diaminobenzidina y analizadas visualmente. Las semillas atacadas del genotipo Davis presentaron una significativa disminución en el peso fresco a diferencia de IAC-100 que no mostró cambios entre control y tratado. El genotipo resistente (IAC-100) fue más activo que el genotipo susceptible (Davis) en poner en funcionamiento el metabolismo antioxidante, evidenciando una mayor actividad de todas las enzimas estudiadas y una disminución en el contenido de H2O2. En paralelo se realizó un estudio transcriptómico mediante RNA-Seq para analizar la expresión diferencial de genes en los tratamientos mencionados. Se encontró una significativa inducción de la expresión de numerosas proteínas intervinientes en procesos rédox, como, por ejemplo, peroxidasas, glutatión-S-transferasas y tiorredoxinas. Salamanca 2016 P08-6 Thymus mastichina from Spain: Aromatic profile and enantioselective gas chromatography-mass spectrometry Ana Belén Cutillas-Gomariz1, Alejandro Carrasco-Ruiz1, Ramiro Martínez-Gutiérrez2, Virginia Tomás-Martínez3, José Tudela-Serrano1 1 Universidad de Murcia, GENZ-Grupo de Investigación Enzimología, Departamento de Bioquímica y Biología Molecular-A, Campus de Excelencia Internacional Regional Campus Mare Nostrum, Murcia, ES, 2NOVOZYMES SPAIN S.A, Madrid, ES, 3Universidad de Murcia, Departamento de Quimica Analitica, Murcia, ES Introduction: Thymus mastichina or wild marjoram is a perennial herb of the Lamiaceae family. It is endemic from Spain and Portugal andhas aromatic, food and medical uses. The essential oil of T. mastichina, has been obtained by Clevenger hydrodistillation. Experimental (www.um.es/genz/e00605/serv03.htm): Fast gas chromatography on a non-polar fast column SLB-5ms 15m x 0.1mm x 0.1μm, and an enantioselective chiral column Chiraldex B-DM 30m x 0.25mm x 0.12μm, were carried out using an Agilent 7890 chromatograph, with hydrogen as gas carrier (PDH), and an Agilent 5975 MSD-EI-SQ. Sandwich split/splitless injections were performed (Gerstel MPS-2XT). Results: The main biomolecules of T. mastichina oil from Murcia were (% concentration, % enantiomers): α-Pinene (2.49,+77,-23), β-Pinene (3.14,+50,-50), Limonene (2.57,+3,-97), 1,8-Cineole (63.74), Linalool (20.75,+4,-96), Camphor (0.33,+0,-100), Borneol (0.81,+0,-100), Terpinen-4-ol (0.94,+100,-0), α-Terpineol (2.86,+66,-34), δ-Terpineol (0.21), Linalyl acetate (1.55,+5,-95), E-β-Caryophyllene (0.62,+0,-100), and other minor components. Discusion: T. mastichina essential oil shows high concentrations of 1,8-Cineole>Linalool>β-Pinene>α-Terpineol>Limonene>α-Pinene> Linalyl acetate, as main components. Near pure (-)-enantiomers are Limonene, Linalool, Camphor, Borneol and E-Caryophyllene, whereas mainly (+)-enantiomers are α-Pinene, Terpinen-4-ol and Terpinyl acetate. Conclusions: The essential oil of T. mastichina from Murcia has components, according to ISO4728:2003 standard. Acknowledgements: Study partially supported by grants from: University of Murcia, Spain (UMU-15452, UMU-17766) and Fundacion Seneca, CARM, Murcia, Spain (19545/PI/14). A.B.C. has a fellowship from the Spanish government (MECD/FPU13-04013). P08-7 PHB3 and NOA1 interact to maintain the root stem-cell niche in Arabidopsis Noel Blanco-Touriñán1, Tamara Lechón2, Virginia Palomares2, Ivett Barany3, Miguel Ángel Blázquez1, Miguel Moreno-Risueño4, Pilar Testillano3, Mari Carmen Risueño3, Óscar Lorenzo2, Luis Sanz Andreu2 1 Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Valencia, ES, 2Instituto Hispano Luso de Investigaciones Agrarias (CIALE). Universidad de Salamanca, Salamanca, ES, 3CIB Centro de Investigaciones Biológicas, Madrid, ES, 4Centro de Biotecnología y Genómica de Plantas, UPM-INIA, Madrid, ES Prohibitins (PHB) are highly conserved proteins across different taxa that are present in mitochondrial multimeric complexes. The Arabidopsis thaliana genome contains five PHB genes. The PROHIBITIN3 (PHB3) gene is broadly expressed in the root and it is necessary for meristematic cell production (1). Recently, PHB3 has been reported to influence nitric oxide (NO) levels in different tissues (2). In addition, the mouse Nitric Oxide-Associated protein 1 (mNOA1) has been reported to interact with several members of the PHB complex (3). Given the scarce knowledge on PHB in plants, we have investigated the putative functional connection Pósters / Posters between PHB3 and NOA1 in Arabidopsis thaliana. Careful examination of phb3 roots showed dramatically disorganized meristems, confirming a function for PHB3 in mitotic cell activity in Arabidopsis primary roots. This effect seems to be linked to the negative regulation by PHB3 of WUSCHEL RELATED HOMOEOBOX5 (WOX5) expression, a gene specifically expressed in the quiescent center (QC) niche which maintains its stem cell nature. Moreover, meta-analysis of transcriptomic defects in phb3 and noa1 mutants showed that 29% of the genes misregulated in phb3 mutant were also affected in the noa1 mutant, and most of these genes were regualted in the same direction by both NOA1 and PHB3, suggesting a positive correlation between the functions of these two proteins in root development. In agreement with this, the double phb3noa1 mutant displayed a much more severe disorganization in the root meristem than the single phb3 mutant, probably due to the increased expression domain of WOX5. Therefore, we conclude that PHB3 and NOA1 represent two interacting pathways that maintain the integrity of the QC by preventing ectopic expression of WOX5. References [1] Van Aken et al. (2007) Plant J 52:850-64. [2] Wang et al. (2010) Plant Cell 22:249-59. [3] Heidler et al. (2011) J Biol Chem 286:32086-93. P09. Biotecnología molecular / Molecular biotechnology P09-1 Cianómica: una visión holística de la biodegradación de cianuro y residuos industriales cianurados Conrado Moreno Vivián, Purificación Cabello, Lara P. Sáez, Isabel Ibáñez, Mª Paz Escribano, Isabel Manso, Víctor M. Luque Almagro, Mª Dolores Roldán Universidad de Córdoba, Córdoba, ES El cianuro se utiliza en la industria química, en la minería y en la joyería, actividades que generan residuos muy tóxicos y peligrosos que deben ser tratados mediante costosos métodos físico-químicos. El cianuro, a pesar de su toxicidad, puede ser tolerado o incluso asimilado por algunas bacterias, por lo que la biodegradación es una solución alternativa para el tratamiento de residuos industriales cianurados. La bacteria Pseudomonas pseudoalcaligenes CECT5344 utiliza cianuro, cianato o complejos cianuro-metálicos como única fuente de nitrógeno para su crecimiento a pH alcalino, lo que evita la formación de HCN volátil. La integración de aproximaciones ómicas para el estudio de la biodegradación de cianuro y residuos industriales cianurados por esta bacteria está proporcionando una visión global del proceso, que resulta de gran interés biotecnológico. La estirpe CECT5344 ha sido el primer organismo cianotrofo cuyo genoma se ha secuenciado completamente. Mediante 454-pirosecuenciación se estableció un borrador del genoma, con un tamaño de 4,65 Mb y 4.321 genes (1). Mediante secuenciación SMRT (Pacific Biosciences) se ha cerrado totalmente el genoma, con un tamaño final de 4.696.984 pb y 4.514 genes, y se ha establecido el patrón de metilación de bases o metiloma (2). También se ha realizado un análisis transcriptómico de la respuesta al cianuro, al residuo de la industria joyera y a la carencia de nitrógeno mediante DNA arrays, que ha permitido identificar genes inducidos por el cianuro y/o el residuo cianurado y caracterizar la expresión de genes de interés biotecnológico, como los implicados en la producción de polihidroxialcanoatos utilizables en la fabricación de bioplásticos (3). Actualmente se están realizando estudios proteómicos mediante 2D-PAGE y cromatografía líquida-espectrometría de masas (LC-MS/MS), cuya integración con los resultados del análisis transcriptómico y la cuantificación de la expresión génica por PCR cuantitativa proporcionarán una visión holística de las capacidades degradativas y biotecnológicas de esta bacteria. 75 Pósters / Posters Agradecimientos: Ministerio de Economía y Competitividad (BIO2015-64311-R), Junta de Andalucía (P11-CVI-7560) y empresas Magtel, Gemasur-Ámbito FCC, Saveco y Avenir. Referencias [1] Luque-Almagro et al. (2013). Environ. Microbiol. 15:253-270. [2] Wibberg et al. (2016). J. Biotechnol., en prensa. [3] Luque-Almagro et al. (2015). J. Biotechnol. 214:171-181. P09-2 Selección de levaduras salvajes y obtención de nuevos híbridos con buenas propiedades para panificación Rosana Chiva Tomás1, Lorena Celador2, José Antonio Uña2, Encarna Velázquez2, María Ángeles Santos García2, Mercedes Tamame González1 1 Instituto de Biología Funcional y Genómica (IBFG). CSIC / Universidad de Salamanca, Salamanca, ES, 2Departamento de Microbiología y Genética Universidad de Salamanca Campus Unamuno, Salamanca, ES Las levaduras confieren al pan gran parte de sus propiedades organolépticas y sensoriales. Las cepas comerciales de levaduras de panificación proceden de híbridos seleccionados por su alta capacidad para fermentar harinas de trigo y son muy similares genéticamente. Existe otro grupo de levaduras presentes en masas madre naturales con características fisiológicas y origen filogenético diferente que están emparentadas con levaduras vínicas. Las masas madre son mezcla de harina, agua y comunidades simbióticas de bacterias ácido lácticas (BAL) y levaduras salvajes (LEV), cuya biodiversidad final viene determinada por varios parámetros físicos y biotecnológicos. Las masas madre confieren al pan propiedades organolépticas y sensoriales más apreciadas que las de productos fabricados a gran escala con levadura industrial. La selección y caracterización de nuevas cepas BAL y LEV autóctonas de MM desarrolladas con harinas innovadoras permitirá formular nuevos starter mixtos para conseguir fermentaciones reproducibles a gran escala y productos industriales más diferenciables y con propiedades similares a los artesanales. Para satisfacer la reciente demanda de incrementar la biodiversidad y el repertorio de las levaduras de panificación disponibles, (i) se han aislado 535 LEV a partir de grano, harinas o masas madre naturales desarrolladas por expertos; (ii) se han caracterizado genética, bioquímica y fisiológicamente; (iii) se han seleccionado nuevas cepas de Saccharomyces capaces de fermentar eficazmente harinas de cereales tradicionales o innovadores (Tritordeum); y (iv), se han aislado cepas de especies no convencionales (ncLEV) con potencial interés biotecnológico. Además se han obtenido mediante “rare mating”, por primera vez, nuevas levaduras híbridas (LEVHY), entre cepas comerciales de panificación y vínicas; de algunas de éstas con cepas de laboratorio o/y con LEV aisladas de masas madre. Las LEV, ncLEV y LEVHY no están modificadas genéticamente y poseen propiedades deseables para la innovación en la industria agroalimentaria. XXXIX Congreso SEBBM sequences or the loss of viral endogenous regions have been described and it may cause viral attenuation. The CRISPR/Cas9 genome-editing system has been proposed as a powerful alternative to generate efficiently recombinant viruses. We have selected the US4 gene from HSV-1 and HSV-2, encoding glycoprotein G (gG), as the target locus. gG binds chemokines and neurotrofic factors, enhancing their activities, and represents an excellent candidate to study viral immune modulation. Knock-in viruses, in which the US4 gene was replaced by an eGFP cassette as a selection marker, were generated using the CRISPR/Cas9 tool through the transfection-infection method. We have specifically replaced the US4 gene by eGFP, selecting fluorescent plaque-purified recombinant viruses. We have sequenced by next generation sequencing (NGS) the recombinant viral genomes and confirmed the correct insertion of eGFP and the absence of alterations elsewhere in the genome. These results support the CRISPR/Cas9 genome editing system as a useful approach to generate HSV-1 and HSV-2 recombinants. P09r-4 Effect of p73 deficiency in adipocyte differentiation using an in vitro iPSC cell model Laura Maeso-Alonso1, Marta Martín-López1, Sandra Fuertes-Álvarez1, Alberto J. Villena2, M. Margarita Marques3, María C. Marín4 1 Institute of Biomedicine (IBIOMED) University of León, León, ES, 2 Department of Molecular Biology, University of León, León, ES, 3 Department of Animal Production University of León , León, ES, 4 Institute of Biomedicine (IBIOMED) University of León , León, ES The study of adipocyte differentiation has become very important in the developed countries due to the increased prevalence of obesity and related diseases. In particular, the early phases of the adipogenic differentiation process are not well characterized. Stem cells, either embryonic or induced pluripotent stem cells (iPSCs) can recapitulate different stages of differentiation. Therefore, they are emerging as an excellent tool to improve our understanding of the mechanisms that regulate adipogenesis. The p53 gene family comprises the transcription factors p53, p73 and p63. The members of this family share a similar structure and sequence identity and have been described as regulators of several differentiation processes. Lack of p73 has been related to myeloid and erythroid differentiation. In addition, our group has demonstrated that p73 is a positive regulator of endothelial cell differentiation and vasculogenesis. To further address p73 role in mesodermal cell fate determination, we analyzed the effect of p73 deficiency during the adipogenesis process.The effectiveness of adipogenesis was evaluated in wild type and p73 knockout (KO) iPSCs by immunofluorescense and transcriptional analysis. Lipid droplet accumulation was assessed by oil red O staining. Our results indicate that the dynamics of adipocyte marker expression and lipid droplet accumulation was impaired in differentiated p73KO-iPSCs, suggesting that lack of p73 alters the adipocyte differentiation process. This work was supported by: Grant SAF2015-71381-R (Spanish Ministerio de Ciencia e Innovación). Laura Maeso-Alonso is a recipient of an AECC doctoral fellowship. P09-3 Efficient and specific genome editing of herpes simplex viruses by CRISPR/Cas9 Alberto Domingo López-Muñoz, Alberto Rastrojo, Rocío Martín, Antonio Alcamí Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, ES Herpes simplex viruses (HSVs) are prevalent human pathogens of clinical relevance, frequently used as a model to study viral pathogenesis and modulation of the host immune response. Traditionally, homologous recombination was used as the main method for the construction of viral mutants, with low efficient rates. Bacterial artificial chromosomes (BACs) have also been used to generate recombinant viruses, but residual BAC 76 P09r-5 p73 regulates BMP signaling and its lack affects the mesenchymal to epithelial transition during the initiation phase of somatic cell reprogramming Marta Martín López1, Laura Maeso Alonso1, Sandra Fuertes Álvarez1, Inmaculada Diez Prieto2, Pablo Menéndez Buján3, Timo Otonkoski4, Margarita Marqués Martínez5, M.C. Marín1 1 Institute of Biomedicine (IBIOMED), León, ES, 2Cirugia y Anatomía Veterinaria, León University, León, ES, 3Josep Carreras Leukaemia Salamanca 2016 Research Institute, School of Medicine, Barcelona University, Barcelona, ES, 4Biomedicum Stem Cell Centre, Helsinki, FI, 5 INDEGSAL, León University, León, ES Somatic cell reprogramming to generate induced pluripotent stem cells (iPSCs) occurs in three sequential phases: initiation, maturation and stabilization. The initiation phase starts with a mesenchymal-to-epithelial transition (MET) triggered by BMP signaling and is characterized by the activation of an epithelial gene expression program. The maturation phase is considered the major bottleneck of the process, since cells must activate endogenous expression of the pluripotency core circuitry (Oct4, Sox2, and Nanog); thus, only a small number of cells progress to the final stabilization phase to become fully reprogrammed iPS cells. p73 is a member of the p53 transcription factor gene family, which also includes p53 and p63. The members of this family have been involved in the regulation of stem cell biology. Indeed, p53 acts as an important barrier to somatic cell reprogramming. However, p73 role in this process has never been addressed. Therefore, we hypothesized that p73 may play a role in the cellular reprogramming process. To address this, we reprogrammed WT and p73KO-mouse embryonic fibroblast (MEFs) and observed that lack of p73 negatively affects the reprogramming efficiency. Moreover, our data indicates that BMP signaling is impaired in the absence of p73 affecting the MET transition during the initiation phase of reprogramming, and resulting in an altered maturation and stabilization phase. Our results revealed, for the first time, that p73 is necessary for appropriate BMP signaling during the MET transition of somatic cell reprogramming. This work was supported by the grants: SAF2012-36143 and SAF2015-71381-R. P09-6 Construcción de levaduras capaces de utilizar glucosamina Carmen-Lisset Flores, Carlos Gancedo Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Madrid, ES La glucosamina es un aminoazúcar (2-amino-2-deoxi-glucosa) que forma parte de polisacáridos y de proteínas o lípidos glicosilados. En la levadura Saccharomyces cerevisiae la glucosamina es transportada al interior y, posteriormente, fosforilada por la hexokinasa. Sin embargo la levadura no puede usar ese azúcar como fuente de carbono. De hecho, es un análogo tóxico de la glucosa y se ha usado para obtener mutantes resistentes a represión catabólica, alguno de los cuales genera complejas formas priónicas. Nos hemos preguntado por qué S. cerevisiae no crece en ese aminoazúcar. Una búsqueda in silico de genes codificantes de enzimas que podrían participar en la vía de utilización de la glucosamina mostró que S. cerevisiae carece del gen codificante de la glucosamina-6-P desaminasa que cataliza la desaminación-isomerización deglucosamina-6-P a fructosa-6-P. Hemos clonado el gen que codifica esa proteína en la levadura no convencional Yarrowia lipolytica y lo hemos expresado en S. cerevisiae. La cepa transformada es capaz de utilizar glucosamina mostrando que la deficiencia de esa enzima es la responsable de la falta de crecimiento de una cepa silvestre. Estamos estudiando diversos efectos de la utilización de la glucosamina sobre la levadura. Curiosamente, aunque Y. lipolytica tiene las enzimas que le permitirían utilizar la glucosamina, no crece en este azúcar. La causa de ese defecto podría estar relacionada con las características del equipo de fosforilacion de azúcares de Y. lipolytica. Estamos trabajando para dilucidar el defecto metabólico que impide a Y. lipolytica utilizar la glucosamina. Agradecimientos: Trabajo parcialmente realizado con la ayuda BFU2010-19628-C02-02 del MINECO. Pósters / Posters P09r-7 Regulación de la expresión de los genes de síntesis de polifosfatos (ppk2A, ppk2B) y su degradación (ppx1 y ppx2) en Corynebacterium glutamicum Alfonso G. de la Rubia, Laura Marcos-Pascual, Álvaro Mourenza, José A. Gil, Luis M. Mateos Delgado Facultad Biología-Ambientales Campus de Vegazana Universidad de León, León, ES Corynebacterium glutamicum constituye un buen prototipo para los análisis moleculares de bacterias corimeformes y otros relacionados: Mycobacterium, Rhodococcus. Estos suelen presentar resistencia a agentes generadores de estrés oxidativo por factores tales como (i) pared celular con ácidos micólicos; (ii) sistemas antioxidantes como los pares tiorredoxina y su reductasa Trx/TrxR, y el exclusivo par redox de actinobacterias Micotiol (MSH)/Micorredoxinas (Mrx). Otro mecanismo de resistencia frente a estrés podría ser el basado en presencia de acúmulos de polifosfatos (Poli-P), típico de representantes de Corynebacterium. Estos polímeros, formados por uniones fosfoanhidro de residuos de fosfato inorgánico (Pi) en supervivencia frente a cambios osmóticos y de pH, formación de biofilms, procesos de virulencia y acción como chaperoninas, además de los típicos reserva de fosfato y fuente alternativa de energía. En C. glutamicum hay dos enzimas polifosfato quinasas (PPK2A y PPK2B) que participan en la formación de Poli-P, y dos actividades exopolifosfatasas (PPX1 y PPX2) implicadas en movilizar Poli-P. En mutantes de Corynebacterium afectado en alguno de estos genes, se observan diferentes niveles de supervivencia frente a agentes oxidantes (peróxido de hidrógeno) y otros agentes estresantes. Los análisis de expresión génica usando técnicas de qPCR han puesto de manifiesto que los genes ppk2A y ppk2B se regulan de forma coordinada. Además, en este proceso de regulación global, la carencia de movilización de los gránulos de poli-P (como ocurre al interrumpir los genes ppx1 y ppx2) parecen reprimir transcripcionalmente a los genes para PPKs. Esto puede estar relacionado con el tamaño del gránulo de polifosfato y su posible función de chaperonina primitiva. Además de los estudios transcripcionales de expresión se está evaluando su correlación con análisis cuantitativos de polifosfatos mediante técnicas espectrofotométricas. P09r-8 Label-free single-molecule analysis of protein-DNA complexes with nanopores Garbiñe Celaya1, Jéssica Rodríguez2, Judit Perales-Calvo1, José Luis Mascareñas2, Arturo Muga1, Fernando Moro1, David Rodríguez-Larrea1 1 Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco UPV/EHU and Instituto Biofisika (CSIC, UPV/EHU), Leioa, ES, 2Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS), Departamento de Química Orgánica, Universidade de Santiago de Compostela, Santiago de Compostela, ES Protein interactions with specific DNA sequences is crucial in the control of gene expression and the regulation of replication. Single-molecule methods offer unique capabilities to unravel the mechanism and kinetics of these interactions. Here we develop a nanopore approach where the target DNA sequence is contained in a hairpin followed by a ssDNA reporter. This system allows to distinguish DNA-protein complexes from bare DNA molecules, giving both the equilibrium and kinetic constants of the interaction. We show that this approach can be used to test the inhibitory effect of small molecules, and their action mechanism. In a proof of concept, we use different dsDNA-ssDNA reporters to probe the ability of the nanopore to distinguish the effect of an inhibitor in a complex mixture of target DNAs and proteins. We anticipate that the use of this 77 Pósters / Posters technology in chips carrying thousands of pores will led to new inhibitors of transcription factor binding. P09-9 Reactivos de transfección de células eucariotas basados en polietilenimina (PEI) y colorantes fluorescentes infrarrojos (NIR) María Dolores Girón González1, Eduardo de los Reyes Berbel2, Francisco Reche Pérez1, Mariano Ortega Muñoz2, Francisco Javier López Jaramillo2, Fernando Hernández Mateo2, Francisco Santoyo González2, Rafael Salto González1 1 Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, Universidad de Granada, Granada, ES, 2Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Granada, Granada, ES La terapia génica se basa en el desarrollo de sistemas de transfección eficientes que posibiliten el tráfico extra e intracelular de ácidos nucleicos. La transfección basada en el uso de polímeros catiónicos, una técnica denominada polifección, es adecuada en la transfección de células en cultivos así como en animales de experimentación No obstante existe la necesidad de desarrollar reactivos menos tóxicos y con una alta eficiencia, fundamentalmente en las aplicaciones in vivo. La posibilidad de incluir en la estructura de los reactivos de transfección una serie de moléculas que permitan su seguimiento durante la transfección en animales, lo que se denominan reactivos teragnósticos, es una poderosa herramienta para el desarrollo de reactivos de transfección más eficaces. Por ello, en este trabajo se presenta la evaluación biológica de reactivos de transfección basados en polietilenimina de diferentes pesos moleculares unidos a fluoróforos en el infrarrojo cercano. Los reactivos así sintetizados presentan una alta capacidad de unión y protección del DNA durante la transfección, una elevada eficiencia de transfección en diferentes tipos celulares en cultivo y una baja toxicidad. Además, en modelos experimentales de ratones es posible el seguimiento y localización de los reactivos de transfección mediante IVIS utilizando el fluoróforo infrarrojo asociado y la eficiencia de transfección utilizando sistemas reporteros basados en la luciferasa. En la actualidad estamos extendiendo el uso de estos reactivos para el transporte de fármacos hacia células diana. Investigación subvencionada por el Proyecto CTQ2014-55474-C2-2-R (Ministerio de Economía y Competitividad). P09-10 Actividad hidrolítica de bacterias halotolerantes aisladas de pozas salinas Ángel Wuilder Gárate-Palomino1, Amparo Iris Zavaleta2, Pilar Foronda Rodríguez3, Basilio Valladares Hernández3, Maria Antonieta Quispe Ricalde1 1 Escuela profesional de Biología, Facultad de Ciencias. Universidad Nacional de San Antonio Abad del Cusco, Cusco, PE, 2Laboratorio de Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad Nacional Mayor de San Marcos, Lima, PE, 3Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias. Universidad de La Laguna, La Laguna, ES Los ambientes salinos son fuentes de microorganismos productores de enzimas que resisten a procesos biotecnológicos exigentes de salinidad y temperatura. Así, se han descrito microorganismos halófilos con gran actividad lipolítica y proteolítica. La búsqueda y aislamiento de tales microorganismos son el paso inicial para la obtención de enzimas resistentes a procesos industriales escalables. En el presente trabajo se describe el aislamiento y la identificación de bacterias halotolerantes de las aguas salinas de la Provincia de Urubamba, del Departamento de 78 XXXIX Congreso SEBBM Cusco (Perú). Las muestras fueron obtenidas de 4 pozas saladas y de la fuente de alimentación. Se realizaron 3 diluciones de cada muestra y el aislamiento se realizó en medio HM (Extracto de levadura 1%, peptona 0,5%, glucosa 0,1%, MgSO4 0,025%, KCl 0,05%, CaCl 0,009%). La caracterización de cada aislado se determinó en base a sus características morfológicas, fisiológicas y bioquímicas. La identificación molecular se realizó mediante secuenciación del gen ribosómico 16S. Las reacciones hidrolíticas evaluadas fueron: la proteolisis de gelatina y caseína, lipólisis de tween 80 y la hidrólisis de almidón y esculina. Las bacterias aisladas fueron 9 cocos grampositivos, pertenecientes al género Staphylococcus. Los aislados crecieron en NaCl 5%, 10% y 15% (p/v), sus temperaturas de crecimiento fueron entre 25ºC a 42ºC. En cuanto al pH los aislados crecieron entre 5 y 9. Las actividades lipolíticas y proteolíticas se observaron en 5 y 7 aislados respectivamente, además 4 hidrolizaron la esculina. Financiación: Científicos INC-Círculos de Investigación en Ciencia y Tecnología. Convenio Nº007-2014-FONDECYT. CONCYTEC-Perú. P09-11 The protein phosphatase CnPpz1/CnHal3 system from Cryptococcus neoformans Chunyi Zhang IBB-Universitat Autonoma de Barcelona, Cerdanyola de Vallès, ES The Saccharomyces cerevisiae protein phosphatase (PPase) Ppz1 is involved in multiple cellular processes because its important role in the regulation of monovalent cation (K+ and Na+) homeostasis. In budding yeast, Ppz1 is inhibited by two regulatory proteins, Hal3 and Vhs3, which also have moonlighting properties related to CoA biosynthesis. Ppz1 is found only in fungi, including pathogenic ones and, when overexpressed in high-copy number Ppz1, it appears to be the most toxic protein in budding yeast. These characteristics define Ppz1 as a possible target for antifungal therapies. Cryptococcus neoformans is a pathogenic fungus responsible for a high rate of mortality in invasive infections. Exploration of the C. neoformans genome has identified a single putative gene coding for Ppz1 (CNAG_03673) and two genes encoding possible Hal3-like proteins (CNAG_00909 and CNAG_07348). We are in the process of functionally characterize all three proteins by expression in E. coli and in S. cerevisiae, as a first step towards the characterization of the function of these protein sin C. neoformans. Although characterization of CnPpz1 has been hampered by its extremely G/C rich N-terminal region, we have recently succeeded in expressing the protein in S. cerevisiae. Preliminary results indicate that, in high copy number, CnPpz1 can partially replace endogenous budding yeast Ppz1. P09-12 Single mutants that tolerate the overexpression of Ppz1 protein phosphatase Carlos Alberto Calafí Pascual, María López Malo, Antonio Casamayor Gracia, Joaquín Ariño Carmona Institut de Biotecnologia i Biomedicina - Departamento de Bioquímica, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, ES The Saccharomyces cerevisiae PPZ1 gene encodes a protein phosphatase involved in multiple cellular processes, such as cation homeostasis, cell cycle progression and cell wall integrity maintenance (Clotet et al., 1996; Posas et al., 1995). PPZ1 is a non-essential gene, but its overexpression promotes cell cycle delay (de Nadal et al., 1998) and it appears to be a highly toxic gene when overexpressed (Makanae et al., 2013). Since Ppz1 is only present in fungi, including pathogenic ones, it represents a Salamanca 2016 possible antifungal target. In an attempt to clarify its toxicity mechanism we are trying to identify genes whose absence abolish or alleviate the Ppz1-dependent slow growth phenotype. For this purpose we screened the EUROFAN KO mutant collection transformed with a plasmid in which the Ppz1 expression iscontrolled by the GAL1-10 inducible promoter. We expect that single mutants found by this strategy will be involved in: 1) mechanism of induction of the GAL1-10 promoter by galactose, or 2) mutants related to the mechanism of toxicity of Ppz1 when it is overexpressed. The screen of the mutant library has revealed 6 mutants that tolerate the overexpression of Ppz1 from the used high-copy plasmid. Cells lacking GAL3 or GAL4 are probably involved in the galactose-dependent expression of Ppz1. We also found gal11, pgd1, med2 and tcm62 mutant cells, which are being tested as suppressors of the toxicity triggered by overexpression of this phosphatase. P09-13 Caracterización de nanopartículas dirigidas a marcadores de Cancer Stem Cells en el cáncer microcítico de pulmón Carmen Garnacho Montero, Ana Sarrias Giménez, Miguel Ángel González Reyes, Elena Ábalos Marco, Inés de la Rosa Zurera Departamento de Citología e Histología Normal y Patológica. Facultad de Medicina, Universidad de Sevilla, Sevilla, ES El cáncer de pulmón es la principal causa de muerte por cáncer en todo el mundo y, a pesar de los avances en el diagnóstico y tratamiento, el pronóstico de los pacientes sigue siendo pobre. Diversos trabajos han propuesto que muchos tipos de cáncer, incluyendo el cáncer de pulmón, pueden ser promovidos por las células madre del cáncer (CSC, del inglés Cancer Stem Cell), un subconjunto de células indiferenciadas con la capacidad de autorrenovarse y al mismo tiempo dar lugar a una progenie heterogénea diferenciada. Estas células serían las responsables no solo de la iniciación del tumor, sino también de la recurrencia, resistencia a quimioterapia y radioterapia, y metástasis. Los sistemas de direccionamiento de fármacos con nanopartículas pueden ofrecer un enfoque innovador para superar la resistencia de las CSC, así como reducir la frecuencia de recidivas. En este estudio hemos caracterizado los marcadores ABCG2, CXCR4 y CD44 para su uso como dianas en un sistema de direccionamiento de fármacos con nanopartículas que eliminaría selectivamente las CSC en un modelo in vitro de cáncer microcítico de pulmón. Nuestros resultados revelan la presencia de los tres marcadores en la superficie de las líneas celulares pulmonares estudiadas, aunque con diferentes patrones de expresión. Además, tanto las NP anti-CXCR4, anti-ABCG2, como anti-CD44 se unieron de forma específica a las células e indujeron la endocitosis de las mismas. Por lo tanto, los receptores CXCR4, ABCG2 y CD44 podrían ser una buena diana para el desarrollo de un sistema dirigido de fármacos mediante nanopartículas para el tratamiento del cáncer microcítico de pulmón. Financiación: Proyectos de Investigación en Salud. Consejería de Igualdad, Salud y Políticas Sociales 2013 (PI-0051-2013). P09r-14 Modulación de la producción de hidrógeno a través del control del consumo de acetato en Chlamydomonas José Luis Jurado Oller, Alexandra Dubini, Aurora Galván, Emilio Fernández Reyes, David González Ballester Universidad de Córdoba, Córdoba, ES Chlamydomonas es un alga verde unicelular ampliamente utilizada como organismo modelo para el estudio de multitud de procesos relacionados con plantas (fotosíntesis, metabolismo del nitrógeno y del azufre, etc.). Sin embargo, en los últimos años ha recibido especial atención Pósters / Posters debido a su capacidad para producir triacilglicéridos, hidrógeno y otros compuestos susceptibles de ser empleados como biocombustibles. Entre ellos, el hidrógeno (H2) destaca debido a que puede ser combustionado obteniendo rendimientos energéticos similares a los combustibles fósiles sin liberar CO2 a la atmósfera. La condición de cultivo más ampliamente utilizada y estudiada para promover la producción de H2 por Chlamydomonas es la deficiencia de azufre, aunque la ausencia de otros nutrientes (nitrógeno, fósforo, magnesio, etc.) en la composición del medio de cultivo también conduce a este fenómeno. Otro factor importante para la producción de H2 en Chlamydomonas es la presencia de acetato, una fuente de carbono reducida asimilable por el alga. Aunque se conoce que la presencia de acetato potencia la producción de H2 en cultivos adaptados a anoxia y luz, no ha sido realizado un estudio en profundidad del papel de este compuesto en el proceso. En este trabajo se discute el papel del acetato en la producción de H2 y se describe una metodología alternativa a la deficiencia nutricional para la obtención simultánea H2 y biomasa a través del control del consumo de acetato. Asimismo, se propone un modelo para la producción de H2 vinculado a la asimilación de acetato con la participación de la cadena fotosintética. Founded by MICINN (Proyecto IEO-Plan-E) and BFU2015-70649-P) with support of the FEDER program, Junta de Andalucía (BIO-128 and BIO-502). P09-15 La flora intestinal como fuente de “nuevos” microorganismos probióticos Borja Sánchez Instituto de Productos Lácteos de Asturias (CSIC), Villaviciosa, ES La definición de probiótico más aceptada actualmente es la de “microorganismos vivos que cuando se administran en cantidades adecuadas confieren un beneficio para la salud del hospedador”. El estudio de los probióticos ha avanzado de manera notable durante los últimos años, espoleado por los grandes avances en el conocimiento de la implicación de la microbiota intestinal en el condicionamiento de la salud y la enfermedad. A pesar de que los microorganismos comensales de la microbiota intestinal son frecuentemente fuente de cepas probióticas, éstas no pueden denominarse como tal si no son convenientemente aisladas, caracterizadas y si no se presentan estudios clínicos que avalen sus efectos beneficiosos sobre la salud, así como su seguridad. La evidencia científica disponible a día de hoy sugiere que ciertas especies de microorganismos comensales representan buenos candidatos para identificar nuevos probióticos. Muchos estudios metagenómicos han puesto en evidencia que algunas de estas especies están subrepresentadas en la microbiota intestinal de grupos con distintas situaciones fisiológicas y enfermedades, como la enfermedad inflamatoria intestinal o la obesidad. De hecho, se sospecha que la ausencia de ciertos de estos comensales, como las bacterias productoras de ácido butírico, podría predisponer a padecer estas enfermedades, aunque esta relación causa-efecto es un foco de discusión candente entre la comunidad científica. Algunos de estos microorganismos comensales, de los que sin duda oiremos hablar mucho en el futuro, son las especies Akkermansia muciniphila, Faecalibacterium prausnitzii, Roseburia spp. o Eubacterium hallii. El consenso actual entre las distintas asociaciones internacionales implicadas es considerarlos como probióticos, aunque condicionado a la presentación de cepas bien caracterizadas, estudios de intervención sólidos sobre su eficacia y seguridad, y también estudios de ciencia básica donde se describa el mecanismo de acción preciso a nivel de cepa. Palabras clave: microbiota intestinal, probióticos, comensales, metagenómica, salud. 79 Pósters / Posters P09-16 Syngas as feedstock for polyhydroxyalkanoate production in Rhodospirillum rubrum Olga Revelles1, J.L. García2, M.A. Prieto2 Department of Biosystems Science & Engineerin (D-BSSE), Basel, CH; Centro de Investigaciones Biológicas, CSIC, Madrid, ES, 2 Centro de Investigaciones biológicas, CSIC, Madrid, ES 1 The potential use of municipal solid wastes (MSW) as feedstock for the production of polyhydroxyalkanoates (PHAs) by a process known as syngas fermentation is an attractive bio-economic strategy to reduce urban and agricultural wastes. The anaerobic photosynthetic bacterium Rhodospirillum rubrum, is an organism particularly attractive for the bioconversion of syngas into PHAs. In this work, a quantitative physiological analysis of R. rubrum was carried out by implementing GC-MS and HPLC techniques to unravel the metabolic pathway operating during syngas fermentation that leads to PHA production. Further, detailed investigations of the central carbon metabolites using 13C-labelled substrate showed significant CO2 assimilation (of 40%) into cell material and PHA from syngas carbon fraction. By a combination of quantitative gene expression and enzyme activity analyses, the main role of carboxylases from the central carbon metabolism in CO2 assimilation was shown, where the Calvin-Benson-Bassham cycle (CBB) played a minor role. Finally, the potential of this strain as microbial cell factory for the synthesis of PHA from syngas produced from municipal solid waste is also evaluated. In summary this work provides valuable information to further optimize the fermentation process, as well as, it reveals the strong robustness of this syngas fermentation where the purity of the syngas is not a critical constraint for PHA production. XXXIX Congreso SEBBM warming suggest an increase in the severity and frequency of these adverse climate events. Developing new biotechnological strategies to increase crop productivity is essential to face a climate change scenario and secure food supply to a continuously growing population. Understanding how plants perceive and transduce adverse environmental conditions into biochemical signals is critical to fulfill this objective. Here, we report the identification of RCI5, an Arabidopsis flavine monooxigenase (FMO) whose levels increase in response to abiotic stress and is implicated in the biosynthesis of TMAO, a new molecule not described in plants so far. We show that Arabidopsis has basal levels of TMAO under control conditions and that these levels augment when exposed to low temperature, high salt or water stress. Analyses of stress tolerance in Arabidopsis transgenic plants containing constitutive high levels of TMAO and in wild-type Arabidopsis plants treated with TMAO demonstrate that this molecule is a positive regulator of plant response to different abiotic stress conditions. Our data also indicate that TMAO induces a wide transcriptomic reprograming and a subsequent change in the metabolic profile that accounts for its role in plant tolerance to abiotic stress. Interestingly, we have observed a similar role for TMAO in the tolerance of different crops to abiotic stress. We propose that TMAO is a new plant signalling molecule with a high biotechnological potential that mediates abiotic stress responses. P10. Estructura y función de proteínas / Protein structure and function P10r-1 P09-17 Explorando la existencia de lipid rafts en bacterias Daniel López Centro Nacional de Biotecnología, CSIC, Madrid, ES La existencia de microdominios funcionales de membrana en bacterias, estructural y funcionalmente similares a los conocidos lipid rafts de celular eucariotas es un campo emergente en microbiología que desarrollaré en mi presentación utilizando el patógeno Staphylococcus aureus como modelo bacteriano. Mi presentación delineará tres líneas de trabajo que están actualmente siendo desarrolladas en mi laboratorio. Una línea estructural, para dilucidar la composición y el proceso de ensamblaje de estas plataformas de membrana. Una línea funcional que investiga su significado biológico y, en este caso particular de S. aureus, su implicación en mecanismos celulares de infección. Finalmente presentaré una línea más aplicada, utilizando moléculas capaces de alterar estas plataformas de membrana como terapia antimicrobiana. La existencia de microdominios funcionales de membrana en bacterias implica que los organismos procariotas son mucho más complejos de lo que anteriormente habíamos anticipado. P09-18 An Arabidopsis flavine monooxygenase provides new biotechnological strategies to improve crop tolerance to abiotic stress Rafael Catalá1, Rosa Mª López-Cobollo2, M.A. Berbis1, J. Jiménez-Barbero3, J. Salinas1 1 Centro de Investigaciones Biológicas, CSIC, Madrid, ES, 2Imperial College London, South Kensington Campus, London, UK, 3Centro de Investigaciones Biológicas, CSIC, Madrid, ES; CIC bioGUNE, Bizkaia Technological Park, Derio, ES Drought, salinity and extreme temperatures severely limit crop production accounting for more than 40% of losses in yield. Predictions about global 80 The ribotoxin α-sarcin cleaves the sarcin/ricin loop within late 60S pre-ribosomes Miriam Olombrada1, Cohue Peña1, Olga Rodríguez-Galan2, Purnima Nerurkar1, Martin Altvater1, José G. Gavilanes3, Álvaro Martínez-del-Pozo3, Jesús de La Cruz2, Lucía García-Ortega3, Vikram G. Panse1 1 Institute of Biochemistry, ETH Zurich, Zurich, CH, 2Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/ Universidad de Sevilla, Sevilla, ES, 3Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, Madrid, ES The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin targets the SRL only within mature 60S subunits in vivo remains unresolved. Here, we show that α-sarcin can cleave SRLs within late 60S pre-ribosomes poised for nuclear export as well as final cytoplasmic maturation. In vitro cleavage assays revealed that mature 25S rRNA, but not nucleolar/nuclear 27S pre-rRNA containing 60S pre-ribosomes, are effective substrates of α-sarcin. Conditional expression of α-sarcin in budding yeast is lethal, but does not impede nuclear export and/or the cytoplasmic maturation pathway of 60S pre-ribosomes. Thus, defective SRL containing pre-ribosomes escape nuclear and cytoplasmic proofreading. Polysome analyses revealed that these faulty 60S subunits form 80S initiation complexes, but are impaired in translation elongation. We propose that the functional integrity of the SRL might be assessed only later during translation. P10-2 Halophilic protein adaptation results from synergistic residue-ion interactions in the folded and unfolded states Gabriel Ortega Quintanilla1, Tammo Diercks2, Óscar Millet2 UC Santa Barbara, Santa Barbara, US, 2Structural Biology Unit, CIC bioGUNE, Bilbao, ES 1 Salamanca 2016 The slow but unstoppable progress of evolution has enabled the conquest of virtually any environment on Earth, even those with conditions far from “standard” in which the presence of organisms seems unthinkable. Extremely high salt concentrations exert a huge osmotic stress on living organisms ultimately leading to their death by dissecation. That is why salt is a common food preserver, and also why salines and lakes such as the Dead Sea have been traditionally thought devoted of life. But some organisms – haloarchaea and some bacteria – are capable of surviving in these media at near saturating extracellular salt concentrations. They are the so-called halophiles.How do they do it? They balance the osmotic pressure at both sides of the cell membrane by accumulating large quantities of intramolecular cosolutes. In particular, extreme halophilic organisms accumulate KCl in their cytoplasm up to concentrations of 4M. This is a huge shock for the intracellular machinery, which has undergone severe reengineering along evolution in order to remain native and functional under such harsh conditions. The basis for halo-adaptation relies in a strong evolutionary selection of certain amino acid types. While maintaining the same structure as its mesophilic homologues, the amino acid composition of halophilic proteins is markedly different: acidic residues – especially aspartic acid – and short, polar side chains – threonine – are favored over bulky, hydrophobic residues. This reduction of the overall hydrophobic content is mainly achieved through a drastic decrease in the number of lysines, which have a long, disordered, aliphatic chain. As a result, halophilic proteins are very stable, soluble and functional at high salt concentrations. We have studied the relationship between amino acid composition and halophilic adaptation. We have mimicked in vitro the evolutionary process in order to design proteins with different degrees of haloadaptation, and conducted a comprehensive biophysical characterization thereof under conditions simulating hypersaline environments. The use of atomic-resolution NMR has allowed us to study the structural and dynamic properties of folded and unfolded states of halophilic proteins at the level of individual residues. We have found how the action of two opposite mechanisms confers great stability to halophilic proteins in the presence of high amounts of salt: -The unspecific preferential interactions between highly abundant carboxylates in the surface and K+ cations in solution stabilize the folded state, -Whereas the increase in polar surface – and the concomitant decrease in hydrophobic area – promotes preferential ion exclusion from the surface of the unfolded halophilic proteins, enhancing unfolded state destabilization upon cosolute addition. These two opposite but complementary mechanisms, both dependent on the amino acid composition and the amount of the area exposed, constitute the basis of halophilic adaptation. P10-3 Tumor specific mutations in the N-terminal domain of the Tumor Suppressor ING5 decrease protein stability and cell growth inhibition Georgina Ormaza Hernandez1, Nekane Merino1, Maider Villate1, Ignacio Palmero2, Robert Kypta1, María dM. Vivanco1, Francisco J. Blanco1 1 CIC bioGUNE, Derio, ES, 2IIB (CSIC-UAM), Madrid, ES The INhibitor of Growth (ING) family of tumor suppressors consists of five homologous proteins that regulate the transcriptional state of chromatin by recruiting histone acetyl trasferase and histone deacetylase complexes to chromatin regions with histone H3 trimethylated at K4 (H3K4me3)1. This modification is recognized by the Plant HomeoDomain (PHD) that is present at the C-terminus of the five ING proteins2. ING5 dimerizes through its N-terminal domain, with a symmetric antiparallel coiled-coil structure. The PHD fingers are connected with the dimerization domain by a disordered flexible region that makes them chemically independent and equivalent as they sample a large volume around the dimerization domain. Three point mutations have been described for ING5 in oral squamous carcinoma primary tumors: Q33R, I68V, and, C75R. The three of them are Pósters / Posters located in the N-terminal domain, and point to ING5 as a tumor suppressor in this type of cancer 3. We have found that the N-terminal domains of the three ING5 mutants form dimeric coiled-coils like the wild type but with different stability, as measured by thermal denaturation. While the stability of Q33R mutant is similar to the wild type, mutants I68V and C75R are strongly destabilized, suggesting a disruption of ING5 function. Cell-based assays indicate that the structural alterations have a functional impact. We have found that NIH3T3 cell cultures stably expressing any of the three ING5 mutants display large changes in their cell cycle profile, compared to wild-type, most dramatic in the case of C75R mutant, which is also the most unstable one. References [1] Y. Doyon et al. (2006) Mol Cell 21, 51-64. [2] P.V. Peña et al. (2006) Nature 442, 100-3. [3] B. Cengiz et al. (2010) Int J Cancer 127(9), 2088-2094. P10-4 Structural basis for Gαi activation by a non-receptor protein Mikel García-Marcos1, Alain Ibáñez de Opakua2, Kshitij Sharma1, Nekane Merino2, Lien T. Nguyen1, Maider Villate2, Anthony Leyme1, Arthur Marivin1, Vincent DiGiacomo1, Francisco J. Blanco3 1 Boston University School of Medicine, Boston, US, 2CIC bioGUNE, Derio, ES, 3CIC bioGUNE, Ikerbasque, Bilbao, ES Non-receptor Guanine nucleotide Exchange Factors (GEFs) have emerged as important G protein activators and signaling components in diverse cellular processes. Their involvement in human disease makes them attractive therapeutic targets. Here we used nuclear magnetic resonance (NMR) to study residue-level conformational changes in Gαi3 upon binding of a non receptor GEF named GIV (Gα-interacting, vesicle associated protein a.k.a. Girdin). Changes in many NMR signals of Gαi3 could be measured upon GIV binding in solution. Signal perturbations occurred not only in the predicted binding region (SwII/α3, based on a molecular model) but also in the nucleotide binding pocket and a GPCR binding loop. Based on these observations we carried out an extensive site-directed mutagenesis study from which we conclude that GIV binding on Gαi does not overlap with the GPCR binding region and that it allosterically induces conformational changes in the nucleotide binding pocket. These conformational changes only partially overlap with those observed upon GPCR activation, indicating that GIV and GPCRs utilize markedly different molecular mechanisms to activate G proteins. These results suggest that at least some non-receptor GEFs can be specifically targeted without affecting GPCR-G protein coupling and explain why receptor and non-receptor GEFs have different potency in activating G proteins. P10r-5 Structural and biochemical characterization of Mycobacterium tuberculosis replicative DNA polymerase DnaE1 and its unique PHP-exonuclease Soledad Baños-Mateos, Ulla F. Lang, Sarah Maslen, Mark Skehel, Meindert H. Lamers MRC Laboratory of Molecular Biology, Cambridge, UK High fidelity DNA synthesis is essential for all organisms and depends on a proofreading 3’-5’ exonuclease that is associated with the replicative DNA polymerase. Contrary to humans and the model organism Escherichia coli, the proofreading activity in the major pathogenic bacteria Mycobacterium tuberculosis (Mtb) is not performed by the canonical DEDD exonuclease. Instead, the exonuclease active site in Mtb is located in an intrinsic 81 Pósters / Posters domain of the replicative DNA polymerase DnaE1, the PHP domain. The mechanism of action of the PHP-exonuclease is unknown. Understanding the mechanisms that control fidelity and mutation rates in M. tuberculosis is crucial as point mutations drive antibiotic resistance in Mycobacteria, an issue that is a global health problem. We have solved the crystal structure of the Mtb DnaE1 polymerase and biochemically characterized its PHP-exonuclease. The structure shows the presence of a narrow groove that is likely to bind ssDNA, connecting the polymerase and the exonuclease active sites. This groove potentially leads the DNA into the PHP domain and positions it for removal of a misincorporated nucleotide. Moreover, we have identified a tri-nuclear Zn center in the PHP domain coordinated by nine conserved residues. Interestingly, the PHP active site in DnaE1 shows remarkable similarities to the active site of E. coli endonuclease IV (Endo IV), an enzyme that also cleaves DNA. All these observations allow us to propose a mechanism for DNA hydrolysis by the PHP-exonuclease based on the mechanism of action of Endo IV. Finally, the unique PHP-exonuclease active site of Mtb appears an attractive target for specific inhibition as is not found in eukaryotes, making the likelihood of cross reactivity to human exonucleases small. Therefore, this work provides new insights in understanding replication fidelity in Mtb and will be a valuable tool in the development of novel treatments that target DNA replication in Mycobacterium tuberculosis. P10-6 Recognition of NES cargoes by CRM1 exportin is altered in certain leukemias Igor Arregi1, María Ángeles Urbaneja1, Iraia García-Santisteban2, Marián Alonso-Mariño1, Juan Jesús García-Vallejo3, José Antonio Rodríguez2, Sonia Bañuelos1 1 Biofisika Institute (UPV/EHU, CSIC) and Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Leioa, ES, 2Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Leioa, ES, 3Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, NL Nucleocytoplasmic distribution crucially modulates protein function, and is often deregulated in pathologies. Nuclear export of many proteins relies on the recognition of their nuclear export signal (NES) by the exportin CRM1. Nucleophosmin (NPM), a CRM1 cargo that is normally enriched in nucleoli, is frequently mutated in acute myeloid leukemia (AML) and aberrantly located in the cytoplasm of leukoblasts from those patients. The AML-linked mutation of NPM implies acquisition of a novel NES, responsible for a more efficient nuclear export. A detailed description of the interaction between CRM1 and different AML-linked mutants should better explain their pathogenic behavior. In another hematological malignancy, chronic lymphocytic leukemia (CLL), a recurrent point mutation (E571K) of CRM1 is found in 5% of patients. The E571K mutation results in a charge inversion near the NES binding groove of CRM1. However, the functional consequences of such change remain unexplored. Novel structural insights into the basis of CRM1/NES interaction allow experimentally addressing the binding mechanism. We have analyzed the formation of complexes between wild type and mutant forms of both CRM1 and NES cargoes, trying to understand the effect of different pathogenic mutations of NPM and CRM1. P10-7 Analysis of the Binding of the N-terminus of Neuronal Nitric Oxide Synthase to various cellular targets Carlos Costas Insua, Javier Merino Gracia, María Ángeles Canales Mayordomo, José Ignacio Rodríguez Crespo Universidad Complutense de Madrid, Madrid, ES 82 XXXIX Congreso SEBBM Neuronal Nitric Oxide Synthase (nNOS), unlike the endothelial (eNOS) and inducible isoforms (iNOS), presents an N-terminal extension of approximately 300 amino acids which contains a PDZ domain and a beta hairpin, moieties implicated in protein-protein interactions that regulate its subcellular localization. PDZ domains consist of ~100 amino acids which share a common beta-sandwich fold with 6 beta-strands and 2 alpha-helices. Between the beta2-strand and the alpha2-helix, there is a hydrophobic groove where the C-termini of interacting proteins bind as antiparallel beta strands. This is typically directed by the very last four amino acids of the target protein and has led to a simplistic classification of PDZ domains into class I, II or III relying on the target sequence. The PDZ domain of nNOS was initially described as a class III PDZ domain, but recent data have proposed ligands falling within all classes. Nevertheless, the best characterized protein known to bind to this motif, NOS1AP, presents a class II motif. Hence, the molecular basis of these interactions remains controversial. Using a recombinantly produced PDZ domain, in vitro binding techniques, NMR spectroscopy and confocal microscopy we have shed light in these interactions. We have demonstrated that the PDZ domain of nNOS behaves as a hybrid module, capable of binding multiple class sequences, but an acidic residue is always necessary. Furthermore, we suggest a differential binding mode between class II and class III peptides that would implicate more than the typical last four residues. Our data has led us to the identification and characterization of the interaction with the tight-junction protein Claudin-3. Moreover, we propose a binding regulated through tyrosine phosphorylation, a mechanism not previously reported for any nNOS PDZ ligand. Finally, we have developed an affinity chromatography/ mass spectrometry approach which will allow us to identify novel binding partners not only to the nNOS PDZ domain but also to the nNOS beta-hairpin. P10-8 Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes Ben Murray1, V. Svetlana Antonyuk2, Alberto Marina3, Shelly C. Lu4, José M. Matod5, S. Samar Hasnain3, Adriana L. Rojas3 1 Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, UK; Structural Biology Unit, Center for Cooperative Research in Biosciences, Derio, ES, 2Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, UK, 3Structural Biology Unit, Center for Cooperative Research in Biosciences, Derio, ES, 4Division of Gastroenterology, Cedars-Sinai Medical Center, Los Angeles, US, 5CIC bioGUNE, CIBERehd, Parque Tecnológico Bizkaia, Derio, ES The principal methyl donor of the cell, S-adenosylmethionine (SAMe), is produced by the highly conserved family of methionine adenosyltranferases (MATs) via an ATP-driven process. These enzymes play an important role in the preservation of life, and their dysregulation has been tightly linked to liver and colon cancers. We present crystal structures of human MATα2 containing various bound ligands, providing a “structural movie” of the catalytic steps. High- to atomic-resolution structures reveal the structural elements of the enzyme involved in utilization of the substrates methionine and adenosine and in formation of the product SAMe. MAT enzymes are also able to produce S-adenosylethionine (SAE) from substrate ethionine. Ethionine, an S-ethyl analog of the amino acid methionine, is known to induce steatosis and pancreatitis. We show that SAE occupies the active site in a manner similar to SAMe, confirming that ethionine also uses the same catalytic site to form the product SAE. Salamanca 2016 Pósters / Posters P10-9 P10-11 Repositioning Tolcapone as a potent inhibitor of transthyretin amyloidogenesis and its associated cellular toxicity Architecture of hemidesmosomes Ricardo SantAnna1, Natàlia Reixach2, María Rosario Almeida3, Raul Insa4, Adrián Velázquez-Campoy5, David Reverter1, Nuria Reig4, Salvador Ventura1 1 Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, ES, 2Molecular and Experimental Medicine Department, The Scripps Research Institute, La Jolla, US, 3 Molecular Neurobiology, IBMC- Instituto de Biologia Molecular e Celular, ICBAS- Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, PT, 4SOM-Biotech, Barcelona, ES, 5 Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Unit IQFR-CSIC-BIFI, Universidad de Zaragoza, Zaragoza, ES Background: Transthyretin (TTR) is a plasma homotetrameric protein implicated in fatal systemic amyloidoses. TTR tetramer dissociation precedes pathological TTR aggregation. Native state stabilizers are promising drugs to treat the TTR amyloidoses. Material and Methods: Here, we used biophysical, cell biology and in vivo studies to repurpose Tolcapone, an FDA-approved molecule for Parkinson’s disease, as a very potent TTR aggregation inhibitor. Results and Discussion: Tolcapone binds specifically to TTR in human plasma, stabilizes the native tetramer in vivo in mice and humans and inhibits TTR cytotoxicity. The crystal structures of Tolcapone bound to wild type TTR and to the V122I cardiomyopathy-associated variant explain why this molecule is a better amyloid inhibitor than Tafamidis, so far the only drug in the market to treat the TTR amyloidoses. Conclusions: Overall, Tolcapone, already in clinical trials, is a strong candidate for therapeutic intervention in these diseases, including those occurring in the central nervous system, for which no small molecule approach exist. José Antonio Manso1, Arturo Carabias1, María Gómez-Hernández1, Inés García Rubio2, Arnoud Sonnenberg3, José María de Pereda1 1 Instituto de Biología Molecular y Celular del Cáncer (CSIC-USAL), Salamanca, ES, 2Centro Universitario de la Defensa, Academia Militar General, Zaragoza, ES, 3Netherlands Cancer Institute, Amsterdam, NL Hemidesmosomes (HDs) are junctional complexes that mediate stable attachment of epithelial cells to the basal lamina, linking the extracellular matrix to the cytokeratins. In stratified epithelia HDs are composed of integrin α6β4, the bullous pemphigoid antigen 2 (BPAG2), tetraspanin CD151, and two proteins of the plakin family: plectin and BPAG1e. The integrin α6β4 is a receptor for laminins and a central hub of the HD protein network. The intracellular interactions of α6β4 are mediated by the cytodomain of the β4 subunit. Plectin and BPAG1e bind to β4 via their N-terminal regions and to cytokeratins via their C-terminal domains. Defects in HD-proteins cause several types of the blistering disease epidermolysis bullosa. Our long-term goal is to understand the organization and regulation of HDs. Previously, we had characterized the structure of the plectin-integrin β4 complex. Now we have elucidated the structural basis of the BPAG1e-β4 interaction. Binding occurs between an N-terminal segment of BPAG1e and the third and fourth fibronectin type III domains (FnIII-3,4) of β4. Ser residues in BPAG1e are involved in the binding interface, suggesting that the interaction may be regulated by phosphorylation. Complementing the analysis of hetero-interactions, we have elucidated the structure of dimeric plakins. Similarly to most plakins, plectin has an N-terminal “plakin domain” formed by spectrin repeats that adopt an elongated structure, which is followed by a central coiled-coil rod domain that mediates dimerization. The plakin domains are arranged in parallel in the homo-dimer. Thus, the distance between the β4 binding-sites is restricted, suggesting that the avidity-mediated stabilization of the interaction might be linked to the clustering of α6β4 in the membrane. P10-12 P10-10 Structural changes induced by acidic pH in human Apolipoprotein B-100 César Martín1, José Ángel Fernández-Higero1, Asier Benito-Vicente1, Aitor Etxebarria1, José Carlos G. Milicua2, Ostolaza Helena1, José Luis R. Arrondo1 1 Instituto Biofisika (UPV/EHU, CSIC) y Dpto. Bioquímica y Biología Molecular, UPV/EHU, Bilbao, ES, 2Dpto. Bioquímica y Biología Molecular UPV/EHU, Bilbao, ES Acidification in the endosome causes lipoprotein release by promoting a conformational change in the LDLR allowing its recycling and degradation of LDL. Not with standing conformational changes occurring in the LDLR has expanded considerably, structural changes occurring in LDL particles have not been fully explored yet. The objectives of the present work were to study structural changes occurring in ApoB100 by infrared spectroscopy (IR) and also LDL size and morphology by dynamic light scattering (DLS) and electron microscopy (EM) at both pH 7.4 and 5.0. We found that pH acidification from 7.4 to 5.0, resembling that occurring within endosomal environment, induces a huge reversible structural rearrangement of ApoB100 that is characterized by a reduction of beta-sheet content in favor of alpha-helix structures. These structural changes, which are likely implied in particle release from lipoprotein receptor, also compromise the apoprotein stability what would facilitate LDL degradation. In conclusion, the obtained results reveal a more dynamic picture of the LDL/LDLR dissociation process than previous perceived and provide new structural insights into LDL/LDLR interactions than can occur at endosomal low-pH milieu. Towards a specific cADPR phosphohydrolase by designed mutagenesis of human ADPRibase-Mn María Jesús Costas1, José Canales1, Alicia Cabezas1, Rosa María Pinto1, Joaquim Rui Rodrigues2, João Meireles Ribeiro1, José Carlos Cameselle1 1 Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, ES, 2Escola Superior de Tecnologia e Gestão, Instituto Politécnico de Leiria, Leiria, PT The NAD+ metabolite cyclic ADP-ribose (cADPR) is a messenger for Ca2+ mobilization. cADPR turnover is believed to occur by enzymatic glycohydrolysis to ADP-ribose(1). Mn2+-dependent ADP-ribose/ CDP-alcohol diphosphatase (ADPRibase-Mn) is the only enzyme known to act as cADPR phosphohydrolase (kcat/KM ≈ 4 × 103 M-1s-1) but it is much less efficient (150-, 40- or 6-fold lower kcat/KM) than its major activities on, respectively, ADP-ribose (A), CDP-choline (B) and 2´,3´-cAMP (C). Mutagenesis of the Phe37, Leu196and Cys253 residues of ADPRibase-Mn changes enzyme specificity in relative terms, such that the best substrate of the triple mutant F37A+L196F+C253A is cADPR, with 3-, 1.6- or 9-fold higher efficiencies than on A, B and C. By comparison to the F37A+L196F mutant, C253A is essential for acquisition of preference for cADPR(2). Aiming to improve this preference, we have tested new mutations of Cys253 and Val252. The triple mutant F37A+L196F+C253G, with a smaller side chain at residue 253 (Ala>Gly), showed an efficiency increase with cADPR and decreases with A, B and C. The quadruple mutant F37A+L196F+V252A+C253G, with another side chain made smaller (Val>Ala), showed additional increase (cADPR) and decreases (A and C) of efficiencies. The quadruple ADPRibase-Mn mutant 83 Pósters / Posters displayed kcat/KM ≈ 6 × 104 M-1s-1 on cADPR, which was about 60-, 16or 57-fold higher than on A, B and C, respectively, and represents a 15-fold efficiency increase compared to the wild type. This specificity was demonstrated in incubations with multiple nucleotides. Further enhancing of cADPR phosphohydrolase specificity may be obtained by design of new mutations. Support: Junta de Extremadura and FEDER (GR15143). References [1] Lee, 2012, J Biol Chem 287:31633. [2] Cabezas et al., 2015, PloS ONE 10(2):e0118680. P10-13 The principle of microscopic reversibility and detailed balance for cyclic and acyclic systems Milagros Molina Alarcón1, Francisco García-Sevilla1, María Llanos Amo-Saus1, Manuela García-Moreno1, Francisco García-Cánovas2 1 UCLM, Albacete, ES, 2Universidad de Murcia, Murcia, ES In this contribution, we do not examine neither genesis of the microscopic reversibility and detailed balance nor their equivalence or difference, in its conventional and classical expression, which has been widely studied in the literature(1-3). Considerable confusion and contradictory opinions surround these concepts in cyclic systems. So, in this communication, we suggest a general and global formalism, supported by the well-known Botts and Morales’s cyclic mechanism of enzyme systems. Our analysis attempts to unify the different positions about these concepts, by means of generalised valid interpretations of formal enzyme kinetics. The limitations of the present approach are discussed, and consequences for universally valid interpretations of phenomenological chemical kinetics are suggested. As a particular case of this general formalism results the classical and conventional expression of the microscopic reversibility and detailed balance. Finally, some considerations about the rapid equilibrium and steady-state approaches are made. Although our analysis is focussed to enzyme systems, it could be extended to any cyclic or acyclic system, enzymatic or not. XXXIX Congreso SEBBM ppGpp(1). This molecule takes part in the transition from a regular transcriptional program to a stress-adapted one. It is known that RelA is activated to synthesize (p)ppGpp in complex with ribosomes bearing deacylated-tRNA(2). It is also known that RelA binds to the ribosome stabilizing an unusual distorted form of the deacylated-tRNA in the A-site(3). As stringent response is crucial for bacteria survival in stress condition and virulence, and (p)ppGpp mediated stringent response system is absence in eukaryotes(4), the enzymes involved in this mechanism are new targets for drug development, and structural studies of their activation are needed. We present a cryo-EM structure of RelA in complex with Thermus thermopilus 70S ribosome in presence of the antibiotic relacin with an average resolution around 4 Ǻ. Despite the low occupancy of the factor, the structure reveals RelA bound to the distorted tRNA in the A-site of the ribosome. Besides, the map reveals a unique interaction between RelA and the t-RNA, in which a region of the factor encloses the deacyalted-tRNA. References [1] Potrykus, K. et al., Annu Rev Microbiol (2008) 62: 35-51. [2] Cashel, M. Annu Rev Microbiol. (1975) 29, 301-318. [3] Aguirrezabala, X. et al., EMBO Rep. (2013) 14, 811-816. [4] Hauryliuk,V. et al., Nat. Rev. Microbiol. (2015) 13, 298-309. P10-15 Molecular characterization of a novel catalytic macrodomain from Fusobacterium sp Rubén Zapata Pérez1, Ana Belén Martínez Moñino1, Antonio Ginés García Saura1, Fernando Gil Ortiz2, Álvaro Sánchez Ferrer1 1 University of Murcia. Campus of International Excellence “Mare Nostrum”. Departament of Biochemistry and Molecular Biology. Faculty of Biology, Espinardo, Murcia, ES, 2ALBA Synchrotron Radiation Facility, Cerdanyola del Vallès, Barcelona, ES Miguel Zamora Porras, Xabier Agirrezabala, Mikel Valle CIC bioGUNE, Bilbao, ES Post-translational modification of proteins by lysine acylation or deacylation has profound effects on the physiological function of enzymes. This deacylation, carried out by sirtuins, renders O-acyl-ADP-ribose (OAADPr) as a product. This latter compound is substrate for macrodomains, which are conserved structural domains found in proteins with the ability to bind NAD+ metabolites. Macrodomains substrate (OAADPr) has been implicated as a signalling molecule, modulating several cellular processes. A deep in silico analysis of macrodomain proteins reveals the presence of a putative macrodomain protein from Fusobacterium sp (FspMacroD) similar to the human OARD1. In the present poster, the cloning, expression, purification and activity of the recombinant macrodomain from this microorganism is described. The gene was cloned first in pET28a vector, but low expression was achieved. Thus, a multi-tag cloning approach was carried out, finding that, with a maltose binding protein tag, its soluble expression was high at 25 ºC. An affinity chromatography purification method was developed. The molecular mass of purified FmMacroD was determined by SDS-PAGE (60 kDa). The enzyme activity was analyzed by HPLC, showing that this macrodomain was active towards OAADPr, rendering ADPr, the final product of the reaction. A comparative analysis at sequence level was carried out to shed light on the classification of this enzyme. The stringent response is a central adaptation mechanism to the environment in gram-negative bacteria. This response links nutrient starvation with the transcriptional control of genes. When levels of amino-acid are low in bacteria, the amount of deacylated-tRNAs increases in the cell, and those are detected by stringent factor RelA which converts ATP and GTP/GDP to the signaling molecule (p) This study was partially supported by MINECO-FEDER (BIO2013-45336-R) and by Fundación Séneca (Ayudas Grupos de Excelencia Científica Región de Murcia, project 19893/GERM/15). R.Z.-P. and A.G.G.-S. are holders of a predoctoral research contract (FPU) from University of Murcia, Murcia, Spain. A.B.M.-M. is a holder of research contract associated with the above MINECO-FEDER project. In Memoriam. We want to recognize to our fellow, friend and father in science Ramon Varon who, unexpectedly, passed away when he was working in finishing this contribution. Rest in peace. References [1] Cornish-Bowden, A., Cardenas, M.L. Control of metabolic processes, Plenum Press, New York, 1990. [2] Gorban, A.N., Radulescu, O. (2007). Dynamic and static limitation in multiscale reaction networks, revisited. Physics.Chem-ph arXiv: physics/0703278v3. [3] Purich, D.L. Enzyme kinetics. Catalysis and control: A reference of theory and best-practice methods. Academic Press, London, 2010. P10-14 Structural study of restriction factor RelA interaction with bacterial ribosome 70S 84 Salamanca 2016 P10-16 Structural basis for the substrate channel formation upon dimerization of the hyperthermophilic Pf2001 esterase Nathalia Varejao1, Rafael De Andrade2, Rodrigo Almeida2, Cristiane Ano Bom2, Débora Fogel3, David Reverter1 1 Institut de Biotecnologia i de Biomedicina and Departament de Bioquimica i Biologia Molecular, Cerdanyola del Valles, ES, 2Instituto de Química, Programa de Engenharia Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, BR, 3 Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro, BR Carboxylic ester hydrolyzing enzymes constitute a large group of enzymes spread among all forms of life that catalyze the hydrolysis or synthesis of ester bonds. Among members of esterases, a major biotechnological interest constitutes the group of hyperthermophilic esterases from bacteria and archaea, due to its high intrinsic stability at high temperatures. Here, we describe the structural and functional properties of the Pf2001 esterase from Pyrococcus furiosus. The crystal structure of the Pf2001 esterase has been solved at high resolution in two different conformations: monomer and dimer. Our results reveal important rearrangements in the structure of the “cap” domain between monomer and dimer, by the formation of an extensive intertwined helical interface. Moreover, contacts in the dimer interface are essential for the formation of the hydrophobic substrate channel in the active site and for the substrate selectivity displayed by the Pf2001 esterase for medium-size fatty acids (C7-chains). Mutagenesis and kinetic analysis confirm the major role of the dimer interface in the enzymatic properties of this esterase. Thus, we propose an activation mechanism of the Pf2001 esterase by dimerization, which probably occurs at high temperatures and is necessary for the correct formation of the substrate selectivity channel in the active-site cleft. P10-17 Proteínas quiméricas de Chs3 como modelo para entender el tráfico intracelular de proteínas de la membrana Noelia Sánchez Núñez, Carlos Antón Plágaro, Rosario Valle Vicente, César Roncero Maíllo Universidad de Salamanca, Salamanca, ES La proteína Chs3 de S. cerevisiae se ha revelado como un paradigma en el estudio del tráfico intracelular de proteínas ya que su transporte está regulado a todos los niveles: salida del retículo endoplásmico (RE), tráfico desde el Complejo de Golgi, endocitosis, reciclaje endocítico o degradación en la vacuola. Chs3p es una proteína de membrana politópica con 6 dominios transmembrana que definen 3 dominios citosólicos y uno extracelular, cada uno con funciones específicas en la regulación de su transporte y función. Para abordar el estudio de estas funciones se han construido una serie de proteínas quiméricas derivadas de Chs3p que contienen los diferentes dominios de la proteína fusionada a la GFP. La mayoría de éstas se acumulan en el RE, confirmando la importancia de la estructura terciaria de Chs3 en su plegamiento y salida del RE. En muchos casos se observaba también una tinción citoplásmica, comprobándose que ésta estaba asociada a su degradación en el citosol. La caracterización de estas proteínas en diversos mutantes de S.cerevisiae ha permitido demostrar como su degradación es dependiente en algunos casos de la ruta ERAD (ER-associated protein degradation) y como la interacción de las quimeras con la proteína silvestre puede prevenir parcialmente esta degradación. Excepcionalmente, una de las proteínas mostró una distribución punteada a lo largo de la célula, que no co-localizaba significativamente con marcadores de cis-Golgi, TGN (Trans-Golgi Network) o ERES (ER exit sites), por lo que su localización Pósters / Posters precisa esta todavía por determinar. Los conocimientos obtenidos de estas construcciones podrían ofrecer nuevas ideas en los modelos de plegamiento proteico, extrapolablesa otros eucariotas. P10-18 Aislamiento y caracterización de albúminas 2S: desarrollo de herramientas clínicas para el diagnóstico de la alergia Cristina Bueno Díaz1, Laura Martín-Pedraza1, Sara Abián1, Pablo San Segundo-Acosta1, Juan Carlos López-Rodríguez1, Rodrigo Barderas1, Eva Batanero1, Javier Cuesta-Herranz2, María Teresa Villalba1 1 Departamento de Bioquímica y Biología Molecular, Faculta de Ciencias Químicas, Universidad Complutense de Madrid, Villanueva del Pardillo, ES, 2Departamento de Alergología, Fundación Jiménez Díaz, Madrid, ES Objetivos: Identificación de diez albúminas 2S de semillas y frutos secos, caracterización de su estructura y ensayos inmunológicos con sueros de pacientes alérgicos. Métodos: Las albúminas 2S se aislaron por métodos cromatográficos a partir del extracto proteico. Tras la confirmación de su naturaleza química por espectrometría de masas MALDI-TOF, se realizó su caracterización estructural con métodos electroforéticos (PAGE-SDS mono y bidimensional); la estructura secundaria y su estabilidad térmica fueron analizadas mediante dicroísmo circular. Por último, se llevaron a cabo inmunoensayos para comprobar su capacidad alergénica, empleando sueros de pacientes alérgicos a la mostaza. Resultados: Las proteínas fueron purificadas a partir de los extractos en dos pasos cromatográficos, una cromatografía de penetrabilidad seguida de una de fase reversa por HPLC. Los estudios de espectrometría de masas las identificaron como albúminas 2S y se determinó la secuencia de algunos péptidos. Presentan una estructura secundaria generalmente helicoidal, siendo estables a 85ºC y con intervalos de pI y tamaño muy variados Discusión: Las proteínas purificadas exhiben características similares a otras albúminas 2S previamente descritas. La caracterización estructural reveló su baja masa molecular y pI variados; algunas de ellas presentan la estructura típica de las proteínas de esta familia, con dos cadenas polipeptídicas de diferente tamaño, mientras que otras, como la de la pipa de girasol, la de cacahuete y la de piñón están formadas por una sola cadena. Los estudios de dicroísmo circular pusieron de manifiesto la alta resistencia de estas proteínas al tratamiento térmico, a excepción de la albúmina 2S de pistacho. Conclusiones: Se disponen de 10 albúminas 2S caracterizadas que pueden ser empleadas como herramientas clínicas en Diagnóstico por Componentes Puros (CRD) con la tecnología ADVIA-Centauro, en poblaciones alérgicas españolas, lo que permitirá un diagnóstico más eficaz y un tratamiento más efectivo de las alergias a alimentos vegetales. P10-19 Pulmonary surfactant protein SP-B promotes purinergic-mediated exocytosis of surfactant by type II pneumocytes Marta Martínez-Calle1, Bárbara Olmeda1, Paul Dietl2, Manfred Frick2, Jesús Pérez-Gil1 1 Departamento Bioquímica y Biología Molecular I. Facultad Biología. Universidad Complutense de Madrid, Madrid, ES, 2 Institute of General Physiology. Ulm University, Ulm, DE Pulmonary surfactant is a lipoprotein complex essential to lower surface tension at the respiratory air-liquid interface, stabilizing the lungs against physical forces tending to collapse alveoli. Surfactant complexes are synthesized by alveolar type II cells and stored in specialized secretory organelles, called 85 Pósters / Posters lamellar bodies (LBs). Surfactant is stored in LBs in the form of multilamellar membrane arrays, where surfactant phospholipids and the hydrophobic proteins SP-B and SP-C sustain highly packed and dehydrated states. Stimulation of alveolar type II cells leads to LB exocytosis and surfactant release. Secreted pulmonary surfactant forms a surface active film along the air-liquid interface in the alveoli. Surfactant protein SP-B is crucial to promote formation of the surface active film, and to maintaining their proper structure along the respiratory dynamics. We report a novel activity of SP-B that may suppose an important contribution to the homeostasis of pulmonary surfactant at the alveolar spaces. Membrane-associated SP-B is able to efficiently induce secretion of pulmonary surfactant from alveolar type II cells. The presence in the extracellular medium of lipid-protein complexes containing SP-B activates the P2Y2 purinergic signaling pathway that ultimately triggers exocytosis of lamellar bodies from alveolar type II cells. This implies that SP-B may not only be an essential component for the biophysical function of surfactant but a central element to ensure that a proper density of surfactant complexes is developed at the respiratory surface. P10-20 Hydrophobic pores and lipid transfer: a model for the structure of pulmonary surfactant protein SP-B and the saposin-like proteins Barbara Olmeda1, Begoña García-Alvarez1, Manuel J. Gómez2, Marta Martínez-Calle1, Antonio Cruz1, Jesús Pérez-Gil1 1 Universidad Complutense de Madrid, Madrid, ES, 2Centro de Astrobiología (INTA-CSIC), Madrid, ES Surfactant protein SP-B is essential to facilitate the formation and proper performance of surface active pulmonary surfactant films at the respiratory air-liquid interface, decreasing the work of breathing and avoiding collapse of alveoli at expiration. Despite its physiological importance, neither a structural model nor a molecular mechanism of SP-B is available. SP-B belongs to the saposin-like family of proteins, which share a common structural fold even though they show a broad range of different functions, including colipase, cytolytic and antibiotic activities. In the present work we have purified native SP-B supramolecular assemblies using detergent-solubilized surfactant and atomic force and electron microscopy reveals the presence of ring-shaped particles. This structure of SP-B assemblies agrees with the structure predicted by a theoretical modeling based on the crystallographic structure of saposin B, supporting a model that is consistent with most structure-function features described for SP-B. Docking of rings from neighboring surfactant membranes would lead to formation of SP-B-based hydrophobic tubes, competent to facilitate the rapid flow of surface active lipids both between membranes and between surfactant membranes and the interface. The existence of these SP-B complexes not only sustain the dynamic behavior and mechanical stabilization of the film required to support breathing, but also explain the permeability of native surfactant membranes to both polar and non polar molecules. Besides this, the SP-B oligomeric structure provides a common pattern to sustain the mechanism of action of other members of the saposin family, based on potential oligomerization-triggered activities of these proteins. P10r-21 dUTPasas: la necesidad de lo no esencial J.Rafael Ciges-Tomas1, Suzanne Humphrey2, José R. Penadés2, Alberto Marina1 1 Instituto de Biomedicina de Valencia-CSIC, Valencia, ES, 2Institute of Infection, Immunity and Inflammation. University of Glasgow, Glasgow, UK Las dUTPasas (Duts) son enzimas ubicuas que catalizan la hidrólisis de dUTP generando dUMP y PPi. Anteriormente demostramos que las Duts de fagos de Staphylococcus aureus actúan como proteínas moonlighting, 86 XXXIX Congreso SEBBM participando en la derepresión de islas de patogenicidad (SaPI). Dicha actividad señalizadora requiere de un motivo propio de este grupo de Duts, que denominamos motivo VI, para diferenciarlo de los cinco motivos catalíticos conservados en todas las Duts. Este motivo VI no está conservado a nivel de secuencia entre las Duts de fagos de S. aureus, pero sí a nivel estructural. En base a esta observación hipotetizamos que la actividad moonlighting de las Duts está regida por la adquisición de inserciones específicas, motivos VI, cuyas diferencias en secuencia deben ser responsables de la especificidad por las proteínas diana a las que regula en cada tipo celular. Para demostrar nuestra hipótesis, y generalizar la capacidad señalizadora de las Duts, se analizaron las secuencias disponibles en bases de datos en búsqueda de aquellas que presentasen inserciones propias de especie. En esta búsqueda se observó que las Duts de enterococos presentaban una inserción localizada en una zona característica, aunque varía en tamaño en función de la especie. Esta característica nos ha hecho pensar que las Duts de estos organismos deben presentar actividad señalizadora, y que esta inserción debe jugar un papel importante en esa función. Para confirmar nuestra propuesta llevamos a cabo una aproximación inicialmente estructural, resolviendo la estructura tridmensional de las Duts de Enterococcus faecalis y E. faecium, así como el pariente cercano Lactococcus lactis que carece de dicho motivo VI. Estas estructuras nos permitieron conocer y delimitar la topología del motivo VI que, de forma similar a lo observado en las Duts de S. aureus, presenta una arquitectura estructural similar entre ambos enterococos aunque su secuencia proteica es radicalmente diferente. En base a estos datos estructurales hemos podido diseñar una batería de mutantes deleccionales, así como quimeras entre ambas especies de enterococos, que han sido evaluadas in vitro e in vivo. Los resultados funcionales y estructurales, que serán presentados en la comunicación, apoyan el papel señalizador de las Duts y el papel que juega el motivo VI en esta función. P10-22 EgcA, bases moleculares de la actividad de una D-L endopeptidasa implicada en infección bacteriana Laura Miguel-Romero1, Gadea Rico-Pérez2, Patricia Casino3, Francisco García-del Portillo2, Alberto Marina1 1 Instituto de Biomedicina de Valencia-CSIC, Valencia, ES, 2 Centro Nacional de Biotecnología-CSIC, Madrid, ES, 3 Departamento de Bioquímica, Universitat de València. Estructura de Recerca Interdisciplinar en Biotecnologia i Biomedicina (ERI BIOTECMED), Burjassot, ES En las bacterias, procesos tan importantes como el crecimiento, la división, y la adaptación al medio o infección requieren de cambios constantes de su pared celular, donde el peptidoglicano es su componente principal. En el patógeno bacteriano intracelular Salmonella enterica serovar Typhimurium, la DL-endopeptidasa EcgA (endopeptidase responding to cessation of growth) es una de las enzimas encargadas de remodelar esta pared. EcgA hidroliza el enlace γ-D-glutamil-meso-diaminopimélico (D-Glu-m-Dap) de las cadenas laterales aminoacídicas del peptidoglicano. EcgA se comporta como un factor de virulencia, ya que su expresión esta aumentada en bacteria intracelular durante la infección de células epiteliales y fibroblastos, y es necesaria para producir enfermedad en el modelo animal de ratón. Como otros factores de virulencia de S. Typhimurium ausentes en bacterias no patógenas, la expresión de EcgA está regulada por el sistema de dos componentes PhoQ-PhoP. Para conocer las bases moleculares de la actividad de este enzima y su participación en los procesos de virulencia, abordamos su caracterización estructural. Utilizando técnicas de cristalografía de proteínas hemos resuelto la estructura tridimensional de la proteína salvajeasí como del mutante C350A donde la Cys catalítica ha sido substituida por Ala. El análisis comparativo de estas estructuras entre sí y con otras proteínas de la misma familia nos han dado indicaciones sobre el reconocimiento y especificidad por substrato, mecanismo catalítico y regulación del enzima, así como Salamanca 2016 de su posible mecanismo de regulación. Estas propuestas están siendo evaluadas por diferentes aproximacionesque van desde análisis in silico hasta la generación de variantes y su caracterización funcional y biofísica. Los resultados y su interpretación en el contexto de la actividad del enzima y su participación en la biología del S. Typhimurium serán presentados en la comunicación. Pósters / Posters given concentrations can rescue the stability of EGFP-UROS mutants. All together, this indicates that pharmacological chaperones can be effectively used as a novel therapeutic intervention line against rare diseases, when derived from protein misfolding. P10-25 P10-23 Designer Optical Neurotransmitter Sensors (DOTS) Inmaculada Sánchez Romero, Harald Janovjak Institute of Science and Technology Austria (IST Austria), Viena, AT The foundation of learning lies in synaptic plasticity, the ability of synaptic connections between nerve cells to change in strength. In its most common form, activation of N-methyl-D-aspartate receptors (NMDARs) by chemical neurotransmitters Glutamate, Glycine and or D-Serine plays a critical role in synaptic plasticity. The limited knowledge of neurotransmitter signaling at NMDARs prevents us from completely understanding synaptic plasticity and in turn learning. Our goal is to engineer a suite of Designer Optical neuroTransmitter Sensors (DOTS) that can quantitatively and non-invasively visualize these neurotransmitters. With the exception of Glutamate, no sensors for neurotransmitters exist. Following the previous approaches to develop sensors for Glutamate, we have generated novel sensors for D-Serine and Glycine. These specific and non-invasive optical sensors would provide novel and highly effective means to directly ‘watch’ neurotransmitter signaling during synaptic plasticity in situ and in real-time. P10-24 Pharmacological chaperones as a novel therapeutic approach against congenital erythropoietic porphyria (CEP) Pedro Urquiza Ortiz1, Ana Laín1, Arantza Sanz-Parra1, JM. Blouin2, E. Richard2, Juan M. Falcon-Perez1, Oscar Millet1 1 CIC BioGUNE, Bilbao, ES, 2Biothérapies des Maladies Génétiques et Cancers, Institut National de la Santé et de la Recherche Médicale U1035, Université Victor Segalen, Bordeaux, FR Congenital erythropoietic porphyria (CEP) is a rare genetic disease produced by deleterious mutations in UROS gene. Among the most aggressive mutations, C73R drastically reduces the activity and stability of uroporphyrinogen III synthase enzyme (UROS), the fourth enzyme of the haem biosynthetic pathway and it is present in almost one of third of all the reported CEP cases. As we demonstrated in previous studies, we can restore the catalytic activity UROS by incorporating residues prone to interact with R73. Based on this proof-of-concept now we propose a new therapeutic approach based on the use of pharmacological chaperones to intracellularly modulate and increase the kinetic stability of the enzyme. Ideally, the molecules should be able to upregulate the protein’s homeostasis upon binding the enzyme and stabilizing the folded conformation. Currently, we have screened a library of 2500 compounds and, interestingly, some hit compounds stabilize the hostspot C73R in vitro, as monitored by circular dichroism (CD) and nuclear magnetic resonance (NMR). Additionally, the intracellular activity for a set of hit compounds was monitored through the analysis of EGFP-tagged version of UROS(C73R) mutant in M1 Fibroblast human cells. The results obtained at HC automated fluorescent microscope suggest that the compounds enter into the cells and are able to interact with the protein UROS(C73R). This result has been further confirmed using two deletereous mutations resulting in a loss of kinetic stability (C73R and P248Q) and in a plethora of cells lines (M1, Human Embryonic Kidney 293 cells (HEK 293) and erythroleukemia k562 cells). In all cases, we found that the treatment with the selected molecules at Cloning, purification and characterization of human Nicotinamide N-Methyltransferase Ana Belén Martínez Moñino, Rubén Zapata Pérez, Victoria García Plana, Antonio Ginés García Saura, Álvaro Sánchez Ferrer University of Murcia. Campus of International Excellence Mare Nostrum. Departament of Biochemistry and Molecular Biology. Faculty of Biology, Espinardo, ES Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines, and other analogues using S-adenosyl-L-methionine as donor (EC 2.1.1.1). NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. This work describes the cloning, overexpression, purification as well as the molecular characterization of a soluble human NNMT (hNNMT). We cloned the NNMT gene in pET28a with an N-terminal His-tag and expressed it under several expression conditions. After that, an efficient purification chromatography method was designed with a good enzyme yield. The molecular mass of purified hNNMT was determined by SDS-PAGE (~ 25 kDa), and by gel filtration (~ 25.10 kDa), confirming the monomeric nature of hNNMT. The enzyme was active towards nicotinamide in the presence of S-adenosyl-L-methionine as donor, rendering 1-methylnicotinamide and S-adenosyl-L-homocysteine as its products, when assayed by HPLC. The enzyme was also kinetically characterized with both substrates, and its optimum temperature and pH determined. In addition, a thermal melt assay was developed using SyPro Orange in order to test new modulators.Finally, a phylogenetic analysis of this enzyme and other close related NNMTs found in the databases was also carried out to found conserved blocks. This study was partially supported by MINECO-FEDER (BIO2013-45336-R) and by Fundación Séneca (Ayudas Grupos de Excelencia Científica Región de Murcia, project 19893/GERM/15). R.Z.-P. and A.G.G.-S. are holders of a predoctoral research contract (FPU) from University of Murcia, Murcia, Spain. A.B.M.-M. is a holder of research contract associated with the above MINECO-FEDER project. P10-26 A mechanism for cargo-specific sorting from endosomes Maria Lucas1, David Gershlick2, Ander Vidaurrazaga1, Adriana Rojas1, Juan Bonifacino2, Aitor Hierro1 1 CIC bioGUNE, Derio, ES, 2Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, US Endosomes undergo extensive spatiotemporal rearrangements as proteins and lipids flux through them in a series of fusion and fission events. These controlled changes enable the concentration of cargo for eventual degradation while ensuring the proper recycling of other components. A growing body of studies has now defined multiple recycling pathways from endosomes to the trans-Golgi network (TGN) which differ in their molecular machineries. The recycling process requires specific sets of lipids, coats, adaptors, and accessory proteins that coordinate cargo selection with membrane deformation and its association with the cytoskeleton. Retromer is a multi-protein complex that associates with the cytosolic face of endosomes, where it functions to recycle receptors, transporters, adhesion 87 Pósters / Posters molecules, and other proteins to the trans-Golgi network (TGN) and the plasma membrane, through sorting into tubular-vesicular carriers. Defects in retromer alter the subcellular distribution of its transmembrane cargos and underlie some forms of Alzheimer’s disease and Parkinson’s disease. The retromer complex comprises a VPS26-VPS29-VPS35 heterotrimer that was previously implicated in cargo recognition, and various combinations of sorting nexin (SNX) proteins that contribute to membrane recruitment and formation of recycling tubules. P10-27 A comparative study of assay methods for screening human Poly(ADP-ribose) Polymerase inhibitors Antonio Ginés García Saura, Rubén Zapata Pérez, José Francisco Hidalgo Céspedes, Ana Belén Martínez Moñino, Álvaro Sánchez Ferrer University of Murcia. Campus of International Excellence Mare Nostrum. Departament of Biochemistry and Molecular Biology. Faculty of Biology, Murcia, ES Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR) is a large, structurally complex polymer of repeating ADP-ribose units, and it is biosynthetized from NAD by poly(ADP-ribose) polymerases (PARPs). PARP1 is activated by DNA damage and plays a pivotal role in the repair of DNA strand breaks. The inhibition of PARP1 could have therapeutic interest in treatment of cancer and ischemia. Thus, the developing of high-throughput assays (HTS) to screening libraries of potential inhibitors is of biomedical interest. This work describes the cloning, overexpression and purification of human PARP1 (hPARP1) to be used in a comparative study of HTS screening methods due to the high discrepancy between results found in the bibliography. Two methods, one chemical and another enzymatic were tested. The first based on reaction with acetophenone at high temperature and the second, based on the use of alcohol dehydrogenase/ diaphorase with resazurin. The first chemical method was useful in the NAD+ concentrations range between 0-10000 nM, whereas the enzymatic was useful between 0-100 nM. The enzyme coupled method was simple and with less data dispersion. Therefore, the enzymatic method was tested with two known hPARP1 inhibitors (Rucaparib and Veliparib), finding a good correlation with described values. Finally, the enzymatic method was used to test new inhibitors previously selected by molecular docking, obtaining a new set of hPARP1 inhibitors. This study was partially supported by MINECO-FEDER (BIO2013-45336-R) and by Fundación Séneca (Ayudas Grupos de Excelencia Científica Región de Murcia, project 19893/GERM/15). R.Z.-P. and A.G.G.-S. are holders of a predoctoral research contract (FPU) from University of Murcia, Murcia, Spain. A.B.M.-M. is a holder of research contract associated with the above MINECO-FEDER project. P10-28 Visión sobre los mecanismos de direccionalidad cis/trans en la autofosforilación de histidinas quinasas Cristina Mideros Mora1, Alberto Marina1, Patricia Casino2 Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, ES, 2 Departamento de Bioquímica. Universitat de València. Estructura de Recerca Interdisciplinar en Biotecnologia i Biomedicina (ERI BIOTECMED), Universitat de València, Valencia, ES 1 Las histidinas quinasas (HK) son proteínas sensoras y transductoras de señales que junto con las proteínas efectoras denominadas reguladores de la respuesta (RR), forman los denominados sistemas 88 XXXIX Congreso SEBBM de dos componentes (TCS). Los TCS son el principal mecanismo de señalización en bacterias generando respuestas adaptativas a cambios ambientales; también están presentes en hongos y plantas pero ausentes en mamíferos. Para transducir la señal las HK se autofoforilan utilizando ATP en un residuo conservado de His, un proceso regulado por el estímulo. Después, el grupo fosforilo es transferido a un residuo de Asp en el RR, activando así la transcripción de genes. Los TCS se apagan cuando el RR es desfosforilado, un proceso que se da por autohidrólisis o mediado por la HK. Recientemente se ha demostrado que las HK pueden autofosforilarse en cis o trans, una característica derivada de su naturaleza dimérica. De este modo unas HK pueden autofosforilarse dentro de su propia subunidad, es decir en cis, mientras otras HK se autofosforilan entre subunidades dentro del dímero, es decir en trans. Nuestro grupo ha demostrado que la direccionalidad cis/trans está relacionada con la conexión entre las dos hélices que forman el dominio DHp, donde se encuentra la His fosforilable. Más aún, hipotetizamos que esta diferente conectividad podría tener repercusiones en el reconocimiento entre HK y RR, afectando a las reacciones catalizadas por los TCS. Para profundizar en la relación entre la direccionalidad de la autofosforilación y el resto de reacciones catalizadas por los TCS hemos diseñado y analizado una bateria devariantes deleccionables del dominio DHp en la HK de Thermotoga maritima HK853. Hemos observado que la eliminación de tan solo 2 residuos es capaz de cambiar la direccionalidad de dicha reacción y que dichos cambios afectan la fosfotransferencia y defosforilación de su RR. Finalmente hemos analizado como estos cambios afectan las constantes de unión entre la HK y el RR. Este estudio nos permite obtener una visión de cómo las reacciones catalizadas por los TCS afectan al mecanismo de reconocimiento entre HK y RR. P10r-29 Structural insights of the calcium mediated reorganization of the calmodulin/Kv7.2 channel complex Eider Nuñez Viadero1, Ganeko Bernardo-Seisdedos1, Carolina Gomis-Pérez1, Alessandro Alaimo1, Beatriz Peñalver1, Pilar Areso2, Álvaro Villarroel1 1 Instituto Biofisika (UPV/EHU, CSIC), Leioa, ES, 2Pharmacology, UPV/EHU, Leioa, ES The M-current, which is a key controller of neuronal excitability, is under dynamic control by the phospholipase C cascade, which causes reduction on PIP2 levels and release of Ca2+ from IP3 sensitive intracellular stores. Both branches can be used independently by different G-protein coupled agonists. For instance, whereas M-current inhibition by muscarinic agonists in sympathetic neurons is mediated by PIP2, regulation by bradykinin depends on Ca2+ elevation. While the action of PIP2 in gating is thought to be direct on the channel, Ca2+regulation is thought to be mediated by calmodulin (CaM), which binds to an intracellular site of the channel known as helix A + helix B. The current hypothesis regarding Ca2+ gating posits that CaM wraps around helix B under resting conditions, and, when CaM becomes loaded with Ca2+, it embraces both A+B helices simultaneously, causing a large structural rearrangement. We have examined this issue by monitoring conformational changes triggered by Ca2+ using FRET. Small FRET changes between fluorophores linked to the N- and C-terminus of the same AB domain were observed, which were consistent with minimal differences on the AB module measured by NMR (see communication by Bernardo-Seisdedos). In contrast, Ca2+ did not cause changes on the transfer of energy between fluorophores located at helices A+B (donor) and CaM (acceptor). Our results suggest that the AB module behaves as a rigid body around which CaM accommodates, both loaded with and without Ca2+ Salamanca 2016 P10-30 The Role of RsgA in Ribosomal Maturation Sean Connell, Jorge Pedro López-Alonso, Paola Fucini, David Gill CIC bioGUNE, Derio, ES The bacterial (70S) ribosome consists of 3 RNA molecules and dozens of ribosomal proteins (r-proteins) which assemble together in a complex and highly coordinated process. Although in vitro ribosomes can be assembled by co-incubating their constitutive RNA and protein components, in vivo the assembly process is facilitated by several trans-acting factors called ribosome assembly factors. These factors can serve as checkpoints to monitor the assembly process and/or actively promote rRNA-folding and r-protein/rRNA interactions to shape the folding landscape. Here we present a ~5 Å cryo-EM structure of the ribosome assembly factor, RsgA, bound to the bacterial 30S subunit allowing us to clearly position RsgA on the ribosome and to build a backbone model of RsgA in its ribosome bound state. Most obvious in the reconstruction is that the body and platform domains of the 30S subunit have much higher local resolution than the head. This stems from the fact that RsgA binding to the 30S subunit promotes the release of several r-proteins in the head which increases the flexibility of the rRNA resulting in a loss of order. Notability RsgA releases r-protein S3, a protein whose premature addition leads to inactive 30S subunits. With regards to the structure of RsgA our reconstruction suggests that the ordering of the catalytic GTPase centre of RsgA is dependent on contacts with several distinct rRNA elements (h44, h45, and h24a) where for example residue A790 (h24a) inserts into a pocket in the RsgA structure. Therefore, the maturation checkpoint monitored by RsgA is likely the correct confluence of rRNA helices 44/45/24a and is a prerequisite for activation of RsgA’s GTPase activity which promotes its release from the ribosome. P10-31 Architecture of heteromeric AMPA glutamate receptors Beatriz Herguedas, Javier Garcia-Nafria, Ingo H. Greger MRC Laboratory of Molecular Biology, Cambridge, UK AMPA-type glutamate receptors (AMPARs) are ligand-gated cation channels that mediate fast excitatory transmission and play a role in synaptic plasticity and memory formation. AMPARs predominantly occur as heteromers of the subunits GluA1 to GluA4. So far, AMPAR structures have been limited to GluA2 homomers. Here we report the first structures of AMPAR heterotetramers determined by X-ray crystallography and electron cryo-microscopy. First, we determined the crystal structures of the GluA2/3 and GluA2/4 N-terminal domains (NTD), which constitute the most sequence-diverse half of the receptor and project towards the synaptic cleft. Structures of NTD heterotetramers revealed a novel compact conformation with an alternating arrangement of the four subunits around a central axis. This organization was confirmed by cysteine cross-linking and permitted us to obtain the structure of an intact GluA2/3 heteromer by cryo-EM. Two models at resolutions of 8.25 and 10.3 angstroms were obtained, corresponding to the resting and desensitized states of the receptor. Our data reveal the organizational features of heteromeric AMPARs and provide snapshots of their gating transitions in the absence of ligand. P10r-32 Structural organization and regulation of the guanine nucleotide exchange factor C3G Arturo Carabias del Rey, María Gómez-Hernández, Beatriz Martín-Gracia, Carmen Guerrero, José M. de Pereda Instituto de Biología Molecular y Celular del Cáncer (CSIC-USAL), Salamanca, ES Pósters / Posters C3G is a guanine nucleotide exchange factor (GEF) for some members of the Ras family of GTPases including Rap1 and R-Ras. C3G is involved in the regulation of a wide range of cellular processes such as proliferation, differentiation, cytoskeletal remodelling, transformation, and apoptosis. C3G (120 kDa) is a tripartite protein according to its structural and functional features: (i) The N-terminal part contains an α-helical rich region that binds to the cytoplasmic tail of E-cadherin. (ii) The central segment contains five Pro-rich sequences (P0-P4) that mediate binding to the SH3 domains of at least Crk, p130Cas, Grb2, Hck and c-Abl proteins. Furthermore Tyr 504 in this region is phosphorylated by Src-family kinases during activation of C3G. (iii) The GEF activity of C3G lies in the C-terminal third, which consists of a Ras Exchange Motif (REM) and a catalytic Cdc25H domain. Here we show that C3G adopts a closed conformation stabilized by a head-tail interaction. We have identified some residues involved in this intramolecular interaction. Other GEFs of the Cdc25H family are autoinhibited by analogous head-tail interactions. We are currently analyzing the relationship between the conformational state of C3G and its GEF activity. In this context we are also analyzing the effect of post-transductional modifications on the structural organization of C3G and its GEF catalytic activity. P10-33 Identification of an extranucleolar site of ribosome production in yeast Giulia Moriggi, Sonia G. Gaspar, Blanca Nieto, Mercedes Dosil Centro de Investigación del Cáncer, CSIC/Universidad de Salamanca, Salamanca, ES In budding yeast the nucleolus is a crescent-shaped structure that abuts the nuclear envelope and occupies up to one-third of the nucleus. In mitosis, the nucleolus remains intact and splits just when the rDNA is segregated, at the end of anaphase. Thus, the nucleolar ribosome synthesis machinery persists as a recognizable region throughout mitosis and streams from the mother into the daughter during telophase. We have unveiled that in early mitosis there is a site of ribosome production that is separate from the bulk of the nucleolar material. We found that, during a short time in metaphase, several ribosome-maturation proteins are present both in the nucleolus and at a punctate body located in the vicinity of the nuclear envelope in the emerging daughter cell. Such body is well-separated from the bulk of the rDNA, which is mostly located in the mother cell, and also from the daughter spindle pole body. It is a body that contains rDNA-binding proteins, pre-rRNAs and ribosome maturation factors characteristic of nucleolar and nucleoplasmic pre-ribosomes, but is devoid of late maturation factors. The focal accumulation of pre-export 40S subunits at this discrete site is being used as a tool to study when and how different factors are incorporated into the 40S subunit synthesis pathway. P10-34 New insights into ribosome formation in human cells Blanca Nieto, Giulia Moriggi, Sonia G. Gaspar, Xosé R. Bustelo, Mercedes Dosil Centro de Investigación del Cáncer, CSIC/Universidad de Salamanca, Salamanca, ES The nucleolus is a highly structured subnuclear compartment that is formed around actively-transcribed ribosomal RNA gene repeats. Together with pre-rRNA synthesis, the nucleolus houses the other major processes involved in ribosome formation: the processing of pre-rRNAs and the assembly of ribosomal proteins. Studies in yeast have yielded most of the current knowledge on the pathway. More than 200 proteins (trans-acting factors) are known to be essential for specific steps of ribosomal subunit maturation. The majority of those factors have been found and characterized 89 Pósters / Posters in yeast. The functions of the putative human homologs and of other ribosome biogenesis proteins present only in humans remain largely unexplored. In recent years, a renewed interest for ribosome synthesis in humans has emerged by some efforts to find new cancer treatments thorough inhibition of ribosome production, and by the discovery that several rare human diseases, the so-called ribosomopathies, are caused by defects in ribosome maturation. Progress in the field is, however, being hampered by the lack of success in applying to human cells the techniques successfully used in yeast. In our laboratory we have optimized a series of procedures to study pre-ribosomal complexes in human cells. We are using a combination of microscopy studies, sucrose gradient sedimentation analysis and size-based separation experiments, to study normally-growing cells and cells blocked at specific steps of ribosome maturation. Here we will show our findings on the functions of three trans-acting factors that are essential for nucleoplasmic maturation and nuclear export of 40S subunits in humans. P10-35 Mutations in KCNQ2 causing early infantile epileptic encephalopathy alter Kv7.2 channel PIP2 sensitivity Carolina Gomis-Pérez1, A. Marcé-Grau2, C. Malo1, E. Cuenca-León2, A. Felipe-Rucián2, M. Raspall-Chaure2, A. Macaya2, P. Areso1, A. Villaroel1 1 Instituto de Biofisika (CSIC UPV/EHU), Leioa, ES, 2Hospital Universitari Vall d’Hebron, Barcelona, ES Mutations in the KCNQ2 gene lead to several neonatal onset seizure disorders including an epileptic encephalopathy, with adverse neurodevelopmental outcome. We have found the latter in four patients, carrying de novo (E130K, W270R and G281R) or maternally inherited(L246F) KCNQ2 mutations . Here we describe the functional consequences in cells expressing these channels, alone or in combination with the partner subunit Kv7.3 to mimic the allelic balance found in humans. All mutations exerted a dominant negative effect in homomers, whereas Kv7.3 rescued the function of mutants to different extent. The mutations caused a reduction on current density with no major changes on voltage-dependency or gating kinetics. The use of a voltage-dependent phosphatase revealed that the sensitivity to the essential PIP2 phospholipid is affected in all four mutations to varying degree. Thus, current density and PIP2 sensitivity may contribute to the pathogenic mechanism. XXXIX Congreso SEBBM hCPOΔC in complex with Nerita versicolor carboxypeptidase inhibitor (NvCI) was solved by X-ray crystallography at 1.85 Å resolution. The asymmetric unit of the crystal reveals 2 identical polypeptide chains of the enzyme (but the biological unit is a monomer, based on gel filtration experiments) bound asymmetrically to 1 molecule of NvCI. Comparing the active and inactive forms of the enzyme, we were able to study in detail the structural features of its catalytic mechanism and the arrangement of the active site in both situations. As expected, we can observe the movement of the side chain of Tyr248 of almost 180° from the “up” to the “down” position upon binding of NvCI. Theoverall structure of hCPO displays the classic α/β hydrolase fold typical of MCPs and presents all important residues within the active site involved in substrate binding and catalysis. Remarkably, the key residue responsible of the substrate specificity of MCPs (Ile255 in hCPA1, Ile251 in hCPA2 and Asp253 in hCPB) is replaced in hCPO by an arginine residue (Arg275), which explains the acidic substrate preference of this enzyme unlike other known human digestive MCPs. Finally, inhibition constants (Ki) were calculated for complexes of hCPOΔC andvarious proteinaceous tight-binding inhibitors of M14A subfamily of MCPs. P10r-37 Structural insides of Calmodulin/Kv7.2 channel complex: the calcium effect Ganeko Bernardo-Seisdedos1, Carolina Gomis-Pérez1, Alessandro Alaimo1, Araitz Alberdi1, Eider Nuñez-Viadero1, Covadonga Malo1, Oscar Millet2, Álvaro Villarroel1 1 Instituto Biofisika (CSIC, UPV/EHU), Leioa, ES, 2CIC-bioGUNE, Derio, ES Calmodulin (CaM), a bi-partite protein, binding to the A-B module, a bi-partite target, underlie multiple mechanisms governing the function of Kv7.2 subunits, which are the main component of the non-inactivating K+ M-current, a key controller of neuronal excitability. Simultaneous binding to helix A and B is crucial for trafficking to the plasma membrane, and mediates Ca2+ inhibition. We have resolved [CaM]/[AB-Kv7.2] complex by NMR and characterized the influence of calcium on the structure.The result suggest that the complex structure only suffers minor conformational changes caused by Ca2+ binding. However, the possible mechanism by which the calcium signaling is mediated in the Kv7.2 channel will be discussed. P10-38 P10-36 Enzymatic and structural characterization of human carboxypeptidase O, a novel digestive metallocarboxypeptidase with acidic C-terminal specificity María del Carmen García Guerrero1, Javier García Pardo1, Roberto Fernández Álvarez1, Francesc Xavier Avilés Puigvert1, Peter Lyons2, David Reverter Cendrós1, Julia Lorenzo Rivera1 1 Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular. Universitat Autònoma de Barcelona, Bellaterra, ES, 2Department of Biology. Andrews University, Berrien Springs, US Human carboxypeptidase O (hCPO) belongs to the M14A subfamily of metallocarboxypeptidases (MCPs) and is an N-glycosylated, membrane-bound enzyme that participates in the hydrolysis of C-terminal acidic residues from dietary proteins and peptides, thus complementing the digestive actions of the well-known pancreatic MCPs hCPA1, hCPA2 and hCPB. In this study we report the enzymatic and structural characterization of a recombinant soluble form of hCPO (hCPOΔC). The 3D-structure of 90 Biophysical studies on the interaction among scFv-h3D6, Aβ and apolipoproteins E and J Laia Montoliu-Gaya, Sandra Villegas Protein Folding and Stability Group. Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, ES The antibody fragment scFv-h3D6 has been shown to be effective at the behavioral, cellular and molecular levels in the treatment of the triple-transgenic 3xTg-AD mouse model of Alzheimer’s disease (AD). Previous studies demonstrated that its ability to aggregate into a worm-like fibril pathway make this molecule able to withdraw Aβ oligomers from the amyloid pathway and, in this way, to prevent its toxicity. Since the important role apolipoproteins E (apoE) and J (apoJ) appear to play in Aβ clearance and also the fact that the levels of both apolipoproteins were restored in the 3xTg-mice-AD after scFv-h3D6 treatment, we aimed to study the interactions among scFv-h3D6, Aβ and apolipoproteins E and J. To do so, we have used mimetic peptides (MP) composed by essential structures within these apolipoproteins for binding to other molecules. We have performed biophysical characterization of these proteins/peptides alone or in Salamanca 2016 combination with each other. Our studies show that apoE-MP interacts with Aβ but no with scFv-h3D6, and when the three are combined worm-like fibrils cannot be formed. In the apoJ-MP case, it does not interact with Aβ or neither scFv-h3D6, but when the three molecules are together there is an interaction and the formation of worm-like fibrils is potentiated. More studies are necessary, but with these results we are getting closer to understand how the scFv-h3D6 is able to reverse AD symptoms in vivo and to set the proper treatment with such a promising therapy. Funding: Instituto de Salud Carlos III [FIS-PI113-01330], Generalitat de Catalunya [SGR-GRC-2014-00885], & Generalitat de Catalunya + FEDER (50%) [2014-PROD00032]. PIF-UAB student grant (LMG). P10r-39 From single-molecule protein unfolding to protein degradation by the proteasome Alejandra Aguado, Pablo Martín, David Fernández, David Rodríguez-Larrea Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco UPV/EHU and Instituto Biofisika (CSIC, UPV/EHU), Leioa, ES Biological nanopores are frequently found within many biological structures involved in protein folding/unfolding. In the case of the proteasome, protein degradation requires unfolding and translocation of protein substrates through a narrow pore into the internal catalytic chamber. How protein stability relates to protein degradation remains an unsolved question. Single molecule approaches using protein nanopores with dimensions comparable to the size of biological molecules have the potential to provide insight into the underlying physics of the process. Here we measure the kinetics and energy consumption of the bacterial proteasome ClpXP during degradation of a battery of mutants of SsrA-tagged thioredoxin. Single-molecule measurements of protein unfolding during translocation through the α-hemolysin pore showed excellent correlation with protein degradation. The thermodynamic stability as measured with differential scanning calorimetry and the kinetic stability obtained with urea induced unfolding did not show this degree of correlation. Our results suggest that the unfolding observed during nanopore one-end pulling efficiently captures the denaturation mechanism as it occurs during protein degradation. P10-40 The spindle-stabilizing function of Cdc14 is required to promote recombinational DNA repair María Teresa Villoria López, Facundo Ramos Ochoa, Encarnación Dueñas, Andrés Clemente Blanco Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC), Salamanca, ES Eukaryotic cells are constantly threatened by innumerable sources of genotoxic stresses that cause DNA damage. In order to maintain genome integrity, cells have developed a coordinated signaling network known as the DNA Damage Response (DDR). While numerous kinases have been thoroughly studied during the activation of the DDR, the role of protein phosphatases remains elusive. Previous data coming from our group have revealed the importance of the phosphatase Cdc14 in promoting recombinational DNA repair. However, how this phosphatase exerts its molecular function in the DDR is still unknown. Here we show that a DSB (double strand break) induced by the expression of the HO endonuclease is actively recruited to one of the SPBs (Spindle Pole Bodies). Microtubules destabilization by nocodazole treatment during the induction of the DNA break disrupts SPB-DSB interaction and Pósters / Posters impairs HR (homologous recombination), indicating that SPB integrity and SPB-DSB binding are essential features of the DNA repair process. Curiously, inactivation of Cdc14 during the induction of the DNA lesion causes continuous misalignment of the metaphase spindle, increases oscillatory SPBs movements, and impairs DSB-SPB tethering. The DSB-SPB interaction stimulated by Cdc14 requires the N-terminus domain of the SPB protein Mps3. Overall, this suggests a role of Cdc14 in DNA repair by promoting spindle stability. Supporting this hypothesis, Cdc14 is released to the nucleoplasm upon HO expression and a distinct focus of the phosphatase overlaps with the DSB at the SPBs. In a screen looking for Cdc14 substrates at the SPBs after the induction of a DNA break, we identified Spc110, the intranuclear receptor for the g-tubulin complex. Loss of Cdk-dependent Spc110 phosphorylation during DSB induction causes the same phenotypes as cdc14-1 mutants. Together, our results point to the function of Cdc14 in DNA repair by promoting SPB stabilization and SPB-DSB interaction, and suggest that the relocation of damage sites to the SPBs plays an important role in a naturally occurring repair process that minimizes genome instability. P10-41 Cdc14 is released from the nucleolus under DNA damage and is required for DNA repair by homologous recombination Facundo Nehuén Ramos Ochoa1, María Teresa Villoria López2, Encarnación Dueñas2, Andrés Clemente-Blanco2 1 Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC), Salamanca, ES, 2Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC), Salamanca, ES Endogenous metabolic products, such as reactive oxygen species, and exogenous physical and chemical genotoxic stress, constantly assault the genetic material in the cell. It has been estimated that there are about 105 lesions per cell per day in humans. In response to such a high levels of DNA damage, cells have developed a coordinated signalling network known as the DDR (DNA damage response), which coordinates cell cycle progression with DNA repair. When this system fails or the rate of DNA damage exceeds the capacity of the pathway to repair them, the accumulation of errors can overwhelm the cell and result in early senescence, apoptosis, or cancer. Recently, multiple lines of evidences have suggested a complex role for several kinases in the regulation of the DDR (including the CDK), but little is known about the phosphatases that revert the effects of these kinases. The serine/threonine phosphatase Cdc14 was firstly identify in S. cerevisiae as an essential cell cycle phosphatase required for Cdk inactivation. One special feature of this phosphatase is its predisposition to dephosphorylate targets of the Cdk, therefore is reasonable to think that Cdc14 could be a key candidate to counterbalance the effect imposed by the Cdk in the DDR. In favour of this hypothesis, Cdc14 is required for cell viability under different DNA damage conditions. To determine the role of the phosphatase in the DDR, we used two different recombinational repair pathways: SDSA (Synthensis dependent strand annealing) and dHJ (double Holliday junction repair). We observed that cells lacking Cdc14 activity presented defects in DNA repair in both SDSA and dHJ. Supporting the role of the phosphatase in DNA repair, we observed that Cdc14 was released from the nucleolus after induction of a single DSB or treatment with phleomycin. Finally, by using mass spectrometry in a screen to identify targets of the phosphatase exclusively in DNA damage, we have detected several targets directly implicated in DNA damage repair. Altogether, we have placed Cdc14 at the DNA damage response context by collaborating in the repair throughout the modulation of the homologous recombination repair pathway, and providing new evidences about the role of this phosphatase in the repair of a DNA lesion. 91 Pósters / Posters P11. Genómica y proteómica / Genomics and proteomics P11-1 Estudio molecular y genómico de la contribución de WHSC1 a la patología del Síndrome de Wolf-Hirschhorn en pacientes y en modelos animales César Cobaleda Hernández1, María Luisa Martínez-Frías2, Sergi Beltran3, Elena Campos-Sánchez1 1 Centro de Biologia Molecular Severo Ochoa, Madrid, ES, 2 Centro de Investigación sobre Anomalías Congénitas (CIAC), Estudio Colaborativo Español de Malformaciones Congénitas (ECEMC), Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, ES, 3Centro Nacional de Análisis Genómico (CNAG), Barcelona, ES El Síndrome de Wolf-Hirschhorn (WHS) es una enfermedad poco frecuente causada por la pérdida de material genético en hemizigosis en el cromosoma 4(p). Los pacientes presentan numerosos problemas (malformaciones craneofaciales, discapacidad intelectual, epilepsia, retraso en el crecimiento, etc.) de bases moleculares aún poco claras. Una importante causa de mortalidad en el WHS son las infecciones, causadas por deficiencias del sistema inmune. Uno de los genes delecionados en 4p es el Wolf-Hirschhorn-Syndrome-Candidate-1(WHSC1), un regulador epigenético que también participa en reparación del daño al DNA y cuya sobreexpresión o activación también está implicada en mieloma múltiple y en leucemias linfoblásticas agudas infantiles. En la actualidad estamos estudiando el exoma completo de una muestra de pacientes con WHS, con el fin de determinar si existe acumulación de daño debida a la pérdida de WHSC1 en 4p, lo que podría hacer que los pacientes fuesen más susceptibles a desarrollar tumores con la edad. Además, estamos caracterizando, mediante citometría de flujo y mRNA-seq, el desarrollo hematopoyético y la función inmune en modelos de ratón de pérdida y ganancia de función de Whsc1. De esta manera, combinando la potencia del estudio mediante secuenciación masiva de muestras de pacientes humanos y de modelos animales avanzados, esperamos poder identificar los mecanismos moleculares subyacentes a las alteraciones inmunes y de otros tipos descritas en los pacientes. Esto permitiría, entre otras cosas, mejorar la calidad de la asistencia médica a los pacientes de WHS y conocer su susceptibilidad al daño genético. P11-2 Heterogeneous susceptibility and evolution to breast cancer: analysis using a systems biology approach Jesús Pérez Losada Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC), Universidad de Salamanca/CSIC. Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, ES An essential question in cancer is why patients with the same disease have different clinical outcome. Progress toward a more personalized medicine in cancer patients requires new strategies to analyze underlying factors determining heterogeneous clinical evolution. We integrated genetic, transcriptomic, cell signaling and metabolic profiles to predict clinical outcomes in a population of mice with variable susceptibility to breast cancer. Tumors originated in the heterogeneous population of mice showed similarities with human breast cancer at those different levels highlighting the importance of studies in 92 XXXIX Congreso SEBBM heterogeneous populations of mice to extrapolate additional knowledge to the human population. Consequently, a common gene expression signature between human and mouse ERBB2 positive breast cancer predicted evolution in both species. The global architecture of the studied signaling pathways was similar in breast tumors and livers, and defined both tumor pathophenotypes and mouse clusters of prognosis. Mice with intrinsically high levels of AKT and ERK pathways were more reluctant to develop tumors. Levels of pAKT1(S473) modified tumor dissemination capability. We identified a metabolic profile before the onset of the disease that anticipated breast cancer evolution. We identified the Quantitative Trait Loci associated with the variability at those different levels. With this approach, we generated prediction models that better define the heterogeneous behavior of the disease among individuals considering several molecular layers and tumor traits simultaneously. Here, we demonstrate by a Systems Biology approach an effective strategy to identify the heterogeneous behavior of the ERBB2 positive breast cancer among individuals. This strategy would help to unravel cancer variability, an essential issue to develop more individualized medical approaches. P11-3 Identification of a DNA methylation signature in liquid biopsy for early non-small cell lung cancer (NSCLC) diagnosis Juan Sandoval del Amor1, Ángel Díaz Lagares2, Jesús Mendez-González3, David Hervás4, María José Pajares5, María Saigi3, Diana García4, Ana Belén Crujeiras6, Ruben Pío5, Luis M. Montuenga5, Javier J. Zulueta5, Ernest Nadal3, Antoni Rosell3, Manel Esteller3 1 IISLaFe, Valencia, ES, 2University Hospital of Santiago de Compostela (IDIS-CHUS), Santiago de Compostela, ES, 3 IDIBELL, Barcelona, ES, 4Medical Research Institute La Fe, Valencia, ES, 5University of Navarra, Pamplona, ES, 6Health Research Institute of Santiago (IDIS-CHUS) and CIBERobn, Santiago de Compostela, ES Lung cancer is the leading cause worldwide, mainly due to late diagnosis. Patient outcome is closely linked to tumor stage at diagnosis and unfortunately, most lung cancer patients are diagnosed at late stages when a curative treatment is no longer possible.The aim of this work was to identify a panel of epigenetic biomarkers for improving early diagnosis of lung cancer patients using minimally and non-invasive biological fluids. DNA hypermethylated biomarkers were identified performing a Genome-wide DNA methylation analysis (Infinium 450K array) in non-small cell lung cancer (NSCLC) primary tumors from two different public databases: CURELUNG FP7 Consortium and The Cancer Genome Atlas (TCGA). DNA methylation levels of selected candidates were analyzed by pyrosequencing in non- or minimally invasive samples from three independent cohorts of stage I NSCLC patients and non-tumoral controls: bronchoalveolar aspirates, bronchoalveolar lavages and sputum. Combined Receiver Operating Characteristic (ROC) curve was obtained to evaluate the diagnostic utility of the epigenetic signature. A nomogram was used to represent the final predictive model for lung cancer disgnostic. The herein identified DNA methylation signature could improve, in combination with current diagnostic protocols, the early diagnosis and outcome of NSCLC patients. The high diagnostic accuracy of this signature obtained in liquid biopsy offers a minimally invasive and easy accessible tool for early lung cancer diagnosis. A nomogram based on the results of this model is proposed as a predictive tool for clinical diagnostic use. Salamanca 2016 P11r-4 Histology and transcriptional responses of biotransformation and antioxidant enzymes in the liver and testis of Mus spretus mice exposed to p’,p-DDE Noelia Morales Prieto1, Isabel Lourdes Pacheco2, Sara López Bellón1, Ana Luna Alcántara1, José Pérez2, Carmen Pueyo1, Nieves Abril1 1 Department of Biochemistry and Molecular Biology. University of Córdoba, Córdoba, ES, 2Department of Anatomy and Comparative Pathology. University of Córdoba, Córdoba, ES Prohibited in the early 1970’s, the pesticide DDT is still being used to control insects that carry diseases such as malaria and the Zica virus. Thus, human and animal populations worldwide are exposed to DDT, directly or by consuming contaminated food. DDT metabolism generates p’,p-DDE (1,1-dichloro-2,2-bis(p-chlorophenil)-ethilene), a byproduct described as an androgen receptor antagonist that can produce male genital abnormalities and associated with oxidative stress. The molecular mechanisms and pathological processes underlying the p’, p-DDE toxicity are still poorly understood. We have analyzed the impact of p’,p-DDE on Mus spretus hepatic and reproductive health at histological and transcriptional levels. Mus spretus, the Algerian mouse, diverged just ~3 million years ago from the lab mouse Mus musculus and is emerging as particularly appropriate for studying multigenic diseases, including inflammation and cancer. Histomorphological analysis of testicular sections revealed normal structure of seminiferous tubule with well-organized arrangement of germ cells and somatic cells in control group. The testicular sections of p’,p-DDE exposed mice were, in contrast, discolored and, on histological examination, showed tubular damage. In the testes of the exposed mice the seminiferous tubules were significantly (P<0.001) higher and showed a reduction >30% in the area occupied by germinal cells and spermatozoids. In the liver, p’,p-DDE caused cell swelling with small clear vacuoles in the cytoplasm, probably caused by failure of the Na+/K+ pump. p’,p-DDE differentially altered the testicular and hepatic mRNA abundance profiles ofgenes involved in biotransformation pathways (cytochromes and glutathione S-transferases) and the antioxidant defense (peroxiredoxins, glutathione peroxidases, catalase, superoxide dismutases), but exerted minimal effects on apoptotic genes expression. This work will contribute to uncover the involvement of some detoxifying and antioxidant enzymes on hepatotoxicity and the impairment of male reproductive health by p’,p-DDE. P11-5 Efecto de una dieta funcional enriquecida con probióticos y extracto de dátil sobre la expresión proteica de la dorada (Sparus aurata) María José Prieto Álamo1, Ana Belén Plata Gómez1, Esther Donoso Contreras1, María Ángeles Esteban Abad2, Juan Jurado Carpio1 1 Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Cordoba, ES, 2Departamento de Biología Celular e Histología, Universidad de Murcia, Murcia, ES La acuicultura intensiva es una actividad productiva cuyo desarrollo está condicionado por diversas patologías infecciosas. La prohibición en la Unión Europea del uso de muchos de los fármacos veterinarios clásicos exige la búsqueda de alternativas naturales efectivas y seguras. En este sentido, los probióticos y los extractos vegetales pueden desempeñar un importante papel. Se ha investigado el efecto a nivel proteómico de una dieta suplementada con los probióticos Shewanella putrefaciens Pdp11 y Bacillus sp en combinación con un extracto de dátil de conocidas propiedades antioxidantes sobre especímenes de dorada (Sparus aurata) cultivados tanto en condiciones normales como en presencia de una infección experimental por Photobacterium damselae sbsp. piscicida, uno de los patógenos que diezman las explotaciones de esta importante especie Pósters / Posters para la acuicultura española. El análisis de la expresión proteica diferencial mediante 2-DE ha puesto de manifiesto cambios significativos en 18 proteínas. Algunos son efecto de la dieta mientras que otros son provocados por la infección. En un análisis preliminar de los cambios de expresión se han identificado actinas, conocidos sensores redox fisiológicos, lo que podría relacionarse con el estrés oxidativo inducido por la infección y/o con el papel antioxidante atribuido a la dieta funcional. Así, se ha comparado el grado de oxidación reversible de los tioles de las proteínas sin observar, globalmente, cambios cuantitativos significativos, aunque los patrones de bandas indican oxidación diferencial en proteínas específicas. También se ha investigado la actividad de enzimas relacionadas con el estado redox celular y las defensas antioxidantes: GOR, G6PDH, catalasa y SOD. Financiación: AGL2011-30381-CO3-03. P11-6 Breast cancer phenotypic variability is affected by the biological age María del Mar Sáez Freire1, Adrián Blanco Gómez2, Sonia Castillo Lluva3, Chris Lauber4, María Carmen Patino Alonso5, Purificación Galindo Villardón5, Carmen Martín Seisdedos6, María Isidoro García6, María Eugenia Muñoz Bermejo7, Julie Milena Galvis Jiménez8, Trent Northen9, Andrés Castellanos Martín10, Lars Kaderali11, Jesús Pérez Losada2 1 Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC). Universidad de Salamanca/CSIC; Instituto de Investigación Biosanitaria de Salamanca (IBSAL) y Departamento de Fisiología y Farmacología. Universidad de Salamanca, Salamanca, ES, 2 Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC). Universidad de Salamanca/CSIC; Instituto de Investigación Biosanitaria de Salamanca (IBSAL), Salamanca, ES, 3Departamento de Bioquímica y Biología Molecular I. Facultad de Biología. Universidad Complutense de Madrid, Salamanca, ES, 4Instituto de Biometría y Estadística Médica. Facultad de Medicina. Universidad Tecnológica de Dresden, Dresden, DE, 5Departamento de Estadística. Universidad de Salamanca, Salamanca, ES, 6 Servicio de Bioquímica Clínica. Hospital Universitario de Salamanca, Salamanca, ES, 7Departamento de Fisiología y Farmacología. Universidad de Salamanca, Salamanca, ES, 8 Instituto de Biología Molecular y Celular del Cáncer (IBMCC-CIC). Universidad de Salamanca/CSIC; Instituto de Investigación Biosanitaria de Salamanca (IBSAL), Salamanca, ES; Instituto Nacional de Cancerología de Colombia, Colombia, CO, 9Department of Bioenergy/GTL & Structural Biology, Life Sciences Division, Lawrence, Berkeley, California, US, 10Institute for Research in Biomedicine (IRB), Barcelona, ES, 11Institute for Bioinformatics. University Medicine Greifswald, Greifswald, DE, Breast cancer incidence rates considerably increase with age and young age at diagnosis correlates with worse prognosis due to a more aggressive breast cancer behaviour.(1,2) Biological age estimates the functional status of individuals comparing to other individuals of the same chronological age. (3) We defined the biological age integrating phenotypes of oxidative stress, and calculated Δ biological age as the difference between chronological and predicted (biological) age. Mice with predicted ages older than chronological age, were considered biologically older; and mice with predicted ages younger than chronological age, were considered biologically younger.(4) Our main goals were (i) define biological age using processes that are common to cancer and ageing, such as the oxidative stress, and (ii) analyze breast cancer phenotypic variability according to the biological age. We generated a cohort of mice with different susceptibility and evolution to ERBB2-induced breast cancer, using a backcross strategy, and dissected the disease into different pathophenotypes. We also measured intermediate phenotypes of oxidative stress. Linkage analysis was carried out to identify quantitative trait loci (QTL) associated with these phenotypes, and used multivariate 93 Pósters / Posters models to define biological age. We identified that biologically older mice developed more aggressive disease than biologically younger mice. We also identified QTL simultaneously associated with Δ biological age and tumor pathophenotypes. We identified several genetic and molecular markers that define biological age and observed that ERBB2 breast cancer phenotypic variability is affected by the biological age. References [1] McPherson K, et al., Bmj. 2000; 321(7261):624-8. [2] Anders CK, et al., Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2008; 26(20):3324-30. [3] Karasik D, et al., Biological sciences and medical sciences. 2004; 59(3):218-26. [4] Cho IH, et al., Mechanisms of ageing and development. 2010; 131(2):69-78. (#) (*) Equally contribution as second authors. Equally contribution as senior authors. P11r-7 Connection between PKP1 and oncogenic pathways involving PI3K, NF-κB and H-Ras detected in lung squamous cell carcinoma cell lines by gene expression microarrays Álvaro Andrades1, Joel Martín-Padrón2, Laura Boyero3, Mª Esther Fárez-Vidal4, Pedro P. Medina1 1 Centre for Genomics and Oncological Research (GENYO); Department of Biochemistry and Molecular Biology I, School of Sciences, Granada University, Granada, ES, 2Centre for Genomics and Oncological Research (GENYO); Department of Biochemistry and Molecular Biology III and Immunology, School of Medicine, Granada University, Granada, ES, 3Centre for Genomics and Oncological Research (GENYO); Department of Biochemistry and Molecular Biology I, School of Sciences, Granada University; Department of Biochemistry and Molecular Biology III and Immunology, School of Medicine, Granada University, Granada, ES, 4Department of Biochemistry and Molecular Biology III and Immunology. School of Medicine. Granada University, Granada, ES Previous studies have identified the plakophilin-1 gene (PKP1) as a specific biomarker of lung squamous cell carcinoma (LSCC) in comparison with other types of lung cancer. In order to gain more insight into the role of plakophilin-1 in LSCC, PKP1 expression was modified in three LSCC cell lines for subsequent differential expression studies by gene expression microarrays. In particular, PKP1 expression was inhibited using siRNA in SK-MES-1 and EPLC-272H, which have comparatively high basal PKP1 expression, whereas it was overexpressed by means of lentiviral vectors in NCI-H2170, which has low basal expression of PKP1. The data generated by the microarrays was analyzed statistically and functionally. In the analysis, a positive correlation between PKP1 and key nodes in concogenesis, such as PI3K, NF-κB and H-Ras, was observed. These results suggest a possible connection between PKP1 and oncogenic pathways in LSCC cell lines. XXXIX Congreso SEBBM of Molecular Biology, Massachusetts General Hospital and Harvard Medical School, Boston, US, 4Centro Nacional de Análisis Genómico y Centro de Regulación Genómica, Barcelona, ES Induction of pluripotency in somatic cells by the Yamanaka factors OSKM is typically an inefficient and slow process and demethylation of pluripotency gene promoters has only been detected after about 2-3 weeks. We recently found that B cells exposed to an 18h pulse of C/EBPα and subsequently exposed to OSKM efficiently reprogram into iPSCs. We have therefore taken advantage of this ultra-fast reprogramming system to generate genome wide nucleotide resolution maps for 5mC and 5hmC (its oxidized derivative) at different time points during the path towards pluripotency. Already after the pulse C/EBPα treatment, we observed a gain of 5hmC and a concomitant loss of 5mC at enhancers of pluripotency genes directly bound by C/EBPα (including Klf4, Lefty2, Id1 and Tet2). Furthermore, at these sites chromatin becomes accessible and histones decorated by active marks. The mechanism underlying C/EBPα local demethylation is likely mediated through the recruitment of Tet2. This enzyme mediates oxidation of methylcytosine residues and has been shown to be important for transdifferentiation and reprogramming. Strikingly, only 24 hours after activation of the Yamanaka factors methylation at the promoters of the key pluripotency gene Pou5f1 (Oct4) decreases, correlating with the gene’s rapid activation. Our reprogramming system showing highly dynamic methylation changes may shed new light on mechanisms of early embryo development and cancer. P11-9 Mapping and characterization of R-loops causing genetic instability in S. cerevisiae J. Carlos Cañas, Belén Gómez-González, Andrés Aguilera Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide-Junta de Andalucía, Sevilla, ES R-loops, structures formed by a RNA-DNA hybrid and a displaced single-stranded DNA molecule, are naturally involved in many different cellular processes, such as the replication of E. coli plasmids or mitochondrial DNA, as well as in class-switch recombination of immunoglobulin genes in B-cells. In certain abnormal circumstances, the co-transcriptional formation of R-loops can lead to replication fork impairment with deleterious consequences for genome integrity. To date, such aberrant DNA-RNA hybrids have been detected through a battery of both indirect and direct tools. However, at present, neither their length or the frequency nor whether these hybrids are continuous or discontinuous is known. With the aim of further characterizing R-loops, we have mapped and counted the DNA molecules containing DNA-RNA hybrids at the molecular level by the bisulfite modification assay. We will present the results of the length of R-loops and the frequency at which they are formed in several mutant strains of S. cerevisiae, containing mutations in genes involved in different processes such as mRNA processing or chromatin remodelling. The implications for the role of R-loops in transcription-associated genetic instability will be discussed. P11r-10 P11-8 Rapid demethylation of pluripotency gene regulatory regions during reprogramming of C/EBPα-primed B cells into iPSCs José Luis Sardina1, Antonio Gómez1, Samuel Collombet2, Bruno Di Stefano3, Ralph Stadhouders1, Clara Berenger1, Carolina Segura1, Tian V. Tian1, Simon Heath4, Thomas Graf1 1 Centro de Regulación Genómica, Barcelona, ES, 2Institut de Biologie de l’Ecole Normale Supérieure (IBENS), París, FR, 3Department 94 Proteomic and phosphoproteomic analysis of the rat pancreas identifies novel targets involved in the onset of acute pancreatitis Violeta García-Hernández1, Carmen Sánchez-Bernal1, Domitille Schvartz2, José Julián Calvo3, Jean-Charles Sánchez2, Jesús Sánchez-Yagüe1 1 Department of Biochemistry and Molecular Biology, University of Salamanca, IBSAL, Salamanca, ES, 2Translational Biomarker Group, Department of Human Protein Sciences, University Medical Center, Geneva, CH, 3Department of Physiology and Pharmacology, Salamanca 2016 University of Salamanca, IBSAL, Salamanca, ES Acute pancreatitis (AP) is an increasingly frequent disorder of the pancreas that can sometimes be lethal. The development of AP would be based on early changes in protein expression and signalling pathways modulating at least the phosphorylation of proteins. We reported here a Tandem Mass Tag (TMT)-based proteomic and phosphoproteomic study of rat subcellular fractions of the pancreas during the early phase of experimental AP. This phase was induced by two s.c. injections of 20 μg cerulein(Cer)/kg body weight at hourly intervals. Changes in protein expression were validated by Western blotting, and phosphorylation patterns by Phos-tag SDS-PAGE followed by immunoblot. A bioinformatics enrichment analysis was performed to characterize the modulated processes.Out of the around 1000 identified proteins, a total of 353 unique proteins were found differentially expressed during AP. 84 and 261 of them were respectively over or underexpressed by the Cer-treatment, while 8 proteins pointed to a probable subcellular redistribution. These proteins were mainly related to inflammation and apoptosis (haptoglobin, clusterin, MCTS-1), trafficking (coatomer subunits, Rab-related proteins), serine protease inhibition (serpinA1, A6, B1, A3K) or cytoskeleton impairment (actin, profilin-1). Also, from the onset of AP we have determined changes in the phosphorylation pattern of several interesting targets such as fetuin-A or tristetraprolin. We are currently testing the most promising candidates –including fetuin-A or various members of the SERPIN superfamily- as early biomarkers of post-ERCP pancreatitis in human plasma. These data would provide valuable information for deciphering AP at the molecular level, that may be potentially useful for its clinical management. Support: FIS(PI15/01156). P11-11 Estudio comparativo del estado oxidativo del tejido adiposo subcutáneo y omental de pacientes obesos y su contribución al desarrollo de resistencia a insulina María del Carmen Navarro Ruiz1, José Alberto Díaz-Ruiz Ruiz2, José López Miranda3, Rafael Vázquez Martínez1, Rocío Guzmán Ruiz1, María del Mar Malagón Poyato1 1 IMIBIC / Hospital Reina Sofía / Universidad de Córdoba, CIBERobn, Córdoba, ES, 2Experimental Gerontology Section, Translational Gerontology Branch, National Institute on Aging, National Institutes of Health, Baltimore, US, 3Unidad de Arteroesclerosis y Lípidos del Hospital Universitario Reina Sofía, Córdoba, ES La obesidad se caracteriza por un aumento del tejido adiposo que predispone al desarrollo de resistencia a insulina. A nivel molecular, la disfunción de los adipocitos en obesidad se relaciona, entre otros procesos, con estrés oxidativo. En este trabajo profundizamos en el estudio de los mecanismos que contribuyen a este proceso y en la búsqueda de posibles proteínas diana que actúen como marcadores de disfunción del tejido adiposo en obesidad, teniendo en cuenta la distinta contribución de los dos depósitos grasos mayoritarios, subcutáneo (SC) y omental (OM), a la resistencia a insulina. Para ello, realizamos estudios comparativos de muestras de tejido adiposo SC y OM de pacientes obesos con distintos grados de sensibilidad a insulina [normoglucémicos (NG), insulino-resistentes (IR) y diabéticos tipo II (T2DM)] y evaluamos: i) carbonilación de proteínas mediante derivatización con DNP y análisis 2D-PAGE, ii) peroxidación lipídica mediante trata la medida de 4-HNE por inmunoblot, iii) especies reactivas de oxígeno (ROS) utilizando la sonda 2,7’-diclorofluoresceina diacetato y iv) enzimas antioxidantes [superóxido dismutasa 1 (SOD1), glutatión sintasa (GS)] por inmunoblot. Observamos que el contenido total de proteínas carboniladas fue mayor en IR y T2DM vs NG en tejido adiposo OM mientras que no se hallaron Pósters / Posters diferencias en el depósito SC. El análisis 2D-PAGE también reveló perfiles diferentes de proteínas carboniladas entre depósitos. De igual manera, los niveles de ROS intracelular fueron significativamente diferentes entre los grupos en OM, pero no en SC, aunque los niveles de enzimas antioxidantes muestran un perfil similar en ambos depósitos. En conjunto, nuestros resultados sugieren una respuesta diferencial dependiente de depósito en relación al desarrollo de enfermedad metabólica en obesidad y proporcionan nueva información sobre la contribución fundamental del depósito omental en este proceso. Financiación: MINECO/FEDER (BFU2013-44229-R; BFU2015-70454-REDT); JJAA/FEDER (PI-0200/2013); FIS (PIE14_00005) y CIBERobn (ISCIII) P11r-12 GRAPES: A web-based tool for analyzing, annotating and filtering structural variants from Whole-Exome Sequencing Bernat del Olmo1, Jesús Matés1, Irene Mademont1, Carles Ferrer-Costa2, Catarina Allegue1, Vincenzo Pascali3, Antonio Oliva3, Ramon Brugada4 1 Cardiovascular Genetics Center, Biomedical Research Institute of Girona, IDIBGI, Salt, ES, 2Gendiag.exe SL, Esplugues del Llobregat, Barcelona, ES, 3Institute of Public Health, Section of Legal Medicine, Catholic University, Roma, IT, 4Cardiovascular Genetics Center, Biomedical Research Institute of Girona, IDIBGI; Department of Medical Sciences, School of Medicine, University of Girona; and Cardiac Genetics Unit, Cardiology Service, Hospital Josep Trueta, Girona, ES Although the advent of Next Generation Sequencing (NGS) has led already to breakthrough discoveries, two of the most challenging and limiting aspects that often limit the discovery of new findings using this technology continues to be data analysis and data integration, especially for the thousands of laboratories that lack of computational power and expertise. Here, we report the development of a new web-based platform to analyze, annotate, and filter Structural variants (SVs) for users with only basic computational skills. SVs are genetic variants longer than 50 bp that include Copy Number Variants (CNVs), inversions, tandem duplications and translocations. CNVs have been associated with various diseases, including cancer, cardiovascular disease and neurologic disorders. Whole-Exome Sequencing (WES) is an effective tool to discover common and rare variants within the coding regions of all human genes. Due to its widespread adoption, many tools have been designed to detect CNVs from WES, but only a few include SV discovery support and often they require advanced bioinformatic skills. Our approach, known as GRAPES (Genomic Rearrangement Algorithm for Paired-End Sequencing) integrates coverage signals and misalignment signatures to improve the sensitivity and sensitivity from WES datasets. We predict that this application will improve and facilitate the detection of SVs from WES in the clinical and research fields. Bernat del Olmo and Jesús Matés: Equally contributing authors. P11-13 Análisis del secretoma del tejido adiposo y su papel en la obesidad D. Pérez-Sotelo1, A. Roca-Rivada2, SB. Bravo3, A. Castro4, J. Baltar5, I. Baamonde5, FF. Casanueva6, M. Pardo7 1 Grupo Obesidómica, Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Xestión Integrada de Santiago (XXIS/SERGAS), Santiago de Compostela, ES, 2CIBER Fisiología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Santiago de Compostela, ES, 3Unidad de Proteómica (IDIS), (XXIS/SERGAS), 95 Pósters / Posters Santiago de Compostela, ES, 4CIBER Fisiología de la Obesidad y Nutrición, Instituto de Salud Carlos III; Grupo Obesidómica, Instituto de Investigacion de Santiago de Compostela (IDIS), Xestion Integradad de santiago (XXIS/SERGAS); y Laboratorio de Endocrinología Molecular y Celular, Instituto de Investigación Sanitaria de Santiago (IDIS), (XXIS SERGAS), Santiago de Compostela, ES, 5Servicio de Cirugía General, (XXIS/SERGAS), Santiago de Compostela, ES, 6CIBER Fisiología de la Obesidad y Nutrición, Instituto de Salud carlos III; Laboratorio de Endocrinología Molecular y Celular, Instituto de Investigación Sanitaria de Santiago (IDIS), (XXIS SERGAS), Santiago de Compostela, ES, 7CIBER Fisiología de la Obesidad y Nutrición, Instituto de Salud carlos III; Grupo Obesidómica, Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Xestión Integrada de Santiago (XXIS/SERGAS), Santiago de Compostela, ES La obesidad constituye en la actualidad el mayor problema sanitario a nivel mundial adquiriendo dimensiones epidémicas; se estima que a este ritmo podría alcanzar un 20% de la población adulta en 2025. Hasta el momento el número de fármacos que han sido desarrollados para el tratamiento de la obesidad es muy limitado y no muy exitoso, siendo la cirugía bariátrica el tratamiento más efectivo hasta la fecha. Por esta razón, para el desarrollo de nuevos y mejores tratamientos anti-obesidad, que junto con la dieta y el ejercicio contribuyan a una reducción del peso corporal, se requiere un mejor conocimiento de los mecanismos a nivel molecular que controlan el balance energético. Bajo este contexto, el análisis del perfil secretor del tejido adiposo, como un actor principal en el desarrollo de la obesidad, se considera fundamental para entender los mecanismos de regulación energética. Además del tejido adiposo per se, el lugar anatómico de su acumulación clasificada como central o periférica, tiene gran influencia sobre el metabolismo sistémico y sobre el desarrollo de las patologías asociadas a la obesidad. En el grupo Obesidómica hemos aplicado técnicas proteómicas al estudio del secretoma de explantes completos de tejido adiposo omental, subcutáneo y gonadal procedente de modelos animales de obesidad y anorexia. Además de adipoquinas clásicas, hemos identificado nuevas proteínas y descrito un perfil secretor característico en función de su distribución anatómica. Más recientemente, hemos puesto a punto la técnica CILAIR (Comparison of Isotope-Labeled Amino acid Incorporation Rates) al estudio del secretoma de tejido adiposo humano visceral y subcutáneo de pacientes obesos. Además del alto porcentaje de proteínas identificadas previamente relacionadas con la obesidad o sus comorbilidades, el CILAIR se muestra como muy útil para el estudio del microambiente del tejido adiposo obeso permitiendo analizar factores recientemente implicados en esta patología como la remodelización de la matriz extracelular y el perfil inflamatorio. P11-14 Proteómica funcional para el descubrimiento de biomarcadores y nuevos fármacos Manuel Fuentes Centro de Investigación del Cáncer, Universidad de Salamanca-CSIC, Salamanca, ES En la era post-genomica, una vez secuenciado el genoma humano, uno de los grandes retos en Biomedicina, es comprender la función de las proteínas codificadas por dichos genes. Aunque, el genoma posee las instrucciones codificadas de cómo y cuándo expresar las proteínas, son estas últimas quienes realizan gran parte de las funciones celulares; principalmente mediante su regulación, interacciones entre ellas y pequeñas moléculas (ej. cofactores,…), y el desarrollo de los procesos bioquímicos requeridos para ejercer su función. A pesar de los grandes avances realizados en genómica y biología molecular, solamente una pequeña parte del proteoma se ha caracterizado desde 96 XXXIX Congreso SEBBM el punto de vista bioquímico. Biología de sistemas y proteómica se ha centrado en la creación de modelos predictivos de rutas de señalización en base al comportamiento cuantitativo de las proteínas. De hecho, el conocimiento de estas redes dinámicas de proteínas permitirá describir interacciones aberrantes relacionadas con procesos patológicos como el cáncer,… Históricamente, los métodos empleados para la caracterización bioquímica de estas proteínas solo permiten estudiar de forma simultánea un número relativamente bajo y, en ocasiones, sin aportar información dinámica de dichas interacciones. Nuestro objetivo es mostrar una aproximación metodológica que permite estudiar cientos/miles de proteínas simultáneamente y en tiempo real, mediante la combinación de aproximaciones proteómicas y nanotecnológicas, con el objetivo de identificar biomarcadores y/o nuevos fármacos de utilidad en medicina personalizada. P12. Metabolismo del nitrógeno / Nitrogen metabolism P12r-1 El óxido nítrico en plantas se produce por la Mo-enzima ARC con NR Alejandro Chamizo Ampudia, Ángel Llamas Azua, Aurora Galván Cejudo, Emilio Fernández Reyes Universidad de Córdoba, Córdoba, ES La proteína ARC (Componente de Reducción de Amidoxima) fue descubierta en 2006 como una nueva enzima que contiene cofactor de molibdeno, la primera actividad enzimática que se le asigno fue la de reducción de compuestos N-hidroxilados como la prodroga Amidoxima. Las proteínas de esta familia se distribuyen a lo largo de los tres reinos de los seres vivos. La proteína ARC de plantas se ha caracterizado en Chlamydomonas, donde se demostró que es una proteína capaz de desarrollar su función con una NADH-citocromo b5 reductasa y un citocromo b5-1 para elsuministro de electrones. Curiosamente la estructura formada por estas 3 proteínas muestra similitud con la nitrato reductasa (NR), ya que ambas tienen un grupo cofactor de molibdeno, un grupo hemo y otro grupo FAD, diferenciándose en el sitio activo, donde se encuentra el cofactor de molibdeno, particular para cada enzima, y el dominio de dimerización, que solo está presente en la NR. Además se observó que la NR con Cytb5-1 y Cytb5-R presentan una alta homología de secuencia, lo que hizo pensar que la NR podría reemplazar a las Cytb5-1 y Cytb5-R e interactuar con la ARC. En este sentido, se caracterizó esta interacción y presentó una típica cinética de Michaelis. Además, se estudió como el nitrito inhibe competitivamente la actividad ARC con benzamidoxima, lo que indica que el nitrito puede ser un sustrato de ARC en la producción de óxido nítrico (NO). Se ha determinado que no hay producción de óxido nítrico in vivo en un mutante por deleción de la NR, en cambio sí que hemos visto producción de oxido nítrico en un mutante puntual en la actividad terminal de NR (actividad dependiente de cofactor de molibdeno), que es incapaz de reducir nitrato, pero que sí tiene su actividad diaforasa funcional. Por quimioluminiscencia, se observó que la NR puede producir óxido nítrico a partir de nitrito, pero no en presencia de nitrato (mM). Sin embargo, en esta condición de nitrito más nitrato, ARC más NR sí que puede producir de manera eficiente óxido nítrico a partir de nitrito, con los electrones proporcionadas por NAD(P)H-diaforasa-NR hacia la ARC. Además, toda esta producción de óxido nítrico se ha localizado en el citoplasma. Salamanca 2016 P13. Neurobiología molecular / Molecular neurobiology P13-1 Impact of sexual dimorphism in human hippocampus during brain ageing Daniel V. Guebel1, Néstor V. Torres2 1 Biotechnology Counselling Services, Buenos Aires, AR, 2Systems Biology and Mathematical Modelling Group. Departamento de Bioquímica, Microbiología, Biología Celular y Genética. Universidad de La Laguna, San Cristóbal de La Laguna, ES At the brain level, the effects of sexual dimorphism and normal ageing seem to overlap. Therefore, to better understand the pathogenesis of many important neurodegenerative diseases, a robust discrimination between these effects is required. In this communication we present the results of the transcriptomic data analysis of samples from human hippocampus, which map the transition from adulthood to ageing. For this purpose we have used an optimized technique for microarray post-processing (Q-GDEMAR; Guebel et al. 2015(1)) extended to a factorial design. We have been able to identify large sets of differentially expressed genes. The sets of genes operating only under sex-dependence were disaggregated from those working only under age-dependence. In addition, both sets of gene types were well-separated from those operating under sex and age interaction-dependence. The identified genes were validated against three external, independent sources of data. The highly significant differences observed among the three types of genetic dependences lead to the conclusion that sex dimorphism and sex-for-age interaction should be taken explicitly into account in the studies of the transition from normal ageing to early neurodegenerative pathologies. Acknowledgments: This work has been supported by a project grant from MINECO (ref. BIO2014-54411-C2-2-R) and the IMBRAIN project (ref. FP7-REGPOT-2012-CT2012-31637-IMBRAIN). DVG was awarded with a fellowship for visiting professors of Universidad de La Laguna (ref. 597423). The authors thank Dr. Catalina Feledi for her valuable collaboration. References [1] Guebel DV, Perera-Alberto M, Torres NV. Molecular Biosystems. 2015, 12(1):120-32. P13r-2 Combination of T7 Phage Display and Protein Microarrays for the characterization of the humoral response in Alzheimer Disease Pablo San Segundo Acosta1, Ana Montero Calle1, María Garranzo Asensio1, Carmen Oeo Santos1, Juan Carlos López Rodríguez1, Laura Martín Pedraza1, Cristina Bueno Díaz1, Laura Ruiz2, Alberto Rábano2, Eva Batanero1, Mayte Villalba Díaz1, Rodrigo Barderas Manchado1 1 Complutense University of Madrid, Madrid, ES, 2Fundación CIEN, Madrid, ES Alzheimer’s disease (AD) is a progressive, chronic and neurodegenerative disorder with a high socioeconomic impact. New approaches to study AD are necessary to identify new diagnostic biomarkers and therapeutic targets. Serum autoantibodies from AD patients against altered proteins during the course of the disease might become a new source of AD biomarkers, useful for early diagnosis. A combination of phage display and protein microarrays has been used to identify AD-specific autoantibodies and their target proteins as biomarkers of the disease. We have screened two T7 phage display libraries displaying the cDNA repertoire of the brain of AD patients and healthy individuals’ with Pósters / Posters serum from AD patients and controls provided by the CIEN foundation. Both libraries were iteratively biopanned using serum from healthy individuals to remove non-specific phages and subsequently with AD patients’ sera to enrich the libraries in AD-specific phages. After biopanning, 768 phages from both libraries were printed on nitrocellulose microarrays, probed with sera from AD patients at different Braak stages and controls to identify specific phages displaying peptides and proteins recognized by IgGs from AD patients. Then, specific phages will be sequenced to identify the displayed peptides and proteins; and their immunoreactivity analyzed by complementary immunological approaches using a higher cohort of sera. The identified target proteins will permit the discovery of new disease-specific biomarkers and the elucidation of new altered pathways and cellular processes. Further validation, functional analysis and quantification of the target proteins in sera are planned to determine their usefulness as blood-based biomarkers and to identify new potential targets of intervention. P13-3 Developing a human celular model for the bacterial amyloidosis RepA-WH1 Aída Revilla-García1, María Moreno-del Álamo2, Juan F. Giménez-Abián1, Rafael Giraldo1 1 Department of Cellular and Molecular Biology, CIB-CSIC, Madrid, ES, 2Department of Microbial Biotechnology, CNB-CSIC, Madrid, ES Protein amyloids arise from the conformational conversion of a soluble protein into fibrillar aggregates with a crossed b-sheet backbone, leading to human neurodegenerative and systemic proteinopathies. In bacteria, amyloids assemble as functional extracellular scaffolds but no natural proteinopathic amyloidosis has been found in microorganisms yet. The bacterial protein, RepA can assemble into amyloid fibres upon binding to plasmid-specific DNA sequences. RepA-WH1 causes in E. coli an amyloid proteinopathy, which is vertically transmissible but not infectious, enabling conformational templating by cross-seeding in vitro and in vivo. Bacterial lineages maintain two mutually exclusive types (strains) of RepA-WH1 amyloids. The bacterial Hsp70 chaperone DnaK modulates the vertical propagation of these amyloid strains. We are developing new tools to studying amyloid toxicity elicited by the synthetic RepA-WH1 prionoid in mammalian cells. For this purpose, we are now constructing SH-SY5Y based cell lines for the stable, tetracycline-induced expression of RepA-WH1, targeting it to either the cytoplasm or the nucleus (the latter depending on a nuclear localization signal). This objective will identify the route(s) involved in programmed death of the neuroblastoma cells upon the stable expression of RepA-WH1(A31V)-TagGFP2. This is the most cytotoxic variant of the prionoid both in bacteria and, according to our preliminary data on transient transfections, in human cultured cells too. In parallel, controls carrying just the fluorescent marker (TagGFP2) and two less cytotoxic variants of the prionoid (WT and ΔN37) will be also expressed from stable cells lines. The results presented here empower the bacterial RepA-WH1 prionoid as a synthetic minimalist model system for amyloid proteinopathies. P13-4 The WRAP53 protein modulates neuronal survival after ischemia Irene Sánchez Morán, Cristina Rodríguez, Ángeles Almeida Instituto de Investigación Biomédica de Salamanca (IBSAL), Hospital Universitario de Salamanca; Instituto de Biología Funcional y Genómica (IBFG), CSIC/Universidad de Salamanca, Salamanca, ES The WD40 domain-containing protein Wrap53 (WD40 encoding RNA Antisense to p53) is a scaffold protein implicated in Cajal Bodies maintenance, telomere elongation and DNA repair. WRAP53 loss of function 97 Pósters / Posters has been related to carcinogenesis and premature aging. Double-strands breaks may result from ischemic stroke, thereby contributing to neuronal death and subsequent brain dysfunction. An adequate DNA damage response is essential to survive after cerebral ischemia and preserve the integrity of the transcribed genome in neurons. Although DNA repair pathways are active after ischemia, the molecular mechanisms underlying neuronal survival remain unknown. To investigate the role of Wrap53 in neuronal survival after ischemia, primary mouse cortical neurons were subjected to an experimental protocol of ischemia in vitro (oxygen and glucose deprivation) for 3 hours and were further incubated in regular medium (reoxygenation). We first observed that ischemia promoted DNA damage, as revealed by the accumulation of γH2AX and 53BP-1 in the neurons. Furthermore, we found a time-dependent increase in WRAP53 gene expression and protein abundance from 4 hours after the ischemic insult. In parallel, ischemia induced the traffic of Wrap53 to the nucleus, which has been associated to cell survival in tumor cells. Moreover, depletion of Wrap53 by siRNA increased neuronal susceptibility to ischemia-induced apoptosis. Our results demonstrate that ischemia-induced Wrap53 nuclear accumulation plays an essential role in neuronal survival. Funded by ISCIII (PI15/00473; RD12/0014/0007), FEDER, and Junta de Castilla y León (ISM). P13-5 Alterations in the ribbon synapse of the first Pomt1 conditional knockout mouse model of dystroglycanopathies Marcos Rubio-Fernández1, Mary Luz Uribe2, Javier Vicente-Tejedor3, Cristina Susín Lara1, Francisco Germain3, Pedro de la Villa Polo3, José Martín Nieto2, Jesús Cruces Pinto1 1 Departamento de Bioquímica, Instituto de Investigaciones Biomédicas Alberto Sols UAM-CSIC; Facultad de Medicina, Universidad Autónoma de Madrid; Instituto de Investigación Hospital Universitario La Paz - IdiPAZ, Madrid, ES, 2Departamento de Fisiología, Genética y Microbiología, Facultad de Ciencias. Universidad de Alicante, Alicante, ES; Instituto de Investigación Hospital Universitario La Paz - IdiPaz, Alicante, ES, 3Departamento de Biología de Sistemas, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, ES Protein O-mannosyltransferase 1 (POMT1) is one of the causative genes of dystroglycanopathies (DGPs), a heterogeneous group of congenital recessive neuromuscular diseases. The most severe DGPs course with important muscular, brain and ocular anomalies derived from hypoglycosylation of α−dystroglycan (α−DG), a key protein component of the dystrophin-glycoprotein complex (DGC) in muscular and neural cells. α−DG interacts with extracelular matrix (ECM) proteins by means of its O-glycosylated residues, specifically O-mannosyl glycans, whose initial mannose is covalently added by POMT1. Our group has previously evidenced the embryonic lethality of a Pomt1 constitutive knockout (KO) mutation. Consequently, in this work we generated, by using the Cre-loxP system, a retinal conditional KO (cKO) mouse model selectively undergoing a Pomt1 intragenic deletion in developing photoreceptors. Lack of POMT1 in the retinas of these mice correlated with a loss of α−DG glycosylation and laminin-binding ability. Electroretinographic (ERG) records in Pomt1 cKO mice showed a significantly reduced b-wave amplitude and increased implicit time. By immunohistofluorescence, β-DG and pikachurin (a retinal ECM, α−DG-interacting-protein) were found to be absent in the outer plexiform layer (OPL), and a sprouting of bipolar cell dendrites into the outer nuclear layer was observed. Finally, at the ultrastructural level ribbon synapses exhibited an anomalous organization, with bipolar cells processes being barely visible in the synaptic cleft of cones and rods axon terminals. 98 XXXIX Congreso SEBBM In conclusion, our findings are strongly suggestive of a pivotal role of POMT1 and α−DG glycosylation in the formation and function of ribbon synapses between photoreceptors and bipolar cells in the OPL. P13-6 The function of alpha-CaMKII is altered in a mouse model of trisomy 21 Juan José Casañas, Beatriz Galán-Rodríguez, Alexandra Alves-Sampaio, José Antonio Troca-Marín, María Luz Montesinos Dpto. Fisiología Médica y Biofísica. IBiS-Universidad de Sevilla, Sevilla, ES Local dendritic translation plays an important role in synaptic plasticity. RNA binding proteins such as the Cytoplasmic Polyadenylation Element-Binding protein 1 (CPEB1) and the Fragile X Mental Retardation Protein (FMRP) not only mediate the transport of a number of messengers into dendrites, but also regulate their translation in response to synaptic activity. We previously found that local translation is affected in the trisomy 21 (T21) mouse model Ts1Cje (Alves-Sampaio et al., 2010; Troca-Marín et al. 2011). Now, we have found that the levels of CPEB1 and FMRP are increased in the Ts1Cje hippocampus. Accordingly, the dendritic levels of several localized mRNAs, including the alpha-CaMKII messenger, were augmented. Finally, signalling through alpha-CaMKII in response to synaptic activity was affected, which could be relevant for the synaptic plasticity phenotype of this T21 model. References [1] Alves-Sampaio, Troca-Marín and Montesinos (2010). J Neurosci 30:13537-13548. [2] Troca-Marín, Alves-Sampaio and Montesinos (2011). J Neurosci 231:9445-9455. P13r-7 Is ARMS/Kidins220 involved in the effect of Syt-IV on BDNF release? Ana Julia Sánchez Sánchez, Saray López Benito, Juan Carlos Arévalo Martín Instituto de Neurociencias de Castilla y León, Universidad de Salamanca, Salamanca, ES Neurotrophins are essential for a proper development and functioning of the nervous system. BDNF, one of the members of the neurotrophin family, plays a crucial role in physiological and pathological conditions although the mechanisms underlying its regulated secretion are not completely understood yet. Previously, it has been proposed that Synaptotagmin-IV (Syt-IV) acts as a mediator of the BDNF release in neurons. To check the reliability of Syt-IV in BDNF regulated secretion, we performed secretion assays under depolarising conditions, in cultured cortical neurons where Syt-IV levels were reduced. We observed that Syt-IV depletion potentiated depolarisation-mediated secretion of BDNF. Our studies were extended to NT-3 and NT-4 treatment and we also observed an enhanced BDNF release. Since it has recently been reported that ARMS/Kidins220 modulates regulated secretion (López-Benito, 2016), we decided to study if there was any relationship between this protein and Syt-IV. We have seen an interaction between them both in over-expression (HEK293 cells) and endogenous conditions (cultured cortical neurons), which has been further supported by co-localisation assays. Interestingly, an inverse correlation between their expression and an increased BDNF regulated release in differentiated cultured cortical neurons was noticed. The relevance of the interaction of ARMS and Syt-IV is currently under study. Further experiments will be required to disclose its potential role to fully understand BDNF regulated secretion. Salamanca 2016 P13-8 The MDM2-p53 signalling pathway is involved in neuronal ischemic tolerance Rebeca Vecino Pérez1, Ángeles Almeida Parra2, María Delgado Esteban2 1 Instituto de investigación biomédica de Salamanca (IBSAL), Hospital Universitario de Salamanca, Salamanca, ES; Instituto de Biología Funcional y Genómica (IBFG), Zamora, ES, 2Instituto de investigación biomédica de Salamanca (IBSAL), Hospital Universitario de Salamanca; Instituto de Biología Funcional y Genómica (IBFG), Salamanca, ES Brain ischemic preconditioning (IPC) refers to a state of transient tolerance of the brain tissue to a lethal insult that can be evoked by a prior mild insult. It is thought that IPC may induce different pathways responsible for neuroprotection. These mechanisms can involve the attenuation of cell damage pathways including the apoptotic cell death. In this context, p53 is a stress sensor that accumulates during brain ischemia leading to neuronal death. The mouse double minute2 homolog (MDM2), a p53-specific E3 ubiquitin ligase, is the main cellular antagonist of p53, mediating its degradation by the proteasome. Increased levels of MDM2 inhibit p53 stability and inactivate apoptotic function, although the role of MDM2 in neuroprotection is largely unknown.Here, we study the role of MDM2 and p53 interaction on IPC-induced neuroprotection. Primary cortical neurons were exposed to a moderate subtoxic concentration of N-methyl-D-aspartate (NMDA; 20μM NMDA; IPC condition) for 2 hours, followed by incubation for further 90 min in normoxic (presence of oxygen and glucose) or ischemic (oxygen and glucose deprivation; OGD). In parallel, control neurons were not stimulated with NMDA. After 4 hours of incubation in culture medium (reoxygenation condition), neuronal apoptosis (Annexin-V-staining) was analyzed by flow cytometry. Gene expression and protein levels were determined by RT-qPCR and western blotting, respectively.Our results show that IPC induced the expression of MDM2 in neurons, leading to MDM2 accumulation at 4 hours after OGD. Moreover, IPC prevented OGD-induced p53 stabilization, caspase-3 activation and apoptosis, which were prevented by neuronal treatment with the MDM2 inhibitor nutlin-3. These finding demonstrate the key role of the MDM2-p53 signalling pathway in neuroprotection induced by IPC against a subsequent ischemic insult and poses MDM2 as an essential target in ischemic tolerance. This work was funded by grants from The Instituto de Salud Carlos III (Miguel Servet I CP0014/00010; RD12/0014/0007) and FEDER (European regional development funding). P13-9 Regulation of AMPA receptor trafficking by CPT1C Maria Casas Prat1, Rut Fadó Andrés1, José Rodríguez Álvarez2, Núria Casals Farré3 1 Basic Sciences Department, Facultat de Medicina i Ciències de la Salut, Universitat Internacional de Catalunya, Sant Celoni, ES, 2Institut de Neurociències and Dpt. Bioquímica and Biología Molecular, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, ES, 3Basic Sciences Department, Facultat de Medicina i Ciències de la Salut, Universitat Internacional de Catalunya, Sant Cugat del Vallès, ES Carnitine Palmitoyltransferase 1C (CPT1C) is an isoform exclusively found in the brain that, unlike the other two mitochondrial isoforms, is localized in the endoplasmic reticulum and has a residual catalytic activity. Although CPT1C is not involved in mitochondrial transport of fatty acids for their oxidation, this isoform is also able to bind malonyl-CoA, an intermediary metabolite of fatty acid synthesis that decreases during fasting conditions. It has been suggested that CPT1C might be a sensor of malonyl-CoA levels in the brain. Pósters / Posters Despite the biochemical function of CPT1C remains unknown; recent studies have demonstrated that this protein is one of the constituents of AMPA type glutamate receptors (AMPARs) involved in learning processes. Here, we determined whether basal GluA1 trafficking changed during metabolic stressed conditions in cortical neurons, and whether CPT1C was involved in this regulation. Neither total GluA1 levels nor the strength of CPT1C-GluA1 interaction were altered after 2 hours of TOFA treatment (an inhibitor of malonyl-CoA synthesis). However, biotinylation assays indicated that surface GluA1 levels decreased when neurons were treated with TOFA for 2 hours. By contrast, CPT1C KO cortical neurons did not respond properly to changes in malonyl-CoA levels. These results suggest an interesting role of CPT1C as a sensor of malonyl-CoA and a regulator of GluA1 trafficking during metabolic stressed conditions. P13r-10 USP8 deubiquitinates TrkB and modulates TrkB-BDNF functions Carlos Martín Rodríguez1, Minseok Song2, Begoña Anta3, Francis S. Lee4, Juan Carlos Arévalo1 1 Institute of Biomedical Research of Salamanca (IBSAL), Department of Cell Biology and Pathology, Instituto de Neurociencias de Castilla y León (INCyL), University of Salamanca, Salamanca, ES, 2Synaptic Circuit Plasticity Laboratory, Department of Structure & Function of Neural Network, Korea Brain Research Institute, Daegu, KR, 3Department of Cell Biology and Pathology, Instituto de Neurociencias de Castilla y León (INCyL), University of Salamanca, Salamanca, ES, 4Department of Psychiatry, Weill Medical College of Cornell University, Department of Pharmacology, Weill Medical College of Cornell University, Nueva York, US Ubiquitination of the TrkA neurotrophin receptor is a well-recognized mechanism that plays an important role in nerve growth factor (NGF) functions. Although TrkB ubiquitination has been also reported, the molecular machinery involved in the ubiquitination/deubiquitination of the receptor in response to brain derived neurotrophic factor (BDNF) is completely unknown. Here we describe the identification of the ubiquitin-specific protease 8 (USP8), as a deubiquitinase for TrkB. TrkB and USP8 interact in cultured cortical neurons through the c-terminus of TrkB and the MIT domain of USP8. Overexpression of USP8 increases the total amount of TrkB protein and its deubiquitinating activity is required for this effect. Indeed, in vitro deubiquitination assays show that USP8 deubiquitinates the receptor and kock-down of USP8 protein in neurons increases TrkB ubiquitination in response to BDNF. In addition, USP8 depletion impairs TrkB endocytosis and BDNF-mediated signaling, resulting in a reduced dendritic arborization of hippocampal granule cells in vivo. Altogether these data indicate that the modulation of TrkB ubiquitination by USP8 regulates the trafficking and the signaling of the receptor and, consequently, TrkB-BDNF functions, highlighting the relevance of TrkB ubiquitination. P13r-11 Regulación de las fosfatasas de especificidad dual, DUSP, durante la diferenciación de las neuronas granulares en cultivo. Implicación del receptor P2X7 Mª José Queipo García, Juan Carlos Gil Redondo, Raquel Pérez Sen, Esmerilda García Delicado, Mª Teresa Miras Portugal Departamento de Bioquímica y Biología Molecular. Facultad de Veterinaria. Universidad Complutense de Madrid, Madrid, ES Las neuronas granulares de cerebelo de rata se caracterizan por ser un excelente modelo para estudios de neuroprotección. Éstas constituyen nuestro principal modelo de estudio y presentan una amplia expresión 99 Pósters / Posters de receptores de nucleótidos. Nuestro grupo ha podido comprobar que el receptor ionotrópico de nucleótidos P2X7 es un activador de la señalización de las quinasas activadas por mitógenos (MAPK) en las neuronas granulares. Las MAPK están implicadas en procesos celulares tan trascendentes como proliferación, diferenciación y supervivencia, motivo por el que están sometidas a una estricta regulación, principalmente a través de fosfatasas capaces de inactivarlas. Entre estas fosfatasas cabe destacar las fosfatasas de especificidad dual selectivas de MAPK (DUSP o MKP), capaces de desfosforilar tanto residuos de Ser/Thr como de Tyr. Concretamente, el receptor P2X7 modula de manera significativa la señalización a través de la vía ERK1/2. Por tanto, centramos nuestros estudios en las fosfatasas específicas de ERK1/2, como la DUSP6 que actúa a nivel citoplásmico, y la DUSP1, de carácter nuclear e inducible. Hemos comprobado que la expresión de DUSP6 y del receptor P2X7 se incrementa de manera paralela a lo largo del desarrollo del cultivo, mientras que los niveles de DUSP1 disminuyen. Además, la estimulación del receptor P2X7 con el agonista farmacológico BzATP, modula de manera bifásica la fosfatasa DUSP6. En una primera fase se observa una caída de los niveles de DUSP6 consistente con una degradación inicial vía proteasoma, seguida de una fase de recuperación debida a su inducción transcripcional. Ambos procesos son dependientes del estado de activación de ERK1/2, que a su vez se ven moduladas por DUSP6 en un proceso de retroalimentación negativa. Por otro lado, el receptor P2X7 parece no regular la fosfatasa DUSP1. Sin embargo, su expresión se encuentra potentemente inducida por la estimulación con la neurotrofina BDNF, que también activa de manera muy potente las ERK1/2 en las neuronas granulares. XXXIX Congreso SEBBM P13-13 Resveratrol modulates adenosine receptors endogenously expressed in C6 glioma cells Alejandro Sánchez-Melgar, José Luis Albasanz, Mairena Martín Departamento de Química Inorgánica, Orgánica y Bioquímica. CRIB. Facultad de Ciencias y Tecnologías Químicas / Facultad de Medicina de Ciudad Real. Universidad de Castilla-La Mancha, Ciudad Real, ES Resveratrol (RV) is a polyphenol present in the red wine and others vegetables products. Epidemiological studies have revealed that resveratrol could exhibit some neuroprotective role but the mechanisms by which this polyphenol is acting are still unclear. Adenosine has also a very important role as a neuromodulator through its receptors. Adenosine receptors are G protein coupled receptors (GPCR) that inhibit adenylyl cyclase (AC) activity (A1 and A3) or activate this enzymatic activity (A2A and A2B). The aim of the present work was to study whether resveratrol was able to modulate the adenosine receptors in a rat glioma C6 cell line model which endogenously express these receptors. Our results show a clear and significant modulation of adenosine receptors gene expression after resveratrol treatment at 100 μM during 24 hours assessed by real-time PCR. While A2AR and A2BR genes expression was significantly increased, A1R and A3R genes were decreased. This was accompanied by a significant increase in A1R, but not changes in A2AR level, determined by radioligand binding assays. AC and PKA levels were analyzed by Western blotting assay. While AC was not significantly affected, PKA was also upregulated after RV treatment. Results show that resveratrol modulates adenosine receptors and theirs transduction pathways and suggest a possible role of these receptors in the effect of resveratrol on cells. P13-12 Los receptores nucleotídicos P2X7 y los receptores de EGF regulan los niveles de la proteína fosfatasa DUSP6 en astrocitos cerebelosos de rata Juan Carlos Gil Redondo, María José Queipo García, Esmerilda García Delicado, Raquel Pérez Sen, María Teresa Miras Portugal Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, ES En la regulación de la homeostasis, diferenciación y supervivencia de las células destaca la vía de las proteínas quinasas activadas por mitógenos (MAPK). La regulación de la fosforilación y activación de estas serina/ treonina quinasas depende de varias fosfatasas, entre las que encontramos las proteínas fosfatasas de especificidad dual (DUSP). En concreto, la fosfatasa DUSP6 (o MKP3) es específica del grupo de MAPK denominadas ERK. En el presente trabajo, realizado en astrocitos de cerebelo de rata, se demuestra cómo la estimulación del receptor purinérgico P2X7 es capaz de regular los niveles de proteína de la fosfatasa DUSP6 de una manera similar a la realizada por la estimulación del receptor EGF. La estimulación del receptor P2X7 con el agonista BzATP, o del receptor de EGF, provoca una caída inicial en los niveles de la fosfatasa, con un pico mínimo en torno a los 15-30 minutos, seguido por una recuperación a niveles basales tras una hora de estimulación, superándolos en estimulaciones más largas. La caída inicial es consecuencia de la degradación proteasomal de la proteína fosfatasa, como se pudo comprobar al emplear el inhibidor del proteasoma MG-132. Asimismo, el aumento posterior de los niveles de proteína se debe a la inducción transcripcional del gen dusp6, como se comprobó mediante experimentos de PCR cuantitativa. La disminución inicial de los niveles de fosfatasa coincide en ambos casos con un aumento en los niveles de fosforilación de las ERK. Tanto la degradación inicial como la posterior inducción transcripcional son consecuencia de la fosforilación y activación de las ERK mediante la MAPK quinasa (MEK), como se demostró al emplear su inhibidor U-0126. Asimismo, se comprobó la participación de la proteína quinasa C (PKC) en dichos procesos. 100 P13r-14 ARMS/Kidins220 protein controls regulated secretion of BDNF Saray López Benito1, Ana Julia Sánchez Sánchez1, Laura Calvo Enrique2, Cristina Vicente García1, Verónica Inés Brito3, Seila Fernández Fernández4, Juan Pedro Bolaños4, Silvia Ginés Padrós3, Lino Tessarollo5, Juan Carlos Arévalo Martín1 1 Department of Cell Biology and Pathology, Instituto de Neurociencias de Castilla y León (INCyL), University of Salamanca; Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, ES, 2Scheeles väg 2, MBB departement, Karolinska Institutet, Solna, Stockholm, SE, 3Departament de Biomedicina, Facultat de Medicina, Universitat de Barcelona; Instituto de Investigación Biomédica August Pi i Sunyer (IDIBAPS); Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, ES, 4Department of Biochemistry and Molecular Biology, University of Salamanca; Institute of Functional Biology and Genomics (IBFG), University of Salamanca-CSIC; Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, ES, 5Neural Development Group, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, US Among the growth factors required for the development and correct functioning of the nervous system, BDNF is one of the most studied due to its implication in both physiological and disease conditions. However, the mechanisms controlling the regulated secretion of BDNF are not completely understood yet. Here we describe that BDNF secretion induced by depolarization, NGF, NT-3 or NT-4 is potentiated upon depletion of ARMS/ Kidins220 protein in cultured neurons. To address in vivo the role of ARMS/ Kidins220 in BDNF release, we generated a mouse model to knock-down its expression in an inducible manner. Cortical slices with reduced levels of ARMS/Kidins220 showed enhanced BDNF secretion in response to the aforementioned stimuli. In addition, BDNF levels in the striatum, which come Salamanca 2016 from cortical and hippocampal projections, are increased in mutant mice with reduced ARMS/Kidins220 in cortex and hippocampus. Moreover, the levels of BDNF were further increased in the striatum and hippocampus, but not in the cortex, of mice depleted of ARMS/Kidins220 that experienced physical activity with respect to control mice. Since BDNF secretion is impaired in mouse models of Huntington disease, we sought to check ARMS/ Kidins220 levels in these mice. ARMS/Kidins220 protein is increased in the hippocampus of HD mice and reduction of ARMS/Kidins220 levels rescued the secretion defect increasing BDNF release to that one of control mice. Altogether this data indicated that ARMS/Kidins220 protein controls regulated secretion of BDNF and may play a crucial role in the pathogenesis of Huntington´s disease. P13-15 Effects of febrile seizures on adenosine receptors in neonates María Crespo Gutiérrez, David Agustín León Navarro, Mairena Martín López Facultad de Ciencias y Tecnologías Químicas. Dpto de Química Inorgánica, Orgánica y Bioquímica, Facultad de Medicina de Ciudad Real. CRIB. Universidad de Castilla-La Mancha, Ciudad Real, ES Pósters / Posters and deposits of β amyloid caused by aberrant cleavage of its precursor (APP). Adenosine receptors are G-protein coupled receptors distributed in the CNS. They can inhibit or stimulate adenylyl cyclase activity, mediating different physiological responses in cells, and have a neuroprotective role. It is also known that protein kinase A (PKA) is implicated in learning and memory processes. Previous results of our group showed that adenosine A1 and A2A receptors are significantly increased in plasma membranes from frontal cortex brain in AD. The aim of the present work was to study different components of transduction pathway in the same cortical area. To this end, cytosolic fraction of post-mortem frontal cortex brain from different stages of AD patients and age-matched controls were extracted. PKC activity was significantly increased from early stages of AD (I-II of Braak). However, PKA activity revealed a biphasic profile decreasing in early-medium and increasing in advanced stages of AD. A1 and A2A receptors analysed by Western-blot were both significantly increased in early stages of AD and AC1, the main isoform coupled to these receptors, was also increased in the same stages. Moreover, βA1-40 was significantly increased from early stages while βA1-42 was increased only in advanced stages in AD. These results show modulation of transduction pathways mediated by adenosine and suggest A1 and A2A receptors, ACI, PKA and PKC as promising pharmacological targets in AD. P13r-17 Febrile seizures have been associated with epilepsy development but the underlying mechanism isstill poorly understood. Although febrile seizures are commonly related to the brain cortex, several studies have suggested that other sub-cortical areas, such as the cerebellum, could be also involved. Febrile seizures are one of the most typical convulsive disorders in the children between 3 months and 6 years corresponding to 2 weeks of life in rodents, stageat which the cerebellum is still under development. Adenosine is a nucleoside widely recognized asan endogenous modulator of neuronal excitability, which exerts neuroprotective and anticonvulsantactions in the brain. We previously showed short term modulation ofA1R and A2AR and 5’-nucleotidase (5’-NT) activity in cortex from rat brain following hyperthermia-induced seizures, suggesting a neuroprotective role of adenosine. The aim of the present work was to study whether both receptors and 5’-nucleotidase activity could also be modulated in the cerebellum in response to hyperthermia-induced seizures. Neonates were sacrificed 48 hours, 5 and 20 days after hyperthermia seizures and cerebellar plasma membranes were isolated. The effect of fever seizures on A1R and A2AR was studied by radioligand binding assays using[3H]DPCPX and [3H]ZM241385 as radioligands, respectively. A1Rs were significantly increased after 48h of hyperthermia and no significant differences were observed after 5 or 20 days. However, A2ARs were affected in a biphasic manner being decreased after 48 h and increased after 5 and 20 days of hyperthermia. Changes on receptors were accompanied by affectation of 5’-NT activity. These results suggest that adenosine could exhibit a possible neuroprotective role on hyperthermia seizures alsoin cerebellum. P13-16 Modulation of PKA and PKC in the frontal cortex of Alzheimer’s disease human brains Patricia Alonso Andrés1, José Luis Albasanz Herrero1, Isidro Ferrer Abizanda2, Mairena Martín López1 1 Faculty of Sciences and Chemical Tecnologies. Faculty of Medicine of Ciudad Real. Inorganic, Organic Chemistries and Biochemistry Department. CRIB. Castilla-La Mancha University, Ciudad Real, ES, 2Institute of Neuropathology Pathological Anatomy Service. University Hospital of Bellvitge, L’Hospitalet de Llobregat, ES Alzheimer’s disease (AD) is the main neurodegenerative disease in aging that affects to 24 millions of people around the world. AD is characterized by cognitive, behaviour and memory alterations. In human brain, it has been found neurofibrillary tangles with abnormal phosphorylated tau proteins, p73 is required for ependymal cell ciliogenesis and Planar Cell Polarity Sandra Fuertes Álvarez1, Natalia Robledinos-Antón2, Laura González-Cano3, Isabel Fariñas4, M. Margarita Marqués5, María C. Marín6 1 IBIOMED, Universidad de León, Valencia de Don Juan, ES, 2 Institute of Biomedicine (IBIOMED), University of León (Current address: Alberto Sols Institute, CSIC, University of Madrid, León, ES, 3Institute of Biomedicine (IBIOMED), University of León (Current address: Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-Belval, Luxembourg), Leon, ES, 4 Departament of Cellular Biology and CIBERNED, University of Valencia, Burjassot, ES, 5Department of Animal Production, University of León, Leon, ES, 6Institute of Biomedicine (IBIOMED), University of León, León, ES p73 is a transcription factor that belongs to the p53 family, together with p53 and p63. These genes regulate important processes, like cell differentiation or stem cell renewal. In particular, p53 and p73 are essential regulators of Neural Stem Cell biology. Examination of Trp73 knockout mice revealed TP73 as a key regulator of central nervous system development. These animals have several neurological abnormalities such as hippocampal dysplasia and reduced cerebral cortex. Our group has previously demonstrated the role of TP73 in neural stem/progenitor cells self-renewal. However, this p73-function does not explain some of the neurological defects observed in p73KO mice, like hydrocephalus. The adult subventricular zone contains one of the neurogenic niches of the brain, comprised of neural stem cells (B-cells), proliferating progenitor cells (C-cells), neuroblast (A-cells) and ependymal cells (ECs). This germinal centre is organized in structures called pinwheels in which ECs surround B-cells. We have has demonstrated that p73 deficiency halts ECs maturation hindering SVZ neurogenic cytoarchitecture and disturbing the correct neurogenic function.EC ciliary function is fundamental for appropriate brain function. In this regard, EC ciliogenesis is dependent upon the establishment of Planar Cell Polarity (PCP). We have addressed whether lack of p73 affected EC ciliogenesisand have observed that cilia development is altered in p73KO mice. Strikingly, ECs lacking p73 fail to establish PCP. Our data have revealed a novel p73 function in ependymal cell maturation and ciliogenesis, and moreover, identify p73 as a key regulator of PCP. Work supported by: Grant LE310U14 (Junta de Castilla-León), Grants SAF2012-36143 and SAF2015-71381-R (Ministerio de Ciencia e Innovación). 101 Pósters / Posters P13-18 Alterations in hippocampal cells genome are required for memory formation Ángel Manuel Carrión Rodríguez Universidad Pablo de Olavide, Sevilla, ES The memory formation phenomenon is a complex process that involve information codification and storage. During this process acquired information goes from an initial and labile memory state to and stable memory state by a protein synthesis-dependent mechanism called as consolidation. Alterations of protein synthesis requires of chromatin remodelling, through epigenetic alterations, together with transcription factor activation. In the last decade, we have study the role of some epigenetic modifications in learning and memory processes. Histone acetylation and poly-ADP-ribosylation (PARylation) are processes link to chromatin remodelling and gene expression activation. We demonstrated that histone H3 acetylation and histone H1 PARylation are involved in hippocampal-dependent memory storage. In addition, both epigenetic modifications happen sequentially after training session in order to promote IEG expression. In addition to de novo protein synthesis, it has been postulated that neuronal genome sequence alterations may encode memory trace. This hypothesis seems to be corroborate by two recent discoveries: a, physiologic electric activity provoke transient DNA breaks in hippocampus; and b, neuronal genome is a mosaic. During the last years, we discovered that both DNA metabolism, DNA breaks and repair mechanisms, and LINE1 retrotransposition are induced after a training session. Interesting, pharmacologic or genetic inhibition of both processes produced long-term memory formation impairment. All these data together indicate that transient alterations in epigenetic factors as well as stable DNA sequence alteration contribute to the de novo new protein expression programs required for stabilized the long-term memory trace to from persistent memories. P13-19 Expression and regulation of the methionine cycle genes in the mouse cochlea Néstor Vallecillo1, María A. Pajares2, Steven Zeisel3, Lourdes Rodríguez de la Rosa4, Isabel Varela-Nieto4 1 Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, ES, 2Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM); Instituto de Investigación Sanitaria La Paz (IdiPAZ), Madrid, ES, 3Department of Nutrition, School of Public Health, University of North Carolina Chapel Hill, North Carolina, Chapel Hill, US, 4Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM); Instituto de Investigación Sanitaria La Paz (IdiPAZ); CIBERER, ISCiii , Madrid, ES The impairment in cochlear homocysteine (Hcy) metabolism has been associated with hearing loss(1). Epidemiological studies revealed a correlation between hearing loss and increased plasma Hcy(2). Hcy represents a key point in the remethylation pathway of the methionine cycle. Betaine homocysteine S-methyltransferases (BHMT) are responsible for the remethylation of Hcy. Cochlear BHMT and Hcy metabolism have been reported to have organ-specific traits(1). Hearing was evaluated by auditory brainstem response (ABR) recording of 2-3 month-old mice of different strains. Cochlear samples were used for analysis of selected methionine cycle enzymes by RT-qPCR and Western blotting. The methionine cycle was studied in control and VS noise-exposed C57BL/6J mice (105 dB SPL, 2-20 kHz, 30 min). Two days post-noise, exposed mice showed increased ABR thresholds and reduced cochlear expression of Bhmt and Bhmt2. No changes were observed inMat2a, Mat2b and Ahcy expression or in BHMT, BHMT2, MATα2, MATβ and AHCY levels. As reported(1), a BHMT band of 68 kDa was found in C57BL/6J 102 XXXIX Congreso SEBBM cochleae. In contrast, the canonical BHMT band of 45 kDa was detected in Ola:MF1 mice, suggesting a strain-specific regulation. To further explore the role of BHMT, we studied the hearing phenotype of Bhmt+/+, Bhmt+/-and Bhmt-/-C57BL/6x129sv mice(3). Noise exposure caused a significant and irreversible increase in ABR thresholds in Bhmt-/- with respect to Bhmt+/+ mice. Bhmt-/-but notBhmt+/+ mouse cochleae showed significantly increased levels of Bhmt2, no differences were detected in Mat2a, Mat2b and Ahcy. These data suggest that BHMT plays a central role in cochlear methionine metabolism and that Bhmt2 could carry out a compensatory role in cochlear protection against noise injury in the absence of Bhmt. This work was supported by Spanish “Ministerio de Economía y Competitividad” SAF2014-53979-R and FP7-INNOVA-2-AFHELO grants to IVN. References [1] Martínez-Vega et al., FASEB J. 29: 418–432. 2015. [2] Gok et al., Auris Nasus Larynx, 31:19-22. 2004. [3] Teng et al., J Biol Chem. 286(42):36258-67. 2011. P13-20 The APC/C-Cdh1-Rock2 pathway maintains neuronal network integrity in the adult brain Verónica Bobo-Jiménez, María Delgado-Esteban, Juan P. Bolaños, Ángeles Almeida Institute of Biomedical Research of Salamanca, University Hospital of Salamanca-University of Salamanca and Institute of Functional Biology and Genomics, University of Salamanca-CSIC, Salamanca, ES Dendrite network disruption contributes to the pathology of neurodegenerative disorders, such as Alzheimer’s disease (AD). However, the underlying molecular mechanisms are unknown. Here, we show that postnatal deletion of Cdh1, a cofactor of the anaphase-promoting complex/ cyclosome (APC/C) ubiquitin ligase, in neurons (Cdh1 cKO), disrupted dendrite arborization and caused dendritic spines and synaptic loss in the cortex and hippocampus, concomitantly with memory impairment and neurodegeneration during mouse adulthood. We found that the dendrite destabilizer Rho protein kinase-2 (Rock2), which accumulates in the brain of AD patients, is an APC/CCdh1 substrate in vivo, and that Rock2 protein and activity increased in cortex and hippocampus of Cdh1 cKO mice. In these, inhibition of Rock2 activity, using the clinically approved drug Fasudil, prevented dendritic network disorganization, memory loss and neurodegeneration. Thus, APC/CCdh1-mediated degradation of Rock2 maintains dendritic network, memory formation, and neuronal survival thereby pharmacological inhibition of aberrantly accumulated Rock2 is a suitable therapeutic strategy against neurodegeneration. P13r-21 The p53 Arg72Pro single nucleotide polymorphism determines amyloid-ß neurotoxicity upon Cdk5-induced p53 stabilization Rebeca Lapresa, Juan Pedro Bolaños, Ángeles Almeida Instituto de Biología Funcional y Genómica (IBFG), CSIC-Universidad de Salamanca and Instituto de Investigación Biomédica de Salamanca (IBSAL), Hospital Universitario de Salamanca, Salamanca, ES The p53 tumor suppressor protein functions as a key regulator of cell apoptosis and has been described to accumulate in affected brain areas from Alzheimer’s disease (AD) patients. However, the role of p53 in AD is controversial. This protein naturally occurs in humans in two functional variants with single nucleotide polymorphism (SNP) resulting in Arg or Pro Salamanca 2016 at residue 72 that modulates the apoptotic activity of the p53 protein. Here, we evaluated the impact of the p53 Arg72Pro SNP on amyloid-ß (Aß)-induced neuronal apoptosis. Exposure of cortical neurons in primary culture to Aß triggered cyclin dependent kinase-5 (Cdk5)-induced p53 phosphorylation and stabilization, leading to mitochondrial dysfunction and neuronal apoptosis, which were prevented by genetic inhibition of Cdk5 and p53. In cortical neurons from humanized Tp53 Arg72Pro knock-in mice, we found that neurons expressing the Arg72-p53 polymorphic variant showed a higher susceptibility to Aß-mediated mitochondrial depolarization and neuronal death when compared with the Pro72-p53 variant. This effect was abrogated by ectopically expressing apoε4 –a well-known major risk factor for AD- but not apoε3 and apoε2.These results indicate that neuronal susceptibility to Aß toxicity is conditioned by the Tp53 Arg72Pro SNP, hence suggesting that this SNP may be considered as a genetic biomarker for AD risk in apoε4 non-carriers. P13-22 L-lactate mediated neuroprotection against glutamate-induced excitotoxicity requires ARALAR-MAS pathway Beatriz Pardo Merino, Irene Llorente-Folch, Carlos Rueda, Irene Pérez-Liébana, Jorgina Satrústegui Centro de Biología Molecular Severo Ochoa (CBMSO), CSIC-UAM; Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER); Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Madrid, ES ARALAR/AGC1 is the aspartate-glutamate carrier in neuronal mitochondria and constitutes the regulatory step in the malate-aspartate NADH shuttle (MAS) in brain. The lack of ARALAR expression provokes, in mice, neurological disturbances such as epilepsy, hypomyelination, and deficits in motor coordination; and in human, the rare disease “global cerebral hypomyelination” with similar characteristics to those described for mice. Glutamate excitotoxicity plays a key role in the induction of neuronal cell death occurring in many neuropathologies, including epilepsy. ARALAR deficiency did not aggravate glutamate-induced neuronal cell death in vitro; however, L-lactate acting as an additional metabolic source rescued from glutamate-induced neuronal death only control, but not ARALAR-deficient neurons. L-lactate ameliorated glutamate-stimulated neuronal respiration partially preventing the decrease in the cytosolic ATP/ADP ratio induced by glutamate, and drastically reduced reactive oxygen species formation in mitochondria (measured as accumulation of 8-oxoguanosine) only in ARALAR-expressing neurons. Systemic administration of the glutamatergic agonist kainic acid (KA) is a well characterized model to study epilepsy-induced brain damage. The loss of half-a-dose of ARALAR in aralar+/- mice produced a normal lifespan with no apparent phenotypic disturbances in basal conditions. However, these aralar+/mice suffered enhanced KA-induced seizures and hippocampal neuronal cell death with respect to control animals, in an in vivo model of excitotoxicity in which increased L-lactate levels and L-lactate consumption have been proven. Failure to use lactate is possibly responsible for the exacerbated in vivo excitotoxicity in aralar+/- mice. Pósters / Posters of trisomy Ts16 and euploid mice. We report that in the plasma membrane of euploid cells an increase in phosphatidylcholine concentrations occurs in the presence of oleic acid. However, in trisomic cells oleic acid failed to increase phosphatidylcholine incorporation into the plasma membrane. Gene expression analysis of trisomic cells revealed that the phosphatidylcholine biosynthetic pathway was deregulated. Taken together, these results suggest that the overdose of specific genes in trisomic lines delays differentiation in the presence of oleic acid. The dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) gene is located on human chromosome 21. DYRK1A contributes to intellectual disability and the early onset of Alzheimer’s disease in DS patients. Here we explored the potential role of Dyrk1A in the reduction of phosphatidylcholine concentrations in trisomic cells in the presence of oleic acid. The downregulation of Dyrk1A by siRNA in trisomic cells returned phosphatidylcholine concentrations up to similar levels to those of euploid cells in the presence of oleic acid. Thus, our results highlight the role of Dyrk1A in brain development through the modulation of phosphatidylcholine location, levels and synthesis. P13-24 Toxins and pathogenic LRRK2 cause alterations in secretory vesicle age Antonio Jesús Lara Ordóñez1, Mar Martínez-Salvador1, Jesús Madero-Pérez1, Elena Fernández1, Evy Lobbestael2, Veerle Baekelandt2, Sabine Hilfiker1 1 Instituto de Parasitología y Biomedicina López-Neyra, Granada, ES, 2Laboratory for Neurobiology and Gene Therapy, Leuven, BE Neurons and neuroendocrine cells are packed with secretory vesicles, only a few of which seem to be releasable upon appropriate stimuli. Previous studies have shown that vesicles display functional and spatial segregation according to age, whereby newly synthesized vesicles seem to be preferentially released. Here, using various fluorescent cargo proteins which change colour over time and fused to neuropeptides, we evaluated effects of toxins associated with Parkinson´s disease on secretory vesicle age and distribution in dopaminergic cells in culture. We found alterations in the percentage of old versus newly synthesized vesicles largely consistent with previously observed toxin-induced effects on secretion and/or axonal transport. Various reagents which modulate either proteasome function or macroautophagy indicate that preferentially older secretory vesicles are subject to degradation by autophagy. In addition, older vesicles were preferentially excluded from neurites, indicating the existence of a cellular mechanism able to distinguish vesicles according to their age for intracellular transport as well as degradation. Pathogenic LRRK2 caused similar alterations in vesicle age, with implications for previously described LRRK2-mediated dopaminergic transmission deficits. P13-25 Analysis of the Akt/mTORC1 pathway after glucose deprivation versus oxygen-glucose deprivation in neurons and nervous system Ana Velasco Criado, Marian Hijazi, José María Medina Universidad de Salamanca, Salamanca, ES Mario Villa González1, Carlota Gil Martín1, Laura Mateos2, Francisco Wandosell3, María José Pérez Álvarez4 1 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, ES, 2Centro Nacional de Biotecnología. CSIC-CNB, Cantoblanco, ES, 3Centro de Biología Molecular Severo Ochoa, CSIC-UAM y CIBERNED, Cantoblanco, ES, 4Centro de Biología Molecular Severo Ochoa, CSIC-UAM Dpto. Biologia (Fisiología Animal), Facultad de Ciencias, UAM, Cantoblanco, ES Aberrant formation of the cerebral cortex could be attributed to the lack of suitable substrates that direct the migration of neurons. Previous work carried out at our laboratory has shown that oleic acid is a neurotrophic factor. In order to characterize the effect of oleic acid in a cellular model of Down’s syndrome (DS), here we used immortalized cell lines derived from the cortex Ser/Thr kinase mTOR (mammalian target of rapamycin) with a key role in cell growth and metabolism regulates the balance between anabolic and catabolic processes, linking up extracellular stimuli: growth factors (GFs) and nutrients (glucose, amino acids, etc.) with intracellular processes: transcription, translation, synthesis of lipids/proteins and autophagy. In P13-23 Restrained phosphatidylcholine synthesis in a cellular model of Down’s syndrome is associated with the overexpression of Dyrk1A 103 Pósters / Posters XXXIX Congreso SEBBM neurons, the activation of the Akt/mTOR induces increase of lipids/proteins synthesis and attenuate autophagy; and its downregulation may increase autophagy exacerbated in neurodegeneration processes such as Alzheimer Disease´s (AD). mTOR comprises two different complex: mTORC1 and mTORC2, with differences in their functions and susceptibility to rapamycin. Both mTOR kinases are stimulated by a plethora of GFs, through membrane receptors RTKs or GRPC´s/PI3K/PDK1/Akt at different levels, while mTORC1 is regulated by Akt whereas mTORC2 regulates Akt. Extracellular glucose (Glu), via ratio AMP/ATP, activates AMPK that also regulates mTORC1. We are interested in the analysis of the relative contribution to mTORC1 activation of glucose and/or oxygen in models of cerebral ischemia. Our initial data indicated a differential coupling of AMPK, Akt and mTORC1 in ischemia models. To evaluate the neuronal contribution we asses the status of Akt/mTORC1 activity after deprivation of different combinations of GFs, Glu or oxygen at distinct time points using SHSY5Y neuroblastoma cells. MTORC1 activity levels was inferred by phosphorylation of p706SK and p-EBP1 and Akt activity was assessed by phosphorylation of Thr308. Our data indicated a differential response of Akt, mTORC1 and AMPK after deprivation of GF, oxygen and/or glucose (OGD). These results will be discuss in the context of brain ischemia model. P13r-26 Role of gabra2, GABAA receptor alpha-2 subunit, in CNS development Verónica González Núñez Universidad de Salamanca, Salamanca, ES P13-28 The role of PTEN in Alzheimer’s pathology gabra2 gene codes for the alpha-2 subunit of the GABAA receptor, one of the ionotropic receptors which has been related to anxiety, depression and other behavioural disorders, including drug dependence and schizophrenia. GABAergic signalling also plays a role during development, by promoting neural stem cell maintenance and renewal. To investigate the role of gabra2 in CNS development, gabra2 deficient zebrafish were generated. The pattern of proliferation during the embryonic development was disrupted in morphant embryos, which also displayed an increase in the number of apoptotic nuclei mainly at the mid- and hindbrain regions. The expression of several genes (notch1, pax2, fgf8 and wnt1) known to contribute to the development of the central nervous system was also affected in gabra2 morpholino-injected embryos, although no changes were found for pax6a and shh a expression. The transcriptional activity of neuroD (a proneural gene involved in early neuronal determination) was down-regulated in gabra2 deficient embryos, and the expression pattern of gad1b (GABA-synthesizing enzyme GAD67) was clearly reduced in injected fish. I propose that gabra2 might be interacting with those signalling pathways that regulate proliferation, differentiation and neurogenesis during the embryonic development; thus, gabra2 might be playing a role in the differentiation of the mesencephalon and cerebellum. Given that changes in GABAergic circuits during development have been related to several psychiatric disorders, such as autism and schizophrenia, this work might be helpful to understand the role of neurotransmitter systems during CNS development and to assess the developmental effects of several GABAergic drugs. P13-27 The Mdm2 309T>G polymorphism modulates the MDM2-p53 signaling pathway and conditions the functional outcome of stroke patients 1 1 María Esther Ramos Araque , Cristina Rodríguez González , Irene Sánchez Morán1, José Carlos Gómez Sánchez1, Tomás Sobrino2, José Castillo2, Ángeles Almeida Parra1 1 Instituto de Investigación Biomédica de Salamanca, Hospital Universitario de Salamanca; Instituto de Biología Funcional y Genómica, Universidad de Salamanca-CSIC, Salamanca, ES, 2 Instituto de Investigación Sanitaria de Santiago de Compostela, Hospital Universitario de Santiago de Compostela, Santiago de Compostela, ES 104 The highly variable prediction of functional outcome after stroke could be the effect of different genetic backgrounds to apoptosis. MDM2 protein is the main negative regulator of p53, which plays an important role on neuronal apoptosis after cerebral ischemia. Recently, we found that the Arg72Pro single nucleotide polymorphism (SNP) of p53 regulates the pro-apoptotic activity of the protein and conditions neuronal vulnerability to ischemia-induced apoptosis and functional outcome of stroke patients. In MDM2 a SNP in the promoter gene (309T>G) modulates levels of Mdm2 expression. However, the role of Mdm2 309T>G SNP in stroke prognosis remains unknown. Here we study the association of the Mdm2 309T>G SNP and the functional prognosis after stroke in blood samples from 408 patients with ischemic stroke and 206 with intracerebral hemorrhage. Functional outcome at 3 and 12 months was evaluated by the modified Rankin scale. Mononuclear cells from healthy individuals were collected to measure levels of MDM2 mRNA and protein. We found that mononuclear cells harboring the Mdm2 TT genotype have lower levels of MDM2 mRNA and protein than those with the Mdm2 GG and Mdm2 TG genotypes, which may affect p53 stabilization and then cell vulnerability to ischemia-induced apoptosis. Furthermore, patients harboring the Mdm2 TT genotype showed poor functional outcome at 3 and 12 months following ischemic (p=0.003) and hemorrhagic (p<0.0001) stroke. Our results suggest that the Mdm2 309T>G SNP modulates the MMD2p53 signaling pathway and then cell susceptibility to apoptosis, which conditions the functional outcome of patients after stroke. Shira Knafo Ikerbasque, Basque Foundation for Science, Bilbao, ES Dyshomeostasis of amyloid-β peptide (Aβ) is responsible for synaptic malfunctions leading to cognitive deficits ranging from mild impairment to full-blown dementia in Alzheimer’s disease. Aβ appears to skew synaptic plasticity events toward depression. We found that inhibition of PTEN, a lipid phosphatase that is essential to long-term depression, rescued normal synaptic function and cognition in cellular and animal models of Alzheimer’s disease. Conversely, transgenic mice that overexpressed PTEN displayed synaptic depression that mimicked and occluded Aβ-induced depression. Mechanistically, Aβ triggers a PDZ-dependent recruitment of PTEN into the postsynaptic compartment. Using a PTEN knock-in mouse lacking the PDZ motif, and a cell-permeable interfering peptide, we found that this mechanism is crucial for Aβ-induced synaptic toxicity and cognitive dysfunction. Our results provide fundamental information on the molecular mechanisms of Aβ-induced synaptic malfunction and may offer new mechanism-based therapeutic targets to counteract downstream Aβ signaling. P14. Parasitología molecular / Molecular parasitology P14-1 Aptámeros como herramientas para la detección de Leishmania Víctor Manuel González Muñoz1, Celia Pinto Díez1, Valerio Frezza1, Manuel Soto2, Ana García Sacristán3, M. Elena Martín Palma1 1 IRYCIS-Hospital Ramón y Cajal, Madrid, ES, 2Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, ES, 3Aptus Biotech SL, Madrid, ES Los parásitos del género Leishmania producen la leishmaniasis, la cual afecta a millones de personas en todo el mundo. Existen distintos sistemas Salamanca 2016 diagnósticos para la leishmaniasis, muchos de ellos para uso veterinario. Los comúnmente utilizados en diagnóstico clínico están basados en la detección de anticuerpos generados por el sistema inmunológico del paciente o la amplificación de regiones específicas del genoma del parásito mediante PCR. Ambos sistemas presentan problemas ya que la respuesta inmune puede mantenerse elevada aún bastante tiempo después de que se supere la enfermedad y, por otra parte, aunque la detección del DNA del parásito indica la presencia del mismo, la baja presencia de Leishmania en sangre aumenta el número de falsos negativos. La alternativa actual es realizar PCR de muestras de médula ósea, en las que el número de parásitos es sensiblemente mayor. Sin embargo, se trata de una técnica invasiva que produce molestias y puede resultar peligrosa para el paciente. Los aptámeros son moléculas de RNA o DNA de cadena sencilla que, debido a la estructura terciaria particular que son capaces de adquirir en función de su secuencia, interaccionan con su diana con elevadas afinidad y especificidad. Hasta el momento, hemos obtenido aptámeros frente a diferentes proteínas de L. infantum (H2A, H3 y PABP) y demostrado que son capaces de detectar sus dianas en distintos sistemas como ELONA, western blot o slot blot. Nuestro propósito es desarrollar nuevos sistemas diagnósticos basados en el reconocimiento de proteínas del parásito (más abundantes que el propio genoma) por aptámeros específicos que, eventualmente, pueden ser amplificados por PCR, lo que podría permitir alcanzar mayor sensibilidad que los sistemas actualmente disponibles. P14-2 Predicción de proteínas de membrana en el intestino del argásido Ornithodoros moubata: Selección de antígenos para el desarrollo de vacunas Prosper Obolo-Mvoulouga, Ricardo Pérez-Sánchez, Raúl Manzano-Román, Ana Oleaga Pérez IRNASA, CSIC, Salamanca, ES El argásido Ornithodoros moubata es el principal vector en África de la peste porcina africana y de la fiebre recurrente humana y representa un buen modelo para investigar el proceso digestivo en argásidos a nivel molecular. En el intestino de las garrapatas se expresan proteínas vitales para su fisiología y para la infección y transmisión de patógenos, las cuales son de interés como potenciales dianas para el desarrollo de vacunas anti-garrapata y vacunas bloqueantes de la transmisión de patógenos. En la búsqueda de antígenos vacunales, se prioriza a los expresados en la membrana plasmática del enterocito, porque están expuestos a la luz intestinal y resultan accesibles a los anticuerpos ingeridos con la sangre del hospedador inmunizado. En el presente trabajo, el transcriptoma intestinal de O. moubata se analiza in silico con objeto de definir las proteínas de membrana expresadas por el enterocito, incluyendo su anotación funcional y potencial antigenicidad. Se identifican 1725 transcritos con 1 dominio transmembrana y 1348 con 2 o más, anotándose respectivamente 506 y 601 proteínas. Su caracterización funcional revela diferencias entre ambos grupos. La mayor parte de las proteínas con actividad catalítica y unidora presentan 1 dominio transmembrana y las que tienen función transportadora presentan varios dominios. Se identifican 91 proteínas potencialmente localizadas en la membrana plasmática, la mayoría con múltiples dominios transmembrana y actividad transportadora y, de ellas, 59 son potencialmente antigénicas (score Vaxijen >0,5). Varias de éstas (tetraspanina, aquaporina, la homologa a la Bm86) ya han sido definidos como candidatos vacunales frente a otras garrapatas y otros parásitos y su expresión en el intestino de O. moubata se ha confirmado mediante estudios proteómicos. Estos resultados han permitido seleccionar varios antígenos de interés, que serán validados en pruebas de vacunación en animales. Trabajo financiado por el Ministerio de Economía y Competitividad, Proyecto AGL2013-42745-P. Pósters / Posters P14-3 Análisis del transcriptoma del intestino del argásido Ornithodoros moubata e identificación de genes implicados en la alimentación, digestión de la sangre y la defensa frente a patógenos Prosper Obolo Mvoulouga, Ana Oleaga Pérez, Raúl Manzano Román, Ricardo Pérez Sánchez IRNASA, CSIC, Salamanca, ES Las garrapatas son parásitos hematófagos de importancia médica y veterinaria porque transmiten numerosos patógenos que afectan al ganado, los animales de compañía y las personas. El intestino medio de las garrapatas es el órgano encargado de digerir la sangre y absorber los nutrientes imprescindibles para su supervivencia y reproducción. Además constituye la primera barrera defensiva a la que se enfrentan los patógenos ingeridos con la sangre. El intestino es pues parte de la interfase hospedador-garrapata-patógeno y en él se expresan proteínas vitales para la garrapata y para la transmisión de los patógenos. El estudio de los genes diferencialmente expresados durante la alimentación permite explorar nuevas estrategias de control de las garrapatas y conocer la fisiología de la digestión en argásidos a nivel molecular, asunto sobre el que apenas existen datos. El objetivo del presente trabajo es la identificación de los genes de O. moubata diferencialmente expresados durante la digestión de la sangre, para lo cual se ha secuenciado y analizado el transcriptoma intestinal en condiciones basales (ejemplares no alimentados) y en las primeras fases de la digestión (a las 48 horas de la alimentación). Se han identificado 8.026 genes diferencialmente expresados entre garrapatas alimentadas y no alimentadas. Su clasificación funcional revela que determinados grupos de genes con actividad peptidasa implicados en la digestión de hemoglobina y los implicados en respuestas defensivas, detoxificación y respuesta al estrés asociado a la toma de sangre se encuentran fuertemente regulados y su nivel de expresión aumenta significativamente tras la alimentación. Por el contario, el nivel de expresión de genes involucrados en procesos de endocitosis y transporte intracelular y en el metabolismo y transporte de carbohidratos y lípidos no varía tras la ingesta de sangre. Este trabajo proporciona datos inéditos acerca del efecto de la alimentación en el transcriptoma intestinal de esta especie. Trabajo financiado por el Ministerio de Economía y Competitividad, Proyecto AGL2013-42745-P. P14-4 Análisis de la expresión génica diferencial mediante RNAseq en los promastigotes metacíclicos de Leishmania major Alberto Rastrojo Lastras, José Carlos Solana, Manuel Soto, Begoña Aguado, José M. Requena Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, ES Los protistas del género Leishmania son causantes de las leishmaniasis, un grupo de graves enfermedades que afectan a humanos y otros mamíferos en muchas zonas del planeta. Estos parásitos son particularmente interesantes por su regulación génica postranscripcional. La transcripción policistrónica y los procesos de trans-splicing y poliadenilación son los responsables de generar los niveles adecuados de cada uno de los mRNAs en cada una de las fases de su ciclo vital. El parásito requiere de dos huéspedes, un insecto vector y un mamífero donde multiplicarse, idealmente de forma crónica. Para la transmisión exitosa desde el insecto hasta el huésped mamífero el parásito experimenta un proceso denominado metaciclogénesis en el que adquiere la capacidad de resistir a la lisis por complemento y a otros elementos de defensa del sistema inmunitario. Hemos estudiado los cambios en el transcriptoma del parásito asociados a la diferenciación desde 105 Pósters / Posters las formas procíclicas a las formas metacíclicas, utilizando Leishmania major, por ser la especie en la que el transcriptoma ha sido detalladamente anotado. Se prepararon tres réplicas biológicas de parásitos recién aislados de ratones infectados, separándose promastigotes procíclicos y metacíclicos. Se aislaron las formas metacíclicas (alrededor del 1% de los parásitos en la fase estacionaria de cultivo), utilizando el método de selección negativa con aglutinina de cacahuete. Una vez extraído el RNA, se procedió a la secuenciación masiva de la fracción de poli-A y las lecturas se mapearon frente al transcriptoma de referencia, determinándose los niveles de expresión de cada transcrito. Se han identificado transcritos cuyos niveles varían significativamente durante la metaciclogénesis. Entre los transcritos cuyos niveles disminuyen están los codificantes de las histonas, en consonancia con que en la fase metacíclica el parásito no se divide. Otros transcritos que varían codifican para proteínas de unión a RNA y transportadores de membrana. P14-5 Role of ubiquitin-activating (E1) and ubiquitin-conjugating (E2) genes in the infective stage of Leishmania infantum Jaime Larraga, Ana Alonso, Pedro J. Alcolea, Vicente Larraga Centro de Investigaciones Biológicas (CSIC), Madrid, ES Leishmaniasis, a disease caused by protozoa of the genus Leishmania, affects about two million people all over the world. The main clinical forms are: cutaneous, mucocutaneous and visceral. In Europe, the visceral disease is caused by L. infantum and constitutes a zoonosis transmitted through the bite of sand flies of the genus Phlebotomus. At the infective stage of Leishmania, within the insect vector, a certain number of genes are overexpressed and may be related with the infection ability of the parasite. Between these genes are the ubiquitin-activating enzyme (E1) and the ubiquitin-conjugating enzyme (E2). The ubiquitin-activating enzyme E1 catalyzes the first step of the ubiquitination reaction that marks proteins for degradation via the proteasome. The E2 enzyme accepts ubiquitin from the E1 complex and catalyzes its covalent attachment to other proteins.Both genes have been cloned in pQE-30 vector and expression was carried out in Escherichia coli strain M15. These proteins have been purified using Ni2+ affinity chromatography. For the detection of the abundance of the protein in the growth curve of the parasite, a policlonal antibody has been obtained for both proteins. The modeling of both proteins was performed using PyMol software and the protein structure has been compared with the corresponding human ones. The E1 enzyme seems to be a homodimer structure and is different to the human orthologous. The E2 enzymes is similar to the A chain of the human orthologous. The alignments against the orthologous sequences have been made using ClustalW and BlastP. In the case of the ubiquitin-activating gene (E1), the homology with different species of the Leishmania genusis approximately 90%. With the related in Trypanosoma cruzi the homology is 41%. With Homo sapiens the homology drops to 33%. The ubiquitin-conjugating gene (E2) shows a homology of 92% with L. major and 82% with L. braziliensis. With T. cruzi the homology drops to 68%. In the case of H. sapiens the homology is reduced to 69%. P14-6 Influencia del microambiente en el transcriptoma de promastigotes de Leishmania infantum: cultivo axénico vs flebotomo Ana Alonso Ayala1, Pedro J. Alcolea1, María A. Degayón1, Maribel Jiménez2, Ricardo Molina2, Vicente Larraga1 1 Centro de Investigaciones Biológicas, CSIC, Madrid, ES, 2Instituto de Salud Carlos III, Madrid, ES Los distintos perfiles de expresión génica se han obtenido comparando promastigotes extraidos del medio natural, el vector Phlebotomus 106 XXXIX Congreso SEBBM perniciosus (Pro-Pper), con los de cultivo axénico. Hemos seleccionado tres poblaciones en cultivo: promastigotes en fase estacionaria (Pro-Stat), promastigotes metacíclicos obtenidos por selección negativa con aglutinina de cacahuete (Pro-PNA-) y amastigotes intracelulares, obtenidos a partir de la infección in vitro de células fagocíticas de la línea celular U937 (Amas). El análisis transcriptómico se ha llevado a cabo mediante microarrays genómicos de Leishmania infantum; en el caso de la comparación de los promastigotes obtenidos del medio natural con los de fase estacionaria de cultivo axénico (Pro-Pper/Pro-Stat), también mediante spliced leader RNA seq (SL-RNAseq). Los resultados obtenidos indican una tasa de sobre-expresión menor de genes en amastigotes, lo que apoya la hipótesis de la pre-adaptación de los promastigotes diferenciados para su paso al medio intracelular en el hospedador mamífero. También hemos comprobado que los Pro-Pper son más infectivos que los Pro-PNA-, se sobre-expresan genes relacionados con la infectividad, como los genes que participan en la síntesis de GIPL, LPG y PPG, además de obtener un mayor número de células U937 infectadas in vitro. En la comparación Pper/Pro-Stat la mayoría de los genes diferencialmente expresados están implicados en regulación de la expresión génica y señalización intracelular. Los datos de SL-RNAseq confirman la infectividad de Pro-Pper, se sobre-expresan genes directamente relacionados con la metaciclogénesis como ATG8 (autofagia), HASPA1, HASPB, MBAP, AMA1 y META2. La influencia del cultivo axénico debe ser evaluada caso por caso, ya que puede afectar a los resultados de expresión. Los distintos estudios del transcriptoma permiten laselección de marcadores relacionados con la infectividad del parásito y su posible utilización como posibles dianas terapéuticas o nuevos candidatos de antígenos protectores para el desarrollo de vacunas frente a la leishmaniasis. P14-7 Obtainment and proteomic characterization of exosomes from sheep´s fertile cyst hydatid fluid: definition of potential exosomal markers for Cystic Echinococcosis Raúl Manzano Román1, Carlos Sánchez Ovejero1, Adriano Casulli2, Mar Siles Lucas1 1 Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Salamanca, ES, 2Istituto Superiore di Sanità, Rome, IT Cystic echinococcosis (CE) is a chronic and complex disease with a limited understanding of parasite-host interactions. These interactions can be modulated by a dynamic crosstalk between extracellular vesicles (EVs) harboring proteins and nucleic acids. Diagnosis of CE relies on tools with no satisfactory performances and prognosis-associated follow-up test are still lacking. There is, therefore, a need for the identification of specific markers. The cargo of EVs has been shown to be changed in disease, as is their abundance in the circulation. Studies showing a correlation with disease progression have made these vesicles a favorable target for diagnostic purposes (circulating antigens or miRNAs). In relation with CE, these novel sources for plasma-derived markers may also serve to improve viability assessment and the basis for alternative therapeutics. Floatation into Iodixanol gradients differently separates subtypes of EVs. Thus, in the present study, an exosome enriched EV fraction was isolated from hydatid fluid (HF) of fertile sheep cysts by filtration, ultracentrifugation and further purification by the OptiPrepTM method. GO analyses showed endosome derived EVs -bona fide exosomes- that were further visualized by Nanoparticle Tracking Analyses and parasite specific proteins were further identified by electron microscopy after immuno-gold labelling. Label-free semi-quantitative proteomic analysis of this fraction determined specific parasite exosomal markers of potential clinical interest. This proteomic characterization identified a panel of potentially interesting diagnostic proteins: unique canonical exosomal markers -like TSG101and vesicle-surface proteins such as Protein DJ 1 or beta enolase of parasite origin potentially useful as CE biomarkers. Salamanca 2016 P14r-8 Using attenuated Leishmania parasites lacking for HSP70-II gene as a vaccine against leishmaniasis José Carlos Solana Morcillo, Laura Corvo Villén, José María Requena Rolanía, Manuel Soto Álvarez Centro de Biología Molecular Severo Ochoa (CBMSO-UAM), Madrid, ES Leishmaniases are a group of neglected tropical diseases caused by the infection with the protozoan parasite Leishmania. Since patients cured of leishmaniasis develop protective immunity to reinfection, vaccination should be feasible. The only effective vaccine for controlling the human disease is leishmanization, a procedure abandoned due to safety concerns. During the last decade advances in Leishmania molecular genetics have allowed the design of genetically modified parasites resulting in attenuated strains. A Leishmania infantum knockout, lacking for HSP70 type II gene (LiΔHSP70-II) was developed in our group and used as a live vaccine in a resistant model of cutaneous leishmaniasis. C57BL/6 mice subcutaneously immunized with LiΔHSP70-II did not show any side effects due to vaccination and were able to control an infective challenge with a virulent strain of Leishmania major. Vaccinated mice mounted an early and controlled inflammatory response resulting in the absence of a lesion in the site of infection (ear dermis). These promising results support the strategy of generating a new line of ΔHSP70-II attenuated parasites on cutaneotropic species such as Leishmania major. Two different approaches have been considered: replacing of both alleles, after two rounds of transfection with antibiotic-resistance cassettes, and CRISPR-Cas9-mediated gene knockout. This new technique can be useful to solve some difficulties frequently observed in the traditional method due to Leishmania genomic plasticity, mosaic aneuploidy and creation of extra copies of the targeted gene. Funding: EU-7th. 603181 (MuLeVaClin). FIS-PI14/00366 (FEDER FUNDING). Pósters / Posters as a higher replication rate within macrophages exposed to Leishmania overexpressing YinP. All together, these results strongly suggest a relation between YinP gene expression and leishmaniasis pathogenesis. In order to localize YinP expression in the leishmanial cell, pXG-mCherry34-YinP plasmid was constructed. In this vector, YinP gene was inserted directly near mCherry, to generate fluorescent fusion proteins expressed in the parasites. Fluorescent microscopy disclosed that red fluorescence of mCherry-YinP was localized in the nucleus. Further experiments need to be done to elucidate other cellular location of YinP protein. Finally, we aim to analyze the involvement of YinP in Leishmania virulence in vivo. To that end, we will use the integrative constitutive expression vector pLEXSY to generate new strains (pLEXSY-YinP) also overexpressing the gene. We’ll then assess the infectivity of those mutants in a murine in vivo model. Therefore, a study of both, parasitic burden within target organs such as liver and spleen, and the lesion progression during the infection will be performed. P14-10 Epigenetic variation in malaria parasites: sex, drugs and… adaptation Alfred Cortés Closas ICREA y ISGlobal, Barcelona, ES Two cells with identical genomes can have dramatically different phenotypes. If the differences are transmissible from one generation to the next, the differences are considered epigenetic. Recent research has established that the genes that control key processes in the biology of malaria parasites, such as nutrient uptake, resistance to some toxic compounds or sexual conversion, are regulated at the epigenetic level. Differences between individual parasites in the expression of these genes confer phenotypic plasticity and can mediate the adaptation of parasite populations to changes in their environment. In this talk I will present our latest results in the study of the adaptation of P. falciparum parasites to changes in their environment, with a focus on pfap2-g, the master regulator of sexual conversion, and adaptation to heat-shock. P14-9 YinP gene: New approach for understanding the mechanism of acquiring infectivity by Leishmania parasites? P14-11 The lack of type I IFN may enhance Leishmania major infection in C57BL/6 mice Miriam Algarabel Olona1, Andrés Vacas Oleas1, José Peña Guerrero1, Connar Leeming1, Celia Fernández Rubio1, Ester Larrea2, Socorro Espuelas3, Paul Nguewa1 1 Instituto de Salud Tropical Universidad de Navarra (ISTUN)/ Department of Microbiology and Parasitology, Pamplona, ES, 2 Instituto de Salud Tropical Universidad de Navarra (ISTUN)/ Pathogen Immunology (CIMA) University of Navarra, Pamplona, ES, 3Instituto de Salud Tropical Universidad de Navarra (ISTUN)/ Department of Pharmacy and Pharmaceutical Technology, University of Navarra, Pamplona, ES Celia Fernández-Rubio1, Estanislao Nistal-Villan2, Miriam Algarabel Olona1, Andrés Vacas-Oleas1, José Peña-Guerrero1, Connar Leeming1, Gloria González-Aseguinolaza2, Esther Larrea3, Paul Nguewa1 1 Institute of Tropical Health University of Navarra (ISTUN), Department of Microbiology and Parasitology, Pamplona, ES, 2 Gene Therapy and Regulation of Gene Expression Program, Center for Applied Medical Research (CIMA) , Pamplona, ES, 3Institute of Tropical Health University of Navarra (ISTUN), Pamplona, ES Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. Current drug therapies are unsatisfactory due to their toxicity, long treatment courses and development of resistance. In order to improve existing treatments, the identification and characterization of novel therapeutic targets based on parasite genes involved in Leishmania pathogenesis is essential. One of these genes is YinP. It may play a role in the acquisition of infectivity by Leishmania promastigotes. This gene is highly conserved and has demonstrated to be involved in several cellular processes such as embrionary progress, ribosomal biogenesis, cellular proliferation, genetic transcription and carcinogenesis. Our first assays have shown that YinP reaches its highest expression level in metacyclic promastigotes, the infective stage. Furthermore, we have performed several experiments to analyze the infectivity of parasites overexpressing YinP. Our data reveal a dramatic increase of the ratio of infection as well Leishmania major is the causative agent of the old world cutaneous leishmaniasis currently re-emerging due to the refugee crisis in the Middle East and North Africa. It is widely recognized the anti-parasite role of the type II IFN, but function of type I IFN in the parasite infection still remains unclear. Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF) and IFN-β promoter stimulator-1 (IPS-1) are protein adaptors implicated in the recognition of double-strand RNA that enhances adaptive immune responses.The aim of this study was to investigate the impact of the deficiency in TRIF and IPS-1 protein adaptors during Leishmania major infection. For that, bone marrow-derived macrophages (BM-DM) from C57BL/6 wild type (wt) and double TRIF and IPS-1 knock out (ko) mice were infected with Leishmania major promastigotes. After 6 and 72 hours of infection, a significant increase in the percentage of Leishmania-infected BM-DM 107 Pósters / Posters and in the amount of Leishmania DNA in BM-DM was detected. Such alterations were related to the lack of the mentioned adaptors. Consequently, these results prompted us to study the expression pattern of certain host genes involved in leishmanicidal activity, such as iNOS and IFNβ. We observed a significant decrease of iNOS and IFNβ mRNA expression in BM-DM from double ko mice compared to wt animals. Furthermore, a reduced nitric oxide (NO) production in BM-DM cells from doble ko mice was also observed. An in vivo assay was performed comparing wt and IPS-1 ko mice infected with Leishmania major. Four weeks post-infection, half of animals showed ulceration (50 % IPS-1 ko vs 0 % wt). In addition, there was a higher parasite burden in the liver from the IPS-1 ko group respect to wt. These results point to a role of type I IFN in Leishmania infection. P14-12 Analysis of the protein interactome of Leishmania major ORC1/CDC6 Laura Corvo1, César Poza-Carrión2, María Gómez1, José M. Requena1 Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, ES, 2Centro Nacional de Biotecnología (CSIC), Madrid, ES 1 Eukaryotic DNA replication involves firing of multiple origins licensed by the assembly of pre-replication complexes (pre-RCs), composed of an Origin Recognition Complex (ORC), a six-subunit complex (Orc1-6) that recruits Cdc6 to the replication origin, and the MCM (mini-chromosome maintenance) helicase complex (Mcm2-7) which is loaded, along with Cdt1, onto DNA by the ORC-Cdc6 complex. The assembly of additional factors as Cdc45 and GINS leads to the pre-initiation complex formation, which allows the establishment of bidirectional replication forks. This process is tightly regulated and highly conserved among eukaryotic and archaeal organisms. While nuclear DNA replication has been extensively investigated in yeast and higher eukaryotes, little is known about the replication machinery in protozoans. Trypanosomatids are a group of kinetoplastid parasites which includes the human pathogenic genera Trypanosoma and Leishmania. The latter causes a group of diseases globally known as Leishmaniasis, responsible of 20,000 to 30,000 deaths annually worldwide. Several eukaryotic replication protein orthologues have been annotated in trypanosomatids, although some of them are lacking, and only one ORC orthologue, related to both Orc1 and Cdc6 proteins, have been clearly found in genomic databases, indicating that these parasites may possess a novel or diverged replication machinery. In order to gain insight into Leishmania origin recognition complex architecture and function, we have generated a Leishmania major cell line overexpressing Orc1 fused to mCherry. We have focused on the analysis of Orc1 subcellular location and the identification of Orc1-interacting factors in both wild type and Orc1-mCherry overexpression backgrounds. P14-13 Análisis funcional mediante RNA-seq de la proteína fosfatasa tipo 1 LinJ.15.0240 de Leishmania infantum María A. Degayon1, Ana Alonso1, Pedro J. Alcolea1, Gowthaman Ramasamy2, Guillermo Padilla1, Peter J. Myler2, Vicente Larraga2 1 Centro de Investigaciones Biológicas. CSIC, Madrid, ES, 2Center for Infectious Disease Research. Seattle, US La leishmaniasis visceral es una zoonosis causada por el parásito Leishmania infantum en la Cuenca Mediterránea. Las proteínas fosfatasas tipo 1 (PP1) son las principales serina-treonina fosfatasas presentes en los organismos eucariotas y participan en la regulación de diversos procesos celulares. El papel de dichas proteínas apenas ha 108 XXXIX Congreso SEBBM sido estudiado en el género Leishmania. De acuerdo a los análisis de expresión génica diferencial mediante microarrays (Alcolea y cols. 2010), el gen de la PP1 LinJ.15.0240 está diferencialmente expresado a lo largo del ciclo biológico de L. infantum. El perfil de expresión génica analizado por qRT-PCR mostró una expresión constante de la PP1 LinJ.15.0240 a lo largo de la curva de crecimiento de los promastigotes axénicos, coincidiendo con la abundancia de proteína detectada por Western blot. Además, se observó la sobre-expresión del gen LinJ.15.0240 en promastigotes obtenidos del vector P. perniciosus respecto a promastigotes axénicos. Por otro lado, se generaron transfectantes estables de sobre-expresión de la PP1 mediante el vector integrativo pIRmcs. Los perfiles de expresión génica evaluados por RNA-seq revelaron una sobre-expresión del gen LinJ.15.0240 en todos los días analizados de la curva de crecimiento. Sin embargo, los análisis por Western blot mostraron la sobre-expresión de la PP1 únicamente a día 1. Curiosamente, el 92% de los genes diferencialmente expresados se encuentran sub-expresados según los datos de secuenciación masiva. Estos genes están asociados a funciones moleculares como la expresión génica, transporte y metabolismo de acuerdo a la base de datos Gene Ontology. Entre los genes diferencialmente expresados cabe destacar la sobre-expresión en el día 1 del gen A2, característico de amastigotes, así como la sub-expresión en los días 3 y 5 de genes descritos como marcadores de metaciclogénesis, SHERP y Meta 1. Estas observaciones sugieren que la PP1 LinJ.15.0240 puede tener un papel esencial en los procesos de diferenciación de L. infantum. P14-14 The Enoyl-CoA hydratase/isomerase of Leishmania infantum as a potential therapeutic target Luis Telles de Carvalho Coimbra Martins, Ana Alonso, Pedro Alcolea, Vicente Larraga Centro de Investigaciones Biologicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, ES Leishmaniasis are a group of infectious diseases, caused by protozoan parasites of the genus Leishmania and transmitted by the bite of phlebotomine sand flies. L. infantum is the responsible strain for the zoonotic visceral form of the disease in the Mediterranean Basin, being the dog the main reservoir. The treatments currently available present several disadvantages such as toxicity, drug resistance and recurrence. We have selected a specific gene that encodes for a L. infantum enoyl-CoA protein (LinJ. 29.2420), over-expressed during the most infective stage of the parasite, obtained, from metacyclic promastigotes isolated through negative selection with PNA (Alcolea et. al., 2009). This over-expression may be directly related with the infectivity of this parasite. This protein has both hydratase and isomerase activities which are involved in the -oxidation pathway. Enoyl-CoA hydratase catalyzes the syn hydration of water. Enoyl-CoA isomerase catalyzes the reaction in which cis-3-enoyl-CoA or trans-3-enoyl-CoA is converted into trans-2-enoyl-CoA. This gene has been cloned in the pQE-30 vector and its expression was performed in the Escherichia coli strain M15. Optimal expression conditions of the encoded protein were found to be 2 h at 37 °C. This protein has been purified by Ni2+ using affinity chromatography at native conditions. A polyclonal antibody has also been obtained in order to study the protein abundance trough out the growth curve of the parasite. Using the I-Tasser, PyMOL and BlastP software’s together with the PDB database we were able to predict a protein 3D model. This protein forms hexamers, made up of two trimers. The nucleotide sequence was aligned, using ClustalW with orthologous sequences, giving low percentages of homology with the mammalian proteins, and very low with the human one (6%). These values as well as the predicted protein structure suggest that this could be a good candidate as a therapeutic target. Salamanca 2016 P14-15 Leishmania and its relationship with human skin microbiome.- Leishmanicidal activity of modulins, peptide toxins from Staphylococcus sp M. Ángeles Abengozar Infantes1, Nuria Davó1, Luis Rivas López1, David Andreu Martinez2 1 Centro de Investigaciones Biológicas (CSIC), Madrid, ES, 2 Universitat Pompeu Fabra, Parc de Recerca Biomèdica, Barcelona, ES Leishmania has been traditionally studied as an isolated organism or at most as a mandatory intracellular parasite of the macrophage. However, in cutaneous leishmaniasis (CL), Leishmania is in contact with the components of skin microbiome. Of special relevance, Leishmania skin ulcers often become infected with bacteria, often by Staphylococcus epidermidis, a typical skin commensal, and by S. aureus. Both staphylococci secrete a group of cationic and amphipathic peptides collectively known as phenol-soluble modulins (PSM), with a broad microbicidal spectrum. Our goal was to ascertain the role of modulins in the control of CL as well as their potentiality as new leishmanicidal agents. As a proof of mechanism, PSMα1 and PSαM2, and their respective N-terminal dipeptide truncated forms from S. aureus, together with δ- and γ-toxins from S. epidermidis were assayed both on L. major promastigotes as well as on axenic and intracellular L. pifanoi amastigotes. All these modulins lysed promastigotes at low micromolar concentrations (IC50 <30 μM: ranked in increasing IC50 PSMα2Δ1-2 <pps mα1=psmα1δ1-2≤psma2<δ-toxin<<γ-toxin), with=”” additive=”” effect=”” in=”” combination.=”” axenic=”” amastigotes=”” psmα2=”” was=”” the=”” most=”” active=”” peptide=”” (ic50=3.3 μM), and with the highest selectivity index (SI=3). It also reduced intracellular infection by 30% at sub-IC50 concentrations. </ppsmα1=psmα1δ1-2≤psma2<δ-toxin<<γ-tox in),>PSMs led to plasma membrane permeabilization by an all-or-none mechanism, as assessed by in vivo bioenergetic collapse of promastigotes, plasma membrane depolarization, permeation to vital dyes and transmission electron microscopy. In addition, PSMs were spotted inside Leishmania at subIC50, suggesting additional intracellular targets. In combination with LL-37 or dermcidin, two human skin antimicrobial peptides, PSMs showed antagonism and no effect, respectively. Altogether, PSMs appear to be feasible modulators of infection, as well as new leishmanicidal hits. The role of other microbial actors in the final CL outcome will be discussed. Projects: FIS ISCIII,PI12-02706& RETICS-FEDER, RD12/0018/0007 (LR) & AGL2014-52395 (DA). P14-16 Papel inmunomodulador de la doxiciclina en un modelo murino de malaria cerebral María Linares1, Javier Peiró1, Gabriela Martínez-Chacón1, Daniel Sánchez-Melendo1, Susana Pérez-Benavente1, Antonio Puyet1, José M. Bautista1, Patricia Marín-García2, Amalia Diez1 1 Departamento de Bioquímica y Biología Molecular IV (UCM). Instituto de Investigación Hospital 12 de Octubre, Madrid, ES, 2 Facultad de Ciencias de la Salud (URJC), Alcorcón, ES La malaria cerebral (MC) es la complicación más grave de la infección por Plasmodium falciparum. La variabilidad individual de sus síntomas refleja que es un síndrome heterogéneo lo que dificulta el manejo de la enfermedad, impidiendo su diagnosis temprana y un tratamiento rápido y eficaz. Aunque su patogénesis no está bien definida, su complejidad se relaciona con el secuestro de eritrocitos en la vasculatura cerebral y la respuesta inflamatoria del hospedador. La magnitud y el momento de liberación de mediadores inflamatorios modulan la respuesta inmune a la malaria, resultando en un control adecuado de la parasitemia o por el contrario provocando el desequilibrio de tales mediadores causando un daño letal en el hospedador. En este sentido la inmunomodulación sería una nueva perspectiva para Pósters / Posters el tratamiento de la MC. La doxiciclina, como antibiótico, es una terapia capaz de controlar la malaria, sin embargo, se desconoce si durante el aclaramiento del parásito posee un efecto potenciador del sistema inmune en el cerebro. Por ello, hemos evaluado el posible efecto inmunomodulador de la doxiciclina durante el desarrollo de la malaria cerebral mediante el análisis de la respuesta inmune en el modelo murino C57BL/6 infectado con P. berghei ANKA. Los resultados obtenidos muestran que los animales que desarrollan MC y son tratados con doxiciclina, aumentan los niveles de IgG específicas tras una segunda infección que se relaciona con un patrón diferencial de reconocimiento antigénico. Dicha respuesta inmune adaptativa, aunque es capaz de prolongar la supervivencia de los animales en la primera infección, no les confiere protección suficiente en las sucesivas infecciones. Adicionalmente describimos el papel de la IL10 como elemento condicionante de la MC durante la infección. P14-17 Inhibition of Plasmodium falciparum intraerythrocytic cycle by purine analogs Laura Sánchez Vega1, Susana Pérez Benvente1, Rafael de la Cámara1, David Monedero2, Daniela Ubiali3, Giovanna Speranza4, José Manuel Bautista1, Jesús Fernández Lucas2, Antonio Puyet Catalina1 1 Department of Biochemistry and Molecular Biology IV & Research Institute Hospital 12 de Octubre, Universidad Complutense de Madrid, Madrid, ES, 2Applied Biotechnology Group, Universidad Europea de Madrid, Madrid, ES, 3Drug Sciences Department, University of Pavia, Pavia, IT, 4Department of Chemistry; University of Milan, Milan, IT The purine synthesis pathways are essential for the life cycle of many pathogenic organisms in mammals. Most parasites lack the necessary enzymes for de novo synthesis of purines, using instead the salvage pathways as their only source of these substrates. The transporters and enzymes involved in the salvage pathways are considered good targets for chemotherapy agents and purine analogs, which are used extensively in the treatment of several diseases. The enzymes catalyzing the cleavage of glycosidic bonds of nucleosides have an essential role in the purine salvage pathway of Plasmodium, the parasite causing malaria, and can therefore be considered as potential therapeutic targets for antimalarial drugs. New inhibitors of the plasmodial adenosine deaminase (PfADA), purine nucleoside phosphorylase (PfPNP) and hypoxantine-guanosine-xantine phosphoribosyl transferase (PfHGXPRT) are currently investigated for this purpose. In addition, PfPNP activity can be used for the activation of pro-drugs, which can be channeled through PfHGXPRT. The resulting metabolites would be toxic for the parasite, as they could be incorporated to RNA/DNA hampering transcription/translation or replication. In this work several purine base and purine ribonucleoside analogs displaying substitutions at positions 6, 7 and 8, and chemotherapy agents with substitutions a position 2 of adenine have been tested as potential inhibitors of the intraerythrocytic cycle of Plasmodium falciparumin vitro. The results suggest that fluorinated derivatives of adenine, adenosine and adenosine phosphate can be effective as parasite growth inhibitors at subcytotoxic concentrations. P14-18 Study of YinP, a novel gene involved in Leishmania pathogenesis. A potential new target for drug discovery Miriam Algarabel Olona1, Celia Fernández-Rubio1, Andrés Vacas Oleas1, Esther Larrea2, José Peña Guerrero1, Connar Leeming1, Iñigo Gamboa1, Carlos Berrio1, Carmen Sanmartín3, Socorro Espuelas4, Paul Nguewa1 1 Institute of Tropical Health University of Navarra (ISTUN), Department of Microbiology and Parasitology, Pamplona, ES, 109 Pósters / Posters 2 Institute of Tropical Health University of Navarra (ISTUN), Pamplona, ES, 3Institute of Tropical Health University of Navarra (ISTUN), Department of Organic and Pharmaceutical Chemistry, Pamplona, ES, 4Institute of Tropical Health University of Navarra (ISTUN), Department of Pharmacy and Pharmaceutical Technology, Pamplona, ES Leishmaniasis is a neglected tropical disease caused by Leishmania spp. The improvement of existing treatments and the discovery of new drugs remain ones of the major goals to control and eradicate this disease. Based on parasite genome, we have identified and characterized YinP, a novel therapeutic target involved in Leishmania pathogenesis. YinP is a highly conserved gene which contains two main domains: YinP (N-terminus) and ONC. It has been demonstrated that YinP is involved in several cellular processes such as cellular proliferation and genetic transcription. Firstly, we analyzed the intracellular location of the protein. Fluorescent microscopy disclosed that red fluorescence of mCherry fused with YinP was only localized in the nucleus. In addition, we demonstrated that, in Leishmania promastigotes, it plays a role in the acquisition of infectivity. In fact, YinP reaches its highest expression level in metacyclic promastigotes, the infective forms. After generating mutant parasites overexpressing YinP, we observed that these clones exhibited a dramatic increase of the ratio of cell infection and higher replication rate within macrophages. Furthermore, we are carrying a screening of new compounds that have shown promising leishmanicidal activity. Interestingly, YinP overexpressing parasites are more resistant to these drugs. Consequently, it may be a new therapeutic target. On the other hand, we have obtained more mutant parasites with different levels of ONC domain overexpression. Preliminary data suggest that these cells may show lower growth compared to WT parasites. Moreover in such mutants, YinP gene expression levels seem to be abrogated respect to control. Finally, we have also generated clones with high levels of YinP∆ONC in order to study the role of ONC in YinP. Therefore, further experiments need to be performed to better characterize YinP function and activity. P14-19 Epitope mapping of immunodominant antigens from Plasmodium falciparum as serological markers of malaria infection Patricia Marín-García1, Amalia Diez2, Isabel González-Azcárate2, Alejandra Martínez-Serna2, Ana Abad2, Pedro Reche3, José Miguel Rubio4, Antonio Puyet2, José M. Bautista2 1 Fac. Ciencias de la Salud, URJC, Alcorcón, ES, 2Depto. Bioquímica y Biología Molecular IV, UCM e Instituto de Investigación Hospital 12 de Octubre, Madrid, ES, 3Depto. Inmunología, UCM, Madrid, ES, 4Instituto de Salud Carlos III, Majadahonda, ES Blood-stage malaria immunity can be partially acquired after years of exposure to Plasmodium falciparum infection through the elicitation of protective antibodies. The target antigens of protective antibodies and the basis of their acquisition remain largely unknown. Addressing these knowledge gaps could accelerate malaria vaccine discovery and the development of new rapid diagnostics kits. To this end, we performed immunomics screening of human sera against a clinical P. falciparum isolate. Malaria cases from African patients were classified into different categories according to clinical symptoms and parasitaemia and degree of immunological protection. After 2D-immunoblotting of P. falciparum proteins and peptide mass fingerprinting we identified 17 antigens representing a collection of parasite proteins conferring residual immune protection against clinical malaria. Interestingly, this data included new antigens not previously described. We selected five new antigenic proteins for epitope screening with high-resolution 15-mer overlapping peptide arrays, and identified high immunoreactive epitopes in all five antigens. Next, we selected ten 20-mer peptides from these immunoreactive peptides for further characterization by ELISA with a human sera collection of 110 XXXIX Congreso SEBBM infected and non-infected individuals from endemic areas in Africa. Our preliminary results suggest that some of these epitopes may be useful for detection of subclinical malaria. P14-20 Antimalarial activity of a new family of PfG6PD inhibitors María Linares1, Irene Sola2, Nelson Alencar3, Paloma Abad1, Susana Pérez-Benavente1, Caterina Pont2, Cristina Sampedro2, Jordi Juárez-Jiménez3, F. Javier Luque3, Diego Muñoz-Torrero2, José M. Bautista1 1 Department of Biochemistry and Molecular Biology IV & Research Institute Hospital 12 de Octubre, Universidad Complutense de Madrid, Ciudad Universitaria, Madrid, ES, 2Laboratory of Pharmaceutical Chemistry (CSIC-associated Unit), Faculty of Pharmacy and Institute of Biomedicine (IBUB), Universidad de Barcelona (UB), Barcelona, ES, 3Department of Nutrition, Food Science and Gastronomy, Faculty of Pharmacy (Torribera Campus) and IBUB, UB, Santa Coloma de Gramenet, ES Worldwide malaria caused by Plasmodium falciparum is responsible for nearly 1 million deaths annually. Despite the efforts made in the discovery of new antimalarial agents, the parasite has developed resistance against the vast majority of available therapies. Therefore, there is an urgent need to search for new drugs with new mechanisms of action. In the malaria parasite, the bifunctional glucose-6-phosphate dehydrogenase-6-phosphoglucono-lactonase (PfG6PD) has been validated as a target to inhibit the asexual parasite stages. Since PfG6PD is the evolutionary result of the fusion of the two genes that are separated in most eukaryotes (including the human host hG6PD), their differences could enable the rational design of selective inhibitors against the parasite PfG6PD active site. We have evaluated the antimalarial activity of a new family of inhibitors designed and synthesized upon the structural differences between PfG6PD and hG6PD. A total of nine different molecules have been screened by enzyme kinetics in the parasite and human enzyme, IC50 in P. falciparum culture and cytotoxicity in human cells. Most molecules showed greater selectivity against PfG6PD with IC50 values within the micromolar range. In addition, the most potent inhibitors also showed a greater inhibitory activity of the P. falciparum growth in culture. Furthermore, phenotypic assays at 48h upon treatment determined that schizont was the most sensitive intraerythrocytic stage to this family of compounds, which suggests a different mechanism of action than that of chloroquine, making it interesting for combined antimalarial therapy and enhanced immunomodulation. Finally, the absence of in vitro cytotoxicity complements to these new molecules with an added value as promising antimalarial inhibitors. P14-21 Iron overload delays malaria infection in mice and improves immune and antioxidant responses Sandra Sánchez Jaut1, Isabel González Azcárate1, Patricia Marín García2, Susana Peréz Benavente1, Javier Uceda Fernández1, Marta García Sánchez1, Alí Naghi Kamali1, Antonio Puyet Catalina1, Amalia Díez Martín1, José Manuel Bautista Santa Cruz1 1 Department of Biochemistry and Molecular Biology IV, Universidad Complutense de Madrid, Facultad de Veterinaria, Madrid, ES, 2Faculty of Health Sciences, Universidad Rey Juan Carlos, Alcorcón, ES Anemia is a common public health problem in Africa, where >80% of malaria prevalence occurs, and where 50% anemia cases are due to iron deficiency. Consequently, in malaria endemic regions the iron supplementation is routinely used to prevent iron deficiency anemia and to Salamanca 2016 treat patients with the disease. However, the association of iron levels with malaria tolerance is still controversial. To study the effect of iron supplementation on malaria infection, we inoculated 100mg/kg/day of iron dextran during 21 days to BALB/c mice and challenged with lethal Plasmodium yoelii 17XL. This induced iron overload promoted transitory red skin, weight loss and liver damage, that resumed once finished iron inoculation. Expectedly, hepcidin-1 expression was induced in liver to maintain iron homeostasis. Interestingly, iron overload delayed lethal malaria infection through early mechanisms related to antioxidant and innate immune responses. Thus, iron changed the spleen immune-cell pattern with respect to macrophages, B cells, antigen-presenting cells and dendritic cells (DCs), as well as induced the expression of the anti-inflammatory heme oxygenase-1 in liver. Iron treated mice increased their proportion of macrophages during the parasitemia growth in spleen in comparison to control, providing advantage against the blood-stage malaria infection. Further experiments are required to assess whether additional mechanisms induce beneficial immune responses. P14-22 Caracterización de factores asociados a AMPK que regulan la quiescencia en la forma infectiva de Trypanosoma brucei Gloria Ceballos Pérez, Manuel Saldivia, Jean-Mathieu Bart, Miguel Navarro Consejo Superior de Investigaciones Científicas (CSIC), Granada, ES En mamíferos, la señalización a través de AMPK se encuentra asociada a respuestas celulares frente al estrés oxidativo, metabolismo glicosídico y diferenciación. En Trypanosoma brucei, nuestro grupo ha observado mediante herramientas moleculares y farmacológicas que la proteína AMPK regula de manera específica la diferenciación del parásito hacia formas quiescentes mediante ensayos in vivo e in vitro. Además, el análisis proteómico del complejo TbAMPK identificó una posible asociación de esta quinasa con proteínas responsables de la respuesta oxidativa del parásito. Con el objetivo de determinar la posible relación entre la regulación de AMPK, el metabolismo oxidativo y la diferenciación de T. brucei, se analizó la capacidad de respuesta aestrés oxidativo del parásito mediante la regulación de AMPK. Nuestros resultados sugieren que la activación de TbAMPKα1 es capaz de regular la respuesta oxidativa de T. brucei. Además, se observó mediante RT-qPCR que el tratamiento con H2O2 es capaz de regular la expresión de genes asociados a diferenciación hacia formas quiescentes. En conjunto, estos datos sugieren que TbAMPKα1 tiene una función antioxidante asociada a diferenciación en T. brucei. P14-23 La localización de la RNA polimerasa II mediante Chip-Seq identifica secuencias promotoras funcionales en el genoma de Trypanosoma brucei Carlos Cordón Obras1, Sara Torres Rusillo1, Diana López Farfán1, Jean Mathieu Bart1, Fabian Lorenzo2, Basilio Valladares2, Mark Carrington3, Nicholas J. Dickens4, Miguel Navarro1 1 Instituto de Parasitología y Biomedicina López-Neyra, CSIC, Granada, ES, 2Universidad de Canarias, Las Palmas de Gran Canaria, ES, 3Department of Biochemistry. University of Cambridge, Cambridge, UK, 4Wellcome Trust Centre for Molecular Parasitology. University of Glasgow, Glasgow, UK La diversificación de los kinetoplástidos, incluyendo Trypanosoma brucei, a partir de otros eucariotas dio lugar a la adquisición de nuevas características moleculares y celulares a nivel de transcripción y genoma. En concreto, la transcripción policistrónica de los genes codificantes de Pósters / Posters proteínas revela un sistema ancestral semejante al de los procariotas. Estudios previos han sugerido que el inicio de la transcripción puede estar localizado en las regiones conocidas como regiones de cambio de hebra (Strand Switch Regions –SSR-), situadas entre unidades transcripcionales. Sin embargo, estas secuencias SSR son de gran tamaño y aún no está claro su papel en el inicio y regulación de la transcripción. Para estudiar la ocupación de la ARN polimerasa II (pol II) en el genoma de Trypanosoma brucei, realizamos ensayos combinando inmunoprecipitación de cromatina (ChIP) y secuenciación masiva (ChIP-Seq). La alta resolución de esta técnica permite definir de forma precisa las regiones en las que la pol II se acumula. Nuestros resultados revelan que existe una acumulación significativa de la pol II en 306 picos a lo largo del genoma, la mayoría de ellos asociados con SSRs (52,7%) y regiones intergénicas (27,4%), con un tamaño medio de 1955 bases. A continuación, analizamos la actividad promotora de las secuencias ocupadas por la pol II en el cromosoma 7 mediante un ensayo de transfección in transient con un gen reportero (luciferasa) en formas procíclicas de T. brucei. Estudiamos una de estas secuencias generando deleciones para definir la mínima región necesaria para el inicio de la transcripción y mutaciones a través de este mínimo fragmento. Así, determinamos la posición +1 mediante Primer Extension e identificamos los principales elementos reguladores, la mayoría de ellos situados aguas abajo del +1. Nuestro trabajo demuestra el potencial de estas regiones como secuencias promotores y proporciona la primera descripción de elementos reguladores asociados a las unidades transcripcionales policistrónicas de T. brucei. P15. Radicales libres y estrés oxidativo / Free radicals and oxidative stress P15r-1 Absence of PGC-1α induces cellular senescence and early immortalization Ignacio Prieto, Alberto Zambrano, Ana Aranda, María Monsalve Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, ES Peroxisome Proliferator Activated Receptor (PPAR) γ Coactivator 1α (PGC-1α) is a master regulator of oxidative metabolism; being responsible of the Reactive Oxygen Species (ROS) detoxification, and the lack of PGC-1α leads to oxidative stress due to ROS accumulation. It is well-known that elevated concentrations of ROS can cause Double Strand Breaks (DSB) in the DNA. Oxidative stress and DNA damage are, among others, triggers of Cellular Senescence, a cellular process characterized by cell cycle arrest in response of aberrant proliferation and tumor progression. The aim of the study was to determine the possible role of PGC-1α in cellular senescence. To test this idea, we used PGC-1a+/+ and PGC-1a-/- MEFs that were serially passed till they reach cellular senescence and immortalized spontaneously. Our results show that, in the absence of PGC-1a, MEFs produce higher levels of mitochondrial reactive oxygen species, accumulate more oxidative DNA damage measured by DNA damage repair complexes formation and showed delayed DNA repair, leading to an early induction of senescence markers and induction of cell cycle inhibitors. However, cell proliferation rate was not significantly different between PGC-1a+/+ and PGC-1a-/MEFs regardless of passage number. Importantly, PGC-1a-/- MEFs showed and early escape from senescence, compared to PGC-1a+/+ MEFs. These results suggest that the lack of PGC-1α leads to enhanced DNA damage and induction of senescence, but is also associated with an early escape from cellular senescence, supporting a tumor suppressor role for PGC-1α. 111 Pósters / Posters P15-2 PGC-1α downregulation in the steatotic liver enhances ischemia-reperfusion injury and impairs ischemic preconditioning Cristina Sánchez Ramos1, Alberto Tierrez2, Javier Laso2, Ramón Bartrons3, Joan Roselló-Catafau4, María Monsalve1 1 Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, ES, 2CNIC, Madrid, ES, 3Universitat de Barcelona, Barcelona, ES, 4Institut d´Investigacions Biomèdiques de Barcelona (CSIC), Barcelona, ES Liver steatosis is associated with mitochondrial dysfunction, elevated ROS levels and a enhanced sensitivity of ischemia-reperfusion (IR) injury and a very limited response to preconditioning protocols. In this study we aimed to determine if the downregulation in the steatotic liver of peroxisome proliferator activated receptor co-activator 1 (PGC-1), a master regulator of mitochondrial metabolism and reactive oxygen species (ROS), that is known to play a relevant role in liver metabolic control, could be responsible for the sensitivity of the steatotic liver to ischemic damage.We found that PGC-1 is induced in the normal liver, following an IR protocol and this induction correlates with increased levels of antioxidant proteins. This induction is severely curtailed in the steatotic liver, and results in a limited induction of antioxidant proteins. PGC-1-/- mice under chow diet do not have steatotic livers, but show an enhanced sensitivity to IR injury and a lack of response to ischemic preconditioning, that recapitulates the behaviour of the steatotic liver in terms of liver damage, although the inflammatory response differers between both models. We used an in vitro model to analyze how PGC-1 expression is regulated by changing O2 tensions, and found that hypoxia (1% O2) downregulates PGC-1 while reoxygenation (3% O2) induces it, and is responsible for the recovery of antioxidant gene expression following the ischemic period. Conclusion: PGC-1 plays an important role in the protection against IR injury in the liver that is likely associated with its capacity to induce antioxidant gene expression. P15-3 Daily supplementation with an almond beverage enriched with docosahexaenoic, vitamin E and olive oil alters inflammatory state in response to acute exercise Xavier Capó1, Miquel Martorell2, Antonio Sureda1, Joan Riera3, Franchek Drobnic3, Josep Antoni Tur1, Antoni Pons1 1 CIBER: CB12/03/30038 Fisiopatología de la Obesidad la Nutrición, CIBEROBN, Instituto de Salud Carlos III (ISCIII). Research Groupon Community Nutrition and Oxidative Stress, Science Laboratory of Physical Activity, Department of Fundamental Biology and Health Sciences. University of Balearic Islands, Palma de Mallorca, ES, 2Departamento de Nutrición y Dietética, Facultad de Farmacia, Universidad de Concepción, Concepción, CL, 3Sports Physiology Dept. CAR, Sant Cugat del Valles, ES Omega 3polyunsaturated fatty acids (n3-PUFA) diet supplementation exerts healthy effects against oxidative stress-based diseases. High PUFA levels enhance lipid peroxidation altering oxidative stress balance. Ageing is also associated with oxidative stress with higher values of oxidative damage markers in elderly than in young. The aim was to determine the effects of an almond and olive oil-based DHA and vitamin E-enriched beverage dietary supplementation, acute exercise and age on circulating inflammatory markers. The diet of five young and five senior athletes were supplemented during five weeks with a functional beverage. Athletes performed a maximal exercise test at the beginning and at the end of the nutritional intervention. Plasma and erythrocytes were isolated from blood taken immediately before and 1 hour after the exercise. Functional beverage diet supplementation increased the DHA erythrocytes content but attenuated the increased plasma non-esterified 112 XXXIX Congreso SEBBM fatty acids levels induced by acute exercise. Dietary functional beverage supplementation significantly decreased sL-selectin, ICAM3 but increased TNFα plasma levels; age increased ICAM3 and acute exercise decreased lipoxin, PGE2 plasma levels. Some interaction between exercise, age and supplementation were observed in sL-selectin, ICAM, IL6 and TNFα plasma levels. Young athletes presented the lowest sL-selectin and ICAM3 and the highest IL6 plasma levels after acute exercise after dietary beverage supplementation respect the other situations. The TNFα plasma levels were the highest in senior athletes after exercise and dietary supplementation. Finally, dietary functional beverage supplementation of young athletes enhances a pro-inflammatory circulating environment in response to acute exercise that was minus evident in senior athletes. Acknowledgments: CIBEROBN CB12/03/30038. We hereby acknowledge the PhD grant provided by the University of the Balearic Islands. P15-4 Serum and follicular fluid distribution of human PON1 and PON3 enzymes Irantzu Pérez-Ruiz1, Susana Meijide1, Zaloa Larreategui2, Marcos Ferrando2, José Ignacio Ruiz-Sanz1, M. Begoña Ruiz-Larrea1 1 Department of Physiology, Medicine and Nursing School, University of the Basque Country, UPV/EHU, Leioa, ES, 2Valencian Institute of Infertility (IVI) – Bilbao, Leioa, ES Physical and mental health is compromised in women suffering from infertility and is currently considered a sociological problem. Assisted reproduction techniques (ART) are helping these women to overcome this problem, so that improvements in the area are of great interest. It is known that ROS and antioxidants play an important role in reproduction; therefore the control of redox balance and the enzymes which are responsible for its preservation is essential. The human paraoxonase (PON) gene family consists of three members (PON1, PON2, and PON3) that exhibit antioxidant activities. The aim of this study was to characterize the extracellular paraoxonase system (PON1 and PON3) in serum and follicular fluid (FF). Samples were retrieved from the same women undergoing an ovarian stimulation cycle. To this end, PON1 (arylesterase and paraoxonase) and PON3 (simvastatinase) activities were measured by spectrophotometric and HPLC methods, respectively. Protein expression was detected by quantitative western blotting using specific antibodies. The results indicate that all analyzed PON1 activities were significantly higher (p<0.05) in serum than in FF. However, PON1 expression did not show significant differences between both fluids. Regarding PON3 activity, no differences were found between serum and FF, but, in contrast, its expression was significantly higher (p<0.05) in serum than in FF. In conclusion there are different regulatory mechanisms governing the specific activity of these extracellular enzymes in serum and follicular fluid. This work was supported by the Basque Country Government (Dep. Education, Universities and Research, ref. IT687-13), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (M10_2015_180_RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (October 5, 2015). P15-5 Training enhances immune cells mitochondrial dynamics and its antioxidant capabilities synergistically with dietary docosahexaenoic supplementation Carla Busquets-Cortés1, Xavier Capó1, Miquel Martorell2, Josep Antoni Tur1, Antonio Sureda1, Antoni Pons1 1 CIBER: CB12/03/30038 Fisiopatología de la Obesidad la Salamanca 2016 Nutrición, CIBEROBN, Instituto de Salud Carlos III (ISCIII). Research Groupon Community Nutrition and Oxidative Stress, Science Laboratory of Physical Activity, Department of Fundamental Biology and Health Sciences. University of Balearic Islands, Palma de Mallorca, ES, 2Departamento de Nutrición y Dietética, Facultad de Farmacia, Universidad de Concepción, Concepción, CL Exercise training induces adaptations in mitochondrial metabolism, dynamics and oxidative protection. Omega-3 fatty acids change membrane lipid composition and modulate mitochondrial function. The aim was to investigate the effect of 8-weeks training and docosahexaenoic acid (DHA) supplementation (1.14 g/day) on the mitochondria dynamics and antioxidant status in peripheral blood mononuclear cells (PBMCs) from sportsmen. Subjects were assigned to an intervention (N=9) or placebo groups (N=7) in a randomized double-blind trial (ClinicalTrial. gov NCT02177383). Nutritional intervention significantly increased the DHA content in erythrocyte membranes from the experimental group. No significant differences were reported in circulating PBMCs, in their capability to produce reactive oxygen species and in Mn-superoxide dismutase protein levels. The proteins related with mitochondrial dynamics generally increased after 8-weeks training and tha was enhanced by DHA supplementation. Mitofusin (Mtf)-1 and -2, optic atrophy protein-1 (Opa-1) and mitochondrial transcription factor A (Tfam) were significantly higher in the DHA-supplemented group after intervention. Cytochrome c oxidase (COX IV) activity and uncoupling proteins (UCP)-2 and 3 protein levels increased after training, with higher UCP-3 levels in the supplemented group. In conclusion, training induced mitochondrial adaptations which may contribute to improved mitochondrial function. This mitochondrial response is modulated by DHA supplementation. Acknowledgments: Acción Estratégica en Salud del Ministerio de Ciencia e Innovación DPS2008-07033-418 C03-03. The authors hereby acknowledge the FPI/1648/2014 grant provided by Conselleria d’Educació, Cultura i Universitats, Direcció General d’Universitats i Recerca, Govern de les Illes Balears. P15r-6 Dietary polyphenols and their role in coenzyme Q metabolism Lucía Fernandez del Rio1, Rosa Mª Nevado García1, María Isabel Burón Romero1, Anish Nag2, Catherine F. Clarke2, José Manuel Villalba Montoro1 1 University of Córdoba, Córdoba, ES, 2University of California, Los Angeles, US Coenzyme Q (Q) is a lipophilic organic molecule well known as a membrane-soluble electron carrier and antioxidant. Due to its antioxidant properties and its use as dietary supplement, the interest in this molecule has increased in recent years. However, our knowledge of its metabolism is quite limited. p-Hydroxybenzoic acid (pHB) was the only confirmed precursor of Q ring for more than 40 years until Pierrel et al. (2010) and Marbois et al. (2010) characterized p-aminobenzoic acid (pABA) as a novel Q precursor in yeasts. Later, Block et al. (2014) showed that Arabidopsis is able to use p-coumarate, but not pABA, as another ring precursor in Q biosynthesis. Recently, Xie et al. (2015) have described that human and E. coli cells do not utilize pABA as precursor in the biosynthesis of Q while p-coumarate and resveratrol, another polyphenol structurally similar to p-coumarate, serve as ring precursors of Q in E. coli, yeast and human cells. We hypothesized that other natural polyphenols could also behave as biosynthetic precursors of Q in mammalian cells. Mouse cells isolated from kidney proximal tubule (TKPTS) were used to test seven polyphenols: resveratrol, kaempferol, quercetin, piceatannol, luteolin, naringenin and apigenin. Our results showed an increase in Q levels in cells treated with some of these polyphenols, with kaempferol having the strongest effect. Pósters / Posters The increase of Q was related with upregulation of its biosynthetic rate because it was totally inhibited by pABA, a well-characterized inhibitor of Coq2 activity in mammalian cells. Additionally, we studied directly the role of kaempferol in Q biosynthesis by a competitive assay with radiolabelled 14 C-pHB. Kaempferol competed with 14C-pHB as ring precursor of Q and inhibited incorporation of 14C-pHB while simultaneously increasing Q levels. Moreover, we have performed studies with U-13C-kaempferol and show that this polyphenol is efficiently used as ring precursor in the biosynthesis of Q. Further experiments will be needed to understand the metabolic steps of how kaempferol is utilized as a ring-precursor in the Q biosynthetic pathway. P15r-7 Alterations of mitochondrial biogenesis, physiology and autophagy flux in an in vitro model by a dietary polyphenol Elena Gutiérrez Casado, Lucía Fernández del Río, José Manuel Villalba Montoro Departamento de Biología Celular, Fisiología e Inmunología. Universidad de Córdoba, Córdoba, ES Many antioxidant substances are able to suppress the deleterious effect of reactive oxygen species (ROS). Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a flavonoid found in many edible plants and used in traditional medicine, which can behave as antioxidant or pro-oxidant in a concentration-dependent manner. At high concentrations it can also induce autophagy and apoptosis, whereas at low concentrations it is capable of neutralizing superoxide anion. Recent results obtained by our research group indicate that this substance produces a significant increase in the levels of coenzyme Q, an endogenous antioxidant, in mouse kidney cells. The goal of the present study was to determine how mitochondrial physiology, as well as, the autophagy flux were affected under the same experimental conditions previously tested. Moreover, since this polyphenol can also activate the mitochondrial sirtuin Sirt3, we wanted to test out if in the presence of nicotinamide, a Sirt1 (NAD+-dependent deacetylase which plays a role in the autophagy flux) activator at low doses, we observed an increased response added to the flavonoid treatment. Cultures of a mouse kidney proximal tubule cell line (TKPTS) were treated with 10 μM kaempferol during 48 hours in the presence of absence of 10 mM nicotinamide. After that, different mitochondrial and oxidative stress-related parameters were measured, including mitochondrial abundance, intracellular levels of superoxide and peroxides, mitochondrial superoxide and mitochondrial membrane potential. Proteins related with autophagy were also measured by western blot. Our results indicate that kaempferol can modulate ROS levels and is able to modify the expression of different autophagy markers. In addition this polyphenol can alter some parameters of the mitochondrial function in TKPTS cells. P15-8 Efecto de un sazonador de hollejo de uva sobre vías de señalización asociadas a la hipertensión en células HUVEC Mónica Gisela Gerardi, Raquel Del Pino García, Mónica Cavia-Saiz, Dolores Rivero-Pérez, Mª Luisa González San José, Pilar Muñiz Rodríguez Departamento de Biotecnología y Ciencia de los Alimentos, Facultad de Ciencias, Universidad de Burgos, Burgos, ES La disfunción endotelial es el principal mecanismo implicado en el desarrollo de la hipertensión arterial. Es resultado de un desbalance entre los niveles de óxido nítrico (NO) y de radicales libres (ROS) en el endotelio vascular como consecuencia del estrés oxidativo. Diversas vías de señalización celular 113 Pósters / Posters regulan los niveles de estas dos moléculas. Entre ellas, se encuentran las que incluyen los factores de transcripción NF-κB y Nrf2. Los polifenoles de la uva poseen efectos moduladores sobre estos factores, lo que le otorgan efectos protectores manteniendo la buena funcionalidad del endotelio vascular. Nuestro trabajo consistió en caracterizar un producto de hollejo de uva con características funcionales para ser usado como sazonador. Para evaluar sus efectos funcionales el hollejo de uva se sometió a dos digestiones in vitro (gastrointestinal y fermentación colónica). Se evaluó la capacidad antioxidante por el método ABTS y su efecto protector en la línea de células endoteliales (HUVEC) sometida a estrés oxidativo con una alta concentración de glucosa. Se evaluó la viabilidad celular (mediante el ensayo de MTT) observándose un efecto citoprotector de las fracciones de digestión gastrointestinal y de fermentación colónica. Asimismo, el análisis de la expresión de los factores de transcripción (NF-κB y Nrf2) mediante real-time PCR y los niveles de activación de las kinasas (p38-MAPK y Akt) y de la vía del NF-κB (NF-κB e ikk) mediante inmunoblot. Se observó que el producto digerido en forma completa posee efectos protectores al aumentar la expresión de Nrf2 y disminuir la de NF-κB en las células sometidas a estrés oxidativo así como también al aumentar los niveles de p38-MAPK y Akt fosforilados y disminuir los de NF-κB e ikk. P15-9 Las glutaredoxinas monotiólicas Grx3/4 regulan Sir2 mediante glutationilación. Efecto en la resistencia a estrés y envejecimiento Núria Vall-llaura, Elisa Cabiscol Català IRB Lleida. Dept. Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida, ES En S. cerevisiae, las glutaredoxinas (Grx) monotiólicas son esenciales en la homeostasis del hierro pero, a diferencia de las Grx ditiólicas, se considera que no presentan una actividad oxido-reductasa relevante, dado que solo cuentan con 1 cisteína en su centro catalítico. Por otra parte, los mecanismos de regulación de la sirtuína Sir2, una histona deacetilasa NAD-dependiente implicada en la respuesta al estrés oxidativo y el envejecimiento, son desconocidos. En este estudio se investigó la regulación redox de Sir2 en situación de estrés y el papel e impacto fisiológico de Grx3 y Grx4 como tiol-reductasas de Sir2. Demostramos tanto in vitro como in vivo, que Sir2 se conjuga con glutatión (glutationilación) bajo estrés, lo que disminuye su actividad deacetilasa. Niveles fisiológicos de Grx3/4 pueden revertir esta modificación postraduccional. Grx3/4 interaccionan con Sir2 reduciéndolo y restableciendo su actividad de silenciamiento en el telómero. Mediante mutagénesis dirigida, se identificaron residuos de cisteína claves, localizados en el dominio catalítico de Sir2, como dianas de glutationilación. La mutación de esos residuos generó cepas con mayor resistencia tanto al estrés redox como al envejecimiento cronológico. Estos hallazgos ofrecen una nueva perspectiva, a nivel molecular, de las Grx monotiólicas en los procesos regulados por cambios redox, siendo Sir2 un sustrato fisiológico regulado por S-glutationilación. Teniendo en cuenta que la familia de las sirtuínas está conservada desde bacterias hasta humanos, estos resultados pueden ampliar nuestra comprensión del envejecimiento y sus enfermedades asociadas. P15-10 Dietary supplementation with almond and olive oil based beverage enriched with docosahexaenoic and vitamin E affects inflammatory gene expression in immune cells Antoni Pons Biescas1, Xavier Capó1, Miquel Martorell2, Antoni Sureda1, Joan Riera3, Francisco Drobnic3, Josep A. Tur1 1 CIBER: CB12/03/30038 Fisiopatología de la Obesidad la Nutrición, CIBEROBN, Instituto de Salud Carlos III (ISCIII), Research Group on Community Nutrition and Oxidative Stress, Science Laboratory of Physical Activity, Department of Fundamental 114 XXXIX Congreso SEBBM Biology and Health Sciences, University of Balearic Islands, Palma de Mallorca, ES, 2Departamento de Nutrición y Dietética, Facultad de Farmacia, Universidad de Concepción, Concepción, CL, 3Sports Physiology Dept. CAR, Sant Cugat del Valles, ES Exercise alters the number and function of circulating cells of the immune system. Omega-3 diet supplementation and exercise influence cytokine production and protect against chronic inflammation. The aim was to analyse the effects of an almond and olive oil beverage enriched with α-tocopherol and docosahexaenoic (DHA) on inflammatory and immune gene expression in peripheral blood mononuclear cells (PBMCs) and its effect on physical performance in sportsmen. Five young athletes and five senior athletes were supplemented for five weeks with a functional beverage. Blood samples were taken before and after supplementation and immediately before and 1 hour after the exercise test and PBMCs were isolated. Diet supplementation increased DHA erythrocytes content. Exercise test significantly increased body temperature, lactate blood levels and fatigue perception (Borg index) but no effects of dietary supplementation were observed on physical performance parameters. Age significantly reduced the values of Borg index during exercise test. NFκβ activation significantly increased as consequence of acute exercise mainly after dietary functional beverage supplementation; however no effects of exercise, supplementation or age were observed on NFκβ gene expression. Dietary beverage supplementation significantly increased 15LOX2 PBMCs gene expressions mainly in senior group. IL1β and IL8 expressions were enhanced after supplementation in young group mainly in post-exercise but not in senior group. To sum up, diet supplementation do not affect athletic performance. PBMCs are primed to pro-inflammatory response after exercise as indicated by its increased NFkβ active levels. Exercise enhances pro-inflammatory genes expressions mainly in young group after supplementation. This reinforces a pro-inflammatory effect of the functional beverage supplementation in young athletes. We hereby acknowledge the PhD grant provided by the University of the Balearic Islands and CIBEROBN CB12/03/30038. P15-11 La pérdida de DNA mitocondrial en células de cáncer hepático desequilibra la autofagia inducida por hipoxia química mediante la desregulación de la vía de señalización AMPK-ULK1 independientemente de HIF-1 Elisa Lozano1, Maitane Asensio2, Silvia Di Giacomo3, Annelies Noorlander2, Silvia Jiménez2, Felipe Jiménez1, Elisa Herráez1, José Juán García Marín1, María José Pérez García1 1 Hepatología Experimental y Vectorización de Fármacos (HEVEFARM), IBSAL, Universidad de Salamanca. Centro de Investigación Biomédica En Red para el Estudio de Enfermedades Hepáticas y Digestivas (CIBERehd), Salamanca, ES, 2Hepatología Experimental y Vectorización de Fármacos (HEVEFARM), IBSAL, Universidad de Salamanca, Salamanca, ES, 3Dept. Physiology and Pharmacology Vittorio Erspamer, Sapienza University of Rome, Roma, IT Antecedentes: La activación de la autofagia por factores exógenos del microambiente tumoral, como hipoxia, o endógenos como alteraciones en el DNA mitocondrial (DNAmt) es frecuente en células tumorales. Objetivo: Estudiar el efecto de la pérdida del genoma mitocondrial sobre la autofagia inducida por hipoxia en células SK-Hep-1 de cáncer hepático. Métodos: La hipoxia se simuló químicamente añadiendo cloruro de cobalto (CoCl2) o deferoxamina (DFO) al cultivo. Resultados: En las células silvestres (WT) y deficientes en DNAmt (Rho), estos agentes aumentaron la actividad lactato deshidrogenasa y la expresión de HIF-1 y p21, e indujeron la parada del ciclo celular. La hipoxia química activó la autofagia en las células WT pero no en las Rho. Se reprimió la Salamanca 2016 expresión de inhibidores de autofagia dependientes de HIF-1, como Bcl-2 y mTOR, en ambos tipos de células mientras que, solo en las WT, la hipoxia química activó la ruta pro-autofágica mediada por AMPK-ULK1. La generación de especies reactivas de oxígeno constitutiva e inducida por hipoxia química fue menor en las células Rho. En las células WT, el antioxidante N-acetilcisteína bloqueó la activación de AMPK inducida por CoCl2 y DFO, pero no el estrés del retículo endoplásmico inducido por CoCl2. Estos compuestos aumentaron la tasa de muerte en las células WT y en menor medida en las Rho. Al bloquear la activación de la autofagia con 3-metiladenina, la muerte celular inducida por DFO se redujo, mientras que la inducida por CoCl2 aumentó en las células WT, pero no en las Rho. Conclusión: La disfunción mitocondrial debida a la pérdida de DNAmt interfiere con las rutas de señalización que desencadenan la autofagia en células tumorales hepáticas, lo que puede favorecer la carcinogénesis. Pósters / Posters species (ROS) as metabolic byproducts of oxidative phosphorylation. Acute hypoxia produces a superoxide burst, which can signal downstream cell adaptations. Here we show that hypoxia triggers acute deactivation of mitochondrial complex I, changing its catalytic activity from an oxidoreductase to a Na+/H+ antiporter. The subsequent stimulation of mitochondrial Na+/Ca2+ exchanger (NCLX) activity promotes mitochondrial depolarization and superoxide production and underlies the participation of both complex I and NCLX in acute oxygen sensing. NCLX inhibition is associated with the suppression of downstream ROS-driven responses to hypoxia such as hypoxic pulmonary vasoconstriction, HIF-1α stabilization and ischemic brain damage in vivo. These results provide biological explanations for superoxide production in acute hypoxia and highlight complex I and NCLX as potential therapeutic targets in diseases involving mitochondrial ROS signalling, hypoxia and oxidative stress. Cofinanciado ISCIII-FEDER (PI15/00179). P15-14 P15-12 Las redoxinas y la promiscuidad de los intercambios redox entre cisteínas José Antonio Bárcena Universidad de Córdoba, Córdoba, ES Existen dos rutas antioxidantes de tioles en la mayoría de los organismos, una basada en Tiorredoxina (Trx) y otra en Glutarredoxina (Grx) y glutatión (GSH). Cada una tiene una flavoenzima oxidorreductasa única para mantenerlas reducidas con el poder reductor del NADPH (también Ferredoxina en plantas). Originalmente se consideró a la Trx ajena al mundo del GSH, que era exclusivo de Grx y las Peroxirredoxinas (Prx) realizaban su acción antioxidante con ayuda exclusiva de Trx. El panorama es distinto hoy día pues se han descrito y se siguen descubriendo interacciones entre ambos sistemas, el glutatión y las cisteínas de otras proteínas, dando lugar a una diversidad de mecanismos, no solo antioxidantes, sino de regulación y señalización. En esta charla se hará un recorrido sobre los descubrimientos de estas interacciones “inesperadas”, con énfasis en las contribuciones históricas y recientes del grupo de investigación sobre redoxinas de la Universidad de Córdoba y de otros grupos españoles. Entre las primeras cabe destacar la detección en los ’90 de isoformas de Grx en tejidos de mamífero y su localización en el núcleo de algunas células; la distribución de la Grx2 de levadura, producto de un único gen con dos sitios de iniciaciónde la traducción, entre el citosol y la mitocondria; la conexión entre ácido lipoico, Grx y GSH; la participación de Grx2 en el ciclo catalítico de la Prx1 de levadura; la resolución de Prx1 por GSH, mediante un mecanismo novedoso a concentraciones muy bajas, sin consumirse durante el ciclo catalítico y protegiendo a Prx1 de la sobreoxidación; la actuación de Trx como desglutationilasa completando el ciclo; finalmente, las dos actividades contrapuestas desnitrosilante y transnitrosilante de Trx1 humana funcionando simultáneamente. P15r-13 Acute hypoxia produces a superoxide signal through oxygen sensing by mitochondrial complex I and sodium/calcium exchange via NCLX Pablo Hernansanz Agustín1, Laura Moreno1, Elena Ramos2, Tamara Villa-Piña2, Elisa Navarro1, Esther Parada1, Alicia Izquierdo-Álvarez2, Nuria Sánchez-López1, Laura Pelaez-Aguado1, Daniel Tello1, Izaskun Buendía1, Anabel Marina1, Ángel Cogolludo3, Javier Egea1, Manuela G. López1, Anna Y. Bogdanova4, Antonio Martínez-Ruiz2 1 Universidad Autónoma de Madrid, Guadalajara, ES, 2Hospital Universitario de la Princesa, Madrid, ES, 3Universidad Complutense, Madrid, ES, 4University of Zurich, Zurich, CH Oxygen is a key molecule of aerobic life, but can give rise to reactive oxygen Evaluación del efecto citotóxico y bioactividad de un producto derivado de residuos de vinificación sobre las células HUVEC Mónica Gisela Gerardi, Mónica Cavia Saiz, Dolores Rivero Pérez, Ana Castañeda, Raquel Del Pino, Mª Luisa González San José, Pilar Muñiz Rodríguez Departamento de Biotecnología y Ciencia de los Alimentos, Facultad de Ciencias, Universidad de Burgos, Burgos, ES La hipertensión arterial (HTA) es uno de los factores de riesgo cardiovascular más prevalentes en los países occidentales. Numerosos estudios epidemiológicos sugieren que dietas ricas en polifenoles mejoran la función endotelial y disminuyen la presión arterial. Estos efectos positivos se han relacionado con la reducción del estrés oxidativo por su efecto directo sobre los ROS resultando en un aumento en la biodisponibilidad de NO y la inhibición de la enzima convertidora de la angiotensina (ACE), clave para el control de la presión arterial. Una fuente importante de polifenoles son los productos derivados de residuos de vinificación, descritos como fuentes de fibra dietética y antioxidantes. El objetivo de este estudio fue evaluar el efecto sobre biomarcadores de estrés oxidativo e hipertensión arterial del tratamiento con un producto derivado de residuos de masas de vinificación sobre células endoteliales de la vena umbilical (HUVECEA.hy926) normoglicémicas e hiperglicémicas. Para ello se trataron las células HUVEC con los compuestos liberados tras la digestión gastrointestinal in vitro de los extractos y se evaluó la citotoxicidad, el estado redox, cambios en la permeabilidad endotelial, el efecto inhibitorio sobre el sistema renina- angiotensina. Los resultados muestran que el producto no presenta citoxocidad y mejora el estado redox celular en células sometidas a un medio hiperglucémico. Sus efectos antihipertensivos se asocian a cambios observados en la regulación de las enzimas ACE y eNOS. Asimismo, el tratamiento con el producto de hollejo regula la permeabilidad de la monocapa de células endoteliales modificada por el TNF-α y el INF-γ en condiciones normo como hiperglucémicas. En resumen, nuestros resultados sugieren que los productos derivados de residuos de masas de vinificación presentan un alto potencial antioxidante y un efecto protector de la función endotelial, lo que implica su posible uso como alimento funcional o nutracéutico para la prevención y/o tratamiento de patologías cardiovasculares. P15-15 Control redox de tioles proteicos por redoxinas en una línea celular de hepatocarcinoma en un contexto de señalización por óxido nítrico endógeno María José López-Grueso1, Raúl González2, Carlos A. Fuentes-Almagro3, Raquel Requejo-Aguilar1, Emilia Martínez-Galisteo1, Jordi Muntané2, 115 Pósters / Posters C. Alicia Padilla1, J. Antonio Bárcena1 Dpto. Bioquímica y Biología Molecular e IMIBIC. Universidad de Córdoba, Córdoba, ES, 2Dpto. Cirugía General, Hospital Universitario Virgen del Rocío. IBIS. Universidad de Sevilla, Sevilla, ES, 3Unidad de Proteómica, SCAI. Universidad de Córdoba, Córdoba, ES 1 Los efectos del óxido nítrico (NO) se deben en su mayor parte a la modificación por S-nitrosilación de restos de cisteína estructural y funcionalmente importantes en proteínas clave. La Tiorredoxina (Trx) y la Glutarredoxina (Grx) son los principales sistemas celulares para el control del estado redox cisteínico habiéndose demostrado actividad desnitrosilasa para la Trx. Para definir el papel de Trx y Grx en la homeostasis redox y sus proteínas diana en condiciones nitrosilantes, se han silenciado Trx1 y Grx1 con siRNA específicos en células HepG2 que sobreexpresan NO Sintasa-3 (NOS3) y se han determinado cuantitativamente los proteomas global (Progenesis QIp) y redox tiólico (MRM-Skyline) diferenciales. Se ha aplicado la técnica de “Biotin Switch” para comprobar que los cambios redox se deben a S-nitrosilación. La sobreexpresión de NOS3 provoca muchos cambios a nivel del proteoma global. A nivel de proteoma redox, se han asignado valores de la relación “Cys reducida/Cys oxidada” para cada péptido cisteínico identificado. Así se han determinado dianas de la actividad desnitrosilasa de Trx1. La propia Trx1 se encuentra nitrosilada en su Cys73. Sorprendentemente, algunas Cys están menos oxidadas cuando la Trx1 está silenciada, indicando que posiblemente son sustratos de la actividad transnitrosilasa de Trx1. Por tanto, se demuestra que la Trx1 actúa en sentido antioxidante eliminando nitrosotioles proteicos mediante el sitio activo que tiene el ditiol Cys32-Cys35 y en sentido prooxidante transfiriendo un grupo nitroso a otros sustratos cisteínicos desde un segundo sitio activo en el que está Cys73. Estas actividades contrapuestas de Trx son conocidas, pero hasta ahora nunca se había descrito su funcionamiento simultáneo en las mismas condiciones celulares. P15r-16 Complex I assembly into supercomplexes regulates mitochondrial ROS production in neurons and astrocytes Irene López-Fabuel1, Juliette Le Douce2, Gilles Bonvento2, Andrew M. James3, Michael P. Murphy3, Angeles Almeida4, Juan Pedro Bolaños1 1 Institute of Functional Biology and Genomics (IBFG), University of Salamanca-CSIC, Salamanca, ES, 2CEA, DSV, I2BM, MIRCen, CNRS UMR 9199, Université Paris-Sud, Université Paris-Saclay, Fontenay-aux-Roses, FR, 3Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Cambridge, UK, 4 Institute of Biomedical Research of Salamanca (IBSAL), University Hospital of Salamanca, Salamanca, ES Reactive oxygen species (ROS) abundance is the result of the balance between their biosynthesis and degradation. In the brain, ROS detoxification is taken over by astrocytes through a robust antioxidant system that protects neighbor neurons; however, the mechanisms that control ROS production are unknown. Here, we found that astrocytes produce ROS several-fold faster than neurons by mitochondrial complex I. In astrocytes, complex I is mainly present as an individual complex, while in neurons it is predominantly assembled into supercomplexes. Using a complexomics approach, we identified complex I subunit, NDUFS1, to be less abundant in astrocytes than in neurons. Interestingly, NDUFS1 knockdown in neurons limited the association of complex I into supercomplexes, leading to impaired mitochondrial oxygen consumption and increased mitochondrial ROS. Conversely, over-expression of NDUFS1 in astrocytes promoted complex I incorporation into supercomplexes leading to decreased ROS. Thus, dynamic complex I assembly into supercomplexes is unveiled as a novel biological mechanism governing ROS abundance and contributing to the bioenergetics shapes of neurons and astrocytes. 116 XXXIX Congreso SEBBM P15-17 Estudio de las características bioenergéticas y del estrés oxidativo en líneas de cáncer de colon de diferentes estadios y su relación con la sensibilidad a la quimioterapia con 5-fluorouracilo e irinotecán Mª del Mar Blanquer Rosselló, Jordi Oliver Oliver, Ádamo Valle Gómez, María del Pilar Roca Salom Grupo Multidisciplinar de Oncología Traslacional, Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBERObn, CB06/03), ISCIII, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, ES Las células tumorales de estadios primarios son más vulnerables al estrés oxidativo por presentar bajos niveles de enzimas antioxidantes. En tumores de estadios más avanzados, en cambio, los sistemas antioxidantes se encuentran incrementados por una presión selectiva hacia la adaptación al estrés confiriéndoles resistencia a los tratamientos quimioterapéuticos que inducen estrés oxidativo. El objetivo fue analizar parámetros bioenergéticos y comparar el efecto citotóxico de los tratamientos antitumorales 5-fluorouracilo (5-FU) e irinotecán (IRI) y en combinación con resveratrol (RSV) en líneas de cáncer de colon en diferentes estadios: tumor primario (Caco-2 y HT29) y metastásica (SW620). Para ello, se determinó la velocidad de crecimiento, los niveles de estrés oxidativo y la expresión de proteínas implicadas en la funcionalidad mitocondrial y metabolismo celular (SIRT1, SIRT3, PGC1α, NRF1, NRF2, TFAM, UCP2, PDH y LDH). Para determinar los efectos citotóxicos de los tratamientos, se analizó la viabilidad celular y los niveles de estrés a diferentes dosis y tiempos. La línea metastásica fue la que presentó un mayor crecimiento, mayores niveles de estrés, así como mayor expresión de SIRT3 y TFAM. Se observó que el IC50 del 5-FU es menor que el del IRI en HT29 y Caco-2 mientras que en la línea SW620 se da el perfil opuesto. Además, dosis de IRI de 5 a 40 μM a 48 h provocaron un 50 % más de estrés que el 5-FU en las SW620. Estos resultados sugieren que el IRI tiene un mayor efecto citotóxico en la línea metastásica que en las primarias. Por otra parte, el tratamiento conjunto de 5-FU y RSV en la línea SW620 resultó en un efecto citotóxico sinérgico. Beca FPU del MECD, ISCIII (PI12/01827 y PI14/01434) y FEDER-Unión Europea “Una manera de hacer Europa”. P15-18 La importancia de la UCP2 en el control del estrés oxidativo en líneas de melanoma Margalida Torrens-Mas1, Daniel González-Hedström2, Mario Moreno-Fernández2, Jordi Oliver1, Pilar Roca1, Jorge Sastre-Serra1 1 Grupo Multidisciplinar de Oncología Traslacional, Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBERObn, CB06/03), ISCIII, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, ES, 2Dept. Biología Fundamental y Ciencias de la Salud, Universitat de les Illes Balears, Palma de Mallorca, ES El receptor de melanocortina 1 (MC1R) es clave para el desarrollo del melanoma, ya que está implicado en la pigmentación de la piel y la protección contra la radiación ultravioleta. Cuando se activa, el receptor induce la expresión de PGC-1α, que a su vez controla genes relacionados con la biogénesis mitocondrial y las enzimas antioxidantes, así como la proteína desacoplante UCP2, regulando así los niveles de especies reactivas de oxígeno (ROS). Estudios recientes sugieren que la UCP2 podría conferir Salamanca 2016 a las células resistencia frente al estrés oxidativo, siendo importante para la promoción tumoral. Nuestro objetivo fue estudiar la importancia de la UCP2 sobre el estrés oxidativo en distintas líneas de melanoma con distintas características del MC1R (mutado y no mutado). Para ello, se determinó la expresión de los genes PGC-1α, enzimas antioxidantes y UCP2, así como las actividades de las enzimas antioxidantes y la producción de ROS en las líneas HBL, A375 y MeWo. Las líneas HBL y MeWo, no mutadas para MC1R, presentaron unos mayores niveles de PGC-1α respecto a la línea con el receptor mutado A375. La producción de ROS fue superior en las líneas A375 y MeWo comparado con HBL. Sin embargo, las células MeWo mostraron unos mayores niveles tanto de la expresión como de la actividad de las enzimas antioxidantes que las otras dos líneas, mientras que la expresión de UCP2 fue mayor en la línea HBL. Estos resultados sugieren que la UCP2 puede jugar un papel clave en la producción de radicales libres en líneas de melanoma y, por tanto, en la evolución del tumor, siendo un factor importante a tener en cuenta junto a las características de PGC-1α y MC1R. Financiado: beca FPU del MECD yAECC (Asociación Española contra el Cáncer). P15-19 4-n-Butylresorcinol, a depigmenting agent used in cosmetics, reacts with tyrosinase Antonio García Jiménez1, José Antonio Teruel Puche2, Carmen Vanessa Ortiz Ruiz1, José Berna Cánovas3, José Tudela Serrano1, Francisco García Cánovas1 1 GENZ-Group of research on Enzymology (www.um.es/genz), Department of Biochemistry and Molecular Biology-A, Regional Campus of International Excellence Campus Mare Nostrum, University of Murcia, Espinardo, ES, 2Group of Molecular Interactions in Membranes, Department of Biochemistry and Molecular Biology-A, University of Murcia, Espinardo, ES, 3Group of Synthetic Organic Chemistry, Department of Organic Chemistry, Faculty of Chemistry, University of Murcia, Espinardo, ES Introduction: 4-n-Butylresorcinol (BR) is considered the most potent inhibitor of tyrosinase, which is why it is used in cosmetics as a depigmenting agent. However, some of these compounds previously considered inhibitors have recently been described to act as alternative substrates. Materials and methods: Spectrophotometric assays were carried out to determine the nature of BR, and to characterize it kinetically. Chemical shift values (δ) in 13C were determined by NMR. The chemical structures of these ligands were constructed with PyMOL 1.5.0.1. Results: The values obtained provided the Michaelis-constant (60.26 ± 8.76 μM) and catalytic constant (8.49 ± 0.20 s-1). Studies of BR docking to the Em (met-tyrosinase) form of the enzyme show that the hydroxyl group in C-1position is oriented toward the copper atom A (CuA), as in it is L-tyrosine. As regards Eox (oxy-tyrosinase), BR is oriented with the carbon in C-6position ready to be hydroxylated. Discussion: The Em form is not active on BR, but Eox can act on this molecule, hydroxylating it to o-diphenol. In turn, this is oxidized to an o-quinone, which isomerizes to a red p-quinone. Thus, for tyrosinase to act on this compound, a mechanism to generate Eox in the medium is required, which can be achieved by means of hydrogen peroxide or ascorbic acid. Conclusion: The results obtained show that BR is an alternative substrate of tyrosinase, originating o-quinones, which isomerize to p-quinones that can react with thiol compounds, a finding that could have important implications in pharmacology and cosmetics. Acknowledgements: This work was partially supported by: Projects 19545/ PI/14, 19304/PI/14 and 19240/PI/14 (Fundación Seneca, CARM, Spain); Projects SAF2013-48375-C2-1-R and CTQ2014-56887-P (MINECO); Projects UMU15452 and UMU17766 (University of Murcia); C.V. Pósters / Posters Ortiz-Ruiz has a MEC-FPU fellowship (AP2010-4300), A. García-Jiménez has a FPU fellowship from University of Murcia. P15-20 Efecto del resveratrol sobre la proliferación celular, ciclo celular y estrés oxidativo en la línea celular de cáncer de colon SW620 María del Mar Blanquer-Rosselló1, José Alberto Guerrero Castillo2, Reyniel Hernández López1, Ádamo Valle1, Pilar Roca1, Jordi Oliver1 1 Grupo Multidisciplinar de Oncología Traslacional, Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBERObn, CB06/03), ISCIII, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, ES, 2Dept. Biología Fundamental y Ciencias de la Salud, Universitat de les Illes Balears, Palma de Mallorca, ES El resveratrol (RSV) es un estilbenoide, presente en la piel de las uvas y otros frutos rojos, del que se han descrito efectos antioxidantes y antinflamatorios; además de efectos anticancerígenos, al inhibir la proliferación y la progresión del cáncer. Algunos estudios han relacionado estos efectos por ser un activador de la función mitocondrial y los mecanismos de antioxidación. El balance de estrés oxidativo juega un importante papel en la regulación del ciclo celular. Por ello, nos planteamos estudiar el efecto de dosis fisiológicas de RSV (10 μM) sobre el estrés oxidativo, el ciclo celular y la proliferación de la línea de cáncer de colon SW620. Se ha evaluado la viabilidad celular (cristal violeta), ciclo celular (citometría), el potencial de membrana (TMRM), la producción de ROS (Amplex red) y los niveles de UCP2, la apoptosis (Annexin V y PARP/PARP rota) y autofagia (MDC). También se estudió el efecto del RSV como adyuvante al tratamiento con 5-fluorouracilo (5FU) sobre la viabilidad y la producción de ROS. El tratamiento con RSV en las células de colon SW620 tuvo un efecto inhibidor de la proliferación celular induciendo la apoptosis (ciclo celular y PARP/PARP rota) sin aumento de la autofagia (MDC). Este efecto podría ser debido al incremento en la producción de ROS, ligado en parte a la reducción de la expresión de la UCP2. El efecto del RSV potenció los del 5FU inhibiendo la viabilidad y el nivel de estrés oxidativo. Los resultados obtenidos permiten concluir que el RSV aumenta la susceptibilidad de las células cancerígenas frente a la quimioterapia, al modificar la capacidad de las células de colon SW620 a hacer frente a un estrés oxidativo. Financiado: beca FPU del MECD, ISCIII (PI12/01827 y PI14/01434) y FEDER-Unión Europea “Una manera de hacer Europa”. P15-21 HO-1 induction via α7 nicotinic acetylcholine receptors: implications in the neuroinflammatory response Manuela G. López1, Elisa Navarro1, Cristina Fernández-Mendívil1, Enrique Luengo1, Jonathan Godbout2 1 Instituto Teófilo Hernando. Departamento de Farmacología. Facultad de Medicina. Universidad Autónoma de Madrid. Instituto Universitario de Investigacion la Princesa. Hospital de la Princesa, Madrid, ES, 2Department of Neuroscience. Institute for Behavioral Medicine Research. Center for Brain and Spinal Cord Repair. The Ohio State University Wexner Medical Center, Ohio, US We have previously shown that microglial HO-1 induction, via α7nAChR, controls inflammation to provide neuroprotection in stroke. Now, we have evaluated the implication of the α7nAChR/Nrf2/HO-1 117 Pósters / Posters axis in inflammatory models and its functionality with aging. Exposure of organotipic hippocampal cultures to the combination of antimycin-A (0.1 μM) and lipopolysaccharide (1 ng/ml) caused synergic neurotoxicity, in addition to mitochondrial depolarization, overproduction of reactive oxygen species, Nox4 overexpression and aberrant protein aggregation. The α7 nicotinic receptor agonist, PNU282987, prevented all these alterations by a mechanism that implicated HO-1 induction. The implications of HO-1 in controlling neuroinflammation, via α7nAChR stimulation, were further corroborated in WT and LysMCreHmox1∆/∆ mice, that do not express HO-1 in myeloid cells, treated with LPS. Furthermore, BALB/c mice were injected with LPS and the α7nAChR agonist, PNU282987 (i.p. or i.c.v). As expected, LPS (i.p.) challenge reduced body weight and induced lethargy and social with drawal in adult mice. Peripheral administration of the α7nAChR agonist improved these parameters. The α7nAChR agonist also reduced microglial activation with suppression of IL-1β and TNFα mRNA levels 4 and 24 h after LPS injection. Nonetheless, when PNU282987 was administered (i.p.) two hours after LPS challenge there was no effect. These data were interpreted to indicate that peripheral enhancement of α7nAChR signaling after microglia became activated was an ineffective intervention. When the α7nAChR agonist was administered centrally (i.c.v.) 2 h after LPS injection in adult and aged mice, in adult mice, central augmentation of nAChR signaling attenuated LPS-induced sickness behavior and microglial activation; however, in aged animals there was no improved recovery or decreased microglial activation following LPS challenge. In conclusion, we provide evidence that stimulation of α7nAChR signaling is anti-inflammatory via HO-1, but this cholinergic-dependent regulation is absent in microglia of aged mice. XXXIX Congreso SEBBM P15r-23 Friedreich ataxia cell model to screen new therapeutic drugs Elena Britti, Fabien Delaspre, Joaquim Ros IRB Lleida, Lleida, ES Friedreich´s ataxia (FA) is the most common hereditary autosomal recessive ataxia, affecting one of 50,000 people. It has been linked to expansion of a GAA-triplet repeat in the first intron of the FXN gene, leading to a reduced level of frataxin. The main affected cell types are sensitive neurons from dorsal root ganglia, cardiomyocytes and β cells. There is currently no effective cure or treatment for Friedreich ataxia. Life expectancy is around 40-50 years. During over 20-30 years the management of individuals with FA will require a multidisciplinary approach, from requirement of mobility aids to surgery, significantly impacting upon quality of life. Our lab has developed a FA cell model using primary culture of dorsal root ganglion sensory neurons (DRGs) from neonatal rat in which frataxin level was reduced by lentivirus transduction of shRNA sequences targeting frataxin mRNA. Frataxin deficiency in DRGs results in altered calcium homeostasis, neurite degeneration and apoptotic cell death. The analyse of this model point out several altered Ca2+-dependent pathways in response to reduced level of frataxin. Based on this model we set up a drug screen in order to identify potential new therapeutic approaches that could be used to treat FA patients. We characterized the ability of altered pathway targeting drugs in protecting frataxin deficient cells from neurite degeneration, calcium homeostasis alteration and apoptosis. In parallel, we used lymphoblastoid cell lines from FA patients to decipher the mechanism of action of the drugs. P15-24 P15r-22 Redox state of lipoic acid is altered in Frataxin-deficient cardiac myocytes Rosa Purroy Lledós, Anna Carrera Salinas, Joaquim Ros Salvador, Jordi Tamarit Sumalla Ciències Mèdiques Bàsiques, Universitat de Lleida, IRB Lleida, Lleida, ES Friedreich ataxia is a neurodegenerative disease accompanied by hypertrophic cardiomyopathy. This hereditary disease is caused by deficient frataxin expression, a mitochondrial protein that has been related to iron homeostasis, energy metabolism, and oxidative stress. We have recently set up a cardiac cellular model of Friedreich Ataxia based on neonatal rat cardiac myocytes and lentivirus-mediated frataxin RNA interference. As these frataxin-deficient cardiomyocytespresent signs of oxidative stress, we decided to explore the presence of protein thiol modifications in this model. With this purpose, the presence of reversible oxidized cysteine residues was investigated using the thiol-reactive fluorescent probe Bodipy-iodoacetamide and 2D-gel electrophoresis. We identified three spots with altered redox status in frataxin deficient cardiomyocytes that were identified by mass spectrometry as Electron transfer flavoprotein-ubiquinone oxidoreductase (ETFD), Dihydrolipoyl dehydrogenase (DLDH) and ATP synthase subunit alpha (ATPA). As DLDH is involved in lipoic acid regeneration, we hypothesized that frataxin-deficient cells could present lipoic acid in a more oxidized from. Lipoic acid is found bound to the E2 subunit of pyruvate dehydrogenase (PDH) and 2-ketoglutarate dehydrogenase complexes. Thus, we analyzed the western-blot signal of lipoic acid in cells treated or not with iodoacetamide. We observed a clear decrease in the iodoacetamide reactive form of lipoic acid bound to PDH E2, indicating that lipoic acid was more oxidized in frataxin-deficient cells. 118 Transformation of plum plants with cytosolic antioxidants leads to enhanced abiotic stress tolerance Pedro Díaz Vivancos CEBAS-CSIC, Murcia, ES In order to understand the role of cytosolic antioxidant enzymes in abiotic stress protection, transgenic plum (Prunus domestica cv. Claudia verde) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) and/ or ascorbate peroxidase (cytapx) were produced. First, we examined the potential functions of cytSOD and cytAPX in in vitro plum plants against salt stress. Several transgenic plum plantlets expressing cytsod and/or cytapx showed an enhanced tolerance to salt stress, mainly lines C5-5 and J8-1 (expressing several copies of sod and apx, respectively). Interestingly, these plants showed higher regeneration efficiency and enhanced vigour. Transformation as well as NaCl treatment influenced the antioxidative metabolism of plum plantlets, including enzymatic and non-enzymatic antioxidants. Then we tested the water stress (WS) tolerance under greenhouse conditions of two transgenic lines: line C3-1, co-expressing both transgenes simultaneously; and line J8-1, harbouring 4 copies of cytapx. Line J8-1 exhibited an enhanced stress tolerance that correlated with better photosynthetic performance and a tighter control of water use efficiency. Furthermore, this WS tolerance also correlated with a higher enzymatic antioxidant capacity than non-transformed and line C3-1 plum plants. On the other hand, line C3-1 displayed an intermediate phenotype between non-transformed plants and line J8-1 in terms of WS tolerance. Moreover, proteomic analysis revealed differences between non-transformed and J8-1 plants, mainly in terms of the abundance of proteins related with carbohydrate metabolism, photosynthesis, antioxidant defences and protein fate. Overall, our results suggest that transgenic plum plants are able to cope more efficiently with the induced oxidative stress under unfavourable environmental conditions. Salamanca 2016 P15-25 Sobrecarga de hierro y estrés oxidativo en la médula ósea de pacientes con síndromes mielodisplásicos: evolución tras el tratamiento con deferasirox Tamara Jiménez Solas1, María Díez Campelo2, Irene Aires Mejía2, Juan Carlos Caballero Berrocal2, Sandra Muntión Olave2, Félix López Cárdenas2, Luis García Martín2, Alba Redondo Guijo2, Mª Consuelo del Cañizo2, Ángel Hernández Hernández3 1 Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca. Servicio de Hematología, Hospital Universitario de Salamanca. Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, ES, 2Servicio de Hematología, Hospital Universitario de Salamanca. Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, ES, 3Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca. Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, ES Los síndromes mielodisplásicos (SMD) son una serie heterogénea de desórdenes hematológicos, caracterizados por hematopoyesis inefectiva en médula ósea asociada a citopenias, incluyendo anemias severas. Un alto número de pacientes de SMD de bajo riesgo recibe soporte transfusional como única terapia. Éstos presentan sobrecarga férrica, lo que provoca aumento en la producción de especies reactivas del oxígeno (ROS), al ser el hierro un catalizador de las reacciones de Fenton y Harber-Weiss. Esto podría implicar un alto nivel de estrés oxidativo para las células de la médula ósea (MO) de los pacientes. En base a estos datos, nuestra hipótesis de trabajo es la siguiente: La sobrecarga férrica involucrada en el incremento de ROS en la MO potencia la disfunción de las células hematopoyéticas (HSPC) y del estroma. Nuestro objetivo es analizar si el tratamiento quelante con deferasirox (DFX) puede reducir el estrés oxidativo en HSPC, así como el daño asociado en el DNA, y estudiar su efecto sobre la capacidad hematopoyética. Al comparar muestras de pacientes de SMD antes del tratamiento frente a controles sanos, nuestros resultados preliminares muestran mayores niveles de ROS en las poblaciones de HSPC de MO de los pacientes, mayor daño en el DNA y cierto incremento de la apoptosis temprana. Tras el tratamiento con DFX, se aprecia una tendencia hacia la reducción del daño en el DNA en la mayoría de pacientes. Los ensayos clonogénicos realizados antes del tratamiento indican una capacidad hematopoyética disminuida de las HSPC de pacientes. Mientras que tras el tratamiento, la relación cluster/CFU se aproxima a los valores control. Globalmente, estos resultados sugieren una mejora de la funcionalidad de la MO de los pacientes tras el tratamiento quelante. Pósters / Posters Whilst it is known the neuroprotective benefits of subtle stimulation of cannabinoid receptor-1 (CB1), its over-stimulation during cannabis abuse causes psychosis, memory loss and neurodegeneration. Recently, it has been demonstrated the occurrence of mitochondrial CB1 (mtCB1) in neurons and astrocytes where, by down-regulating protein kinase A and complex I activities, neuronal functions are physiologically regulated. However, whether over-activation of mtCB1 during cannabis abuse contributes to the detrimental effects of cannabis abuse on neurons is elusive. Given that neuronal survival depends on astrocyte metabolic support, we studied the impact that over-stimulation of astrocyte mtCB1 exerts on mitochondrial energy metabolism of neurons. We show that long-term (24 h) exposure of the CB1 agonists ∆9-tetrahydrocannabinol and HU-210 to astrocytes causes dephosphorylation of the complex I-assembly factor NDUFS4 and loss of complex I protein. In consequence of this, the production of mitochondrial reactive oxygen species and glycolytic-released lactate significantly decreased in astrocytes. Co-incubation of the CB1 agonists-pretreated astrocytes with untreated neurons caused mitochondrial membrane depolarization, energy impairment and apoptotic death of neurons. Noticeably, the changes triggered by the CB1 agonists were abolished in astrocytes obtained from CB1 knockout mice or treated with the plasma membrane-permeable CB1 antagonists AM251 and SR141716A, but not the plasma membrane-impermeable CB1 antagonist hemopressin. Thus, the astrocyte-dependent metabolic support of neurons is disrupted by over-stimulating mtCB1 in astrocytes, likely contributing to the neurological disabling caused by cannabis abuse. (Funded by the NIH/NIDA grant number 1R21DA037678-01). P15-27 Hydroxytyrosol reduces oxidative damage on oxidative stress in an experimental model of multiple sclerosis Eduardo Agüera1, Cristina Conde1, Evelio Luque2, Macarena Aguilar Luque3, Manuel LaTorre3, Ana Isabel Giraldo3, Montse Feijoó3, Rafael Lillo4, Begoña Mª Escribano5, Alberto Galván3, Isaac Túnez3 1 Unidad de Gestión Clínica de Neurología, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC)/Hospital Universitario Reina Sofía (HURS), Córdoba, ES, 2Sección de Histología, Departamento de Ciencias Morfológicas, Facultad de Medicina y Enfermería, IMIBIC/Universidad de Córdoba (UCO), Córdoba, ES, 3 Departamento de Bioquímica y Biología Molecular, Facultad de Medicina y Enfermería, IMIBIC/UCO, Córdoba, ES, 4Sección de Psiquiatría, Departamento de Ciencias Sociosanitarias y Radiología Clínica y Medicina Física, Facultad de Medicina y Enfermería, IMIBIC/UCO, Córdoba, ES, 5Sección de Fisiología, Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Veterinaria, IMIBIC/UCO, Córdoba, ES (Col.: Novartis) P15r-26 Over-stimulation of mitochondrial cannabinoid receptor-1 (mtCB1) in astrocytes disrupts astrocyte-dependent metabolic support of neurons Daniel Jiménez Blasco1, Mónica Resch-Beucher1, Mónica Carabias-Carrasco1, Eva Resel2, Manuel Guzmán2, Juan P. Bolaños1 1 Institute of Functional Biology and Genomics (IBFG), University of Salamanca-CSIC; and Institute of Biomedical Research of Salamanca (IBSAL), University Hospital of Salamanca, Salamanca, ES, 2Department of Biochemistry and Molecular Biology I, Instituto Universitario de Investigación Neuroquímica, Complutense University, Madrid, ES Objective: The aim of the present work was studied the effects of administration of hidroxytyrosol (HT) in a myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) model, similar to reproduce human multiple sclerosis (MS). Material and methods: Twenty male Dark Agouti rats, weighing 180-200 g and 2 months old were used in the study. The rats were divided into 4 groups of 5 as follow: i) control; ii)vehicle; iii) EAE; and iii) EAE+HT. Rats were injected with an emulsion containing myelin oligodendrocyte glycoprotein (MOG) supplemented with heat-inactivated Mycobactirium tuberculosis H37Ra. HT was administered orally using catheter during 65 days, starting 14 days after inoculation with MOG. Results: Our data showed a significant decrease score as well as a reversion towards to normality in the oxidative stress biomarkers after HT supplementation Conclusions: The HT administration shows positive and protective effects in EAE animals. However, more experimental and clinical studies in this line are required. 119 Pósters / Posters P15-28 Papel de la proproteína convertasa subtilisina/ kexina de tipo 9 (PCSK9) en el macrófago César Eduardo Rosales-Mendoza1, Marina Mojena2, Silvia González-Ramos2, Marta Paz-García2, Emilio Acosta-Medina3, Paloma Martín-Sanz2, Lisardo Boscá2 1 Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, ES; Departamento de Bioquímica y Medicina Molecular, Universidad Autónoma de Nuevo León, Monterrey, MX, 2Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, ES, 3 Centro de Estudios Avanzados de Cuba, La Habana, CU PCSK9 es una serin proteasa perteneciente a la familia de las proproteínas convertasas que se libera principalmente por el hepatocito. Su función es regular la degradación del receptor del colesterol LDL (LDL-R), uniéndose a este receptor e impidiendo su reciclaje e induciendo su degradación lisosomal así como la disminución de su expresión en la superficie de los hepatocitos. Esto impide el aclaramiento hepático del colesterol LDL (LDL-C) sérico, aumentando sus niveles, lo que supone un importante factor de riesgo cardiovascular. PCSK9 se ha convertido en una proteína de interés relevante desde la identificación de su papel en el metabolismo lipídico. A su vez, PCSK9 ha sido implicada en la inflamación. Se ha documentado que los niveles de PCSK9 correlacionan con los de LDL-C en condiciones patológicas como la sepsis. Los macrófagos, que son participantes activos de la respuesta inflamatoria, así como en los procesos antiinflamatorios y de pro-resolución de la inflamación, son de gran interés en el desarrollo de aterosclerosis. El proceso inflamatorio de la aterosclerosis parece estar más vinculado a PCSK9 que en solo la regulación del LDL-R. En el presente trabajo presentamos que tanto macrófagos humanos como murinos no expresan PCSK9. Sin embargo, su presencia modula la activación y fenotipo de estas células inmunes, tanto a nivel transcripcional como funcional, entre ellos la disminución del LDR-L, así como la expresión de genes inflamatorios y la generación de radicales libres. Por tanto, además de su papel en el metabolismo lipídico, PCSK9 ejerce cambios en el macrófago que pueden contribuir al desarrollo de aterosclerosis. P16. Regulación de la expresión génica y dinámica del genoma / Regulation of gene expression and genome dynamics P16-1 Thermodynamic signature for drug binding in the minor-groove of DNA XXXIX Congreso SEBBM in the minor groove of either C+G-rich or A+T–rich DNA sequences is entropically driven while intercalation is enthalpically driven. To gain insights into the different binding signatures, we have used UV-thermal denaturation and ITC (isothermal titration calorimetry), as well as computations of the hydrophobic free energy of binding, to determine the thermodynamic profile of drug binding to G+C-rich DNA sequences. The thermodynamic analysis shows that the binding in the minor groove of DNA is, regardless of the DNA sequence involved, always entropically-driven, dominated by the hydrophobic transfer of drug molecules from solution to the DNA-binding site. Direct molecular recognition between drugs and DNA through hydrogen bonding and van der Waals contacts also contributes significantly to drug-DNA complex formation. References [1] Chaires, J. B. (2006) Arch. Biochem. Biophys. 453, 24-29. [2] Barceló, F., Scotta, C., Ortiz-Lombardía, M., Méndez, C., Salas, J. A. & Portugal, J. (2007) Nucleic Acids Res. 35, 2215-2226. P16r-2 Efecto sobre el envejecimiento celular de la regulación traduccional mediada por la fosforilación del factor eIF2alfa Tamara Jiménez Saucedo, Juan José Berlanga Chiquero, Miguel Ángel Rodríguez Gabriel Centro de Biología Molecular Severo Ochoa, Madrid, ES El resultado de la respuesta a estrés (daño, muerte o adaptación y supervivencia) depende fundamentalmente de los procesos tempranos que tienen lugar en las células tras la exposición al mismo. Estos eventos tempranos incluyen la detección de estrés, la transducción de señales bioquímicas y los cambios transcripcionales y postranscripcionales. La hipótesis central de este proyecto es que los primeros cambios postranscripcionales de la expresión génica tras el estrés, especialmente la reprogramación de la traducción por la fosforilación del factor 2 de iniciación eucariota (eIF2), generan las señales y el conjunto de proteínas necesarias para promover la adaptación y la supervivencia de las células y organismos. Tras la aplicación de estrés, se induce la fosforilación de la serina 52 de la subunidad α del factor eIF2 lo que conlleva una disminución de la tasa de traducción general, así como la traducción de mRNAs específicos de respuesta a estrés. Además, estos eventos tempranos pueden condicionar procesos a largo plazo tales como el envejecimiento celular, y la aparición de patologías asociadas a una exposición crónica al estrés. En nuestro laboratorio hemos utilizado la levadura de fisión Schizosaccharomyces pombe como modelo y hemos llevado a cabo experimentos de privación de nutrientes, lo cual produce un efecto positivo sobre la longevidad. Además, se ha estudiado el efecto de varios inhibidores de la traducción sobre el envejecimiento así como el estado de la traducción en ambas condiciones. Detallamos los análisis bioquímicos de los resultamos en nuestra comunicación. Francisca Barceló Mairata1, José Portugal2 Universidad de les Illes Balears, Palma de Mallorca, ES, 2Instituto de Biología Molecular de Barcelona, CSIC, Parc Cientíific de Barcelona, Barcelona, ES P16-3 1 A mechanism for the recruitment of recombination proteins during DNA damage tolerance We have obtained complete thermodynamic profiles consisting of binding constants, free energy, enthalpy, and entropy for the binding of drugs in the G+C-rich regions of DNA. Such profiles are important in anticancer drug design because they provide information on the balance of driving forces that is not accessible by structural or computational methods alone [1, 2]. Despite most of the small drugs that bind to C+G-rich sequences are intercalating agents, a variety of molecules bind directly and selectively to C+G-rich DNA tracts through the minor groove without intercalation. Interestingly, many of those molecules are active as chemotherapeutic agents by inhibiting replication or transcription. The binding of drugs María J. Cabello-Lobato, María I. Cano-Linares, Román González-Prieto, Félix Prado Cabimer-CSIC, Sevilla, ES 120 The recombination proteins Rad52 and Rad51 help the fork to pass through blocking lesions and to fill in the gaps of ssDNA generated during the process of lesion bypass. Their recruitment to the ssDNA lesions has obligatorily to occur during S phase. Here we show that Rad52 and Rad51 display the same kinetics of chromatin binding as the helicase Mcm2-7: they accumulate in G1, are released during replication, and Salamanca 2016 Pósters / Posters Exploring the contribution of human prefoldin to gene expression gene expression. To achieve this PipX establishes physical complexes with PII or NtcA in response to low or high 2-oxoglutarate levels, respectively. Transcriptome analyses carried out with mutant strains are consistent with NtcA-dependent gene expression modulation of PipX. The results also suggest regulatory roles of PipX in an NtcA-independent way, implicating novel proteins in its network. In most cyanobacteria, pipX forms an operon with pipY. PipY is a member of the widely distributed COG0325 protein family, PLP-binding proteins of unknown function but related to amino acid metabolism and Vitamin B6 homeostasis, as inferred by null mutant analyses. To try to understand the function of COG0325 proteins and the regulatory connections between PipX and PipY proteins in cyanobacteria, we are characterizing the pipX-pipY operon of Synechococcus elongatus PCC7942, with an emphasis on the in vivo functions of PipY. Results from bioinformatics, transcriptomic and genetic analyses will be discussed. Laura Payán-Bravo1, Silvia Jimeno-González2, Xenia Peñate1, José Carlos Reyes2, Sebastián Chávez1 1 Instituto de Biomedicina de Sevilla (IBiS), Hospital Virgen del Rocío-CSIC-Universidad de Sevilla, Sevilla, ES, 2Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), CSIC, Sevilla, ES Contribution of TFIIS to nucleosome positioning revealed by improved MNase-seq remain bound to chromatin in the presence of replicative blocking lesions. This coordinated response requires Cdc7-dependent physical interactions between Rad51/Rad52 and the Mcm2-7 helicases that are not at the fork. Accordingly, reducing Cdc7 activity impairs sister-chromatid junction formation and ssDNA filling by recombination. Therefore, the loading of Rad51 and Rad52 in G1, together with a Mcm2-7-mediated mechanism that couples the fork advance with Rad52/Rad51 binding to replicative damage, provides a strategy to ensure the filling of the ssDNA lesions through an error-free repair mechanism. P16r-4 Prefoldin is a heterohexameric complex composed of six different subunits (PFDN1 to PFDN6) present from archaea to higher eukaryotes. It is a co-chaperone that cooperates with the cytoplasmic chaperonin CCT in the folding of actin and tubulin monomers. Eukaryotes also display a prefoldin-like complex, which contains PFDN2 and PFDN6 in addition to other non-canonical prefoldin subunits.We have previously showed that prefoldin is also localized in the nucleus of the yeast Saccharomyces cerevisiae where it plays a role in chromatin dynamics during transcription elongation (Millán-Zambrano, et al., 2013). We have also demonstrated that defective histone dynamics provokes changes in transcription elongation and co-transcriptional splicing in human cells (Jimeno-González et al., 2015). Here, we report that depletion of the canonical complex-specific PFDN5 subunit caused co-transcriptional splicing defects in CTNNBL1, a long human gene commonly used to analyze transcription elongation. However, we did not detect major alteration in the transcription rate of RNA polymerase II or its processivity (indirectly measured by pre-mRNA appearance) when PFDN5 was depleted. On the whole, these results suggest a possible role of prefoldin in human gene expression by contributing to co-transcriptional events. References [1] Millán-Zambrano G, Rodríguez-Gil A, Peñate X, de Miguel-Jiménez L, Morillo-Huesca M, et al., (2013) The prefoldin complex regulates chromatin dynamics during transcription elongation. PLoS Genet 9(9): e1003776. [2] Jimeno-González S, Payán-Bravo L, Muñoz-Cabello AM, Guijo M, Gutierrez G, et al., (2015). Defective histone supply causes changes in RNA polymerase II elongation rate and cotranscriptional pre-mRNA splicing. Proc Natl Acad Sci U S A. 112(48):14840-5. P16-6 Gabriel Gutiérrez1, Gonzalo Millán-Zambrano2, Xenia Peñate Salas2, Sebastián Chávez de Diego2 1 Departamento de Genética, Universidad de Sevilla, Sevilla, ES, 2 Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, ES Understanding chromatin dynamics is a key to other related processes, including DNA replication and transcription. As a first step, recently, an increasing amount of effort has been devoted to precisely define nucleosome positioning in different organisms. The most popular method to do so involves chromatin digestion by micrococcal nuclease (MNase), nowadays followed by ultrasequencing of the generated fragments. Although the sequence bias of MNase has been known for a long time, a data-based way of correcting this bias for sequencing experiments is still missing. Here we provide such a method, based on the digestion of naked DNA and the use of the bioinformatic tool DANPOS. Using Saccharomyces cerevisiae as a model, we demonstrate that the overall quality of the data is better after the correction proposed here. The characteristics of the method make it perfectly applicable to any other organism. We found that the correction affects more to those genes containing a canonical TATA element in their promoter than to TATA-like genes. Importantly, the improved method helped to uncover a new role for the TFIIS transcription elongation factor in chromatin dynamics. The absence of TFIIS caused a general increase in nucleosomal fuzziness, and a stronger occupancy of the -1 nucleosome at some promoters. Considering that TFIIS acts on RNA polymerase II to solve backtracking events, our results suggest that RNA polymerase II backtracking stabilizes nucleosomes out of their usual location and, by preventing it, TFIIS contributes to shape nucleosome positioning. P16-5 P16r-7 Expanding the roles of PipX, a cyanobacterial global regulator Búsqueda de nuevos reguladores de Kin4 y análisis de su función en el punto de control de posicionamiento del huso José Ignacio Labella, Javier Espinosa, Raquel Cantos, Asunción Contreras Universidad de Alicante, San Vicente, ES Cyanobacteria are ubiquitous autotrophic microorganisms that perform oxygenic photosynthesis. They response efficiently to changes in the Carbon/Nitrogen ratio through the highly conserved signal transduction protein PII and the global nitrogen regulator NtcA, a cyanobacterial CRP-like transcription factor. In the model cyanobacterium Synechococcus elongatus PCC 7942, PipX, a small protein exclusive of cyanobacteria, works as a mechanistic link to connect PII signalling with NtcA-regulated Laura Matellán1, Ana María Rincón2, Fernando Monje-Casas3 1 Universidad de Sevilla / CABIMER, Sevilla, ES, 2Universidad de Sevilla, Sevilla, ES, 3CSIC, Sevilla, ES Durante la división celular por mitosis es esencial mantener la fidelidad en la transmisión del material genómico para evitar que las células resultantes adquieran un cariotipo aberrante. Con este fin, las células cuentan con distintos mecanismos de vigilancia que controlan la segregación de los cromosomas. Entre estos sistemas, en Saccharomyces cerevisiae es particularmente interesante la existencia del punto de control 121 Pósters / Posters de posicionamiento del huso (SPOC), un mecanismo de vigilancia que evita que las células con el huso mitótico mal orientado salgan de mitosis, lo cual es esencial dada la naturaleza asimétrica de la división celular en la levadura de gemación. La proteína quinasa Kin4 juega un papel central en el SPOC. Sin embargo, los mecanismos que regulan la actividad de Kin4 tras la activación del SPOC todavía no se conocen en profundidad. Nuestro objetivo es la búsqueda de proteínas que interaccionen con Kin4 de forma dependiente del estado de activación del SPOC, y que por tanto puedan jugar un papel importante en la regulación de este mecanismo de vigilancia. Con este fin, hemos generado una estirpe de levaduras en la que podemos activar constitutivamente el SPOC de forma condicional. Usando esta estirpe, hemos conseguido purificar Kin4 tanto en condiciones de crecimiento normal como en condiciones de activación del SPOC, y hemos estudiado el conjunto de proteínas que interaccionan con esta quinasa de forma diferencial en ambas condiciones. El análisis de estas proteínas y de su papel en la regulación de la actividad y/o la localización de Kin4 nos permitirá descifrar los mecanismos moleculares de regulación del SPOC. Los resultados de esta investigación serán importantes para, por medio del estudio de los homólogos de las proteínas identificadas, entender cómo se coordina el correcto posicionamiento del huso con la progresión del ciclo celular en divisiones asimétricas en eucariotas superiores. P16-8 Histone depletion prevents telomere-telomere fusions in pre-senescent cells Marta Barrientos-Moreno1, Marina Murillo-Pineda2, Ana M. Muñoz-Cabello3, Félix Prado1 1 CABIMER-CSIC, Sevilla, ES, 2Instituto Gulbenkian de Ciência, Oeiras, PT, 3IBIS, Sevilla, ES Nucleosome assembly during DNA replication establishes the primary chromatin structure, which is essential for genome organization and stability. Defective chromatin assembly causes replication fork instability and hyper-recombination, and impairs sister-chromatid decatenation and chromosome bi-orientation during mitosis. The importance of nucleosome assembly in genome integrity is also highlighted by the fact that replicative aging in yeast and replicative senescence in yeast and humans are associated with a severe reduction in the amount of histones. Telomere shortening can prevent tumour development by inducing senescence of transformed cells; however, it can also lead to cancer initiation by inducing chromosomal instability because cells divide during pre-senescence with critically short telomeres that can be recognize as unrepaired breaks and be fused to other telomeres (telomere-telomere fusions; T-TFs) or DSBs, thus causing gross chromosomal rearrangements. Here we show that histone depletion prevents T-TFs in mec1∆ tel1∆ cells defective in telomere maintenance. Since pre-senescence in cells lacking the telomerase has been shown to be associated with a reduction in the pool of available histones through a Mec1-dependent mechanism, we hypothesize that histone depletion is an active mechanism to protect telomeres that is lost in mec1∆ tel1∆ cells. This mechanism is independent of the telomerase but strictly dependent on homologous recombination. We propose that the increase in recombination by replication fork collapse as a consequence of the deficit of histones protects short telomeres during pre-senescence. P16-9 Sod1-activity is linked to selenite toxicity in yeast Hayat Heluani Gahete1, Elisabet Fernández García1, Carlos Ruiz Rodríguez1, Prince Saforo Amponsah2, Bruce Morgan2, Ralf Wellinger1 1 CABIMER, Sevilla, ES, 2TU Kaiserslautern, Kaiserslautern, DE Selenium (Se) is a trace element that is essential for human health. Reduction of inorganic selenium (sodium selenite; Na2SeO3) involves 122 XXXIX Congreso SEBBM glutathione (GSH) oxidation and the formation of reactive oxygen species (ROS). ROS then have to be detoxified in order to avoid ROS-mediated DNA damage and genomic instability1. We find that the budding yeast sod1Δ mutant rescues growth of selenite hypersensitive yeast mutants. This is a surprising observation, given the fact SOD1 encodes for a superoxide dismutase needed for the tolerance to ROS by the conversion of ion superoxide (O2-) into oxygen (O2) and hydrogen peroxide (H2O2). Here, we will present new data on factors involved in selenite resistance pointing to a complex interplay between mechanisms involved in selenite detoxification and oxidative stress response. Keywords: ROS, Sod1, Sodium Selenite References [1] Herrero E, Wellinger RE. Review: “Yeast as a model system to study metabolic impact of selenium compounds”. Microbiol Cell (2015). P16r-10 Role of morphine, miR-212/132 and μ opioid receptor in the regulation of Bdnf in zebrafish embryos Ada Jiménez González1, Adrián García Concejo1, Saray López Benito1, Juan Carlos Arévalo2, Raquel E. Rodríguez3 1 Instituto de Investigación Biomédica de Salamanca; Instituto de Neurociencias de Castilla y León (Universidad de Salamanca), Salamanca, ES, 2Instituto de Investigación Biomédica de Salamanca; Instituto de Neurociencias de Castilla y León (Universidad de Salamanca); Departamento de Biología Celular y Patología, Salamanca, ES, 3Departamento de Bioquímica y Biología Molecular; Instituto de Investigación Biomédica de Salamanca; Instituto de Neurociencias de Castilla y León (Universidad de Salamanca), Salamanca, ES Morphine is one of the first-line therapies for the treatment of pain despite its secondary effects. It modifies the expression of post-transcriptional regulators like miRNAs. In the present study, we analyzed miR-212 and miR-132 and their implication in morphine effects in the zebrafish Central Nervous System (CNS) through the regulation of Bdnf expression. We used control and knock-down zebrafish embryos to assess the effects of morphine in miRNAs212/132 and mitotic or apoptotic cells by qPCR, immunohistochemistry and TUNEL assay, respectively. Bdnf and TrkB were studied by western blot and through a primary neuron culture. A luciferase assay was performed to confirm the binding of miRNAs 212/132 to mecp2. Our results showed that morphine exposure decreases miR-212 but upregulates miR-132, as wells as Bdnf and TrkB, and it changes the localization of proliferative cells. However, Bdnf expression was downregulated when miRNAs 212/132 and oprm1 were knocked-down. Furthermore, we proved that these miRNAs inhibit mecp2 expression by binding to its mRNA sequence. The described effects were corroborated in a primary neuron culture from zebrafish embryos. Taking these results into consideration we propose a mechanism in which morphine alters the levels of miRNAs 212/132 increasing Bdnf expression through mecp2 inhibition. oprm1 is also directly involved in this regulation. The present work confirms a relationship between the opioid system and neurotrophins pointing to miRNAs 212/132 as novel regulators of the effects produced by morphine on the CNS. Both miRNAs have a key role on morphine effects through the regulation of Bdnf pathway which is also controlled by oprm1. Salamanca 2016 Pósters / Posters P16r-11 P16-13 μ opioid receptor expression after morphine administration is regulated by miR- 212/132 cluster Regulación de la replicación por el checkpoint de fase S en Saccharomyces cerevisiae Adrián García Concejo, Ada Jiménez González, Raquel E. Rodríguez Instituto de Neurociencias de Castilla y León (Universidad de Salamanca); Instituto de Investigación Biomédica de Salamanca, Salamanca, ES Since their discovery, miRNAs have emerged as a promising therapeutical approach in the treatment of several diseases. miR-212 has already been related to cocaine intake and to addiction, and here we prove that the miR-212/132 cluster can be regulated by morphine through the activation of the mu opioid receptor (Oprm1). The molecular pathways triggered after morphine administration also induce changes in the levels of expression of oprm1. In addition, miR-212/132 cluster is actively repressing the expression of mu opioid receptor by targeting a sequence in the 3’’ UTR of its mRNA. These findings suggest that this cluster is closely related to opioid signaling and function as a post-transcriptional regulator, modulating morphine response in a dose dependent manner. The regulation of miR-212/132 cluster expression is mediated by the MAP kinase pathway, CaMKII-CaMKIV and PKA, through the phosphorylation of CREB and methyl cytosine binding protein 2 (MeCP2). In addition, the regulation of both oprm1 and of the miRNAs cluster promoter is mediated by MeCP2, acting as a transcriptional repressor on methylated DNA after prolonged morphine administration. This mechanism explains the molecular signaling triggered by morphine as well as the regulation of the expression of the mu opioid receptor mediated by morphine and the implication of miR-212/132 in these processes. P16r-12 Caracterización bioquímica de la resolvasa de uniones de Holliday Yen1 Francisco Javier Aguado Domínguez, Vanesa Hurtado Nieves, Miguel González Blanco Centro de Investigación en Medicina Molecular y Enfermedades Crónicas (CiMUS), Universidade de Santiago de Compostela, Santiago de Compostela, ES La recombinación homóloga (HR) es un mecanismo esencial para la reparación de roturas en la doble cadena del DNA. Durante este proceso, se pueden generar intermediarios recombinatorios ramificados, que en algunos casos pueden madurar hasta formar uniones estables constituidas por cuatro cadenas de DNA denominadas uniones de Holliday (HJ). Dichas conexiones deben deshacerse oportunamente para permitir una correcta segregación cromosómica durante la mitosis. Para ello, la célula dispone de varios mecanismos de resolución de HJ, siendo una de las enzimas implicadas en este proceso la nucleasa selectiva de estructura Yen1. La actividad de Yen1 está altamente regulada a lo largo del ciclo celular mediante modificaciones post-traduccionales. Concretamente, su fosforilación por parte de Cdk en fase S conlleva su expulsión del núcleo y disminuye su afinidad por el DNA, reduciendo la actividad biológica del enzima. En anafase, el enzima es defosforilado por la fosfatasa Cdc14, que permite su reactivación e importación al núcleo, donde podrá eliminar aquellos intermediarios de recombinación más persistentes. Recientemente hemos demostrado la toxicidad de expresar una versión mutante de Yen1 constitutivamente activa. Sin embargo, desconocemos cuál de las posibles actividades nucleásicas de Yen1 puede ser responsable de dicha toxicidad ya que aún no se han caracterizado a fondo las propiedades bioquímicas de este enzima. Aquí presentamos un análisis bioquímico de Yen1 que revela actividades inesperadas de esta nucleasa. Tales actividades distintas de la resolución canónica de las HJ podrían explicar los efectos deletéreos de la desregulación de este enzima y, por tanto, de la importancia de su adecuado control. Esther C. Morafraile1, Alberto Bugallo1, Mónica Segurado2 Instituto de Biología Funcional y Genómica, Salamanca, ES, 2 Departamento de Microbiología y Genética (USAL), Salamanca, ES 1 Para mantener la integridad genómica, el DNA debe ser protegido del daño provocado por agentes genotóxicos o por alteraciones surgidas durante la replicación. El checkpoint de fase S, mediado por Mec1 y Rad53 en Saccharomyces cerevisiae (ATR y Chk2 en células humanas, respectivamente) responde a perturbaciones en la replicación y coordina una respuesta celular global necesaria para mantener la integridad del genoma. Estamos interesados en entender cómo el checkpoint de fase S regula la replicación en estas condiciones, y más específicamente, en los mecanismos moleculares que preservan la estabilidad de las horquillas de replicación y promueven el reinicio de la síntesis de DNA tras bloqueos en la replicación. Como la quinasa Rad53 estabiliza las horquillas de replicación mediante un mecanismo dependiente de Exo1, hemos estudiado la regulación de esta nucleasa, y nuestros resultados indican que Exo1 es fosforilada por Rad53 en respuesta a estrés replicativo o daño en el DNA. Mutantes fosfomiméticos de EXO1 suprimen el colapso de horquillas de replicación de mutantes rad53, al igual que la deleción de la nucleasa, lo que sugiere fuertemente que la fosforilación de Exo1 tiene un efecto inhibitorio en la actividad de la proteína. Sin embargo, la deleción de EXO1 no suprime el defecto de reiniciación de horquillas observado en el mutante rad53, lo que sugiere que la reactivación de las horquillas bloqueadas es regulada mediante otro mecanismo. Nuestros resultados indican que Rad53 promueve el reinicio de horquillas de replicación a través de la inducción transcripcional de genes de respuesta a daño en el DNA. Por tanto, además de prevenir la degradación de las horquillas, Rad53 promueve el reinicio de la replicación induciendo la expresión de los genes de la ribonucleótido reductasa (RNR) y regulando la localización subcelular del complejo tras estrés replicativo. P16r-14 Búsqueda de nuevos reguladores de Bfa1 y análisis de su papel en la inhibición de la salida de mitosis tras la generación de daños al ADN Inés García de Oya1, Fernando Monje-Casas2 1 Universidad Sevilla/ CABIMER, Sevilla, ES, 2CSIC, Sevilla, ES La fidelidad de la transmisión de la información genética durante el ciclo celular es controlada por la acción de distintos mecanismos de vigilancia, que previenen la generación de productos aneuploides. En Saccharomyces cerevisiae, estos puntos de control (checkpoints) controlan, entre otros aspectos, la integridad del genoma, la unión anfitélica de los cromosomas al huso mitótico, o el correcto posicionamiento del huso. A pesar de que los tres mecanismos de vigilancia anteriores responden a distintas señales y en diferentes momentos del ciclo, todos ellos conducen a una inhibición de la salida de mitosis a través de la acción del complejo Bfa1-Bub2. Estas proteínas son reguladores negativos de la ruta MEN (mitotic exit network), una cascada de señalización que determina la salida de mitosis. La actividad del complejo Bfa1-Bub2 es regulada mediante su fosforilación. En concreto, Bfa1 es fosforilada gradualmente a lo largo del ciclo celular por las quinasas Cdc28 y Cdc5, alcanzando su máximo nivel de fosforilación en anafase. La fosforilación de Bfa1 por Cdc5 en anafase inhibe la actividad de Bfa1-Bub2, promoviendo así la salida de mitosis. Por otro lado, en condiciones de activación del checkpoint de posicionamiento del huso (SPOC), Kin4 fosforila a Bfa1 para mantenerla en un estado activo e inhibir la salida de mitosis hasta que el huso se alinee correctamente. Estudios recientes desarrollados por nuestro grupo de investigación (Valerio-Santiago et al., 2013) demuestran que, además de por las quinasas anteriores, el estado de fosforilación de Bfa1 es 123 Pósters / Posters también regulado por una quinasa aún no identificada que mantiene a Bfa1 fosforilado tras la activación del checkpoint de daño al ADN. El objetivo de mi investigación es la identificación de la quinasa o quinasas responsables de la fosforilación de Bfa1 tras la generación de daños en el ADN y de su papel en el control de la ruta MEN en estas condiciones. P16r-15 Mechanisms underlying gene repression in hypoxia María Tiana Cerrolaza, Benilde Jiménez Cuenca, Luis del Peso Ovalle Universidad Autónoma de Madrid, Madrid, ES In response to hypoxia, cells activate a stereotyped gene expression pattern that allows them to cope with the restricted oxygen supply. This response involves the induction of hundreds of genes by a mechanism that requires the recruitment of the hypoxia inducible factors (HIFs) to their cis-regulatory regions. On the other hand, the adaptation to reduced oxygen also implies the repression of a large number of genes, but the mechanisms by which hypoxia leads to gene repression have not been fully elucidated yet. In this work we aimed to identify the general mechanisms that underlie gene downregulation in response to hypoxia. To this end, we firstly determined the relative contribution of transcription and decay rates to the hypoxic gene expression and found that transcription is the major factor contributing to the gene expression changes triggered by hypoxia, whereas modification of half-lives had marginal impact. Next, by focusing on transcriptional changes, we found that, although EPAS1 binding was restricted to the fraction of the genes induced by hypoxia, it was required for both positive and negative changes of transcription. Taken together, these results suggested that transcriptional repression is indirectly mediated by EPAS1 and consequently we concluded that it should be mediated by transcription factor(s) downstream of EPAS1. Next, we exploited the genome-wide transcription factor binding sites generated by the ENCODE project to identify transcription factors enriched in the regulatory regions of the transcriptionally repressed genes. This analysis revealed a reduced set of transcription factors as key molecules that could act downstream of EPAS1 to repress gene expression in hypoxia. P16-16 Activation-induced cytidine deaminase interacts with SUV4-20H and targets H4K20 methylation to class-switch recombination sites Virginia Carolina Rodríguez Cortez1, Paloma Martínez-Redondo*2, Francesc Català-Moll*1, Javier Rodríguez-Ubreva1, Antonio García-Gómez1, Laura Ciudad Garrido1, Henar Hernando Arrabal1, Carlos Company1, José Miguel Urquiza1, Javier Di Noia3, Alejandro Vaquero2, Esteban Ballestar Tarín1 1 Chromatin and Disease Group, Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, ES, 2Chromatin Biology Group, Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, ES, 3Institut de Recherches Cliniques de Montréal, Division of Immunity and Viral Infections, Montreal, CA Activation-induced cytidine deaminase (AID) triggers antibody diversification in B cells by catalysing deamination and subsequently mutating immunoglobulin genes. The biological roles of this enzyme with respect to its potential relationship with active DNA demethylation are controversial. Association of AID with RNA Pol II suggests a possible relationship with gene regulatory functions. AID is mutated in hyper-IgM type 2 (HIGM2) syndrome. In this study, we investigated the potential role of AID in the acquisition of epigenetic changes. We discovered that 124 XXXIX Congreso SEBBM the binding of AID to the IgH locus, a prominent AID target, causes an increase in H4K20me3. We also demonstrate for the first time that AID interacts with the three SUV4-20 enzymes, and that SUV420H1.2, which is bound to the Sμ site of the IgH locus, is replaced by SUV420H2 upon AID binding. Analysis of HIGM2 mutants indicates that the interaction with SUV4-20 enzymes takes places at the N-t of AID, as even a truncated form of AID lacking two C-t thirds of the protein shows a specific interaction. Our results provide a new perspective on the interplay between AID and the histone modification machinery in setting the epigenetic status of class-switch recombination sites. *Equal contributors Correspondence to: Esteban Ballestar, Chromatin and Disease Group, Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical Research Institute (IDIBELL) L’Hospitalet de Llobregat, Barcelona, Spain. Key words: AID, Hyper-IgM, H4K20me3, SUV420. P16-17 Meiotic recombination variability in male mice: genetic and environmental effects Elena de la Casa Esperón1, Esteranía San Martín Pérez2 1 Parque Científico y Tecnológico de Castilla-La Mancha. C.R.I.B. Universidad de Castilla-La Mancha, Albacete, ES, 2C.R.I.B. Universidad de Castilla-La Mancha, Albacete, ES Meiotic recombination is not only important for reshuffling the alleles passed to the next generation, but also for ensuring proper chromosome segregation to the gametes. Changes in the frequency and distribution of crossovers can increase the risk of producing aneuploid gametes that result in fertility and developmental defects. Hence, recombination is very tightly controlled. Crossover frequency and location depend on both genetic and epigenetic factors, but little is know about both of them in mammals. Recent studies have shown that the endocrine disruptor bisphenol A can modify the levels of recombination, possibly through epigenetic changes. We have initiated a study for characterizing the effects of other environmental factors on male crossover frequency and distribution, particularly of those that have epigenetic and phenotypic effects through generations, such as certain diets. Because recombination levels show some degree of variation in mouse, we expect that the environment might have different impact on recombination in different genetic backgrounds. Therefore, we have first characterized the frequency and distribution of crossovers in males of diverse Mus musculus inbred strains. Our studies confirm that recombination levels depend on the genetic background and, hence, are genetically controlled. These levels are particularly high in strains of M. m. musculus subspecies origin. We are initiating our studies of environmental impact on recombination by testing whether diverse diets have an effect on male recombination levels. P16r-18 Papel de la eIF2alfa quinasa GCN2 en la supervivencia celular en respuesta a estrés nutricional y a radiación ultravioleta Teresa García Prieto, César de Haro Castella, Juan José Berlanga Chiquero Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, ES La respuesta al estrés permite a los seres vivos responder y adaptarse a un ambiente cambiante que los expone a radiación, cambios de temperatura, agentes tóxicos y a la eventual carencia de nutrientes. Una adecuada respuesta al estrés no solo determina las posibilidades de sobrevivir y adaptarse, sino que también puede afectar al proceso natural de envejecimiento y al desarrollo de algunas patologías Salamanca 2016 Pósters / Posters asociadas al mismo (cáncer, diabetes, enfermedades neurodegenerativas y cardiovasculares). La naturaleza de la respuesta al estrés en mamíferos es fundamentalmente adaptativa, y debe de entenderse como un intento del organismo de restablecer la homeostasis celular y sistémica que ha resultado alterada. Además, episodios de estrés de moderada o baja intensidad a lo largo de la vida pueden preparar al animal para responder mejor a futuras situaciones de estrés agudo. Este concepto, se ha denominado hormesis. La fosforilación transitoria del factor de iniciación de la traducción eIF2 en su subunidad alfa, por eIF2alfa quinasas específicas, provoca una parada instantánea de la síntesis general de proteínas y, al mismo tiempo, activa la traducción de algunos ARNm implicados en promover y coordinar la respuesta al estrés. La eIF2alfa quinasa GCN2 se activa en respuesta a privación de aminoácidos y a irradiación con luz ultravioleta. En este estudio hemos comprobado que la falta de GCN2 en fibroblastos embrionarios de ratón hace a estas células más sensibles a dichas formas de estrés, disminuyendo su capacidad de supervivencia a corto plazo y su capacidad proliferativa a largo plazo. Además, hemos tratado de probar el concepto de hormesis, sometiendo a las células a privación de aminoácidos previamente a su tratamiento con radiación ultravioleta. development. Data from our lab indicate that DYRK1A is recruited to proximal promoter regions of genes characterized by the presence of a conserved palindromic motif. DYRK1A-dependent transcriptional regulation of its targets appears to be due to phosphorylation of the Carboxy-Terminal Domain (CTD) of the RNA polymerase II. However, we know little on how DYRK1A is recruited to its genomic loci. By using electrophoretic mobility assays, we have found that bacterially expressed and purified DYRK1A binds to single-stranded DNA probes containing the palindromic sequence, suggesting that DYRK1A might bind to unwound DNA stabilizing single strands at the chromatin level. Analysis of the ChIP-Seq data from ENCODE reveals that several transcription/ chromatin-related factors can be recruited to the DYRK1A-consensus motif. The list includes the co-repressor ZBTB33/KAISO and the tumor suppressor BRCA1. We have validated the presence of ZBTB33/KAISO and BRCA1 at several DYRK1A-bound regions; in addition, the interaction of DYRK1A with KAISO or BRCA1 is detected in nuclear extracts by co-immunoprecipitation experiments. These findings raise the possibility of a functional cross-talk between DYRK1A and any of these proteins in the regulation of their target genes. P16-19 Oncogene or tumor suppressor? New roles in tumorogenesis for the mitotic kinase Plk1 P16-21 An X family DNA polymerase protects mitochondrial DNA in the infective filament of Ustilago maydis María Tenorio-Gómez, José Pérez-Martín Instituto de Biología Funcional y Genómica, Salamanca, ES Ustilago maydis is a phytopathogenic fungus thatinfects the maize plant to provoke the corn smut disease. A morphological switch from yeast to infective filament is an indispensable prerequisite for enabling the infection to take place. This filament is a cell cycle arrested dikaryotic hypha that arises from the conjugation of two compatible yeasts, grows on the plant surface and eventually develops an specific structure called appresorium, required to penetrate the plant tissue. The formation of the filament in U. maydis is driven by a complex transcriptional programme which is mainly orchestrated by a transcriptional regulator called b-factor. One of the direct targets of the b-factor is PolX, a gene encoding a putative DNA polymerase which is strongly expressed upon filament formation (Brachmann et al., Mol Microb 42, 1047). According to its sequence, this polymerase would belong to the X family of DNA polymerases, implicated in DNA repair processes. We have analysed the ability of polX null mutant dikaryotic hyphae to cope with DNA damaging agents and we have found only slight defects in response to oxidative stress. Nevertheless, we have come across that PolX seems to be required for the ability of the dikaryotic hyphae to resume proliferation once the b-programme is repressed. Interestingly, we have also found that PolX is a mitochondrial protein, most likely involved in the repair of damaged mitochondrial DNA. In this communication we will discuss why the induction of the infective hypha needs the presence of a DNA polymerase to protect the mitochondria. Pablo Sanclemente1, Rocio Sotillo2, Marcos Malumbres1, Guillermo de Cárcer Díez1 1 CNIO, Madrid, ES, 2DKFZ, Heidelberg, DE Mitosis is the ultimate step of the cell cycle, allowing the dividing cell to segregate their DNA content equally to the two daughter cells, and it is tightly regulated by many kinases. Interestingly, several mitotic kinases are overexpressed in different type of tumors. Therefore, mitotic kinases are bona fide anti-cancer targets, and several of them are currently in clinical trials. Polo-like kinase 1 (Plk1) is a cell cycle regulator kinase, which controls a wide variety of process throughout cell cycle progress, being essential for the mitosis accomplishment. Plk1 modulates centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. When Plk1 kinase activity is inhibited, it leads to mitotic arrest due to spindle malformation, and eventually to cell death. Since early days, many efforts have been done to depict the Plk1 relationship with cancer disease. In 1997 Plk1 was catalogued as oncogene, and subsequent studies demonstrate its interactions with other cancer-related proteins. Indeed, Plk1 is overexpressed in many tumor types, and this feature often confers poor prognosis. However, the possible oncogenic mechanism of Plk1 is still not well described. We aimed to reevaluate the tumorigenic potential of Plk1 overexpression, by generating a precise Plk1 induction knock-in mouse model. Surprisingly, our in vitro results revealed that when Plk1 is overexpressed, it halts cell proliferation, induces polyploidy and moreover prevents cell transformation driven by other major oncogenes. Concomitantly, our in vivo data show that Plk1 does not lead to transformation events in the mouse. Therefore, our data indicates that Plk1 might play as a tumor suppressor, leading to a completely new scenario for this mitotic kinase. P16-20 Chromatin bound-DYRK1A: recruitment and regulatory mechanisms Laura Barba Moreno, Chiara Di Vona, Susana de la Luna Gargantilla Gene Regulation, Stem Cells and Cancer Programme, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, and Universitat Pompeu Fabra, Barcelona, ES DYRK1A (Dual-specificity tyrosine-(Y)-Regulated Kinase 1A) belongs to a highly conserved family of protein kinases -DYRK- present in all eukaryotes. DYRK1A is known to participate in cell proliferation and differentiation decisions, and in particular, to fulfill key roles in brain P16-22 Common genetic polymorphisms are excluded from functional hypoxia response elements Daniel Martínez Alcázar, Luis del Peso Ovalle Instituto de Investigaciones Biomédicas Alberto Sols, Madrid, ES A wide range of diseases course with an unbalance between the consumption of oxygen by tissues and its supply. This situation triggers a transcriptional response, mediated by the hypoxia inducible factors (HIFs), that aims to restore oxygen homeostasis. Little is known about the inter-individual 125 Pósters / Posters variation in this response and its role in the progression of disease. Herein, we sought to identify common genetic variants affecting hypoxia response elements (HREs) and characterize their effect on transcription. To this end, we constructed a list of genome-wide HIF-binding regions from publicly available experimental datasets and studied the genetic variability in these regions by integration of human variability deposited in the dbSNP database. Our analysis revealed that, although we found 141 SNPs that disrupt potential HREs in HIF-bound regions, the proportion of functional HREs affected by variants is significantly lower than expected by chance. These data strongly suggest that functional HREs are under negative selection. P16-23 Non-coding variants in human cardiac disease Mel·lina Pinsach Abuin1, Bernat del Olmo1, Jesús Mates1, Jing Zhang2, Daria Merkurjev2, Catarina Allegue1, Sergiy Konovalov2, Farah Sheikh2, Ramon Brugada1, Ivan Garcia-Bassets2, Sara Pagans1 1 Department of Medical Sciences, University of Girona, Girona, ES, 2 Department of Medicine, University of California San Diego, La Jolla, US Each human individual is unique in 0.1% of the genome sequence (known as genetic variation). This small but important fraction of distinct sequence largely contributes to phenotypic disparity among individuals and their distinct susceptibility to disease and response to pharmacological treatments. Thereby understanding how the “0.1% fraction” leads to phenotypic disparity and predict at least part of its contribution to pathology would be essential to understand and treat human disease. Here, we present an integrative and comprehensive approach that combines genomic, epigenomic, experimental, and deep-learning-based studies to decipher rules that govern the translation of genetic variation into phenotypic heterogeneity in noncoding regions, and apply this approach for the study of human cardiac diseases, in general, but also Brugada Syndrome (BrS), in particular. Until now, BrS is a heart disease mainly associated with genetic variation within coding regions of cardiac ion channels. We have identified and characterized >30,000 genomic regions in human cardiac cells in which genetic variation is more likely to have a functional impact, and have sequenced a cohort of BrS patients to associate genetic variation within some of these regions to disease. We used maps of transcription factor binding combined with a deep-learning-based approach to predict functional effects of genetic variation. We captured and genotyped 1,300 (over the total of >30,000) open chromatin regions related to cardiac ion channels, identifying 6,584 variants that were unique for each individual and short haplotypes shared by several patients. Focused on transcription factor CTCF for validation purposes, we found 68 variants overlapping with CTCF binding sites, and predict that 49/16/3 will decrease/increase/ no-change CTCF binding, respectively. Experimental validation of these predictions allows us the extrapolation of our approach to the full set of thousands of noncoding variants, and for the prediction of functionally relevant variants in any type of cardiac and non cardiac disease. P16-24 Use Yen1ON as a tool to understand the dynamics of replication/early recombination intermediates XXXIX Congreso SEBBM must be eliminated to safeguard sister chromatid segregation. In budding yeast, the Yen1 nuclease acts as a last resource to eliminate JMs that have persisted until late stages of mitosis. This nuclease is under a strict cell cycle-dependent control, which regulates both its subcellular location and biochemical activation. During S phase, Cdk-mediated phosphorylation of Yen1 promotes its nuclear exclusion and its catalytic inactivation. In anaphase, Cdc14 drives Yen1 dephosphorylation leading to its nuclear relocalization and enzymatic activation. The relevance of such an stringent regulation is underscored by the genome instability that ensues after the expression of a constitutively active and nuclear version of Yen1 (Yen1ON). Indeed, overexpression of such mutant leads to cell death, despite the cause of such lethality remains unclear. Incidentally, we have observed the appearance of spontaneous suppressors of this phenotype, which we intend to use as a tool to unveil the cellular targets of Yen1 unregulated activity and their availability for cleavage. Here we will lay out the strategy and preliminary results of an unbiased screening for suppressors of the lethality of Yen1ON overexpression. P16-25 Caracterización funcional del promotor del gen humano TIMM8A Héctor Merenciano González, Guillermo Suay Montagud, Sandra García Benlloch, Beltrán Ortolá Navarro, Rafael Alís Pozo, Jesús Ángel Prieto Ruiz, José Rafael Blesa Blesa Grupo Medicina Molecular y Mitocondrial. Facultad de Medicina y Odontología. Universidad Católica de Valencia San Vicente Mártir, Valencia, ES Fundamentos y objetivos: El síndrome de Mohr-Tranebjaerg (MTS) fue la primera enfermedad descrita debida a alteraciones en alguna translocasa mitocondrial. La base molecular corresponde a alteraciones en la proteína TIMM8A, un miembro de las tiny TIMMs. El objetivo del trabajo es realizar la caracterización funcional del promotor de TIMM8A para comprender los mecanismos de expresión génica, con el fin de abrir nuevas vías hacia la búsqueda de estrategias para la actuación y tratamiento sobre esta enfermedad. Materiales y métodos: Se realizó análisis in silico mediante el programa TESS y la base de datos TRANSFAC® 6.0. Mediante técnicas de biología molecular (PCR, clonación, etc.) se generaron diferentes constructos, que se transfectaron en células HeLa y SH-SY5Y. Se hicieron ensayos de Luciferasa y estudios in vitro de retardo en gel (EMSA). Resultados: En el análisis in silico se encontraron sitios de unión para los factores de transcripción Sp1, NRF-1 e IRF-2, cuya funcionalidad fue comprobada mediante EMSAs. De los ensayos con Luciferasa se concluyó que no existen sitios de unión para factores de transcripción relevantes en las regiones en dirección 5’ desde el sitio de unión para IRF-2. Discusión y conclusiones: Los resultados obtenidos nos permiten afirmar que Sp1 y NRF-1 son los principales activadores de la expresión de TIMM8A. El hallazgo de la secuencia de unión para IRF-2 y la comprobación de su funcionalidad abre un nuevo campo de estudio acerca de la regulación de las translocasas mitocondriales. Palabras clave: Translocasas de Membrana Mitocondrial Interna (TIMM), proteína Translocasa de Membrana Mitocondrial Interna 8 (TIMM8), gen de Translocasa Mitocondrial Interna 8 (TIMM8A), Factor regulador de interferón 2 (IRF-2), factor de transcripción Sp1, Factor Respiratorio Nuclear 1 (NRF-1), Terapia génica mitocondrial. Vanesa Hurtado Nieves DNA repair and Genome Intregity, Santiago de Compostela, ES P16-26 Homologous recombination (HR) is one of the main pathways for double strand break (DSB) repair. HR involves a series of strand exchanges that result in branched DNA intermediates named joint molecules (JMs). JMs are usually disrupted and repaired at early cell-cycle stages by anti-recombinogenic helicases. However, JMs can occasionally mature into four-way DNA intermediates known as Holliday Junctions (HJs), which 126 Expression of the human TIM23 gene is regulated by the GABP (NRF-2) transcription factor Jesus A. Prieto-Ruiz1, Sara Sáez-Atiénzar1, Rafael Alis Pozo2, Ignacio Ventura1, Sandra Garcia-Benlloch1, Beltran Ortola-Navarro1, José Hernández-Yago1, José R. Blesa1 Salamanca 2016 1 Facultad de Medicina, Universidad Católica de Valencia San Vicente Mártir, Valencia, ES, 2Universidad Católica de Valencia, Valencia, ES The Tim23 protein is the key component of the mitochondrial import machinery in all mitochondrion-bearing eukaryotes. It is essential in S. cerevisiae and N. crassa, and Tim23-/- knockout mice are not viable. In this paper, we characterize the promoter region of the humanTIM23 gene and establish the essential role of the GA-binding protein (GABP or NRF-2) transcription factor in activating TIM23 expression. In particular, we found four GABP sites cooperating in the global expression of TIM23 although with different contributions. In addition, expression of TIM23 also appears to be regulated by the repressor activity of the transcriptional factor RBP-JKappa. These results interestingly are in the way to complete the picture where the main subunits of the human mitochondrial translocases (TOM and TIM) are regulated by GABP transcription factor. P16-27 Dynamics of cell fate decisions in embryonic stem cells Sergi Aranda Aragón Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology; and Universitat Pompeu Fabra (UPF), Barcelona, ES The self-renewing nature of embryonic stem cells (ESCs) is a consequence of their ability to proliferate indefinitely while maintaining pluripotency, by means the capacity to differentiate into all the adult cell types. Despite remarkable progress in understanding the mechanisms controlling ESCs pluripotency, little is known about how ESCs cell division is coordinated with self-renewal and differentiation. ESCs are characterized by a unique cell cycle structure, with a short G1 phase and enlarged S phase. The singularity of the ESCs cell cycle is proposed to be a hallmark of pluripotency. Indeed, recent studies suggest that the shortening of the cell cycle is required for pluripotency and that the extension of the G1 phase is a cause, rather than a consequence, of cell differentiation. We have performed comprehensive proteomic and transcriptomic analyses and have found that pluripotency gene networks and the activity of chromatin modifiers oscillate during cell cycle progression. These oscillations correlate with changes in epigenetic marks throughout the different phases of the cell cycle. Finally, our functional studies indicate that ESCs are primed for a specific cell fate by a cell cycle-linked mechanism. In sum, our data suggest that the cell cycle acts as segregation clock,by dictating stem cell fate towards distinct cell types. Our findings provide fundamental insight into how cell fate decisions take place during embryo development, and suggest a potential framework whereby oscillations during cell cycle can be utilized to differentiate ESCs towards a specific cellular outcome, which is a major goal in regenerative medicine. P16-28 eIF5A translation elongation factor function and post-transcriptional regulation Esther Gamero1, Verónica Muñoz Soriano2, Carlos Villarroel1, Alice Stanciu1, Tianlu Li1, María T. Martínez-Pastor3, Sergi Puig4, Nuria Paricio2, Paula Alepuz1 1 Departamento de Bioquímica y Biología Molecular; ERI Biotecmed, Universitat de València, Burjassot, ES, 2ERI Biotecmed; Departamento de Genética, Universitat de València, Burjassot, ES, 3 Departamento de Bioquímica y Biología Molecular, Universitat de València, Burjassot, ES, 4Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Paterna, ES eIF5A is an essential and evolutionary conserved translation elongation factor, which has been proposed to be involved in the translation of proteins Pósters / Posters with consecutive prolines. In yeast, eIF5A is required for the shmoo formation that initiates cell fusion during mating. In this process, cells require eIF5A for the localization of polarisome components, induction of cell fusion proteins and actin assembly. We discovered eIF5A to be needed for the translation of the formin Bni1, which is involved in the formation of actin cables from the shmoo tip and, therefore, required for the polarized growth during shmoo formation. The function of eIF5A in controlling polarized growth seems to be conserved among eukaryotes. We show that Drosophila eIF5A is able to restore growth of yeast cells depleted of endogenous eIF5A, and allows efficient translation of yeast Bni1 formin and therefore, shmoo formation. In flies, we discovered eIF5A to be involved in dorsal closure (DC) during Drosophila embryogenesis through the translation of formin Diaphanus. In humans, overexpression of eIF5A has been involved in tumor development and associated with poor survival and metastasis. In many eukaryotes, such as yeast and humans, eIF5A is encoded by two paralogous genes which are differentially regulated and contain putative AU-rich elements (AREs) in the 3’-UTR of their mRNAs. We are investigating the role of these ARE-elements in the regulation of the expression of eIF5A isoforms and the ARE-binding proteins involved. This post-transcriptional regulation of eIF5A could be important for the polarized growth of cells during cancer progression. P17. Regulación metabólica / Metabolic regulation P17-1 Osteopontin: a key regulator of liver lipid metabolism Maitane Núñez-García1, Beatriz Gómez-Santos1, Xabier Buqué2, Juan L. García-Rodríguez1, Marta R. Romero3, José J.G. Marín3, Beatriz Arteta4, Carmelo García-Monzón5, Wing-kin Syn6, Olatz Fresnedo1, Patricia Aspichueta2 1 Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country, UPV/EHU, Leioa, ES, 2 Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country, UPV/EHU, Leioa, ES; BioCruces Health Research Institute, Barakaldo, ES, 3Experimental Hepatology and Drug Targeting (HEVEFARM), IBSAL, University of Salamanca, Salamanca, ES; Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, ES, 4Department of Cellular Biology and Histology, Faculty of Medicine and Dentistry, University of the Basque Country, UPV/ EHU, Leioa, ES, 5Liver Research Unit, Santa Cristina University Hospital, Instituto de Investigación Sanitaria Princesa; Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, ES, 6Regeneration and Repair, Institute of Hepatology, Foundation for Liver Research, London, UK; Division of Gastroenterology and Hepatology, Medical University of South Carolina, Charleston, US; Section of Gastroenterology, Ralph H Johnson Veteran Affairs Medical Center, Charleston, US Osteopontin deficiency protects from obesity-related hepatosteatosis and liver fibrosis. Here we have investigated the role of osteopontin (OPN) as a direct regulator of liver metabolism and whether OPN could be a serum-biomarker of liver metabolic dysfunction in non-alcoholic fatty-liver (NAFL) disease patients. For this purpose, metabolic pathways were analyzed by lipidomic and radiometric techniques in hepatocytes and total livers from OPN-KO mice and WT littermates and from WT mice that received recombinant (r)OPN. OPN-KO mice were also treated with intragastric atorvastatin for two weeks. Liver biopsies 127 Pósters / Posters taken during programmed laparoscopic cholecystectomy and matched serum samples from non-obese subjects with NAFL (n=13) or with normal liver (n=13) were analyzed. We demonstrate that OPN regulates the crosstalk between liver cholesterol and phosphatidylcholine metabolism, being the key process the conversion of cholesterol into bile acids through the action of cholesterol 7-alpha-hydroxylase, frequently decreased in NAFL. In OPN-deficient mice enhanced cholesterogenesis in hepatocytes leads to decreased de novo lipogenesis and in vivo inhibition of cholesterogenesis by atorvastatin normalizes liver phosphatidylcholine content. Finally, serum OPN levels correlate with liver phosphatidylcholine and cholesterol concentration in non-obese NAFL patients, in whom adipose tissue metabolic deregulation is not evident and conversion of cholesterol into bile acids is decreased. In conclusion, OPN is a key regulator of liver lipid metabolism and may be an early serum-biomarker of metabolic deregulation in non-obese NAFL patients. Supported by the Basque Government (IT-336-10) and the UPV/EHU (UFI11/20). P17-2 Optimization and validation of sample preparation procedures for high throughput untargeted liver lipid profiling by UHPLC-ESI-Q-TOF MSE Beatriz Abad1, Laura Guevara2, Daniel Bailera2, José Andrés Fernández3, Begoña Ochoa2 1 Central Analysis Service, Faculty of Science and Technology, UPV/EHU, Leioa, ES, 2Department of Physiology, Faculty of Medicine and Dentistry, UPV/EHU, Leioa, ES, 3Department of Physical-Chemistry, Faculty of Science and Technology, UPV/EHU, Leioa, ES Lipids are deregulated in the onset and progression of many diseases, and are functionally involved in mechanisms of disease etiologies. This requires the development of high throughput analyses capable of dealing with large numbers of samples which are necessary to reach statistical significance in clinical studies. In this work, a rapid, selective and sensitive untargeted ultra high performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF MS) strategy using automatic and simultaneous acquisition of exact mass at high and low collision energy, MSE have been used for determination the complex profile of lipid species forming a tissue in a single run injection of 13 min. However, a lipidomics analysis by UHPLC-MS relies on the quality and reproducibility of the extraction procedure of lipids. We now know that there is not a unique procedure able to successfully extract all lipid classes and molecular species, and also that guidelines are offered in several published papers and the LIPID MAPS website. Thus, adoption of one protocol requires neat election of criteria according to the objective of the analytical study, of the equipment available and of the staff training. Here we compare reproducibility and efficiency of extraction of two sample preparation protocols for liver lipid extraction and profiling by UHPLC-ESI-Q-TOF MSE using 17 glycerolipid and 10 sphingolipid non-natural standards. The protocols are a liquid-liquid extraction procedure (method A) and a protein precipitation method (method B). The two methods were then benchmarked using a set of criteria selected to be adopted in high throughput analytical workflow, including simplicity and cost, reproducibility, lipid recovery and coverage. In addition, comparison with benchmarking for targeted lipid profiling was performed. Our data show that the method of choice for liver extract preparation depends on the coverage and human and equipment available and that the two methods A and B meet good quality levels. 128 XXXIX Congreso SEBBM P17-3 Las gotas lipídicas como centro coordinador del metabolismo lipídico y su desregulación en obesidad María del Mar Malagón, Yoana Rabanal-Ruiz, Andrés Trávez, Rocío Guzmán-Ruiz, Rafael Vázquez-Martínez IMIBIC/Hospital Universitario Reina Sofía, Universidad de Córdoba, Córdoba, ES El tejido adiposo juega un papel crucial en la gestión de las reservas energéticas del organismo, que se almacenan en forma de lípidos neutros en las gotas lipídicas características de los adipocitos. En obesidad, el tejido adiposo sufre importantes cambios moleculares que conducen a la deposición ectópica de lípidos y al desarrollo de resistencia a insulina por un proceso de lipotoxicidad. La capacidad de los adipocitos para almacenar y liberar lípidos depende en gran medida de la maquinaría molecular asociada a las gotas lipídicas, que incluye, entre otras, enzimas relacionadas con la síntesis de triglicéridos y fosfolípidos, lipasas, perilipinas, y proteínas relacionadas con el tráfico intracelular y la fusión de membranas, como las GTPasas de bajo peso molecular Rab y Arf, y v- y t-SNARE. De hecho, las gotas lipídicas son orgánulos altamente dinámicos que interaccionan entre sí y con otros orgánulos relacionados con el tráfico y metabolismo de lípidos, aunque no se conocen los mecanismos que median dichas interacciones ni la relevancia de las mismas en la función de los adipocitos. En este contexto, nuestros estudios ponen de manifiesto que una de las proteínas Rab asociadas a gotas lipídicas, Rab18, que interviene en la lipólisis mediada por receptores β-adrenérgicos y en la lipogénesis inducida por insulina, coordina la interacción de las gotas lipídicas con retículo endoplásmico, mitocondrias y peroxisomas. De esta manera, Rab18 permitiría ajustar la incorporación, movilización y uso de lípidos con el remodelado de las gotas lipídicas, tanto de los lípidos neutros del interior de las gotas como de los fosfolípidos de su cubierta. Los cambios observados en la expresión de Rab18 y de uno de sus reguladores, GDI2, en el tejido adiposo en condiciones de obesidad podrían contribuir a la desregulación del almacenamiento y movilización de lípidos observado en dichas condiciones. Financiación: MINECO/FEDER (BFU2010-17116; BFU2013-44229-R; BFU2015-70454-REDT); J. Andalucia/FEDER (PI-0200/2013; CTS-6606); FIS (PIE14-00005) y CIBERobn (ISCIII). P17m-4 Role of MKK3 in High-Fat Diet induced diabetes Edgar Bernardo Vasco1, Nuria Matesanz1, Leticia Herrera1, Sonia Pérez-Sieira2, Elena Rodríguez1, Miguel Marcos3, Lourdes Hernández-Cosido4, Rubén Nogueiras2, Ángel Nebreda5, Guadalupe Sabio1 1 Fundación Centro Nacional de Investigaciones Cardiovasculares CNIC, Madrid, ES, 2Department of Physiology, CIMUS, University of Santiago de Compostela-Instituto de Investigación Sanitaria; CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn), Santiago de Compostela, ES, 3Department of Internal Medicine, University Hospital of Salamanca-IBSAL; Department of Medicine, University of Salamanca, Salamanca, ES, 4Bariatric Surgery Unit, Department of General Surgery, University Hospital of Salamanca; Department of Surgery, University of Salamanca, Salamanca, ES, 5Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology; Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, ES Obesity is an imbalance between food intake and energy expenditure characterized by an excess in fat accumulation and by a chronic inflammation. Obesity is also related to other diseases such as type 2 diabetes, dyslipidemia and hypertension. MAPK are serine/threonine Salamanca 2016 specific protein kinases, include ERK1/2, p38 MAPK and c-jun N-terminal kinase (JNK), they are involved in several cellular processes such as inflammation, growth and differentiation. JNK is recently described to have a role in diabetes however the role of p38 in obesity is poorly understood. p38 is activated by two upstream kinases, MAPK kinase 3 and MAPK kinase 6 (MKK3 and MKK6). In this work we study the upstream kinase MKK3 in order to understand the role of p38 MAPK in obesity and diabetes. Pósters / Posters stages of adipogenesis blocks the differentiation. Furthermore it has been demonstrated that p38 take a part in glucose uptake stimulated by insulin both in adipocytes and myocytes. Treatment of those cells with p38 alpha and beta inhibitors moderate the glucose uptake via GLUT4 but has no effects on the translocation of this transporter to the cell membrane. Since, the role of p38 delta kinase in the adipose tissue is not well understood, our goal in this study is to examine the contribution of p38 delta specifically in the adipose tissue by selectively knocking-down this p38 isoform in adipose tissue, as well as the impact of p38 delta in the communication of adipose tissue with other metabolic organs. P17-5 MKK6 controls T3-mediated browning of white adipose tissue Nuria Matesanz1, Edgar Bernardo1, Rebeca Acín-Pérez1, Beatriz Caballero1, Sonia Pérez-Sieira2, Lourdes Hernández-Cosido3, María del Valle Montalvo1, Alfonso Mora1, María Elena Rodríguez1, Luis Leiva-Vega1, Ana Victoria Lechuga1, Clara Álvarez2, Jesús Ruiz-Cabello1, Jorge L. Torres3, Francisco Centeno4, Miguel Marcos3, José Antonio Enríquez1, Rubén Nogueiras2, Guadalupe Sabio1 1 Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, ES, 2University of Santiago de Compostela-Instituto de Investigación Sanitaria, A Coruña, ES, 3Hospital Universitario de Salamanca, Salamanca, ES, 4Universidad de Extremadura, Badajoz, ES Obesity, a major health problem that has become pandemic, is characterized by fat accumulation, alteration of different metabolic pathways, and insulin resistance in peripheral tissues. Increase of body temperature by activating adipose tissue has emerged as a possible tool to fight obesity. Previous studies in metabolism showed that p38 MAPK family, a group of kinases that respond to stimuli such as metabolic and oxidative stress, may be implicated in brown adipose activation, but little is known about its role in obesity. We found that expression of MKK6, an upstream kinase that controls phosphorylation and activation of p38, increased in adipose tissue of wild-type (WT) mice on a high-fat diet (HFD). To study the participation of MKK6 in the development of obesity and diabetes, the effect of a HFD on knockout mice lacking MKK6 (Mkk6-/-) was investigated. We thank the staff at the CNIC Animal facility. G.S. is an investigator of the Ramón y Cajal Program.This work was funded by the following grants to G.S.: ERC 260464, EFSD 2030, MICINN SAF2013-43506-R, and Comunidad de Madrid S2010/BMD-2326; to L.H.C: Junta de Castilla y León GRS 681/A/11; to R.N: MICINN BFU2012-35255, Xunta de Galicia EM 2012/039 and 2012-CP069. The CNIC is supported by the Ministry of Economy and Competition and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). We thank the staff at the CNIC Genomics and Bioinformatics units for technical support and help with data analysis. G.S. is an investigator of the Ramón y Cajal Program. I.N. is supported by a CNIC IPP FP7 Marie Curie Programme (PCOFUND-2012-600396). This work was funded by the following grants to G.S.: ERC 260464, EFSD 2030, MICINN SAF2013-43506-R, and Comunidad de Madrid S2010/BMD-2326. The CNIC is supported by the Ministerio de Economía y Competitividad and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). P17m-7 Role of p38 δ in liver metabolism Valle Montalvo-Romeral, Luis Leiva, Victor Bondía, Juan Antonio López, Jesús Vázquez, Guadalupe Sabio Fundación Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, ES Obesity is currently a serious health problem throughout the world, which prevalence has reached epidemic proportion (Mokdad et al. 2001). This is due to the obesity is associated with others disorders like impaired glucose tolerance, dyslipidemia and hypertension. All these are collectively known as “metabolic syndrome” (Isomaa et al., 2001). Stress activated protein kinases (SAPKs), an important family of kinases implicated in the transduction of stress signals into the cell, could be key regulator of the development of diabetes and cancer. While the role of c-Jun N-terminal kinases (JNK) in metabolic diseases has been study for a long time (Sabio et al., 2008; Sabio and Davis 2010; Sabio et al., 2010), less is known about the role of the other stress kinases, p38 mitogen-activated stress kinases (p38 MAPK). In mammals, four p38 MAPK isoforms have been identified: p38α, -β, -γ, and -δ. The role of the p38γ/δ remains elusive, although recently we have founded that these kinases (γ/δ) control liver steatosis (González-Terán, Matesanz and Nikolic et al., 2016). As liver is an essential metabolic organ which control body energy metabolism, playing a key role in controlling blood glucose, protein and fat levels, we will discuss the role of p38 δ in liver metabolism. P17r-6 Role of p38 delta protein kinase in adipose tissue Ivana Nikolic, Alfonso Mora, Luis Leiva Vega, Victor Bondia, Elena Rodríguez, Guadalupe Sabio Stress kinases in Diabetes, Cancer and Cardiovascular Disease, Myocardial Pathophysiology Area, Spanish National Center for Cardiovascular Research, Madrid, ES Mitogen-activated protein kinases (MAPKs) are serine-threonine protein kinases which detect a variety of extracellular stimuli and response by activating signaling cascade pathways resulting as specific cellular reaction. This protein family includes the ERK, predominantly activated by mitogens and differentiation signals and JNK and p38 MAPKs, primarily activated by stress stimuli. Up to now it has been identified 4 different isoforms of p38: p38 alpha, beta, gamma and delta. It has been shown that p38 kinases are involved in differentiation of preadipocytes into adipocytes and chemical inhibition of p38 alpha and beta in the early P17m-8 The metabolic pathways involved in maintaining liver glycerolipid homeostasis are dysregulated in early stages of obesity promoted hepatocellular carcinoma Daniela Mestre Congregado1, Igor Aurrekoetxea1, Larraitz Fernández-Ares1, Xabier Buqué1, Juan L. García-Rodríguez2, Beatriz Gómez-Santos2, Diego Sáenz de Urturi-Indart2, Maitane Núñez-García2, Jon Ander Fernández-Rodríguez2, Ainhoa Iglesias3, Ana Zubiaga3, Patricia Aspichueta1 1 Dpt. of Physiology, Fac. of Medicine and Nursery, University of Basque Country and BioCruces Health Research Institute, Cruces University Hospital, Bilbao, ES, 2Dpt. of Physiology, Fac. of Medicine and Nursery, University of Basque Country, Bilbao, ES, 3Dpt. of Genetic, Physical Anthropology and Animal Physiology, Fac. of Science and Technology, University of Basque Country, Bilbao, ES 129 Pósters / Posters Obesity prevalence is increasing and with it the patients with non-alcoholic liver disease and the risk of suffering hepatocellular carcinoma (HCC). HCC, the primary malignant liver cancer, is the third leading cause of cancer-related deaths. The aim here was to analyze, during the first stages of liver disease, the metabolic changes involved in obesity promoted HCC. C57B16:129Sv mice were injected intraperitoneally with diethylnitrosamine (DEN) (25mg/ kg) or PBS when they were 14 days old and fed with high fat diet (HFD) or chow diet until sacrificed at 3 (3m) or 9 months old (9m). As expected, mice exposed to the mix of HFD+DEN exhibited higher number and size of tumors at 9m than those exposed only to DEN. At 3m, mice treated with HFD+DEN gained more body weight and white adipose tissue comparing to any other group. They also lost sensitivity to glucose, responded higher to piruvate after fasting than any other group and exhibited increased serum fatty acids, all of it suggesting the development of insulin resistance with increased gluconeogenesis. HFD+DEN treatment induced a decrease in liver phosphatidylethanolamine and phosphatidylcholine (PC) content and a concomitant increase in that of diglyceride and triglyceride (TG). Besides, liver TG secretion was reduced in mice exposed to HFD+DEN when compared to any other group. Although 14C-glycerol incorporation into hepatic glycerolipids maintained unaltered, 3H-choline incorporation into PC increased in mice treated with HFD+DEN, which suggest PC synthesis through CDP-choline pathway. As conclusion, the increased fatty acid uptake, decreased VLDL secretion and altered glycerolipid metabolism in liver together with the high insulin resistance in the first stages of liver disease will promote HCC development in obesity. P17-9 An aging model in calorie-restricted mice following a range of dietary fats reveals significant changes of key ultrastructural and physiological parameters in kidney Miguel Calvo-Rubio Barrera1, María Isabel Burón Romero1, Guillermo López Lluch2, Plácido Navas Lloret2, Rafael de Cabo3, Jon J. Ramsey4, José Manuel Villalba Montoro1, José Antonio González Reyes1 1 Departamento de Biología Celular, Fisiología e Inmunología, Campus de Excelencia Internacional Agroalimentario, ceiA3, Universidad de Córdoba, Córdoba, ES, 2Universidad Pablo de Olavide-CSIC, Sevilla, ES, 3Translational Gerontology Branch, National Institute of Aging, National Institutes of Health, Baltimore, US, 4VM Molecular Biosciences, University of California, Davis, US The Membrane Theory of Aging proposes that lifespan is inversely related to the level of unsaturation in membrane phospholipids. Calorie restriction (CR) has been repeatedly reported to prevent age-related diseases in a wide range of animals, including nonhuman primates and humans. In rodents CR also increase life span, which may be related to alterations in membrane phospholipids fatty acids, constituting a powerful tool to study the aging process. Here we have studied the effect of these dietary fats on structural and physiological parameters of kidney from mice submitted to 40% CR for 6 and 18 months. We have established four dietary groups: one control group fed 95 % of a predetermined ad libitum intake, and three CR groups fed 40 % less than ad libitum intake. Lipid source for the control and one of the CR groups was soybean oil whereas the two remaining CR groups were fed diets containing fish oil, or lard. Analyses were performed using quantitative electron microscopy techniques and protein expression in western blots. CR seems to mitigate most of the analyzed parameters related to aging in kidney. These measured parameters included glomerular basement membrane and foot processes thickness as well as filtration slits width in podocytes, along with mitochondrial mass in convoluted proximal tubules. Markers of mitochondrial dynamics and autophagy were also evaluated in renal homogenates. The significance of our results in this study are reinforced by additional approaches developed by our group in different key organs such as skeletal muscle and liver. These studies have independently indicated that lard as a fat source shows the optimal effect in 130 XXXIX Congreso SEBBM all of them, which could be due to significant increases of monounsaturated fatty acids levels in the tissues. P17m-10 GLUT1 correlates with aggressiveness while diabetes is inversely related with prostate cancer Pedro González Menéndez, David Hevia, Iván González-Pola, Juan C. Mayo, Rosa M. Sáinz Instituto de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, Avilés, ES Cancer cells show an increase in glucose uptake, mainly associated with an overexpression and/or membrane translocation of GLUT transporters. GLUT1 is the main responsible in most of the tumors but others can be involved such as the insulin-dependent GLUT4. Interestingly, diabetes decreases the risk of prostate cancer (PCa) while the insulin signalling has an important role in PCa progression. Thus, the aim of this study was to find evidences in vivo about the participation of GLUT transporters in PCa progression as well as its relation with diabetes and insulin blood levels. For that, TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice were employed. First, we found that there is a direct association between GLUT1 and GLUT4 protein levels and PCa progression. GLUT4 levels progressively increased with tumour progression while GLUT1 levels were enhanced predominantly in poorly-differentiated tumours and seemed associated to agressiveness. This increase correlated with Bcl-2, HIF1α and cytoplasmatic AR levels. After castration, GLUT1/4 levels decreased and the treatment with testosterone for 5 days restored both GLUT1 and GLUT4 mRNA levels and GLUT4 protein expression. This recovery was related with AMPK phosphorylation. TXNIP, an inhibitor of GLUT1 translocation, was overexpressed in castrated mice. Blood Insulin levels were higher in TRAMP mice compared to WT mice. Its levels decreased with malignancy, as well as in castrated mice. In addition, streptozotocin-induced diabetic TRAMP mice showed an inhibition of tumor development. In conclusion, our work confirms that diabetes is inversely related with PCa in mice while GLUT1 protein levels are associated with tumour aggressiveness. P17-11 Alteración en la homeostasis de los ácidos biliares por interferencia de los glucocorticoides con la vía de señalización enterohepática FXR/FGF19 Faten A. Al-Aqil1, María J. Monte1, María A. Serrano1, Raquel González2, Carlos J. Aranda2, Borja Ocón2, Olga Martínez-Augustín2, Fermín Sánchez de Medina2, Iker Uriarte3, Matías A. Ávila3, José J.G. Marín1, Marta R. Romero1 1 Hepatología Experimental y Vectorización de Fármacos (HEVEFARM), Instituto de Investigación Biomédica de Salamanca (IBSAL), Universidad de Salamanca. Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Salamanca, ES, 2Facultad de Farmacia, Universidad de Granada. Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Granada, ES, 3 Hepatología, Centro de Investigación Médica Aplicada (CIMA), IDISNA, Universidad de Navarra. Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, ES Antecedentes: El tratamiento con glucocorticoides (GC) puede alterar la homeostasis de los ácidos biliares (AB), regulada por FXR, a través del péptido intestinal FGF19 (Fgf15 en roedores). Objetivo: Evaluar los efectos de los GC de uso clínico: dexametasona (DEX), prednisolona y budesonida, sobre la vía de señalización enterohepática FXR/ FGF19 y su repercusión en el metabolismo de los AB. Salamanca 2016 Métodos: Se utilizaron ratones C57BL/6, Fxr-/-, Fgf15-/- y GR-/(selectivamente para el receptor de GC intestinal) y células Alexander de hepatoma humano transfectadas con FXR/RXR. Resultados: El tratamiento con GC redujo de forma dosis-dependiente la expresión intestinal de Fgf15 en ratones, mientras que elevó la del transportador de AB Asbt. A dosis no hepatotóxicas la DEX disminuyó la expresión intestinal de Fgf15 sin alterar la hepática de Cyp7a1 (clave en la síntesis de AB), que es inhibida por Fgf15. Tampoco se alteró la de genes implicados en el transporte (Bsep, Ntcp, Mrp2) y el metabolismo (Baat) de AB. En ratones Fxr-/- y GR-/-, con baja expresión basal de Fgf15, la DEX causó una represión adicional de Fgf15. A pesar de lo cual, la expresión de Cyp7a1 estaba inhibida en ambas cepas. Por el contrario, en ratones Fgf15-/-, los GC estimularon la expresión de Cyp7a1. En células Alexander los GC redujeron la expresión de FGF19, aún en presencia de GW4064 (agonista de FXR), y estimularon, de forma FXR-independiente, la expresión de CYP27A1 (clave en la síntesis de AB). Conclusión: Los GC interfieren con la regulación de la síntesis hepática de AB, alterando el control que ejerce FXR a través de la vía de señalización mediada por FGF19, lo que puede contribuir a la aparición de alteraciones en la función hepatobiliar en pacientes que reciben tratamientos prolongados con GC. P17-12 Ghrelin reduces GABAergic output through carnitine palmitoyltransferase (CPT) 1A Joan Francesc Mir1, Matthieu Lichtenstein2, Judit García-Villoria3, Minéia Weber1, María Calderón-Domínguez1, Blanca Balanyà1, David Sebastián4, Antonio Zorzano4, Núria Casals5, Antònia Ribes3, Cristina Suñol2, Laura Herrero1, Dolors Serra Cucurull1 1 Departament de Bioquímica i Fisiologia, Facultat de Farmàcia, Institut de Biomedicina de la Universitat de Barcelona. Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Barcelona, ES, 2IIBB - CSIC. CIBEResp, Barcelona, ES, 3Sección de Errores Congénitos del Metabolismo – IBC, Servicio de Bioquímica y Genética Molecular, Hospital Clínic, IDIBAPS, Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Raras, Barcelona, ES, 4Institute for Research in Medicine (IRB Barcelona), Barcelona, ES, 5Departament de Ciències Bàsiques, Facultat de Medicina i Ciències de la Salut, Universitat Internacional de Catalunya (UIC). Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Barcelona, ES Introduction: Ghrelin modulates neuropeptides in arcuate hypothalamus and it is essential for the control of feeding and glycaemia. Nonetheless, little is known about its effect on amino acid neurotransmitters. We assessed ghrelin effect on gamma-aminobutyric acid (GABA) metabolism and release and the involvement of carnitine palmitoyltransferase (CPT) 1A asa downstream effector in primary cortical neurons (CTX) and hypothalamic GT1-7 cell line. We used a pharmacological inhibition with etomoxir, a CPT1A-specific inhibitor, and genetic inhibition with Ad-shCPT1A and total ablation in CTX from CPT1A(loxP/loxP) mice. Results: We observed that ghrelin reduces GABA release in cortical neurons under physiological glucose concentration similar to that of the cerebrospinal fluid. This effect is mediated by CPT1A, since its pharmacological inhibition with etomoxir or genetic ablation restores this reduction. Ghrelin also produces an increase in mRNA of cpt1a, gadx, vgat and GABA shunt genes. Moreover, we observed that ghrelin induces changes in the levels of TCA intermediates, as well as it modifies mitochondrial function by reducing oxygen consumption rate and the production of reactive oxygen species. These three effects are reversed by genetic inhibition of CPT1A. Conclusion: All these observations indicate that ghrelin treatment of cortical/hypothalamic neurons might modulate GABA metabolism and release and highlight CPT1A as a key mediator of ghrelin’s effect. Pósters / Posters P17-13 Generation of a CPT1C mutated protein insensitive to malonyl-CoA Cristina Miralpeix1, Rosalía Rodríguez-Rodríguez1, María Casas1, Laura Herrero2, Dolors Serra2, Núria Casals3 1 Basic Sciences Department, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, Sant Cugat del Vallès, ES, 2CIBER Fisiopatología de la Obesidad y la Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid, ES. Department of Biochemistry and Molecular Biology, Institut de Biomedicina de la Universitat de Barcelona (IBUB), Universitat de Barcelona, Barcelona, ES, 3CIBER Fisiopatología de la Obesidad y la Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid, ES; Basic Sciences Department, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, Sant Cugat del Vallès, ES Carnitine palmitoyltransferase 1 (CPT1) C is a brain specific isoform of the CPT enzymes family, which is localized in neuronal endoplasmic reticulum. Despite its minimal CPT1 catalytic activity, CPT1C is able to bind malonyl-CoA and long-chain acyl-CoAs. CPT1C KO mice present impaired spatial learning and cognition, motor deficits and deregulation of food intake and energy homeostasis. However, the exact molecular mechanisms mediating these functions and if they depend on malonyl-CoA are still unknown. Therefore, our aim was to evaluate whether CPT1C function in neurons is regulated by malonyl-CoA. To carry out this study we constructed the mutant CPT1CM589S by site directed mutagenesis to alter its binding site to malonyl-CoA. Methionine 589 had been previously described to be crucial for malonyl-CoA binding to CPT1A. We first determined the loss of sensitivity to malonyl-CoA by performing binding assays between CPT1CM589S and malonyl-CoA in microsome samples. Secondly, we studied the involvement of malonyl-CoA in the interaction of CPT1C with the subunit 1 of glutamate AMPA receptors (GluA1), a recently well-known interaction, and in the regulation of GluA1 expression levels. For this purpose, CPT1C and CPT1CM589S were overexpressed in cortical neurons and immunoprecipitation and western blot assays were performed. We found that CPT1CM589S maintained the interaction with GluA1 and increased GluA1 protein levels similarly to CPT1C overexpression. These results suggest that the CPT1C role in GluA1 receptor expression regulation is malonyl-CoA-independent, and hence confirming the functionality of CPT1CM589S. In conclusion, we have developed a tool to study whether CPT1C is acting as a malonyl-CoA sensor in neurons and how this interaction further regulates its binding with other neuronal proteins. P17-14 Detecting lipid variance in cellular models of Parkinson’s disease Aoife Bannon1, Massimo Valoti2 University of Siena, Siena, IT, 2Supervising Associate Professor, Univeristy of Siena, IT 1 Background: Maintaining lipid homeostasis is essential for neuronal function and evidence suggests that lipid metabolism alterations are involved in Parkinson’s disease (PD). Lipid profiling can be used to investigate lipid pathways that are essential in cell biology and in specific disease processes. Aim: To develop a method in which SHSY5Y cells can be used to detect lipid variance in PD. Methods: The lipid profile of SHSY5Y cells was obtained using a newly developed one-phase lipid extraction method and Q-TOF LC/MS analysis. Cells were differentiated either by retanoic acid(RA) or RA and phorbol ester (PE), the latter pushing further towards a dopaminergic phenotype. Lipid extraction procedures are being optimised to compare the lipid profiles of undifferentiated and differentiated cells. Neurotoxin toxicity is being investigated on different cell types. 131 Pósters / Posters Results: Preliminary trials suggest the new lipid extraction method provides satisfactory recovery of lipids from cell samples compared to the gold standard methods. Western blot analysis of dopaminergic proteins confirmed differentiation. Analysis of undifferentiated, RA-differentiated and RA/PE differentiated cells suggests there is variance in lipid profiles of the cell types, though further experiments must be carried out to confirm this. This can provide a profile against which cells treated with toxic/ protective agents can be compared. Conclusions: These experiments could provide a method by which the SHSY5Y cell line can be used to detect lipid variance caused by toxins/ neuroprotective compounds, allowing identification of lipid biomarkers for specific diseases and development of neuroprotective drugs. Acknowledgements: The presented research is funded by TINTIN, a Marie Curie ITN funded project 2013-17(GA no:608381). P17r-15 Effect of cell penetrating peptides based on connexin43 on the rate of glucose uptake in glioma stem cells Sara Gutiérrez-Pelaz, Elvira Criado-Moronati, José María Medina, Arantxa Tabernero Instituto de Neurociencias de Castilla y León (INCYL), Universidad de Salamanca, Salamanca, ES Connexin43 (Cx43), the main gap junction channel-forming protein in astrocytes, is downregulated in malignant gliomas. These tumors are composed of a heterogeneous population of cells, some of which, termed glioma stem cells (GSCs), display stem-cell-like properties and high tumorigenic potential. Restoring Cx43 in GSCs reverses their phenotype through the inhibition of c-Src and consequently reduces GSCs tumorigenicity. We have developed a cell-penetrating peptide (TAT-Cx43266-283) containing the region of Cx43 that interacts with c-Src that mimics the effect of Cx43 on GSC phenotype. In terms of catabolism, GSC phenotype is characterized by the Warburg effect - part of the metabolic reprogramming considered a cancer hallmark - in which mitochondrial OXPHOS is decreased in favor of aerobic glycolysis and glucose uptake is increased. In the tumor microenvironment, GSCs can reprogram their metabolism to compete for glucose resources through expression of GLUT-3, the high affinity glucose transporter. Expression of GLUT-3 and other glucose metabolism proteins is regulated by transcription factor HIF-1-alpha, which can in turn be regulated by c-Src. In this study we have analysed the effect of TAT-Cx43266-283 on the rate of glucose uptake in human GSCs and in non-tumoral cells. Our results showed that glucose uptake in human GSCs treated with TAT-Cx43266-283 is decreased compared to that of control human GSCs. Furthermore, we have determined that glucose uptake in astrocytes from rat brain primary cultures treated with TAT-Cx43266-283 shows no differences compared to that of control astrocytes. In conclusion, our results suggest that TAT-Cx43266-283 reduces the rate of glucose uptake specifically on GSCs. P17m-16 Adipose tissue expansion in obesity. Is oxidative stress friend or foe? Martín Alcalá Díaz-Mor1, Dolors Serra Cucurull2, Laura Herrero Rodríguez2, María del Pilar Ramos Álvarez1, Marta Viana Arribas1 1 Departamento de Química y Bioquímica, Facultad de Farmacia, Universidad San Pablo CEU, Madrid, ES, 2Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Barcelona, Barcelona, ES Adipose tissue expansion plays a key role in the physiopathology of obesity. Changes in adipocytes size and number, macrophage infiltration 132 XXXIX Congreso SEBBM and extracellular matrix fibrosis are key features that define the expansion of the adipose tissue. A healthy expansion may prevent the lipotoxicity in other tissues. Thus, reducing oxidative stress, which is present in the molecular basis of all the mechanisms of adipose tissue remodeling, may improve the pathological profile of obesity. Objectives: To determine, in an animal model, the role of oxidative stress in the adipose tissue expansion. Methods: CBL57/6J mice were divided into 3 groups: control group (C), fed on standard diet; an obese group (O), fed on high-fat diet (45% energy from fat) and a third group, fed on high-fat diet and a vitamin E supplementation (OE). Animals were sacrificed after 14 and 28 weeks of treatment. Adipose tissue structure, macrophage infiltration and extracellular matrix were analyzed and related to oxidative stress parameters. Results: Vitamin E supplementation for 14 weeks in obesity caused adipose tissue fibrosis, adipocyte hypertrophy and macrophage accumulation, which may account for the insulin resistance observed in these animals. However, after 28 weeks, when obesity caused a marked oxidative stress, vitamin E reduced adipose tissue fibrosis, improving the metabolic, oxidative and inflammatory profile. Conclusions: The preventive use of vitamin E in short-term obesity causes adipose tissue remodeling that leads to lipotoxicity, probably due to a reduction in the physiological levels of ROS. However, in mid-term obesity, vitamin E reduced the pre-existing oxidative stress, enhancing the expansion of the adipose tissue, preventing lipotoxicity and improving the metabolic profile. P17r-17 G protein-coupled receptor kinase 2 plays a role in the development of non-alcoholic steatohepatitis Marta Cruces Sande1, Rocío Vila-Bedmar1, Elisa Lucas1, Ángela M. Valverde2, Águeda González-Rodríguez2, Cristina Murga1, Federico Mayor Menéndez1 1 Centro de Biologia Molecular Severo Ochoa UAM-CSIC, Madrid, ES, 2Instituto de Investigaciones Biomédicas Alberto Sols (CSIC/UAM), Madrid, ES Introduction: Insulin resistance (IR) and obesity are major health problems and important risk factors for the development of non-alcoholic fatty liver disease, a disease spectrum that may include hepatic steatosis, non-alcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. G protein-coupled receptor kinase 2 (GRK2), first identified as a regulator of G protein-coupled receptors (GPCRs), has been described to play a relevant role in the development of IR and obesity in vivo. However, the effect of GRK2 in the development of NASH had not been addressed so far. Since the deletion of GRK2 prevents excessive body weight gain, we fed WT and GRK2 global hemizygous mice (GRK2+/-) with a methionine and choline-deficient diet (MCD), a well stablished model of NASH that is independent of fat mass accretion. Results: Even though the MCD diet induced similar metabolic alterations and a comparable elevation in plasma transaminase activity in WT and GRK2+/- mice, other negative effects of the MCD were partially alleviated in GRK2 +/- animals. The increase in hepatic triglyceride content caused by this diet was significantly lower in GRK2+/- mice and, interestingly, MCD feeding induced an increase in GRK2 protein levels in WT but not in GRK2+/- livers. Moreover, GRK2+/- mice presented protection from some deleterious effects of MCD in the liver as indicated by reduced markers of endoplasmic reticulum stress, and conversely maintained some hepatic protective mechanisms such as autophagy or mitochondrial fusion. Accordingly, these results provide a link between GRK2 levels and hepatic lipid homeostasis leading to NASH. Conclusion: Taken together, these results suggest a role for GRK2 in the establishment and/or development of NASH. Salamanca 2016 P17m-18 Papel dual de APC/C-Cdh1 en obesidad Rubén Rodríguez, Irene García-Higuera, Sergio Moreno Instituto de Biología Funcional y Genómica (IBFG), CSIC-Universidad de Salamanca/Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, ES Nuestro grupo lleva años estudiando el papel biológico de Cdh1, una proteína activadora del Complejo Promotor de la Anafase, o ciclosoma (APC/C), utilizando para ello modelos murinos de inactivación génica. El complejo APC/C-Cdh1 dirige, durante la fase G1 del ciclo de división celular, la poliubiquitinación de ciclinas y otros reguladores de ciclo, para su posterior degradación por el proteasoma. Nuestro trabajo ha desvelado que APC/C-Cdh1 promueve diversos procesos de diferenciación, como neurogénesis o eritropoyesis. Sin embargo, nada se sabe acerca de su posible papel en adipogénesis, un proceso de diferenciación complejo e inusual, del que aún queda mucho por descifrar. Realizando ensayos de diferenciación in vitro, con MEF y preadipocitos primarios, hemos comprobado que la ausencia de Cdh1 provoca un aumento significativo de la adipogénesis, y estimula tanto la expansión clonal de los preadipocitos, como su diferenciación terminal. Por otro lado, también hemos observado que los adipocitos diferenciados carentes de Cdh1 presentan ciertas características de grasa marrón y expresan niveles incrementados de UCP1 tras la inducción del proceso de browning. In vivo, la deleción de Cdh1 restringida a tejido adiposo da lugar a ratones con depósitos de grasa blanca y marrón de mayor tamaño, lo que confirma que APC/C-Cdh1 ejerce una sorprendente y novedosa actividad inhibitoria en la formación del tejido adiposo. En conjunto nuestros resultados apuntan a un posible papel dual del complejo APC/C-Cdh1 en obesidad, modulando la formación de los depósitos de grasa blanca y marrón. P17-19 La falta de osteopontina está vinculada a la esteatosis precoz durante el envejecimiento Beatriz Gómez-Santos1, Maitane Núñez-García1, Diego Sáenz de Urturi-Indart1, Larraitz Fernández-Ares2, Daniela Mestre Congregado2, Juan L. García-Rodríguez1, Igor Aurrekoetxea2, Xabier Buqué2, Patricia Aspichueta2 1 Departamento de Fisiología, Facultad de Medicina y Enfermería, Universidad del País Vasco, Bilbao, ES, 2BioCruces Health Research Institute, Hospital Universitario Cruces y Departamento de Fisiología, Facultad de Medicina y Enfermería, Universidad del País Vasco, Bilbao, ES El envejecimiento se caracteriza por la pérdida progresiva de la integridad fisiológica. La osteopontina (OPN) es una glicoproteína multifuncional que muestra expresión incrementada en patologías derivadas del síndrome metabólico y varios tipos de cáncer, siendo la prevalencia de estas patologías mayor con la edad. Nuestro objetivo fue determinar si OPN ejerce algún papel en la modulación del metabolismo hepático durante el envejecimiento y en tal caso definir el mecanismo implicado. Se usaron ratones deficientes en OPN (OPN-KO) y sus controles de 3, 10 y 20 meses. Parte de los ratones de 10 y 20 meses fueron alimentados 16 semanas con dieta rica en grasa. Se estudió el contenido lipídico hepático y su síntesis de novo. Se analizaron parámetros séricos y de señalización ligadas a procesos de síntesis y oxidación. Los resultados mostraron que en animales control los niveles séricos de OPN aumentaban de 3 a 10 meses y se mantenían de 10 a 20. El mismo patrón se observó en la expresión proteica de OPN en hígado. Los ratones de 20 meses con dieta rica en grasa presentaban niveles séricos de OPN incrementados respecto a los de dieta control. Sin embargo, el incremento inducido por la dieta no se produjo en animales de edad intermedia. En animales de 10 meses la falta en OPN resultó en el aumento del índice hepático y el contenido hepático en colesterol esterificado (CE), triglicérido (TG) y distintos glicerofosfolípidos (PL) que Pósters / Posters se mantenía constante a los 20 meses. Sin embargo, los animales salvajes mostraron un aumento paulatino del contenido de TG y CE a lo largo del envejecimiento, produciéndose el mayor incremento a los 20 meses. La síntesis de novo de PL, DG, TG y CE en hígado estaba aumentada en los animales OPN-KO de 10 meses que también mostraron mayor expresión de la acetil-CoA carboxilasa, enzima limitante en la síntesis de novo de ácidos grasos, compatible con la mayor síntesis de novo de ácidos grasos. Como conclusión, la falta de osteopontina a lo largo del envejecimiento está asociada al incremento precoz del almacén lipídico hepático, principalmente debido a la mayor la síntesis de novo de lípidos. P17r-20 The autophagic protein DOR/TP53INP2 is a novel regulator of brown adipose tissue metabolism Alba Sabaté Pérez1, Montserrat Romero de Pablos1, Xavier Testar Ymbert2, Antonio Zorzano Olarte1 1 Institute for Research in Biomedicine (IRB Barcelona); CIBER in Diabetes and Associated Metabolic Diseases (CIBERDEM); Departament of Biochemistry and Molecular Biomedicine, Faculty of Biology, University of Barcelona, Barcelona, ES, 2CIBER in Diabetes and Associated Metabolic Diseases (CIBERDEM); Departament of Biochemistry and Molecular Biomedicine, Faculty of Biology, University of Barcelona, Barcelona, ES Brown adipose tissue-mediated thermogenesis is an important effector on energy dissipation and can have an impact on total energy balance. Due to the recent discovery that adult humans have active brown adipose tissue depots, this tissue has emerged as a novel target for the treatment of metabolic disorders. Diabetes and obesity-regulated protein (DOR), also called TP53INP2, is a nuclear cofactor that exits to the cytosol and stimulates autophagy in cellular models, in Drosophila melanogaster and in skeletal muscle under in vivo conditions. Moreover, we found that DOR mRNA levels are markedly induced in brown adipose tissue under situations characterized by enhanced thermogenic activity such as cold exposure. DOR is also a negative modulator of white adipogenesis and its ablation causes augmented adiposity in mice. In this regard, tamoxifen-inducible total DOR knockout (DOR KO) mice presented increased brown adipose tissue weight and lipid droplet size, downregulation of key genes involved in brown adipogenesis and thermogenesis, and cold intolerance. Using brown preadipocytes, we also found that DOR is required for brown fat differentiation. DOR loss-of-function in these cells resulted in reduced lipid accumulation, mitochondrial content and expression of brown adipocyte marker genes, as UCP1 and PGC1α. Therefore, we generated a Myf5-DOR knockout mice to study the impact on tissue homeostasis of DOR ablation in brown adipose precursor cells. Preliminary data on brown adipose tissue from Myf5-DOR knockout mice showed a gene expression profile comparable to the global DOR KO mice model. All these results suggest that DOR has a crucial role controlling whole body energy homeostasis by modulating brown adipose tissue metabolism. P17-21 Desregulación de la colesterogénesis por sobreexpresión de SND1 en células de hepatoma Hiart Navarro Imaz, Yuri Rueda Estévez, Olatz Fresnedo Aranguren UPV/EHU, Leioa, ES SND1 es una proteína muy conservada cuya función aún no ha sido esclarecida. Se considera una proteína multifuncional, que a través de interacciones muy diversas con RNA y proteínas parece que participa en procesos dispares. Nuestros estudios funcionales en torno al metabolismo lipídico han mostrado que podría jugar un papel relevante en la homeostasis celular del colesterol. En este trabajo hemos profundizado en los mecanismos 133 Pósters / Posters implicados en la sobreacumulación de ésteres de colesterol (CE) en células de hepatoma debida a la sobreexpresión de SND1. Para ello se han empleado células McA-RH7777 que sobreexpresan SND1 de forma estable (McA-S). Estas células no muestran la respuesta homeostática de las células control (transformadas con el cDNA de la β-galactosidasa; McA-L) tras el suministro exógeno de colesterol, acumulando mayores cantidades de CE. Esta alteración es debida a una activación constitutiva de la proteína de unión al elemento de respuesta a esteroles tipo 2 (SREBP2), causada por un menor contenido en colesterol libre (CL) de las membranas del retículo endoplasmático (RE). El tratamiento de los cultivos con un inhibidor de la esterificación del colesterol y el consecuente aumento del contenido en CL del RE revierten la activación de SREBP2 y moderan la velocidad de síntesis de novo del colesterol. Los resultados de este trabajo sugieren que el papel de SND1 en la homeostasis del colesterol está vinculado a la modulación de su distribución celular, en particular al mantenimiento del contenido de CL del RE. Subvencionado por el GV (SA-2010/00050, IT-336-10 and AE-2012-1-69), la UPV/EHU (UFI11/20) y la Fundación Jesús de Gangoiti Barrera. P17-22 Characterization of Endothelial Cells metabolism in normal and pathological conditions Marta Cascante Serratosa1, Erika Zodda1, Anusha Jayaraman1, Pedro de Atauri1, Ramon Messeguer2, Olga Tura-Ceide3, Timothy Thomson4 1 Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, Universitat de Barcelona, Barcelona, ES, 2Biomed Division, Leitat Technological Center, Parc Científic Barcelona, Barcelona, ES, 3Department of Pulmonary Medicine, Hospital Clínic-Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, ES / Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, ES, 4Department of Cell Biology, Molecular Biology Institute, National Research Council (IBMB-CSIC), Barcelona, ES Atherosclerosis is considered a disease associated with premature biological aging, as atherosclerotic plaques show evidence of cellular senescence. Endothelial dysfunction is a primary factor in the onset and progression of atherosclerosis and other vascular related diseases as well as in the thrombus formation and stabilization. Not just the size but rather the stability of atherosclerotic plaques is a determinant for acute clinical implications. Emerging evidences indicate that pathological blood vessel responses and dysfunctionality of Endothelial Cells (ECs) are associated with metabolic alterations in ECs. Preliminary data from our group have suggested that ECs derived from patients show an altered hyperproliferative phenotype and a resistance to apoptosis when compared to controls. In this study we aim to establish an in vitro model of endothelial pathology, using patient-derived endothelial cell lines which are subjected to a systematic evaluation against control cells in order to determine the metabolic profile and to understand if endothelial dysfunctionality is a consequence of an abnormal endothelial cell metabolism. The endothelial cell lines are studied in terms of morphology, proliferation and metabolic profiling. P17-23 La deficiencia en MAT1A confiere resistencia al desarrollo de obesidad y a la resistencia a insulina en ratones Diego Sáenz de Urturi-Indart1, Juan L. García-Rodríguez1, Igor Aurreoketxea2, Daniela Mestre2, Beatriz Gómez-Santos1, Maitane Núñez-García1, Larraitz Fernández-Ares2, Virginia Gutiérrez-de Juan3, M. Luz Martínez-Chantar3, José María Mato3, Xabier Buqué2, Patricia Aspichueta2 134 XXXIX Congreso SEBBM 1 Departamento de Fisiología, Facultad de Medicina y Enfermería, Universidad del País Vasco, Bilbao, San Sebastián, ES, 2 Departamento de Fisiología, Facultad de Medicina y Enfermería, Universidad del País Vasco, Bilbao y BioCruces Health Research Institute, Hospital Universitario Cruces, Barakaldo, ES, 3CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Parque Tecnológico de Bizkaia, Elexalde Derio, ES La S-adenosilmetionina (SAMe) es sintetizada en la primera reacción del metabolismo de metionina a través del enzima Metionina adenosiltransferasa (MAT), producto del gen MAT1A. El descenso hepático de SAMe, provocado por la falta de MAT1A, induce el desarrollo de la enfermedad de hígado graso no alcohólica (NAFLD). Sin embargo, poco se sabe sobre la implicación de MAT1A en el desarrollo de obesidad. El objetivo fue investigar si la falta del gen MAT1A podría conferir resistencia al desarrollo de obesidad y de resistencia a insulina así como a la desregulación metabólica de tejido adiposo blanco (WAT). Se utilizaron animales control (WT) y deficientes en MAT1A (MAT1A-KO). Los cuales recibieron dieta control (CD) o dieta rica en grasa (HFD) durante 10 semanas. Sorprendentemente, la HFD provocó obesidad solo en los WT. Los animales WT y MAT1A-KO con dieta control presentaban una tolerancia a la glucosa similar sin resistencia a insulina. La HFD provocó únicamente la resistencia a insulina esperada en animales WT. Además, los MAT1A-KO, presentaron una mayor respuesta en la señalización de insulina aumentando la actividad de AKT en WAT. El tratamiento con HFD provocó el incremento en los niveles de triglicéridos (TG) y ácidos grasos (AG) en WAT de los ratones WT. Sin embargo, no lo logró en los MAT1A-KO, que por su parte mostraron un aumento de cuerpos cetónicos (KB) séricos y un descenso en los de AG con respecto a los WT. Además, los MAT1A-KO alimentados con CD tenían una menor lipólisis que sus respectivos controles. Esto no se observó en animales alimentados con HFD. La síntesis de novo de TG no cambiaba en los animales MAT1A-KO alimentados con HFD con respecto a sus WT. Sin embargo, la esterificación y la oxidación lipídica estaban aumentadas, junto con una mayor fosforilación de AMPK. Como conclusión, la resistencia al desarrollo de obesidad y resistencia a insulina en los ratones MAT1A-KO está ligada al incremento en la oxidación lipídica de WAT más que a cambios en su lipolisis o síntesis de novo de lípidos. P17-24 TGF-β enhances glucose metabolism through PFKFB3 gene expression in T98G glioblastoma cell line Ana Rodríguez1, Paula Samsó1, Pere Fontova1, Anna Manzano1, Helga Simon1, Esther Castaño2, Francesc Ventura1, Àurea Navarro-Sabaté1, Ramon Bartrons1 1 Departament de Ciències Fisiològiques, Universitat de Barcelona, Barcelona, ES, 2Centres Científics i Tecnològics, Universitat de Barcelona, Barcelona, ES The aim of this study was to investigate the mechanisms connecting TGF-β, glucose metabolism and PFKFB3 in a glioblastoma cell line (T98G). It is known that TGF-β expression in malignant gliomas renders survival advantages to the tumor cells by enhancing cell growth, migration, invasion, angiogenesis, immune suppression and stem cell properties. PFKFB3 is an homodimeric bifunctional enzyme, belonging to the family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases, that controls the conversion of fructose-6-phosphate (Fru-6-P) to fructose-2,6-bisphosphate (Fru-2,6-P2). This metabolite is important for the dynamic regulation of glycolytic flux by allosterically activating the rate-limiting enzyme of glycolysis phosphofructokinase-1 (PFK-1). It has been shown that PFKFB3 serves as an essential regulator of glycolysis during cell cycle progression and growth. Its activity is regulated by HIF-1α, Akt, p38 and PTEN, and required for the survival and growth of multiple cancer types. On the other hand, in human cancers, TGF-β plays a dual role by acting both as a tumor suppressor Salamanca 2016 and as a promoter of tumor metastasis. Also, TGF-β overexpression has been related to the Warburg effect induction. However, little is known about the molecular mechanisms connecting TGF-β with enhanced glycolysis. Here, we demonstrate that TGF-β upregulates PFKFB3 mRNA and protein expression resulting in an increase in Fru-2,6-P2 concentration. Transcription and translation-blockage experiments confirm that PFKFB3 is regulated at the transcriptional level by TGF-β treatment. Accordingly, Smad3 and Smad4 overexpression also show an increase in endogenous PFKFB3 protein expression. In contrast, PFKFB3 protein expression is reduced in presence of siRNA transfection against Smad2/3 and Smad4 after TGF-β treatment. In conclusion, this study demonstrates that PFKFB3 gene is a direct downstream target of TGF-β and, that Smad signaling pathway would be the link between TGF-β and PFKFB3 induction in T98G cancer cells. This work was supported by Instituto de Salud Carlos III-FIS (PI13/0096) and Fondo Europeo de Desarrollo Regional (FEDER). P17-25 Efectos de la realimentación sobre la translocación de la glucógeno sintasa hepática y el metabolismo del glucógeno en ratas sanas ayunadas Josep M. Fernández Novell1, Carmen López Iglesias2, Joan J. Guinovart Cirera3 1 Universitat de Barcelona, Barcelona, ES, 2Serveis cientifico-tècnics. Parc Científic de Barcelona, Barcelona, ES, 3 Universitat de Barcelona. Institut de Recerca Biomèdica. Parc Científic de Barcelona, Barcelona, ES Se ha descrito que la incubación de hepatocitos aislados de ratas sanas ayunadas 24 h en presencia de glucosa induce la síntesis de glucógeno de una forma espacialmente ordenada. Comienza en la periferia celular, desde la membrana celular hacia el interior del hepatocito. Esta síntesis se produce de forma simultánea con el movimiento, la translocación, de la glucógeno sintasa hepática (GS), desde el citoplasma hacia la periferia celular. El presente estudio se ha llevado a cabo para determinar si la concomitancia entre ambos procesos se produce en situaciones fisiológicas. Para ello se utilizaron ratas adultas sanas que se mantuvieron en ayuno durante las 24 horas anteriores al experimento de realimentación, se sacrificaron a las 0h, 1h, 2h y 4h de realimentación y se obtuvo de cada animal un trozo de hígado, una muestra se procesó inmediatamente para obtener los parámetros bioquímicos. Otra muestra más, se fijo inmediatamente para posterior procesamiento y análisis mediante microscopía confocal y, una tercera, también se fijó inmediatamente, para su análisis mediante microscopía electrónica. En ambos análisis por microscopía se utilizó el mismo anticuerpo primario contra la GS. Así, en la microscopía confocal se localizó la GS mediante un anticuerpo secundario marcado con un fluorocromo, mientras que en la microscopía electrónica se localizó mediante la técnica de inmunogold. Los análisis bioquímicos y microscópicos muestran que la realimentación de ratas sanas ayunadas 24 h aumenta la síntesis de glucógeno hepático y, al mismo tiempo, produce una acumulación progresiva de la GS hepática en la membrana celular. Nuestros resultados indican que el fenómeno de la translocación de la glucógeno sintasa es, en las ratas estudiadas, un proceso fisiológicamente activo y necesario para la síntesis del glucógeno hepático. P17-26 Análisis del efecto de inhibidores de enzimas metabólicas sobre la proliferación y viabilidad de una línea celular modelo de leucemia mieloide aguda Carla Ijurko Valeta, Marta Romo González, Ángel Hernández Hernández Pósters / Posters Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca (USAL). Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, ES Desde hace décadas se conoce que la mayoría de las células cancerosas y, en particular, las células leucémicas, presentan una incrementada dependencia de la glucólisis como fuente principal de obtención de ATP, incluso en presencia de suficiente oxígeno (mecanismo conocido como “efecto Warburg”). En esencia, este hecho no es más que un fundamento bioquímico exclusivo de dichas células; por tanto, resultaría interesante utilizar, como tratamiento anticáncer, inhibidores de este mecanismo. En este trabajo se ha empleado una línea celular modelo de leucemia mieloide aguda (LMA), MV4-11, para estudiar la importancia del metabolismo glucídico en las células leucémicas. Se ha analizado la proliferación y viabilidad de estas células frente a inhibidores del transportador de glucosa (phloretin), la hexoquinasa (2-DG), o el complejo piruvato deshidrogenasa (DCA). Los tres inhibidores han demostrado frenar la proliferación celular, con IC50 de 85μM, 2,5mM y 30mM, respectivamente, e inducir la apoptosis de las células MV4-11. Estos datos demuestran la importancia de la vía glucolítica para la supervivencia de esta línea celular, que ha resultado ser más sensible a los tratamientos que otras líneas celulares de origen leucémico como por ejemplo K562. De entre las tres estrategias probadas, phloretin ha sido la más eficaz. DCA, sin embargo, ha resultado ser el tratamiento menos eficaz, probablemente por no ser sustrato de una enzima directamente implicada en la glucólisis. A pesar de que estos inhibidores aún están lejos de ser los fármacos de elección para el tratamiento del cáncer, parece claro que la particular adaptación metabólica de las células cancerosas ofrecerá nuevas posibilidades terapéuticas en el futuro. P17-27 La UCP2 inhibe la cadena respiratoria y el consumo de oxígeno en adenocarcinoma pancreático Margalida Torrens-Mas1, Jorge Sastre-Serra1, Massimo Donadelli2, Ilaria Dando2, Jordi Oliver1, Pilar Roca1 1 Grupo Multidisciplinar de Oncología Traslacional, Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBERObn, CB06/03), ISCIII, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, ES, 2Department of Neuroscience, Biomedicine and Movement, Biochemistry Section, University of Verona, Verona, IT La proteína desacoplante UCP2 está sobreexpresada en el cáncer de páncreas. Estudios recientes sugieren que la UCP2 puede proteger a las células cancerosas del estrés oxidativo y participar en la reprogramación metabólica hacia la glucólisis, siendo de esta manera importante para la promoción del tumor y el desarrollo de la quimioresistencia. Nuestro objetivo fue determinar el efecto de silenciar mediante un siRNA la UCP2 sobre la funcionalidad mitocondrial y el consumo de oxígeno en las células de cáncer de páncreas PANC-1. Para ello, se determinaron los niveles proteicos de los complejos de la cadena respiratoria OXPHOS por Western Blot, así como las actividades enzimáticas de los complejos IV y V, la cantidad de DNA mitocondrial por PCR y el consumo de oxígeno mediante un electrodo de Clark. El silenciamiento de la UCP2 provocó un incremento general en todos los complejos de la cadena respiratoria mitocondrial. Además, las actividades enzimáticas de los complejos IV y V incrementaron un 27% y un 25% respectivamente, sin alterar el número de copias del DNA mitocondrial, sugiriendo una mayor funcionalidad manteniendo el número de mitocondrias. Por otra parte, se observó que la tasa de consumo de oxígeno basal fue significativamente mayor en las células con el siRNA para UCP2, así como la capacidad máxima y la producción de ATP. 135 Pósters / Posters Estos resultados parecen indicar que la UCP2 podría mediar un cambio energético en las células cancerosas, inhibiendo la respiración mitocondrial y favoreciendo así el efecto Warburg. Financiado: beca FPU del MECD, ISCIII (PI12/01827 y PI14/01434) y FEDER-Unión Europea “Una manera de hacer Europa”. P17m-28 Analyzing the etiology of metabolic disorders by means of lipidomic analysis Virginia López Gómez-Carreño1, Sandra Fernández Gallego2, Nilda Gallardo Alpizar1, Christer S. Ejsing2, Antonio Andrés Hueva1 1 Facultad de Ciencias y Tecnologías Químicas, (UCLM), Ciudad Real, ES, 2University of Southern Denmark (SDU), Odense M, DK There are substantial evidences suggesting that hypothalamic inflammation due to excessive accumulation of long-chain fatty acids and/or ceramides exacerbates the development of central and systemic insulin resistance. Wistar rats like humans, increase body weight and adiposity, and become hypertriglyceridemic and hyperleptinemic with aging. Our previous results point out aging associated alterations are responsable of this situation and could be implicated in the development of insulin and leptin resistance. Besides, based on recents publications and in our results we hypothesized that hypothalamus is the first tissue affected. Thus, we aim to analyze whether ceramide levels involved on the sphingolipid metabolism in hypothalamus is altered with aging, and whether food restriction modifies ceramide content. To this end, lipidomic enables to analyze the lipidome, the entire collection of chemically distinct lipid species, in a cell, an organ or a biologycal system. For this reason lipidomic plays an essential role in defining the biochemical mechanisms underlying lipid-related disease process through the determination of alterations in celular lipid signaling, metabolism, trafficking and homeostasis. Hypothalamic lipidomic analysis were carried out using shotgun lipidomics in 3, 8 and 24 months old Wistar rats in order to analyze the effect of aging in the hypothalamus of these groups of rats. Moreover we have analyzed the hypothalamic lipidome of 8 and 24 months old Wistar rats subjected to a moderate food restriction during 3 months. Unexpectally, the total amount of Cer and GM3 decrease with aging. On the other hand, we found that SHexCer, HexCer and Hex2Cer are increased with aging, while food restriction reduced significantly their levels but only in 8 months old rats.This comprehensive analysis provide information about the changes in total content and relative abundance of ceramides and its derivatives (amongst other lipid species) in the hypothalamus during aging and food restriction. With aging and food restriction there are changes not only in ceramides, also in its derivatives which shows that the complex sphingolipid pathway can not be only focused in ceramides. Extend the analysis to all its derivatives would provide more information that will allow us for a better understanding in the mechanisms involved in the development of central and peripheral insulin and leptin resistance. P17r-29 NADPH oxidasas como reguladoras del metabolismo intermediario en leucemia mieloide crónica Marta Romo González, Carla Ijurko, Rodrigo Prieto Bermejo, Alejandro Pérez Fernández, Ángel Hernández Henández Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca (USAL). Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, ES Tanto la sobreproducción de especies reactivas del oxígeno (ROS) como la desregulación del metabolismo (efecto Warburg) son características propias del cáncer, que están íntimamente ligadas a su transformación 136 XXXIX Congreso SEBBM oncogénica. Por una parte, la excesiva producción de ROS en el cáncer se ha relacionado de un modo directo con los procesos de promoción de la supervivencia, metástasis, proliferación e incluso con la resistencia al tratamiento. En la leucemia mieloide crónica (LMC) se ha descrito que la activación constitutiva de la quinasa BCR-ABL desencadena la producción de ROS vía NADPH oxidasas, una familia de enzimas cuya única función conocida es la producción de ROS. Por otra parte, las células cancerígenas al estar sujetas a un crecimiento y proliferación descontrolada, se ven forzadas a readaptar su metabolismo, recurriendo a la fermentación de la glucosa a lactato como fuente para obtener de energía. Además, es importante destacar, que son las mismas rutas de señalización las que inducen la producción de ROS vía NADPH oxidasas y las que provocan un cambio metabólico en la célula tumoral. Por todo ello, en este trabajo nos hemos propuesto estudiar el efecto que ejercen las ROS producidas vía NADPH oxidasas en la particular adaptación metabólica de las células tumorales. Para ello, empleamos dos aproximaciones: el uso del inhibidor químico DPI y el silenciamiento mediante ARN de interferencia de la subunidad p22phox (proteína común a cuatro de los siete miembros de la familia NADPH oxidasa) o del miembro NOX2 en la línea celular K562 de LMC. Nuestros resultados muestran que las NADPH oxidasas afectan a parámetros clave del metabolismo tumoral como son la captación de glucosa, la actividad lactato deshidrogenasa y los niveles de ATP/ADP, NADH/NAD+ y NADPH/NADP+. P17-30 El resveratrol promueve la respiración mitocondrial y la oxidación de ácidos grasos en la línea de cáncer de colon SW620 María de Mar Blanquer-Rosselló, Jordi Oliver Oliver, Pilar Roca Salom, Ádamo Valle Gómez Grupo Multidisciplinar de Oncología Traslacional, Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBERObn, CB06/03), ISCIII, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, ES El resveratrol (RSV) es un polifenol presente en la piel de la uva conocido por sus propiedades anticancerígenas. Varios estudios han descrito que el RSV actúa en las etapas de iniciación, promoción y progresión del cáncer. Aunque su biodisponibilidad es baja debido a su rápida metabolización, su efecto preventivo en el cáncer de colon merece especial atención puesto que se trata de un tejido altamente expuesto a los componentes de la dieta, incluido el resveratrol. Una de las principales características metabólicas de las células tumorales es su voraz consumo de glucosa mediante la vía glucolítica en detrimento de la fosforilación oxidativa (efecto Warburg). Aunque el RSV ha sido descrito como un mimético de la restricción calórica, hay controversia en cuanto a su efecto sobre el metabolismo de las células tumorales. El objetivo del estudio fue analizar los efectos del RSV sobre el metabolismo energético de la línea de cáncer de colon SW620. Para ello se analizó el efecto sobre el consumo de oxígeno en respuesta a inhibidores específicos, los niveles de ATP, la producción de lactato y los niveles de proteínas clave en la regulación del metabolismo energético. El RSV aumentó la biogénesis mitocondrial y en consecuencia la tasa de consumo de oxígeno en las células SW620. La mayor respiración se asoció a un mayor uso de los ácidos grasos como combustible metabólico. No hubo diferencias en la tasa de producción de lactato ni en los niveles de enzimas clave del metabolismo del piruvato. Estos datos sugieren que el efecto citotóxico del RSV en las células de cáncer de colon puede estar asociado a un shift metabólico, revertiendo parcialmente el efecto Warburg. Financiado: beca FPU del MECD, ISCIII (PI12/01827 y PI14/01434) y FEDER-Unión Europea “Una manera de hacer Europa”. Salamanca 2016 P17-31 El 17β-estradiol ejerce un efecto antiinflamatorio en el hígado de ratas ovariectomizadas Bel M. Galmés-Pascual, Miquel Sbert-Roig, Marco Bauzá-Thorbrügge, Francisco J. García-Palmer, Ana M. Proenza, Isabel Lladó, Magdalena Gianotti Grup de Metabolisme Energètic i Nutrició, Dept. Biologia Fonamental i Ciències de la Salut, IUNICS, Universitat de les Illes Balears. IdISPa, Palma de Mallorca. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBERobn, CB06/03/0043), Instituto de Salud Carlos III, Palma de Mallorca, ES Los estrógenos protegen de la acumulación hepática de triacilglicéridos (TAG) y son importantes agentes antioxidantes. Su deficiencia favorece la esteatosis hepática y el aumento del estrés oxidativo, alteraciones que pueden desembocar en inflamación hepática. El objetivo de este estudio fue determinar la modulación que ejerce el 17β-estradiol (E2) en la respuesta inflamatoria hepática, así como elucidar los mecanismos implicados en la mejora del perfil lipídico hepático y en la atenuación del estrés oxidativo. Se realizó un estudio in vivo en ratas Wistar de 14 semanas divididas en tres grupos: hembras control, ovariectomizadas (OVX) y OVX suplementadas con E2 (OVX+E2). Se determinaron la acumulación de TAG hepáticos y los niveles de los principales marcadores de las vías de oxidación y síntesis de ácidos grasos, así como la actividad de la superóxido dismutasa (SOD), el daño oxidativo y los niveles de proteínas involucradas en la respuesta inflamatoria. En las ratas OVX, se puso de manifiesto un desajuste entre la oxidación y la síntesis de ácidos grasos que provocó la acumulación hepática de TAG. Además, se incrementó el estrés oxidativo y, paralelamente, se activaron las vías inflamatorias. La suplementación con E2 revirtió la acumulación de TAG y aumentó las defensas antioxidantes, lo que supuso una disminución de las proteína quinasas activadas por mitógenos (MAPK) JNK y P38. En conclusión, el E2 ejercería efectos antiinflamatorios en el hígado a través de la mejora del perfil lipídico y de la atenuación del estrés oxidativo. Financiado por MECD (SAF2010-21792 y beca FPU), UIB (contrato predoctoral), CAIB (PCTIB-31/2011, AAEE002/2012, AAEE52/2015 y beca FPI), FSE y FEDER-UE “Una manera de hacer Europa”. P17-32 La inflamación del tejido adiposo generada por una dieta hiperlipídica responde al tratamiento con rosiglitazona de manera dependiente del sexo en ratas Marco Bauzá-Thorbrügge, Miquel Sbert-Roig, Bel M. Galmés-Pascual, Francisco J. García-Palmer, Magdalena Gianotti, Isabel Lladó, Ana M. Proenza Grup de Metabolisme Energètic i Nutrició, Dept. Biologia Fonamental i Ciències de la Salut, IUNICS, Universitat de les Illes Balears. IdISPa, Palma de Mallorca. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBERobn, CB06/03/0043), Instituto de Salud Carlos III, Palma de Mallorca, ES Existe un dimorfismo sexual en la funcionalidad mitocondrial del tejido adiposo blanco (TAB) en ratas. Sin embargo, estas diferencias se atenúan en respuesta a una dieta hiperlipídica (HL). El objetivo ha sido determinar si la rosiglitazona (Rsg), un fármaco que mejora la sensibilidad a la insulina y aumenta la funcionalidad mitocondrial de los adipocitos, ejerce efectos antiinflamatorios también dependientes del sexo en el TAB. Pósters / Posters Se utilizaron ratas Wistar de ambos sexos alimentadas durante 16 semanas con dieta control o dieta HL. Durante dos semanas previas al sacrificio, la mitad de los animales HL fueron tratados con Rsg (100 mg/kg dieta). En el TAB retroperitoneal se determinaron parámetros marcadores de inflamación, apoptosis, hipoxia y movilización de lípidos, así como PPARg y PGC1a. El aumento de PPARg y de lipasa sensible a hormonas en las hembras HL es indicativo de una mayor movilización y oxidación de lípidos. En los machos, esta respuesta solo se observa con el tratamiento con Rsg. Los marcadores de hipoxia, inflamación y apoptosis fueron más elevados en los machos HL y disminuyeron de forma más acusada por efecto de la Rsg. En conclusión, la mayor capacidad de movilizar y oxidar combustibles lipídicos del TAB de las hembras las protege de la apoptosis e inflamación inducidas por la dieta HL. Al ser la Rsg un agonista del PPARg, el dimorfismo sexual observado en los niveles de PPARg con la dieta, condiciona una mayor respuesta de los machos al tratamiento con este fármaco, mejorando el perfil inflamatorio generado por la dieta y reforzando el papel modulador de las hormonas sexuales. Financiado por MECD (SAF2010-21792 y beca FPU), UIB (contrato predoctoral), CAIB (PCTIB-31/2011, AAEE002/2012, AAEE52/2015 y beca FPI), FSE y FEDER-UE “Una manera de hacer Europa”. P17-33 El 17β-estradiol mejora la función y biogénesis mitocondrial hepática a través de PGC-1β Bel M. Galmés-Pascual, Miquel Sbert-Roig, Marco Bauzá-Thorbrügge, Francisco J. García-Palmer, Ana M. Proenza, Isabel Lladó, Magdalena Gianotti Grup de Metabolisme Energètic i Nutrició, Dept. Biologia Fonamental i Ciències de la Salut, IUNICS, Universitat de les Illes Balears. IdISPa, Palma de Mallorca. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBERobn, CB06/03/0043), Instituto de Salud Carlos III, Palma de Mallorca, ES Los estrógenos, y en concreto el 17β-estradiol (E2), se han postulado como moduladores de la función y biogénesis mitocondrial en el hígado. La familia de coactivadores 1 del receptor activado por proliferadores peroxisomales gamma (PGC-1) juega un papel central en la regulación de estos procesos en diferentes tejidos, aunque la contribución de sus principales miembros, PGC-1α y PGC-1β, no ha sido elucidada en el hígado. El objetivo de este estudio fue investigar el efecto del 17β-estradiol (E2) sobre la función y la biogénesis mitocondrial en el hígado, así como evaluar la implicación de PGC-1α y PGC-1β como principales moduladores de estos efectos. Se realizó un estudio in vitro en hepatocitos HepG2 tratados con E2, en combinación con el silenciamiento de PGC-1α o PGC-1β mediante un siRNA específico, y se evaluaron marcadores de función y biogénesis mitocondrial. El tratamiento de HepG2 con E2 incrementó la función y biogénesis mitocondrial. El silenciamiento de PGC-1β disminuyó los niveles de TFAM y de ATPasa, tanto en situación basal como con el tratamiento con E2. No obstante, el silenciamiento de PGC-1α no supuso la disminución de estos marcadores en ninguna de las condiciones estudiadas, sino que se incrementaron con la administración de E2. Nuestros resultados aportan evidencias de que los efectos beneficiosos del E2 en la función y biogénesis mitocondrial en el hígado están mediados por el PGC-1β. Por tanto, la activación de PGC-1β podría constituir una diana terapéutica contra los desórdenes mitocondriales hepáticos. Financiado por MECD (SAF2010-21792 y beca FPU), UIB (contrato predoctoral), CAIB (PCTIB-31/2011, AAEE002/2012, AAEE52/2015 y beca FPI), FSE y FEDER-UE “Una manera de hacer Europa”. 137 Pósters / Posters P17-34 El 17β-estradiol mejora la resistencia hepática a la insulina asociada a la inflamación. Papel de la vía de JNK Bel M. Galmés-Pascual, Miquel Sbert-Roig, Marco Bauzá-Thorbrügge, Melanie Raquel Martínez-Cignoni, Francisco J. García-Palmer, Ana M. Proenza, Magdalena Gianotti, Isabel Lladó Grup de Metabolisme Energètic i Nutrició, Dept. Biologia Fonamental i Ciències de la Salut, IUNICS, Universitat de les Illes Balears. IdISPa, Palma de Mallorca. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBERobn, CB06/03/0043), Instituto de Salud Carlos III, Palma de Mallorca, ES La ingesta de dietas hiperlipídicas (HL) conduce a alteraciones metabólicas que se asocian al desarrollo de numerosas patologías. En el hígado, la acumulación de triacilglicéridos (TAG) se relaciona con un estado de resistencia a la insulina, potenciada por la activación de las vías inflamatorias. Destaca el papel de c-Jun-N-terminal kinase (JNK), cuya activación produce una disminución de la respuesta a la insulina. El objetivo de este estudio fue determinar las diferencias entre sexos en la resistencia hepática a la insulina y el estado de inflamación inducidos por una dieta HL y, en particular, el papel modulador del 17β-estradiol (E2). Se realizó un estudio in vivo en ratas Wistar de ambos sexos alimentadas durante 16 semanas con una dieta HL (22,5% materia grasa), paralelamente con el desarrollo de experimentos in vivo en hepatocitos HepG2 tratados con palmitato (PA) y E2. En ambos modelos se cuantificaron elementos de las vías de señalización de la insulina y de las proteína quinasas activadas por mitógenos (MAPK). En respuesta a la dieta HL, las ratas macho mostraron, en comparación con las hembras, una mayor resistencia sistémica y hepática a la insulina, que correlacionó con una mayor activación de JNK. La implicación del E2 en estos efectos fue confirmada en los estudios realizados en los hepatocitos HepG2, en los que esta hormona atenuó los efectos lipotóxicos del PA, disminuyendo la activación de JNK. En conclusión, el E2 protege frente al desarrollo de resistencia hepática a la insulina disminuyendo la inflamación, a través de JNK. Financiado por MECD (SAF2010-21792 y beca FPU), UIB (contrato predoctoral), CAIB (PCTIB-31/2011, AAEE002/2012, AAEE52/2015 y beca FPI), FSE y FEDER-UE “Una manera de hacer Europa”. P17-35 Los efectos antinflamatorios del estradiol en la lipotoxicidad cardíaca no dependen de la activación de AMPK Miquel Sbert-Roig, Marco Bauzá-Thorbrügge, Bel M. Galmés-Pascual, Agustí González-Vicens, Francisco J. García-Palmer, Isabel Lladó, Magdalena Gianotti, Ana M. Proenza Grup de Metabolisme Energètic i Nutrició, Dept. Biologia Fonamental i Ciències de la Salut, IUNICS, Universitat de les Illes Balears. IdISPa, Palma de Mallorca. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBERobn, CB06/03/0043), Instituto de Salud Carlos III, Palma de Mallorca, ES La lipotoxicidad cardíaca supone una disfunción metabólica que conduce a la activación de las vías inflamatorias en los cardiomiocitos, entre las cuales cabe destacar las MAPK. Se ha visto que los estrógenos atenúan la activación de las MAPK en el corazón y, además, pueden activar la proteína AMPK. Esta proteína ejerce un efecto beneficioso sobre el corazón lipotóxico, entre otros mecanismos, a través de la disminución de la respuesta inflamatoria. El objetivo de este estudio fue analizar si los efectos antinflamatorios del 17β-estradiol (E2) en cardiomiocitos sometidos a un estrés lipotóxico 138 XXXIX Congreso SEBBM dependían de la activación de la AMPK por parte de esta hormona. Para ello se realizó un estudio in vitro con cardiomiocitos H9c2 diferenciados y se activaron las vías inflamatorias mediante el tratamiento con palmitato (PA). Además, las células fueron tratadas con A769662 (activador de AMPK), E2 y E2 en combinación con el compuesto c (inhibidor de AMPK). Finalmente, se determinó el nivel de activación de las quinasas AMPK, ACC, ERK, JNK y p38 por western blot. Se observó una activación de las MAPK ERK, JNK y p38 en respuesta al tratamiento con PA, la cual disminuyó mediante la activación de AMPK por parte de A769662. Este efecto inflamatorio del PA disminuyó también en presencia de E2 y no se vio afectado por el compuesto c. En conclusión, el E2 atenúa la activación de las vías inflamatorias de las MAPK en cardiomiocitos H9c2 en una situación lipotóxica, pero estos efectos no implican a la AMPK pese a los efectos antiinflamatorios de esta proteína. Financiado por MECD (SAF2010-21792 y beca FPU), UIB (contrato predoctoral), CAIB (PCTIB-31/2011, AAEE002/2012, AAEE52/2015 y beca FPI), FSE y FEDER-UE “Una manera de hacer Europa”. P17-36 Efectos de la ovariectomía y del tratamiento con 17β-estradiol sobre la función y biogénesis mitocondrial hepática Bel M. Galmés-Pascual, Miquel Sbert-Roig, Marco Bauzá-Thorbrügge, Francisco J. García-Palmer, Ana M. Proenza, Magdalena Gianotti, Isabel Lladó Grup de Metabolisme Energètic i Nutrició, Dept. Biologia Fonamental i Ciències de la Salut, IUNICS, Universitat de les Illes Balears. IdISPa, Palma de Mallorca. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBERobn, CB06/03/0043), Instituto de Salud Carlos III, Palma de Mallorca, ES Estudios previos han puesto de manifiesto la existencia de un dimorfismo sexual en la función y biogénesis mitocondrial en hígado de rata, apuntando a las hembras como poseedoras de mitocondrias más diferenciadas y con mayor capacidad oxidativa. El objetivo de este estudio fue profundizar en estas diferencias de sexo, investigando el efecto de la ovariectomía y del reemplazamiento con 17β-estradiol (E2) en la biogénesis y función mitocondrial en el hígado. Se usaron ratas Wistar hembra de 14 semanas de edad divididas en tres grupos experimentales: control, ovariectomizadas (OVX) y OVX suplementadas con E2 (OVX+E2). En el hígado, se determinaron las actividades citocromo c oxidasa (COX) y citrato sintasa (CS), así como los niveles de PGC-1α, PGC-1β, TFAM, NRF1, COX I, ATPasa y del ADN mitocondrial (ADNmt). En respuesta a la ovariectomía, la expresión de TFAM disminuyó, acompañado de un descenso del ADNmt y el consecuente empeoramiento de la capacidad oxidativa, reflejado por una disminución de la actividad CS y de los niveles de PGC-1β y ATPasa. El tratamiento con E2 revirtió estos efectos a través de un incremento del ADNmt,de la actividad COX y de los niveles de PGC-1β, NRF1, COX I y ATPasa. En conclusión, el tratamiento con E2 revierte los efectos deletéreos que la ovariectomía provoca en la función y biogénesis mitocondrial en el hígado, aumentando el ADNmt y la capacidad oxidativa. Estos resultados están de acuerdo con el dimorfismo sexual previamente descrito en este tejido, y atribuyen un papel clave en estas diferencias a las hormonas ováricas, y en concreto al E2. Financiado por MECD (SAF2010-21792 y beca FPU), UIB (contrato predoctoral), CAIB (PCTIB-31/2011, AAEE002/2012, AAEE52/2015 y beca FPI), FSE y FEDER-UE “Una manera de hacer Europa”. Salamanca 2016 P17r-37 Subcellular distribution of α and β adrenergic receptors in liver of Wistar rats: effects of central leptin and caloric restriction Lorena Mazuecos Fernández-Pacheco1, Virginia López Gómez-Carreño1, Alejandro Fernández Briones1, Carmen Arribas Mocoroa2, Nilda Gallardo Alpízar1, Antonio Andrés Hueva1 1 Facultad de Ciencias y Tecnologías Químicas, Ciudad Real (UCLM), Ciudad Real, ES, 2Facultad de Ciencias Ambientales y Bioquímica (UCLM), Toledo, ES Leptin convey information to the hypothalamus regarding to long-term energy stores. The brain delivers signals to peripheral organs, including the liver, to keep homeostasis. Sympathetic hepatic nerves can modulate hepatocyte function by direct action of their neurotransmitter noradrenaline on α- and β- adrenergic receptors. Alterations in the connections between brain and liver through the adrenergic system could favor the accumulation of hepatic fat and the development of diseases such as fatty liver. Focus on anti-steatotic and anti-diabetic functions of leptin, the indirect effects of this hormone on the hepatic adrenergic receptors were analyzed in order to reveal the involvement of the autonomic nervous system in the central leptin regulation of liver function. To carry out this study, osmotic pumps were implanted, by stereotaxic surgery, in the brain of male 8-month-old Wistar rats fed ad libitum and rats under a moderate caloric restriction. Animals were infused with leptin (0.2 μg/d) or its vehicle (PBS) for 7 days. After that, rats were sacrificed, metabolites and hormones were measured in serum and adrenergic receptor levels were analyzed by Western blot in total liver extract and sub-cellular fractions, in plasma and inner membranes. Our results show that an intra-cerebroventricular leptin administration alters subcellular distribution of α- and β- adrenergic receptors in the liver. Leptin tends to increase α1A adrenergic receptors levels in plasma membrane, which could be associated with increased hepatic glycogenolysis, fatty acid oxidation and ketogenesis. Furthermore, leptin decreases the presence of α2A receptor in the plasma membrane, which could improve lipolysis and attenuate the de novo synthesis of fatty acids. In parallel, central leptin downregulates the hepatic β-adrenergic receptors (β1 and β2) at the plasma membrane and increased their levels in the internal membranes, suggesting that the β-adrenergic receptors play a crucial role in limiting hepatic lipid accumulation. These findings suggest that brain leptin contribute to the prevention of hepatic steatosis throughout activation of the neuronal brain– liver connection. P17-38 La microbiota intestinal como factor determinante del dimorfismo sexual de enfermedades metabólicas José Antonio Santos Marcos1, Oriol Alberto Rangel Zúñiga1, Sonia García Carpintero1, Juan Francisco Alcalá Díaz1, J osé Andrés Morales Martínez1, Manuel Tena Sempere2, Blanca Landa del Castillo3, José López Miranda1, Francisco Pérez Jiménez1, Antonio Camargo García1 1 Unidad de Lípidos y Aterosclerosis. IMIBIC/Hospital Universitario Reina Sofía /Universidad de Córdoba; CIBER Fisiopatología Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Córdoba, ES, 2Departamento de Biología Celular, Fisiología e Inmunología, IMIBIC/Hospital Universitario Reina Sofía / Universidad de Córdoba; CIBER Fisiopatología Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Córdoba, ES, 3 Instituto de Agricultura Sostenible (IAS), Consejo Superior de Investigaciones Científicas (CSIC), Córdoba, ES En los últimos años, el estudio de la microbiota intestinal ha cobrado especial interés tras descubrirse que la transferencia a modelos animales Pósters / Posters carentes de microbiota intestinal, de un sistema bacteriano relativamente pobre en Bacteroidetes y rico en Firmicutes, conllevaba un aumento de peso en estos animales. De hecho, actualmente se considera a la microbiota intestinal como un órgano totalmente integrado en la fisiología del organismo hospedador que interviene en procesos como la regulación de la inmunidad innata y adaptativa, la extracción de la energía, y el control del balance energético. Estudios en modelos animales han identificado una relación directa entre microbiota intestinal, hormonas sexuales y desarrollo de enfermedad. Este hecho sugiere que las diferencias en la composición de la microbiota entre géneros podría estar relacionada con las diferencia que existe en la incidencia de enfermedades metabólicas y sus comorbilidades, las cuales son sexualmente dimórficas y en mujeres, aumentan después de la menopausia. En este estudio comparamos la microbiota intestinal de mujeres pre- y post-menopáusicas, y de 2 grupos control de hombres con la misma edad e índice de masa corporal que los grupos de mujeres. Nuestro estudio mostró que los géneros Prevotella, Ruminococcus y Oscillospira y las familias Erysipelotrichaceae y Desulfovibrionaceae son más abundantes en mujeres pre-menopaúsicas que en mujeres post-menopaúsicas y en ambos grupos de hombres. Nuestros resultados sugieren que diferencias entre la microbiota intestinal de hombres y mujeres podría ser un factor determinante en la prevalencia en el desarrollo de enfermedades metabólicas como diabetes, síndrome metabólico y enfermedades cardiovasculares. P17r-39 Metabolic adaptations of neurons and astrocytes to physiological oxygen levels Moussa Wardé, Juan P. Bolaños Institute of Functional Biology and Genomics (IBFG), University of Salamanca-CSIC, Salamanca, ES Brain energy metabolism is highly dependent on the oxygen levels. The vast majority of biochemical studies aimed to understand the metabolic interactions between astrocytes and neurons, using cultured systems, have been performed at atmospheric, 21% oxygen concentrations. However, it has been estimated that neural cells in vivo are exposed to oxygen concentrations ranging from 1 to 5%, depending on the distance from the brain vessels. Accordingly, it is unknown whether our current knowledge on brain cells energy metabolism is the result of an adaptation to cultured cells to non-physiological high oxygen levels conditions. To understand this issue, here we have established a long-term primary culture of cortical neurons and astrocytes grown on 2.0% oxygen levels using an oxygen-controlled incubator chamber. We found that these cells, when chronically incubated under a 2.0% oxygen concentration, adapt their mitochondrial energy metabolism giving rise to significant changes in glucose metabolism and mitochondrial reactive oxygen species (mROS) production when compared with cells incubated under atmospheric oxygen conditions. We believe that these observations may be of critical relevance when understanding different pathophysiological features of the brain and the astrocyte-neuronal metabolic interactions. P17r-40 Impact of mitochondrial ROS on brain cells energy metabolism in vivo Carlos Vicente-Gutiérrez, Nicoló Bonora, Juan Pedro Bolaños Universidad de Salamanca, Salamanca, ES A substantial body of evidence suggests that mitochondrial reactive oxygen species (mROS) generation is a hallmark of certain neural diseases. However, the underlying molecular mechanisms involved are not fully understood, and whether they play physiological roles is still controversial. We are interested in studying how mROS orchestrate alternative signaling pathways dictating specific changes in cell metabolism. To explore this, we 139 Pósters / Posters have generated an inducible, floxed knock-in mouse model that expresses a version of catalase fused to a canonical mitochondrial localization signal (mCat). We believe that the adequate use of mCat mice would allow us to probe tissue- or cell-specific functions of mROS in vivo. Here, we present data showing the efficacy of mCat at reducing mROS levels in a cell-specific manner, and that this is sufficient to induce changes in astrocyte gene expression as well as their metabolism in vitro. We are currently characterizing the changes at the protein level with the aim to elucidate the impact of mROS in different energy metabolism-related pathways both in astrocytes and in neurons in vivo. We expect to reveal the functional physiological roles for mROS in the brain and to contribute to ascertain novel mechanisms whereby astrocytes and neurons cooperate in the brain energy homeostasis. P17-41 Modelo de células quiescentes para el estudio del papel de DCTPP1 en la homeostasis mitocondrial de nucleótidos Blanca Martínez Arribas, Antonio E. Vidal, Dolores González-Pacanowska Instituto de Parasitología y Biomedicina López Neyra (CSIC), Granada, ES En mamíferos, el pool de desoxirribonucleótidos trifosfato (dNTP) se encuentra físicamente separado en el citosol y la mitocondria. En células en división, el pool citosólico, producido principalmente por la ribonucleótido reductasa (RNR), proporciona los precursores necesarios para la síntesis tanto del DNA nuclear como mitocondrial (mtDNA). A diferencia de lo que ocurre en células proliferativas, en células post-mitóticas de tejidos diferenciados no hay replicación del DNA nuclear pero sí existe síntesis y reparación de DNA mitocondrial por lo que el aporte de nucleótidos debe ser constante. En estas células, el aporte de precursores mitocondriales proviene fundamentalmente de la ruta de salvamento mitocondrial ya que la síntesis de novo y las vías de salvamento citosólico están apagadas. Así, mutaciones en los genes que intervienen en la ruta de salvamento mitocondrial se han asociado con diversas patologías humanas denominadas en conjunto como síndromes de depleción de DNA mitocondrial (MDS) y caracterizadas por la disminución del número de copias de mtDNA y disfunción mitocondrial en los tejidos afectados. En este trabajo se describe un modelo de fibroblastos de pulmón en quiescencia como herramienta para el estudio del papel de DCTPP1 en la homeostasis de nucleótidos mitocondrial. DCTPP1 es una NTP pirofosfatasa de la familia de las todo-α encargada de catabolizar el dCTP produciendo dCMP y pirofosfato. En células en división DCTPP1 se expresa de forma ubicua en núcleo, citosol y mitocondria, mientras que en células quiescentes la expresión disminuye pronunciadamente en citosol y núcleo, y se mantiene constante en mitocondria. Proponemos que DCTPP1 tiene un papel significativo en el mantenimiento de la homeostasis de nucleótidos pirimidínicos mitocondrial proceso que es esencial para la correcta replicación del mtDNA. P17-42 Liver and serum lipidomic analysis of mice overexpressing carnitine palmitoyltransferase 1AM in liver Sandra Recalde Luna1, Minéia Weber1, Fina Casas2, Gemma Fabrias2, Joan Francesc Mir Bonnín1, María Calderón Domínguez3, Eduviges Bustos1, Núria Casals4, Josefa Badia1, Laura Herrero3, Dolors Serra3 1 Department of Biochemistry and Physiology, School of Pharmacy, Institut de Biomedicina de la Universitat de Barcelona (IBUB), Universitat de Barcelona, Molins de Rei, ES, 2Research Unit on Bioactive Molecules, Department of Biomedicinal Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), 140 XXXIX Congreso SEBBM Barcelona, ES, 3Department of Biochemistry and Physiology, School of Pharmacy, Institut de Biomedicina de la Universitat de Barcelona (IBUB), Universitat de Barcelona, Barcelona, ES/ Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y la Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid, ES, 4Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y la Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid, ES/ Department of Basic Sciences, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, Barcelona, ES Obesity is strongly associated with several metabolic disturbances, including insulin resistance and type 2 diabetes. Excessive lipid storage in several tissues leads to dysregulation of intracellular lipid metabolism. In liver, abnormally large accumulations of triacylglycerides produce hepatic steatosis and insulin resistance. Previous studies from our group have shown that an enhanced fatty acid oxidation (FAO) by overexpressing a permanent activated CPT1A isoform (CPT1AM) in liver of high-fat diet (HFD) mice reduces hepatic steatosis, blood glucose and insulin levels. In an attempt to identify lipid species that could be involved in the improvement of HFD-treated mice we performed a lipidomic analysis in liver and serum. Our results show that liver of HFD CPT1AM-expressing mice have a decrease in the major part of triacylglycerol (TAG) and diacylglycerol (DAG) species compared to HFD control mice, which agrees with the reduced steatosis observed in the HFD CPT1AM-treated mice. However, this massive TAGs and DAGs decrease is not observed in serum, only C54:5, C54:4, and C54:3 TAGs were reduced in CPT1AM-treated mice respect to HFD control mice. Among the phosphatidylcholines (PC)s, PC 36:2 and PC 38:3 in liver and PC 36:3 and PC 38:3 in serumwere reduced compared to normal chow diet mice. Lysophosphatidylcholine (LPC)s 16:0 and 18:0 were also reducedin liver but no changes were observed in serum of HFD CPT1AM-expressing mice. Finally, the analysis of sphingolipids showed a significant reduction of liver ceramides 16:0, 18:0, 20:0, 22:0 and 24:2 and dihidrocerimide 22:0. However, ceramide 18:1 was the only one reduced in the serum. All these results show that the enhancement of FAO in liver of HFD mice reduces mainly neutral lipids such as TAGs and DAGs in liver. In contrast, few lipid species showed changes in serum. These serum metabolites may serve as valuable “biomarker candidates” for the steatosis reversion and insulin resistance improvement in future studies. P18. Señalización celular / Cellular signalling P18r-1 Gαq interactions with PB1 domain-containing proteins open new avenues for Gq-coupled GPCR signaling Sofía Cabezudo Violero, Guzmán Sánchez Fernández, Federico Mayor Menéndez, Catalina Ribas Núñez Centro de Biología Molecular Severo Ochoa CBMSO, Universidad Autónoma de Madrid, Madrid, ES Heterotrimeric G proteins play an essential role in the initiation of G protein-coupled receptor (GPCR) signaling through specific interactions with a variety of cellular effectors. The Gαq/11 family canonically activates PLCβ. However, additional effectors may underlie other Gαq functions. We have reported that GPCR activation promotes a direct interaction between Gαq and protein kinase C (PKC) through a novel acidic region in Gαq, different from the classical effector-binding region, Salamanca 2016 and the PB1 domain of PKC. This Gαq/PKCinteraction leads to the stimulation of the ERK5 pathway and to the induction of apoptotic cell death independently of PLCβ. G protein-coupled receptor kinase 2 (GRK2), a known Gαq negative modulator, abrogates ERK5 stimulation by disrupting the complex. Interestingly, we have uncovered that Gαq interacts with another PB1 domain-containing protein, the key p62/SQSTM1 signaling hub, and the implication of this complex in the modulation of autophagy. We find that Gαq and p62 associate in cells and are able to directly interact. Furthermore, their interaction increases upon Gαq activation by GPCR or in the presence of constitutively active Gαq mutants, and is disrupted by GRK2, consistent with p62 being a functional Gαq effector. Gαq/p62 association involves the PB1 domain of p62 and the acidic region of Gαq described to be essential for PKCbinding, although PKCis not required for the formation of the complex. On the other hand, we uncover that Gαq controls autophagy by a mechanism dependent on the modulation of the mTORC1 pathway under different nutrient stress conditions. Given the key reported role of p62 in these processes, we postulate a central role of this novel Gαq/p62 pathway in the control of autophagy. Overall, our studies indicate that PKCand p62 act as functional effectors of Gαq through the engagement of a PB1-type I-like binding region in this subunit. Our data also point out a relevant role for Gαq/11 as a link between nutrient-sensing GPCRs and autophagy modulation. Finally, we suggest that the interaction of Gαq with additional PB1 domain-containing proteins may participate in the control of cellular functions by Gq-coupled GPCR. P18r-2 Role of C3G in hepatocarcinoma tumor growth and progression. Regulation of stemness capacity Celia Sequera1, Sara Manzano1, María Arechederra1, Cristina Baquero1, Carmen Guerrero2, Almudena Porras1 1 Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, UCM, IdISSC, Madrid, ES, 2Centro de Investigación del Cáncer IBMCC (USAL-CSIC), IBSAL, Salamanca, ES C3G is a guanine nucleotide exchange factor for Rap1 and R-Ras. It is essential for embryonic development and it regulates several cellular functions such as cytoskeletal remodeling, differentiation and cell death. However, its role in cancer is controversial acting either as a tumor promoter or suppressor. Using different hepatocarcinoma (HCC) cell models with different degrees of epithelial phenotypes (Hep3B and HLE), our group found that C3G knock-down increased invasion and migration through induction of an epithelial-mesenchymal transition (EMT) process. Accordingly, C3G knock-down cells expressed higher levels of mesenchymal markers (Vimentin, N-Cadherin) and EMT transcription factors (Snail, Zeb1). All these changes induced by C3G silencing were similar to those elicited by TGF-β, a well known EMT inducer. Based on this pro-migratory effect of C3G knock-down, we have evaluated the role of C3G in tumorigenesis in HCC cells. Anchorage-dependent assays showed reduced number of foci and greater dispersion of cells within a focus in C3G knock-down HCC cells, as compared to parental cells. This correlates with the low adhesion of C3G silenced Hep3B cells. In contrast, anchorage independent assays in soft agar revealed an increase in the number and size of foci in C3G-silenced Hep3B cells, as compared to parental cells. However, each individual focus was composed of few and scattered cells. To further understand the function of C3G in tumor growth, we studied its role on cell survival and found that C3G knock-down decreased cell death. Moreover, our preliminary studies on the role of C3G on stemness capacity of HCC cells showed that C3G knock-down favors the induction of a stem cell like phenotype in Hep3B cells. Thus, C3G silenced cells form more spheres and increase the expression of some stemness markers. All these data support a role for C3G as an inhibitor of migration and invasion, while its function in tumor growth remains unclear. We are currently evaluating in vivo the role of C3G in HCC tumor growth. Pósters / Posters P18r-3 Concomitant deletion of H-Ras and N-Ras leads to pulmonary immaturity, respiratory failure and neonatal death in mice Rocío Fuentes Mateos1, David Jimeno2, Carmela Gómez2, Eugenio Santos de Dios2, Alberto Fernández Medarde2 1 Laboratorio 1, Centro de Investigación del Cáncer. Campus Miguel de Unamuno, Fundación de Investigación del Cáncer Universidad de Salamanca, Salamanca, ES, 2Laboratorio 1, Centro de Investigación del Cáncer. Campus Miguel de Unamuno, Instituto de Biología Molecular y Celular del Cáncer. Universidad de Salamanca/CSIC, Salamanca, ES The canonical members of the Ras gene family (H-ras, N-ras, and K-ras) encode four protein isoforms (H-Ras, N-Ras, K-Ras4A and 4B) regulating cell proliferation, differentiation or death/survival. Whereas genomic disruption of K-ras4B causes embryonic lethality, H-ras, N-ras and K-ras4A knockout mice are viable. H-ras and N-ras double KO (DKO) mice are also viable but show increased perinatal mortality suggesting critical physiological roles of those proteins around birth. Our characterization of DKO (Hras-/-;Nras-/-) mice uncovered a significant mortality rate due to respiratory failure during the first two postnatal days. Although DKO mice develop normal lung branching, they show delayed pulmonary maturation, a defect affecting both bronchiolar (Ciliated and Clara Cells) and alveolar lineages (type I and type II Pneumocytes), plus a reduced alveolar space and thicker septa. Clara Cells showed abnormal morphology and lack of proteoglycans production, whereas Ciliated Cells showed incorrect cilia structure. Additionally, these cells were not evenly juxtaposed in the bronchiolar space but formed a continuous layer in which markers for both cell types merged. Alveolar cells showed also abnormal phenotypes. Whereas type II Pneumocytes were wrongly localized inside a cell mass instead of surrounding the alveolar spaces, the type I cells did not exhibit typical flat shape, pointing to a defect in differentiation. The delay in lung maturation was further supported by the observation of increased number of precursors cells of alveolar lineage (more RCA/SftpC+ cells) as well as by higher proliferation rates. Our data uncover important, specific functional roles of H-Ras and N-Ras for lung maturation and neonatal survival that cannot be substituted by K-Ras action. P18-4 The Rac-GAP β2-chimaerin has a dual function in breast carcinogenesis Victoria Casado-Medrano, María José Caloca Roldán CSIC/IBGM, Valladolid, ES β2-chimaerin is a Rac1-specific negative regulator and a candidate tumor suppressor in breast cancer. Despite the relevance of this function, the precise contribution of β2-chimaerin to this pathology has been largely unknown. We show evidence of a differential role of β2-chimaerin in the migration and invasion of breast cancer cells depending on whether they are epithelial or mesenchymal. Expression of β2-chimaerin in LM2 cells, a mesenchymal-like breast cancer cell line, significantly reduced cell migration (analyzed by wound healing assays) and invasion (analyzed by trasnswell assays). However, expression of β2-chimaerin in the epithelial breast cancer cell line MCF7 had no effect in the migratory and invasive properties of these cells. This cell context dependency of the effects of β2-chimaerin raised an important question about the exact role of β2-chimaerin in breast cancer, a pathology that arises from epithelial cells that switch to a mesenchymal phenotype during cancer progression. To answer this question we analyzed the effect of the genetic ablation of β2-chimaerin in the MMTV-Neu/ErbB2 mouse model of breast cancer. We demonstrate that loss of β2-chimaerin accelerates tumor onset and, importantly, delays tumor progression, most likely by delaying the 141 Pósters / Posters epithelial to mesenchymal transition of breast cancer cells. Finally, we show evidence that our findings can be extrapolated to the human pathology, since analysis of breast cancer databases shows that patients with low β2-chimaerin expression have reduced relapse free survival but develop metastasis at similar times. Financial support: JCYL (BIO/VA34/15). P18-5 Mitochondrial dynamics: blasting off to pluripotency Javier Prieto1, Marian León1, Xavier Ponsoda1, Carlos López1, José Manuel Torres Ibáñez2 1 UVEG, Burjassot, ES, 2Universitat de Valencia, Meliana, ES During the process of reprogramming to induced-Pluripotent Stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganisation. Neither the importance of mitochondrial remodelling for cell reprogramming nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is not associated with mitophagy but rather with an increase in organelle biogenesis. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to down-regulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency. The role of the reprogramming factors in triggering mitochondrial reorganisation and function will be discussed. P18r-6 C3G promotes angiogenesis through the regulation of the differential release of pro-/anti-angiogenic factors from thrombin-activated platelets Víctor Martín-Granado1, Sara Ortiz-Rivero1, Sara Gutiérrez-Herrero1, Celia Sequera2, José Ramón González-Porras3, Francisco Martín-Herrero4, Almudena Porras2, Carmen Guerrero1 1 Centro de Investigación del Cáncer IBMCC (USAL-CSIC), IBSAL, Salamanca, ES, 2Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, UCM, IdISSC, Madrid, ES, 3Departamento de Hematología, IBSAL, Hospital Universitario de Salamanca, Salamanca, ES, 4Departamento de Cardiología, IBSAL, Hospital Universitario de Salamanca, Salamanca, ES C3G is a guanine nucleotide exchange factor (GEF) of several proteins within the Ras superfamily, including Rap1a/b and R-Ras. Rap1 is known to be involved in critical aspects of platelet function, including aggregation, adhesion and spreading, through the activation of the platelet integrin αIIbβ3. Using transgenic mouse models expressing C3G or C3G∆Cat (a mutant lacking the catalytic domain) in megakaryocytes and platelets, our group has identified C3G as a mediator of Rap1 actions leading to platelet activation and aggregation. Furthermore, C3G promotes α-granule release, evidenced by the increase in P-selectin exposure on the platelet surface, following its activation. In addition, preliminary immunofluorescence analysis suggests that C3G modulates the release of VEGF and endostatin from thrombin-activated platelets. The goal of the present study was to further characterize the underlying mechanisms that regulate the function of C3G in the differential release of pro- and anti-angiogenic factors upon platelet activation, and to evaluate 142 XXXIX Congreso SEBBM the angiogenic potential of the platelet releasate. We have found that VEGF release was up-regulated in thrombin-activated mouse platelets from both, C3G and C3GΔCat, transgenic mice. Additionally, thrombospondin release was also up-regulated in the latter. The releasate of TgC3G platelets had an overall pro-angiogenic effect as evidenced in a capillary-tube formation assay with HUVECs and in heterotopic tumor mouse models using B16 and 3LL cells. On the other hand, platelet spreading analysis revealed an up-regulation of this function in both transgenic mouse platelets upon thrombin activation, with TgC3G platelets showing the highest extension area. A preliminary co-IP analysis revealed an interaction between C3G and VAMP-7 upon activation, which could explain this result. Taken together, our data indicate the co-existence of RapGEF-dependent and independent mechanism through which C3G is regulating platelet secretion, and suggest an overall pro-angiogenic effect of platelet C3G. P18r-7 C3G: a new player in the control of invasiveness, stemness and tumorigenesis in glioblastoma Sara Manzano1, Celia Sequera1, María Arechederra1, Cristina Baquero1, Neibla Priego1, Carmen Guerrero2, Almudena Porras1 1 Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, UCM, IdISSC, Madrid, ES, 2Centro de Investigación del Cáncer IBMCC (USAL-CSIC), IBSAL, Salamanca, ES C3G is a guanine-nucleotide exchange factor (GEF) for some Ras family members, although it can act through GEF independent mechanisms. It is essential for embryonic development due to its role in adhesion and regulates different cellular functions. The role of C3G in human cancer is controversial, acting as either a tumour suppressor or mediator. Although the function of C3G in migration depends on the context, C3G was shown to inhibit migration in various cell types. We also found that C3G acts as a migratory and invasive repressor in MEFs and HCT116 cells through down-regulation of p38α MAPK activity. Based on this, we have studied the role played by C3G in human glioblastoma through gene silencing, using the U87 cell line as an in vitro model. We found that C3G knock-down enhances invasion and reduces cell adhesion. In fact, upon C3G silencing, U87 cells re-organize actin cytoskeleton, leading to neurite-like extensions. These functional and morphological changes correlate with the expression of transcription factors and proteins involved in the epithelial-mesenchymal transition (EMT). Moreover, C3G knock-down induces anchorage-dependent and -independent growth of U87 cells, suggesting that C3G could inhibit tumour growth. C3G may also play a role in the control of stemness and the acquisition of a glioblastoma initiating cell-like phenotype, as its silencing favours spheres formation. C3G silencing also induces changes in different signalling pathways, such as p38 MAPKs, ERKs, STAT3 or c-Met, which might contribute to regulate the observed functional changes. These data indicate that C3G is a new player in the regulation of glioblastoma progression, as it would inhibit tumour growth and invasion. Therefore, its down-regulation might promote tumour growth and the generation of metastasis through a mechanism involving an EMT process and stemness induction. However, more studies are required to fully characterize the function of C3G in glioblastoma, as well as to determine the molecular basis of this regulation and its relevance in clinic. P18-8 PPAR-α-FGF21 signaling is induced by a combined docosahexaenoic acid and thyroid hormone protocol Luis Videla Cabrera, Romina Vargas Villagrán, Virginia Fernández Arancibia Universidad de Chile, Facultad de Medicina, ICBM, Programa de Farmacología, Santiago, CL Salamanca 2016 Combined omega-3 and thyroid hormone (T3) treatment prevents ischemia-reperfusion liver injury. Considering that energy requirements of this protection may entail peroxisome-proliferator activated receptor-α (PPAR-α)-fibroblast growth factor 21 (FGF21) signaling, a combined protocol of docosahexaenoic acid (DHA) (300 mg/kg for 3 days) plus T3 (0.05 mg/kg, single dose) was administered to fed rats. Enhanced liver DHA content and serum T3 levels were paralleled by increased PPAR-α mRNA and its DNA binding. Higher PPAR-α target genes mRNA expression (carnitine-palmitoyl transferase 1α, acyl-CoA oxidase, and 3-hydroxyl-3-methylglutaryl-CoA synthase 2) were achieved, effects that were mimicked by 0.1 mgT3/kg or the PPAR-α agonist WY-14632. As increased mRNA expression of retinoic X receptor-α (RXR-α), accompanied by augmented liver mRNA and protein FGF21 levels and those of serum FGF21, were also achieved, it is concluded that PPAR-α-FGF21 induction by DHA combined with T3 may involve ligand activation of PPAR-α by DHA and enhanced expression of PPAR-α by T3, with consequent upregulation of the FGF21 that is controlled by PPAR-α. Supported by FONDECYT 1150104. P18r-9 Función de PKCδ en el tráfico polarizado de cuerpos multivesiculares y secreción de exosomas por los linfocitos T en la sinapsis inmune Gonzalo Herranz Gómez, David Fernández Moreno, Sergio Dávila Martínez, Laura Márquez Expósito, Álvaro Merchán Martín, Raúl de Martín Esteban, Jesús Gómez Alonso, Ioana Bianca Stancu, Teresa Fernández Caballero, Mario Quintanilla Álvarez, Víctor Calvo López, Manuel Izquierdo Pastor Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Madrid, ES La formación de la sinapsis inmune (SI) es crucial para garantizar una respuesta inmune eficaz. La SI entre una célula presentadora de antígeno y un linfocito T, induce en este último el tráfico de los cuerpos multivesiculares (MVB), que se fusionan a la membrana plasmática en la SI. Tras la fusión, las vesículas intraluminales contenidas en los MVB se liberan al exterior celular en forma de exosomas. Los exosomas son vesículas de doble membrana lipídica que intervienen en la comunicación intercelular, ya que contienen diversas moléculas bioactivas. En linfocitos T, los exosomas contienen proteínas proapoptóticas que median la citoxicidad de linfocitos T citotóxicos y la muerte celular inducida por reactivación (AICD) de linfocitos T. Por tanto, dilucidar los mecanismos que controlan su secreción permitiría modificar la función y la homeostasis en el sistema inmune. Se ha documentado que el tráfico polarizado de MVB hacia la SI se controla de forma directa por reorganización del citoesqueleto de actina, e indirectamente por el enzima diacilglicerol quinasa α (DGKα) y diacilglicerol (DAG), su sustrato. Sin embargo, las bases moleculares de este fenómeno están sin aclarar. Hemos observado que la proteína quinasa C delta (PKCδ), un isotipo de PKC activado por DAG, se acumula cerca de los MVB. Cuando se interfiere la expresión de PKCδ en linfocitos T disminuye la eficiencia de la polarización de los MVB hacia la SI, y también se reduce la secreción de exosomas y la AICD. La reorganización del citoesqueleto de actina cortical que ocurre en la SI está afectada en los clones interferidos en PKCδ, tanto espacial como temporalmente. Esto sugiere que PKCδ y su control del citoesqueleto de actina median el efecto de DGKα en el tráfico secretor polarizado del linfocito T. Pósters / Posters P18-10 Papel de la AMPK (proteína quinasa activada por AMP) como promotor tumoral en cáncer de colon Ana Chocarro-Calvo1, José Manuel García-Martínez1, María Gutiérrez-Salmerón1, Antonio de la Vieja2, Custodia García-Jiménez1 1 Universidad Rey Juan Carlos, Alcorcón, ES, 2Instituto de Salud Carlos III, Majadahonda, ES Estudios epidemiológicos asocian una mayor incidencia de cáncer de colon con la diabetes. La AMPK permite a la célula adaptarse al estrés metabólico y es una diana molecular importante en diabetes. El papel de AMPK en la progresión tumoral y su potencial como diana terapéutica es controvertido ya que puede actuar como un supresor tumoral condicional o como un oncogen contextual. Objetivo: Caracterización del papel de la AMPK durante la adaptación al estrés metabólico impuesto por la captura facilitada de glucosa en cáncer de colon. Metodología: Usamos células de colon tumorales (HCT 116) y sanas (CCD-18Co) en normo o hiper-glucemia. Los tratamientos incluyen inhibidores/activadores y siRNA específicos de AMPK. Las alteraciones en las proteínas se miden por western Blot e inmunoprecipitaciones. Resultados: La hiperglucemia aumenta la actividad de AMPK en células tumorales de colon induciendo su fosforilación en la T172 e inhibiéndola en la S485. La activación de AMPK correlaciona con la fosforilación de p300 y su activación y con autofagia aumentada, pero no con la inhibición de la vía de mTORC1. La inhibición o depleción de AMPK bloquean estos efectos pero la Metformina no los reproduce. Conclusiones: La fosforilación de AMPK en la S485 puede determinar su papel como oncogén o supresor tumoral en células de colon. Los resultados sugieren un nuevo mecanismo de adaptación de algunas células tumorales al estrés metabólico. P18-11 Analysis of the function of Sp6 and Sp8 transcription factors in the limb ectoderm Rocío Pérez Gómez1, Marc Fernández-Guerrero1, Sandra Zunzunegui2, Juan F. López-Giménez1, Marian Ros1 1 Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Consejo Superior de Investigaciones Científicas (CSIC), Santander, ES, 2Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria (UC), Santander, ES We have recently identified the crucial role played by Sp6 and Sp8, two transcription factors members of the Sp family, during limb development. In their absence or substantial reduction no limbs form and their progressive dose reduction associates a spectrum of limb malformations ranking from syndactyly through Split-hand/split-foot malformation, to olygodactyly. When digits form, they exhibit bidorsal tips. We have postulated that Sp6 and Sp8 are necessary, downstream of Wnt/B-catenin, to activate Fgf8 and downstream of Bmp signaling, possible cooperating with Smads, to activate En1, the marker of the ventral ectoderm. Currently we are exploring these suspected protein-protein interactions by co-immunoprecipitation (CoIP) and by bimolecular fluorescence complementation (BiFC). To this end, Sp6, Sp8 and Smads were tagged with Myc or FLAG epitopes to their N-terminal end and with YFP full length or YFP moieties to their C-terminal end. We show by CoIP and BiFC that Sp8 homodimerizes. To determine the region involved in Sp8 interaction, we have generated different truncated versions of Sp8. Our results show that Sp8 homodimerizes at its C-terminal domain. Our study underscores the relevance of Sp6/8 in mediating B-catenin and Bmp signaling pathways in the limb ectoderm. 143 Pósters / Posters P18-12 El avance del ciclo celular en G1/S está regulado por la estabilidad de PLK1 vía CDK1/βTrCP María Galindo-Moreno1, Servando Giráldez1, M. Cristina Limón-Mortés1, Joaquín Herrero-Ruiz1, Mar Mora-Santos1, Carmen Sáez2, Miguel Á. Japón2, María Tortolero1, Francisco Romero1 1 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Sevilla, ES, 2Instituto de Biomedicina de Sevilla (IBIS) y Departamento de Anatomía Patológica del Hospital Universitario Virgen del Rocío de Sevilla, Sevilla, ES Polo-like kinase 1 (PLK1) es una quinasa de serina/treonina implicada en diversas etapas del ciclo celular, entre las que destacan la entrada y salida de mitosis y la citocinesis. Además, tiene un papel esencial en la regulación de la replicación y, junto con ciclina A/CDK, fosforila a la subunidad CDH1 del APC/C, induciendo su ubicuitilación y degradación por el proteasoma. Esto permite la acumulación de los sustratos correspondientes y el avance del ciclo celular. En muestras de tumores de pacientes de muy diversos orígenes se han detectado niveles elevados de PLK1, comparados con el tejido normal, lo que ha llevado a proponer a PLK1 como un marcador pronóstico de los cánceres humanos. De hecho, se usa como diana de fármacos anticancerígenos y ya se han descrito una serie de inhibidores que están siendo probados en ensayos clínicos. Todos estos datos nos han llevado a estudiar la regulación de la estabilidad de esta quinasa y a profundizar en su papel en el ciclo celular. En resultados previos de nuestro grupo determinamos que la proteína PLK1 nuclear es ubicuitilada por SCFFBXW7 y degradada por el proteasoma en respuesta a los daños en el ADN para impedir la formación de los complejos de pre-replicación y evitar la proliferación celular. En el presente trabajo mostramos que la proteína PLK1 citoplasmática es ubicuitilada y degradada por SCFβTrCP/proteasoma cuando no está correctamente plegada, poniéndose esto de manifiesto al tratar las células con geldanamicina, un inhibidor de la chaperona HSP90. Además, identificamos a CDK1 como la principal quinasa implicada en este proceso. Finalmente demostramos que la inhibición de HSP90 detiene la progresión del ciclo en la transición G1/S a través de la degradación de PLK1 que, a su vez, evita la destrucción de CDH1. En estas condiciones, los sustratos del APC/CCDH1 no se acumulan y el ciclo se detiene. Las consecuencias de estos resultados serán discutidas. María Galindo-Moreno y Servando Giráldez han contribuido igualmente a este trabajo. P18r-13 Characterization of VRK1 and Atxn1 interaction and its functional implications Elena Martín Doncel, Lara Cantarero, Pedro A. Lazo Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca; Instituto de Investigación Biomédica de Salamanca (IBSAL), Hospital Universitario de Salamanca, Salamanca, ES Atxn1 is the protein involved in the inherited neurodegenerative disease SCA1, an autosomal dominant disorder caused by the expansion of a triplet CAG, encoding a polyglutamine tract (polyQ) which confers toxic properties to the protein. Nevertheless, polyQ is necessary but not sufficient to cause pathology. Indeed, is also important the nuclear localization signal, the AXH domain and a serine 776 residue. VRK1 is a Ser-Thr kinase associated with multiple processes, including the regulation of cell cycle progression, chromatin remodeling or Cajal Bodies dynamics. Also VRK1 is mutated in neurodegenerative diseases. Previously we identified Atxn1 like a potential target for VRK1, through the use of linear peptide chips. Our aim is to characterize the interaction VRK1-Atxn1 and its 144 XXXIX Congreso SEBBM potential functional implication. Through immunoprecipitation and nuclei isolation assays, we detected an interaction between both proteins. Besides, by HPLC fractionation is proved that Atxn1 and VRK1 elute in the same high molecular size fractions. By immunofluorescence, we did not detect any colocalization, which may indicate a transient interaction between both proteins. In a kinase assay, we observed distinct phosphorylation levels between Atxn1 with a physiological polyQ and the protein with an expanded polyQ. Only wild-type Atxn1 can be phosphorylated by VRK1. Atxn1 is degraded via ubiquitin proteasome system. Here, we showed that different mutants of Atxn1 have a similar protein accumulation in the nuclear inclusions when proteasome is inhibited with MG-132. Yet, we have evidence that VRK1 downregulation enhances the degradation of Atxn1. We believe other proteins, like VCP, which colocalized with Atxn1 nuclear inclusions with expanded polyQ, might be involved in this process. P18-14 Chromatin dynamics in response to DNA Damage Ignacio Campillo-Marcos1, Marcella Salzano1, Raúl García-González2, Pedro A. Lazo1 1 Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca; Instituto de Investigación Biomédica de Salamanca (IBSAL), Hospital Universitario de Salamanca, Salamanca, ES, 2 Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca, Salamanca, ES Introduction: Genome integrity is continuously challenged by exogenous and endogenous agents causing DNA damage, both in euchromatin and heterochromatin. The repair of these lesions involves changes in the histone organization, such as recruitment and/or incorporation of histone variants or covalent modifications of histones, in order to promote the formation of open, relaxed chromatin structures. Recently, a growing number of chromatin components, remodelers and modifications has been identificated as key players in DNA repair, emphasizing the complex role of chromatin in this process. On the other hand, defects in DNA repair pathways enable cancer cells to survive DNA damage. For this reason, the combination of DNA-damaging agents with specific inhibitors of these pathways could be used in cancer treatment. Currently, specific inhibitors against chromatin remodelers have been developed, but it is still unclear how they act in tumor cells. Our aim is to detemine the effect of histone acetylation/deacetylation and methylation/demethylation inhibition on DNA repair foci. Results and conclusion: We use pharmacological inhibitors of HATs (C646 and MG149); HDACs (Entinostat and SAHA); KMTs (Chaetocin); and KDMs (JMJD2 inhibitor). Some of them are being employed in preclinical regulatory studies. In this analysis, we can observe that closed chromatin induced by HATs inhibitors seems to affect the formation of γH2AX and 53BP1 foci, and the dynamics of foci formation corresponds with the increase of fluorescence level of H4 acetylation after inducing DNA damage. Based on these results, we conclude that chromatin relaxation is an essential early step in DNA repair, which can be blocked by specific inhibitors against HATs, sensibilizing cells to treatments. P18r-15 Functional interaction between Met and TGF-β pathways during EMT in mouse liver progenitor cells Laura Almalé1, María García-Álvaro1, Adoración Martínez-Palacián1, Nerea Lazcanoiturburu1, Annalisa Addante1, César Roncero1, Margarita Fernández1, Eduardo Rial2, Isabel Fabregat3, Blanca Herrera1, Aránzazu Sánchez1 Salamanca 2016 1 Dep. Bioquímica y Biología Molecular II, Facultad de Farmacia, Universidad Complutense de Madrid; Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, ES, 2Departamento de Medicina Molecular y Celular, Centro de Investigaciones Biológicas, CSIC, Madrid, ES, 3Laboratori d´Oncologia Molecular, Universitat de Barcelona, Institut d´Investigació Biomèdica de Bellvitge, L´Hospitalet de Llobregat, ES Adult progenitor liver cells (oval cells, OC) constitute a bipotential cell population that contribute to liver regeneration in chronic liver disease (CLD). However, recent evidence supports a pro-fibrogenic role of OC in the context of the CLD. They are also prone to malignant transformation having been proposed as the cell of origin of a hepatocellular carcinoma subgroup. To better understand the mechanisms behind OC fate decisions, we have analyzed the effect of TGF-β-driven EMT (Epithelial-Mesenchymal Transition) in OC function and the potential interaction between TGF-β and HGF/Met signalling pathways in this context. For that, we have used an in vitro model of OC lines harbouring an active (Metflx/flx cells) or inactive (Met-/-cells) Met tyrosine kinase. After a chronic TGF-β treatment, we obtained cells with a stable mesenchymal phenotype (TβT-OC, TGF-β-treated oval cells). Interestingly, TGF-β-induced EMT did not result in up-regulation of cancer stem cell markers (Epcam, CD44 and CD133) or in acquisition of anchorage-independent growth capacity. Nevertheless, TβT-OC show profound changes in their proliferative and invasive properties and acquire resistance to apoptosis. Importantly, Met signaling seems to be necessary for OC expansion after EMT, since Met-/- cells suffer an irreversible senescence process associated with the induction of cell cycle inhibitors. We are now exploring whether the effect of the exacerbated oxidative stress detected in TβT-Met-/- cells could be the key mechanism for senescence induction. On the other hand, TGF-β-OC no longer respond to HGF in terms of mitogenic or pro-invasive activities and an altered signaling response to HGF is observed post-EMT. These changes could be partly explained by amplification of the HGF autocrine loop detected in OC. In conclusion, our results evidence profound changes in OC phenotypic and functional properties after EMT and an interesting crosstalk between HGF and TGF-β pathways during and post-EMT induction. The molecular mechanisms involved and the consequences for oval cell fate are under study. P18-16 MCI inhibition and acute O2 sensing by peripheral chemoreceptors: Effect of regeneration of electron acceptors Ignacio Arias Mayenco, Hortensia Torres Torrelo, Patricia González Rodríguez, Lin Gao, Patricia Ortega Sáenz, José López-Barneo López Barneo Instituto de Biomedicina de Sevilla, Sevilla, ES The carotid body contains chemoreceptor O2-sensitive glomus cells, which in response to hypoxia release transmitters to elicit compensatory cardiorespiratory reflexes. Understanding the mechanisms underlying the detection of acute changes in O2 tension is of broad biomedical interest.We have recently reported the involvement of mitochondrial complex I (MCI) in acute O2 sensing (Fernández-Agüera et al., Cell Metab. 2015). Knockout mice lacking Ndufs2 (a MCI subunit required for ubiquinone reduction) in catecholaminergic cells (TH-NDUFS2) failed to hyperventilate under hypoxic conditions. Hypoxia-induced increases in NADH and reactive oxygen species (ROS), inhibition of K+ channels and increase in cytosolic Ca2+ were also selectively abolished in glomus cells from these mice. In Ndufs2-deficient glomus cells NADH was accumulated, which could inhibit Kreb’s cycle dehydrogenase activity. Therefore, to determine whether these phenotypes result from a developmental generalized metabolic disarrangement or they are a direct consequence of MCI dysfunction, we have generated the ESR-NDUFS2 mice, in which the Ndufs2 gene was ablated during adulthood. A time-dependent loss of hypoxic ventilatory response with a parallel decrease in MCI activity Pósters / Posters were observed in ESR-NDUFS2 mice. The effects of hypoxia on glomus cell parameters (NADH, ROS, and cytosolic Ca2+) were also abolished. Addition of electron acceptors (alpha ketobutyrate or pyruvate), to regenerate NAD+ and activate Kreb’s cycle, in combination with succinate treatment, to increase mitochondrial complex II activity, failed to recover responsiveness to hypoxia. These results further demonstrate the essential role of MCI in acute O2 sensing and the involvement of NADH and ROS in the signalling pathway leading to K+ channel inhibition, glomus cell depolarization and the trigger of cardiorespiratory reflexes. P18-17 Hypoxia reduces mitochondrial complex IV activity via post-transcriptional control of NDUFA4 by IRP1 Eduardo Balsa1, Bárbara Acosta-Iborra1, Pablo Hernansanz-Agustín2, Daniel Tello1, Adela Guarás3, Nora Cascante-Estepa1, Ángel Ordóñez-Navadijo1, Elia López-Bernardo4, Susana Cadenas4, José Antonio Enríquez3, Manuel O. Landázuri5, Antonio Martínez-Ruiz6 1 Unidad de Investigación, Hospital Universitario Santa Cristina, Universidad Autónoma de Madrid, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, ES, 2Servicio de Inmunología, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP). Departamento de Bioquímica, Universidad Autónoma de Madrid, Madrid, ES, 3Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, ES, 4 Servicio de Inmunología, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP). Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, ES, 5 Unidad de Investigación, Hospital Universitario Santa Cristina, Universidad Autónoma de Madrid, Instituto de Investigación Sanitaria Princesa (IIS-IP) Servicio de Inmunología, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, ES, 6Servicio de Inmunología, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, ES Cytochrome c oxidase or complex IV (CIV) of the mitochondrial oxidative phosphorylation system (OXPHOS) is the principal oxygen-consuming enzyme in eukaryotic cells. Although global OXPHOS and CIV activity are diminished in hypoxia, a hypoxia inducible factor-1 (HIF-1)-dependent exchange of critical CIV subunit isoforms optimizes CIV efficiency. Herein, we demonstrate that CIV abundance is progressively reduced when oxygen supply becomes limiting. Hypoxia does not alter the mRNA levels of CIV subunits, but instead induces degradation of the entire complex, diminishing CIV-containing supercomplexes and reducing oxygen consumption. We show that CIV stability depends on the abundance of the CIV subunit NDUFA4, which is in turn regulated by iron regulatory protein 1 (IRP1) activity. Hypoxia decreases IRP1 binding to an iron response element (IRE) within the 3’-UTR mRNA of NDUFA4, reducing NDUFA4 protein translation. Thus HIF-dependent and independent mechanisms cooperate to fully adapt CIV to fluctuating oxygen availability. - Hypoxia triggers a series of adaptive responses in cell metabolism, including the enhancement of glycolysis and inhibition of different steps of mitochondrial oxidative phosphorylation (OXPHOS). - In higher organisms, the only mechanism described for direct regulation of mitochondrial complex IV (CIV), the main oxygen-consuming system, is a switch of CIV subunits that increases CIV efficiency in hypoxia, driven by the hypoxia-inducible factor 1 (HIF-1). - Several reports have shown that CIV activity decreases in hypoxia, a plausible adaptation to reduce oxygen consumption. - We describe a novel mechanism by which hypoxia reduces CIV activity, mediated by iron regulatory protein-1 (IRP1) binding to the mRNA of NDUFA4, a protein that has been recently described to be a CIV subunit. 145 Pósters / Posters XXXIX Congreso SEBBM P18-18 P18r-20 Activation of arterial chemoreceptor cells by extracellular lactate; role in acute oxygen sensing Regulation of mitochondrial respiration in astrocytes Hortensia Torres-Torrelo, Lin Gao, Patricia González-Rodríguez, José López-Barneo, Patricia Ortega-Sáenz Instituto de Biomedicina de Sevilla, Sevilla, ES Ines Juaristi1, Irene Llorente-Folch1, Araceli del Arco2, Jorgina Satrústegui1 1 Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Departamento de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM); Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER); Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD), ISCIII, Madrid, ES, 2 Centro regional de Investigaciones Biomédicas, Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla La Mancha, Madrid, ES The carotid body (CB) mediates rapid adaptive responses to hypoxia. Glomus cellsin the CB contain K+ channels, which are inhibited by intracellular ROS and NADH generated in mitochondria during hypoxia leading to cell depolarization, transmitters secretion and activation of afferent fibers impinging upon respiratory centers. Recently, it has been reported that glomus cells contain lactate receptors and that lactate released from these cells during hypoxia activate neighboring cells, in an auto/ paracrine phenomenon, that has been suggested to be the mechanism responsible for acute responsiveness to lowering O2 tension. We have investigated the effects of lactate on single glomus cells dispersed or in CB slices. Extracellular lactate increased glomus cells secretion rate, evaluated by amperometry, with a half-maximal activation at 15 mM, without occluding the secretory response to hypoxia; both stimuli (hypoxia and lactate) had additive effects. In glomus cells, studied with perforated patch clamp, lactate had no effect on resting K+ conductance and produced a small but significant increase in voltage-dependent K+ current. This contrasts with the effects of hypoxia, which normally inhibits both resting and voltage-dependent K+ channels. In transgenic TH-NDUFS2 mice, with mitochondrial complex I function almost completely abolished and selective suppression of hypoxic response, lactate induced normal secretory activity. These data indicate that although extracellular lactate can exert a significant stimulatory effect on CB glomus cells it does not seem to play a major role in acute O2 sensing by arterial chemoreceptors. Ongoing studies will clarify the role of extracellular lactate, mainly generated in anaerobic skeletal muscle fibers during exercise, in CB-mediated regulation of respiration. P18-19 Macrophages lacking lipin-2 Enhance IL-1β production through the overactivation of NLRP3 inflammasome Gema Lordén Losada1, Jesús Balsinde2, María Balboa2 1 Universidad de Valladolid, Valladolid, ES, 2CSIC, Valladolid, ES Lipin-2 is a member of the lipin family of proteins, which catalyze the enzymatic conversion of phosphatidic acid to diacylglycerol. Mutations found in the human LPIN2 gene are known to cause Majeed Syndrome, an autoinflammatory disorder in which IL-1βis involved. Since NLRP3 inflammasome orchestrate innate immune responses through activation of caspase-1 leading to the maturation of pro-inflammatory cytokines pro-IL-1β and pro-IL-18, it was hypothesized that lipin-2 could modulate its production and thereby be involved in the regulation of inflammasome activity. We found out that both, classic (LPS and ATP) and metabolic (LPS and palmitic acid) activation of NLRP3 inflammasome, promote an increased production of the proinflammatory cytokine IL-1β in macrophages lacking lipin-2, which depends on the overactivation of NF-κB by LPS. Besides, depletion of lipin-2 in macrophages alter cellular responses to ATP, what leads to overstimulation of the NLRP3 pathway triggering the IL-1β and IL-18 production in an ASC and caspase-1 dependent manner. These effects may be due to the fact that absence of lipin-2 modifies lipid membrane levels, leading to an increase of P2X7 receptor activity, triggering potassium efflux from the cell, and increasing the assembly of the inflammasome and its activity. Collectively, these studies confirm a protective role for lipin-2 in the pathology of inflammatory diseases mediated by activation of inflamasome NLRP3. 146 In the brain, calcium regulates mitochondrial respiration both by activation of ATP consumption and by Ca signaling. Ca signaling in mitochondria may regulate respiration: i) after Ca entering through mitochondrial uniporter (MCU), activating mitochondrial dehydrogenases and F0F1-ATPsynthase; or ii) activating Ca-binding mitochondrial carriers, ARALAR and SCaMCs from the outer face of the inner mitochondrial membrane, which involves metabolite supply. In neurons, ARALAR contributes significantly to workload and Ca-induced respiration (1). In brain astrocytes, ARALAR levels are undetectable and this work aims to study the metabolic response of cultured astrocytes to different workloads and the possible role of Ca in response, using different ligands, glutamate (L-Glu) and ATP. L-Glu is transported into the astrocytes resulting in increased Na+ uptake, which drives ATP breakdown, and also acts on metabotropic L-Glu receptors. L-Glu (200 μM – 500 μM) produced a small rise in Cai in cultured astrocytes, increased glucose utilization, lactate production and stimulated oxygen consumption in an acute and stable way (OCR, by Seahorse XF24 Extracellular Flux Analyzer) and maximal uncoupled respiration (MUR) in the presence of 2.5 mM glucose. A transportable but non-metabolizable glutamate analog, D-aspartate (D-Asp) (500 μM), which also increases Cai, stimulated respiration in a way similar to L-Glu, but failed to increase MUR, clearly showing that L-Glu is used as respiratory substrate by cultured astrocytes. L-Glu and D-Asp-stimulation of respiration was only slightly reduced in the absence of Cao, consistent with a major role of ATP demand in stimulation of OCR.ATP (100 μM - 1 mM) activates metabotropic and ionotropic purinergic receptors and induces a larger Cai rise than L-Glu in cultured astrocytes. ATP caused an increase in glucose utilization and lactate production, and also an acute and transient rise in OCR, which was dependent on Cao. Whether this is due to increased Ca-dependent workload or to the effects of Ca on upregulation of respiration is part of the ongoing work. Remarkably, MUR was also Ca-dependent, especially in astrocytes exposed to high ATP concentrations. It is likely that glycogen breakdown via Ca-dependent adenylate cyclase and PKA (2) plays a role in increasing MUR in the presence of Ca. Further studies are required to dissect the role of MCU and SCaMC-3 and Ca-dependency with each stimulus. References [1] Llorente-Folch et al., J Neurosci (2013) 33 (35). [2] Müller MS et al., Glia (2014) 62(4). P18-21 Cross-regulation between GPCRs and glutamate NMDA receptors: Role of HINT1 proteins and of σ 1 receptors Elsa Cortés Montero, María Rodríguez Muñoz, Pilar Sánchez Blázquez, Javier Garzón Niño Laboratorio Neurofarmacología, Departamento de Neurobiología Molecular, Celular y de Desarrollo, Instituto Cajal, CSIC, Madrid, ES The ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR) is a ligand-gated cation channel highly permeable to Ca2+. This receptor plays an essential role in neuronal plasticity, development, differentiation, learning and Salamanca 2016 memory consolidation. The activity of NMDARs is under the influence of G protein-coupled receptors (GPCRs) which can dampen (as cannabinoid 1 receptor CB1R) or enhance (as mu-opioid receptor MOR) NMDAR function. The cross-regulation between these GPCRs and NMDARs requires of the physical interaction of NMDAR NR1 subunits with the C termini of the GPCRs (1, 2). The present study shows that the interaction between NMDARs and MOR/CB1R reaches physiological relevance in the presence of the HINT1 protein and of the σ1R and in the absence of these proteins, the GPCR-NMDAR cross-regulation is disrupted. Within σ1R-MOR-HINT1 complex, the cytosolic segment C1 of the NMDAR NR1 subunit binds simultaneously to HINT1 and to σ1R, but σ1R weakens HINT1-NR1 association and impedes HINT1 transfer to the NMDAR. MOR activation leads to PKC-mediated phosphorylation of the NR1 C1 reducing the affinity of the coupled NMDAR to the MOR-HINT1 complex, and consequently the active NMDAR-σ1R tandem separates. To avoid hiperactivation of NMDARs by MORs, the antagonists of σ1R prevents σ1R binding to NR1 subunits and thus promote the swapping of HINT1 from the MOR to the NMDAR. Alternatively, within CB1R-HINT1-σ1R-NR1 complex the cannabinoids restrain NMDAR activity and could provoke its hypofunction, thus when necessary NMDARs must be disconnected from the negative influence of the CB1R by transfer of HINT1 from the CB1R to the NMDAR NR1 C1 subunit. The σ1R and its endogenous regulators (neurosteroids) play an essential role in this regulatory process, which serves to maintain NMDAR excitatory activity within physiological limits. Therefore, HINT1 and σ1R form a molecular switch that connect and disconnect the activity or GPCRs such as the MOR and CB1 with that of NMDARs. References [1] Rodríguez-Muñoz, M., et al., (2012). Neuropsychopharmacology, 37, 338. [2] Sánchez-Blazquez, P., et al., (2013). Antioxidants & Redox Signaling, 19, 1766. Pósters / Posters Universitario de Salamanca, Salamanca, ES, 3Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, UCM, IdISSC, Madrid, ES C3G is guanine nucleotide exchange factor for Rap1b, a protein of the Ras family involved in critical aspects of platelet function through the activation of integrin aIIbb3. Using transgenic mouse models, with specific expression in platelets, of full length C3G (tgC3G) or mutant lacking the GEF domain (tgC3GDCat), our group has unveiled an important role of C3G in primary hemostasis. Thus, C3G-Rap1 participates in thrombin-triggered platelet activation, aggregation and clotting through a PKC-mediated pathway. In addition, C3G-overexpressing platelets also show a stronger response to ADP, as compared to wt platelets. Based on these results, we have deepened into the role of C3G in platelet signaling by using several inhibitors of different platelets signaling pathways. This includes: clopidogrel and 2MeSAMP (for P2Y12 ADP receptor), MRS2179 (for P2Y1 ADP receptor), SB203580 (for p38a/b MAPK), U0126 (for ERK1/2), aspirin (for TXA2 synthesis) and wortmannin (for PI3K). We have performed activation, aggregation, and Rap1 activity assays in platelets stimulated with thrombin or ADP/fibrinogen in the presence or absence of the above referred inhibitors. Our results indicate that thrombin- or ADP-stimulated tgC3G platelets are more sensitive to the action of clopidogrel, SB203580, U0126 and aspirin, as compared to wild type platelets. In contrast, tgC3GDCat platelets are more resistant to these inhibitors than their corresponding control platelets. No differences were observed with the rest of inhibitors. These results suggest that C3G may participate in the activation of Rap1 mediated by ADP-P2Y12, but independent of PI3K. Furthermore, our results support a role for C3G in the thromboxane synthesis pathway, probably through modulating ERKs and p38a MAPK activities. P18r-24 P18-22 Pharmaco-phosphoproteomics disection of isoform-selective PI3K signalling in a cell model of acute myeloid leukaemia Maruan Hijazi, Pedro R. Cutillas Cell Signalling & Proteomics Group, Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Phosphatidylinositol 3-kinases (PI3Ks) are a family of lipid kinases that coordinate intracellular signalling in response to extracellular stimuli. Alterations in PI3K signalling cascades are common events in human cancers, making this class of enzymes a prime drug target for anti-cancer therapy. PI3K isoforms have distinct roles in mediating signalling in physiological and oncogenic contexts. However, the precise roles of specific PI3K isoforms in terms of biological responses remain poorly defined. In order to assemble a database of phosphorylation sites annotated with the signalling pathways they belong to, here we classified phosphorylation sites by their patterns of modulation by a panel of class Ia isoform-selective PI3K inhibitors in HL-60 cell line (human myeloid leukaemia cells). Their phosphoproteomes were analysed relative to DMSO control. We derive kinase activity by using label-free phosphoproteomics and bioinformatics tools. We show different patterns of activity modulated in an isoform-dependent manner, suggesting the presence of isoform-specific functions. P18-23 Contribution of the Rap-GEF C3G to signaling pathways in platelets Sara Gutiérrez-Herrero1, Víctor Martín-Granado1, Sara Ortiz-Rivero1, José Ramón González-Porras2, Almudena Porras3, Carmen Guerrero1 1 Centro de Investigación del Cáncer IBMCC (USAL-CSIC), IBSAL, Salamanca, ES, 2Departamento de Hematología, IBSAL-Hospital C3G regulates megakaryocytic differentiation and pro-platelet formation in mice Sara Ortiz-Rivero1, Sara Gutiérrez-Herrero1, Víctor Martín-Granado1, José Ramón González-Porras2, Almudena Porras3, Carmen Guerrero1 1 Centro de Investigación del Cáncer IBMCC (USAL-CSIC), IBSAL, Salamanca, ES, 2Departamento de Hematología, IBSAL-Hospital Universitario de Salamanca, Salamanca, ES, 3Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, UCM, IdISSC, Madrid, ES C3G is an activator of members of the Rap subfamily of Ras proteins. Several studies suggest that C3G plays a role in different processes of differentiation acting through its main target Rap1. C3G is induced during neural differentiation and monocytic differentiation to macrophages. Similarly, an increase in C3G protein levels and its association with Crk is required for adipocyte differentiation. There are also evidences supporting the involvement of C3G in the differentiation of megakaryocytes to platelets through Rap1 activation, although its function in early stages of megakaryocytic differentiation has not yet been addressed. Using transgenic mouse models for C3G and C3GΔCat (C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 gene promoter, we have found that C3G plays a role in the differentiation of immature hematopoietic cells to megakaryocytes. Bone marrow cells from transgenic C3G, but not those from transgenic C3GΔCat mice, showed increased CD41 and CD61 expression upon thrombopoietin (TPO) treatment. Polyploidization analysis supports the hypothesis of a positive role of C3G in megakaryocytic differentiation. Thus, C3G overexpression increased the levels of CD41+ megakaryocytes with 16N and 32N ploidy. In addition, preliminary studies using fresh bone marrow explants, incubated with Tyrode´s Buffer containing 5% mouse serum, showed an increased migration from the osteoblastic niche to the vascular niche and an enhanced ability to form pro-platelets in C3G transgenic megakaryocytes, as compared to wild-type 147 Pósters / Posters ones. These results suggest the participation of C3G in different key aspects of megakaryopoiesis by a mechanism mediated by its GEF activity. XXXIX Congreso SEBBM son determinantes para controlar la quiescencia y características de célula madre de las HSC. Nuestros resultados preliminares apuntan a que esto sucede efectivamente, y que la HSC es más quiescente cuando los niveles de dichas proteínas se reducen en las células mesenquimales. P18r-25 Targeted expression of a Spry1 Y53A mutant precludes replicative senescence Carlos Anerillas Aljama1, Marta Vaquero Susagna1, Sara Cuesta Sancho2, Joaquim Egea Navarro2, Mario Encinas Martín3 1 Universitat de Lleida, Quicena, ES, 2IRB Lleida, Lleida, ES, 3 Universitat de Lleida/ IRB Lleida, Lleida, ES Genes of the Sprouty family (Spry1-4) are feedback inhibitors of receptor tyrosine kinase (RTK) signaling. As such, they restrain proliferation of many cell types and have been proposed as tumor suppressor genes. Data from our group indicate that Spry1 behaves as a tumor suppressor independently of ERK activity by promoting cellular senescence, a fail-safe mechanism opposing cellular transformation. However, the structural domains critical for such function remain poorly understood. Different studies relying on ectopic overexpression point to an important role of a conserved tyrosine in the N-terminus of Spry family members (Tyr53 in Spry1). To confirm the role of this tyrosine in a more physiological setting we have analyzed skin fibroblasts from knockin mice bearing a Tyrosine to Alanine mutation in residue 53 of Spry1. When placed in a 3T3 scheme, skin fibroblasts from wild type but not those from Y53A knockin mice undergo replicative senescence as judged by cell counting, 5’ Bromo-deoxyuridine uptake and senescence-associated beta galactosidase staining. Interestingly, skin fibroblasts from Spry1 null mice do not escape replicative senescence, suggesting that Spry1 Y53A functions in a dominant negative fashion. Finally, in line with our previous data, no differences on ERK phosphorylation were found between wild type and knockin fibroblasts, indicating that induction of senescence by Spry1 is independent of this pathway. P18r-26 β-catenina y PTP-BL están implicadas en la regulación de la quiescencia de las HSC por parte de las células mesenquimales del estroma Alejandro Pérez Fernández, Rodrigo Prieto Bermejo, Marta Romo González, Carla Ijurko, Ángel Hernández Hernández Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca (USAL). Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, ES La hematopoyesis en el adulto se desarrolla en la médula ósea, en un entorno compuesto por distintos tipos celulares y condiciones fisico-químicas que en su conjunto se denomina nicho hematopoyético. Durante los últimos años se ha establecido de forma sólida que las células de linaje distinto al hematopoyético influyen en la biología de las células madre hematopoyéticas (HSC) tanto a través de factores solubles como por contacto directo. Una de las vías de señalización más estudiadas en el contexto de la hematopoyesis, entre otras razones por los resultados contradictorios que arrojan los distintos estudios realizados, es la denominada ruta canónica de Wnt, cuyo efector final es el factor de transcripción y molécula de adhesión β-catenina. Nuestro grupo de investigación ha demostrado recientemente que esta ruta es importante en la diferenciación megacariocítica, y que existe un control de la actividad transcripcional y estabilidad de β-catenina mediado por la fosfatasa PTP-Bas/PTP-BL. Además, estos estudios ponen de manifiesto que los niveles de estas dos proteínas en las HSC son un factor determinante a la hora de regular la quiescencia de estas células, fenómeno que parece ser mediado por la adhesión de las HSC a las células mesenquimales del nicho. Con los anteriores antecedentes, nos propusimos estudiar si los niveles de β-catenina y PTP-Bas/PTP-BL a nivel de células mesenquimales 148 P18-27 USP29 a novel upstream HIF-α activator Amelie Schober, Teresa Martín-Mateos, Encarnación Pérez-Andrés, Onintza Carlevaris, Sara Pozo, Edurne Berra CIC bioGUNE, Derio, ES The transcription factor HIF is the central regulator of the adaptive cellular program in response to hypoxia. In healthy cells, HIF signalling is tightly controlled via the availability of its α-subunit. HIF-α is degraded by the ubiquitin-proteasome system (UPS) in well-oxygenated cells but the protein is stable in hypoxia, dimerises with HIF-β, binds to RCGTG motives of target genes, and transcriptionally drives their expression. HIF-targets involve among many others, genes that enhance glycolysis and metabolic rewiring, angiogenesis and resistance to apoptosis. HIF-α is also stabilised in a variety of cancer cells of different origin due to genetic alterations independently of the present oxygen level. Accordingly, sustained expression of HIF-α in tumours has been associated with higher aggressiveness, migratory and metastasis-initiating potential and therefore worse prognosis. To better understand which proteins might be responsible for pathological activation of HIF signalling, the family of deubiquitinating enzymes (DUBs) was silenced in an RNAi screen. We identified a new non-canonical positive regulator of HIF-α stability that we further characterised using standard biochemical methods and confocal fluorescence live cell imaging techniques. USP29 belongs to the family of ubiquitin-specific proteases (USPs) and efficiently stabilised HIF-α in an O2/PHD/pVHL-independent and a proteasome-dependent way. Furthermore, USP29 expression correlated with disease progression in prostate cancer, suggesting that the maternally imprinted USP29 gene might act as an oncogene. In the light of DUBs being a class of highly druggable proteins, our results might open up possibilities for new therapeutic approaches. P18r-28 A new non-canonical pathway of Gα protein regulating Alex3 in the mitochondria Ismael Izquierdo Villalba1, Serena Mirra2, Cristiane Benincá1, José Antonio Izquierdo3, Eduardo Soriano2, Anna Aragay Combas1 1 IBMB-CSIC, Barcelona, ES, 2Department of Cell Biology, University of Barcelona, Barcelona, ES, 3CNIC, Madrid, ES A novel localization of heterotrimeric G proteins at the mitochondria and their implications on the physiology of the organelle has been recently reported (Beninca et al., 2014; Zang et al., 2010;). In particular, the Gq subfamily is required to keep the proper balance between mitochondria fusion and fission acting at both outer and inner membrane dynamics (Beninca et al., 2014; Sánchez-Fernández G et al., 2014). Together with Gβ (that binds to Mfn1), Gαq stabilizes elongated mitochondria and cristae structure. Gqis also necessary for the maintenance mitochondrial membrane potential and the activity of the respiratory chain and mitochondrial ATP synthesis. Surprisingly, Gαq is necessary for the supercomplex assembly at the inner membrane. The molecular mechanism of action of heterotrimeric G proteins at the mitochondria is still unknown. A recent MS-proteomic analysis has helped us to decipher the Gq-interactome (“Gq-mitoproteome”). We have utilized mitochondrial enriched fractions from four different cell lines, among them the Gq/11-MEF knockout, the recover Gαq-MEF-knockout, MEFs wild type and NIH3T3 cells, as well as, two different anti-Gq antibodies. The new candidate binding proteins are being analyzed by their capacity to interact to Gαq. Salamanca 2016 Among the candidates we found Armadillo-like-domain containing protein Armcx3, also known as Alex3. This mitochondrial outer-membrane protein is involved in the regulation of mitochondrial aggregation and trafficking, among other processes (López-Doménech et. al., 2012). IP studies showed that the constutitive-active mutant of Gq, GqR183C, interacts specifically with Alex3 as well as the mitochondrial Rho-GTPase Miro1, one of the main regulators of mitochondrial transport in neurons. In summary, our group postulates a new non-canonical mitochondrial-function of heterotrimeric G proteins that involves their translocation to the mitochondria and the interaction with several mitochondrial partners. P18-29 La tetraspanina TSPAN33 controla la activación de los macrófagos por los receptores Toll a través de la modulación de Notch Susana López López, Almudena Ruíz-García, José Javier García-Ramírez, Victoriano Baladrón, Laura López-Sanz, María José Ruíz-Hidalgo, Ángela Ballesteros, Jorge Laborda, Eva María Monsalve, María José M. Díaz-Guerra Facultad de Medicina, UCLM, Albacete, ES Los receptores Notch modulan la activación de los macrófagos por los receptors Toll, sin embargo los diferentes componentes y vías que controlan la señalización de estas proteínas todavía no se conocen en detalle. Nuestro grupo ha estudiado la expresión y la función de la proteína Tspan33, una tetraspanina implicada en la maduración de la proteasa ADAM10, en la activación proinflamatoria de los macrófagos. La expresion de Tspan33 aumenta en los macrófagos tras la activación de diferentes receptores Toll, como TLR4, TLR3 y TLR2, siendo aun mayor con un coestímulo de INFγ. La inducción de Tspan33 mediante TLR/ INFγ depende de la activación de Notch, disminuyendo su expresión en macrófagos deficientes en Notch1 y Notch2 y aumentando tras la sobreexpresión de la forma intracelular activa de Notch1. Tspan33 es la tetraspanina de la familia TspanC8 que más se induce durante la activación de los macrófagos, y su sobrexpresión favorece el procesamiento de ADAM10 y su activación en la membrana. De esta forma, Tspan33 incrementa la señalización de Notch en los macrófagos activados. Además, Tspan33 modula la expresión de genes proinflamatorios inducidos por los receptores Toll mediante el aumento de la actividad transcripcional de NF-kB. Nuestros resultados sugieren que Tspan33 es un nuevo punto de control en el desarrollo de la inflamación que podría ser utilizado como diana farmacológica. P18m-30 Papel de Sam68 en las vías de señalización de insulina y leptina en células de la granulosa Teresa Vilariño García1, Antonio Pérez-Pérez2, Flora Sánchez-Jiménez2, Esther Santamaría-López3, Nicolás Prados-Dodd3, Manuel Fernández-Sánchez3, Víctor Sánchez-Margalet2 1 Fundación Pública Andaluza para la Gestión de la Investigación en Salud de Sevilla (FISEVI), Sevilla, ES, 2Bioquímica Médica y Biología Molecular e Inmunología, Facultad de Medicina, Hospital Universitario Virgen Macarena, Sevilla, ES, 3Instituto Valenciano de Infertilidad de Sevilla (IVI), Sevilla, ES Antecedentes y objetivo: La obesidad es un problema médico, no solo por estar asociada a un aumento del riesgo de padecer Diabetes tipo 2 o enfermedades cardiovasculares, sino porque también se asocia con problemas de reproducción cuando afecta a mujeres en edad fértil; de hecho, se asocia con el Síndrome de ovario poliquístico (PCOS), resistencia a la insulina durante el embarazo y disminución de la fertilidad. Se ha demostrado la expresión de Sam68, una proteína de unión a RNA, Pósters / Posters en ratones hembras y se ha observado que los ratones hembra knockout para dicha proteína son subfértiles con problemas de ovulación. Dado que previamente hemos encontrado que Sam68 interviene en las vías de señalización de los receptores de insulina y leptina, planteamos estudiar la posibilidad de participación de Sam68 en dichas vías de señalización en las células de la granulosa. Además pretendemos estudiar la expresión de Sam68 en respuesta a leptina e insulina in vitro. Material y métodos: La señalización fue estudiada por inmunoprecipitación e immunoblot de proteínas fosforiladas. La expresión de Sam68 fue inhibida mediante la estrategia antisentido. Los niveles de expresión de Sam68 y de receptores de insulina y leptina, fueron cuantificados mediante qPCR e immunoblot. Resultados: Hemos encontrado que Sam68 es fosforilado en tyrosina por estimulación con insulina y leptina en células de la granulosa, reclutando Sam68 para complejos de señalización y disminuyendo así su capacidad de unión a RNA. Por otra parte, tanto insulina como leptina incrementan la expresión de Sam68 en las células de la granulosa. Finalmente se evidencia que se requiere la total expresión de Sam68 para que insulina y leptina activen las vías de señalización PI3K y MAPK en células de la granulosa. Conclusión: Sam68 es reclutado para las vías de señalización activadas por insulina y leptina, su expresión es inducida por ambas hormonas y es necesaria para la completa activación de la transducción de la señal de los receptores de insulina y leptina en células de la granulosa. Sam68 podría ser un nuevo elemento importante en la resistencia a la insulina en le ovario y descenso de la fertilidad encontrado en mujeres obesas. P18r-31 Análisis de la función de NADPH oxidadas en hematopoyesis in vivo mediante ARNi frente a p22phox Rodrigo Prieto Bermejo, Guillermo López Ruano, Alejandro Pérez Fernández, Marta Romo González, Carla Ijurko Valeta, Ángel Hernández Hernández Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca (USAL); Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, ES Las especies reactivas de oxígeno (ROS) son compuestos que se producen de manera natural en las células como consecuencia de su metabolismo aerobio, y debido a su gran reactividad siempre se han considerado como agentes nocivos. En las últimas décadas se ha demostrado que esto no es así, y que las ROS están relacionadas con el control de múltiples procesos celulares (como la señalización celular o la expresión génica) en los que actuarían como segundos mensajeros. Esto es debido en gran parte a su capacidad de oxidación reversible de proteínas señalizadoras y reguladoras, y a la capacidad de la familia NADPH oxidasa de generar ROS de forma específica y en respuesta a diferentes estímulos. Actualmente se conoce que la señalización redox interviene en la diferenciación de múltiples tipos de células (cardíacas, neuronales,…). Nuestro laboratorio también ha demostrado la importancia de la producción de ROS por medio de NADPH oxidasas para un correcto proceso de diferenciación megacariocítica. Para esto utilizamos experimentos de ARNi frente a p22phox, proteína necesaria para la activación de varios miembros de la familia, tanto en líneas celulares como en progenitores hematopoyéticos. En este trabajo, hemos querido profundizar aún más en el papel de estas enzimas en el proceso de diferenciación, de manera que hemos analizado el efecto del silenciamiento de p22phox en la hematopoyesis in vivo mediante trasplantes de medula ósea en ratón. Los resultados nos demuestran que el silenciamiento de p22phox en células Lin- de ratón provoca un descenso en las poblaciones celulares de la rama mieloide, en favor de un aumento de las células linfoides. Además, el número de HSC no se ve alterado, pero sí hay un aumento de células LT-HSC compensado por un descenso en células ST-HSC. 149 Pósters / Posters P18-32 AIRAPL: a novel tumor suppressor that regulates IGF1R proteostasis José María Pérez Freije Departamento de Bioquímica, Universidad de Oviedo, Oviedo, ES Protein homeostasis is essential for cell function and involves complex regulatory mechanisms of protein quality control. AIRAPL (arsenite-inducible regulatory particle-associated protein-like, encoded by ZFAND2B) is a highly conserved endoplasmic reticulum protein whose deficiency had been previously reported to cause shortened lifespan in Caenorhabditis elegans as a consequence of proteostasis impairment. We have recently generated AIRAPL-deficient mice, which do not exhibit any obvious development or fertility alterations. However, we have observed that these mice suffer a fully-penetrant myeloproliferative neoplastic process, characterized by early hypercellularity, progressive myeloid skewing and, at later stages, trilineage dysplasia and myelofibrosis. Proteomic analyses of AIRAPL-deficient mouse cells revealed that AIRAPL exerts its antineoplastic function by interacting with newly synthesized IGF1R isoforms, promoting their ubiquitination and degradation in the proteasome. In agreement with this fact, genetic and pharmacological targeting of IGF1R prevented the hematological disease developed not only by AIRAPL-deficient animals, but also by Jak2V617F mutation carriers, demonstrating the relevance of this signaling pathway in the pathogenesis of myeloproliferative neoplasms (MPN). Moreover, we have found that AIRAPL expression is suppressed in human MPN and we have identified mir-125a-3p as the main factor responsible for AIRAPL suppression in these disorders. Based on these findings, we propose that the identification of this novel pathway involving AIRAPL, IGF1R and miR-125a-3p offers new therapeutic opportunities for the management of myeloproliferative malignancies. P18r-33 Regulación del receptor de insulina por la sirtuína 6 María Gutiérrez-Salmerón1, Ana Chocarro-Calvo1, José Manuel García-Martínez1, Antonio De la Vieja2, Custodia García-Jiménez1 1 Facultad Ciencias de la Salud. Universidad Rey Juan Carlos, Alcorcón, ES, 2Instituto de Salud Carlos III, Majadahonda, ES Introducción: La hiperglucemia favorece el desarrollo tumoral a través de mecanismos sistémicos y directos sobre la célula tumoral, potenciando la acetilación de cromatina y la proliferación y migración tumoral. La sirtuína SIRT6 es nuclear y tiene actividades enzimáticas de desacetilación, mono-ADP-ribosilación y demiristoylación. El silenciamiento de SIRT6 en modelos animales permite concluir que es indispensable para la regulación de la homeostasis de la glucosa y como supresor tumoral. Por ello su estudio despierta interés en las áreas de diabetes y cáncer y las posibles relaciones entre ambos. Objetivo: Evaluar la influencia de la hiperglucemia sobre SIRT6 en células tumorales (HCT 116) y no tumorales humanas (CCD18-Co) de colon y en tejido tumoral y no tumoral humano y analizar las consecuencias sobre la señalización por el receptor de insulina (IR). Metodología: Células humanas estimuladas con glucosa en condiciones control o tras depleción o sobre-expresión de SIRT6. Se evalúan cambios en expresión génica (qPCR), exhibición del IR (citometría de flujo), señalización y niveles de SIRT6 e IR (Western-Blot e inmunofluorescencia). Resultados: La hiperglucemia aumenta los niveles de SIRT6 e IR, pero atenúa la señalización de la vía a través de PI3K/Akt. La sobre-expresión de SIRT6 aumenta los niveles totales de IR y la depleción (siRNA) los disminuye. SIRT6 media la regulación por glucosa IR, ya que el silenciamiento de la sirtuína disminuye tanto los niveles como la funcionalidad del receptor. Conclusiones: SIRT6 podría representar un sensor metabólico que media los cambios inducidos por la hiperglucemia sobre el receptor de insulina, 150 XXXIX Congreso SEBBM posiblemente para mantener la homeostasis de la glucosa. Por ello, SIRT6 podría representar una buena diana antitumoral. P18r-34 Impact of mitochondrial ROS on astrocyte antioxidant defense in vivo Nicolò Bonora, Carlos Vicente Gutiérrez, Juan Pedro Bolaños University of Salamanca, Salamanca, ES A substantial body of evidence suggests that mitochondrial reactive oxygen species (mROS) generation is a hallmark of certain neural diseases. However, the underlying molecular mechanisms involved are not fully understood, and whether they play physiological roles is still controversial. Our group revealed that the astrocytes robustly express a plethora of antioxidant enzymes compared to neurons, as a consequence of the major activity of the Nrf2 pathway, the master transcriptional regulator of the antioxidant response, in glial cells. However, the molecular mechanism responsible for the up-regulation of the antioxidant defenses in astrocytes is unknown. We hypothesized that astrocytic mROS would play an important role in neuronal antioxidant protection through the Nrf2 pathway. Nowadays, the vast majority of studies on ROS-mediated cell signaling have used a strategy based on increasing ROS, either by exogenous or endogenously-supplied ROS. We believe that this approach might mask the beneficial roles of physiological mROS. Hence we plan an alternative approach aimed to down-modulate endogenous mROS. To explore this, we have designed and generated an inducible, floxed knock-in mouse model that expresses a mitochondrial version of catalase (mCat). We believe that the analysis of mCat mice would allow us to probe the astrocyte-specific functions of mROS at regulating the antioxidant defense in vivo. We present results showing the efficacy of mCat at reducing mROS levels in an astrocyte-specific manner. Now we have found data that suggest a role of astrocytic mROS maintaining active the Nrf2 pathway. We think that these results may reveal the functional physiological relevance of mROS in astrocytes at contributing to the brain defense against oxidative stress. P18-35 Papel de la ruta de señalización PI3K/AKT en la infección del virus de la enfermedad de Newcastle Javier Blanco1, Cristina Cameirão1, M. Carmen López-Cuesta2, Isabel Muñoz-Barroso1 1 Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, Edificio Departamental Lab.106, Salamanca, ES, 2Departamento de Microbiología Genética, Universidad de Salamanca, Edificio Departamental Lab.236, Salamanca, ES La ruta de señalización fosfatidilinositol-3-quinasa-Akt (PI3K/Akt) regula varios procesos celulares importantes como la síntesis de proteínas, el crecimiento celular, el metabolismo de la glucosa y la inflamación. Muchos tipos celulares utilizan esta ruta para inhibir la apoptosis celular e incrementar su supervivencia. Existen numerosos ejemplos de virus que han desarrollado mecanismos para modular la ruta de señalización PI3K/ Akt con objeto de incrementar su replicación. El virus de la enfermedad de Newcastle (NDV) es un virus patógeno aviar con RNA de polaridad negativa cuyo ciclo se desarrolla completamente en el citoplasma de la célula infectada. En este trabajo, hemos estudiado el efecto del inhibidor de quinasas LY294002 en la infección del NDV en células de cultivo y el efecto del NDV sobre la ruta de señalización PI3K/Akt. Observamos un aumento del efecto citopático en células pretratadas con el inhibidor e infectadas con NDV. Asimismo, la infectividad del virus disminuyó en presencia el inhibidor. La infección del NDV de células HeLa determinó la Salamanca 2016 Pósters / Posters activación de la ruta de señalización PI3K/Akt, detectándose un incremento los niveles de fosforilación de Akt respecto a Akt de hasta cuatro veces a las 6 y 12 h postinfección. Nuestros datos sugieren la implicación de la ruta de señalización PI3K/Akt en la infección del NDV. player in cellular ROS control. The identification of these novel binding partners for Gαq will help to understand the molecular mechanisms of ROS-dependent GPCR signaling and provide new therapeutic targets in cardiovascular diseases. P18-36 P18-38 Cell cycle regulation of Whi7, a new insight in Start progression Mastl (Greatwall)-PP2A: a new cell cycle pathway with therapeutic implications in cancer Mercè Gomar Alba, Ester Méndez Belinchón, Inma Quilis Bayarri, M. Carmen Bañó Aracil, Juan Carlos Igual García Departament de Bioquímica i Biologia Molecular, ERI Biotecnologia i Biomedicina, Universitat de València, Burjassot, ES Mónica Álvarez-Fernández, Belén Sanz-Castillo, María Sanz-Flores, María Salazar-Roa, Miguel Ángel Quintela-Fandino, Marcos Malumbres Spanish National Cancer Research Centre (CNIO), Madrid, ES Start is a key point of cell cycle control in eukaryotic cells. Executing Start is an irreversible decision that implies accurate molecular mechanisms of control. In S. cerevisae the process begins when the Cln3-Cdc28 cyclin-CDK complex is released from its association to the endoplasmic reticulum (ER) in late G1 and accumulates in the nucleus in order to activate a transcriptional program mediated by the SBF and MBF activators and Whi5 and Stb1 repressors. As a result, Cln1/Cln2-Cdc28 complexes appear and initiate a positive feedback on the transcriptional program that gives coherence to the process. The Whi5 paralog Whi7 is a negative regulator of Start controlling cell cycle entry by retaining Cln3 in the ER in a phosphorylation-dependent manner (Yahya et al., 2012). In this work, we describe a new function of Whi7 in Start regulation. Whi7 is a cell cycle regulated protein whose protein level and phosphorylation state increases from late G1 to G2-M. It is an unstable protein which is degraded by the SCFGrr1 ubiquitin-ligase. Whi7 degradation requires phosphorylation by any Cyclin-CDK complex, remaining the unphosphorylated protein stable only in early G1. More important, Whi7 is found associated to CLN2 and RNR1 promoters, mostly in a SBF dependent manner. The interplay with the main effectors of Start suggests a double repressive function of Whi7 through Cln3 retention in the ER and SBF transcriptional program repression, and confirms the necessity of overlapping security mechanisms to ensure a model of robust control in cell division. Protein phosphorylation is an essential mechanism to control progression throughout the different phases of the cell cycle. During the last years, the Greatwall-PP2A axis has emerged as a new pathway implicated in control of mitosis. Greatwall kinase, also known as Mastl in mammals, specifically inhibits PP2A complexes containing the B55 family of regulatory subunits (PPP2R2A-D). Inhibition of PP2A-B55 complexes by Greatwall occurs in a kinase-dependent manner through the phosphorylation of Ensa and ARPP19, the only two Greatwall substrates identified to date. Depletion of Greatwall results in chromosome segregation and condensation defects, which are partially rescued by concomitant depletion of B55 regulatory subunits. Whereas the relevance of Greatwall in cancer is mostly unknown, the phosphatase PP2A is a well-known tumor suppressor, frequently inactivated in cancer. Since PP2A reactivation has been proposed as a therapeutic strategy for cancer treatment, inhibition of Greatwall may have therapeutic benefits through the reactivation of PP2A-dependent pathways. Interestingly, PPP2R2A has been found to be deleted in breast tumors, suggesting that the Greatwall-PP2A pathway may be relevant for breast tumor progression. Indeed, we have found that Greatwall is overexpressed in breast tumors, and its expression level correlates with poor prognosis, both in hormone-positive as well as triple-negative breast tumors. Moreover, Greatwall depletion results in defective proliferation of specific breast tumor cell lines. By using a Mastl conditional knockout model, we have found that Greatwall deletion also impairs the growth of breast tumors in vivo. Altogether, these data suggest Greatwall as a new therapeutic target for breast cancer. P18-37 Gq-interactome and oxidative stress signaling pathways P18-39 Álvaro Caballero, Federico Mayor Menéndez, Catalina Ribas Núñez Centro de Biologia Molecular Severo Ochoa (CSIC-UAM) and Instituto de Investigación Sanitaria La Princesa, Madrid, ES Caracterización de NLRC5 y su relación con la respuesta inmune antiviral en branquias de salmónidos Activation of G protein-coupled receptors (GPCR) coupled to the Gq/11 family of heterotrimeric G proteins plays a paramount role in cardiovascular physiopathology. In addition to the canonical PLCß effector, additional proteins are being identified to underlie some of the functions of Gq. Recently, we have reported that GPCR activation promotes a direct interaction between Gαq and protein kinase C ζ (PKCζ) through a novel acidic region in Gαq, different from the classical effector-binding region, and the PB1 domain of PKCζ. This Gαq/PKCζ interaction leads to the stimulation of the ERK5 pathway and is required for angiotensin-induced cardiac hypertrophy in mice, and is also able to trigger the induction of apoptotic cell death independently of PLCß. We hypothesize that the interaction of Gαq with additional PB1 domain-containing proteins may participate in the control of cellular functions by Gq-coupled GPCR. GPCR activation can lead to increased production of reactive oxygen species (ROS) in a way depending on specific agonists and cell type. Gq-GPCR are known to promote an excess of ROS during hypertension, ischemia-reperfusion injury and heart failure, thus contributing to cell death and apoptosis. In this work, we have uncovered that Gαq associates with another PB1 domain-containing protein, the Nox2 activator subunit p67PHOX. Moreover, Gαq functionally interacts with Keap1, also a relevant Felipe Ramírez, Claudio Álvarez, Daniel Oyanedel, Paula Santana, Nicole Canto, Byron Morales-Lange, Jimena Cortés, Luis Mercado Vianco Pontificia Universidad Católica de Valparaíso, Valparaíso, CL La creciente investigación acerca de receptores de reconocimiento de patrón (PRR), activados por patrones moleculares asociados a patógenos (PAMP), no solo ha concitado el interés en vertebrados superiores, sino que también en invertebrados y vertebrados inferiores. De hecho en estos grupos animales han venido a explicar la fortaleza de la inmunidad en sistemas sin una inmunidad adaptativa robusta como sucede en aves y mamíferos. Entre los PRR, en peces se ha identificado un número mayor de receptores tipo Toll (TLR) en comparación a mamíferos, y para la mayoría de ellos no se ha establecido con claridad cuál es su ligando. Con respecto a los PRR solubles o citosólicos, la información es mucho menos abundante para peces. Con la idea de conocer la capacidad de respuesta inmune no relacionada con el tejido linfático asociado a branquias (GiALT), iniciamos la caracterización de PRR citosólicos en epitelio branquial, identificando su potencial ligando, así como también la respuesta inmune que es capaz de desarrollar. Cuatro nuevas secuencias de receptores tipo NOD (NLR) fueron 151 Pósters / Posters caracterizadas en trucha arcoíris, y se pudo establecer la sobreexpresión de NLRC5 en respuesta a Poly I:C, en la línea celular de epitelio branquial RT-Gill de O. mykiss. Inicialmente además se pudo demostrar que Poly I:C induce la sobreexpresión de TNF-alpha e IFN de tipo I en este mismo tipo celular. Mediante la técnica de siRNA, se inhibió la transcripción de NLRC5 en células RT-Gill, lo que provocó que en presencia de Poly I:C disminuyó la sobreexpresión de TNF-alpha e IFN tipo I. Paralelamente y mediante IF pudimos establecer que Poly I:C induce el incremento de NLRC5 e IFN tipo I en RT-Gill, a nivel de proteína. Para confirmar estos resultados in vitro, un grupo de peces salmónidos fueron desafiados con el virus ISA, en éstos se detectó una sobreexpresión de NLRC5, seguida de un incremento de IFN de tipo I. Estos resultados confirman lo observado in vitro y constituyen las primeras evidencias de respuesta inmune antiviral vía NLR en branquias de salmónidos. P18-40 Dual role of the pseudokinaseTRIB3 in the development of breast cancer Alba Orea-Soufi1, María del Mar Lorente1, Sonia Castillo1, María Salazar-Roa1, Elena García-Taboada1, José Miguel Lizcano2, Endre Kiss-Toth3, Guillermo Velasco1 1 Complutense University; Department of Biochemistry and Molecular Biology. School of Biology, Madrid, ES, 2Institut de Neurociències and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, ES, 3 Department of Cardiovascular Science, University of Sheffield, Medical School, Beech Hill Road, Sheffield, UK Tribbles pseudokinase 3 (TRIB3) participates in the regulation of multiple signaling pathways that are involved in the regulation of cell survival, Emerging evidences obtained during the last few years suggest that TRIB3 is a crucial modulator of tumorigenesis. Recent observations by our research group have shown that TRIB3 plays a tumor suppressor role in several models of cancer through a mechanism that relies on the regulation of the MTORC2/AKT axis Although there is limited and often conflicting literature concerning to the role of TRIB3 in breast cancer, the existing data suggest that this pseudokinase could be deregulated during the development of this pathology. In this work, we have investigated the effect of the genetic inactivation of TRIB3 in the malignant properties of several human breast cancer cell lines. Our results show that TRIB3 genetic inhibition increases proliferation and invasiveness of BT474 (ER+, PR+, HER2+) whereas it decreases both parameters in MDA-MB-231 (ER-, PR-, HER2-), and MCF7 (ER+, PR+, HER2-) cells. Moreover, Trib3 silencing accelerated the growth of tumor xenografts generated with BT474 and produced the opposite effect in tumour xenografts generated from MDA-MB-231 and cells. Our data also show that Trib3 genetic inhibition enhances Akt and FOXO phosphorylation in BT474 and decreases the phosphorylation of these proteins in MDA-MB-231 cells. suggesting that the ability of Trib3 to modulate the AKT/FOXO pathway determines its tumor suppressor or oncogenic role in breast cancer cells. Ongoing work is currently investigating the role of postranslational modifications and its influence on the suncellular location of TRIB3 on the dual role of this pseudokinase in breast cancer. P18-41 El plasma de pacientes con tumor pulmonar activa la expresión de VCAM e ICAM-1 en la superficie de células endoteliales en cultivo Alejandra Romero, Andrea Veiga Barrientos, Ana Dione Ibáñez, Isabel Vaquero, Juan-Carlos Murciano, Fátima Esquivel Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Madrid, ES 152 XXXIX Congreso SEBBM El cáncer es una patología muy asociada con múltiples procesos que benefician el desarrollo de la enfermedad y la aparición de otras patologías de riesgo elevado. Los procesos inflamatorios pueden asociarse a metástasis, mediante la cual pueden aumentar aquellos receptores o moléculas involucradas en la traslocación celular. El paciente con tumor pulmonar posee un cáncer de muy mal pronóstico y evolución pronunciada, hecho que se ve potenciado por su virulencia, pero también, por su diagnosis en periodos del cáncer con estadiaje avanzado. Nuestro grupo viene realizando una colección de muestras sanguíneas de pacientes diagnosticados de un tumor en sus primeros periodos tras el diagnóstico. Estos pacientes aun no han comenzado un tratamiento antitumoral activo. Junto con las muestras, la base de datos recoge datos analíticos, epidemiológicos y de laboratorio. Los datos generales representativos de los pacientes muestran una edad próxima a los 70 años y un porcentaje de pacientes masculinos cercanos al 70 %, datos muy similares en cuanto a la distribución y diagnóstico de este tipo de tumores independientemente de su localización geográfica. Es interesante resaltar que cuando exponemos células endoteliales de cordón umbilical en cultivo a la presencia del plasma de estos pacientes, se produce un aumento de la expresión de VCAM e ICAM-1 en su superficie, obteniendo valores superiores a aquellas células expuestas únicamente al plasma de controles sanos. Esta expresión se encuentra más elevada tras 10 horas de exposición, siguiendo cinéticas de expresión de dichas moléculas muy similares a aquellas obtenidas con inductores proinflamatorios como el LPS. Estos resultados parecen incluso asociarse al tipo de histología del tumor, viéndose más incrementada la expresión de estas moléculas en los adenocarcinomas, que en los epidermoides o en los microcíticos. Este estudio trata de establecer la relación del citado incremento de moléculas proinflamatorias que podrían ir asociadas a un mayor riesgo metastático del tumor, o cuando menos de peor pronóstico. Agradecimientos: Este estudio ha sido realizado con cargo al proyecto PI11/0668 donde J-C.M es el IP. Este proyecto recibe financiación del Instituto de Salud Carlos III (Plan Estatal de I+D+i 2013-2016) y ha sido cofinanciado por el Fondo Europeo de Desarrollo Regional «Una manera de hacer Europa», FEDER. La Dra. FE recibe su salario a cargo de este proyecto. P19. Transgénesis en mamíferos / Transgenesis in mammals P19-1 Implicación de Igf1r en la respuesta inflamatoria pulmonar aguda a la bleomicina y en la hiperreactividad de las vías respiratorias tras inducción de asma con extracto de ácaros del polvo doméstico Sergio Piñeiro Hermida1, Icíar P. López1, Raquel Torrens1, Joshua A. Gregory2, Mikael Adner2, José G. Pichel1 1 Centro de Investigación Biomédica de La Rioja (CIBIR), Fundación Rioja Salud, Logroño, ES, 2Karolinska Institute, Institute of Environmental Medicine (IMM), Unit of Experimental Asthma and Allergy Research, Estocolmo, SE IGF1R (Insulin-like Growth Factor type 1 Receptor) es un receptor transmembrana de expresión ubicua con funciones biológicas esenciales como la supervivencia, proliferación, diferenciación y adhesión celular. En el pulmón, participa tanto en el desarrollo embrionario como en el mantenimiento de la homeostasis en el adulto. También se ha relacionado con el estrés oxidativo asociado al proceso inflamatorio y con el remodelado de Salamanca 2016 las vías respiratorias. Con el objeto de determinar su función en la respuesta al daño pulmonar, los ratones mutantesUBC-CreERT2; Igf1rfl/fl con deleción de Igf1r inducida posnatalmente, se sometieron a dos tipos de challenges respiratorios. i) Tras la administración intratraqueal de bleomicina, los mutantes presentaron mejor tasa de supervivencia que los controles. A los tres días sus pulmones mostraron menos congestión pulmonar y contenido de células inflamatorias, así como un descenso de marcadores inflamatorios (TNF, Il1b, Il6, Ly6g, Cxcl1 y Adgre1), de adhesión (Icam, Pecam1) e hipoxia (Hif1a), y un incremento del marcador de antiestrés oxidativo Gpx8. ii) Después de la administración intranasal de extracto de ácaros durante un mes, los mutantes revelaron mejor función pulmonar y menos eosinófilos en lavados bronquioalveolares. Sus pulmones estaban menos inflamados y contenían menos células mucosecretoras, colágeno, eosinófilos y niveles reducidos de marcadores de producción de moco (Spdef y Muc5ac). La menor hiperreactividad bronquiolar en el mutante se reflejó en la reducción del grosor del músculo liso y en la alteración morfológica del epitelio bronquiolar distal. Estos resultados demostraron que Igf1r está implicado en la respuesta inflamatoria y en la hiperreactivad respiratoria tras el daño pulmonar tanto agudo como sostenido. P19-2 The role of fibronectin synergic site in cutaneous wound healing Irene Gimeno Lluch, María Benito Jardón, Mercedes Costell Universidad de Valencia, Almacera, ES In humans, there are several pathological conditions where wound healing is delayed, such as in diabetes, or excessive healing as occurs in the hypertrophic and keloid scars. Fibronectin (FN) is thought to be one of the most important extracellular matrix proteins involved in cutaneous wound repair. FN is an extracellular matrix glycoprotein formed by two identical subunits joined at the C-terminus by two disulphide bonds. Its structure includes three different types of repeating units named type I, type II and type III. FN binds several heterodimeric transmembrane receptors called integrins. The main integrin binding site is located at the FN type III10 module. In this region, an Arg-Gly-Asp (RGD) motif joins α5β1, αIIbβ3, α8β1 and the αv family of integrins. Adjacent to the RGD motif there is a Pro-His-Ser-Arg-Asn (PHSRN) sequence which has been described to enhance FN adhesion to α5β1and αIIbβ3 integrins and so-called synergic site (FNsyn). The in vitro role of FNsyn has been intensively studied and is thought to have a crucial role in situations where the FN-integrin bond is tensioned. We studied wound healing capacity in mice carrying point mutations in the FN gene that impair the synergy site function (FNsyn/syn) and compared with wild type littermates (FN+/+). Our results show a delay in the gap closure in the FNsyn/syn mice at early times. During re-epithelialization, the keratinocytes, confined in the epidermis, start to migrate from both sides of the wound through a FN-fibrin provisional matrix. We analysed in vitro the keratinocyte migration on substrates of either purified wild type FN or FN-syn. We observed that keratinocytes migrated at lower speeds on substrates of FN-syn. These results confirm the mechanistic role of synergic site in the integrin-FN engagement. This is the first time that the synergic site is studied in vivo and we will discuss its implication during cutaneous wound healing. P19r-3 Destrucción in vitro del oncogen BCR-ABL p210 mediante nucleasas de edición genómica CRISPR/Cas9 Ignacio García-Tuñón1, Lucia Méndez-Sanchez2, Luis Hernández-Cano2, María Hernández-Sánchez1, Miguel Quijada1, José Luis Ordóñez1, Verónica Alonso-Pérez1, Rocio Benito1, Carmen Guerrero3, Jesús María Hernández-Rivas1, Manuel Sánchez-Martín2 1 Centro de Investigación del Cáncer (CIC), IBMCC, Instituto de Investigación Biomédica de Salamanca (IBSAL), Servicio de Hematología, Hospital Universitario, Salamanca, ES, 2Servicio Pósters / Posters de Transgénesis, Nucleus. Universidad de Salamanca, Salamanca, ES, 3Departamento de Medicina. Facultad de Medicina. Universidad de Salamanca, Salamanca, ES La caracterización molecular de las alteraciones génicas implicadas en la transformación celular así como el desarrollo de nuevas herramientas de edición genómica cambiarán radicalmente el panorama terapéutico de algunas neoplasias en los próximos años, especialmente el de aquellas asociadas a mutaciones “drivers” bien caracterizadas, como sucede en la leucemia mieloide crónica (LMC). Esta enfermedad se caracteriza por la presencia de la translocación t(9;22)(q34;q11) que genera el oncogén de fusión BCR/ABLp210 y que produce una proteína con actividad tirosinquinasa (TK) constitutiva. Esta actividad TK es crucial y se considera indispensable en el mecanismo de transformación celular al que conduce. El diseño de fármacos inhibidores de TK específicas ha constituido uno de los mayores logros terapéuticos en la medicina actual. Sin embargo, su administración es continua y prolongada en el tiempo, la acción terapéutica se limita a la anulación del efecto a nivel proteico y en ningún caso hay reparación o anulación del daño a nivel genético, lo que en ocasiones conlleva a la aparición de resistencias. Afortunadamente, la aparición de las nuevas herramientas de edición genómica, tipo CRSIPR-Cas9, solventaría todos estos inconvenientes se podrían corregir/eliminar mutaciones a nivel genómico lo que abre un nuevo panorama terapéutico. Este estudio explora el uso de esta tecnología CRSIPR-Cas9 en un modelo celular tumoral específico del oncogén p210 y demuestra su eficacia y utilidad como posible nueva herramienta terapéutica para la LMC. Financiación: HUS272413, PI15/01471 y Junta de Castilla y León. P19-4 Shortage of dNTPs underlies replication defects and genome instability in the absence of the APC/C cofactor Cdh1 Javier Garzón Hidalgo, Sergio Moreno Pérez, Irene García-Higuera Instituto de Biología Funcional y Genómica (IBFG), CSIC / Universidad de Salamanca. Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, ES The anaphase promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that promotes proteasomal degradation of mitotic cyclins and other critical cell-cycle regulators. Its activity is limited to the cell cycle period comprised between anaphase and the end of the G1 phase, and is controlled by the regulated binding of two co-factors: Cdc20 or Cdh1. APC/C-Cdc20 is active during anaphase, while APC/C-Cdh1 is active from anaphase until the end of G1.Over the last few years, our group has been addressing the biological function of the APC/C-Cdh1 complex using mouse models of Cdh1 deficiency. Cells derived from mutant embryos (MEFs) accumulate DNA breaks and show increased levels of RPA foci and phospho-Chk1, suggestive of replication stress. Direct analysis of the DNA replication process on stretched DNA fibers (DNA fiber assays) revealed reduced rates of replication fork progression and increased frequency of origin firing in the absence of Cdh1. Interestingly, addition of exogenous dNTPs or nucleosides, normalized replication dynamics in mutant cells, and prevented DNA breakage. Consistently, quantification of intracellular dNTP pools confirmed decreased levels of all four dNTPs in Cdh1-deficient MEFs. Moreover, overexpression of RRM2, the regulatory subunit of Ribonucleotide Reductase (the rate-limiting enzyme for dNTP synthesis), restored efficient replication and genome stability in cells lacking Cdh1 expression. These results highlight the importance of APC/C activity during the G1 phase to prevent replication defects, and provide evidence that, in the absence of Cdh1, insufficient supply of dNTPs to the replisome leads to replication stress, which, in turn, causes chromosomal breaks. 153 Pósters / Posters P20. Transportadores de membrana / Membrane transporters P20-1 Deciphering the role of the exomer along the evolution Carlos Antón Plágaro, Irene Arcones Ríos, Rosario Valle Vicente, César Roncero Maíllo Universidad de Salamanca, Salamanca, ES In order to reach the plasma membrane (PM) receptors, channels and enzymes use the secretory pathway. Along this route, the main sorting station is the Trans-Golgi-Network (TGN). Here, proteins can be sent to multiple destinations: vacuole/lysosomes, PM or earlier Golgi compartments. To address these purposes, cells use the lipid language, the cytoskeleton, several small GTPases, adaptors and coats, although the precise mechanisms involved in the process are not still fully understood. One of the best studied platform for cargo sorting at the TGN is the yeast exomer, originally described in S. cerevisiae as an effector of Arf1 necessary for the transport of the chitin synthase Chs3 to the PM. It is formed by two molecules of Chs5 bound to a dimer of any of the four ChAPs: Chs6, Bud7, Bch1 and Bch2. The ChAP proteins will act not only as adaptors for the three cargoes described: Chs3, Fus1 and Pin2, but they will play additional roles in exomer function. Here we will show that exomer is involved in the intracellular transport of several other proteins not previously identified as cargoes. These include several PM transporters as Ena1, Gap1 or Tat2, whose transport is only impaired in the absence of exomer. Moreover, we will show that the absolute requirement of exomer for the bona fide cargos seems linked to their interaction to AP-1 complex. This interaction has been probably acquired later in the fungal evolution, being a rather unique characteristic of the Saccharomyces genus. Our results thus suggest that exomer has a general and evolutionary conserved function in protein sorting at the TGN which has later evolved to more specialized function in cargo selection in some yeast. P20-2 Bases moleculares de la quimiorresistencia en el cáncer gástrico: papel del transportoma Ruba Al-Abdulla1, Rocío I.R. Macías1, Elisa Lozano1, Oscar Briz1, Marta Alonso1, Beatriz Castaño1, Francisco González San-Martín1, Jesús M. Bañales2, Luis Bujanda2, José J. G. Marín1 1 Hepatología Experimental y Vectorización de Fármacos (HEVEFARM), IBSAL, Universidad de Salamanca. Centro de Investigación Biomédica En Red para el Estudio de Enfermedades Hepáticas y Digestivas (CIBERehd), Salamanca, ES, 2Biodonostia, Hospital Universitario de San Sebastián, Universidad del País Vasco, Ikerbasque; Centro de Investigación Biomédica En Red para el Estudio de Enfermedades Hepáticas y Digestivas (CIBERehd), San Sebastian, ES XXXIX Congreso SEBBM de confirmación, se determinó el nivel de expresión de proteínas capaces de transportar 5-FU o cisplatino. Tanto en tumores como en tejido no tumoral la expresión de transportadores de la familia SLC22 fue baja, mientras que las de la familia SLC28 y del transportador SLC31A1 fue elevada. Resultados: Las bombas de la superfamilia ABC con mayor expresión en ACG fueron MRP5>MRP3>BCRP. No se observaron diferencias según la localización y estadio del tumor. El análisis inmunohistoquímico confirmó una elevada expresión de MRP5 en ACG, mientras que la de BCRP fue muy variable. En células AGS derivadas de ACG humano, la inhibición de las bombas MRP con probenecid incrementó la sensibilidad de las células al efecto citotóxico del 5-FU. Conclusión: El transportoma, y en particular algunas bombas exportadoras de la familia MRP, está implicado en la quimiorresistencia del ACG, lo que permitiría identificar potenciales dianas moleculares para el desarrollo de nuevas estrategias sensibilizantes a la quimioterapia del ACG. P20r-3 Structural determinants of glycine neurotransporter 2 (GlyT2) selective inhibitor ALX1393 Cristina Benito-Muñoz1, Almudena Perona2, Enrique Núñez1, Carmen Aragón3, Beatriz López Corcuera3 1 Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, ES, 2 Smartligs, Parque Científico de Madrid, Campus de Cantoblanco, Madrid, ES, 3Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid; IdiPAZ-Hospital Universitario La Paz, Universidad Autónoma de Madrid, Madrid, ES Glycine transporters are plasma membrane proteins that remove the neurotransmitter glycine from the synaptic cleft promoting the termination of the glycinergic signal (GlyT1) and the pre-synaptic terminal reloading (GlyT2). GlyT1 and GlyT2 have been associated to the pathogenesis of nervous system disorders, including schizophrenia and related affective and cognitive disturbances, alcohol dependence, pain, epilepsy, breathing disorders and hyperekplexia or startle disease. GlyTs belong to the solute carrier 6 (SLC6) family of sodium and chloride-dependent transporters, and their three dimensional structure has been recently modeled by homology with the crystal structure of the Drosophila melanogaster dopamine transporter. Since the study of the molecular determinants of the binding of selective GlyT inhibitors may have therapeutic consequences, in this report our aim is to localize the binding site of the GlyT2-specific inhibitor ALX1393. By using computational and biochemical docking on GlyT2 models subjected to dynamic simulation, we have identified the key residues involved in the binding of the inhibitor. The effect of the N-glycosylation of the transporter has also been evaluated P20-4 Antecedentes: El adenocarcinoma gástrico (ACG) es un tipo de tumor muy resistente a la quimioterapia, generalmente basada en la combinación de 5-fluorouracilo (5-FU) y cisplatino, lo que justifica que el tratamiento farmacológico fracase en >95% de los pacientes con ACG no operable. Objetivo: Investigar el papel de las proteínas transportadoras que median la capacidad de las células tumorales de captar fármacos antitumorales o el aumento de su expulsión como mecanismos que contribuyen a la falta de respuesta del ACG a la quimioterapia. Métodos: Se obtuvieron muestras quirúrgicas pareadas (n=24x2) de tejido tumoral y adyacente no tumoral de pacientes con ACG en estadios II, III y IV, que no habían recibido previamente quimioterapia antitumoral. Mediante Taqman Low Density Arrays (TLDA) y RT-QPCR convencional 154 GLUT12: functional mechanism and pathophysiological implications Eva Gil-Iturbe, Jonai Pujol-Giménez, Carla Valriveras, María Pilar Lostao Universidad de Navarra, Pamplona, ES GLUT12 belongs to the class III of the facilitative glucose transporter family SLC2A. It was cloned from the mammary cancer cell line MCF-7, and its expression was found in human crude membranes of adipose tissue, small intestine and skeletal muscle, where it responds to insulin. GLUT12 was also found in mammary gland and prostate Salamanca 2016 tumors, where it may act as a key in the energy supply to malignant cells. Studies from our group, expressing GLUT12 in Xenopus laevis oocytes, have characterized some of its functional properties: GLUT12 is able to transport a wide variety of sugars, including α-methyl-glucose (αMG), a characteristic substrate of the SGLT family. Sugar transport is increased in the presence of extracellular Na+, is voltage dependent and up-regulated by PKA. Strikingly, GLUT12 is electrogenic and the glucose-induced currents are driven by a chloride conductance, which is uncoupled to the substrate transport. In small intestine, GLUT12 is located in the apical cytoplasm, below the brush border membrane of rat and human enterocytes; in the perinuclear region of human enterocytes and in rat basolateral cytoplasm. Interestingly, GLUT12 is expressed in brush border membrane vesicles of Caco-2 cells where it is upregulated by its substrates. The pro-inflammatory cytokine TNFα increases αMG uptake by GLU12 through the translocation of the transporter to the apical membrane. GLUT12 protein is also expressed in retroperitoneal fat from mouse and rat and 3T3-L1 cells. Immunohistochemistry assays localize GLUT12 around the nuclei of the adipocytes, nerves and arterial myocytes. Uptake of αMG by GLUT12 in 3T3-L1 cells is increased by insulin. Finally, GLUT12 is overexpressed in the frontal cortex of Alzheimer disease patients and in several distinct cancer cell lines, supporting GLUT12 role in the glycolytic metabolism of cancer cells. Overall, the functional features found for GLUT12 are very different from those of the other GLUT isoforms, indicating that it must have a specific physiological role within glucose homeostasis, still to be discovered. P20r-5 Tráfico intracelular y regulación de variantes del transportador neuronal de glicina GlyT2 asociadas a hiperplexia humana Andrés de la Rocha Muñoz1, Enrique Núñez1, Carmen Aragón2, Beatriz López-Corcuera2 1 Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, ES, 2 Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid; IdiPAZ-Hospital Universitario La Paz, Universidad Autónoma de Madrid, Madrid, ES La hiperplexia o enfermedad de sobresalto es un síndrome clínico raro caracterizado por reacciones motoras exageradas frente a estímulos triviales auditivos o táctiles. Se origina por disfunción de la neurotransmisión glicinérgica inhibidora, pudiendo producir la muerte del neonato por problemas cardiorrespiratorios. El transportador neuronal de glicina GlyT2 aporta neurotransmisor a la presinapsis desde la hendidura sináptica para el rellenado de vesículas y su posterior reutilización. Por ello, algunas mutaciones en este gen humano (SLC6A5) producen una forma presináptica de la enfermedad. Se han asociado a hiperplexia unas 30 mutaciones en GlyT2. La mayoría son autosómicas recesivas y causan la inactividad del transportador o su ausencia de la membrana. En este trabajo se estudian las propiedades de tráfico intracelular y regulación de cuatro mutantes puntuales de GlyT2 presentes en pacientes de hiperplexia humana que son parcialmente activos. Mediante ensayos de acumulación de glicina tritiada y biotinilación de superficie de los transportadores expresados en cultivos celulares (COS7, MDCK II, neuronas primarias), hemos demostrado que las variantes difieren en la expresión en membrana y en la velocidad máxima del transporte, lo que sugiere una alteración de su tráfico intracelular. Utilizando técnicas inmunoquímicas se estudiarán los puntos de retención intracelular y su capacidad de ubiquitinación, así como las alteraciones en la unión a proteínas del interactoma de GlyT2. Pósters / Posters P20-6 Participation of brain aquaporins in adult hydrocephalus associated to hypoxia and aging Miriam Echevarría, José Luis Trillo-Contreras, Reposo Ramírez-Lorca, Ismael Sánchez-Gomar, Ana Galán-Cobo, Nela Suárez-Luna, Magdalena Olivares Blanco, María Oliver, Juan José Toledo-Aral, Javier Villadiego Instituto de Biomedicina de Sevilla (IBiS), Campus Hospital Universitario Virgen del Rocío, Sevilla, ES Aquaporins (AQPs) are integral membrane proteins that allow transport of water across cell membranes. The demonstration that AQP1 and AQP4, expressed in choroid plexus (CP) epithelial cells (AQP1) and ependymal cells bordering the intraventricular compartments (AQP4) play an important role in the cerebrospinal fluid (CSF) production, make us to think that both proteins may have an important role on the onset of adult hydrocephalus. The description by our group that AQP1 expression is up-regulated in the CP of animals exposed to low oxygen tension (10% O2 for 48h), led us to propose that overexpression of brain AQPs during hypoxia may yield to an increment in CSF production an thus may participate in the origin of adult hydrocephalus. We explored in mice exposed to hypoxia for 2-5 days whether expression levels of AQP1 and AQP4 in different regions of the brain were affected. Cortex, striatum, ependyma and CP, were analyzed separately both for mRNA and protein levels under control conditions and after hypoxia. Magnetic resonance images (MRI) and intracranial pressure were evaluated in both conditions using young and aged animals. Moreover, in patients with hydrocephalus of different origins, we evaluated by ELISA assays the amount of AQP1 and AQP4 shed to the CSF to establish a correlation between amount of AQPs and hydrocephalus condition in humans. A general increaseof mRNA and protein for brain AQPs was observed with the hypoxic treatment, evaluated by RT-qPCR, western blot and immunocytochemistry assays. In animals exposed to hypoxia ventricular dilatation was observed by MRI being clearly larger the ventricle volume in aged animals. AQPs levels in human CSF also varied upon the hydrocephalus condition. Our results indicate a likely correlation between AQPs and hydrocephalus in humans and animals. As a working hypothesis both conditions, hypoxia and aging, acting together could be crucial to explain the developing of the hydrocephalus state in adult patients. However, more experiments are needed to strength this association and further explore the role of AQPs on adult hydrocephalus. P20-7 An assay to quantify, at the cellular scale, the membrane binding profile of protein assemblies involved in vesicle transport Irene Pazos Capell, Ana García, Carla Belmonte, Nereida Jiménez, Oriol Gallego IRB Barcelona, Barcelona, ES Vesicle trafficking is fundamental to distribute correctly lipids and proteins among cellular compartments. A complex network of interconnected pathways transports vesicles between organelles according to the cellular demand. However, the study of vesicle trafficking is often limited to local routes between two organelles and ignores its contribution at the global cellular scale. PICT (Protein interactions from Imaging of Complexes after Translocation) is a live-cell imaging technique to study the interaction between two proteins. Here we have used an assay based on PICT to quantify the membrane binding profile of protein assemblies in yeast. We have further developed the method to increase the sensitivity two orders of magnitude and to quantify the association between two proteins bound to the same membrane. MTCs (Multisubunit Tethering Complexes) are a group of 9 protein assemblies conserved from yeast to human that work as long distance connectors between the vesicle and the acceptor organelle, providing specificity and directionality to vesicle trafficking. We used a set 155 Pósters / Posters of 19 proteins that localize in specific cellular compartments as markers to quantify the binding of MTCs to each of these membrane compartments of the cell. We then quantified the effect on MTCs membrane binding profile at the cellular scale upon perturbing locally the trafficking in the trans-Golgi– endosomes transport. Overall, these results shed light on the physiological role of MTCs and the dependency among distant vesicle transport routes. This assay could find applications in the study of other protein assemblies and to understand the interplay between different trafficking pathways. P20r-8 Papel del transportoma en la sensibilidad al sorafenib en líneas celulares de leucemia mieloide aguda Anabel Sánchez-Martín1, Óscar Briz2, Gabriela Rodríguez-Macías3, Luis I. Sánchez-Abarca4, María D. Odero5, José J.G. Marín2, Rocío I.R. Macías2 1 IBSAL, Salamanca, ES, 2Laboratorio de Hepatología Experimental y Vectorización de Fármacos, Universidad de Salamanca, IBSAL. CIBERehd , Salamanca, ES, 3Servicio de Hematología, Hospital Gregorio Marañón, Madrid, ES, 4Servicio de Hematología, Hospital Universitario de Salamanca, IBSAL, Salamanca, ES, 5Departamento de Bioquímica y Genética, Universidad de Navarra, Pamplona, ES Introducción: Las mutaciones en el receptor FLT3 justifican la aparición de resistencia al sorafenib en pacientes con leucemia mieloide aguda (LMA), aunque otros mecanismos aún desconocidos contribuyen a la falta de respuesta. El objetivo fue investigar si los niveles de expresión de las proteínas implicadas en la captación y el eflujo de sorafenib se relacionan con la sensibilidad de líneas celulares al fármaco. Métodos: El efecto sobre la viabilidad celular se determinó mediante el test de formazán y con el kit anexina V/7-AAD tras 72 h de exposición a concentraciones crecientes de sorafenib. La expresión de los genes de interés se determinó por RT-PCR y la funcionalidad de las proteínas por citometría de flujo. Resultados: La sensibilidad de las líneas celulares al sorafenib fue MOLM-13>>HL-60>HEL≈K-562. Los niveles del transportador OCT1 fueron mayores en células MOLM-13 que en HL-60 y no se detectaron en HEL y K-562. En cuanto a las bombas de eflujo, la MDR1 estaba sobre-expresada en células HEL>K-562≈MOLM-13 y no se detectó en HL-60. La expresión de BCRP fue moderada en células HEL>K-562>HL-60>MOLM-13, los niveles de MRP3 fueron bajos en las células MOLM-13 y K-562 y no se detectaron en HL-60 y HEL y todas las líneas presentaron una elevada expresión de MRP4. Las células MOLM-13, además de la mutación FLT3-ITD presentaron niveles 1000 veces superiores de FLT3 al resto de líneas. Los resultados de funcionalidad de los transportadores confirmaron la relación entre la mayor capacidad de las células de retener sorafenib y su sensibilidad a este fármaco. Conclusión: Junto con los niveles de las dianas del fármaco y la presencia de mutaciones, la expresión de proteínas de captación y eflujo de sorafenib contribuye a la sensibilidad de las células de LMA a este fármaco. P21. Grupo invitado: Biología química / Invited group: chemical biology P21-1 Generation of ubiquitinated p15 protein for structural studies Amaia González Magaña1, Nekane Merino1, Alfredo De Biasio2, Francisco J. Blanco3 1 CIC bioGUNE, Derio, ES, 2Elettra Sincrotrone, Trieste, IT, 3CIC bioGUNE; IKERBASQUE Basque Foundation for Science, Bilbao, ES 156 XXXIX Congreso SEBBM p15PAF (proliferating-cell-nuclear-antigen-associated factor, hereafter p15) is an intrinsically disordered protein of 111 amino acids with increased expression in tumors(1). It binds PCNA through its conserved PCNA-interacting protein motif, the PIP-box, located in the central region. It also binds DNA, mainly through its N-terminal region. The binding to PCNA and DNA occurs simultaneously and independently with similar affinity (Kd = 1.1 μM at 25 ºC). This mode of binding suggests that p15 acts as a flexible regulator of the velocity of PCNA sliding on the DNA(2). Recent studies have shown that p15 facilitates the switch from replicative to translesion synthesis polymerases at stalled replication forks. This role is related with the existence in the cell of a fraction of PCNA-bound p15 ubiquitinated at Lys 15 and Lys 24 during normal S-phase progression(3). Monoubiquitination at these two Lys residues should not affect PCNA binding but would reduce the affinity of p15 for the DNA as it removes two positive charges and introduces steric hindrance. To confirm this hypothesis we are preparing milligram amounts of ubiquitinated p15. We are following a non-enzymatic strategy that results in a disulfide directed ligation mimicking the isopeptide bond between the p15 Lys side chain and the carboxyl group at ubiquitin C-terminus(4). We have produced a p15 mutant with Lys 15 and 24 mutated to Cys and two native Cys residues mutated to Ser. An ubiquitin with a C-terminal reactive thiol group is prepared from a fusion protein with an intein that is cleaved with cysteamine. The ubiquitinated p15 protein has been generated and purified with high homogeneity but low yields. Structural analysis by circular dichroism and multiangle light scattering indicate that the p15 chain remains disordered and monomeric while the bound ubiquitin is cooperatively folded. P21r-2 Alteraciones en la estructura y función de la Conexina 43 en melanoma Adrián Varela-Vázquez1, Paula Carpintero-Fernández1, María L. Díaz2, María C. Vázquez-Blanco1, Ángel Fernández-Flores3, Carmen Rivas2, Eduardo Fonseca1, María D. Mayán1 1 Grupo de Investigación CellCOM. Instituto de Investigación Biomédica de A Coruña (INIBIC). Complexo Hospitalario Universitario de A Coruña (CHUAC-XXIAC). Universidade da Coruña. Sergas, ES, 2Grupo Virus and Cancer. CIMUS, USC, Santiago de Compostela, ES, 3Servicio de Anatomía Patológica. Hospital del Bierzo, Ponferrada, ES Las conexinas (Cx) son proteínas transmembrana que forman canales que permiten la comunicación directa célula-célula a través de uniones comunicantes (UC) y célula-matriz a través de hemicanales. En las distintas capas de la epidermis se han detectado diferentes Cxs, siendo la Cx43 la más ampliamente expresada. La Cx43 es esencial para la función de barrera de la piel, y ha sido descrita como factor condicional supresor de tumores presentando diferente actividad durante el desarrollo del tumor y el proceso de metástasis. Resultados de co-IP, inmunofluorescencia y western-blot (líneas celulares y tejidos de pacientes) evidenciaron que la Cx43 en melanoma se encuentra sumoilada, junto con disminución en los niveles de la proteína y alteraciones en la localización subcelular. Ensayos de scrape loading demostraron una pérdida total de comunicación celular a través de UC. La restauración de los niveles de la Cx43 en líneas celulares de melanoma rescató la comunicación célula-célula reduciendo los niveles de migración y proliferación celular. Los resultados obtenidos sugieren que alteraciones en la estructura y niveles de la Cx43 asociados con cambios en la localización celular de la proteína podrían impedir la comunicación directa célula-célula a través de UC, alterar proliferación y migración celular y por tanto estar jugando un papel importante en el desarrollo o malignidad del melanoma. Salamanca 2016 P21r-3 The invasion of glioblastoma stem cells is reduced by the sequence of the connexin43 that interacts with c-Src Myriam Jaraíz Rodríguez1, Mª Dolores Tabernero2, María González-Tablas3, Álvaro Otero4, Alberto Orfao3, José M. Medina1, Arantxa Tabernero1 1 Instituto de Neurociencias de Castilla y León, Universidad de Salamanca, Salamanca, ES, 2Instituto de Estudios de Ciencias de la Salud de Castilla y León (IECSCYL); Research Laboratory of the University Hospital of Salamanca, Instituto de Investigación Biomedicina de Salamanca (IBSAL); Centre for Cancer Research (CIC-IBMCC; CSIC/USAL; IBSAL), Salamanca, ES, 3Centre for Cancer Research (CIC-IBMCC; CSIC/USAL; IBSAL); Department of Medicine, Universidad de Salamanca, Salamanca, ES, 4 Neurosurgery Service of the University Hospital of Salamanca and IBSAL, Salamanca, ES Glioblastoma stem cells (GSC) constitute a niche of cells with self-renewal ability and resistance to conventional therapies. Thus, they are responsible for relapse in these aggressive brain tumors when their resection is not complete. The expression of connexin43 (Cx43), the main gap junction channel-forming protein, and c-Src, a non receptor tyrosine kinase, is inversely related in glioblastoma cells. Interestingly, restoring Cx43 in GSC reverses GSC phenotype. The sequence responsible for this effect is the region that interacts with c-Src (amino acids 266-283). The Focal Adhesion Kinase (FAK) requires the c-Src-mediated phosphorylation in tyrosines 576 and 577 in order to be fully active and promote cell migration. In this study, fresh human tumour biopsies were disaggregated to obtain human primary GSCs. These cells were treated with a cell-penetrating peptide containing the Cx43-Src interacting sequence (Tat-Cx43266-283). The migration was analyzed by tracking individual cell trajectories in cultures recorded by Time-Lapse Live-cell Imaging. Matrigel-treated transwell inserts were used to analyze the invasion and the levels of phosphorylation of FAK tyrosines 576 and 577 were evaluated by western Blot. Our results showed that Tat-Cx43266-283 reduced the migration and invasion of human primary GSC. The analysis of c-Src and FAK proteins suggests that the mechanism by which this reduction takes place includes the inhibition of c-Src/FAK axis. In conclusion, Tat-Cx43266-283 inhibits c-Src with the subsequent reduction in FAK phosphorylation required to establish appropriate focal adhesions for migration. All together, these results indicate an important antivasive role of the sequence of Cx43 that interacts with c-Src in glioma stem cells. P21-4 Whole body imaging and therapeutic targeting of aggressive cancers: focus on premetastatic niches Maria Soengas, David Olmeda Melanoma Laboratory, Molecular Oncology Programme, Spanish National Cancer research Centre (CNIO), Madrid, ES Cutaneous melanoma is the deadliest form of skin cancer. Although the presence of tumor cells in lymph nodes is a poor prognosis indicator, the specific contribution of lymphangiogenesis to the mortality associated with melanoma metastasis remains unclear. This is mainly due to the lack of tumor markers and experimental models for non-invasive imaging of lymphangiogenesis in vivo. To address molecular mechanism(s) regulating metastatic spread of melanoma, we developed a series of genetically engineered mouse strains derived from a “lymphoreporter model” in which a luciferase cassette is expressed as a reflection of the endogenous Flt4 (VEGFR3) promoter. These “lymphoreporter” melanoma models allowed tracing of metastatic dissemination from very early stages of melanoma development. In particular, we have identified distinct patterns Pósters / Posters of melanoma-driven neo-lymphangiogenesis activated at distal sites before the actual colonization by tumor cells. Proteomic analysis of the melanoma secretome revealed new pro-lymphangiogenic factors that promote tumor metastasis. Here we will present the prognostic value of these metastatic genes and discuss the use of the lymphoreporter mice for the screening of anti-metastatic agents. Together, these results support the VEGFR3-luc mouse melanoma models as a cost-effective strategy for gene discovery in the context of otherwise aggressive and intrinsically metastatic tumors. P21-5 IFN-γ receptor signaling is critically dependent on its location in lipid nanodomains Ornella Morana1, Cedric Blouin2, Dalila Ciceri1, Maier Lorizate1, Christophe Lamaze2, Xabier Contreras3 1 Instituto Biofisika (CSIC-UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, ES, 2Institut Curie - Centre de Recherche, Membrane Dynamics and Mechanics of Intracellular Signaling Laboratory. Centre National de la Recherche Scientifique (CNRS), UMR 3666, París, FR, 3Instituto Biofisika (CSIC-UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco. IKERBASQUE, Basque Foundation for Science, Leioa, ES Interferon-γ (IFN-γ) is a cytokine that orchestrates many critical cell functions and signaling processes through transcriptional activation and regulation of a vast number of genes. Among others, INF-γ plays crucial roles in controlling host defense, immunopathological processes, and fighting tumors. IFNγ mediates its pleiotropic effects on cells binding to a receptor (IFNGR) expressed on the membrane surface of a large variety of cells. However, understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge mainly due to a lack of accurate methodology. Here, we show that IFNGR localizes, in vivo, in lipid nanodomains and that IFN-γ addition induces a conformational change in IFNGR that leads to a specific IFNGR-sphingolipid interaction that could play a role as a docking site for receptor phosphorylation and JAK-STAT signaling cascade activation. This experiment of nature established the critical importance of dynamic interactions with lipid nanodomains in IFN-γR signaling. P21-6 NMR, molecular recognition and chemical glycobiology Jesús Jiménez-Barbero CIC BIOGUNE, Derio, ES Molecular recognition by specific targets is at the heart of the life processes. In recent years, it has been shown that the interactions between proteins (lectins, enzymes, antibodies) and carbohydrates mediate a broad range of biological activities, from fertilization, embryogenesis, and tissue maturation, to pathological processes. The elucidation of the mechanisms that govern how glycans are accommodated in the binding sites of these receptors is currently a topic of interest. Thus, the determination of the structural and conformational factors and the physicochemical features that govern the molecular recognition of these molecules is of paramount importance. This presentation is focused on the application of NMR methods both from the ligand and receptor’s perspective (especially HSQC-based chemical shift perturbation analysis and 19F-based Saturation Transfer Difference) to the study of molecular recognition processes between a variety of polypeptides of biomedical interest and carbohydrate-based molecules, drugs and inhibitors. The use of novel lanthanide-binding tags[1, 2] and 19F-containing glycomimetics [3] as probes for monitoring interactions from the ligand’s point of view will be highlighted. Different glycan receptors, both wild type and mutants, 157 Pósters / Posters have been used as receptors with the final aim to know and to evaluate the relative importance of polar (hydrogen bonding, electrostatic interactions) and non polar (van der Waals, CH-π) forces in the molecular recognition processes and to design novel molecules with improved binding properties. As examples, the structural and conformational details of the effect of branching in the glycan chain and how this affects its recognition by lectins, enzymes, and antibodies will be shown. References [1] Á. Canales, Á. Mallagaray, M.Á. Berbís, A. Navarro-Vázquez, G. Domínguez, F.J. Cañada, S. André, H.J. Gabius, J. Pérez-Castells, J. Jiménez-Barbero, J Am Chem Soc. 136, 8011-7 (2014). [2] A. Canales, A. Mallagaray, J. Pérez-Castells, I. Boos, C. Unverzagt, S. André, H.J. Gabius, F.J. Cañada, J. Jiménez-Barbero, Angew Chem Int Ed Engl. 52, 13789-93 (2013). [3] L.P. Calle, B. Echeverria, A. Franconetti, S. Serna, M.C. Fernández-Alonso, T. Diercks, F.J. Cañada, A. Ardá, N.C. Reichardt, J. Jiménez-Barbero, Chem Eur J. 21, 11408-16 (2015). P21-7 Toll-like receptors modulation: computational studies and drug repositioning Sonsoles Martín-Santamaría, Lucía Pérez-Regidor, Joan Guzmán-Caldentey, Jean-Marc Billod, Laura Ortega, Malik Zarioh, Alessandra Lacetera Centro de Investigaciones Biológicas, CIB-CSIC, Madrid, ES Innate immunity is the first defensive wall a pathogen needs to beat to flourish in our body and, due to the enormous diversity of microorganisms present in our environment, it is composed by a number of agents able to respond in a highly effective way. Toll-like receptors (TLR) family can recognize a wide variety of pathogens, making them an interesting target to help our body fight the disease. Each TLR is specialized in the recognition of a particular pathogen-associated molecular pattern arisen from a bacteria or virus. TLR modulators have the potential to be used with different biomedical applications, especially in the field of infection, inflammation and autoimmune diseases, but also in central nervous system disorders such as Alzheimer´s disease, and cancer. However, just a few of them are currently under clinical development. Therefore, it is imperative to find new chemical entities as TLR modulators with drug-like properties in order to facilitate their development as drugs. Our group has directed its efforts towards the finding of novel TLRs modulators (in particular, TLR1/TLR2, TLR2/TLR6, and TLR4/MD-2 systems) by virtual screening and drug repositioning approaches, leading to the identification of novel TLR4 antagonists with advantageous drug-like properties. We have also applied different computational and molecular modeling tools to the study of the mechanism of the TLR4/MD-2 system and the molecular recognition by different ligands: we have studied the dynamics of the MD-2 binding pocket and its ability to accommodate lipid A,(1) and we have also provided a molecular model for activation of the human TLR4/MD-2 complex by penta-acylated lipid A from B. cenocepacia LPS.(2) Our computational approaches have also assisted to the rational design of glycolipid-based TLR4 modulators and fluorescent probes.(3) Finally, we also here report the computational study of the dynamics of the full TLR4/MD-2 system (extracellular + intracellular + transmembrane domains) inserted in the membrane environment by MD simulations aiming to understand the dimerization mechanism at atomic detail. References [1] J. Vasl; A. Oblak; T. T. Peternelj; J. Klett; S. Martín-Santamaría; T. L. Gioannini; J. P. Weiss; J. Jerala. J. Immunol. 2016, 196, 2309-18. [2] F. Di Lorenzo; L. Kubik; A. Oblak; N. I. Lore; C. Cigana; R. Lanzetta; M. Parrilli; M. A. Hamad; A. De Soyza; A. Silipo; R. Jerala; A. Bragonzi; M. A. Valvano; S. Martin-Santamaria; A. Molinaro. J. Biol. Chem. 2015, 290, 21305-19. 158 XXXIX Congreso SEBBM [3] C. Ciaramelli; V. Calabrese; S. E. Sestito; L. Pérez-Regidor; J. Klett; A. Oblak; R. Jerala; S. Martín-Santamaría; F. Peri. Chem. Biol. & Drug Des., 2016, doi: 10.1111/cbdd.12749. P21-8 Synthetic probes and tools for biological intervention José Luis Mascareñas Cid Universidade de Santiago de Compostela, Santiago de Compostela, ES The ability to synthesize molecules is at the heart of the progress in many branches of science. Therefore there is a continuum need to devise practical and efficient synthetic methods that allow to construct relevant products from readily available starting materials in a sustainable and reliable manner. In this context, one of our research lines is focused to the development of new and efficient synthetic processes.(1) As molecular makers we are also highly interested in the design and synthesis of molecules that can lead to new type of biomolecular recognition processes, or promote specific biological and cellular responses. An important part of our work has been focused to devise molecular agents and strategies for the interaction with specific DNAs. Some of these derivatives take advantage of the coordination capability of late transition metals to attain their recognition properties. We have also developed variations that not only bind the designed targets but are also capable of emitting a signal upon that interaction, therefore working as selective biosensors.(2) References [1] F. López, J.L. Mascareñas Chem. Soc. Rev. 2014, 2904. M. Gulías, J. L. Mascareñas Angew. Chem. Int. Ed. 2016, 10.1002/anie.201511567R1. [2] M. I. Sánchez, J. Martínez-Costas, F. González, F., M. A. Bermúdez, M.E. Vázquez, J. L Mascareñas ACS Chem. Biol. 2012, 7, 1276. C. Penas, E. Pazos, J. L. Mascareñas, M. E. Vázquez J. Am. Chem. Soc. 2013, 135, 3812. M. I. Sánchez, C. Penas, M. E. Vázquez, J. L. Mascareñas Chem. Sci. 2014, 5, 1901. J. Rodríguez, J. Mosquera, J. R. Couceiro, M. Eugenio Vázquez, J. L. Mascareñas Chem. Sci. 2015, 6, 4767. P21-9 Protein-targeted dynamic combinatorial chemistry in drug discovery Ruth Pérez-Fernández1, L. Martínez-González1, M.J. Sánchez-Barrena2, A. Martínez1 1 Centro de Investigaciones Biológicas, CIB-CSIC, Madrid, ES, 2 Instituto de Química Física Rocasolano, IQFRS-CSIC, Madrid, ES The development of synthetic molecules that mimics the precision of nature in molecular recognition represents a huge challenge to chemists. Dynamic combinatorial chemistry (DCC) holds enormous potential for drug discovery speeding the identification and optimization of novel ligands for biological targets. In DCC, building blocks react with one another using reversible chemical reactions giving mixtures of oligomers (Dynamic Combinatorial Libraries, DCLs). If a protein is added to the system and one or more molecules show affinity to it, these building blocks will, according to the Le Chatelier’s principle, be amplified on the expense of the other non-bonding constituents. We present the discovery of novel ligands of Frequenine-2 (Frq2), a high-affinity Ca2+-binding protein conserved from yeast to humans (named Neuronal Calcium Sensor-1). Frq2 is involved in pathologies that result from an abnormal synapse number such as Fragile X syndrome (FXS). Fragile X syndrome is the most common inherited cause of intellectual disability and a common known single gene cause of autism spectrum disorders. Salamanca 2016 P21r-10 pH-labile PGA-Doxorubicin based combination conjugates with enhanced antitumor activity in breast cancer models Juan José Arroyo Crespo, David Charbonnier, Ana Armiñán, María Jesús Vicent Docón Centro de Investigación Príncipe Felipe, Valencia, ES Introduction: It has been already demonstrated that coupling of small drugs in a synergistic ratio to the same polymer ensures the arrival of both drugs to the same cell at the same time, yielding to an improved therapeutic index, and allowing to control the drug release kinetics profile and reduce side effects1,2. We have previously reported a novel PGA-AGM-DOX conjugate family, bearing the combination of Aminoglutethimide (AGM) with Doxorubicin (Dox), in which both drugs were attached to the polymer through different protease-cleavable linkers, such as G, GG or GFLG, being PGA-GAGM10-DOX5 the conjugate showing the greatest antitumor activity in an orthotopic 4T1 breast cancer model3.We now propose the use pH-labile hydrazone linkers as a strategy to delivery DOX in a new family of PGA-AGM-DOX conjugates. Pósters / Posters Results and discussion: PGA-GAGM10-hyd-DOX5 and PGA-GAGM10-EMCH-DOX5 families have been developed bearing pH-sensitive hydrazone linkers. Looking for P.Q. descriptors, a deep characterization of the conjugates has been carried out. Conjugates have a concentration-dependent behavior, showing the capacity to self-assemble in aggregates up to 200 nm in aqueous solution, with differences in either their critical aggregation concentration and/or size, depending on the linker and on the presence of one or both drugs. CD analysis revealed a clear trend for some of the conjugates to self-assemble as an alpha helix with greater rigidity, data in agreement with the release kinetics observed. Cell viability in a representative model is coherent with such release profiles. Conjugates showing the greatest in vitro activity were chosen for further in vivo evaluation in an orthotopic and metastatic 4T1 murine breast tumor model to analyze, not only the antitumor activity, but also their capacity to reduce metastasis progression. References [1] Duncan R. J Control Release. 2014, 190:371-80. [2] Greco, F, Vicent M.J, Adv. Drug. Deliv. Rev. 2009, 61(13):1203-13. [3] C. Deladriere, J.J. Arroyo, E. Masiá, V.J. Nebot, A. Armiñán, M.J. Vicent. Submitted. PW1. Enseñanza de la bioquímica / Teaching biochemistry PW1-1 PW1-2 El origen de la vida en la Tierra y fuera de ella: una perspectiva bioquímica Implementación del modelo flipped classroom y videotutoriales como herramientas para la mejora del autoaprendizaje del alumno en el grado de Bioquímica y estudios de posgrado relacionados Juan Antonio Aguilera Mochón Dpto. Bioquímica y Biología Molecular I, Universidad de Granada, Granada, ES Como dijo Günter Wächtershäuser, “Una teoría de la evolución temprana debe tener como propósito principal el problema de explicar la Bioquímica”. Lo mismo cabe decir, por supuesto, respecto a las teorías sobre el origen de la vida. No cabe, pues, eludir los aspectos bioquímicos en estas materias, ni siquiera cuando se trate de divulgarlas. Con estas ideas de principio, el autor ha intentado trasladar, en un lenguaje lo más asequible posible, su experiencia de más de una década de enseñanza de “Bioquímica evolutiva” en un libro divulgativo acerca de “El origen de la vida en la Tierra” (RBA, 2016). ¿Cuándo ocurrió, y en qué escenario?, ¿cómo se pudo lograr la complejidad que alcanzó el último pasado común universal (LUCA)?, ¿cómo se gestó el código genético y se desarrolló el metabolismo?, ¿cómo aparecieron los eucariotas?… Carecemos de respuestas firmes, pero las hipótesis — avaladas en lo posible por datos experimentales y filogenéticos— son fascinantes. Este análisis sobre la biogénesis en nuestro planeta es imprescindible de cara a especular con alguna base sobre las posibilidades de vida en otros. ¿Qué condiciones se necesitan para el desarrollo de una bioquímica similar a la nuestra en otros mundos? Y, además, ¿son posibles otras bioquímicas, basadas en la química del carbono o no? Todas estas preguntas las ha abordado el autor en otro libro de divulgación científica, “La vida no terrestre” (RBA, 2016), en el que intenta acercarse con rigor a unos temas fácilmente propensos a la pseudociencia. Los dos textos, aunque parten de una base sencilla, podrían (sobre todo merced a recuadros más técnicos) servir de recurso al profesorado de Bioquímica para ofrecer a los alumnos un enfoque evolutivo, imprescindible (recordemos a Dobzhansky) en cualquier explicación biológica. Joan Ribot, Paula Oliver, Juana Sánchez Laboratorio de Biología Molecular, Nutrición y Biotecnología (Nutrigenómica y Obesidad), Universidad de las Islas Baleares (UIB) y CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Palma de Mallorca, ES Hemos incorporado el uso del modelo de flipped classroom y videotutoriales tratando de potenciar el interés del estudiante en la resolución y ejecución de actividades propuestas y favorecer un ambiente de aprendizaje colaborativo e interactivo en estudios de grado y posgrado. En la asignatura “Bioinfomática de Sistemas Aplicada”, los alumnos debían analizar los resultados de un microarray (actividad obligatoria), para lo que disponían de bibliografía y de vídeo tutoriales preparados por el profesor. En la asignatura “Bioquímica Analítica y Clínica”, los alumnos (actividad grupal y optativa) debían preparar un vídeo-presentación sobre “nuevos biomarcadores” de una enfermedad. Finalmente, en la asignatura de posgrado “Nutrición Molecular”, los alumnos (actividad grupal y obligatoria) debían preparar un proyecto de investigación y defenderlo delante de sus compañeros, que los evaluaron. Se realizó una encuesta de satisfacción a los alumnos de grado y se valoró el impacto de las actividades propuestas sobre sus notas. Participaron 19 alumnos (actividad obligatoria) y 7 de 45 (optativa). Contestaron la encuesta un 75% de los alumnos participantes. La aceptación de los videotutoriales por parte de los alumnos fue excelente. Los alumnos consideraron las actividades de dificultad media invirtiendo las mismas o menos horas que en otras asignaturas con carga lectiva similar. Las actividades resultaron muy interesantes, motivadoras e útiles para los alumnos que no dudarían en repetir (9 de cada 10), siendo 159 Pósters / Posters el grado de satisfacción global muy alto (8,5 sobre 10). La realización de estas actividades les supuso un incremento significativo en la nota final. Destaca que las evaluaciones de los propios alumnos a sus compañeros fueron muy homogéneas y más exigentes que las del profesor. Estos resultados reflejan la buena aceptación entre los alumnos y nos anima a continuar con el uso del modelo de flipped classroom y videotutoriales como herramientas para la mejora del autoaprendizaje en los estudios de Bioquímica. Financiado por Institut de Recerca i Innovació Educativa de la UIB. XXXIX Congreso SEBBM docentes, investigadoras y de gestión de los profesores universitarios como requisito previo para la contratación de éstos en alguna de las figuras establecidas en la mencionada Ley. En particular, establece que la contratación de Profesores Ayudantes Doctores (PAD) y Profesores Contratados Doctores (PCD) se celebrará entre doctores que hayan obtenido la evaluación positiva por parte de la ANECA o del órgano de evaluación externo que la Ley de la Comunidad Autónoma determine. La ponencia pretende aportar información práctica sobre la estructura de los programas, los criterios de evaluación establecidos en las correspondientes resoluciones de la Dirección General de Universidades para los campos de las Ciencias Experimentales y de las Ciencias de la Salud y el funcionamiento de los Comités de Evaluación. PW1-3 Uso de YouTube como recurso didáctico en el aprendizaje de la Bioquímica Inmaculada Ballesteros-Yáñez1, Beatriz Rodríguez-Martín2, Carlos Alberto Castillo Sarmiento2 1 Facultad de Medicina, Universidad de Castilla-La Mancha, Ciudad Real, ES, 2Facultad de Terapia Ocupacional, Logopedia y Enfermería, Universidad de Castilla-La Mancha, Talavera de la Reina, ES Introducción: Desde la implantación del Espacio Europeo de Educación Superior, la formación integral del estudiante se basa en la adquisición de competencias, actitudes y habilidades desde el inicio de sus estudios de Grado y no solo en conocimientos teóricos. Objetivo del profesor: Fomentar la adquisición de competencias potenciando el uso de herramientas gratuitas de publicación en red, que permitan a nuestros alumnos un contacto más directo con la sociedad, en este caso la red social YouTube. Objetivo de los alumnos: Realizar un vídeo divulgativo de la resolución de un caso práctico con los medios de grabación y edición que consideraran convenientes. Metodología: Como trabajo grupal se propuso resolver un caso práctico a elegir entre un listado de 33 problemas complejos que incluían 1 o varios temas de la asignatura. Tras la entrega del caso, se debía grabar y editar en formato audiovisual un material que explicara de forma sencilla el caso resuelto. La única restricción era que el vídeo sería público (soportado en YouTube) y no debía durar más de 10 minutos. Resultados y conclusiones: Se presentan fragmentos de vídeos realizados por los alumnos que demuestran que, además de competencias específicas de la asignatura, se trabajaron de forma satisfactoria competencias básicas (ej: aplicar conocimientos a su vocación), transversales (ej: dominio de las TIC) y generales (ej: demostrar dotes de innovación), algunas de las cuales no estaban contempladas en la guía de la asignatura. Estos trabajos ponen de relevancia el valor a explotar de este tipo de herramientas no solo como potenciadores del aprendizaje sino también como medios de información para orientar y cubrir las necesidades de la comunidad en la que los alumnos desarrollarán sus trabajos. PW1-4 Orientaciones para superar la evaluación de Profesor Ayudante Doctor y Profesor Contratado Doctor Begoña Ochoa Departamento de Fisiología, Facultad de Medicina y Enfermería, Universidad del País Vasco UPV/EHU, Leioa, ES La Ley Orgánica 4/2007, de 12 de abril, por la que se modifica la Ley Orgánica 6/2001, de 21 de Diciembre, de Universidades, atribuye a la Agencia Nacional de Evaluación de la Calidad y Acreditación (ANECA) funciones relacionadas con la evaluación de las actividades 160 PW1-5 El contrato de aprendizaje en la enseñanza de la Bioquímica. ¿Un elemento motivador para el alumnado universitario de Ciencias? Ángel Luis García-Ponce1, Ángel Blanco-López1, Ana Mª Rodríguez Quesada2, Francisco J. Alonso Carrión2, María Fernanda Suárez2, Miguel Ángel Medina Torres2 1 Universidad de Málaga, Andalucía Tech, Departamento de Didáctica de la Matemática, de las Ciencias Sociales y de las Ciencias Experimentales, Facultad de Ciencias de la Educación, Málaga, ES, 2Universidad de Málaga, Andalucía Tech, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Málaga, ES La Bioquímica es una materia que, organizada en sus múltiples asignaturas, se imparte a lo largo del currículo de algunos grados de Ciencias (Biología, Química, Bioquímica). Es habitual que la primera aproximación a esta disciplina que el alumnado afronta sea cursando una asignatura de Fundamentos de Bioquímica o Bioquímica General, que a su vez puede estar organizada semestralmente en forma de dos asignaturas independientes y sucesivas, Bioquímica I y II. En bastantes casos, los contenidos de esta disciplina, y en concreto los correspondientes a Bioquímica II, habitualmente dedicada al estudio del metabolismo, su regulación e integración, es percibida por los estudiantes como una disciplina muy exigente y de notable dificultad. Ello hace que, ya casi desde el inicio de curso, muchos estudiantes le muestren un cierto desapego inicial que se traduce en un retardo en afrontar su estudio, llegando incluso al abandono temporal de la misma. En los últimos años, es conocido que, en algunas de nuestras universidades españolas, las tasas de éxito y rendimiento para esta asignatura son sensiblemente menores comparadas con las de otras cursadas en los mismos cursos. La propuesta pedagógica que se presenta se ha desarrollado en el marco de un Proyecto de Innovación Educativa de la Universidad de Málaga (PIE15-163) que se centra en el empleo de una estrategia pedagógica, el contrato de aprendizaje. Se considera que –sin ser extremadamente novedosa– esta estrategia puede constituir un elemento motivador para ese sector del alumnado que encuentra una especial dificultad en el aprendizaje y asimilación de los contenidos de los fundamentos de la Bioquímica en general, y del metabolismo y su regulación en particular. PW1-6 Medida de glucosa en refrescos: una útil herramienta para ilustrar el aprendizaje de los fundamentos del análisis enzimático Beatriz Martínez-Poveda1, Ángel Luis García-Ponce2, Ángel Blanco-López2, Miguel Ángel Medina Torres1, Ana Mª Rodríguez Quesada1 Salamanca 2016 1 Universidad de Málaga, Andalucía Tech, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Málaga, ES, 2Universidad de Málaga, Andalucía Tech, Departamento de Didáctica de la Matemática, de las Ciencias Sociales y de las Ciencias Experimentales, Facultad de Ciencias de la Educación, Málaga, ES Es indudable la relevancia que el análisis enzimático tiene en el ámbito de la Bioquímica, la Química Clínica y la Química de la Alimentación, entre otras. En la asignatura “Bioquímica Aplicada” de cuarto curso del grado en Química de nuestra Universidad, los alumnos aprenden los fundamentos teóricos del análisis enzimático, que luego ponen en práctica en el laboratorio, en un contexto cotidiano, mediante la determinación de glucosa en diferentes refrescos carbonatados. Aunque existen diversos métodos enzimáticos para la valoración de la concentración de glucosa, hemos elegido para la sesión práctica el que se basa en las reacciones acopladas de la glucosa oxidasa (EC 1.1.3.4.) y la peroxidasa (EC 1.11.1.7.) por ser especialmente ilustrativo a la hora de afianzar los conceptos teóricos desarrollados en clase, así como para despertar su espíritu crítico al enfrentarles a la resolución de problemas “del mundo real” y hacerles elegir qué método analítico puede ser mejor para un caso concreto. Los alumnos usan dos protocolos distintos (métodos cinético y a punto final) para determinar la concentración de glucosa, inicialmente en una muestra de glucosa en agua, familiarizándose con ambos métodos y permitiéndoles construir las rectas de calibrado, mediante las que deberán establecer el rango de aplicación para cada método. La medida de las variaciones de absorbancia con el tiempo, que en el método cinético son proporcionales a la concentración de glucosa, permite ilustrar aspectos metodológicos de la estimación de la velocidad inicial de una reacción enzimática. Posteriormente los alumnos se enfrentan al problema de la medida de la concentración de glucosa en una serie de bebidas que incluyen refrescos de cola y tónicas, con o sin azúcar (refrescos “cero”), encontrándose con nuevas dificultades como son la necesidad de diluir en varios órdenes de magnitud las muestras (lo que les familiariza con el uso de las diluciones seriadas), y la aparición de interferencias debidas al color de la muestra, decantándose por uno u otro método en función de la naturaleza del problema analítico planteado. PW1-7 Memoria de 20 años del curso “¿Y tú? Yo, Bioquímica?” Josep M. Fernández Novell1, Joan J. Guinovart2 1 Departamento de Bioquímica y Biomedicina Molecular, Universitat de Barcelona, Barcelona, ES, 2Departamento de Bioquímica y Biomedicina Molecular, Universitat de Barcelona; Institut de Recerca Biomèdica, Parc Científic de Barcelona, Barcelona, ES El curso Iniciación a la Bioquímica y Biología Molecular para Alumnos de Secundaria, conocido con el lema “¿Y tú? Yo, Bioquímica”, cumple este 2016 su 20 aniversario. Al hacer un balance de estos veinte cursos el resultado numérico es muy alentador así, se debe remarcar que de las más de 8000 solicitudes presentadas se han preseleccionado y entrevistado a más de 2000 de los cuales, finalmente, se han escogido 480 excelentes alumnos, 24 por curso. Se han realizado más de 140 conferencias con la participación del profesorado universitario involucrado en dicha área. Estas charlas, con títulos muy sugerentes para los jóvenes participantes, les han mostrado los últimos avances de la Bioquímica. Además, las sesiones de laboratorio han estado preparadas con alto rigor científico por parte de nuestro alumnado predoctoral y pregraduado, donde les han mostrado algunas de las técnicas que actualmente utilizan los investigadores. Cada curso termina con el acto de clausura y entrega de certificados que otorga la UB. El balance “afectivo” nos dice que Pósters / Posters ha sido un trabajo coral, sin la ayuda de tantas personas e instituciones esta aventura habría fracasado. Pero, debemos reconocer que uno de los pilares han sido y son los centros de secundaria, en especial su profesorado y es bueno señalar que se ha producido un aumento constante a lo largo de estos 20 años de la buena relación entre la educación universitaria y la secundaria. Hemos sido capaces de convertir en realidad el título del artículo “Bridging the gap in biochemistry between secondary school and university”. PW1-8 La coevaluación como recurso formativo. Aplicación y resultados en la enseñanza de la cinética enzimática Néstor V. Torres, Guido Santos Systems Biology and Mathematical Modelling Group. Departamento de Bioquímica, Microbiología, Biología Celular y Genética. Universidad de La Laguna, San Cristóbal de La Laguna, ES La coevaluación o evaluación entre pares es reconocida como una estrategia eficaz para fomentar el papel activo del estudiante en el proceso de aprendizaje. El alumnado tiene la oportunidad de revisar el trabajo de sus compañeros/as de clase frente a su propia evaluación y este ejercicio tiene reflejo en su propio proceso de aprendizaje, provocando la reorientación de sus propias estrategias de aprendizaje y el reforzamiento y la mejor comprensión de las materias. En esta comunicación se muestran los resultados de un ejercicio de coevaluación llevado a cabo con un grupo de 90 estudiantes del Grado en Biología, en la asignatura de Bioquímica, en el tema cinética enzimática. Una vez realizada la actividad práctica cada estudiante redactó un informe de la misma de acuerdo en un formato previamente determinado. Cada informe (anónimo) fue evaluado individual y anónimamente por uno, dos o tres compañeros/as de curso y por el profesorado. Los resultados de las evaluaciones muestran un nivel alto de convergencia entre las evaluaciones del alumnado y de estas con las del profesorado. Se muestra la percepción del alumnado, se señalan los problemas detectados y las propuestas de mejora. Agradecimientos: Este trabajo ha sido realizado gracias a la financiación del Proyecto del MINECO (ref. BIO2014-54411-C2-2-R). PW1-9 Utilizando Jmol para explicar la bioquímica y biología molecular del cáncer Jordi Sastre-Serra, Jordi Oliver, Margalida Torrens-Mas, Pilar Roca Grupo Multidisciplinar de Oncología Traslacional, Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBERObn, CB06/03), ISCIII, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, ES La Bioquímica y Biología Molecular del Cáncer (3 ECTS) es una asignatura optativa del grado de Bioquímica en la Universitat de les Illes Balears. La asignatura tiene el objetivo de introducir al alumno en las bases moleculares de esta patología, explicando las causas, así como las consecuencias de las alteraciones de las proteínas claves implicadas en el desarrollo del proceso carcinogénico. Se presta especial interés a las alteraciones del control del ciclo celular (con el papel de la proteína del retinoblastoma y su control por las proteínas quinasas dependientes de ciclina), las vías de activación de las MAPK quinasas, el papel central del p53 en el control de la apoptosis, los mecanismos de la apoptosis, tratamientos del cáncer. 161 Pósters / Posters Jmol nos proporciona una herramienta que permite acercarnos a nivel molecular a los cambios que sufren proteínas implicadas en el desarrollo de esta patología. Así hemos desarrollado una serie de guiones de diferentes proteínas implicadas en el proceso carcinogénico para ayudar a los alumnos a comprender los fenómenos moleculares y los efectos de las mutaciones en las proteínas que intervienen en el control del ciclo celular, apoptosis y vías de señalización de las MAPK quinasas: Proteína Retinoblastoma, Proteínas quinasas dependientes XXXIX Congreso SEBBM de ciclinas, p53, Caspasas (estructura, activación y mecanismo enzimático), Scr, Ras y la familia de las proteínas Bcl-2. (Más información en: http://gmot.uib.es/moleculas/molec_cancer.html.) Jmol y sus guiones realizados se han demostrado como una herramienta de gran ayuda para que los alumnos puedan adentrarse en los procesos moleculares implicados en el cáncer, donde pueden observar los cambios que se producen al interaccionar varias proteínas, o los cambios ocasionados por la presencia de mutaciones en proteínas como Ras, p53. PW2. Workshop: Investigador emergente / Workshop emergent investigator PW2-1 Bringing light to receptors with LOV Álvaro Inglés Prieto, Harald Janovjak Institute of Science and Technology Austria, Viena, AT Receptor tyrosine kinases (RTKs) are a large family of membrane receptors that sense growth factors and hormones and regulate a variety of cell behaviors in health and disease. We engineered RTKs that can be selectively activated by low-intensity blue light. We screened light-oxygen-voltage (LOV)-sensing domains for their ability to activate RTKs by light-activated dimerization. Incorporation of LOV domains resulted in robust activation of relevant RTKs and the induction of cellular signaling in human cancer and endothelial cells with high spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behavior induced by growth factors. Finally, we applied these light-activated RTKs in an optogenetics-assisted drug screen. This platform obviates the addition of chemical activators and reporters, which reduces the number of operational steps in a cell-based drug screen against human protein kinases including an orphan RTK. PW2-2 Ligand engaged TCR is triggered by Lck not associated with CD8 coreceptor Javier Casas Requena1, Joanna Brzostek2, Veronika I. Zarnitsyna3, Jin-sung Hong4, Quianru Wei2, John A.H. Hoerter5, Guo Fu5, Jeanette Ampudia5, Rose Zamoyska6, Cheng Zhu4, Nicholas R.J. Gascoigne2 1 Instituto de Biología y Genética Molecular (IBGM), CSIC-Universidad de Valladolid, Valladolid, ES, 2Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore, SG, 3Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, US, 4George W Woodruff School of Mechanical Engineering, Georgia Institute of Technology and Emory University, Atlanta, US, 5Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, US, 6 Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK Recognition of antigen by the T cell receptor (TCR) is the key event for the T cell activation. The earliest molecular events in T cell recognition have not yet been fully described, and the initial TCR triggering mechanism remains a subject of controversy. By using supported lipid bilayers to 162 present peptide MHC class I complexes to CD8+ cells, we can monitor the molecular events occurring at the immune synapse. The lipid bilayer technology mimics the signaling environment for T cell activation as judged by Ca2+ flux, total phospho-Tyr levels, and IL-2 secretion. Using TIRF/FRET microscopy, we observe a two-stage interaction between TCR, CD8, and MHCp. There is an early (within seconds) interaction between CD3ζ and the coreceptor CD8 that is independent of the binding of CD8 to MHC, but that requires CD8 association with Lck. Later (several minutes) CD3ζ-CD8 interactions require CD8-MHC binding. Lck can be found free or bound to the coreceptor. This data suggests that, upon antigen recognition, TCR may be initially phosphorylated by Lck not associated with coreceptor, followed by MHC dependent recruitment of CD8-Lck complexes. This work indicates that the initial TCR triggering event is induced by free Lck. PW2-3 Understanding the role of RAS-PI3K signalling in lung cancer Esther Castellano Sánchez1, Clare Sheridan2, Miriam Molina-Arcas2, Julian Downward2 1 Centre for Cancer and Inflammation, Barts Cancer Institute, Londres, UK, 2Oncogene Biology, The Francis Crick Institute, Londres, UK Non-small cell lung cancer (NSCLC) is the most frequent type of lung cancer. The prognosis is poor with a 15% overall 5year survival rate for patients with advanced disease. This is attributable to the presence of metastasis at the time of diagnosis. KRAS mutations occur in approximately 25% of NSCLC patients. Unfortunately, understanding oncogenic changes in NSCLC has not provided effective long-term therapeutic strategies. Thus, there is a profound need to uncover the molecular mechanisms of RAS-driven lung tumours to develop new therapies to target NSCLCs. We have shown in a mouse model of lung cancer that disruption of RAS signaling through PI3-Kinase (PI3K), one of its main effectors, is essential for RAS driven tumor initiation and maintenance. We have also shown that genetically blocking the interaction of RAS with p110α causes partial regression of established tumors, followed by long-term tumor stasis. Interestingly, simultaneous inhibition of p110α together with inhibition of ERK, another major RAS effector pathway, causes major tumor regression.Proteomic analysis of the regressing lung tumors lacking RAS-PI3K signaling showed significant changes in proteins involved in cell adhesion and actin cytoskeleton reorganization. This correlated with a decrease in cell migration via regulation of Reelin (Reln) expression. We found that RAS-PI3K regulation of Reln is essential for proper cell migration and failure of regulation of this pathway results in loss of Reln expression Salamanca 2016 and increased migratory phenotype both in in vitro and in vivo settings. Furthermore, loss of Reelin correlates with decreased survival of lung and breast cancer patients. Reelin thus plays a role in restraining RAS and PI3-kinase promotion of cell motility and potentially tumour metastasis. PW2-4 Interplay between SUMOylation and ubiquitination during DNA replication Emilio Lecona1, Sara Rodríguez-Acebes2, Julia Specks1, Antonio Galarreta1, Patricia Ubieto1, Sergio Ruiz1, Andrés López-Contreras3, Isabel Ruppen4, Matilde Murga1, Javier Muñoz4, Juan Méndez2, Óscar Fernández-Capetillo1 1 Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid, ES, 2DNA Replication Group, Spanish National Cancer Research Centre (CNIO), Madrid, ES, 3Center for Chromosome Stability, University of Copenhagen , Copenhagen, DK, 4Proteomics Unit, Spanish National Cancer Research Centre (CNIO), Madrid, ES Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication and genome stability. Our previous results show that the chromatin around replisomes is rich in SUMO and depleted in Ub, whereas an opposite pattern is observed in mature chromatin. The mechanism responsible for the generation and maintenance of this SUMO-rich/Ub-low environment at sites of DNA replication is not known. We have identified USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. The chemical inhibition or genetic deletion of USP7 blocks both new origin firing and the activity of active forks. The analysis of the targetome of USP7 revealed SUMO2/3 as a new substrate for this deubiquitinase and USP7 counteracts the ubiquitination of SUMO2/3 and SUMOylated proteins on chromatin. The blockade of USP7 leads to the ubiquitination of SUMOylated proteins and their displacement from the replisomes, in cooperation with the VCP/p97 segregase. Our findings provide a model to explain the accumulation of SUMO and depletion of Ub at replication forks through a new essential function of USP7 in DNA replication. Currently, we are exploring the potential ubiquitin ligases that target SUMOylated proteins on the replisome and the mechanisms that direct VCP/p97 to proteins modified by SUMO and ubiquitin. Given the potential of targeting USP7 for cancer treatment, this new role of USP7 should be taken into account in the application of USP7 inhibitors as anticancer agents. PW2-5 Coordinated actin and septin cytoskeletal rearrangements modulate mechano-transduction and the emergence of Cancer-associated fibroblasts Fernando Calvo1, Romana Ranftl1, Aaron J. Farrugia1, Steven Hooper2, Erik Sahai2 1 Institute of Cancer Research, London, UK, 2Crick Institute, Londres, UK Cancer-associated fibroblasts (CAFs) are non-malignant cells found in tumours that can favour tumour progression, dissemination and therapeutic resistance through remodelling of the extracellular matrix and signalling to cancer, endothelial and immune cells. To investigate the mechanisms governing the tumour promoting phenotype of CAFs, we isolated fibroblasts from different stages of breast cancer progression and normal counterparts, and analysed their phenotype, function and gene Pósters / Posters expression. Our analyses reveal that enhanced mechano-transduction and activation of the transcriptional regulator YAP are signature features of CAFs. YAP regulates the expression of cytoskeletal genes that are required for CAFs to remodel the extracellular matrix and promote cancer cell invasion and angiogenesis. Matrix stiffening further enhances YAP activation, thus establishing a feed-forward self-reinforcing loop that helps to maintain the CAF phenotype. In addition, we identify Cdc42EP3, a previously uncharacterised Cdc42 effector, as a YAP target gene in CAFs. Using super-resolution microscopy and biochemical approaches we demonstrate that Cdc42EP3 functions by coordinating the actin and septin networks, which change dramatically during CAF emergence. In CAFs, Cdc42EP3 and septin fibres are required for mechano-transduction, force-mediating matrix remodelling and promoting cancer cell invasion, angiogenesis and tumour growth. To conclude, our studies underlie the relevance of mechano-transduction and coordinated cytoskeletal rearrangements in modulating the aggressive behaviour of stromal fibroblasts in cancer, and define a novel molecular and biological role for the septin cytoskeleton. PW2-6 Early events in ribosome biogenesis Christina Schmidt1, Fanny Boissier2, Sebastién Fribourg2, Jorge Pérez-Fernández1 1 University of Regensburg, Regensburg, DE, 2Univ. Bordeaux, IECB, F-33607, Pessac, FR. INSERM, U1212 & CNRS UMR 5320, F-33077, Pessac, FR Ribosome synthesis is an essential multistep process which involves the correct folding of the mature ribosomal (r)RNAs and the incorporation of the ribosomal proteins. In eukaryotic cells, more than 150 biogenesis factors associate transiently with the pre-rRNA to promote the correct formation of ribosomes. During the early events of ribosome biogenesis, a large set of proteins associates to the nascent rRNA to form the first pre-ribosomal particle or SSU-processome (small subunit). Formation of the SSU-processome occurs by the stepwise association of the early assembly factors to the rRNA in a highly hierarchical process. Some of these early-acting factors have been isolated as small protein complexes (UTP complexes) and they are the primary binders of the pre-rRNAs. However, the molecular mechanism behind the hierarchical relationships that control the formation of the SSU-processome remains elusive. To uncover this aspect, we have studied the role of Pwp2, a component of the UTP-B complex which may play a central role during the formation of the SSU-processome. The crystal structure of the N-terminal domain of Pwp2 shows a classical tandem β-propeller folding. Analysis of different mutants revealed that the N-terminal domain of Pwp2 is not enough to support the formation of the UTP-B complex. Instead, the C-terminal domain of Pwp2 establishes interactions with three other UTP-B components and is required for the stabilization of the full complex and the stable formation of the SSU-processome. Overall, our data suggest that the UTP-B complex works as a platform for the association of some assembly factors. PW2-7 Carnitine palmitoyltransferase 1 increases lipolysis, UCP1 protein expression and mitochondrial activity in brown adipocytes Laura Herrero1, María Calderón-Domínguez1, David Sebastián2, Raquel Fucho1, Minéia Weber1, Joan F. Mir1, Esther García-Casarrubios3, María Jesús Obregón3, Antonio Zorzano2, Ángela M. Valverde4, Dolors Serra1 1 Department of Biochemistry and Physiology, Institut de Biomedicina de la Universitat de Barcelona (IBUB), Universitat 163 Pósters / Posters de Barcelona, Barcelona, ES; Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y la Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid, ES, 2Institute for Research in Biomedicine (IRB Barcelona) and Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, ES; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III , Madrid, ES, 3Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM) and Instituto de Investigación Sanitaria La Paz, Madrid, ES, 4Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM) and Instituto de Investigación Sanitaria La Paz; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, ES The discovery of active brown adipose tissue (BAT) in adult humans and the fact that it is reduced in obese and diabetic patients have put a spotlight on this tissue as a key player in obesity-induced metabolic disorders. BAT regulates energy expenditure through thermogenesis; therefore, harnessing its thermogenic fat-burning power is an attractive therapeutic approach. We aimed to enhance BAT thermogenesis by increasing its fatty acid oxidation (FAO) rate. Thus, we expressed carnitine palmitoyltransferase 1AM (CPT1AM), a permanently active mutant form of CPT1A (the rate-limiting enzyme in FAO), in a rat brown adipocyte (rBA) cell line through adenoviral infection. We found that CPT1AM-expressing rBA have increased FAO, lipolysis, UCP1 protein levels and mitochondrial activity. Additionally, enhanced FAO reduced the palmitate-induced increase in triglyceride content and the expression of obese and inflammatory markers. Thus, CPT1AM-expressing rBA had enhanced fat-burning capacity and improved lipid-induced derangements. This indicates that CPT1AM-mediated increase in brown adipocytes FAO may be a new approach to the treatment of obesity-induced disorders. PW2-8 Regulation of human Polλ by post-translational modification José F. Ruiz Universidad de Sevilla, Sevilla, ES The genome integrity in mammalian cells is constantly threatened by a wide variety of lesions, the repair of which often require a DNA synthesis step either to replace damaged nucleotides or DNA strands or to bypass them provisionally until their subsequent removal. DNA synthesis during these repair or tolerance processes is mainly performed by specialized DNA polymerases belonging to the X and Y families. Among these enzymes DNA polymerase lambda (Polλ) is one of the most versatile, having relevant roles in the repair of oxidative DNA damage through Base Excision Repair (BER) as well as in the repair of DNA double-strand breaks by Non-Homologous End Joining (NHEJ). The involvement of Polλ in these different processes suggests a complex regulation, which remains largely unknown so far. In this work we will discuss on the complex interplay between post-translational modifications (PTMs) and human Polλ, describing novel modifications that we have recently identified and showing their relevance in different aspects like the subcellular localization or its activity during the repair of IR-induced DSBs by NHEJ. Our work will contribute to the mechanistic understanding of how DNA polymerases lambda can influence genome integrity through its participation in different repair pathways, with an emphasis on the means by which these pathways intersect human diseases. 164 XXXIX Congreso SEBBM PW2-9 Clot formation as a potential diagnostic tool for Alzheimer’s disease Marta Cortés Canteli, Jesús Ruiz-Cabello, Valentín Fuster Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, ES Alzheimer’s disease (AD) is a multifactorial disorder with no effective treatment available. Accumulating evidence shows that AD presents an important vascular component. Epidemiological studies show a correlation of AD with vascular risk factors. There are also profound alterations of cerebrovascular structure and function present in AD, and deposition of beta-amiloid in the cerebral blood vessels, that could affect brain circulation. Indeed, a decrease in cerebral blood flow has been reported in AD patients which could have a very detrimental effect on neuronal viability and cerebral function in general. Several pieces of evidence indicate increased thrombosis, decreased fibrinolysis and increased obstruction of the cerebral blood vessels in the AD brain. These obstructions in AD cerebral vasculature could initiate and/or aggravate the brain hypoperfusion and inflammation present in the AD brain. Cerebral hypoperfusion seems to be a good indicative for dementia conversion, which suggests that the occlusion of vessels might be happening at very early stages of the disease, many years before the clinical manifestation of AD. We are using state-of-the-art imaging technology to develop an in vivo non-invasive method to identify these occlusions. This research could serve as a novel biomarker since it will identify the subpopulation of AD patients with early dysregulation of cerebral blood flow and might prove to be a useful tool to identify patients at very early stages of the AD pathology. PW2-10 Cristae shape determines mitochondrial bioenergetics under compromised respiration Rubén Quintana Cabrera, Mauro Corrado, Luca Scorrano Dpto. Biología. Universidad de Padua; Venetian Institute of Molecular Medicine (VIMM), Padua, IT Mitochondrial cristae function as a membrane platform where respiratory (super)complexes sit to account for respiration. A growing body of evidence shows that maintaining the architecture of the cristae is key to assemble respiratory supercomplexes and improve respiration rates; however, how mitochondrial ultrastructure controls mitochondrial bioenergetics remains obscure. Capitalizing on genetic modulation of Opa1 as a master regulator of cristae shape, we show that cristae determine F0F1-ATP synthase stability and mitochondrial bioenergenetic parameters such as matrix pH, ATP or membrane potential. Narrowing of cristae by Opa1 stabilizes F0F1-ATP synthase to account for sustained mitochondrial function and cell survival under compromised respiration or metabolic reliance on mitochondrial ATP production. Electron microscopy analysis of inner membrane and apoptotic cristae remodeling by Bid confirmed that keeping cristae tight is essential to preserve F0F1-ATP synthase stability and function. As a result of improved F0F1-ATP synthase composition and activity, OPA1 accounts for lower reactive oxygen species production under compromised mitochondrial respiration. Interestingly, the pro-survival and bioenergetic advantage from OPA1 overexpression are lost when F0F1-ATP synthase dimerization is impaired, thus indicating a need for dimers as a link for structural and functional consequences of cristae remodeling. In sum, our results prove a connection of mitochondrial ultrastructure and bioenergetics, offering a proof of principle for therapies aimed at restoring mitochondrial function in disease. Salamanca 2016 PW2-11 Caracterización funcional y estructural del regulador transcripcional pleiotrópico RcsB de Salmonella typhimurium Patricia Casino1, Laura Miguel-Romero2, Alberto Marina2 Departamento de Bioquímica, Universitat de València. Estructura de Recerca Interdisciplinar en Biotecnologia i Biomedicina (ERI BIOTECMED), Universitat de València , Burjassot-Valencia, ES, 2Instituto de Biomedicina de Valencia, CSIC, Valencia, ES 1 El sistema RcsCDB es un sistema de dos componentes complejo presente en Enterobacteriacea, que regula la formación de biofilm así como otros genes implicados en el mantenimiento de la pared celular, la división celular, el factor sigma σS, motilidad y virulencia. El sistema RcsCDB está formado por la histidina quinasa híbrida RcsC, la fosfotransferasa RcsD y el regulador de la respuesta pleiotrópico RcsB que se activan mediante múltiples pasos de fosforilación, también denominados como “phosphorelay”. Como respuesta a cambios ambientales, RcsC se autofosforila en un residuo de His conservado y fosfotransfiere el grupo fosforilo a un residuo de Asp conservado presente en su misma cadena polipeptídica. Posteriormente el grupo fosforilo se transfiere de nuevo desde el Asp a otro residuo de His presente en la fosfotransferasa RcsD. Finalmente, el grupo fosforilo se transfiere desde RcsD a un residuo de Asp presente en RcsB, el cual controla la transcripción de múltiples genes bien de manera independiente o junto con otros reguladores transcripcionales auxiliares. Así pues, la complejidad en la respuestaa cambios ambientales generada por el sistema RcsCDB viene determinada principalmente por la actividad pleiotrópica que presenta el regulador de la respuesta RcsB para regular la transcripción de múltiples genes. Con el fin de comprender las bases moleculares del mecanismo de “phosphorelay” en RcsCDB así como el mecanismo pleiotrópico de regulación transcripcional, hemos realizado estudios funcionales con el complejo formado por RcsD y RcsB, así como estudios estructurales y funcionales con RcsB. Ello nos han permito resolver la estructura tridimensional de RcsB en dos estados diferentes de activación arrojando así luz sobre su activación y regulación transcripcional. Pósters / Posters a strict cell-cycle regulatory mechanism that depends on changes in its phosphorylation status by the Cdk kinase and the Cdc14 phosphatase. Such control implies that Yen1 will display low activity and cytoplasmic localization in S-phase, while in anaphase it will re-enter the nucleus and become highly active. Interestingly, expression of a mutant that is constitutively active and nuclear (Yen1ON) results in detrimental effects for the cell. These range from increased genome instability when Yen1ON is expressed at endogenous levels to cell death when it is overexpressed. Here, we will present the lab strategy and novel data where we are using Yen1ON as a tool to explore the importance of cell cycle control on SSE activation as well as the dynamics of early recombination/replication intermediates under unperturbed conditions. PW2-13 Understanding the structurally dynamic AMPA receptors Javier García Nafria, Beatriz Herguedas, Ingo H. Greger MRC Laboratory of Molecular Biology, Cambridge, UK AMPA receptors (AMPARs) are glutamate-gated ion channels localized at the post-synaptic density. AMPARs are not only involved in synaptic transmission, but also have a central role in synaptic plasticity, which is believed to be the basis of human cognitive functions and memory formation. In the brain, the channel is mainly a hetero-tetramer, formed by combinations of four different subunits that yield functionally different channels. Structurally, AMPAR is divided in three domain layers with a transmembrane region (TMD), acting as the ion channel, a ligand binding domain (LDB), which binds glutamate, and the membrane-distal N-terminal domain (NTD). Using a variety of techniques including Anisotropic Network Model, cysteine crosslinking, X-ray crystallography and single-particle electron microcopy, we were able to 1) predict and confirm new conformations of AMPA receptor homomers, 2) show that, although flexible, all AMPARs subunits can adopt a common set of discrete conformations, 3) describe two new conformations using single-particle cryo-electron microscopy, one of which is the description of the first AMPAR heteromer, and 4) speculate about the potential of these different conformation in regulating synaptic protein-protein interactions. PW2-12 Too early to prune: Premature activation of structure-selective endonucleases destroys essential branched DNA structures PW2-14 Miguel González Blanco, Francisco Javier Aguado Domínguez, Vanesa Nieves Hurtado Centro de Investigación en Medicina Molecular y Enfermedades Crónicas. Universidade de Santiago de Compostela, Santiago de Compostela, ES Jorge Alegre-Cebollada Fundación CNIC, Madrid, ES The transmission of genetic information from mother to daughter cells depends on the faithful duplication of their DNA content and its accurate distribution into two equal genomic packages. Despite conceptually simple, such a task requires the coordinated actions from different molecular machineries, like those responsible for DNA replication, repair and segregation. In most cases, this synchronization relies on the master cell cycle regulator kinase Cdk, which dictates the main order of events in the cell. However, in situations of high genotoxic stress, a signaling cascade known as the DNA damage response (DDR) can trigger a checkpoint that arrests cell cycle progression, providing enough time for the cell to repair DNA before committing to the next cell cycle stage. Yen1 is a structure-selective endonuclease (SSE) involved in the removal of persistent homologous recombination intermediates. The activation of this nuclease, rather than depending on the DDR, is under Single-molecule mechanobiochemistry laboratory at CNIC We use biochemistry and biophysics techniques to study the mechanics of heart tissue and its regulation in health and disease. Our lab takes a multidisciplinary approach to try to learn more about molecular determinants of the mechanical properties of the heart. We specialize in single molecule methods using atomic force microscopy (AFM), which are able to measure the effects of mechanical forces on cardiac structural proteins. Our main lines of research are: 1. Regulation of protein elasticity by redox posttranslational modifications (PTM). Using in vitro experiments, we have recently described that PTM of buried cysteines in the giant protein titin makes heart tissue more elastic (Cell 2014, 156:1235). We are using mass spectrometry techniques to detect native redox PTMs from heart tissue. Once relevant redox PTMs are identified, we use single-molecule methods by AFM to determine their effect in protein elasticity. We want to explore how these mechanical PTMs are differentially present during heart disease. 2. Genotype/phenotype relations in familial cardiomyopathies. The majority of familial cardiomyopathies are caused by mutations in 165 Pósters / Posters sarcomeric genes that code for proteins with mechanical roles. We are exploring the connection between dysregulation of protein mechanics and development of cardiomyopathy. Using single-molecule AFM, we measure the mechanical properties of proteins whose mutation cause cardiomyopathy. We plan to examine several other cellular functions that may be affected by mutations. 3. Development of engineered muscle-mimicking biomaterials. Using a directed crosslinking strategy, we are able to polymerize proteins into hydrogels whose stiffness can be measured in the lab using tensile testers built by us (Macromolecular Materials and Engineering 2015, 300, 369). We are developing experimental platforms that allow us to predict the stiffness of the biomaterial from the mechanical properties of the constituent proteins measured at the single-molecule level. PW2-15 Microenvironment-induced metabolic switch promotes metastasis in pancreatic cancer Sara Trabulo, Andrés Cano, Shanthini Crusz, Petra Jagust, Andrew Palfreeman, David Barneda, Christopher Heeschen, Patricia Sancho Centre for Stem Cells in Cancer & Ageing, Barts Cancer Institute, Queen Mary University of London, London, UK Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers worldwide due to its chemoresistance and early onset of metastases, characteristics that, at least in part, can be attributed to the presence of cancer stem cells (CSCs). We have established that CSCs are eminently OXPHOS-dependent, with MYC/PGC1A expression ratio as the main determinant for their sensitivity to mitochondrial targeting (i.e. metformin) (Sancho et al., Cell Metabolism 2015). We now show that, concomitant with the acquisition of migratory and invasive capacities, microenvironmental factors such as Tumour-Associated Macrophages (TAMs), modulate the metabolic phenotype of PDAC cells (Sainz et al., Gut 2015). Specifically, incubation of PDAC cells with TAM-conditioned medium or direct co-culture with TAMs in either 2D or 3D conditions, increased their MYC/PGC1A ratio, leading to a metabolic shift towards glycolysis. As expected, cells that underwent EMT became unresponsive to the mitochondrial inhibitor metformin, which was attributed to upregulation of MYC, as previously described (Sancho et al., 2015). Importantly, MYC knockdown blocked these metabolic and phenotypic changes induced by TAMs, implying a crucial role for MYC in EMT induction via modulation of metabolism. Interestingly, single-cell gene expression analysis of circulating-tumour cells (CTCs) demonstrated an increased MYC/PGC1A ratio in the CTC population as compared to cell derived from primary tumours. This was even more pronounced in the subset of circulating CSCs, suggesting that CSCs evaded from the primary tumour have switched their preferential metabolism from OXPHOS to glycolysis via upregulation of MYC. In summary, our results suggest that in PDAC an increased MYC/ PGC1A ratio promotes a pro-metastatic programme through metabolic reprogramming of CSCs. Inhibition of this process may provide a novel approach for combating metastasis in PDAC. PW2-16 Identification of protective cellular pathways involved in ischemic preconditioning María Delgado Esteban Instituto de Investigación Biomédica de Salamanca (IBSAL)/ Hospital Universitario de Salamanca; Instituto de Biología Funcional y Genómica (IBFG)/Universidad de Salamanca (CSIC/ USAL), Salamanca, ES 166 XXXIX Congreso SEBBM Ischemic preconditioning (IPC) is one of the most important endogenous mechanisms responsible for the increased tolerance of brain and heart after an ischemic insult. Although the molecular mechanisms underlying IPC-induced cell protection are still unknown, several studies suggest that IPC promotes the activation of endogenous antioxidant systems and modulates apoptosis. Then, IPC is an effective tool for identifying molecular targets involved in cell survival. In this context, two transcription factors, p53 -the key regulator of apoptotic cell death- and the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein -the key regulator of antioxidant response-, have been described to play an essential role in the progression of cerebral ischemia and heart failure. Our work is focused on the study of the role of p53 and Nrf2 on the IPC-induced cell protection against an ischemic insult. Indeed, cellular redox status is directly regulated by p53 through modified expression of pro- and anti-oxidant proteins. For this purpose, we used two different models of IPC insults: i) primary cultured cortical neurons were stimulated with moderate concentrations of N-methyl-D-aspartate (20 μM NMDA); and ii)human endothelial cells were incubated upon sublethal oxygen and glucose deprivation (OGD) conditions for 20 min. After the corresponding IPC insult, cells were incubated for further 2 h either in the presence (IPC) or absence (IPC+OGD) of oxygen and glucose (lethal OGD). Our results show that IPC modulated p53-mediated neuronal apoptosis by interfering with the caspase-3 pathway. IPC prevents the OGD-induced p53 mRNA levels abrogating the accumulation of p53 and its target p21 in cortical neurons.Furthermore, IPC modulated mRNA levels of Nrf2 and its targets hemeoxygenese-1 (HO-1) and NADPH quinona reductase NQO1 in endothelial cells. Our results demonstrate that IPC promotes cell survival through the control of the p53 signaling pathway and the Nrf2-antioxidant pathway. Then, IPC plays a key role in the control of cellular redox homeostasis and survival. Funded by grants from the Instituto de Salud Carlos III (Miguel Servet I CP0014/00010; RD12/0014/0007); FEDER (European regional development fund). PW2-17 Iron overload causes endolysosomal deficits modulated by NAADP regulated two pore channels and RAB7A Belén Fernández López1, Elena Fernández Fernández1, Patricia Gómez Suaga1, Fernando Gil Hernández2, Isabel Molina Villalba2, Isidro Ferrer3, Sandip Patel4, Grant C. Churchill5, Sabine Hilfiker1 1 Instituto de Parasitología y Biomedicina López-Neyra (CSIC), Armilla (Granada), ES, 2Universidad de Granada. Facultad de Medicina. Departamento de Medicina Legal, Toxicología y Antropología Física, Granada, ES, 3Universidad de Barcelona. Instituto de Nueropatología. IDIBELL-Hospital Universitario Bellvitge, L’Hospitalet de Llobregat, ES, 4University College London. Dept. of Cell and Developmental Biology, Londres, UK, 5 Dept. of Pharmacology, University of Oxford, Oxford, UK Various neurodegenerative disorders are associated with increased brain iron content. Iron is known to cause oxidative stress, which concomitantly promotes cell death.Whereas endolysosomes are known to serve as intracellular iron storage organelles, the consequences of increased iron on endolysosomal functioning, and effects on cellviability upon modulation of endolysosomal iron release remain largely unknown. Here,we show that increasing intracellular iron causes endolysosomal alterations associated with impaired autophagic clearance of intracellular protein aggregates, increased cytosolic oxidative stress and increased cell death. These effects are subject Salamanca 2016 to regulation by NAADP, a potent second messenger reported to target endolysosomalTPCNs (two pore channels). Consistent with endolysosomal iron storage, cytosolic iron levels are modulated by NAADP, and increased cytosolic iron is detected when overexpressing active, but not inactive TPCNs, indicating that these channels can modulate endolysosomal iron release. Cell death triggered by altered intralysosomaliron handling is abrogated in the presence of an NAADP antagonist or when inhibiting RAB7A activity. Taken together, our results suggest that increased endolysosomal iron causes cell death associated with increased cytosolic oxidative stress as well as autophagic impairments, and these effects are subject to modulation by endolysosomalion channel activity in a RAB7A-dependent manner. These data highlight alternative therapeutic strategies for neurodegenerative disorders associated with increased intracellular iron load. PW2-18 Immunoregulatory properties of human adipose derived stem cells (hASCs) in inflammatory diseases. Differences between Crohn´s disease and obesity Carolina Serena, Noelia Keiran, Ana Madeira, Joan Vendrell, Sonia Fernández-Veledo Instituto de Investigación Sanitaria Pere Virgili. Hospital Joan XXIII de Tarragona. CIBERDEM, Tarragona, ES Background: Inflammation and dysbiosis are common in several highly prevalent diseases, such as Cohn’s disease (CD), obesity and type 2 diabetes (T2D). Visceral fat depot plays a decisive role in the inflammatory environment observed in these pathologies. Specifically, in CD subjects there is frequently an increase of mesenteric fat-tissue which is called “creeping fat”. However, CD patients show an aggressive local inflammatory response whereas obese and T2D patients develop a subtle chronic inflammatory status. The main objective of this study was to identify key factors involved in triggering the immune innate response of adipose tissue (AT). Specifically, we focus on the potential role that human adipose-derived stem cells (hASCs) play in local and systemic inflammation in the setting of CD and obesity. Methods: We isolated hASCs from subcutaneous (SAT) and visceral AT (VAT) of a well-characterized cohort including 4 groups: inactive CD (in remission), active CD, obese, and obese subjects with diabetes (T2D). hASCs were immunophenotyped by flow cytometry. Gene expression profile, migration, invasion, as well as phagocytic capacity were analyzed in hASCs obtained from different donors. Results: hASCs obtained from CD subjects shown a significantly greater migratory and invasive capacity than those derived from obese subjects, regardless of the presence of diabetes. This distinctive difference in phenotype between CD versus obese hASCs was observed in both active and inactive CD donors and in both depots SAT and VAT. The phagocytic capacity was also higher in CD subjects compare obese subjects but in this occasion in inactive CD donors showed lower phagocityc activity to compare active CD donors. Accordingly, ATinflammation markers (IL-6, TNFa, IL-1b, MCP-1) indicated that both active and inactive CD subjects present a higher proinflammatory environment compared to obese subjects. Remarkably, we demonstrate that CD produces a detrimental effect not only on mesenteric adipose tissue-resident stem cells, but also on the subcutaneous fat depots. Conclusions: hASCs may have different immuno-modulatory capacities depending on the inflammatory event. Pósters / Posters PW2-19 Peripheral modification of disease pathogenesis in skeletal muscle affects systemic Huntington´s disease Silvia Corrochano Sánchez1, Debbie Williams1, Gonzalo Blanco2, Michelle Simon1, Michael R Duchen3, Henning Wackerhage4, David C Rubinsztein5, Steve D.M. Brown1, Abraham Acevedo-Arozena6 1 MRC Mammalian Genetics Unit, Harwell, Oxfordshire, UK, 2 York University, York, UK, 3University College London (UCL), London, UK, 4University of Aberdeen, Scotland, UK, 5Cambridge University, Cambridge, UK, 6MRC Mammalian Genetics Unit, Harwell, Oxfordshire, UK, and Unidad de Investigación, Hospital Universitario de Canarias, Tenerife, ES Polyglutamine expansions in huntingtin (HTT) cause Huntington’s disease (HD). HD is primarily a neurodegenerative condition; however, HTT is ubiquitously expressed, leading to pathological alterations also in peripheral organs, as well as general metabolic abnormalities. To identify new HD modifiers, we performed a genetic screen using N-ethyl-N-nitrosourea (ENU) mutagenesis in mice, identifying a mutation in the skeletal muscle voltage-gated sodium channel (Scn4a) that exacerbated the overall HD phenotype. Huntingtin and Scn4a are only highly expressed together in skeletal muscle, leading to cell autonomous effects of their genetic interaction. That in turn causes systemic effects, including metabolic alterations associated with energy imbalance leading to further weight loss and a dramatic reduction in survival in double mutants. These systemic alterations, including further changes in the kynurenine system, ultimately perturb the expression of key HD genes in the brain. Our results reveal that local metabolic changes in skeletal muscle can modulate the systemic nature of HD pathogenesis. PW2-20 Positioning of centrioles is a conserved readout of Frizzled planar cell polarity signalling José María Carvajal González, Sonia Mulero-Navarro Universidad de Extremadura, Badajoz, ES Planar cell polarity (PCP) signalling is a well-conserved developmental pathway regulating cellular orientation during development. An evolutionarily conserved pathway readout is not established and, moreover, it is thought that PCP mediated cellular responses are tissue-specific. A key PCP function in vertebrates is to regulate coordinated centriole/cilia positioning, a function that has not been associated with PCP in Drosophila. Here we report instructive input of Frizzled-PCP (Fz/PCP) signalling into polarized centriole positioning in Drosophila wings. We show that centrioles are polarized in pupal wing cells as a readout of PCP signalling, with both gain and loss-of-function Fz/PCP signalling affecting centriole polarization. Importantly, loss or gain of centrioles does not affect Fz/PCP establishment, implicating centriolar positioning as a conserved PCP-readout, likely downstream of PCP-regulated actin polymerization. Together with vertebrate data, these results suggest a unifying model of centriole/cilia positioning as a common downstream effect of PCP signalling from flies to mammals. PW2-21 Structural basis of TubZ filament assembly and dynamic modulation María A. Oliva Blanco1, María Eugenia Fuentes Pérez2, Rafael Núñez Ramírez1, Alejandro Martín Gonzalez2, David Juan Rodríguez1, José Manuel Andreu Morales1, Oscar Llorca1, Fernando Moreno Herrero2 1 CSIC-CIB, Madrid, ES, 2CSIC-CNB, Madrid, ES 167 Pósters / Posters Accurate DNA segregation is essential for genome transmission during cell division and partition systems are key on low copy plasmids maintenance. These systems require only three elements to effectively produce DNA movement: a cis-acting centromere-like DNA sequence, a CBP (centromere-binding protein) and a motor protein. Based on the nature of the motor protein partition systems are classified into type I (Walker A-like ATPases), II (actin-like ATPase) and III (tubulin-like GTPase). In type III systems, the motor protein (TubZ) grows in a helical fashion following treadmilling or dynamic instability, although the underlying molecular mechanism is not clear. Using a biochemical approach in combination with electron and atomic force microscopy we have studied the structural changes in TubZ filament upon GTP hydrolysis and how those drive the disassembly process. We present phage CbTubZ primary assembly into a 4-stranded helical arrangement, which is just supported by the presence of GTP caps. The transition from GTP to GDP-bound state propagates along the filament only when GTP caps are released and involves the stiffening of the filament. Our results point to the TubZ C-terminal flexible tail as an unstructured domain directly involves in the filament dynamics because is essential on the formation of a 4-stranded helical filament and helps in the conformational change upon hydrolysis for depolymerization. Further, the C-tail also modulates the interaction with the partner regulator TubY and can induce t