Session 3

Transcription

Session 3

XXXIX Jornadas Portuguesas da Genética
Scientific Committee
Cecília Arraiano (Universidade Nova de Lisboa)
Isabel Sá-Nogueira (Universidade Nova de Lisboa)
Jorge Vieira (Universidade do Porto)
Jorge Canhoto (Universidade de Coimbra)
Manuel Santos (Universidade de Aveiro)
Margarida Casal (Universidade do Minho)
Leonor Cancela (Universidade do Algarve)
Sandra Viegas (Universidade Nova de Lisboa)
Organizing Committee
Centre of Molecular and Environmental Biology
Department of Biology, University of Minho
Margarida Casal (Coordinator)
Ana Preto
Artur Ribeiro
Björn Johansson
Maria João Sousa
Pedro Santos
Pedro Soares
Raul Machado
Ricardo Franco-Duarte
Sandra Paiva
Susana Chaves
Teresa Almeida
Teresa Matamá
Tony Collins
1
Universidade do Minho - Campus de Gualtar 25-27th May 2015
Institutional Support
SOCIEDADE PORTUGUESA DE GENÉTICA
www.spgenetica.pt
CENTRO DE BIOLOGIA MOLECULAR E AMBIENTAL
cbma.bio.uminho.pt
DEPARTAMENTO DE BIOLOGIA
UNIVERSIDADE DO MINHO
CAMPUS DE GUALTAR
4710-057 BRAGA
bio.uminho.pt
2
Schedule
Monday, 25th May
Registration
Tuesday, 26th May
Wednesday, 27th May
Plenary Session 2
Sónia Melo
Session 5
Human Genetics
Session 3
Gene regulation and expression I
Coffee Break
Coffee Break
Session 3
Gene regulation and expression II
Poster Session
(all)
Lunch
Lunch
Session 6
Genetics and Biotechnology I
Lunch
Session 1
Evolutionary Genomics
Session 4
Plant genetics I
Session 6
Genetics and Biotechnology II
Poster Session
(odd numbers)
Poster Session
(even numbers)
16:00
Coffee Break
Coffee Break
16:30
Session 2
Microbial Genetics
Session 4
Plant Genetics II
Plenary Session 3
Rita Costa
09:00
09:30
10:00
10:30
11:00
Opening Session
11:30
Plenary Session 1
Martin Richards
12:00
12:30
13:00
13:30
14:00
14:30
15:00
Plenary Session 4
Jorge Pacheco
15:30
Awards and Closure
17:00
17:30
18:00
General Assembly SPG
3
Detailed Programme
1st day: 25th May
9.00: Registration
11.00: Opening session
11.30: PL1. The Archaeogenetics of West Eurasia
Martin Richards
Coordinator: Pedro Soares
12.30: Lunch
Session 1: Evolutionary Genomics
Coordinators: Cláudio Sunkel and Maria João Sousa
14.00: OS1. Drosophila americana diapausing females show features typical of young flies
Micael Reis, Felipe B. Valer, Cristina P. Vieira and Jorge Vieira
14.18: OS2. Genetic ancestry of Austronesian-speaking populations in Island Southeast Asia and the Pacific
Pedro Soares, Teresa Rito, Bruno Cavadas, Andreia Brandão, Maria Pala, Stephen Oppenheimer, Luísa Pereira, Martin
B Richards
14.36: OS3. Intracellular endosymbiont selection contributes to Drosophila adaptation to viral infection
Vitor G. Faria, Nelson E. Martins, Sara Magalhães, Viola Nolte, Christian Schlötterer, Luis Teixeira and Élio Sucena
15.00: Poster session 1 (odd numbers)
16.00: Coffee break
Session 2: Microbial Genetics
Coordinators: Manuel Santos and Tony Collins
16.30: OS4. Multitask ATPases: a common theme in bacteria?
Mário J. Ferreira, Aristides L. Mendes and Isabel de Sá Nogueira
16.48: OJ1. Establishment of the MurT/GatD complex as a new virulence factor – role in host evasion
Bárbara Gonçalves, Ana Madalena Ludovice, Hermínia de Lencastre, Luís Jaime Mota and Rita Sobral
17.00: OJ2. Unravelling the role of Escherichia coli BolA, a pleiotropic protein that turns off motility and turns on
biofilm formation
Susana Barahona, Ricardo Neves Moreira, Clémentine Dressaire, António Pedro Alves de Matos and Cecília Maria
Arraiano
17.12: OJ3. Time series analysis of the transciptome of a Staphylococcus aureus mutant with an impaired cell Wall
Raquel Portela, Ana Madalena Ludovice, Rui Catarino, Francisco Pinto and Rita Sobral
17.24: OJ4. Identification of HIV-1, HCV and HBV using multiplex PCR
Adriana Resende, Ana Constança, Isabel Pereira-Castro, Amanda Bradley Stewart, Rory N. Gunson, Helena Ramos and
Filipe Pereira
17.36: OJ5. Microbiome Characterization of a Striped Dolphin (Stenella coeruleoalba) Of the Coast of Portugal
Ana Luísa Alves, Filipa Gonçalves, Cristina Sousa Mesquita, Pedro Soares-Castro, Marisa Ferreira, Ana Marçalo, José
Vingada, Catarina Eira, Filipa Godoy-Vitorino, B. Cabrera, A. Rodriguez, J. Fabre and Pedro Miguel Santos
4
17.48: OS5. Genetic determinants responsible for the adaptation of Saccharomyces cerevisiae strains to different
ecological niches
Ricardo Franco-Duarte, Paula Sampaio and Célia Pais
18.00: General Assembly SPG
2nd day: 26th May
9.00: PL2. The Biology and Functional Contribution of Exosomes in Cancer Progression and Metastasis
Sónia Melo
Coordinator: Ana Preto
Session 3: Gene regulation and expression
Coordinators: Isabel Sá-Nogueira and Sandra Paiva
10.00: OJ6. Cellular networks that regulate adaptation to protein synthesis errors in HEK293 cells
Ana Sofia Varanda, Mafalda Santos, Carla Oliveira and Manuel A. S. Santos
10.12: OJ7. A long non-coding RNA regulating stemness and cellular reprogramming
Sara Barros, Catarina Alves-Vale, Bruno Bernardes de Jesus and Maria Carmo-Fonseca
10.24: Coffee break
11.00: OS6. Regulation of transcriptional reactivation of the oocyte during meiosis
Paulo Navarro-Costa, Pedro Prudêncio, Jörg D. Becker and Rui Gonçalo Martinho
11.18: OJ8. A novel protein complex involved in ribosome biogenesis and rRNA quality control
Ricardo F. dos Santos, José M. Andrade, Joana Pissarra and Cecília M. Arraiano
11.30: OJ9. Measurement of Long Noncoding RNA kinetics in T-cells
Soraia da Silva, Rita Vaz‐Drago, Maria Carmo‐Fonseca and Noélia Custódio
11.42: OJ10. Functional characterization of a putative tRNA/rRNA methyltransferase on somatic embryogenesis
Sandra Correia, Patrícia Fernandes, Bruno Casimiro, Paula Veríssimo, Jorge Canhoto
11.54: OJ11. The role of mitochondrial phospholipid composition in Saccharomyces cerevisiae acetic acid-induced
apoptosis
Catarina Afonso, Tânia Fernandes, Elisabete Maciel, Rosário Domingues, Manuela Côrte-Real, Maria João Sousa
12.06: OJ12. Functional analysis of String/CDC25 phosphatase in post-mitotic neurons
Rui Gonçalves and Carla S. Lopes
12.18: OJ13. Candida glabrata susceptibility to antifungals and phagocytosis is modulated by the carbon source
Sandra Mota, Rosana Alves, Catarina Carneiro, Sónia Silva, Fabian Istel, Karl Kuchler, Paula Sampaio, Margarida Casal,
Mariana Henriques and Sandra Paiva
12.30: Lunch
Session 4: Plant Genetics
Coordinators: Raquel Chaves and Manuela Costa
14.00: OS7. Functional characterization of Arabidopsis thaliana SUMO proteases as new development and
environmental stress determinants
Pedro Humberto Castro, Tiago Lourenço, Miguel Ângelo Santos, Sara Freitas, Javier Ruiz-Albert, Rui Manuel Tavares,
Eduardo Rodrigues Bejarano, Herlander Azevedo
14.18: OS8. Genes from distinct lineages determine gametophytic self-incompatibility in Malus and Prunus
Bruno Aguiar, Jorge Vieira, Ana E. Cunha, Nuno A. Fonseca, Amy Iezzoni, Steve van Nocker and Cristina P. Vieira
5
14.36: OJ14. The sex of the plants...
Rómulo Sobral, Helena Silva, Joana Magalhães and M. Manuela R. Costa
14.48: OJ15. Somaclonal variation: structural composition at the berry color locus in Vitis vinifera L.
Vanessa Ferreira, Isaura Castro, Olinda Pinto-Carnide, David Carrasco, Rosa Arroyo-García
15.00: Poster session 2 (even numbers)
16.00: Coffee break
16.30: OJ16. Transcriptomic atlas of Physcomitrella patens to decipher the evolution of epigenetic mechanisms in
land plants
Pereira, Sónia, Hernandez-Coronado, M., Ortiz-Ramírez, C. and Becker, J.D
16.42: OS9. Label Free Biosensor and HRM used in SNP detection in Vitis vinifera L. for Authenticity Purposes
Leonor Pereira, Luís Moreira, Helena M.R. Gonçalves, Cláudia Castro, Pedro Jorge, José R.A. Fernandes and Paula
Martins-Lopes
15.00: PL3. How genetic and genomic tools may improve resistance to Phytophthora cinnamomi in chestnut
Rita Costa
Coordinator: Cecília Arraiano
3rd day: 27th May
Session 5: Human Genetics
Coordinators: Rui Martinho and Manuela Côrte-Real
9.00: OJ17. How the “major” satellite ncRNA of Felis catus is regulated in cancer?
Daniela Ferreira, Ana Escudeiro, Filipa Chaves, Filomena Adega and Raquel Chaves
9.12: OJ18. Targeting KRAS oncogene promoter by G-quadruplex ligands in human colon cancer cells
Hugo Brito, João Lavrado, Rui Moreira, Alexandra Paulo, Cecília M. P. Rodrigues and Pedro Borralho
9.24: OJ19. Testing short LNA-modified oligonucleotides for Duchenne Muscular Dystrophy gene therapy
Vanessa Borges Pires, Maria Carmo-Fonseca and Célia Carvalho
9.36: OJ20. Misreading tRNAs promote tumor progression in vivo
Patricia M. Pereira, Mafalda Santos, Ana Sofia Varanda, Joana Carvalho, Nuno Mendes, Patricia Oliveira, Marta
Teixeira Pinto, Fátima Carneiro, Carla Oliveira and Manuel A. S. Santos
9.48: OJ21. Transcription-coupled RNA quality control in human genetic diseases
Vaz-Drago Rita, Pinheiro MT, Martins S, Enguita FJ, Carmo-Fonseca M and Custódio N
10.00: OJ22. A role for the carboxyl-terminal domain of RNA Pol II in pre-mRNA splicing
Joana Tavares, Noélia Custódio and M. Carmo-Fonseca
10.12: OJ23. Exploring New Therapeutic Approaches for Hypertrophic Cardiomyopathy
Rita Mendes de Almeida, Teresa Carvalho, Sandra Martins and Maria Carmo-Fonseca
10.24: OJ24. Characterization of Notch signaling pathway at early stages of chick lung development
Carla Silva-Gonçalves, Patrícia Vaz-Cunha, Jorge Correia-Pinto and Rute S Moura
10.36: Coffee break
11.00: Poster session 3 (all)
6
Session 6: Genetics and Biotechnology
Coordinators: Sandra Viegas and Raul Machado
12.00: OS8. Genetic adaptive mechanisms mediating response and tolerance to acetic acid stress in the human
pathogen Candida glabrata: role of the CgHaa1-dependent signaling pathway
Ruben T. Bernardo, Diana V Cunha, Can Wang, Hiroji Chibana, Sónia Silva, Isabel Sá-Correia, Joana Azeredo, Geraldine
Butler and Nuno Pereira Mira
12.18: OJ25. Pseudomonas-based chassis towards à la carte biotransformation of plant-derived volatiles
Pedro Soares-Castro and Pedro Miguel Santos
12.30: Lunch
14.00: OJ26. Metagenomics-guided bioprospection of monoterpene biocatalysts in the rhyzosphere of coark oak
trees.
Filipa Gonçalves, Pedro Soares-Castro, Diana Afonso, Francisca Reis, Teresa Lino-Neto and Pedro Miguel Santos
14.12: OJ27. Synthetic Protein Biotechnology approaches for the creation of antimicrobial biopolymers
André da Costa, Raul Machado, Artur Ribeiro, Tony Collins, Viruthachalam Thiagarajan, Maria Teresa Neves-Petersen,
José Carlos Rodríguez-Cabello, Andreia C. Gomes and Margarida Casal
14.24: OJ28. High-level Biosynthesis of a Silk-Elastin-Like Protein in E. coli: Reducing Acetate Accumulation and
Plasmid Instability
Mário Barroca, Paulo Rodrigues, Fernando Branca, Margarida Casal and Tony Collins
14.36: OJ29. Lathyrus sativus L. Application of Biotechnological and Biochemistry techniques toward plant breeding
Letice Gonçalves, Carlota Vaz Patto, Rui Carvalho and Jorge Canhoto
14.48: PL4. Cancer, Genetics & Ecology
Jorge Pacheco
Coordinator: Margarida Casal
15.48: Awards and closing session
Prize Prof. Doutor Amândio Sampaio Tavares - Best Junior Oral Presentation (sponsored by Centro de Genética Clínica)
Prize Prof. Doutor Luís Archer - Best Poster Presentation (sponsored by Sociedade Portuguesa de Genética)
16.45: Closure
7
Plenary
Sessions
9
PL1.
The Archaeogenetics of West Eurasia
Martin B. Richards
1
1
Department of Biological Sciences, School of Applied Sciences, University of Huddersfield
The past thirty years have witnessed the birth of a new academic discipline. Archaeogenetics stems from the
realisation that we each carry a record of our evolutionary history within us, written in the language of DNA, with
genetic variants gradually accumulating over time. We can use these variants as markers to trace our ancestry back
into the past, tracking lineages back to modern humanity’s source in Africa hundreds of thousands of years ago. For
example, evidence from the maternally inherited mitochondrial DNA (mtDNA) suggests that modern non-Africans are
mainly descended from a single, small pioneer group who initially dispersed from eastern Africa along the tropical
coasts of southern Asia to Australia, around 60,000 years ago. However, the archaeogenetics of West Eurasian
ancestry remains deeply controversial. The revolution in DNA sequencing technologies in the last ten years has also
meant that we can now recover genetic sequences, and even whole genomes, from long-extinct species of human,
such as Neanderthals, leading both to new insights and new debates. The long-discussed extent to which European
Mesolithic forager populations versus Neolithic pioneers from the Near East contributed to the extant gene pool of
Europeans has received new impetus but continues to be contested. Moreover, ancient DNA evidence is beginning to
substantiate dispersals in Europe that could not be address using extant patterns. These new discoveries are
promising to once again shake up our ideas about human evolution in important ways.
PL2. The Biology and Functional Contribution of Exosomes in Cancer Progression and
Metastasis
Sónia Melo
1
1
Institute of Pathology and Molecular Immunology of the University of Porto, IPATIMUP
Exosomes are extracellular vesicles released by all cell types, which carry proteins, mRNA, ncRNAs and DNA. We have
recently shown intercellular trafficking via exosomes contributes to horizontal reprogramming and functional reeducation of recipient cells. More specifically we have shown that breast cancer associated exosomes contain
microRNAs (miRNAs) associated with the RISC Loading Complex (RLC) and display cell-independent capacity to process
precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present
in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer
exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes
derived from cells and sera of patients with breast cancer instigate non-tumorigenic epithelial cells to form tumors in
a Dicer-dependent manner. Additionally, and because the specific detection and isolation of cancer cell-derived
exosomes in circulation is currently lacking, we have used mass spectrometry analyses to identify a cell surface
proteoglycan, glypican-1 (GPC1), specifically enriched on cancer cell-derived exosomes. GPC1+ circulating exosomes
(crExos) were monitored and isolated using flow cytometry from the serum of cancer patients and mice with cancer.
GPC1+ crExos were detected in the serum of patients with pancreas cancer with absolute specificity and sensitivity,
distinguishing healthy subjects and patients with a benign pancreas disease from patients with early and late stage
pancreas cancer. Levels of GPC1+ crExos correlate with tumor burden and survival in patients pre- and post-surgical
tumor resection. GPC1+ crExos from patients and from mice with spontaneous pancreas tumors driven by oncogenic
KRAS contained RNA with specific KRAS mutation, and it emerges as a reliable biomarker for the detection of PanIN
lesions despite negative signal by MRI in mice. GPC1+ crExos may serve as a potential non-invasive diagnostic and
screening tool to detect early stages of pancreas cancer to facilitate possible curative surgical therapy. Currently we
are working on the hypothesis that within a heterogeneous tumor, exosomes-mediated transfer of information
modulates the cooperation between cancer cell subpopulations and the overall dynamics of the tumor.
11
PL3. How genetic and genomic tools may improve resistance to Phytophthora cinnamomi in
chestnut
Rita Costa
1
1
Unidade de Sistemas Agrários, Florestais e Sanidade Vegetal, Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Lisboa
The genus Castanea belongs to Fagaceae, a plant family that dominates much of the climax hardwood forests of the
Northern Hemisphere. The European chestnut (Castanea sativa Mill.) is considered to be the only native species of
Castanea in Europe. Chestnuts are multipurpose trees being used in the food industry, for its edible nuts that present
high quotation in international markets, in the wood industry, as timber, and also for ecological and landscaping
purposes, having a major economic importance in the Mediterranean region. Portugal is a major exporter of nuts
being the economy based on nut production very important in northern regions of Portugal. Root rot (caused by
Phytophthora cinnamomi Rands) is the most destructive disease affecting European chestnut, reducing by half the
productivity of chestnut orchards in Portugal. Therefore breeding for resistance to this pathogen is essential for
improving resistance of Castanea genus, with a positive impact in the economy of the agricultural sector which is
flourishing in Portugal. This sector is one where Portugal can be competitive in Europe, with a turnover of 3.4% in
2013-2014, when compared with 2011-2012.
A research programme was implemented using combined genetic and genomic approaches for mapping quantitative
trait loci (QTLs) linked to disease resistance, for a better understanding of the molecular mechanisms underlying
resistance of Castanea spp to Phytophthora cinnamomi, in order to improve the resistance of the host. In this talk the
conceptual and the logical framework of the research programme will be presented, pointing out the most important
results obtained so far and the goals to achieve in the near future. The idea is to apply the knowledge obtained from
fundamental research, taking profit of the recent technological advances and the new tools available, for speeding up
the selection of improved genotypes of the breeding programme with increased resistance to the pathogen. The final
goal is the introduction of improved genotypes into the market, to overcome the high deficit that exists nowadays in
Europe of improved chestnut plants for plantation.
PL4.
Cancer, Genetics & Ecology*
Jorge Pacheco
1,2
1
Centre of Molecular and Environmental Biology & Departamento de Matemática e Aplicações, Universidade do Minho
ATP-Group - Instituto para a Investigação Interdisciplinar, Universidade de Lisboa
2
The etiology of cancer is multifactorial, with genetic and environmental factors, among others, contributing to
produce a given malignancy. Although knowledge of cancer genetics is rapidly improving our understanding of cancer
biology, to understand the dynamics of tumor growth and response to therapy we also need to investigate the
interactions between malignant cells and normal cells in the environment in which they take place. Here I will provide
concrete examples demonstrating the need for such an understanding of the ecology of cancer, discussing also how
Evolutionary Game Theory (EGT) constitutes a useful and novel framework in which to capture the frequencydependent nature of the dynamics of such ecosystems. In particular, I will show how EGT can be applied to the study
of multiple myeloma bone disease, allowing one to predict the types and timings of interventions that can alter the
natural evolution of the disease.
* Work carried out with David Dingli (Mayo Clinic)
12
Session 1:
Evolutionary Genomics
Coordinators:
Cláudio Sunkel (IBMC) and Maria João Sousa (CBMA)
13
OS1.
Drosophila americana diapausing females show features typical of young flies.
12
13
12
Micael Reis , Felipe B. Valer , Cristina P. Vieira , and Jorge Vieira
12
1
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal
Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto - Portugal
3
Instituto de Biologia, Universidade Federal de Pelotas - UFPel, Pelotas, Rio Grande do Sul, Brazil
2
Diapause is a period of arrested development which is controlled physiologically, preprogrammed environmentally
and characterized by metabolic depression that can occur during any stage of insect development. Nevertheless, in
the genus Drosophila, diapause is almost always associated with the cessation of ovarian development and
reproductive activity in adult females. In this work, we show that, in D. americana (a temperate species of the virilis
group), diapause is a genetically determined delay in ovarian development that is triggered by temperature and/or
photoperiod. Moreover, we show that in this species diapause incidence increases with latitude, ranging from 13% in
the southernmost to 91% in the northernmost range of the distribution. When exposed to diapause inducing
conditions, both diapausing and non-diapausing females show a 10% increase in lifespan, that is further increased by
18.6% in diapausing females, although senescence is far from being negligible. ActinD1 expression levels suggest that
diapausing females are biologically much younger than their chronological age, and that the fly as a whole, rather than
the ovarian development alone, which is phenotypically more evident, is delayed by diapause. Therefore, diapause
candidate genes that show expression levels that are compatible with flies younger than their chronological age may
not necessarily play a role in reproductive diapause and in adaptation to seasonally varying environmental conditions.
OS2. Genetic ancestry of Austronesian-speaking populations in Island Southeast Asia and the
Pacific
1,2
2,3
2
Pedro Soares , Teresa Rito , Bruno Cavadas , Andreia Brandão
2,7
4
Pereira and Martin B Richards
2,4,5
4
6
, Maria Pala , Stephen Oppenheimer , Luísa
1
CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Braga, Portugal
Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal
3
ICVS/3B’s - PT Government Associate Laboratory, School of Health Sciences, University of Minho. Portugal
4
Department of Biological Sciences, School of Applied Sciences, University of Huddersfield
5
Instituto de Ciências Biomédicas da Universidade do Porto (ICBAS), Porto, Portugal
6
University of Oxford, Institute of Social and Cultural Anthropology, UK
7
Faculdade de Medicina da Universidade do Porto, Porto, Portugal.
2
Austronesian languages are spoken throughout a vast area including Island Southeast Asia (ISEA), part of New Guinea
and the Pacific Islands. The high diversity of the language family within the aboriginal tribes of Taiwan inspired a
model that proposes a major Neolithic expansion of Austronesian speakers from Taiwan within the last 5000 years –
the Out-of-Taiwan model. However genetic data has often supported a genetic pool of ISEA structured by
autochthonous dispersals triggered by the drastic shaping on the land due to sea-level rises in the postglacial period.
We combined data from the maternal mitochondrial DNA (mtDNA) and paternal Y-chromosome lineages with
published and new genome-wide data to obtain a comprehensive picture of the region with consistent results in the
three systems. Although most of the common genetic ancestry of Taiwan and ISEA was already established in the
Post-Glacial period, two independent minor Neolithic genetic inputs from Mainland Southeast Asia and Taiwan are
visible in the data. The later one, strongly signalled by mtDNA haplogroup M7, matches an Out-of-Taiwan dispersal
that could have mediated the spread of Austronesian languages. However, considering the small-scale migration
detected, it implies that language shift by the autochthonous people, rather than large population replacement, was
the main route for the spread of Austronesian languages.
Acknowledgments: FCT ( PTDC/IVC-ANT/4917/2012, SFRH/BD/69353/2010, Investigador FCT)
15
OS3. Intracellular endosymbiont selection contributes to Drosophila adaptation to viral
infection
1
1
2
3
3
1
Vitor G. Faria , Nelson E. Martins , Sara Magalhães , Viola Nolte , Christian Schlötterer , Luis Teixeira and Élio
1,4
Sucena
1
Instituto Gulbenkian de Ciências, Apartado 14, 2780-901 Oeiras, Portugal.
cE3c: Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa,
Portugal.
3
Institut für Populationsgenetik, Vetmeduni Vienna, 1210 Wien, Austria.
4
Universidade de Lisboa, Faculdade de Ciências, Departamento de Biologia Animal, edifício C2, Campo Grande, 1749-016 Lisboa, Portugal.
2
Bacteria of the genus Wolbachia are intracellular symbionts of many animal species. In Drosophila, Wolbachia has
been shown to confer protection to RNA virus infections in a strain-dependent manner. Through experimental
evolution, we have selected an outbred Wolbachia-infected population of D. melanogaster for increased resistance to
DCV, a natural viral pathogen. Whole-genome sequencing of this population, upon 20 generations of selection,
revealed that a Wolbachia sub-strain was fixed. Moreover, we show that challenged inter-population hybrids carrying
either Wolbachia variant differ in their fitness, confirming the adaptive value to the host of the selected
endosymbiont. In addition we are presently re-assessing host genome evolution upon Wolbachia clearance and DCV
infection over 20 more generations. These findings demonstrate that the presence of protective endosymbiont plays a
role in shaping the host genome and its own evolution may have a profound effect on host adaptation.
16
Session 2:
Microbial Genetics
Coordinators:
Manuel Santos (UAveiro) and Tony Collins (CBMA)
17
OS4.
Multitask ATPases: a common theme in bacteria?
1
1
Mário J. Ferreira , Aristides L. Mendes and Isabel de Sá Nogueira
1
1
UCIBIO, REQUIMTE, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica,
Portugal.
Transport across biological membranes is fundamental for cell survival and is mainly accomplished by specialized
membrane proteins known as transporters. The ABC-type transporters constitute one of the largest and most diverse
transporter superfamilies characterized by a highly conserved ATP-binding cassette. These systems are widespread
among living organisms and have been found in all species from the microbe to man, with a high conservation of the
primary sequence of the ATP-hydrolyzing domain and in the modular architecture comprising two transmembrane
domains (TMDs) that form the translocation pore and two nucleotide-binding domains (NBDs) that hydrolyse the
necessary ATP for the translocation process. Until the last few years, NBDs were thought to be exclusive of each
transporter complex, however it was discovered that distinct bacterial ABC type I importers share the same energygenerating component. In the Gram-positive model organism Bacillus subtilis ten ABC transporters are predicted to be
involved in sugar uptake and eight of these systems do not display a gene encoding a putative NBD protein in their
vicinity. Recent studies, performed in our laboratory demonstrated that MsmX interacts with several of these eight
distinct ABC sugar importers. Thus, unlike other NBDs, MsmX was shown to be multitask serving as energy-generating
component to several sugar importers. The implications of sharing energy-generating components among ABC
importers in bacteria will be discussed.
OJ1. Establishment of the MurT/GatD complex as a new virulence factor – role in host
evasion.
1,2
2,3
2,4
1
1
Bárbara Gonçalves , Ana Madalena Ludovice , Hermínia de Lencastre , Luís Jaime Mota , Rita Sobral
1
UCIBIO-REQUIMTE, Departamento de Ciências de Vida (DCV), Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa (FCT/UNL), Monte
da Caparica, Portugal;
2
Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica (ITQB), Oeiras, Portugal.
3
Departamento de Ciências de Vida (DCV), Faculdade de Ciências e Tecnologias Universidade Nova de Lisboa (FCT/UNL), Monte da Caparica,
Portugal;
4
Laboratory of Microbiology and Infectious Diseases, The Rockefeller University, New York, USA
Staphylococcus aureus is a dangerous opportunistic pathogen responsible for a wide variety of infections and high
morbidity and mortality. The clinical importance of S. aureus is mainly due to its high capacity to accumulate
resistance mechanisms to virtually all antibiotics. Therefore, despite the large number of antibiotics available, new
strategies to treat S. aureus infections are urgently needed.The murT-gatD operon encodes the enzymes responsible
for the amidation of the D-glutamate residue of peptidoglycan into D-iso-glutamine. Glutamate amidation, a
secondary modification of peptidoglycan, is essential for S. aureus viability and is involved in the mechanisms of
resistance to β-lactams and lysozyme, being an excellent target for the development of new antimicrobial compounds
[1].Resistance to lysozyme, a muramidase produced by the innate immune system that causes cell wall degradation
and cell lysis, is an important intrinsic characteristic of S. aureus allowing its establishment as an intracellular
pathogen. In fact, lysozyme resistance has an impact on the efficiency of bacterial killing by macrophages.
Furthermore, the presence of D-iso-glutamine in the stem peptide influences the recognition of peptidoglycan by the
innate immune system via NOD1/NOD2, suggesting an involvement of amidation in immune evasion. In this way,
peptidoglycan amidation is the only modification known to be implicated in both antibiotic resistance and
virulence.We are currently studying the role of MurT-GatD in the interaction of S. aureus with mammalian immune
cells by comparing the survival of S. aureus wild-type strains with murT-gatD isogenic mutants within human (THP-1)
and mice (RAW264.7) macrophage cell lines. The results of these experiments will be presented and discussed.
[1] Figueiredo et al. 2012. Identification of genetic determinants and enzymes involved with the amidation of glutamic acid residues in the peptidoglycan of S. aureus.
PLoS pathog. 8, e1002508.
19
OJ2. Unravelling the role of Escherichia coli BolA, a pleiotropic protein that turns off motility
and turns on biofilm formation
1
1
1
2
Susana Barahona , Ricardo Neves Moreira , Clémentine Dressaire , António Pedro Alves de Matos , and Cecília Maria
1
Arraiano
1
Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
Egas Moniz – Cooperativa de Ensino Superior, CRL-Campus Universitário – Quinta da Granja – Monte de Caparica, 2829-511 Caparica, Portugal.
2
Bacteria are extremely versatile organisms, which rapidly adapt to changing environments. When bacterial cells switch
from planktonic growth to biofilm, flagellum formation is turned off, and the production of fimbriae and extracellular
polysaccharides is switched on. BolA is present in most Gram-negative bacteria and homologues can be found from
proteobacteria to eukaryotes. Here we show that BolA is a new bacterial transcription factor, which modulates the
switch from planktonic to sessile lifestyle. It negatively modulates flagellar biosynthesis and swimming capacity in
Escherichia coli. Furthermore, BolA overexpression favors biofilm formation, involving fimbriae-like adhesins and curli
production. Our results also demonstrate that BolA is a protein with high affinity to DNA, and is able to regulate many
genes on a genome-wide scale. Herein we propose that BolA is a motile/adhesive transcriptional switch, specifically
involved in the transition between the planktonic and the attachment stage of biofilm formation.
[1] Dressaire* C., Moreira* R.N., Barahona S., Alves de Matos A.P., and Arraiano C.M., "BolA is a transcriptional switch that turns off motility and turns on biofilm
development". * equal contribution. mBio (2015); doi:10.1128/mBio.02352-14
OJ3. Time series analysis of the transciptome of a Staphylococcus aureus mutant with an
impaired cell wall
1
Raquel Portela , Ana Madalena Ludovice
1,2
3
3
, Rui Catarino , Francisco Pinto and Rita Sobral
1
1
Laboratory of Molecular Microbiology of Bacterial Pathogens, UCIBIO@REQUIMTE, Departamento de Ciências da Vida, Faculdade de Ciências e
Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal
2
Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Oeiras, Portugal
3
Biosystems and Integrative Sciences Institute (BioISI), Faculdade de Ciências, Universidade de Lisboa, Portugal
Bactericidal antibiotics, independently of the primary target, were described to promote cell death by stimulating the
production of hydroxyl radicals through TCA cycle, production of NADH and the fenton reaction. The transcriptomic
profile of a Staphylococcus aureus conditional cell wall mutant (COLspacmurF) suggested that the same physiological
alterations are occurring in response to cell wall damage, involving shift of energy producing pathways away from O 2
respiration and TCA cycle by stimulating fermentation. To identify the metabolic steps which link the primary damage
to the production of hydroxyl radicals, we determined a time scale of expression events for COLspacmurF.
COLspacmurF was grown with and without inducer and total RNA was extracted for 3 biological replicates, at 6
consecutive time points, along the exponential and early stationary phase. The 36 RNA samples were tested for
integrity and hybridized against Affymetrix microarrays. The peptidoglycan of COLspacmurF grown with and without
inducer was extracted along time, purified and analysed by RP-HPLC. The incorporation of the abnormal muropeptide
was quantified and, unexpectedly, found to accumulate constantly along growth reaching over 30% of the total
peptidoglycan.The transcriptome profile of each time-point was associated to the respective HPLC pattern, providing a
relation between the cell response and the degree of cell wall damage. The microarray data was integrated through
an in silico reconstructed metabolic network model for S. aureus strain COL. So far, the results indicate that two main
metabolic pathways are affected in the mutant along time: the wall teichoic acids and lipids biosynthetic
pathways.Such observations are currently being validated through the analysis of the cell wall teichoic acids and lipids
of the mutant along time.
20
OJ4.
Identification of HIV-1, HCV and HBV using multiplex PCR
1,2
3
4
5
5
Adriana Resende , Ana Constança , Isabel Pereira-Castro , Amanda Bradley Stewart , Rory N. Gunson , Helena
3
1
Ramos and Filipe Pereira
1
CIIMAR/CIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Porto, Portugal
Faculty of Sciences, University of Lisbon, Lisbon, Portugal
3
Microbiology Laboratory, Santo António Hospital, Porto, Portugal
4
Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal
5
West of Scotland Specialist Virology Centre, Glasgow, Scotland UK
2
The human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV) and hepatitis B virus (HBV) are a significant
threat to public health worldwide, making it necessary to improve clinical diagnosis and extend epidemiological
studies. Diagnostic methods based on nucleic acid testing are more sensitive than traditional serological testing and
allow the direct viral identification. However, the design of efficient nucleic acid-based assays is challenged by the high
genetic diversity characteristic of viruses, particularly RNA viruses (HIV-1 and HCV). Consequently, methods that rely
on the analysis of one or two genomic regions for the viral identification are more susceptible to produce falsenegative results due to sequence variation at primer or probe binding sites. Here we describe three multiplex PCR
assays for the amplification of five HIV-1, six HCV and five HBV genomic regions. The assays are designed to base all
identifications on the analysis of multiple regions, reducing the likelihood of false-negative results due to
polymorphisms. PCR primers were designed to avoid polymorphisms that may inhibit the PCR amplification and virus
detection. The selected regions were first validated by singleplex PCR and then combined in multiplex reactions. No
false negative results were observed, i.e., all tested samples yielded positive amplifications. The assays detected the
viral infection in samples with low viral loads and no cross-reactivity were observed with other viruses. Overall, these
assays can be used to study HIV-1, HCV and HBV with high specificity and robustness using low cost laboratory
equipment and the markers selected here could be used in other assays or sequenced for genetic analysis.
OJ5. Microbiome Characterization of a Striped Dolphin (Stenella coeruleoalba) of the Coast of
Portugal
1
1
1
1
1,2,3
3,4
Ana L Alves , Filipa Gonçalves , Cristina S Mesquita , Pedro Soares-Castro , Marisa Ferreira , Ana Marçalo , José
1,2,3,4
3,4
5
5
5
5
1
Vingada
, Catarina Eira , Filipa Godoy-Vitorino , B Cabrera , A Rodriguez , J Fabre and Pedro M. Santos *
1
CBMA – Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, Campus de Gualtar, Braga, Portugal
Department of Biology, University do Minho, Braga, Portugal
3
Portuguese Wildlife Society (SPVS), Quiaios Field Station, Figueira da Foz, Portugal,
4
CESAM and Department of Biology, University of Aveiro, Aveiro, Portugal
5
Inter American University of Puerto Rico, Metropolitan Campus, San Juan, Puerto Rico
2
The Iberian Peninsula has one of the highest marine mammal stranding rates in the world. These animals are a key
group acting as sentinels of disturbance of the marine environment with a variety of infectious diseases with epizootic
consequences recently divulged. Recent developments in NGS technology have extraordinarily enhanced the
possibility to compare the microbiome in health and disease status. This study is the first systematic effort for
evaluation of dolphin microbiome in different body sites. One striped female, underweight dolphin stranded in
Algarve (Portugal) was necropsied at the Center for Marine Animal Rehabilitation of Quiaios (CRAM-Q). Samples from
a variety of body niches including blowhole, skin, oral cavity, oral mucosa, tongue, stomach, intestines and genital
mucosa were collected for DNA extraction with a bead beating method. The 16S rRNA V3-V4 region was sequenced
with Illumina MiSeq and data analysis done using the QIIME pipeline and the R package. Analysis of nearly 1 million
sequences with an average length of 466 bp identified 37,303 OTUs. A total of 16 bacterial phyla were detected, with
a dominance of Proteobacteria (skin, blowhole and mouth); Fusobacteria (genital and oral samples); Firmicutes and
Bacteroidetes (colon, genital mucosa and duodenum) and Tenericutes (stomach). Pathogenic, gram-negative,
facultative or obligate anaerobic taxa were significantly detected, including Pasteurella, Cetobacterium (both
dominated oral-type samples), Cardiobacteriaceae (skin and blowhole) or Mycoplasma (depicts more than 50% of
stomach bacteria) representing over 50% of the entire microbial community. The preliminary results highlight
variation in structure and diversity according to organ type and a prevalence of gram-negative anaerobic pathogens in
such high dominance confirms the animal diseased status. Comparisons between healthy and stranded striped
dolphins are underway as between multiple dolphin species.
21
OS5. Genetic determinants responsible for the adaptation of Saccharomyces cerevisiae strains
to different ecological niches
1
1
1
Ricardo Franco-Duarte , Paula Sampaio and Célia Pais
1
Centro de Biologia Molecular e Ambiental (CBMA), Departamento de Biologia, Universidade do Minho, Braga, Portugal
The maintenance of microbial species in different environments is associated with adaptive microevolutionary
changes that are not completely understood. Saccharomyces cerevisiae strains undergo genomic changes associated
with generation of phenotypic heterogeneity useful for different technological applications. However, continuous
utilization of yeast strains for industrial purposes introduced artificial selective pressures that are reflected by genome
polymorphisms and consequent phenotypic diversity. One example is the emerging of S. cerevisiae strains as human
pathogens, capable of causing clinically relevant infections. The new approaches of integrative data mining developed
with great potential to relate genotype with phenotype may contribute to understand the genomic variations in
strains from these environments. From our strain collection, four clinical isolates and four isolates from natural
environments were selected. Principal component analysis revealed a complete separation between both groups of
strains, regarding both the 30 phenotypic tests considered and a set of 11 polymorphic microsatellites. Genomic DNA
was sequenced using an Illumina HiSeq2000 analyzer. All reads were aligned with the yeast reference genome and
comparative analysis was performed to evaluate intra-genomic variation. Exclusive nucleotide polymorphisms were
identified in the genome of the clinical strains, in comparison with strains isolated from the environment, both in the
form of non-synonymous SNP and/or frameshift insertions and deletions, showing a stochastic distribution along the
genome. Copy-number variations in some genome segments were also identified between clinical strains and
environmental isolates. In conclusion, results show genetic changes that could be used to explain the adaptation of
yeast strains to new ecological niches, in particular as determinants of the yeast clinical phenotype.
This work was financed in part by the project TRANSBIO (FP7 / nº289603).
22
Session 3:
Gene regulation and
expression
Coordinators:
Isabel Sá-Nogueira (UCIBIO, REQUIMTE) and Sandra Paiva
(CBMA)
23
OJ6.
Cellular networks that regulate adaptation to protein synthesis errors in HEK293 cells
Ana Sofia Varanda
1,2,3
, Mafalda Santos
1,2,3
, Carla Oliveira
3,4
and Manuel A. S. Santos
1
1
iBiMED&Health Sciences, University of Aveiro, 3810-193 Aveiro, Portugal
CESAM, Biology Department, University of Aveiro, 3810-193 Aveiro, Portugal
3
Institute of Molecular Pathology and Immunology, University of Porto (IPATIMUP), 4200-465 Porto, Portugal. 4Faculty of Medicine, University of
Porto, 4200-465 Porto, Portugal
2
Protein synthesis is a highly regulated process and maintenance of its fidelity is essential for life. However, errors do
occur and their clearance requires protein quality control systems that prevent the accumulation of misfolded and
aggregated proteins [1,2]. In order to elucidate how human cells respond to the accumulation of protein synthesis
errors, we have modified the anticodon of a human serine tRNA to misincorporate the amino acid serine at alanine,
leucine and histidine sites [2]. These engineered tRNAs were stably transfected into HEK293 cells.Cells expressing
misreading tRNAs showed normal proliferation and viability and did not show an increase in aggregates or
ubiquitinated proteins after several generations in culture. These suggest that HEK293 cells adapted to protein
synthesis errors through activation of protein quality control mechanisms that allow the clearance of aberrant
proteins. We show that activation of quality control pathways, i.e., ubiquitin-proteasome system, unfolded protein
response and molecular chaperones expression, are dependent on the type of amino acid substitution in the
proteome. These data provide new insight into protein synthesis errors and human cellular pathways that are relevant
for proteome homeostasis.
[1] Reynolds, N. M., Lazazzera, B. A. & Ibba, M. Cellular mechanisms that control mistranslation. Nature Reviews Microbiology 8, 849–856 (2010). ; [2] Geslain, R. et al.
Chimeric tRNAs as tools to induce proteome damage and identify components of stress responses. Nucleic Acids Research 38, e30 (2010).
OJ7.
A long non-coding RNA regulating stemness and cellular reprogramming
1,2
1
1*
Sara Barros , Catarina Alves-Vale , Bruno Bernardes de Jesus and Maria Carmo-Fonseca
1*
1
Instituto de Medicina Molecular, Lisboa, Portugal
Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal
2
Understanding the aging process is critical to prevent and counteract the effects of age-related decline of cells and
tissues. Throughout time, due to the accumulation of genetic and epigenetic changes, stem cells tend to lose their
capacity to replace damaged tissues. A major breakthrough advance was the identification of a combination of four
transcription factors which expression reverts somatic to pluripotent cells (named cellular reprogramming). However,
as differentiated cells grow old, they become more and more difficult to be converted back to pluripotency. Our
project delves into the interesting world of long non-coding RNAs (lncRNAs) and its implications in stemness (selfrenewal and pluripotency) and cellular reprogramming. We are particularly interested on natural antisense transcripts
(NATs), transcripts transcribed from the opposite strand of coding genes, since they are less studied than other noncoding transcripts. We selected Zeb2NAT, the natural antisense transcript of Zeb2, involved in mesenchymal epithelial
transition, an early event during cellular reprogramming of fibroblasts. The first observation was that the downregulation of Zeb2NAT in mouse embryonic stem cells leads to an increase of proliferation and pluripotency, as
observed by higher levels of Oct4. For the reprogramming assay we used adult fibroblasts of a transgenic mouse
model carrying a doxycycline-inducible cassette with the four Yamanaka’s factors. Down-regulation of Zeb2NAT led to
a significant increase in the number of induced pluripotent stem cells (iPS cells) in culture. At the moment we are
characterizing the resulting iPS cell clones for pluripotency. We demonstrated for the first time that a lncRNAs NAT
can enhance stemness and cellular reprogramming of aged cells, highlighting the novelty and possible contribution of
this work to improve the development of stem cell-based therapies.
25
OS6.
Regulation of transcriptional reactivation of the oocyte during meiosis.
Paulo Navarro-Costa
1,2,3
, Pedro Prudêncio
1
1,2,3
3
, Jörg D. Becker and Rui Gonçalo Martinho
1,2,3
2
Departamento de Ciências Biomédicas e Medicina, and Center for Biomedical Research, Universidade do Algarve, Campus de Gambelas, 8005-139
Faro, Portugal.
3
Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, Oeiras 2781-901, Portugal.
During oogenesis oocytes transit from stages of transcriptional activity to those of transcriptional quiescence, and
such transitions are believed to be essential for proper gamete formation. Although the temporal regulation of these
transitions has been well documented across diverse organisms, the molecular mechanisms underlying these
processes remain largely unknown.We will present evidences that in Drosophila the histone demethylase KDM5 is
required for the correct temporal reactivation of the oocyte’s transcriptional activity. Germline-specific depletion of
dKDM5 results in severely reduced female fertility. Oocytes display precocious transcriptional reactivation and an
equally precocious chromatin remodeling, leading to the premature acquisition of an open chromatin configuration.
Both effects are a likely consequence of the significant up-regulation of H3K4me3 levels in the ovary, particularly in
the meiotically arrested oocyte. Increased H3K4me3 levels seem to solely impact the transcriptional status of the
oocyte, as no evidence for either the activation of the DNA damage checkpoint or meiotic maturation defects were
recorded. Since dKDM5 is evicted from the oocyte’s chromatin by early oogenesis and the oocyte transcriptional
defects are observed approximately 24 hours later, we propose that the temporal regulation of the transient
transcriptional reactivation of the maturing oocyte relies on H3K4me3-based epigenetic memory mechanism.
OJ8.
A novel protein complex involved in ribosome biogenesis and rRNA quality control
1
1
1
Ricardo F. dos Santos , José M. Andrade , Joana Pissarra and Cecília M. Arraiano
1
1
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB/UNL), Oeiras, Portugal
The ribosome is the universal macromolecular decoder responsible for protein synthesis. The prokaryotic ribosome is
built of three ribosomal RNAs (5S, 16S and 23S rRNA) and several ribosomal proteins. The maturation and assembly of
all these components is a tightly regulated process, leading to the formation of the functional 70S ribosomal particle.
In this work we describe a new quality control pathway involved in the degradation of defective rRNA and surveillance
of ribosome biogenesis that requires the RNA-binding proteins Hfq and RNase R. The RNA chaperone Hfq and the 3’-5’
exoribonuclease R interact and this novel protein complex is critical for the elimination of aberrant rRNA fragments
and processing of rRNA precursors. Moreover, the ribosomal profile of the Δhfq Δrnr strain is significantly altered. In
the absence of Hfq and RNase R the amount of immature 30S and 50S subunits increase and the levels of functional
70S ribosomes are drastically decreased, which is consistent with the existence of severe defects in ribosome
assembly. This is the first demonstration that the well-conserved Hfq and RNase R proteins interact in vivo, unraveling
an unknown mechanism of RNA-quality control. This cooperation adds a new layer of regulation for the multi-stepped
process of ribosome biogenesis.
26
OJ9.
Measurement of Long Noncoding RNA kinetics in T-cells
1
1
1
Soraia da Silva *, Rita Vaz‐Drago , Maria Carmo‐Fonseca and Noélia Custódio
1
1
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028, Lisboa, Portugal
* Master student of Oncobiology, Universidade do Algarve, Portugal
Long non‐coding RNAs (lncRNAs) are transcripts with more than 200 nt in length and no evident open reading frames
(ORFs). These transcripts have been implicated in a variety of developmental processes such as imprinting and
pluripotency. Several classes of lncRNAs have been described according to their genomic location in relation to
protein‐coding genes, including natural antisense transcripts (NATs). In our lab we are interested in identifying
lncRNAs as potential targets for RNA therapeutics. Here, we present a methodology to monitor synthesis and lifetime
of lncRNAs in a cellular mouse model of T cell acute lymphoblastic leukemia (T‐ALL). We are using cultured mouse Tcells derived from induced T-ALL tumors. To measure lncRNA stability we carried out pulse labeling with the uridine
analogue 4- thioridine (4sU) followed by purification of labeled nascent and total cellular RNA. Real-time RT-PCR (qRTPCR) quantification of the obtained RNA subsets allows the estimation of the selected lncRNA’s half-life. For this study
we selected a list of lncRNAs that are expressed in T‐cells and may have a relevant role in leukemogenisis. The
lncRNAS analyzed are: Malat1, Neat1, Airn, Ens91 (BC028471-004), Ens87 (Gm16534) and Zeb2-NAT. We determined
by qRT-PCR the lncRNA’s expression levels in the T-ALL cell line and Malat1 is the most expressed followed by Airn,
Neat1 and Ens87. Ens91 and Zeb2-NAT are expressed at very low levels. The half-life results indicate that Malat1 is the
most stable with a half-life around 4.5h and the remaining have half-lives of 2-3h. It has been proposed that the halflife of each lncRNA is related to its physiological function. The major expected outcome of this project is gaining a
novel insight into the biology of lncRNAs in the context of leukemogenesis with the ultimate goal of finding possible
transcript targets to RNA therapeutics for T‐ALL.
OJ10. Functional characterization of a putative tRNA/rRNA methyltransferase on somatic
embryogenesis
1
1
1
2
Sandra Correia , Patrícia Fernandes , Bruno Casimiro , Paula Veríssimo , Jorge Canhoto
1
1
Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal
Centre for Neuroscience and Cell Biology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal
2
Somatic embryogenesis (SE) is an important biotechnological tool for large-scale clonal propagation. Different aspects
related to SE induction and somatic embryo development in tamarillo, a woody plant of the Solanaceae family, have
been studied in our laboratory. The experiments performed led to the development of a protocol for the in vitro
establishment of embryogenic and non-embryogenic calli of tamarillo. Previous comparative proteomic analyses
identified a protein (NEP-TC, 26.5 kDa) consistently present in non-embryogenic calli, with a high degree of identity
with the Arabidopsis thaliana tRNA/rRNA methyltransferase (SpoU) family proteins. The role of NEP-TC was assessed
by the evaluation of NEP-TC orthologous gene knockout lines of Arabidopsis thaliana, and methyltransferase activity
analysis during the somatic embryogenesis process. The results obtained demonstrated that NEP-TC orthologous
showed expression levels 3-fold lower than in leaves or callus explants. Although tamarillo’s NEP-TC down-regulated
lines did not show significant differences in terms of development or SE induction rates, Arabidopsis NEP-TC
orthologous knockout plants exhibited enhanced levels of somatic embryogenesis and secondary root induction. The
confirmation that CNE samples with high NEP-TC expression have enhanced methyltransferase activity also supports
the role of this protein on SE induction. The data obtained associates (for the first time) a putative member of the
SpoU methylase super family with plant morphogenesis, in particular with the SE induction process.
27
OJ11. The role of mitochondrial phospholipid composition in Saccharomyces cerevisiae acetic
acid-induced apoptosis
1
1
2
2
1
Catarina Afonso , Tânia Fernandes , Elisabete Maciel , Rosário Domingues , Manuela Côrte-Real , Maria João Sousa
1
1
CBMA – Centro de Biologia Molecular e Ambiental, Universidade do Minho, Braga, Portugal
Mass Spectrometry Centre, UI QOPNA, Department of Chemistry, University of Aveiro, Aveiro, Portugal
2
Endoplasmic reticulum-mitochondria contact sites (ER-CS), found from yeast to mammals, are interface structures
where membranes of the two organelles are maintained in close proximity, by tethering protein complexes. These
structures play a prominent role in the communication between the two organelles, including the transport of
phospholipids. Recent studies on cell death have implicated these contact sites in apoptotic signalling but the exact
mechanisms underlying its involvement are still unclear. In the present work we questioned if modulation of
mitochondrial phospholipid content by ER-CS could have a role in Saccharomyces cerevisiae acetic acid-induced
apoptosis. We also aimed to assess if perturbation of mitochondrial phospholipid content by deletion of other
proteins involved in phospholipid synthesis, namely, cardiolipin biosynthesis (Crd1p) and maturation (Taz1p), or
phospholipid transport from the OMM to the IMM (Ups1) could influence acetic acid induced apoptosis. Cell viability
assays and assessment of cell death markers were conducted with S. cerevisiae BY4741 and Δmdm10 and Δmdm12
mutants (deficient in two proteins belonging to the ER-Mitochondria Encounter Structure, the yeast structure
tethering ER and mitochondria membranes together) as well as with the Δcrd1, Δtaz1 and Δups1 strains. Results
showed that all the mutated strains display decreased apoptotic cell death in response to acetic acid treatment, Δtaz1
and Δmdm10 being the most and Δcrd1 the less resistant. Phospholipid composition of mitochondria isolated from all
the strains, before and after acetic acid treatment, was determined by lipid extraction, followed by thin-layer
chromatography (TLC) and lipid quantification by phosphorus measurement. Comparison of the mitochondrial
phospholipid content, suggested that the differences in cell survival observed for the ER-CS deficient strains are not
related with differences in the relative percentages of major classes of phospholipids.
OJ12. Functional analysis of String/CDC25 phosphatase in post-mitotic neurons
Rui Gonçalves
1,2
and Carla S. Lopes
1
1
Neurodevelopment and Degeneration Group, IBMC – Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal.
Institute for Investigation and Innovation in Health, University of Porto, Portugal.
2
The activity of CDC25 phosphatase (PPase) is associated to proliferating tissues, where they play a key role as CDKs
activators. However, expression and activity of mammalian CDC25s has been reported in adult brains and increased
expression levels were associated to Alzheimer’s disease [1]. The physiological relevance and the potential substrates
of CDC25s in a non-proliferative context has never been addressed. We are using Drosophila visual system as a model
to uncover the function and substrates of CDC25 PPases in post-mitotic neurons. Drosophila genome encodes one
CDC25 homologue, string (stg). Previous studies from our group show that stg is expressed in photoreceptor (PRs) and
lamina neurons of the fly visual system. As a means to promote stg downregulation and address its function in PR-cells
we are using the Gal4/UAS system combined with RNAi. Downregulation of stg in PR-cells leads to roughness of the
adult eye. In the larvae, PR-cells fail to project into the brain and their axonal structure is disrupted, which is a
hallmark of neurodegeneration. These results suggest that stg has an essential function in post-mitotic neurons. We
are currently addressing the molecular mechanisms underlying this phenotype. In order to identify potential direct
substrates of Stg in PR-cells, we are combining the functional analysis with a proteomic approach. We will present our
latest results on this subject. Overall our work suggest that Stg/CDC25 may play a fundamental role in nervous system
development and its function may be linked with neurodisease onset.
[1] K.-H. Chang, F. Vincent, and K. Shah, “Deregulated Cdk5 Triggers Aberrant Activation of Cell Cycle Kinases and Phosphatases Inducing Neuronal Death,” J. Cell Sci., pp.
5124–5137, 2012.
28
OJ13. Candida glabrata susceptibility to antifungals and phagocytosis is modulated by the
carbon source
1, 2
1
1
3
4
4
1
Sandra Mota , Rosana Alves , Catarina Carneiro , Sónia Silva , Fabian Istel , Karl Kuchler , Paula Sampaio , Margarida
1
3
1
Casal , Mariana Henriques , Sandra Paiva
1
CISA – Centro de Investigação em Saúde e Ambiente, Escola Superior de Tecnologia de Saúde do Porto, Instituto Politécnico do Porto, Porto,
Portugal
3
CEB – Centro de Engenharia Biológica, Laboratório de Investigação em Biofilmes Rosário Oliveira, Universidade do Minho, Braga, Portugal
4
Medical University Vienna, Max F. Perutz Laboratories, Department of Medical Biochemistry, Vienna, Austria
2
Candida glabrata is considered a major opportunistic fungal pathogen of humans. The capacity of this yeast species to
cause infections is dependent on the ability to grow within the human host environment and to assimilate the carbon
sources available. Previous studies have suggested that C. albicans can encounter glucose-poor microenvironments
during infection and that the ability to use alternative non-fermentable carbon sources, such as carboxylic acids,
contributes to the virulence of this fungus. Transcriptional studies on C. glabrata cells identified a similar response,
upon nutrient deprivation. In this work, we aimed at analysing biofilm formation, antifungal drug resistance and
phagocytosis of C. glabrata cells grown in the presence of acetic acid as an alternative carbon source. C. glabrata
planktonic cells grown in media containing acetic acid were more susceptible to fluconazole and were better
phagocytosed and killed by macrophages than when compared to media lacking acetic acid. Growth in acetic acid also
affected the ability of C. glabrata to form biofilms. Some genes enconding putative carboxylate transporters were
upregulated in C. glabrata planktonic and biofilm cells in the presence of acetic acid. Phagocytosis assays with mutant
strains suggested a potential role of these genes in the phagocytosis process. These results could impact the
treatment of vaginal candidiasis, a niche of acidic pH and rich in alternative carbon sources such as acetic acid.
29
Session 4:
Plant Genetics
Coordinators:
Raquel Chaves (UTAD) and Manuela Costa (UMinho)
31
OS7. Functional characterization of Arabidopsis thaliana SUMO proteases as new
development and environmental stress determinants
1
1,2
1
1
3
Pedro Humberto Castro , Tiago Lourenço , Miguel Ângelo Santos , Sara Freitas , Javier Ruiz-Albert , Rui Manuel
1
3
2
Tavares , Eduardo Rodrigues Bejarano , Herlander Azevedo
1
Biosystems and Integrative Sciences Institute (BioISI), Plant Functional Biology Center, University of Minho, Campus de Gualtar, Braga, Portugal
CIBIO, InBIO - Research Network in Biodiversity and Evolutionary Biology, Universidade do Porto, Campus Agrário de Vairão, Vairão, Portugal
3
Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas
(IHSM-UMA-CSIC), Dept. Biología Celular, Genética y Fisiología, Universidad de Málaga, Campus Teatinos, 29071 Málaga, Spain
2
Environmental stresses condition plant growth and crop yield, therefore unravelling the molecular mechanisms of
plant tolerance to abiotic stresses is a major research topic in current plant biology. Strategies to tolerate stress
include post-translational modifications (PTMs), which act as fast and reversible regulators of key proteins. Small
Ubiquitin-like Modifier (SUMO) is a PTM that has been increasingly associated with the regulation of abiotic stress
responses [1]. SUMO attachment (sumoylation) requires SUMO peptides to first be processed by SUMO proteases
(ULPs), and then conjugated to a target’s lysine via SUMO E1, E2 and E3 proteins. Deconjugation of the SUMO peptide
can subsequently be carried out by ULPs. Unlike the related modifier ubiquitin, SUMO conjugation machinery
components are less abundant in the plant genome, and null mutants of most components display embryonic lethality
or pleiotropic defects. ULPs constitute a relatively larger gene family of SUMO pathway components and may be a
source of specificity in the pathway, yet they are less functionally characterized. We have studied the transcriptional,
developmental and environmental stress responses of previously uncharacterized Arabidopsis T-DNA insertion
mutants disrupting functionally redundant ULPs, resulting in diverse developmental defects and constitutively
increased SUMO-conjugate levels. Results place ULPs as novel determinants of several developmental traits and
environmental stress responses.
[1] Castro PH, Tavares RM, Bejarano ER, Azevedo H. 2012. SUMO, a heavyweight player in plant abiotic stress responses. Cell Mol Life Sci 69(19):3269-3283.
This work is funded by FEDER through the Operational Competitiveness Program - COMPETE - and by national funds through the Foundation for Science and Technology
- FCT - within the scope of project “SUMOdulator” (Refs. FCOMP-01-0124-FEDER-028459 and PTDC/BIA-PLA/3850/2012).
OS8. Genes from distinct lineages determine gametophytic self-incompatibility in Malus and
Prunus
1
1
1
2
3
3
Bruno Aguiar , Jorge Vieira , Ana E. Cunha , Nuno A. Fonseca , Amy Iezzoni , Steve van Nocker , and Cristina P. Vieira
1
1
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal; Instituto de Biologia Molecular e Celular (IBMC), University of
Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal
2
CRACS-INESC Porto, Rua do Campo Alegre 1021/1055, 4169-007 Porto, Portugal European Bioinformatics Institute (EMBL-EBI,) Welcome Trust
Genome Campus, CB10 1SD, Cambridge, United Kingdom
3
Michigan State University, East Lansing, MI 48824-1325, USA
S-RNase-based gametophytic self-incompatibility (GSI) is a widespread mechanism, in flowering plants, to prevent
inbreeding, which has been given as an example of Dollo's law (once a complex trait is lost it is never regained). In
species from Solanaceae, Plantaginaceae and Rosaceae, pistil specificity is determined by a single extracellular
ribonuclease, called S-RNase. Phylogenetic analyses of the T2-RNases, as well as the presence of amino-acidic motifs
characteristic of the S-RNase genes, suggest that S-RNase-based GSI has evolved only once, before the split of the
Asteridae and Rosidae. However, in the family Rosaceae, the size and structure of the S-locus is distinct between
members of Prunus (Amygdaloideae) and Malus (Pyrinae). In Prunus, S-pollen specificity is determined by a single Spollen gene (SFB), and the region of suppressed recombination is restricted to S-RNase and SFB genes. In Malus,
multiple F-box genes (SFBBs) determine S-pollen specificity, and recombination is suppressed within an extended
region. In this study, we characterised the S-RNase and S-pollen lineage genes present in the genome of three
Rosaceae species: Malus domestica (apple), Prunus persica (peach), and Fragaria vesca (strawberry). We find that
Malus and Prunus S-RNase and S-pollen genes belong to distinct lineages, and that only Prunus GSI-lineage genes are
present in Fragaria. Thus, S-RNase based GSI evolved at least twice in the Rosaceae, with the ancestral system being
similar to that of Prunus and does not follow Dollo's law. Expression patterns were also determined for 16 tissues of
Malus and 5 tissues of Prunus using RNA-seq data. The ancestral S-RNase lineage gene is inferred to be expressed in
pistils only, while the ancestral S-pollen lineage gene is inferred to be expressed in tissues other than pollen.
33
OJ14. The sex of the plants...
1
1
1
Rómulo Sobral , Helena Silva , Joana Magalhães and M. Manuela R. Costa
1
1
BioSystems & Integrative Sciences Institute (BioISI), Plant Functional Biology Center, University of Minho, Campus de Gualtar, 4710-057 Braga,
Portugal
Angiosperms exhibit a variety of sexual systems that range from hermaphroditism to separate sexual organs in
different (dioecy) or in the same individual (monoecy). Albeit in minority, monoecious and dioceious species provide
an excellent system to study the specific determinants that underlie male and female flower development. In our lab
we study flower development of two species that contribute greatly to the Portuguese economic tissue, Quercus
suber and Vitis vinifera. Q. suber is the dominant tree of the Portuguese oak woodlands and is a wind-pollinated
monoecious species with several seasons of flowering. Male flowers occur in early spring and autumn, whereas female
flowering buds usually appear only in spring. V. vinifera L., one of the most important fruit crops in the world, has two
subspecies, the cultivated form V. vinifera subsp. vinifera and the wild form V. vinifera subsp.Sylvestris. Flowers of
cultivated plants are hermaphroditic, whereas wild plants have female and male sexes separated in different
plants. Both the female and male flowers follow a hermaphroditic development pattern during early stages of floral
development, with sexual dimorphism occurring in a late stage of plant development. The main objective of our work
is to use the knowledge gathered from Arabidopsis thaliana, a model species in which the genetic mechanisms of
flower development have been well characterised, and use it to puzzle out the genetic networks that control monoecy
in Q. suber and dioecy in V. vinifera. Using Next Generation Sequencing we are able to identify genes that underlie
each type of sexual system. Moreover, using expression and functional analysis we identified genes that may be
determinant to sexual determination, that were not yet studied in the A. thaliana.
Acknowledgments: This work was funded by FCT /COMPETE / FEDER with the project grants FCOMP - 01 - 0124 - FEDER - 019461 / PTDC / AGR-GPL / 118508 / 2010 and
PTDC/AGR-GPL/119298/2010. R.S. was supported by funding from FCT with a Ph.D. grant (ref. SFRH/BD/84365/2012).
OJ15. Somaclonal variation: structural composition at the berry color locus in
Vitis vinifera L.
1,2
1
1
2
Vanessa Ferreira , Isaura Castro , Olinda Pinto-Carnide , David Carrasco , Rosa Arroyo-García
2
1
Center of Agricultural Genomics and Biotechnology (CGBA-UTAD), University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal
Centre for Plant Biotechnology and Genomics (CBGP-INIA), Biotechnology Department, Campus Montegancedo, 28223 Pozuelo de Alarcón,
Madrid, Spain
2
The colored-berry phenotype results from the presence of a single pigment family, anthocyanins, which vary largely in
concentration and composition depending on the grapevine cultivar, whereas white-berried cultivars do not
synthesize these compounds. Most diversity in grape color can be explained by genetic variation in a major locus colocalising with a cluster of four MYB-like genes located on chromosome 2. Within this cluster two Myb-related
transcripton-factor genes, VvMybA1 and VvMybA2, regulate anthocyanin biosynthesis through induction of the key
VvUFGT gene transcription. The white-skinned genotype arose as the result of disruption of these two genes. In the
case of VvMybA1, gene silencing is produced by insertion of Gret1 retrotransposon, whereas a single-nucleotide
mutation in the VvMybA2 coding region leads to a protein truncation rendering it non-functional. In the last decade, a
growing number of reports have related the genetic determinants of skin color mutants in grapevine, once these
somatic mutations affecting the berry color locus can be beneficial to grapevine breeding, generating new
agronomical relevant phenotypes. In this study, the genetic structure of the large locus of chromosome 2 was
characterized in several groups of somatic variants for berry skin color, comprising 37 accessions that belong to 14
Vitis vinifera L. varieties with a wide range of colors, from black to yellow/green (referred as white), including
intermediary shades. Functional and non-functional alleles of VvmybA1 and VvmybA2 genes were detected in the
grape berry skin color mutants analyzed. These results allowed to explain the berry color phenotype in almost all
accessions studied.
Acknowledgments: This work was supported by the RTA2011-00029-C02-01 project and the SFRH/BD/96400/2013 PhD grant.
34
OJ16. Transcriptomic atlas of Physcomitrella patens to decipher the evolution of epigenetic
mechanisms in land plants
1
1
1
Pereira, S. , Hernandez-Coronado, M. , Ortiz-Ramírez, C. , and Becker, J.D.
1
1
Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal.
Cytosine methylation is an epigenetic mark involved in the regulation of gene expression, development and genome
stability. Previous studies on epigenetic changes during sexual reproduction in the angiosperm Arabidopsis thaliana
identified variations on DNA methylation levels during male gametogenesis with a decrease in CHH methylation in
sperm cells [1]. We are interested in the evolution of such epigenetic mechanisms in land plants. As a model organism
for our evo-devo studies we use Physcomitrella patens, a moss with a haplo-dominant life cycle and motile sperm. We
started by generating a transcriptomic atlas covering most P. patens tissues, including antheridia (male sexual organ)
and antherozoids (sperm cells). This enabled us to identify genes that are specifically expressed or enriched in a given
tissue or developmental stage. Focusing on the epigenetic mechanisms acting during P. patens' male gametogenesis,
we observed that only de novo methyltransferases are significantly expressed in the antherozoids, with PpDRM2, a
homolog of A. thaliana’s Domains Rearranged Methyltransferase 2, being expressed exclusively in these cells. In A.
thaliana DRM2 is considered the major de novo DNA methyltransferase, being essential for RNA-directed de novo
methylation of cytosines in all sequence contexts [2].
In order to study DRM2's potential roles in P. patens' male gametogenesis, we generated two PpDRM2 knock-out
mutant lines. No significant differences between their fertilization rate and that of the wild type were detected either
in the F0 or the F1 generations. Possible justifications for the absence of a clear phenotype in this mutant are the
complementation by the other de novo methyltransferases expressed in the antherozoids or that the mutation's effect
manifests itself in other stages of the life cycle. Both scenarios are being considered in ongoing work.
[1] Calarco, J.P., Borges, F., Donoghue, M.T. a, Van Ex, F., Jullien, P.E., Lopes, T., Gardner, R., Berger, F., Feijó, J. A., Becker, J.D., and Martienssen, R. A. (2012).
Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA. Cell 151: 194–205.
[2] Naumann, U., Daxinger, L., Kanno, T., Eun, C., Long, Q., Lorkovic, Z.J., Matzke, M., and Matzke, A.J.M. (2011). Genetic evidence that DNA methyltransferase DRM2 has
a direct catalytic role in RNA-directed DNA methylation in Arabidopsis thaliana. Genetics 187: 977–9.
OS9. Label Free Biosensor and HRM used in SNP detection in Vitis vinifera L. for Authenticity
Purposes
2
1
1
1
3
Leonor Pereira , Luís Moreira , Helena M.R. Gonçalves , Cláudia Castro , Pedro Jorge , José R.A. Fernandes
1,2
Martins-Lopes
1,3
, Paula
1
University of Trás-os-Montes and Alto Douro, P.O. Box 1013, 5000-911 Vila Real, Portugal
Center of Agricultural Genomics and Biotechnology (CGBA), University of Trás-os-Montes and Alto Douro, Vila Real, Portugal
3
INESC TEC, Rua do Campo Alegre n. 687, 4169-007 Porto, Portugal
2
Food and beverage authenticity assessment is a major concern worldwide, demanding the development of quick and
reliable methods suitable for those purposes. DNA-based detection systems are considered to be the most accurate
when intending to certify the presence of a particular species. SNPs are highly conserved and widely distributed in the
genome making them interesting for authenticity applications. A wide range of SNP detection techniques have been
developed most of them require the use of fluorophore label, making them expensive and difficult to use once they
require specialized equipment and materials. The aim of the present study was to compare two methods, a label free
DNA-based biosensor and a fluorophore based technique, High Resolution Melting (HRM), in SNP detection for Vitis
vinifera L. authenticity. The biosensor platform consisted on the functionalization of an optical fiber long-period
grating with single strand-DNA (specific to V. vinifera). The sensing system was exposed to a complementary
sequence, a partial-complementary and a non-complementary DNA in order to assure the occurrence of specifichybridization. The HRM assay was developed based on the sequencing data of 18 grapevine varieties, using a
StepOne™ Real-Time PCR. The hybridization process with the Biosensor platform was only assured in the presence of
a complementary sequence and was able to detect DNA extracted from a V. vinifera sample. The HRM technique
allowed the discrimination of 18 grapevine varieties based on the allele variants identified in the sequencing data. The
potential use of the biosensor in comparison to the HRM platform for authenticity purposes will be highlighted.
Acknowledgments: Portuguese Science Foundation (FCT) PTDC/AGR-ALI/117341/2010 and PhD grant SFRH/BD/44781/2008 and ON2 EnoExcel - NORTE-07-0124-FEDER000032.
35
Session 5:
Human Genetics
Coordinators:
Rui Martinho (IGC) and Manuela Côrte-Real (CBMA)
37
OJ17. How the “major” satellite ncRNA of Felis catus is regulated in cancer?
1
1
1
1
Daniela Ferreira , Ana Escudeiro , Filipa Chaves , Filomena Adega , Raquel Chaves
1
1
University of Trás-os-Montes and Alto Douro (UTAD), Center of Agricultural Genomics and Biotechnology (CGBA), Department of Genetics and
Biotechnology (DGB), Laboratory of Cytogenomics and Animal Genomics (CAG),Vila Real, Portugal
Satellite DNAs were considered, for many years, as “junk” DNA. Recent evidences are refuting this idea, as have been
found the occurrence of transcription of these sequences as non-coding RNAs. It seems that transcription of some of
these sequences is regulated by DNA methylation mechanisms. Since the modifications at this level are a hallmark of
cancer, it is extremely important to analyze how the expression of these sequences is influenced by those
modifications. The transcriptional profile of the “major” satellite DNA (FA-SAT) of Felis catus (FCA, domestic cat) was
already characterized in normal and tumor cells by our group at different levels. In this work, we compared the
methylation profile of two different passages (50 and 80) of a cat mammary tumour cell line - FkMTp with a normal
cell line. Unexpectedly, FA-SAT is equally methylated in normal and cancer cell lines. However, transcripts
quantification by real time RT-qPCR revealed an increase in passage 50 of FkMTp not explained by copy number
variation, suggesting that its regulation can be complex. To analyse the methylation status of this sequence, a global
demethylation of the genome was performed. In normal cells, the analysis of transcription by RT-qPCR and RNA-FISH
revealed that it is increased, supporting the idea that this sequence is regulated by DNA methylation. On the other
hand, in both FkMTp passages, transcription of FA-SAT did not show a significant change. So, different behaviors of
FA-SAT transcription in normal and tumor cells were observed. These data suggest that the regulation of this
sequence in cancer is performed by other processes than only DNA methylation mechanisms, being dysregulated in
FkMTp. This work highlights the importance of characterizing the epigenetic profile of satellite DNA in cancer and,
more particularly, of FA-SAT sequence in tumor cell lines as FkMTp. Nevertheless, to disclose the exact modulation of
the expression of this satellite additional work is mandatory.
OJ18. Targeting KRAS oncogene promoter by G-quadruplex ligands in human colon cancer cells
1
1
1
1
1
Hugo Brito , João Lavrado , Rui Moreira , Alexandra Paulo , Cecília M. P. Rodrigues and Pedro Borralho
1
1
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal
KRAS mutations occur in 35 to 45% of colon tumors, being an important target for cancer treatment, for which there is
no available targeted therapy. We aimed to downregulate KRAS oncogene transcription via novel G-quadruplex
binding compounds, and to elucidate their anticancer mechanism of action and effect on proliferation and death of
colon cancer (CC) cells. Three indolo[3,2-b]quinolines (IQ3A) ligands of G-Quadruplex (G4) DNA structures were
synthesized and evaluated for their effect on cell viability and death by MTS metabolism and LDH release assays,
Hoechst staining, Guava Viacount and Nexin flow cytometry in colon cancer (CC) cells with mutant KRAS (HCT116
p53+/+, p53-/- isogenic cells; and SW620), and in cells with wild-type KRAS (HEK293T and CCD18co). KRAS promoter
activity, mRNA and protein levels were assessed by Taqman Real-time PCR, immunoblotting and dual luciferase
reporter assay, respectively. IQ3A compounds showed high anti-proliferative activity in HCT116 and SW620 CC cells
(IC50 < 2.69 µM) compared to 5-FU (IC50 < 5.39 µM), without causing cell death in human non-malignant HEK293T and
CCD18co colon fibroblasts. Further, IQ3A compounds significantly reduced KRAS mRNA and protein steady-state levels
at IC50 concentrations, and increased p53 protein steady-state levels and cell death by apoptosis in HCT116 (mut KRAS,
wt p53). Furthermore, KRAS silencing in HCT116 p53(+/+) and p53(-/-) isogenic cells induced a higher level of cell
death, and a higher IQ3A-induced cell death, in HCT116 p53(+/+) compared to HCT116 p53(-/-). Importantly, IQ3A
compounds reduced KRAS promoter activity in a dual luciferase reporter assay. Herein we provide evidence that IQ3A
compounds can target G4 motifs present in KRAS promoter, down-regulate the expression of mutant KRAS by
inhibition of transcription and translation, and induce cell death by apoptosis in CC cells. Thus, targeting KRAS at the
genomic level with G4 ligands may be a new anticancer therapy strategy for CC.
Supported by SPG and FCT (EXPL/QEQ-MED/0502/2012)
39
OJ19. Testing short LNA-modified oligonucleotides for Duchenne Muscular Dystrophy gene
therapy
1
1
Vanessa Borges Pires , Maria Carmo-Fonseca and Célia Carvalho
1
1
Unidade de Biologia Celular, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Professor Egas Moniz, 1649-028
Lisboa, Portugal
Locked Nucleic Acid (LNA) modified oligonucleotides carry an altered sugar moiety that forms a methylene bridge
allowing improved properties in terms of increased duplex stability, high sensitivity, good mismatch discrimination,
1
low toxicity and increased metabolic stability , fitting the properties needed for human therapy. Duchenne Muscular
Dystrophy (DMD) is caused mainly by frame-shifting or nonsense mutations in the DMD gene in chromosome X, which
2
encodes for the dystrophin protein, essential for membrane stability in muscle fibers . The antisense oligonucleotides
Drisapersen (2’-O-methyl chemistry) and Eteplirsen (morpholino chemistry) are being currently tested in clinical trials
3
as a therapy for DMD, with limited sucess . These can restore the dystrophin reading frame by promoting skipping of
3
exon 51, being this approach applicable to ~13% of DMD patients . We aim to test if LNA oligonucleotides can be used
for splicing modulation therapies, with increased efficiency. Myoblast derived cell lines from patients were used for in
vitro induction of skipping of DMD-exon 51. We looked for skipping of exon-51 at the transcript level (RT-PCR) and for
restoration of protein production (Western Blot). Promising preliminary tests have shown that we can detect skipping
of exon 51 and restore dystrophin protein production in dystrophic myoblasts when transfected with LNA
oligonucleotides concentrations 10 times lower than the published results with other oligo-chemistries. This works
intends to make a new step in the search for an effective splice modulation therapy in the treatment of DMD.
1. Grϋnweller, A. et al. Locked Nucleic Acid Oligonucleotides. BioDrugs 21, 235–243 (2007)
2. Aartsma-Rus, A. et al. Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum. Mol. Genet. 12, 907–914 (2003)
3. Lu, Q.-L. et al. What Can We Learn From Clinical Trials of Exon Skipping for DMD? Mol. Ther. Nucleic Acids 3, e152 (2014)
OJ20. Misreading tRNAs promote tumor progression in vivo
1,2,3*
1,2,3*
1,2,3
3
3
3
Patricia M. Pereira
, Mafalda Santos
, Ana Sofia Varanda , Joana Carvalho , Nuno Mendes , Patricia Oliveira ,
3
3,4
3,4
1,
Marta Teixeira Pinto , Fátima Carneiro , Carla Oliveira * and Manuel A. S. Santos *
1
iBiMED/Health Sciences, University of Aveiro, 3810-193 Aveiro, Portugal
CESAM, Biology Department, University of Aveiro, 3810-193 Aveiro, Portugal
3
Institute of Molecular Pathology and Immunology, University of Porto (IPATIMUP), 4200-465 Porto, Portugal
4
Faculty of Medicine, University of Porto, 4200-465 Porto, Portugal
*
Equivalent contribution
2
Expression deregulation of translation machinery has been reported in cancer, and raises the hypothesis that
translational accuracy is compromised in this setting. This hypothesis is supported by the recurrent observation of
protein misfolding, proteotoxic stress, overexpression of molecular chaperones and activation of the unfolded protein
response (UPR) in many tumor types [1]. Protein misfolding leads to endoplasmic reticulum (ER) stress, activating
several pathways to alleviate damage and to promote cellular adaptation. Although prolonged ER stress and
consequent UPR activation ultimately leads to apoptosis, cancer cells highjack the ER adaptive measures in order to
thrive [2]. To study the role of translation accuracy in tumorigenesis, we expressed misreading tRNAs in NIH3T3 cells
and studied UPR and cancer-associated signaling pathways. Here we report an unexpected role for overexpression of
WT and misreading tRNAs in tumor progression. Our data show that expression of misreading tRNAs produce tumors
in vivo with a fast growing phenotype, similar to that of K-Ras-induced tumors. Remarkably, cells expressing
misreading tRNAs were selected in vivo, suggesting advantageous features of this phenotype. Our results also show
that the in vivo microenvironment drives cells to activate the Akt pathway and induce the UPR, as these pathways
were not activated in vitro. Accumulating evidence has shown that UPR activation and decrease in translation fidelity
are required for cancer cells to maintain malignancy and acquire therapy resistance, suggesting that compromising
translation fidelity may select adaptative mutations. Our results demonstrate that mistranslation has a role in tumor
growth, worth to explore further.
D. Ruggero, P. Pandolfi, “Does the ribosome translate cancer?” Nat. Rev. Cancer, vol. 3, pp. 179–192, 2003.
W.-A. Wang, J. Groenendyk, M. Michalak, “Endoplasmic reticulum stress associated responses in cancer.,” Biochim. Biophys. Acta, pp. 1–7, 2014.
40
OJ21. Transcription-coupled RNA quality control in human genetic diseases
1
2
1
1
1
Vaz-Drago R , Pinheiro MT , Martins S , Enguita FJ , Carmo-Fonseca M , Custódio N
1
2
1
Instituto de Medicina Molecular, Universidade de Lisboa, Lisboa, Portugal
University of Manchester, United Kingdom
Current estimates indicate that approximately one-third of all disease-causing mutations are expected to disrupt
splicing. Abnormal splicing often leads to disruption of the reading frame with introduction of a premature
termination codon (PTC) that targets the mRNA for degradation in the cytoplasm by nonsense mediated decay (NMD).
In addition to NMD there are RNA surveillance mechanisms that act in the nucleus while transcripts are still associated
with the chromatin template. However, the significance of nuclear RNA quality control in the context of human
genetic diseases is unknown. Here we used patient-derived lymphoblastoid cell lines as disease models to address
1
how biogenesis of mRNAs is affected by splice site mutations . We observed that most of the mutations analyzed
introduce PTCs and trigger mRNA degradation in the cytoplasm. However, for some mutant transcripts, RNA levels
associated with chromatin were found down-regulated. Quantification of nascent transcripts further revealed that a
subset of genes containing splicing mutations (SM) have reduced transcriptional activity. Following treatment with the
translation inhibitor cycloheximide the cytoplasmic levels of mutant RNAs increased, while the levels of chromatinassociated transcripts remained unaltered. These results suggest that transcription-coupled surveillance mechanisms
operate independently from NMD to reduce cellular levels of abnormal RNAs caused by SM.
1
Vaz-Drago R, Pinheiro MT, Martins S, Enguita FJ, Carmo-Fonseca M, Custódio N.Transcription-coupled RNA surveillance in human genetic diseases caused by splice site
mutations. Hum Mol Genet. 2015 Feb 4
OJ22. A role for the carboxyl-terminal domain of RNA Pol II in pre-mRNA splicing
1
1
1
Joana Tavares , Noélia Custódio , M. Carmo-Fonseca
1
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa
In mammals protein-encoding transcripts are formed by short exons separated by much longer introns and it is still
not fully understood how the small exons separated by long introns are correctly juxtaposed for splicing. According to
a recent model, the carboxyl-terminal domain (CTD) of RNA polymerase II (RNA Pol II) serves as a platform that tethers
the upstream exon until it is ligated to the downstream exon in the nascent pre-mRNA. To test this model, we
generated cell lines with mutant versions of the CTD. The transcriptome (polyA + RNA) of cells containing either wildtype, point mutations or deletions of the CTD was analyzed by RNA-seq. Genes that were either differentially
(1)
(2)
expressed or differentially spliced were identified using two bioinformatics tools, MISO and MATS . The results
show that distinct types of CTD mutations have specific effects on splicing profiles. We conclude that the composition
of the CTD is critical for determining splicing decisions.
1. Katz, Y. et al. Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat. Methods 7, 1009–15 (2010).
2. Shen, S. et al. MATS: a Bayesian framework for flexible detection of differential alternative splicing from RNA-Seq data. Nucleic Acids Res. 40, e61 (2012).
41
OJ23. Exploring New Therapeutic Approaches for Hypertrophic Cardiomyopathy
1
1
1
Rita Mendes de Almeida , Teresa Carvalho , Sandra Martins and Maria Carmo-Fonseca
1
1
Unidade de Biologia Celular, Instituto de Medicina Molecular,Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal
Hypertrophic Cardiomyopathy (HCM) is the most common hereditary disease of the heart. At present there is no
curative treatment for this disease. This work aims to establish a proof-of-principle for a RNA-based therapy and also a
CRISPR-Cas9 based gene therapy for HCM. In parallel, the analysis by Next-Generation Sequencing (NGS) of 16
different HCM patient genomes in which none of the most common HCM mutations were identified will allow the
identification of novel HCM-causing mutations. For the RNA-based therapy, we started by transfecting mouse atrial
cardiomyocytes (HL-1 cells) with “therapeutic” human constructs designed to anneal the two introns flanking the
murine TNNT2 exon 11 (or the intron upstream), which in humans contains HCM associated-mutations. The success of
trans-splicing strategy was evaluated by RT-PCR to assess the replacement of murine exon 11 by the “therapeutic”
human one. Later on, the CRISPR-Cas9 system was used to create a double strand break (DSB) in the mTNNT2 exon 10
to favour the insertion of a “therapeutic” cDNA sequence (Ex10-Ex18) and thus correcting a cluster of downstream
HCM mutations. At present, HCM cellular models are being generated also taking advantage of the CRISPR technology.
Furthermore, the variants identified from the NGS patient data are being analysed in order to predict their potential
pathological effect. In the future, we intend to use the CRISPR methodology to induce these variants at the cellular
level and so test their ability to induce an HCM phenotype. All together, these approaches may allow the development
of new therapeutic approaches for HCM. Moreover, novel mutations associated to HCM may be unravelled and
further used as diagnostic and/or prognostic HCM markers.
[1] White SM, et al, Am J Physiol Heart Circ Physiol. Mar; 286(3):H823-9, 2004
[2] Wally, V., et al, Journal of Investigative Dermatology, doi:10.1038/jid.2012.101, 2012
[3] Ran, F. A., et al, Cell 154, 1380–1389, 2013
OJ24. Characterization of Notch signaling pathway at early stages of chick lung development
1,2
1,2
Carla Silva-Gonçalves , Patrícia Vaz-Cunha , Jorge Correia-Pinto
1,2,3
, Rute S Moura
1,2,4
1
Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal;
ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal;
3
Department of Pediatric Surgery, Hospital de Braga, Braga, Portugal;
4
Biology Department, School of Sciences, University of Minho, Braga, Portugal.
2
Notch signaling is a conserved signaling pathway extremely important throughout embryonic development. The
central element of this pathway is the transmembrane Notch receptor, which triggers signaling through interaction
with membrane-bound ligands expressed on adjacent cells. Notch signaling precisely regulates cell-cell
communication during development, where it’s required that multiple cell types control each other's survival,
proliferation, differentiation and patterning [1]. Lung development is a highly regulated and organized process
directed by mesenchymal-epithelial interactions that control temporal and spatial expression of multiple regulatory
factors required for proper lung formation [2]. Notch signaling has been implicated in several aspects of mammalian
lung biology, including a role in airway epithelial cell growth and differentiation. Nonetheless this signaling pathway
has not been described during chick lung development [3]. Despite the differences in their adult state, mammalian
and avian adult develop similarly, and while mammalian lung development is well studied little is known about the
molecular mechanisms underlying chick lung development. The chick model presents advantages over the mammalian
model, and may emerge as a new model system for pulmonary branching studies. The aim of the project was to
characterize, by in situ hybridization, the expression pattern of several components of the Notch pathway, namely:
notch1, notch2, serrate1, serrate2, lunatic fringe, dll1 and dll4 in early stages of chick lung development. In general,
their expression pattern is similar to their mammalian counterparts which indicate that crucial signaling pathways are
conserved among species. Moreover, nrp1, flk1 and VE-cadherin (vascular makers) were also assessed in order to
verify colocalization with Notch genes.
[1] Andersson et al., Development (2011) 138(17): 3593–3612.
[2] Horowitz et al., Circ Res (2009) 104: 784-795.
[3] Kong et al., Am J Physiol-Lung Cell Mol Physiol (2004) 286(5): 1075–1083.
42
Session 6:
Genetics and
Biotechnology
Coordinators:
Sandra Viegas (ITQB) and Raul Machado (CBMA)
43
OS10. Genetic adaptive mechanisms mediating response and tolerance to acetic acid stress in
the human pathogen Candida glabrata: role of the CgHaa1-dependent signaling pathway
1
1
2
3
4
1,5
4
Ruben T. Bernardo , Diana V Cunha , Can Wang , Hiroji Chibana , Sónia Silva , Isabel Sá-Correia , Joana Azeredo ,
2
1,5
Geraldine Butler and Nuno Pereira Mira
1
iBB, Institute for Bioengineering and Biosciences, Av. Rovisco Pais, 1049-001 Lisboa; 2School of Biomolecular and Biomedical Sciences, Conway
Institute, University College of Dublin, Belfield; 3Medical Mycology Research Center, Chiba University, Chiba, Japan;4CEB, Centre of Biological
Engineering, LIBRO – Laboratório de investigação em Biofilmes Rosário Oliveira, University of Minho, Braga, Portugal;5Instituto Superior Técnico,
Department of Bioengineering, Universidade de Lisboa, Lisbon, Portugal;
C. glabrata is a human commensal found in the genitourinary tract but under certain conditions this harmless
colonization can evolve to a mucosal infection and, in more serious cases, to a life-threatening disseminated mycosis.
To thrive in the vaginal tract C. glabrata has to cope with the presence of a competing commensal microbiota that
restrains the overgrowth of pathogens through the production of acetic and lactic acids, among other interference
effects. The main goal of this work was the identification of key players mediating tolerance of C. glabrata to acetic
acid these being an interesting set of targets to be used in the development of new antifungals. Tolerance
mechanisms of C. glabrata to acetic acid are poorly studied but much knowledge has been gathered in Saccharomyces
cerevisiae[1,2,3,4]. In particular, the essential role played by the ScHaa1 transcription factor in mediating S. cerevisiae
tolerance to acetic acid stress was demonstrated[2,3,4]. In this work it is shown that CgHaa1, an orthologue of
ScHaa1, controls an acetic acid-responsive system in C. glabrata. The mechanisms by which the CgHaa1 pathway
mediate tolerance to acetic acid in C. glabrata were further dissected, exploring a transcriptomics approach, being of
notice the involvement of this system in the control of internal pH and in reducing the internal accumulation of the
acid. In the presence of acetic acid CgHaa1 enhanced adhesion and colonization of reconstituted vaginal human
epithelium, this correlating with a positive effect of CgHaa1 over the expression of adhesin-encoding genes. The
results obtained show similarities, but also remarkable differences, in the way by which the ScHaa1 and CgHaa1
pathways mediate tolerance to acetic acid in S. cerevisiae and in C. glabrata, indicating that the network in the later
species has evolved. The role of the CgHaa1-pathway in the extreme acetic acid-tolerance exhibited by vaginal C.
glabrata isolates will be discussed, along with other mechanistic insights
1] Mira NP, Becker J and Sá-Correia I, OMICS:14, 587-601, (2010); [2] Mira NP, Teixeira MC and Sá-Correia I, OMICS:14, 525-40, (2010); [3] Mira NP et al., Nucleic Acids
Res., 16, 6896-907, (2011); [4] Mira NP, Palma M, Guerreiro J and Sá-Correia I, Microb Cell Fact:9-79, (2010)
OJ25. Pseudomonas-based chassis towards à la carte biotransformation of plant-derived
volatiles
1
Pedro Soares-Castro and Pedro Miguel Santos
1
1
CBMA - Centre of Molecular and Environmental Biology, Department of Biology Universidade do Minho, Gualtar, 4710-057 Braga, Portugal.
Pseudomonas sp. M1 is able to mineralize unusual C-compounds of natural and xenobiotic origin, including the
monoterpene myrcene [1]. Since myrcene derivatives are widely used in industrial processes, M1 strain holds great
potential as cellular chassis and source of novel biomolecules for various biotechnological applications, namely in
biocatalysis and biosensing of plant-derived volatiles. Full exploitation of M1 strain enzymatic repertoire requires
detailed information about its biomolecular catalog. The genome of M1 strain was sequenced by NGS technologies [2],
assembled and annotated using customized pipelines. In silico analysis predicted metabolic pathways involved in
utilization/biotransformation of diverse terpene-backbone substrates. RNA-seq and proteomics of M1 cells exposed to
myrcene suggested an extensive molecular reprogramming, involving mechanisms of cell adaption to myrcene
hydrophobicity and catabolism [2]. In particular, a thorough data analysis led to the identification of a 28-Kb genomic
island essential for myrcene catabolism, which includes genes involved in i) myrcene oxidation and bioconversion of
myrcene derivatives via a beta-oxidation-like pathway, ii) regulation of the pathway, iii) myrcene sensing. Time-course
characterization of promoter regions from the island showed a modular expression kinetics, with different specificities
towards other cyclic and acyclic monoterpenes, extending the biotech applicability of this catabolic element.
Integration of our holistic approach will set the adequate knowledge ground to define the minimal set of genetic
elements required for myrcene catabolism. Moreover, Synthetic Biology approaches will allow the refactoring of
entire M1 functional blocks towards an optimized/unique biological system for myrcene (and chemical related
compounds) biotransformation.
[1] Santos PM, Sá-Correia I (2009) Proteomics 9:5101-11.; [2] Soares-Castro P, Santos PM (2014) Genome Biol Evol7:1-17.
45
OJ26. Metagenomics-guided bioprospection of monoterpene biocatalysts in the rhyzosphere of
coark oak trees
1
1
2
3
3
Filipa Gonçalves , Pedro Soares-Castro , Diana Afonso , Francisca Reis , Teresa Lino-Neto and Pedro Miguel Santos
1
1
CBMA - Centre of Molecular and Environmental Biology, Department of Biology, Universidade do Minho, Gualtar, 4710-057 Braga, Portugal.
ICVS/3B’s, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
3
Plant Functional Biology Centre, University of Minho, Gualtar, 4710-057 Braga, Portugal.
2
Soil microbiome is an unlimited reservoir for prospection of biocatalysts and biosensors towards plant-derived
compounds such as monoterpenes, used in several industrial applications. In soil, terpenes are accumulated from
decaying plant matter and root exudates, allowing the specialization of microorganisms into natural degraders of
terpene-backbone molecules. The soil strain Pseudomonas sp. M1 is one of the most well-described bacteria able to
mineralize myrcene and related terpenes. M1 strain core-code for myrcene metabolism is located in a 28-Kb genomic
island. Evolutionary analysis of the island suggested a modular assembly of genetic elements from different sources
(soil bacteria, some of them reported as being able to degrade terpenes or hydrophobic/recalcitrant compounds),
which evolved to its current form within M1 genome [1]. To understand the evolution of myrcene-related genetic
trait, soil samples from rhizosphere of Q. suber L. trees from 2 sites in Peneda-Gerês National Park (Ermida and Rio
Cabril) were evolved in fed-batch cultures towards myrcene utilization. DNA was extracted for 16S-based
metagenomics with Illumina MiSeq at several time-points and data analysed by Mothur and R package. Original
microbiomes from both sites were similar, dominated by about 100 OTUs. Moreover, myrcene enriched community in
Pseudomonas, Achromobacter, Sphingobacterium, Burkholderia and Ralstonia/Cupriavidus genera, predicted in silico
as putative sources of genes from M1 28-Kb island. Pseudomonas, Burkholderia, Sphingobacterium and Rhizobium
strains were isolated as novel candidates for myrcene catalysis (including some that outgrow M1 strain performance).
Molecular analysis suggests the presence of M1-like modules in the isolates. Current biotransformation experiments
and genome sequencing of the isolates will widen the set of biochassis available for biotech exploitation of plantderived volatiles.
[1] Soares-Castro P, Santos PM (2014) Genome Biol Evol7:1-17.
OJ27. Synthetic Protein Biotechnology approaches for the creation of antimicrobial biopolymers
1
1
1
1
André da Costa , Raul Machado , Artur Ribeiro , Tony Collins , Viruthachalam Thiagarajan
3,4
5,6
1
1
Petersen , José Carlos Rodríguez-Cabello , Andreia C. Gomes , and Margarida Casal
2,3
, Maria Teresa Neves-
1
CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
School of Chemistry, Bharathidasan University, Tiruchirappalli − 620 024, India
3
BioPhotonics Group, Nanomedicine Department, International Iberian Nanotechnology Laboratory (INL), P-4715-310 Braga, Portugal
4
Faculty of Medicine, Aalborg University, DK-9220 Aalborg, Denmark
5
Bioforge (Group for Advanced Materials and Nanobiotechnology), Centro I+D, Universidad de Valladolid, Valladolid, Spain
6
Networking Research Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), E-47011 Valladolid, Spain
2
The spread of antimicrobials resistant microorganisms has triggered the search for new ways to treat infections. In the
present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with
broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the
DNA sequence coding for ABP-CM4 in frame with the N-terminus of the elastin-like recombinamer consisting of 200
repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified
nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide
was also purified through the incorporation of a formic acid cleavage site between the peptide and the A200
sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly
effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity
against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed. The antimicrobial
activity of CM4-A200 was dependent on the physical contact of cells with the film surface. Furthermore, CM4- A200
films did not reveal a cytotoxic effect against both normal human skin fibroblasts and human keratinocytes. Finally, we
have developed an optimized ex vivo assay with pig skin demonstrating the antimicrobial properties of the CM4-A200
cast films for skin applications.
Acknowledgments: This work was supported by FEDER through POFC – COMPETE by FCT through the project PEst-OE/BIA/UI4050/2014. By the Spanish Minister of
Economy and Competitiveness (MAT2012-38043-C02-01) and Junta de Castilla y León-JCyL (VA152A12-2 and VA155A12-2), Spain. AC and RM, acknowledge FCT for
SFRH/BD/75882/2011 and SFRH-BPD/86470/2012 grants, respectively.
46
OJ28. High-level Biosynthesis of a Silk-Elastin-Like Protein in E. coli: Reducing Acetate
Accumulation and Plasmid Instability
1
1
1
1
Mário Barroca , Paulo Rodrigues , Fernando Branca , Margarida Casal and Tony Collins
1
1
Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal
Silk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a
high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is
however restrained by their production levels with yields of only 25-85 mg/L being reported. Our previous studies
allowed for the production of 500 mg/L of purified SELP with an optimised batch production approach and identified
acetate accumulation to toxic levels during cell growth as well as plasmid instability on induction as factors limiting
further improved production levels. We attempted to circumvent these limitations by developing and optimising a
fed-batch approach for SELP production as well as investigating various plasmid stabilisation systems. In the former,
pre- and post-induction growth rates, inducer concentration, dissolved oxygen concentration and dry cell weight on
induction were optimised and allowed for reduced acetate production and approximately 4.3 g/L of purified SELP. For
improving plasmid stability, various plasmid stabilisation systems were compared and a toxin/anti-toxin postsegregational suicide systems was found to allow for improved plasmid stability and a further increase in SELP
production. Approximately 13 g/L of purified SELP was obtained in this study, being over 150-fold higher than that
reported by other groups.
Acknowledgements: This work was financed by the European Commission, via the 7th Framework Programme Project EcoPlast (FP7-NMP-2009-SME-3, collaborative
project number 246176), by FEDER through POFC – COMPETE and by national funds from Fundação para a Ciência e Tecnologia (FCT) through PEst project
C/BIA/UI4050/2011C/BIA/UI4050/2011 and EXPL/BBB-BIO/1772/2013.
[1] Collins T., Barroca M., Branca F., Padrão J., Machado R. and Casal M. (2014). High Level Biosynthesis of a Silk-Elastin-like Protein in E. coli. Biomacromolecules,
dx.doi.org/10.1021/bm5005564; [2] Collins T., Azevedo-Silva J., da Costa A., Branca F., Machado R. and Casal M. (2013). Batch production of a silk-elastin-like protein in
E. coli BL21(DE3): key stress factors and parameters for optimisation. Microb. Cell Fact. Feb 27; 12:21. doi: 10.1186/1475-2859-12-21.
OJ29. Lathyrus sativus L. Application of Biotechnological and Biochemistry techniques toward
plant breeding
1,2
2
3
Letice Gonçalves ; Carlota Vaz Patto ; Rui Carvalho and Jorge Canhoto
1
1
CEF – Centro de Ecologia Funcional, Departamento de Ciências da Vida, Universidade de Coimbra, Coimbra, Portugal
ITQB – Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
3
Centro de Neurociências, Departamento de Ciências da Vida, Universidade de Coimbra, Coimbra, Portugal
2
Grass pea (Lathyrus sativus L.) has considerable potential as legume crop in dryland farming systems since it is
superior in yield, protein value, nitrogen fixation, drought, flood and salinity tolerance than other legume crops [1].
However, this potentially nutrition and environmentally friendly species is still underused being considered a NUC
species (Neglected and Underused Crops). This is in part due to the presence of a secondary metabolite β-N-oxayl-Lα,β- diaminopropionic acid (β -ODAP) which may occurs in two isomeric forms  and β-ODAP[2] the last one being
toxic for humans when grass pea is ingested during a long period and in large amounts. In spite of the interest of this
species for some Portuguese regions, little has been done in the evaluation of genetic diversity of the species, as well
as in the development of effective protocols for clonal propagation of selected genotypes. In this work the genetic
diversity of eight landraces populations, collected from Alvaiázere and Penela, was analysed, by molecular approaches
based on DNA analysis carried out with five polymorphic L. cicera-derived EST-SSRs markers. A high degree of genetic
diversity was found among individuals. Besides, a protocol for L. sativus shoot proliferation was achieved though
shoot tip culture of seedlings For shoot multiplication a half-strengh MS containing 0, 1, 2, 4, 8 and 10 M BA
(benzyladenine) were tested. The best concentration to promote shoot proliferation was 10 M BA, with an average
of 4.7 nodes per explant. NMR analysis allowed the detection and relative quantification of -ODAP in the different
populations, showing minor differences between them. The kinetic conversion of -ODAP into -ODAP was also
monitored by this technique.The results so far obtained gave important insights about the genetic diversity and ODAP content on national grass pea landraces, but further studies are necessary to select and propagate genotypes
showing lower content of this metabolite.
[1] Vaz Patto M C, Skiba B, Pang E C K, Ochatt S J, Lambein F, Rubiales D. 2006b. Lathyrus improvement for resistance against biotic and abiotic stresses: from classical
breeding to marker assisted selection. Euphytica 147: 133–147;
[2] Yigzaw Y, Larsson N, Gorton L, Ruzgas T, Solomon T. 2001. Liquid chromatographic determination of total and b-N-oxalyl-La, b-diaminopropionic acid in Lathyrus
sativus seeds using both refractive index and bioelectrochemical detection. Journal of Chromatography A 929: 13-21.
47
Poster
Sessions
49
P01. Isolation and characterization of 20 polymorphic microsatellite loci in Palaemon serratus
by 454 pyrosequencing
ab
c
ab
c
ab
Alejandra Perina , Neus Marí-Mena , Ana M. González-Tizón , Antón Vizcaíno and Andrés Martínez- Lage
a
Department of Cell and Molecular Biology, Evolutionary Biology Group (GIBE), Universidade da Coruña, A Fraga 10, E-15008 A Coruña, Spain
Facultade de Ciencias and Centro de Investigaciones Científicas Avanzadas (CICA) Universidade da Coruña, 15071 A Coruña, Spain
c
AllGenetics, Edificio de Servicios Centrales de Investigación, Campus de Elviña s/n, A Coruña, E-15008, Spain
b
We developed microsatellite markers in the commercially fished shrimp species Palaemon serratus (Pennant, 1777)
through an enriched genomic library pyrosequenced in a Roche 454 GS-FLX. The capture of P. serratus mantains a very
important traditional activity in some fishing communities due to its high commercial value, mainly in the North of
Spain (up to 140€/Kg on Christmas). We sampled the large european distribution range of the species. Twenty loci
were polymorphic in the following tested populations: Artabro Gulf, Guadalquivir Estuary, Conil, and Strait of Gibraltar
from Spain, Calais and Re Island from France, Anglesey from Wales, Mondego Estuary from Portugal, Duinbergen from
Belgium, and Howth and Cork from Ireland. The number of alleles per locus ranged from 4 to 28. The observed and
expected heterozigosity ranged from 0.000 to 1.000 and from 0.016 to 0.934 respectively. Nine microsatellite loci
were successfully amplified in three species of the genus Palaemon, P. elegans Rathke, 1837, P. adpersus Rathke, 1837
and, P. longirostris (Pennant, 1777). These microsatellite markers will be valuable tools for population genetic studies
in populations of Palaemon prawn species.
P02.
Expanding the study of the MED12 gene in Opitz-Kaveggia and Lujan-Fryns syndromes
Cathy Paulino
1,2,*
1,*
2
1
, Isabel Marques , Raquel Chaves , Paula Jorge and Rosário Santos
1
1
Unidade de Genética Molecular, Centro Genética Médica Doutor Jacinto Magalhães, Centro Hospitalar do Porto - EPE, Porto; Unidade
Multidisciplinar de Investigação Biomédica (UMIB), Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto.
2
University of Trás-os-Montes and Alto Douro (UTAD), Department of Genetics and Biotechnology (DGB), Laboratory of Cytogenomics and Animal
Genomics (CAG), Vila Real.
*Equal contributors
Lujan-Fryns syndrome (LFS; OMIM#309520) is a rare form of X-linked Intellectual Disability (XLID). Clinically, the
patients can be recognized by a peculiar facies in addition to the presentation of some of the following features: tall
marfanoid stature, macrocephaly, long hands with hyperextensible digits, mild general hypotonia and mild to
moderate cognitive deficits with several behavioural problems. Opitz-Kaveggia syndrome (FGS; OMIM#305450) is also
a rare form of XLID. Patients show a distinctive facial appearance, a high and prominent forehead, short stature, small
prominent ears with simplified helical pattern, frontal hair upsweep, hypotonia and constipation. Mental retardation
is frequent, with developmental delay and a distinctive behaviour, with hyperactive personality and excessive
talkativeness. There has been a clear association between the MED12 gene and LFS and FGS. While in LFS the single
recurrent mutation c.3020A>G in exon 22 has been reported, in FGS two frequent mutations, c.2873G>A and
c.2881C>T, both in exon 21, have been described. This gene encodes for the mediator of RNA polymerase II
transcription subunit 12 - an essential subunit of the mediator complex, interacting with different developmental
pathways and involved in the regulation of neuronal gene expression. The aim of this work was to expand the study of
the MED12 gene from the routine seven exons to the entire forty-five coding regions, so as to increase the mutation
detection rate. Screening for variants in all MED12 exons by PCR-amplification followed by Sanger sequencing is being
implemented. Preliminary results reveal the presence of several variants with unknown significance (VUS), but none of
the documented recurrent mutations. In order to determine the pathogenicity of these VUS, several in-silico studies
were carried out. Here we discuss the putative pathogenic effect of such variants.
[1] Schwartz, C. E. et al. The original Lujan syndrome family has a novel missense mutation (p.N1007S) in the MED12 gene. J Med Genet 2007, 44, 472–477.
[2] Wang, H., Shen, Q., Ye, L., Ye, J. MED12 mutations in human diseases. Protein & Cell 2013, 1-4.
[3] Berry, K., Mahajan, S., Sahoo, P., Cheeran, S. Lujan–Fryns Syndrome and Psychosis. International J Scientific Study 2014, 2, 105-107.
[4] Risheg, H. et al. A recurrent mutation in MED12 leading to R961W causes Opitz-Kaveggia syndrome. Nat Genet 2007, 39, 451-453.
[5] Rump, P., Niessen, R. C., Verbruggen, et al. A novel mutation in MED12 causes FG syndrome (Opitz–Kaveggia syndrome). Clin Genet 2011, 79, 183–188.
51
P03. The mechanism underlying proteasome-mediated sensitivity to the chemotherapeutic
agent cisplatin
1
1
1
1
1
Nuno Machado , Isa Mendes , António Rego , Lisandra Castro , Manuela Côrte-Real and Susana R. Chaves
1
1
Centro de Biologia Molecular e Ambiental, Departamento de Biologia, Universidade do Minho, Braga, Portugal
Cisplatin is a highly effective chemotherapeutic drug used in the treatment of several tumors. It is a DNA-damaging
agent that induces apoptosis of rapidly proliferating cells. Unfortunately, cellular resistance occurs often. A large
fraction of tumor cells harbor mutations in p53, contributing to defects in apoptotic pathways and drug resistance.
However, cisplatin-induced apoptosis can also occur in p53 deficient cells; thus, elucidation of the molecular
mechanism involved will potentially yield new strategies to eliminate tumors that have defects in the p53 pathway.
Most of the studies in this field have been conducted in cultured mammalian cells, not amenable to systematic genetic
manipulation. Therefore, we established a simplified model devoid of a p53 ortholog to study cisplatin-induced
programmed cell death (PCD), using the yeast Saccharomyces cerevisiae. We showed that cisplatin induces an active
form of cell death accompanied by DNA condensation and fragmentation/degradation, but no significant
mitochondrial dysfunction. We further demonstrated that proteasome inhibition with MG132 or genetically increased
resistance to cisplatin. In this study, we sought to determine which protein(s) stabilized by proteasome inhibition are
important for cisplatin resistance. For this purpose, we tested whether several candidate genes are required for
proteasome-mediated sensitivity to cisplatin. In addition, we determined how cisplatin affects the protein
ubiquitination profile, both in wild-type and cells deficient in proteasome activity. Finally, we analyzed whether
MG132-mediated resistance is accompanied by changes in genomic stability of surviving cells. Proteasome inhibitors
can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Elucidation of this
mechanism will therefore aid in the development of new strategies to increase the efficacy of chemotherapy.
P04. Silencing of Monocarboxylate transporters as a tool to study the role of 3-bromopyruvate
in breast cancer
João Azevedo-Silva1, Fátima Baltazar2, Young H. Ko3, Peter Petersen4, Ana Preto1, Margarida Casal1
1
ICVS/3B’s - PT Government Associate Laboratory, School of Health Sciences, University of Minho. Portugal.
3
KoDiscovery, LLC, University of Maryland BioPark. USA.
4
Departments of Biological Chemistry and Oncology, Johns Hopkins University. USA
2
One of the most interesting features of tumors is their altered metabolism which enables high proliferative growth
rates. To achieve this cancer cells use glycolysis as primary energy source and driven oxidative phosphorylation to
fulfill the anabolic needs related to increased proliferation. Consequently, high levels of lactate are produced and
therefore exported by Monocarboxylate transporters (MCT) MCT-1 and MCT-4 commonly found overexpressed in
cancer cells. Due to this phenotype MCTs can be seen as good therapeutic targets for cancer therapy. 3Bromopyruvate (3BP) is an alkylating agent, analogue of lactate with proven anticancer efficacy, targeting key
glycolytic enzymes such as Hexokinase II and Glyceraldehyde-3-phosphate. Its similarity to lactate point to the
participation of MCTs, especially MCT-1 [1], in the mechanism of 3BP entry in cancer cells. In this work we have
evaluated the influence of MCT-1 and MCT-4 silencing using specific small interfering RNA (siRNA) in three breast
cancer cell lines (ZR-75-1, MCF-7 and SK-BR-3) with different sensitivities to 3BP as well as different levels of MCTs
expression. Silencing of MCT-1 and MCT-4 expression was optimised for lipofectamine and siRNA concentrations as
well as time of cell culture. Furthermore, 3BP uptake was evaluated using radiolabelled 3BP. Our results demonstrate
that despite a successful inhibition of MCT-1 and MCT-4 can be achieved with this methodology after 4 days of cell
culture, 3BP uptake was not prevented. The utilization of the MCT-1 inhibitor, AR-C158858 indicates that even upon
MCT-1 silencing it is still able to block 3BP transport indicating that eventually, vestigial levels of MCT-1 (undetectable
by western blot) may be enough to uptake 3BP. Also, using this system we cannot exclude the contribution of other
transport systems to compensate the absence of MCTs.
[1] - Azevedo-Silva J., et al. (2015) Biochem J (doi:10.1042/BJ20140921)
52
P05. Phylogeography of Silene section Cordifolia in the mountain ranges of north Iberian
Peninsula and Alps
1
1
2
1
3
2
Isaura Castro , Vanessa Ferreira , João Rocha , Olinda Pinto-Carnide , Francisco Amich , Rubim Almeida , António Luís
4
1
Crespí and Valdemar Carnide
1
Center of Agricultural Genomics and Biotechnology (CGBA-UTAD), University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real (Portugal)
Department of Biology, CIBIO, Faculty of Sciences, University of Porto, Rua Campo Alegre s/nº, 4169-007 Porto (Portugal)
3
Evolution, Taxonomy and Conservation Group (ECOMED), Department of Botany, University of Salamanca, E-37008 Salamanca (Spain)
4
Department of Biology and Environment - CITAB, Botanical Garden and Herbarium, University of Trás-os-Montes and Alto Douro, 5001-801 Vila
Real (Portugal)
2
Silene genus belongs to the family Caryophyllaceae and is recognized since the early days of evolutionary biology as an
important model system in ecology and evolution. This genus comprises about 23 sections and 700 species worldwide,
194 of which were reported to be present in Europe. Silene section Cordifolia is a south European high mountain
plant group that is morphologically well characterized. It is currently divided into four taxa, including the Iberian
endemic Silene acutifolia Link., S. foetida Link subsp. foetida and S. foetida Link subsp. gayana Talavera, and the Alps
endemic S. cordifolia All. These species are found in a highly disjoint range in most high mountain ranges from the
Portuguese Central Massif, along the north mountain ranges of Spain to the eastern Alps, providing the opportunity to
investigate phylogeographic relationships across south-western European mountain ranges. In this study, Silene
section Cordifolia was used as a model to address the phylogeographic relationships between Alps and the north
Iberian Peninsula mountain ranges. For this purpose, maternally inherited plastid DNA sequences, in concrete five
chloroplast microsatellites (cpSSRs), were analysed.A total of nine alleles and four different haplotypes were detected.
Chloroplast SSR data revealed a clear specific and intra-specific genetic differentiation reflecting the existence of four
distinct genetic lineages in the germplasm studied. A terminal position in the haplotype network was observed for S.
cordifolia species, from the Alps, that presents the more genetically distant haplotype. Within the remaining three
haplotypes, detected in the Iberia Peninsula species, variation was only in a mononucleotide of one single allele.
Acknowledgments: This work was supported by the SFRH/BD/43167/2008 PhD grant.
P06.
Portuguese ecotypes of Opuntia ficus-indica differ in biomass production
1,2
1,3
Carlos M. G. Reis , Maria Margarida Ribeiro , Luiz Carlos Gazarini
4
1
Instituto Politécnico de Castelo Branco, Escola Superior Agrária, Qt.ª da Sr.ª de Mércules, 6001-909 Castelo Branco, Portugal.
CERNAS-IPCB financiado por Fundos Nacionais através da FCT (Projeto PEst-OE/AGR/UI0681/2014).
3
Centro de Estudos Florestais. Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.
4
Departmento de Biologia, Universidade de Évora, Évora, Portugal.
2
The Opuntia ficus-indica (L.) Miller is a species from the Cactaceae family with the center of origin and domestication
in central Mexico. This species introduction in the Iberia Peninsula occurred, probably, by the end of the 15th century,
after the discovery of America, spreading later throughout the Mediterranean basin. In Portugal, O. ficus-indica is
located, usually, with a typical ruderal behavior, at the edge of roads and paths. In Portugal, as in other Mediterranean
regions, inlands areas are under severe draught during extensive summers, in particular, and global warming is
expected to affect them deeply in the near future. O. ficus-indica, by its morpho-physiological characteristics and
multiple economic uses, represent an alternative crop for those regions. Sixteen Portuguese O. ficus-indica ecotypes
and two ‘Italian’ cultivars ("Gialla" and "Bianca") were evaluated for plant vigor and biomass production, by nondestructive methods, in the two years following planting. Biomass production and plant vigor were measured by
estimating cladode number, cladode area and fresh weight per plant. Linear models to predict the area of cladodes
and fresh weight per plant were previously established using a biometric analysis of 180 cladodes. It was not possible
to establish an accurate linear model for dry matter using non-destructive estimation. Significant differences were
found among populations in the studied biomass-related parameters, and different groups were unfolded. A group of
four Portuguese ecotypes outperformed in terms of biomass production, comparable with the “Gialla” cultivar. This
group could be used to start a breeding program with the objective of deploy material for animal feeding, biomass
and fruit production. Nevertheless, the ‘Gialla’ cultivar showed the best performance, achieving the highest biomassrelated parameters, not surprisingly for it is an improved plant material.
53
P07. Cytotoxic variability among multi-resistant clinical isolates of Pseudomonas aeruginosa
from Hospital de Braga
1,2
3
1,2
1,2
Ana Luísa Alves , Alberta Faustino , Artur Ribeiro , Andreia Castro Gomes , Pedro Miguel Santos
1,2
1
CBMA – Center of Molecular and Environmental Biology, Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga,
Portugal
2
Department of Biology, University of Minho, Braga, Portugal
3
Clinical Pathology Service, Hospital de Braga, Sete Fontes-São Victor, 4710-243 Braga, Portugal
Pseudomonas aeruginosa is an opportunistic and ubiquitous pathogen responsible for multiple infections in clinical
environments. The ability to adapt to different environments and tolerate a variety of physical conditions make it a
complex and versatile organism. The medical treatment against P. aeruginosa is difficult, with its genomic plasticity,
multifactorial pathogenicity and multidrug-resistance presenting serious therapeutic challenges. This study aimed at
evaluating the cytotoxicity of multi-resistant P. aeruginosa isolates in multi-cell lines and correlate with the pathogen’s
proteomic and genotypic pattern. Five clinical isolates from two patients of Hospital of Braga (Portugal) showed
resistance to every class of antibiotic tested (including colistin, the last-resort option in P. aeruginosa antibiotherapy).
Supernatants from stationary phase cultures of the selected isolates were inoculated with the monocyte-like human
cell line THP-1. Levels of key inflammatory cytokine TNF-α were measured by ELISA assay and cytotoxicity was
evaluated by LDH (membrane integrity) and MTT (metabolism) assays. The supernatants of the isolates retrieved from
patient 1 evidenced a virulent, cytotoxic and immunogenic pattern similar to P. aeruginosa reference strain PA14,
whereas the supernatants of the isolates from patient 2 evidenced resemblances with P. aeruginosa PAO1. Moreover,
the results suggest that the isolates lead to cell death through different processes. These findings complement data
regarding differences found in the genotypic profile assessed by RAPD-PCR where these isolates were clustered in
distinct groups, considering a genetic similarity of 80%. Integration of ongoing immunoproteomics, genomics and gene
expression analysis will clarify the variation between the isolates and will allow to perceive host-bacteria interactions
and how it is translated in to clinical outcome.
P08. Fermentative production of succinic acid: promising Saccharomyces cerevisiae strains
identified towards an improved production
1
1
1
Ricardo Franco-Duarte , Daniela Bessa , Paula Sampaio , Célia Pais
1
1
Centro de Biologia Molecular e Ambiental (CBMA), Departamento de Biologia, Universidade do Minho, Braga, Portugal
Succinic acid is a platform chemical playing an important role as a precursor for the synthesis of biodegradable
polyesters, resins, dyestuff, pharmaceuticals and as food industry additive. Presently, succinic acid is mainly produced
through petroleum-based processes, which imply high costs and severe environmental problems. To overcome these
problems, microbial production of succinic acid from renewable resources has seen great developments in recent
years. We used a well characterized Saccharomyces cerevisiae strain collection, with different origins and properties,
to screen for succinate producing isolates. Phenotypic, genetic and metabolic (HPLC) characterizations were carried
out, and principal component analysis integrated all the obtained traits. A clear anti-correlation was observed
between succinic and acetic acids production, suggesting an antagonism in the production pathways of these two
acids.From the dataset integration, a group of 17 strains was chosen and whole genome sequencing was performed
followed by an extensive analysis directed to the study of differences in 39 selected key genes of the succinic acid
metabolic pathways. Genomic differences were found between good and poor succinic acid producer strains, mainly
in SDH and MDH1 genes, regarding the number of SNPs/InDels and gene copy number. Two strains were identified as
promising succinic acid producers, and the production of this acid was studied along the fermentation time, in
comparison with two strains with low capacity to produce this acid. Real-time PCR was used to confirm changes in
altered genes and allowed to obtain a holistic view of the differences in the metabolic pathway of the good succinic
acid producing strains.
Acknowledgments: This work was financed in part by the project TRANSBIO (FP7 / nº289603).
54
P09.
Predicting Radiotherapy Response: Could the answer be in Copy Number Variations?
1
1,2
2,3
2,3
4
Joana Rodrigues ; Ilda P Ribeiro , Ana Margarida Abrantes , Salomé Pires Lourenço , Paulo Simões , Margarida
4
1
2,5
1,2
2,3
1,2
Borrego , Nuno Lavoura , Marília Dourado , Joana B Melo , Filomena M Botelho , Isabel M Carreira
1
Cytogenetics and Genomics Laboratory, Faculty of Medicine, University of Coimbra, Coimbra, Portugal;
CIMAGO - Center of Investigation on Environment, Genetics and Oncobiology - Faculty of Medicine, University of Coimbra, Coimbra, Portugal;
3
Biophysics Unit, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal;
4
Radiotherapy Department, CHUC, Coimbra, Portugal;
5
Faculty of Medicine, University of Coimbra, Portugal.
2
Head and Neck Cancers (HNC) are a group of tumours located in the upper aero-digestive tract. Head and Neck
[1]
Squamous Cell Carcinoma (HNSCC) represent about 90% of all HNC cases . It has been considered the sixth most
malignant tumour worldwide and, despite clinical and technological advances, the five-year survival rate has not
[2]
improved much in the last years . Nowadays, HNSCC is well established as a heterogeneous disease and its
[1]
development is due to accumulation of genetic events .Apart from the majority of the patients being diagnosed in
an advanced stage, HNSCC is also a disease with poor therapeutic outcome. One of the therapeutic approaches is
radiotherapy. However, this approach has different drawbacks like the radioresistance acquired by some tumour cells,
[3]
leading to a worse prognosis . Identification of genetic markers associated to radiotherapy response in patients is an
essential step towards an improved diagnosis, higher survival rate and a better life quality. HSC-3 and BICR-10 cell
lines were cultured in DMEM supplemented with 10% fetal bovine serum and 1% of penicillin and streptomycin. For
BICR-10 cell line, 1% of hydrocortisone was also added. Both lines were exposed through different doses of RX,
ranging from 0,5 to 15 Gy. Cell viability was accessed using the clonogenic assay. The genetic characterization was
performed by Array Comparative Genomic Hybridization. Comparing the results from the clonogenic assay, HSC-3 cell
line appears to be more radiosensitive than BICR-10 cell line since, for the same irradiation, HSC-3 has a higher
decrease in colonies formation. The genetic analysis showed four regions that could be associated to radiotherapy
response: 4p11-pter, 8p23.3, 14q distal and 18q21.2.Taken together all of our data suggests that its chromosomal
alterations could be related to the radioresistance, allowing, in the future, the prediction of patients’ outcome and a
better choice of treatment approaches.
[1].Rothenberg SM, Ellisen LW. The molecular pathogenesis of head and neck squamous cell carcinoma. The Journal of clinical i nvestigation 2012; 122(6): 1951-7;
[2].GLOBOCAN. http://globocan.iarc.fr/Pages/Map.aspx. 22-07-2014; [3].Bussink J, van der Kogel AJ, Kaanders JH. Activation of the PI3-K/AKT pathway and implications
for radioresistance mechanisms in head and neck cancer. The lancet oncology 2008; 9(3): 288-96.
P10.
Genetic diversity of Portuguese Pinus nigra Arn. populations: first molecular study.
1Ϯ
1Ϯ
2
2
Alexandra Dias , Maria Lemos , Ana Carvalho , Maria João Gaspar , José Lima-Brito
,2
1
University of Tras-os-Montes and Alto Douro, 5001-801 Vila Real, Portugal
Centre of Agricultural Genomics and Biotechnology (CGBA), University of Tras-os-Montes and Alto Douro, 5001-801 Vila Real, Portugal
Ϯ
Authors with equal contribution
2
In Portugal, Pinus nigra species have allochthonous origin, presenting small populations located in the north and
centre of the country. This species is a very important conifer with a widespread distribution from North Africa
through the northern Mediterranean and eastwards to the Black Sea, found as well in the Corsica and Sicily islands. P.
nigra suffered different climatic constrains and geological events, presenting morphological, physiological and
molecular variability. However, the Portuguese populations have never been molecularly studied and the provenance
of the seeds used is unknown. In the present study, a total of 127 individuals from the following six populations:
‘Manteigas’, ‘Vale do Zêzere’, ‘Campeã’, ‘Vila Pouca de Aguiar’, ‘Paredes de Coura’ and ‘Caminha’, were analyzed with
inter-simple sequence repeat (ISSR) and Start Codon Targeted (SCoT) polymorphism markers, to evaluate their genetic
diversity and relationships. Eight SSRs and eight SCoT primers were selected, among 60 primers tested, based on their
amplification potential, polymorphism and/or reproducibility.The results obtained showed inter- and intra-population
polymorphism. Through the construction of an UPGMA dendrogram of genetic similarity, the individuals were globally
clustered per population, suggesting a defined genetic structure. Some geographically closest populations were
grouped together, probably due to a common origin of the plant material used in the afforestation of the studied
stands. Additional studies are ongoing in order to extrapolate the probable provenances of the planted Portuguese P.
nigra populations.
Acknowledgements: AC and AD thank the fellowships SFRH/BPD/68932/2010 and SFRH/BD/91781/2012, respectively, both attributed by Fundação para a Ciência e a
Tecnologia (FCT) and co-financed by FSE/POPH-QREN.
55
P11. Genetic Characterization of Two Head and Neck Squamous Cell Carcinoma Cell Lines by
Karyotyping and Array CGH
1
1,2
1
1
1
1
Joana Rodrigues , Ilda P Ribeiro , Cláudia Pais , Alexandra Estevinho , Alexandra Mascarenhas , Nuno Lavoura , Joana
1,2
1,2
B Melo , Isabel M Carreira
1
Cytogenetics and Genomics Laboratory, Faculty of Medicine, University of Coimbra, Coimbra, Portugal;
CIMAGO - Center of Investigation on Environment, Genetics and Oncobiology - Faculty of Medicine, University of Coimbra, Coimbra, Portugal;
2
Head and Neck Cancers represent tumours located in the upper aero-digestive tract, being about 90% Head and Neck
1
Squamous Cell Carcinoma (HNSCC) [ ]. It was considered the sixth most malignant tumour worldwide and it is
2
estimated the occurrence of 600 000 new cases per year [ ]. Despite clinical and technological advances, the five-year
3
survival rate has not improved much in the last years [ ]. The characterization of commercial cell lines has obvious
benefits, since they are one of the most used model in biomedical studies. As such, genetic characterization is a
necessity to have as much information as possible about the cell lines, especially if they are used for translational
4
research [ ]. HSC-3 (metastatic) and BICR-10 (non-metastatic) cell lines were cultured in DMEM supplemented with
10% fetal bovine serum and 1% of penicillin and streptomycin. For BICR-10, 1% of hydrocortisone was also added. The
genetic characterization was performed by karyotyping and array Comparative Genomic Hybridization (aCGH).Our
results showed the presence of copy number variations (CNV) on BICR-10 that were associated with early events and
progression from a dysplasia to a carcinoma in situ stage, as the case of 11q distal loss. The HSC-3 cell line presented
regions associated with a metastatic stage, such as 1q and 3q gains. Both exhibited regions associated to a worse
prognosis. The non-metastatic line showed less CNV than the metastatic one and the latter presented alterations at
nearly every chromosome. The BICR-10 cell line showed on average 40 chromosomes and HSC-3 56. Our results
between aCGH and karyotyping were concordant. We conclude that both aCGH and karyotyping are helpful for
genetic characterizations and their results are complementary. Furthermore, both lines presented CNV that were
associated to the carcinogenesis model suggested to HNSCC. All sum up, these two cell lines represent an important
resource for further investigation into HNSCC’s development.
1.Stadler ME, Patel MR, Couch ME, Hayes DN. Molecular biology of head and neck cancer: risks and pathways. Hematology/oncology clinics of North America 2008;
22(6): 1099-124, vii.;
2.GLOBOCAN. http://globocan.iarc.fr/Pages/Map.aspx. 22-07-2014.
3.Schmitz S, Ang KK, Vermorken J, et al. Targeted therapies for squamous cell carcinoma of the head and neck: current knowledge and future directions. Cancer
treatment reviews 2014; 40(3): 390-404.
4.White JS, Weissfeld JL, Ragin CC, et al. The influence of clinical and demographic risk factors on the establishment of head and neck squamous cell carcinoma cell lines.
Oral oncology 2007; 43(7): 701-12.
P12. Fatty acid production in Saccharomyces cerevisiae by a disassociated fatty acid synthase
system (FAS type II)
1
Flávio de Azevedo , Björn Johansson
1
1
CBMA – Centro de Biologia Molecular e Ambiental, Departamento de Biologia, Universidade do Minho, Braga, Portugal
Microbial biosynthesis of fats and oils from renewable carbon sources has attracted significant attention in recent
years for the potential production of biofuel and other commodities. Almost all organisms synthesize de-novo fatty
acids via a well conserved cyclic series of four reactions involving the condensation, reduction, dehydration and
reduction of carbon-carbon bonds. In nature there are two main types of fatty acid synthase systems (FAS), type I and
type II. FAS I systems utilize a single large, multifunctional polypeptide and are common to both mammals and fungi,
although with some structural differences. On the other hand, Plants and Bacteria’s (as well in mitochondria and
chloroplasts) utilize the disassociated FAS type II system. The reactions of the type II FAS, unlike FAS I, are catalyzed by
discrete and monofunctional enzymes, the proteins are all expressed as individual polypeptides from separated genes.
The organization of FAS II enables the synthesis of several fatty acid products and is more amenable to modification of
chain length. The separation of functions in single polypeptides also facilitates the metabolic optimization of each
reaction step along the sequence, which is impossible with FASI, since the subunits are stoichiometrically fixed. In this
work we implemented a FASII system consisting of 12 individually expressed genes in a S. cerevisiae strain carrying a
conditional allele of the fatty acid synthase genes of the endogenous FAS system. The physiological consequences of
this expression will be discussed.
Acknowledgments: This work was funded by the FCT project MycoFat PTDC/AAC-AMB/120940/2010. F.A. was supported by an FCT fellowship SFRH/BD/80934/2011.
This work was supported by FEDER through POFC – COMPETE and by Portuguese funds from FCT through the project PEst-OE/BIA/UI4050/2014
56
P13.
Validation of monoolein-based nanocarriers for siRNA cancer therapy
1,2
1,2
1,2,3
1,2,3
4,5
4
Ana C. N. Oliveira , Fernando Almeida , Ivo Lopes , Odete Gonçalves , Thomas Martens , Koen Raemdonck ,
2
4,5
1,3
2,3
Marlene Lúcio , Kevin Braeckmans Andreia C. Gomes , Maria Real Oliveira
1
CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus of Gualtar, Braga, Portugal
Centre of Physics, Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
3
Nanodelivery – I&D em Bionanotecnologia, Lda., Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
4
Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium
5
Center for Nano- and Biophotonics, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium
2
The RNA interference (RNAi) mechanism has been used in the context of gene therapy with the aim of modulating
expression of genes involved in a number of pathologies. Although exciting successes have been achieved in the
advance of RNAi based therapies, the ability to deliver siRNA to target cells is still problematic. We have been working
[1]
in the development of an efficient and safe liposomal nanocarrier system for plasmid DNA delivery , which is now
[2]
being validated for siRNA cancer therapy . Here we report the optimization and validation of these nanocarriers for
siRNA delivery using different cellular models. Liposomes formed by the cationic lipid dioctadecyldimethylammonium
bromide (DODAB) and MO were used to encapsulate siRNA. Optimizations to the system included the addition of
poly(ethylene glycol) (PEG) or an extra anionic cargo (PG - polyglutamate). The characterization of the interations
between nanocarriers and biological models was performed by evaluating the cytotoxicity induced in various cell lines,
the silencing efficiency in different cancer models (leukemia and breast cancer), and stability in biological fluids. Our
results show that the nanocarriers have a small size and a highly positive surface charge that was reduced by the
presence of PEG in the formulation. The presence of PG did not alter the physicochemical characteristics of the
nanocarriers. Additionally, the PEGylated nanocarriers exhibited good stability in bodily fluids. Low cytotoxicity and
efficient gene silencing could be achieved by siRNA-loaded nanocarriers in different in vitro cancer cell models.
[1]
Silva, J.P.N.; Oliveira, A.C.N.; Casal, M.P.P.A.; Gomes, A.C.; Coutinho, P.J.G.; Coutinho, O.P.; Real Oliveira, M.E.C.D., Biochim. Biophys. Acta 2011, 1808, 2440–2449.
Oliveira, A.C.N.; Martens, T.F.; Raemdonck, K; Adati, R.D.; Feitosa , E.; Botelho, C.; Gomes, A.C.; Braeckmans, K; Real Oliveira, M.E.C.D., ACS Appl. Mater. Interfaces
2014, 6, 6977−6989.
Acknowledgements: This work was supported by FEDER through POFC – COMPETE and by national funds from FCT (PEst-OE/BIA/UI4050/2014 (CBMA), PEstC/FIS/UI0607/2013 (CFUM) and PTDC/QUI/69795/ 2006). Marlene Lúcio holds a position of Researcher FCT (IF/00498/2012), and Ana Oliveira a FCT scholarship
(SFRH/BD/68588/2010). K. Raemdonck is a postdoctoral fellow of the Research Foundation – Flanders (FWO-Vlaanderen).
[2]
P14.
Detection of endophytic fungus in different cultivars of Olea europaea L
1
1
2
2
Daniela Costa , Telma Fernandes , Fátima Martins , Paula Baptista , Teresa Lino-Neto
1
1
Biosystems & Integrative Sciences Institute (BioISI), Plant Functional Biology Center (CBFP), University of Minho, Campus de Gualtar, 4710-057
Braga, Portugal
2
Mountain Research Center (CIMO), Polytechnic Institute of Bragança, School of Agriculture, Campus Sta. Apolónia, 5300-253 Bragança, Portugal
Olea europaea L. is an important economic resource in many Mediterranean basin countries, including Portugal.
Anthracnose and verticillium wilt are two of the major olive diseases due to their high incidence and related olive
losses. Picual, Galega and Cobrançosa are cultivars from O. europaea with different susceptibilities to the
abovementioned diseases, from which endophytic species (EF) have been isolated. Based on their ecological function
and also in their abundance in tolerant cultivars to these diseases, some EF fungi were selected for further studies
since they could be acting as biocontrol agents. This work pretends to detect and correlate the presence/absence of
the studied EFs with diseases susceptibilities and thus suggest an EF protecting role against diseases. For this, in order
to detect these putative EF antagonistic species, specific primers were designed for each fungal species that will then
be used in diagnostic assays. The internal transcribed spacer (ITS) of nuclear ribosomal DNA (rDNA) of each EF species
was aligned with phylogenetically related sequences and primers were designed to avoid non target species
amplification. Amplification specificity of diagnostic primers is discussed and detection of EF on distinct olive cultivars
is presented.
57
P14.
Detection of olive fruit fly (Bactrocera oleae) DNA for designing a biocontrol strategy
1
2
1
2
2
Telma Fernandes , Ana M. Dinis , Daniela Costa , José A. Pereira , Paula Baptista , Sónia A.P. Santos
1
Neto
2, 3
, Teresa Lino-
1
Biosystems & Integrative Sciences Institute (BioISI), Plant Functional Biology Centre, University of Minho, Campus de Gualtar, 4710-057 Braga,
Portugal.
2
Mountain Research Center (CIMO), Polytechnic Institute of Bragança, School of Agriculture, Campus Sta. Apolónia, Apartado 1172, 5301-855
Bragança, Portugal.
3
Barreiro School of Technology, Polytechnic Institute of Setúbal, Rua Américo da Silva Marinho, 2839-001 Lavradio, Portugal.
Olive tree has a great economic, social and environmental impact in the Mediterranean region, where it is used for
olives and olive oil production. Besides gastronomy, olives can be also used for skin care and medicinal purposes, and
olive tree wood is used for furniture production. These applications make the olive tree one of the most important
crop species in Mediterranean basin. One of the most harmful olives pests is Bactrocera oleae (Rossi) (Diptera:
Tephritidae), a monophagous fruit fly. The female fly lays the eggs into the olive fruits, where the newly hatched
larvae feed upon the pulp, harming the production of olives and the quality of the oil. This could result in economic
losses of up to 600 million euros per year. To avoid the release of large amounts of pesticides into the environment, a
sustainable strategy to control this pest has been attempted by using alternative pest management practices. The use
of B. oleae predators could be an effective strategy to decrease this pest population and putative predators have been
selected from olive groves. In this work, in order to find the most important predators of B. oleae, the digestion profile
of insect species like Calathus granatensis Vuillefroy and Pterostichus globosus Fabricius (Coleoptera: Carabidae) and
Forficula auricularia L. (Dermaptera: Forficulidae) was quantified by RT-qPCR. Using specific primers for B. oleae for
the mitochondrial cytochrome oxidase subunit I (COX1), the digestion profile was followed by recovering the intestinal
content of the predators after different feeding periods. After DNA extraction, B. oleae remains were evaluated and
quantified by RT-qPCR. Results are discussed taking into account the possible use of evaluated predators as biocontrol
agents of olive fruit fly.
Acknowledgments: This work is funded by FEDER funds through COMPETE (Programa Operacional Factores de Competitividade) and by national funds by FCT (Fundação
para a Ciência e a Tecnologia) in the framework of the project EXCL/AGR-PRO/0591/2012.
P16.
Evaluation of Portuguese cork oak´s (Quercus suber L.) ectomycorrhizal community
1
1
2
1
Olival H , Reis F , Baptista P , Tavares R , Lino-Neto T
1
1
Biosystems & Integrative Sciences Institute (BioISI), Plant Functional Biology Center (CBFP), University of Minho, Campus de Gualtar, 4710-057
Braga, Portugal
2
Mountain Research Center (CIMO), Polytechnic Institute of Bragança, School of Agriculture, Campus Sta. Apolónia, Apartado 1172, 5301-855
Bragança, Portugal
Cork oak (Quercus suber L.) forest has a high socio-economic value to Portugal. Approximately 300,000 tonnes of cork
are harvested all over the world each year, of which Portugal contributes with more than 50%. In addition, Portugal is
the world’s greatest importer of raw cork that will be processed to be later exported. Since almost 90% of the
processed cork is for the foreign market, cork-derived annual transactions nearly achieve 1000M €. The microbial
component of forest ecosystems can establish beneficial, neutral or detrimental associations with host plants, playing
an important role in plant health and productivity. In forest trees, ectomycorrhizal (ECM) fungi play an essential role in
water and nutrient supply. As other plant tree species, cork oak is very dependent on ECM community, in order to
face the typical Mediterranean climate with long, hot and dry summers and mild, rainy winters. This work intends to
assess the ECM community in a high density cork oak forest (sobreiral) that does not allow the practice of agriculture
underneath the trees (Mata das Mestras, Alcobaça). Soil core samples were collected (in duplicate) in five plots (100
2
m each). All soil mycorrhized root tips were isolated according to their color, shape and texture. Molecular
identification, using ITS1 region, allowed the identification of the fungal taxa from isolated root tips. The active ECM
fungal diversity will be discussed taking into account the corresponding ecosystem, as well as other well-described oak
forest ecosystems.
58
P17.
Antifungal capacity of aqueous extract of Ribes rubrum, on Aspergillus fumigatus
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,3
Soriano T. , Soares A. S. , Alves E. , Silva I. , Rocha A. C. , Teixeira S. , Matos M. , Coelho A.C. , Leal F.
1,2
1
UTAD - University of Trás-os-Montes e Alto-Douro, Quinta de Prados, 5000-801 Vila Real, Portugal
Centre of Agricultural Genomics and Biotechnology (CGBA), University of Trás-os-Montes e Alto-Douro, Quinta de Prados, 5000-801 Vila Real,
Portugal
3
CECAV- Center for Animal Science and Veterinary, University of Trás-os-Montes and Alto-Douro, Department of Veterinary Sciences, Portugal
2
Aspergillus fumigatus is the most prevalent airborne fungal pathogen in developed countries, and in
immunocompromised patients causes a usually fatal invasive aspergillosis. Aspergillus fumigatus is normally the most
pathogenic for men, of all known species, depending the infection more on the host immunity condition than the
virulence or pathogenesis of the fungus. Ribes rubrum, known as red currant, is rich in phenolic acids, flavonoids and
phytoalexins, compounds studied because of their antifungal activity. The objective of this study was to evaluate the
antifungal potential of the aqueous extract, obtained from Ribes rubrum fruits, against Aspergillus fumigatus. The
evaluation of the extract ability as antifungal was performed by the mycelial growth method. Aspergillus fumigatus
was grown on potato dextrose agar (PDA) and the effect of four concentrations (5, 10, 20 and 30 mg / mL) of the plant
extract on the R. rubrum have been tested. The results indicated that R. rubrum showed antifungal activity against A.
fumigatus in all concentrations tested. The concentrations of 20 and 30 mg / mL were those that were more effective
showing an increase in mycelial growth inhibition around 3 times compared to control. The results suggests that the
fruit extract of Ribes rubrum has concentration-dependent antifungal effect, and further studies will be carried out in
order to enable the use of this plant in the treatment of diseases caused by Aspergillus fumi
P18. Transcriptional profile of the “major” satDNA sequence of Rattus norvegicus in cancer
genomes
1
1
Susana Meles , Filomena Adega , Raquel Chaves
1
1
Center of Agricultural Genomics and Biotechnology (CGBA), Department of Genetics and Biotechnology (DGB), Laboratory of Cytogenomics and
Animal Genomics (CAG), Vila Real, Portugal, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal,
Contrarily to what was believed, it seems that a great part of repetitive sequences have the potential to transcribe.
Moreover, the resulting non-coding RNAs seem to display important cell functions, as heterochromatin
assembly/maintenance, transmission of epigenetic information, cellular differentiation or response to stress. Satellite
non-coding RNAs have also been associated to cancer. In the present work, we defined the transcriptional profile of
the centromeric “major” satellite DNA sequence SatI of Rattus norvegicus (RNO) in cancer versus a normal genome.
The comparative analysis of SatI in a normal cell line of RNO and in three DMBA-induced rat mammary tumor cell lines
was performed, namely: HH-16cl.2/1 (RMF), the HH-16.cl.4 (RMT) and CLS-ACI-1(CLS). We verified, by Real Time RTqPCR, the occurrence of SatI transcripts in normal and in tumor cell lines, displaying however differences in the levels
of expression, namely an increase of transcripts in cancer cells. The cellular location of SatI transcripts accessed by
RNA-FISH, allowed ascertaining their restrict presence in the nucleus both in normal and tumor cells. SatI is organized
in clusters in all the cells analyzed, but in tumor cells is also found dispersed with smaller signals. The cellular profile of
SatI was further characterized in RNO and RMF and it showed the presence of SatI transcripts in cycling cells at all the
mitotic cell cycle phases and in cells outside the cell cycle in the tumoral cell lines, revealing that it isn´t cell cycle
dependent. The response to stress of this repeat in the different genomes revealed an overexpression in all the cell
lines in serum starvation. Confluence stress only induced overexpression in RMT.The exact roles of these transcripts
are now being deeply analysed. Due to the mechanistic similarity between the tumoral process in human and rat, cell
lines derived from RNO tumors can be successfully used as models for fundamental research and new therapy
development.
59
P19.
Satellite DNA - an essential piece to unveil phylogenetic relationships in Bovidae Family
1
1
Ana Escudeiro , Lara Baptista, Raquel Chaves , Filomena Adega
1
1
Biotechnology (DGB), Laboratory of Cytogenomics and Animal Genomics (CAG), Vila Real, Portugal
The family Bovidae (order Cetartiodactyla) comprises approximately 140 species, whose evolutionary relationships are
often obscure, in large part due to morphological convergence among species. Many of these species are of economic
importance (e.g cattle, sheep), while others are endangered or threatened with extinction. The chromosomal
complement of domestic cattle (2n=60) is considered as reflecting the ancestral karyotype condition, forming the
basis from which the recent bovid karyotypes derived. Satellite DNA (satDNA) sequences represent a useful
phylogenetic source of information, since changes in satDNA composition serve as markers of phylogenetic
relationships, in part due to the rapid turnover that characterize most of them. Bovine satDNA families are majorly
found at the centromeric and pericentric regions of the chromosomes and constitute ~25% of the total genomic DNA
content, being quite heterogeneous. Six major satDNA families have already been isolated from Bos taurus (Bovini
tribe) genome, and some of these sequences’ motifs have been interestingly found in different bovid tribes, namely
Tragelaphini. This tribe is represented by medium- to large-bodied species that are distributed through forested
regions of South Africa of the Sahara, and are assigned to two genera: Tragelaphus and Taurotragus. The scope of this
work was the analysis of these satDNA families in the genome of two Tragelaphini species, Taurotragus oryx
(2n=31m/32f, Common Eland) and Tragelaphus angasii (2n=55m/56f, Nyala). To achieve this, six satDNA families were
isolated, cloned and physically mapped onto the chromosomes of these two species. The orthologous sequences
analyzed revealed phylogenetic proximity of the species under study, and the differences detected on the
hybridization patterns provided important information about the evolutionary history of these Tragelaphini species
since the ancestral genome.
P20. PMSat – a novel satDNA conserved across time as a starting point to disclose the role of
centromeric non-coding RNAs
1
1
Ana Mendes-da-Silva , Filomena Adega , Raquel Chaves
1
1
Biotechnology (DGB), Laboratory of Cytogenomics and Animal Genomics (CAG), Vila Real, Portugal
Initially named “junk DNA” or “selfish genetic material”, satellite DNA (satDNA) is currently considered an essential
fraction of eukaryotic genomes. In the last decade increasing evidences have suggested that these repetitive DNA
sequences are transcribed and that the resulting non-coding RNAs (ncRNAs) play important roles in the organization
and regulation of genomes. Transcription of centromeric (CT) and pericentromeric (PCT) satDNA sequences in the
genomes of many eukaryotes is currently undeniable. In the present study we performed the cytogenetic and
molecular analysis of the “major” satellite DNA of Peromyscus eremicus (Cricetidae, Rodentia) - PMSat – in four
species of Peromyscus genus – Peromyscus eremicus (PER), Peromyscus maniculatus (PMA), Peromyscus leucopus
(PLE) and Peromyscus californicus (PCA). Physical mapping of PMsat on PER chromosomes revels that this sequence
presents a predominantly PCT location in all chromosomes. However, on PMA, PLE and PCA chromosomes the same
sequence presents a CT location, which was confirmed by co-localization with centromeric protein-A (CENP-A). To get
some more insights on the probable functional significance of PMsat, the transcriptional activity of this sequence was
also analysed in this work. The quantification of PMsat transcripts through reverse transcription quantitative real-time
PCR (RT-qPCR) showed that PMSat are transcribed and the differences detected between total and small RNA,
revealed that, most probably, PMsat is transcribed mainly in small RNAs (~200bp). As the function(s) and mechanisms
underlying satDNA sequences transcription remain unclear, it is essential to increase our knowledge in this field by
analysing as many of these sat DNA families as possible. The existence of a satDNA that are conserved across time in
sequence, location and transcriptional activity reinforces their functional significance. These findings place the
Peromyscus species and PMSat as a starting point for disclosing the role of a novel centromeric satDNA ncRNAs.
60
P21. Genetic and epigenetic characterization of laryngeal squamous cell carcinoma using array
comparative genomic hybridization and methylation-specific multiplex ligation-dependent
probe amplification
1
1,2
3,4
Vanessa Marques , Ilda P Ribeiro , Jorge Migueis , Francisco Marques
2,5
1,2
1,2
Baptista , Joana B Melo , Isabel M Carreira
2,5,6
1
7
, Nuno Lavoura , Maria J Julião , Isabel P
1
Cytogenetics and Genomics Laboratory, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 2CIMAGO - Center of Investigation on
Environment, Genetics and Oncobiology - Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 3Department of Otorhinolaryngology Head and Neck Surgery, Coimbra Hospital and University Centre, CHUC, EPE, Coimbra,Portugal; 4Department of Anatomy, Faculty of Medicine,
University of Coimbra, Coimbra, Portugal; 5Department of Dentistry, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 6Stomatology
Unit, Coimbra Hospital and University Centre, CHUC, EPE, Coimbra, Portugal; 7Department of Pathology, Coimbra Hospital and University Centre,
CHUC, EPE, Coimbra, Portugal
Introduction: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignant tumour in the
[1,2]
world and it can arise from the larynx . The presence of a tumour in this region leads to impairment of vital
anatomical structures. Also, laryngeal tumours are usually diagnosed in a late stage. Solid tumours, such as laryngeal
[3]
squamous cell carcinoma (LSCC), result from a multistep process where genetic alterations play a major role .
However, those alterations are usually studied as part of HNSCC and so one of the major challenges is to identify
tumour markers that will help to distinguish laryngeal tumours from other cancers included in head and neck family
[2,3]
and to improve its survival rates .Material and Methods: DNA was extracted from eight fresh-frozen tissue samples
of laryngeal tumours, collected from patients with LSCC, after surgery. Copy number variations (CNV) were accessed
by Array Comparative Genomic Hybridization (aCGH) and one sample from palatine uvula was used as control.
Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) was used to access CNV and to
analyse the methylation profile of the same samples. For each MS-MLPA reaction, three different controls, which
were also extracted from palatine uvula, were used. Results: aCGH results showed mainly gains of genetic material,
especially on chromosome 3q, 7q, 11q and 14q13.1. The most common losses were located on chromosomal regions
3p, 8p, 9p, 12p, Yp and Yq. Overall, MS-MLPA results support the alterations found by aCGH. Regarding the
methylation profile, no genes were found to be significantly methylated. Conclusion: Although no significant
epigenetic changes were found, our study revealed several chromosomal alterations that may be implied in molecular
progression of laryngeal cancer. The correlation between genetic alterations and clinic-pathological data has the
power to identify putative biomarkers with possible diagnostic and prognostic value.
[1].Wong, T.-S., Gao, W., Li, Z.-H., Chan, J. Y.-W. & Ho, W.-K. Epigenetic Dysregulation in Laryngeal Squamous Cell Carcinoma. J. Oncol. 2012, 10 pages (2012).;
[2].Todorova, T. A. et al. Mutational Status of CDKN2A and TP53 Genes in Laryngeal Squamous Cell Carcinoma. Pathol Oncol Res (2014).; [3].Almadori, G. et al. Multistep
laryngeal carcinogenesis helps our understanding of the field cancerisation phenomenon: a review. Eur J Cancer 40, 2383–2388 (2004).
P22.
Heterologous expression of D. Hansenii Jen1 homologs in S. cerevisiae
1
1
1
1
1
1
1
David Ribas , Isabel Soares-Silva , Zozefina Foskolou , Beatriz Barata , Daniela Bessa , Sandra Paiva , Odília Queirós ,
1
Margarida Casal
1
Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Campus de Gualtar, Braga , Portugal
The Jen1 proteins in yeast are involved in the uptake of mono/dicarboxylic acids. We have functionally characterized
the four ScJen1 homologs of D. hansenii by heterologous expression in Saccharomyces cerevisiae. The characterization
of the Debaryomyces hansenii carboxylate uptake system revealed the existence of mediated transport systems for
the uptake of lactate, acetate, succinate and malate. The expression of the DhJen genes was detected by RT-PCR in all
the carbon sources, namely lactate, succinate, citrate, glycerol and glucose. The heterologous expression through
p416GPD shuttle vector in the S. cerevisiae W303-1A jen1 ady2 strain demonstrated that the D. hansenii Jen genes
encode four carboxylate transporters. The Dh27 gene is an acetate transporter (Km 0.94 mM; Vmax 0.43 nmol s-1 mg1), the Dh17 a malate (Km 0.27 mM; Vmax 0.11 nmol s-1 mg-1) and Dh18 (Km 0.31 mM; Vmax 0.83 nmol s-1 mg-1)
and Dh24 (Km 0.16 mM; Vmax 0.19 nmol s-1 mg-1) two succinate transporters. Surprisingly no lactate transporter was
found, although D. hansenii presents a mediated transport for this acid. With this work we extended the current
knowledge on yeast carboxylate transporters by characterizing four new plasma membrane transporters in D.hansenii.
Nevertheless, future studies are still necessary to fully characterize the carboxylate transporter since the gene coding
the lactate permease is still unidentified.
61
P23. Signaling Isc1p-regulated cell death: the role of possible upstream and downstream
mitochondrial mediators
1
1
1,2
1
2,3
1
Vanessa de Campos , Rita Cunha , António Rego , Maria João Sousa , Vítor Costa ,Susana R. Chaves and Manuela
1
Côrte-Real
1
Centro de Biologia Molecular e Ambiental, Departamento de Biologia, Universidade do Minho, Braga;
Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto;
3
Departamento de Biologia Molecular, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto.
2
Mitochondria play a vital role in energy production through oxidative phosphorylation and have an important function
in the regulation of various other processes, including stress responses and cell death. In recent years, interest into
the role of sphingolipids in mitochondria function, redox homeostasis and lifespan has greatly increased, since the
regulation of sphingolipid metabolism may be of potential therapeutic relevance in pathologies associated with
oxidative stress and ageing. Ceramide is a bioactive sphingolipid that modulates cellular processes such as apoptosis
and ageing. The yeast Saccharomyces cerevisiae has a well-defined genome and is a genetically tractable organism.
Therefore, studies in this simple genetic model system may be helpful in defining the role of sphingolipids in
mitochondrial function. The neutral sphingomyelinase nSMase2 is considered a major candidate for mediating
ceramide signalling during stress responses and ageing. Deficiency in Isc1p, the yeast orthologue of mammalian
nSMase2, affects redox status and iron homeostasis and increases cell death by apoptosis. This project aims to
uncover the role of possible downstream mitochondrial mediators.
P24.
A Genetic Approach in Canine Periodontal Disease: The TLR4 Gene
1
1
1,2
3,4
5
3,5
Nuno Anjo , Carlos Albuquerque , Francisco Morinha , João Requicha , Isabel Dias , Carlos Viegas , Estela Bastos
1
1
Department of Genetics and Biotechnology, School of Life and Enviromental Sciences, University of Trás-os-Montes e Alto Douro (UTAD), P.O. Box
1013, 5001-801 Vila Real, Portugal
2
Center of the Research and Technology of Agro-Environmental and Biological Sciences, UTAD
3
Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, ICVS/3B’s Research Group, PT Government
Associated Laboratory –Biomaterials, Biodegradables and Biomimetics, University of Minho, Department of Polymer Engineering, AvePark, 4806909 Caldas das Taipas, Guimarães, Portugal
4
Faculty of Veterinary Medicine, University Lusophone, Lisbon, Portugal
5
Department of Veterinary Sciences, School of Agrarian and Veterinary Sciences, UTAD
The aim of this study is to improve the molecular and genetic characterization of Periodontal Disease (PD) in dogs and
[1]
to contribute to the knowledge about this disease, which has a huge incidence in this species and in man too . PD is
[2]
an inflammatory disease caused by bacterial plaque in the periodontium . Associated with the beginning and
[1]
progression of PD, it is evident an increase of Gram-negative bacteria in subgingival microbiota . The existing
bacterial flora in the mouth and their products trigger an immune response. This response is performed by innate
[3]
immune recognition receptors, such as toll-like receptors (TLR) . Based on these facts, a candidate gene, TLR4, was
selected. TLR4 encodes membrane’s receptors that identify lipopolysaccharides (LPS) present in the outer membrane
of Gram-negative bacteria, and mutations in this gene can cause different predispositions in PD. To proceed to this
study, a population of 90 dogs composed by 40 dogs with PD in the case group and 50 healthy dogs in the control
group, was studied. The Statistical Package for Social Sciences (SPSS) version 21.0 helped into detection of differences
in distributions for each TLR4 variation between the two groups. To analyse separately the haplotype frequencies,
RunGC program was resorted. In this program, also significant differences between these two groups were tested
using a likelihood ratio test (LRT). Although there were no significant differences in this study between control cases
and test cases, we consider this gene an interesting candidate. Furthermore, in the haplotype analysis, we found
differences between individuals: TAA individuals were only found in the case group and AGA individuals were only
found in the control group. Based on this, it is necessary to deepen our knowledge with future investigations.
[1]
Niemiec BA. 2008. Periodontal Disease. Topics in Companion Animal Medicine. 23(2):72-80
Albuquerque C, Morinha F, Requicha J, Martins T, Dias I, Guedes-Pinto H, Bastos E, Viegas C. 2012. Canine Periodontitis: The dog as an important model for
periodontal studies. The Veterinary Journal. 191:299-305
[3]
Hajishengallis G. 2014. Immunomicrobial pathogenesis of periodontitis: keystones, pathobionts, and host response. Trends in Immunology. 35:3-11
[2]
62
P25. Genetic identity and variation of local apple varieties (Malus x·domestica Borkh.) from
Algarve using microsatellite markers
1
1
1
2
2
Isaura Castro , Vanessa Ferreira , Olinda Pinto-Carnide , António Marreiros , Margarida Costa , Valdemar Carnide
1
1
Centro de Genómica e Biotecnologia Agrária (CGBA), Universidade de Trás-os-Montes e Alto Douro, 5000-801 Vila Real
Direção de Serviços de Desenvolvimento Agroalimentar e Rural, Direção Regional de Agricultura e Pescas do Algarve, Apartado 282, Patacão, 8001904 Faro
2
Morphological, organoleptic and sensory peculiarities of apple local varieties allow to easily distinguishing them from
commercial varieties being an advantage for farmers and consumers. Many of these local varieties are almost
forgotten, not well characterized and out of the market circuits. It is of the utmost importance the identification,
characterization and conservation of these varieties. Traditionally, identification of varieties of horticultural trees was
based on morphological traits. However, this method has some limitations, including the number of available
characters, environment–genotype interactions, time consuming, and lack of power to discriminate among genetically
close types. For these reasons, morphological descriptors are often supplemented by molecular analysis. Molecular
markers, especially SSRs (Simple Sequence Repeats), have been extensively used to study genetic diversity and
relationships in Malus x domestica Borkh. SSRs are a powerful and informative means of fingerprinting, once they are
abundant in most genomes, generally distributed across the whole genome, hypervariable, co-dominant, and highly
reproducible. The objective of this study was to investigate the level of genetic differentiation among local apple
varieties from Algarve region, to detect possible duplications, misidentifications and introgression of foreign genes or
varieties into this apple gene pool. A set of 25 local varieties and eight reference genotypes (including commercial
varieties and other Malus species) were genotyped at sixteen SSR loci established by the European Cooperative
[1]
Programme for Plant Genetic Resources (ECPGR) that span most of the apple genome , and have been tested on a
set of standard Malus accessions. Our results highlight the genetic diversity encompassed in the set of samples
analysed. A strong similarity between some varieties, namely 'Maçã Ácida'/'Pedregal' and 'Malápio'/'Malápio de Pé
Curto'/'Malápio Pé de Porco' suggests possible synonymy.
[1]
Evans KM, Fernández F, Laurens F, Feugey L, van de Weg WE (2007) Harmonising fingerprinting protocols to allow camparisons between germplasm collections.
Eucarpia XII Fruit Section Symposium, Zaragoza (Spain), pp 16–20.
P26.
Aloe vera decreases colon cancer cell invasion through metalloproteinase-9 inhibition
1,2
2
2
Eduarda Veríssimo , Ana Lima , Sara Monteiro , Ricardo Ferreira
2,3
1
Molecular Biology and Genetics, Faculdade de Ciências, Universidade de Lisboa, Portugal
Disease and Stress Biology Group, Centro de Botânica Aplicada à Agricultura, Instituto Superior de Agronomia, Universidade de Lisboa, Portugal
3
Disease and Stress Biology Group, Instituto de Tecnologia Química e Biologia, Universidade Nova de Lisboa, Portugal
2
Colorectal cancer is the second most common diagnosed cancer and the second cause of death by cancer in the
European Union. During the last decade, several reports demonstrated that extracellular matrix degradation induced
by a subgroup of matrix metalloproteinases (MMPs) called gelatinases (MMP-2 and specially MMP-9) is largely
responsible for colorectal cancer progression/metastasis, suggesting that MMP inhibitors may be a powerful tool to
reduce cancer invasion. The Aloe genus has over 450 species already identified and spread throughout the world.
Among them, Aloe vera is the most studied specie due to its ancient usage as a medicinal agent. The main properties
attributed to this plant are related to anti-inflammatory, wound healing and most importantly anti-cancer properties,
suggesting it may act as a gelatinase inhibitor. Under this context, the goal of the present study was to ascertain if
Aloe vera is an MMP-9 inhibitor and if so to determine its possible mode of action. For that purpose, total leaf extracts
were tested on the colon cancer cell line HT29. Cell migration and proliferation, as well as extracellular gelanolytic
activity were evaluated after 48-hour incubation with leaf extracts of A. vera. Genetic expression profiles of specific
genes associated to cancer invasion were also monitored during exposure by RT-qPCR. A. vera extracts were indeed
able to induce a significant reduction in cell invasion and inhibition of the gelanolytic activity. Results also show that
this reduction was mostly due to specific MMP-9 inhibition, and the expression evaluation on genes related with its
transcription helped to determine if the anticancer mechanism was due to direct enzymatic inhibition or to a
reduction in gene expression. Overall, our data indicate that A. vera extracts have indeed potential in cancer therapy,
being able to reduce colon cancer cell proliferation and migration by conditioning the metabolic cascades that
contributes to MMP-9 transcription.
63
P27.
Genetic trace of postglacial and Neolithic movements in Southeast Asia
Andreia Brandão
1,2,3
, Martin B. Richards
3,4*
, Pedro Soares
1,5*
, Luísa Pereira
1,6*
1
Instituto de Investigação e Inovação em Saúde, Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto,
Portugal
2
Instituto de Ciências Biomédicas da Universidade do Porto (ICBAS), Porto, Portugal
3
Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom
4
School of Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom
5
CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Braga, Portugal
6
Faculdade de Medicina da Universidade do Porto, Porto, Portugal.
* These authors contributed equally to this work.
Island Southeast Asia (ISEA) is still one of the less genetically characterized region in the world, and it is often absent
from ancestry studies of the Pacific even though it is a key location for understanding the population history of that
region. Recent genetic studies suggest that the simplistic and most widely accepted model of an Australo-Melanesian
first settlement ~60 ka followed by an Austronesian expansion around 4.5 ka does not fully capture the complexity of
ISEA demographic history. To clarify the main dispersal routes and their impact in ISEA population prehistory, we
performed a comprehensive study with a total of 114 newly complete mtDNA genomes affiliated in haplogroups
previously associated with several demographic events in SEA. The most-parsimonious phylogenetic trees were
reconstructed including all published data. The statistical analyses included ρ statistics and maximum likelihood (ML)
to estimate the coalescence times of clades, and Bayesian methods to evaluate changes in effective population size
through time. Our results show two main demographic events contributing to the gene pool of ISEA populations. One
was the result of climatic changes and subsequent landscape alterations which lead to the postglacial dispersal of
mtDNA lineages N9a6a, R9ab1a1a, B4c1b2a2, B5b1c, F3b1 and possibly R9c1a from both Mainland and ISEA,
representing the major signal in ISEA. However dispersal of Austronesian-speaking Neolithic populations from Taiwan
towards insular SEA were detected introducing mtDNA lineages D5b1c1, B4b1a2, F1a4a and Y2a1, that together with
previously analysed M7c3c, correspond to ~20% of ISEA lineages.
FCT grants: PTDC/IVC-ANT/4917/2012 and SFRH/BD/69353/2010
P28. The study of the endocytic trafficking of the yeast monocarboxylate transporter Jen1p
from Saccharomyces cerevisiae
1
2
1
Gabriel Talaia , George Diallinas , Margarida Casal , Sandra Paiva
1
1
Center of Molecular and Environmental Biology, Department of Biology, University of Minho, Campus de Gualtar, Braga, 4710-057, Portugal
Faculty of Biology, University of Athens, Panepistimiopolis 15784, Athens, Greece.
2
The cellular plasma membrane contains proteins capable of recognise or transport a variety of compounds, such as
neurotransmitters, nutrients and virus. Their regulatory mechanisms are crucial in the cell. The intracellular trafficking
routes of some of these cargos have been extensively studied and reviewed (Mukherjee et al, 2006; Lauwers et al,
2009; Steinbusch et al, 2011; Scheller, 2013; Liaunardy-Jopeace and Gay, 2014). Their complexity clearly predominates
on multicellular organisms. Therefore, our group has been focusing on the study of a yeast model cargo, the plasma
membrane monocarboxylate transporter Jen1p from the unicellular organism Saccharomyces cerevisiae. Glucose
triggers a rapid endocytosis of Jen1p being the main signal ubiquitylation through Rsp5p ubiquitin ligase (Paiva et al,
2002; Paiva et al, 2009) and the arrestin-like adaptor Rod1p (Becuwe et al, 2012). Recently, it was described that when
Rod1p is active, it is mostly localized at the trans-Golgi network (Becuwe and Leon, 2014). However, it is largely
unknown which Jen1p intracellular domains are targeted by Rod1p and Rsp5p and their specificity for cellular
compartments.
We performed domain swap experiments between Jen1p and other well-known transporters by gap repair (Lundblad
and Zhou, 2001). Here, we will present the successful genetic strategy adopted but also data regarding the
characterization of the chimeras at the protein and subcellular levels.
Acknowledgments: This work was supported by SFRH/BD/86221/2012 and PEst-OE/BIA/UI4050/2014.
64
P29. Isolation of genomic DNA from mature leaves of European Yew suitable for PCR
amplification
1
1
1
1
2
2
3
João Roque , Bruna Mendes , Ivo Pavia , Maria Lemos , Maria João Gaspar , Ana Carvalho , Maria Margarida Ribeiro ,
2
José Lima-Brito
1
University of Trás-os-Montes and Alto Douro, 5001-801 Vila Real, Portugal
Centre of Agricultural Genomics and Biotechnology (CGBA), University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal
3
Department of Natural Resources and Sustainable Development, Instituto Politécnico de Castelo Branco. Escola Superior Agrária, Castelo Branco,
Portugal.
2
European yew (Taxus baccata L.) is a broadly distributed conifer in Europe, Asia and North Africa. However, is
considered an endangered species, due to wildfires, grazing and invasion by exotic species. In Portugal, T. baccata is
autochthonous in Serra da Estrela, Serra do Gerês, Madeira and Azores. The isolation of high-quality genomic DNA
suitable for molecular analyses constitutes a hard task in polyphenols- and polysaccharides-rich species, such as
gymnosperms. Our research group recently tested three protocols for isolation of genomic DNA from mature leaves
of T. baccata in order to proceed with the molecular characterization of the Portuguese populations using different
molecular marker systems. In the present study, the following extraction methods were tested: CTAB-based protocol
described by Doyle and Doyle (1987) with minor modifications; Genomic DNA Purification Kit (Thermo Scientific); and
DNeasy Plant mini kit (Qiagen) in leaf samples of 10 T. baccata individuals from Serra da Estrela. The integrity of the
isolated genomic DNA samples was evaluated after electrophoresis on 0.8% agarose gels, and quantified in the
spectrophotometer Nanodrop ND-1000® (Eppendorf). Afterwards, the DNA samples were used in PCR amplification
experiments using the UBC 835 ISSR primer. The CTAB-based protocol produced the highest DNA concentrations
(average of 828.71 ng/µL) while the average concentrations achieved with the kits ranged from 4.99 ng/µL (Thermo
Scientific) to 17.90 ng/µL (Qiagen). The DNA purity based on the ratio A260/A280 was also higher in samples extracted
with the CTAB method (~1.8) while the kits produced DNA samples contaminated with proteins (A 260/A280 < 1.8). Due
to the low DNA concentration and purity of the samples obtained with the Thermo Scientific kit, we did not use them
for the ISSR-PCR assays. All the DNA samples extracted with the CTAB-method and the DNeasy kit proved to be
suitable for PCR since ISSR markers were amplified. However, based on our results, the genomic DNA of the remaining
individuals sampled in the Portuguese populations of T. baccata will be isolated with the CTAB method to ensure the
achievement of high-quality and amplifiable DNA required for further molecular studies of these genetic resources.
P30. Expression and specificity profile of the major acetate transporter AcpA in Aspergillus
nidulans
1
1
2
Margarida Casal ,Sá-Pessoa J. , Amillis S. , , Diallinas G.
2
1
Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Campus de Gualtar, Braga, Portugal
Faculty of Biology, Department of Botany, University of Athens, Panepistimioupolis, Athens 15781, Greece.
2
AcpA has been previously characterized as a high-affinity transporter essential for the uptake and use of acetate as
sole carbon source in Aspergillus nidulans. Here, we follow the expression profile of AcpA and define its substrate
specificity. AcpA-mediated acetate transport is detected from the onset of conidiospore germination, peaks at the
time of germ tube emergence, and drops to low basal levels in germlings and young mycelia, where a second acetate
transporter is also becoming apparent. AcpA activity also responds to acetate presence in the growth medium, but is
not subject to either carbon or nitrogen catabolite repression. Short-chain monocarboxylates (benzoate, formate,
butyrate and propionate) inhibit AcpA-mediated acetate transport with apparent inhibition constants (Ki) of
16.89±2.12, 9.25±1.01, 12.06±3.29 and 1.44±0.13mM, respectively. AcpA is also shown not to be directly involved in
ammonia export, as proposed for its Saccharomycescerevisiae homologue Ady2p. In the second part of this work, we
search for the unknown acetate transporter expressed in mycelia, and for other transporters that might contribute to
acetate uptake. In silico analysis, genetic construction of relevant null mutants, and uptake assays, reveal that the
closest AcpA homologue (AN1839), named AcpB, is the 'missing' secondary acetate transporter in mycelia. We also
identify two major short-chain carboxylate (lactate, succinate, pyruvate and malate) transporters, named JenA
(AN6095) and JenB (AN6703), which however are not involved in acetate uptake. This work establishes a framework
for further exploiting acetate and carboxylate transport in filamentous ascomycetes.
65
P31. Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through
autophagy-independent degradation of damaged mitochondria
1,2
1
1
1
3,4
1
1
Suellen Ferro , Helena Pereira , Sara Alves , Lisandra Castro , Fátima Baltazar , Susana Chaves , Ana Preto ,
1
Manuela Côrte-Real
1
CBMA- Centre of Molecular and Environmental Biology. Department of Biology, University of Minho, Campus de Gualtar, Braga, Portugal.
ICBAS - Institute of Biomedical Sciences Abel Salazar. University of Porto, 4050-313, Porto, Portugal.
3
Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal
4
ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal
2
Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce
mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces
lysosome membrane permeabilization (LMP) in CRC cells, associated with release of the lysosomal protease Cathepsin
D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that
CatD can play an anti-apoptotic role in this process, since pepstatin A (a CatD inhibitor) increases acetate-induced
apoptosis of CRC cells. These results mimicked our previous data in the yeast system showing that acetic acid activates
a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the
vacuolar protease Pep4p, ortholog of mammalian CatD. This protease was required for cell survival in a manner
dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this
study, we assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, like acetic acid in
yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD
with siRNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased
mitochondrial mass. Using yeast cells, we further show that the delay in mitochondrial degradation of Pep4p-deficient
cells is reverted by expression of either wild-type Pep4p or CatD, but not of a catalytically inactive Pep4p mutant. This
demonstrates the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented
by CatD, indicating this mechanism is conserved.
P32. Phylogeography of mtDNA haplogroup L2: 60,000 years interactions between Central and
Eastern Africa
1,2
1,3
1,4
Marina Silva , Farida Alshamali , Luísa Pereira , Pedro Soares
1,5
1
Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Portugal.
Faculdade de Ciências da Universidade do Porto (FCUP), Portugal.
3
General Department of Forensic Sciences & Criminology, Dubai Police GHQ, Dubai, United Arab Emirates.
4
Faculdade de Medicina da Universidade do Porto (FMUP), Portugal.
5
Centro de Biologia Molecular e Ambiental (CBMA), Departamento de Biologia, Universidade do Minho, Braga, Portugal.
2
Mitochondrial DNA (mtDNA) haplogroup L2 originated in Western Africa but is nowadays spread across the entire
continent, being very frequent also in Eastern and Southern Africa. L2 movements were previously postulated to be
[1, 2]
related to the Bantu expansion, which crossed sub-Saharan Africa in the last 5 thousand years (5 ka)
. However,
previous HVS-I (Hypervariable segment I) analysis showed that L2 expansion eastwards probably occurred much
earlier, during the early Holocene (~10-12 ka). We aimed to reconstruct the phylogeny of haplogroup L2 to provide
insights on the complex net of migrations that occurred in Africa in the last thousand years. Our results show that
lineages in Southern Africa cluster with west-central African lineages at a recent time scale, whereas, eastern lineages
seem to be older, suggesting that L2 expanded eastwards in the early Holocene. Three moments of expansion are
associated to L2: (1) ~70-50 ka, during the upper Palaeolithic, (2) post-glacial movements (~15-10 ka), when most of L2
lineages arrived in Eastern Africa and (3) the Bantu Expansion (<5 ka) that took L2 southwards. A similar pattern is
observed in the phylogeny of subhaplogroup L0a. Complementary population analysis indicates no strong evidence of
mtDNA gene flow between eastern and southern populations, suggesting that Bantu permanence in Eastern Africa did
not result in strong admixture with local populations and the populations that migrated southwards had almost
entirely ancestry in Central African mtDNA gene pool.
[1]
Salas A, Richards M, De la Fe T, Lareu M-V, Sobrino B, Sánchez-Diz P, Macaulay V, Carracedo A: The making of the African mtDNA landscape. Am J Hum Genet 2002,
71:1082–1111.
[2]
Pereira L, Macaulay V, Torroni A, Scozzari R, Prata MJ, Amorim A: Prehistoric and historic traces in the mtDNA of Mozambique: insights into the Bantu expansions
and the slave trade. Ann Hum Genet 2001, 65:439–458.
66
P33. Molecular responses of yeasts to human galectins
1
1
Giulia Cazzanelli , Cândida Lucas ,Hans-Joachim Gabius
1
2
2
CBMA – Molecular and Environmental Biology Centre, University of Minho, Portugal
Physiological Chemistry, Department of Veterinary Sciences, Ludwig-Maximilians-University, Germany.
Galectins from high Eukaryotes form a numerous family of high affinity β-galactosides-binding lectins sharing a
conserved carbohydrate-binding domain. Only in humans, fifteen galectins were described, differing in specificity for
sugar, in protein structure and the ability to multimerize. The most studied is Galectin-3. This is ubiquitously expressed
within a cell, and varies in role depending on the subcellular localization, the cell type and the proliferation state.
Moreover, it also localizes extracellularly, in which case the recognition of specific carbohydrate patterns by its
carbohydrate-binding domain is important for the cell adhesion and morphology and for the clustering of membrane
protein which transfer information inside the cell. Moreover, Gal3 has a role in the recognition of various pathogens,
including Candida albicans. It was reported to bind to β-1,2-mannosides of this yeast cell wall inducing fungicidal
effect. This study analyses the biological responses of the yeast Saccharomyces cerevisiae to the contact with human
galectins, as a means to unveil the molecular responses associated. Several purified galectins were used - Gal-3, Gal-1,
Gal-4 and Gal-7, and the results confronted with identical testing using a structurally and functionally different lectin Concanavalin A. Moreover, the assays were done in parallel in a strain of Candida albicans for confront with the
literature. Parameters associated with viability/cell cycle progression, as well as apoptotic/necrotic cell death, were
accessed. All the galectins affected differently at least one of the assessed parameters, suggesting that each should
interact with a different ligand, putatively located in the cell surface, able to transduce into the cell and stimulate a
different response pathway. The outer cell wall highly mannosylated proteins are probable candidates. Gal-3 showed
to affect more aspects of the yeast cells, decreasing cell viability and increasing cell size, ROS production and
membrane damage in both S. cerevisiae and C. albicans.
Acknowledgments: Marie Curie Initial Training Network Glycopharm (PITN-GA-2012-317297). This work was further supported by FEDER through POFC – COMPETE and
by national funds from FCT through the project PEst-C/BIA/UI4050/2011.
P34. Diversity of Iberian Peninsula cowpea (Vigna unguiculata L. Walp.) varieties assessed by
morphological traits
1
2
2
1
Márcia Carvalho , Isaura Castro , Manuela Matos , Eduardo Rosa , Valdemar Carnide
2
1
Centre for Research and Technology of Agro-Environment and Biological Sciences (CITAB), University of Trás-os-Montes and Alto Douro (UTAD),
5000-801 Vila Real, Portugal
2
Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal
The accurate assessment of genetic variability is important in the preservation and use of germplasm resources and
breeding of varieties. Morphological characterization of plant genetic resources is the first stage in genetic diversity
studies. Cowpea, Vigna unguiculata L. Walp., is a grain legume originated in Africa and cultivate worldwide due to its
good protein quality with high nutritional values, its nitrogen-fixing ability and its adaptation to high temperatures and
drought. A total of 39 local cowpea varieties (29 from Portugal and 10 from Spain) were characterized using eight
morphological traits based on the Bioversity (ex-IBPGR) descriptors for cowpea. Two qualitative characters (growth
habit and flower colour) and six quantitative characters (plant height, height to first pod, days to flowering, number of
pods per plant, number of seeds per plant and seed weight per plant) were recorded during vegetative and
reproductive stages. The morphological characterization using this set of morphological traits revealed a high
variability within the 39 local cowpea varieties. All varieties presented indeterminate growth habit (87.1% semi-erect
and 12.9% climbing growth). White flowers were predominant (65.5%) comparing to purple (34.5%). Average plant
height ranged 28 cm to 200 cm, and average height to first pod between 18.6 cm and 59.2 cm. The earliest variety
reached flowering at 70 days and the latest at 109 days after sowing. Concerning production traits, in average, the
number of pods per plant ranged from 4 to 31.5 pods, the number of seeds per plant varied from 36.1 to 320.8 seeds,
and the seed weight per plant from 3.8 g to 46.5 g. In conclusion, it is possible to affirm that this cowpea collection has
a high variability. This study will be complemented with more cowpea varieties and the development of a molecular
characterization.
Acknowledgements: Project EUROLEGUME nº613781 supported by the European Union’s Seventh Framework Programme for research, technological development and
demonstration.
67
P35.
Genetic diversity assessed by SNP markers in Portuguese cowpea varieties
1
2
2
1
Márcia Carvalho , Isaura Castro , Manuela Matos , Eduardo Rosa , Valdemar Carnide
2
1
Centre for Research and Technology of Agro-Environment and Biological Sciences (CITAB), University of Trás-os-Montes and Alto Douro (UTAD),
5000-801 Vila Real, Portugal
2
Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal
Cowpea, Vigna unguiculata L. Walp., is protein-rich legume crop that plays a key role in sustaining food security for
people and their livestock. This legume is characterized by its good protein quality with a high nutritional value, its
nitrogen-fixing ability and its adaptation to high temperatures and drought. Single Nucleotide Polymorphism (SNP) is a
single base pair site in the genome that is different from one individual to another. These molecular markers are
nowadays the genetic markers of choice since they are virtually unlimited, evenly distributed along the genome, biallelic and co-dominant. Many genotyping platforms are currently available, making the use of SNPs even more
attractive and efficient. An 60,000-SNP array for cowpea was developed at the University of California (Riverside) in
partnership with African countries. This platform is being used to assess the diversity within germplasm of relevance
to Sub-Saharan Africa and US production regions. The main objective of this study is to understand cowpea’s natural
genetic variability and to assign genes for favourable traits in its genome. Leaf tissues from young plants were used for
DNA extraction. The DNA samples are genotyped using the Illumina Cowpea iSelect 60k chip at the SNP data
production service unit of the University of Southern California (USC). Of the 51,128 SNPs that were possible to
analyze on the array, over 48,500 SNPs were of high quality. Preliminary data generated in a reduced number of
Portuguese accessions revealed 4,888 polymorphic SNPs. The SNP markers are a powerful tool in genetic diversity
studies in cowpea and are more effective compared with other molecular markers or morphological traits. The SNPs
analysis of a set of cowpea accessions, from the five continents is being carried out as well as its morphologic
characterization.
Acknowledgements: Project EUROLEGUME nº613781 supported by the European Union’s Seventh Framework Programme for research, technological development and
demonstration.
P36. Population Analysis and Functional Genetic Polymorphisms of C3435T and G2677T MDR1
Gene
1
1
Andrea Cunha , Daniela Moreira , Ricardo Dinis-Oliveira
1,3,4,5
1,2
, Odília Queirós , Roxana Moreira
1,2
1
CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Gandra PRD, Portugal;
CBMA - Center of Molecular and Environmental Biology / Department of Biology / University of Minho, Braga;
3
REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto;
4
Department of Clinical Analysis and Public Health, Center of Research in Health Technologies (CITS)-IPSN-CESPU, CRL, Vila Nova de Famalicão;
5
Department of Legal Medicine and Forensic Sciences, Faculty of Medicine, University of Porto, Porto.
2
Polymorphisms in genes encoding for proteins that mediate their efflux, such as the MDR1 gene encoding Pglycoprotein (Pgp), may affect the efficacy of drug therapy, determining the interindividual variability in drug
resistance. In this context, and since the genes that encode for these proteins are highly polymorphic, it is necessary
to perform detailed genetic and functional analyses. Aiming to predict the incidence of certain genetic variations in
the Portuguese population, the polymorphisms C3435T and G2677T (MDR1 gene) have been studied in samples from
donors of Instituto Superior de Ciências da Saúde-Norte, by HRM assays. The frequency of the homozygous mutant
genotype was the 17.6% and 6.7% for the C3435T and G2677T polymorphisms, respectively. The frequency of the
homozygous mutant genotype was 2,5%, for both polymorphisms in simultaneous. To complement the population
study, and because several studies for these polymorphisms are inconclusive, the specific role of each polymorphism
was determined. For that, S. cerevisiae was used as a model organism to express the MDR1 gene with and without
polymorphism, in order to determine the influence of the polymorphism in Pgp functionality. In this work, for the
heterologous expression of human Pgp, we used the strain AD1-8 S. cerevisiae, deleted in the Pdr pumps. After
confirming the functionality of the gene (by phenotypic assays and by the use of GFP as reporter gene), the effect of
the polymorphism in the expression of Pgp was assessed. We verify the presence of Pgp in the plasma membrane of
transformed cells, allowing to confirm its correct traffic of the protein. Then, we evaluated phenotypically the strain
obtained and we verified that the strain AD1-8 with the polymorphisms showed an improved growth in medium
supplemented with doxorubicin, when compared to strain AD1-8 transformed with the wild type gene. More studies
are ongoing in order to determine the specific role of these polymorphisms in Pgp activity.
68
P37.
Silk-based polymers functionalized with fibronectin type-II for improved cell adhesion
1,2,3,4*
1,2*
1,2,3,4
1,2
1,2
1,2
Ana Margarida Pereira
, Raul Machado , Telma Bernardo
, André da Costa , Artur Ribeiro , Tony Collins ,
1,2
3,4
5
3,4
1,2
Andreia C. Gomes , Isabel Leonor , David Kaplan , Rui L. Reis , Margarida Casal
1
CBMA - Centre of Molecular and Environmental Biology, University of Minho, Braga, Portugal
Department of Biology, University of Minho, Braga, Portugal
3
3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence
on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal
4
ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal
5
Departments of Biomedical Engineering, Chemistry and Physics, Tufts University, Medford, Massachusetts, 02155, USA
2
Recombinant protein-based polymers (rPBPs) are an emerging class of biopolymers inspired by Nature. These
biopolymers exhibit outstanding features such as biocompatibility and unique physical-chemical and mechanical
[1]
properties . Moreover, molecular genetics tools enable us to fine tune the molecular structure of rPBPs with precise
control over its sequence and to combine in the same polypeptide chain the properties of two or more different
[2]
proteins . Taking advantage of advances in recombinant DNA technology, we have created hybrid biopolymers
composed of a recombinant silk-elastin-like protein and a silk functionalized with a cell adhesion domain. After being
processed into films, the polymers were assessed for their cytotoxicity and their capability to induce cell adhesion.
Results unveil novel biopolymers with cell adhesion properties that are promising candidates to be use as biomaterials
for biomedical applications.
[1]
Rabotyagova, O.S., P. Cebe, and D.L. Kaplan, Protein-Based Block Copolymers. Biomacromolecules, 2011. 12(2): p. 269-289; [2] Machado, R., et al., High level expression
and facile purification of recombinant silk-elastin-like polymers in auto induction shake flask cultures. AMB Express, 2013. 3: p. 1-15.
P38.
Developing a recombinant Saccharomyces cerevisiae strain for growth on xylan
1
1
Leila Esteki , Tony Collins , Björn Johansson
1
1
The production of lignocellulosic biofuels has become the focus of intense research with a major social purpose of
shifting from a petroleum to biomass based society. However, microbial fermentation of plant cell wall components
i.e. cellulose, hemicellulose and lignin, requires hosts capable of tolerating lignocellulosic inhibitors and high sugar and
ethanol concentrations. It should also be able to effectively utilise all the sugar monomers, both hexoses and
pentoses. Saccharomyces cerevisiae is a tolerant organism capable of fermenting hexose sugars efficiently but cannot
[1]
utilise D-xylose (pentose) , the main component of xylan. β-1,4-xylans are complex polysaccharides found mainly in
secondary walls of plants and can represent up to 35% of the total dry weight in certain plants. Indeed, complete
degradation of lignocellulosic components requires the concerted action of a number of enzymes. While many
attempts have been made to develop D-xylose fermenting S. cerevisiae strains, we aim to construct a strain capable of
direct xylan utilisation. To this aim the 1278-bp open reading frame of the Pseudoalteromonas haloplanktis endo-1,4[2]
beta-xylanase gene was amplified by PCR from an E. coli expression plasmid . This was then fused with one of two
different secretion signals: that of the mating factor α1 (MFα1s) or the K28 virus toxin in order to produce
extracellular endo-1,4-beta-xylanase. The fusion genes were cloned in a URA3-based shuttle vector by gap repair
cloning using S. cerevisiae CEN.PK113-5D and were expressed under control of the RPS19a promoter and RPL12a
terminator. Transformation of a yeast strain with these vectors, harboring fused MFα-xyl and K28-xyl, resulted in
production and secretion of active endo-1,4-beta-xylanase into the growth medium. The production and activity of
recombinant xylanase protein was confirmed using the DNS assay. The MFα1s protein construct showed twice the
xylanase activity of the K28 protein construct. The β-xylosidase encoding gene xlnD (2,417-bp) was amplified by PCR
from Aspergillus niger genomic DNA. This was cloned with and without the MFα1 secretion signal using the same
strategy as for the xyl gene and expressed under the control of the RPL12 promoter and TEF1 terminator. We have
expressed these genes together with a xylose metabolic pathway including a D-xylose transporter in S. cerevisiae
[3]
CEN.PK113-5D . Here the effects of this co-expression on growth and metabolism of birch wood xylan will be
discussed.
Acknowledgements: This work was financed by national funds from Fundação para a Ciência e Tecnologia (FCT) through Project EXPL/BBB-BIO/1772/2013.
[1]
Kötter, P., and Ciriacy M. 1993. “Xylose Fermentation by Saccharomyces cerevisiae.” Applied Microbiology and Biotechnology; [2] Collins, T., Meuwis M.A., Stals I.,
Claeyssens M., Feller G., and Gerday G. 2002. “A Novel Family 8 Xylanase, Functional and Physicochemical Characterization.” The Journal of Biological Chemistry 277 (38):
35133–39; [3] Romaní, A., Pereira F., Johansson B., and Domingues L. 2014. “Metabolic Engineering of Saccharomyces Cerevisiae Ethanol Strains PE-2 and CAT-1 for
Efficient Lignocellulosic Fermentation.” Bioresource Technology 179C (December): 150–58.
69
P39. Genotypic characterization of a heterogeneous collection of clinical isolates of
Pseudomonas aeruginosa
1,2
3
Cristina Sousa Mesquita , Alberta Faustino , Pedro Miguel Santos
1,2
1
CBMA – Centre of Molecular and Environmental Biology, University of Minho, Gualtar, 4710-057 Braga, Portugal.
Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
3
Clinical Pathology Service, Hospital de Braga, Sete Fontes-São Victor, 4710-243 Braga, Portugal
2
Pseudomonas aeruginosa is an opportunistic pathogen capable to outcompete in both community and hospital
settings, emerging as a leading cause of nosocomial acute and chronic infections. Therapeutic options are becoming
more and more limited with the continued emergence and spread of antimicrobial resistant strains among patients,
equipment and hospital staff. An ongoing collaboration with Hospital de Braga aims to provide relevant information
and tools to circumvent the pathogenicity of P. aeruginosa by a systematic characterization of clinical isolates. The aim
of the present study was to infer the genetic diversity of a heterogeneous collection of clinical isolates, by RAPD-PCR.
A seasonal collection since 2010 resulted in 703 P. aeruginosa clinical isolates from 394 patients, ranging from 1
month to 99 years old, being the elder individuals more susceptible to infections. Most of the isolates were obtained
from urine samples and lung fluids from a variety of infections, 47% of which containing other pathogenic bacteria
(e.g. E. faecalis, E. coli, MRSA and K. pneumoniae). Among the isolates sampled, 24% were classified as multi-resistant
(resistant to 3 or more classes of antibiotics). The P. aeruginosa clinical isolates from this study presented a
phylogenetic distance ranging from 0 to 0.75. Analysis of band pattern profiles resulted in 50 molecular weight band
classes that divided our collection into 663 genotypic profiles with a similarity of 80%, indicating a great genetic
distance between clinical isolates. Working with a genetic similarity of only 30%, the collection was clustered in 15
genotype groups. However, no clear association of a group with a specific metadata (such as patient age and sex,
hospital service, source, etc.) was found, also evidencing the singularity of each P. aeruginosa isolate. Thus, the
heterogeneity of our collection highlights a great isolate singularity, with repercussions in patient segregation policies
and therapeutic strategies.
P40. FoxM1 accounts for increased sensitivity to chemotherapy with anti-mitotics in human
aged cells.
1*
1*
Sara Vaz , Joana Macedo , Elsa Logarinho
1
*
1
Aging and Aneuploidy Lab, Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
these authors contributed equally to the work
[1]
Oncogenic transcription factor FoxM1 is overexpressed in the majority of human solid cancers . Emerging data
suggest that targeting FoxM1 in mono- or combination therapy may have promising therapeutic benefits for the
[2]
treatment of cancer . FoxM1 expression is associated with the proliferative capacity of the cell, consistently with its
role in primarily driving the expression of G2/M specific genes, with associated phenotypic expression of mitotic
[3]
defects and chromosome aberrations when defective . Using high-resolution live cell imaging to observe individual
cell behavior, we found that human elderly dermal fibroblasts reproduce the mitotic defects of FoxM1 repression. In
agreement, expression of FoxM1 and its downstream targets decreases progressively with chronological ageing,
suggesting it counters senescence. Importantly, we also perceived that old fibroblasts are sensitized to cell death
when treated with anti-mitotic drugs commonly used in chemotherapy. Therefore, we not only depleted FoxM1 in
young cells using RNAi but also overexpressed a constitutively active form of FoxM1 in old cells with viral infection, to
follow their cell fate upon anti-mitotic drug treatment. Our results indicate that FoxM1 downregulation accounts for
the increased sensitivity to anti-mitotics in aged cells. Therefore, a combinatorial treatment using FoxM1 inhibition
plus microtubule poisons should be tested for the pro-apoptotic efficacy against tumor cell lines overexpressing
FoxM1.
[1]
. Pilarsky, C. et al. (2004). Identification and validation of commonly overexpressed genes in solid tumors by comparison of microarray data. Neoplasia 6: 744–50.
. Halasi M. and Gartel A.L. (2013). Targeting FOXM1 in cancer. Biochem Pharmacol 85:644-52.
[3]
. Laoukili, J. et al. (2005). FoxM1 is required for execution of the mitotic programme and chromosome stability. Nature Cell Biology 7:126–36.
[2]
70
P41. Association between genetic, epigenetic and clinicopathological features in tongue
squamous cell carcinoma
1
1,2
Sofia Lisboa , Ilda P Ribeiro , Francisco Marques
2,3
1,2
Baptista , Isabel M Carreira
2,3,4
5
1,2
6
, Maria J Julião , Joana B Melo , Artur Ferreira , Isabel P
1
Cytogenetics and Genomics Laboratory, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
CIMAGO - Center of Investigation on Environment, Genetics and Oncobiology - Faculty of Medicine, University of Coimbra, Coimbra, Portugal
3
Department of Dentistry, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
4
Stomatology Unit, Coimbra Hospital and University Centre, CHUC, EPE, Coimbra, Portugal
5
Department of Pathology, Coimbra Hospital and University Centre, CHUC, EPE, Coimbra, Portugal
6
Maxillofacial Surgery Unit, Coimbra Hospital and University Centre, CHUC, EPE, Coimbra, Portugal
2
Background: Tongue squamous cell carcinoma (TSCC) is the most common malignancy in the oral cavity, characterized
by high recurrence rates, reduced overall survival and increasing incidence worldwide. Identification of genetic
markers, as well as epigenetic alterations, involved in tongue malignant transformation and progression is a crucial
step towards a better understanding of the disease biology, thus improving diagnosis and prognosis. Methods: DNA
was extracted from fresh tissue samples of 31 primary tongue tumors, collected from patients with TSCC, upon
resection surgery. Copy number alteration and methylation status were assessed by Methylation-specific Multiplex
Ligation-dependent Probe Amplification (MS-MLPA). Gingival samples from 11 healthy donors were used as controls.
Results: From the 41 genes analyzed in this study, the ones exhibiting a higher frequency of copy number losses were
present at chromosomal arms 9p, 11p and 11q, whereas those exhibiting gains were more frequent at 2q, 11q, 16p,
17q and 19p. DNA methylation was found in 15 of the studied genes. WT1, PAX5 and MSH6 genes were aberrantly
methylated in 80,65%, 48,39% and 35,48% of the tumor samples, respectively. Methylation of MSH6 gene seems to be
associated with more advanced stages of the disease and metastasis. Conclusion: Our study revealed several genetic
and epigenetic alterations that may play a role in TSCC development in association with patient’s clinicopathological
features. The highlighted genes provide a basis for further research that may lead to the identification of candidate
biomarkers allowing for a better diagnosis and prognostication of TSCC patients.
[1]
Nygren, A. O. H. et al., (2005). Methylation-Specific MLPA (MS-MLPA): Simultaneous detection of CpG methylation and copy number changes of up to 40 sequences.
Nucleic Acids Research, 33(14), 1–9. doi:10.1093/nar/gni127
P42.
Engineering a Commercial Cold-adapted Xylanase for Enhanced Biotechnological Value
1
1
1
1
1
Mário Barroca , Leila Esteki , Gustavo Santos , Diogo Silva , Björn Johansson ,Tony Collins
1
1
Xylanases (EC 3.2.1.8) are industrially important glycoside hydrolases which catalyse the random hydrolysis of the
complex plant heteropolysaccharide xylan. We have isolated a highly active cold-adapted xylanase and developed it
for use as a technological aid in baking. This commercial enzyme is unique among xylanases, belonging to a novel
xylanase containing family and being characterised by a high activity at low to moderate temperatures, a specificity
for xylan, the production of short chain xylo-oligomers and an insensitivity to natural xylanase inhibitors. Indeed,
these unique properties point to its suitability for exploitation in various other application areas and in particular in
further baking applications, in the beverages industry, in animal feeds, in biofuel production by simultaneous
saccharification and fermentation, and in the preparation of health promoting ingredients (prebiotics). Recent studies
have already demonstrated its successful application in the latter while its reduced activity and stability at low pHs
have limited its potential for exploitation in the remaining applications listed. Currently we are using modern protein
engineering approaches in rational design and directed evolution to enhance the low pH stability and activity of this
xylanase and thereby overcome the observed limitations and further improve its commercial value.
Acknowledgments: This work was financed by national funds from Fundação para a Ciência e Tecnologia (FCT) through Project EXPL/BBB-BIO/1772/2013.
[1] Collins T., Feller G., Gerday C. and Meuwis M.A. (2012). Family 8 enzymes with xylanolytic activity. US8309336B2.
[2] Dutron A., Georis J., Genot B., Dauvrin T., Collins T., Hoyoux A. and Feller G. (2011). Use of family 8 enzymes with xylanolytic activity in baking. EP1549147B1(2011),
USP8192772(2012), CN1681392B(2010), WO2004/023879, PCT/BE03/00152; Granted and published in Europe (all countries in Europe), United States, Mexico, Japan,
Eurasia, China, Brazil, Australia.
71
P43. Altered mitochondrial function and dynamics imposed by Sirtuin 3 overexpression in
Huntington’s disease striatal cells
1
1,2
1
1
1
Catarina Carmo , Luana Naia , Ana M. Oliveira , Jorge Valero , Tatiana R. Rosenstock , A. Cristina Rego
1,2
1
CNC – Center for Neuroscience and Cell Biology, University of Coimbra (UC), Coimbra, Portugal;
Faculty of Medicine, UC, Coimbra, Portugal.
2
Mitochondrial dysfunction and altered mitochondrial dynamics are hallmarks of the neurodegeneration seen in
+
Huntington’s disease (HD) [1]. Sirtuins are NAD -dependent lysine deacetylases that have shown neuroprotective
effects in several models of neurodegeneration [2]. Here we evaluated the effect of sirtuin 3 (SIRT3), relevant due to
its mitochondrial localization, on the organelle’s function and dynamics using striatal cells derived from HD knock-in
Q111/Q111
mice (STHdh
). Intriguingly, HD cells displayed a significant increase in endogenous SIRT3 protein and mRNA
levels in relation to control cells. Overexpression (OE) of SIRT3 caused decreased lysine acetylation along with
Q7/Q7
Q111/Q111
alterations in mitochondrial membrane potential in both STHdh
and STHdh
-SIRT3 cells. Untransfected
Q111/Q111
STHdh
cells also exhibited a decrease in the levels of mitochondrial fusion proteins Mfn2 and Opa1 and an
increase in fission-related Fis1. Furthermore, the fission-related Drp1 was accumulated preferentially in the
mitochondrial fraction of mutant cells. SIRT3 OE reduced the unbalance between fission/fusion by decreasing the
Q7/Q7
Q111/Q111
Q7/Q7
protein levels of Fis1 in STHdh
and STHdh
cells, and Drp1 in STHdh
cells, with no differences in fusion
proteins. Parkin, a marker of mitophagy, a way to eliminate defective mitochondria, was also assessed; untransfected
HD cells exhibited lower Parkin levels. Although no significant differences in Parkin were found after SIRT3 OE in both
Q111/Q111
cells, increased Parkin phosphorylation at activating Ser65 was detected in STHdh
-SIRT3 cells. Nevertheless, no
conclusions can be drawn regarding an increased or impaired mitophagy under such conditions. Overall, data suggest
that enhanced levels of SIRT3 underlie reduced mitochondrial function in HD striatal cells.
Acknowledgments: Work supported by FCT projects EXPL/BIM-MEC/2220/2013 and PEst-C/SAU/LA0001/2013-2014, COMPETE and FEDER.
[1] BBA, 2012, 1822:101–10; [2] BBA, 2013, 1832:1345-59.
P44. New multiplex PCR based methodology to discriminate clinically important Candida and
Aspergillus species
1
1
1,2
3
1
Joana Carvalho-Pereira , Catarina Vaz , Manoel Marques Evangelista de Oliveira , Ricardo Araújo , Célia Pais and
1
Paula Sampaio
1
Laboratório de Micologia, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.
3
Institute of Molecular Pathology and Immunology (IPATIMUP), University of Porto, Porto, Portugal
2
Fungal pathogens are the major eukaryotic agents invasive infections, in which infections due to Candida and
Aspergillus species are the most frequent[1]. The rapid and correct identification of this species is essential since the
susceptibility to antifungal drugs varies significantly. In clinical microbiology laboratories several methodologies are
available however with low specificity and sensibility[2]. The objective of this work is the development of a new
method, based in a multiplex PCR analysis, for the identification of the most clinically important fungal species. In
order to create a multiplex strategy, nine markers were selected and combined in a single PCR reaction. A total of 126
clinical isolates from different species were used. The influence of serum in this strategy was also analyzed. This
strategy demonstrated 100% of specificity and 100% sensibility. In this study we also determined the limit of detection
3
in the presence of serum, 50x10 cells and 50 ng of isolated DNA. In conclusion, this new strategy is fast, accurate and
reproducible, allowing the correct identification of the nine most important fungal species, covering a spectrum of
more than 95% of the clinical systemic fungal infections.
1. Tortorano, A.M., et al., Epidemiology of candidaemia in Europe: results of 28-month European Confederation of Medical Mycology (ECMM) hospital-based surveillance
study. Eur J Clin Microbiol Infect Dis, 2004. 23(4): p. 317-22.
2. Ellepola, A.N. and C.J. Morrison, Laboratory diagnosis of invasive candidiasis. J Microbiol, 2005. 43 Spec No: p. 65-84.
72
P45. Propionibacterium freudenreichii short-chain fatty acids inhibit the proliferation of
colorectal cancer cells
Marta Casanova
1
2
1,2 *
2
, Lígia Rodrigues and Ana Preto
1
CBMA – Centre of Molecular and Environmental Biology, University of Minho, Braga, Portugal.
CEB – Centre of Biological Engineering, University of Minho, Braga, Portugal.
Propionibacterium freudenreichii is a commercially relevant bacterium that is well-known for its role as ripening
starter in the cheese industry. Recently, there has been an increasing interest in this bacterium due to its potential
anti-neoplastic effects particularly against colorectal cancer (CRC) as showed by our group, via the production of short
chain fatty acids (SCFA). The aim of this work was to evaluate the growth and biotransformation performance of the
bacteria under different culture conditions, as well as to evaluate its effects on CRC cells. P. freudenreichii adapted to
the digestive stress was cultivated in two bacterial culture media, cancer cells medium and medium mimicking the
content of human colon. Moreover, the effect of the propionibacteria fermentation broths on CRC cells and the effect
of the CRC cells conditioned medium on the bacteria performance were studied. P. freudenreichii showed different
performances when grown in different media, exhibiting a great potential to produce high amounts of acetate and
propionate in bacterial media followed by CRC cells medium. The effect of CRC cells conditioned medium on bacterial
performance was positive, being the bacteria able to grow while increasing its metabolic activity. Furthermore, the
inhibition of CRC cells proliferation when exposed to the SCFA produced by bacteria was confirmed. Results gathered
in this work suggest that P. freudenreichii could potentially be used in CRC prevention/treatment via their ability to
produce SCFAs.
P46.
Identification of the partners of Gup1, the yeast putative Hedgehog-like morphogen
1§
1
Joana Tulha , Célia Ferreira and Cândida Lucas
1
1
Microbes can form organized multicellular structures in which cells behave differently according to their shape and
localization within the community. In the case of yeasts, the associated morphogenesis and differentiation has
multiple meanings corresponding to colony or biofilm formation, to differentiation in pseudo/true hyphae, and to cell
shape maintenance during budding, mating and sporulation in response to environmental cues. The Saccharomyces
cerevisiae membrane-bound O-acyltransferase Gup1 is the yeast orthologue of mammalian HHATL, the negative
regulator of Hedgehog morphogen secretion. The deletion of S. cerevisiae GUP1 is associated with, namely, plasma
membrane and cell wall structure, lipid metabolism, trafficking, cytoskeleton organization and budding pattern, and in
Candida albicans with morphological switching, biofilm formation, virulence and antifungal resistance. The present
work aimed at identifying and characterizing in S. cerevisiae the intracellular partners of Gup1, as a first step to unveil
the molecular role of this protein and devise weather yeasts harbour any Hedgehog-like morphogenic pathway.
Several proteins were described to putatively interact with Gup1. These have diverse cellular localizations, which point
to the possible existence of different partners for Gup1 according to its multiple intracellular localizations previously
found. Using co-imunoprecipitation assay, we report a novel physical interaction with Gup1 – the mitochondrial Porin
(Por1). Por1 is a voltage-dependent anion channel required for maintenance of mitochondrial osmotic stability and
mitochondrial outer membrane permeability. Accordingly, cellular fractionation and western blotting confirmed the
mitochondrial localization of Gup1. We also observed that the absence of Gup1p seems to affect the cellular levels of
Por1p. Making use of single and double deletions of GUP1 and POR1, detailed phenotyping will unveil the common
associated processes.
FCT PhD Student (SFRH/BD/76025/2011).
This work was further supported by FEDER through POFC – COMPETE and by national funds from FCT through the project PEst-C/BIA/UI4050/2011.
73
P47.
A new method for the detection of genetically modified plants using multiplex PCR
1
Filipa Moreira and Filipe Pereira
1
1
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto, Portugal
Genetically modified organisms (GMOs) are those who have undergone changes in their genetic structure by means of
a biotechnological process of recombination, which allows the introduction of new features in a species [1]. The most
recently launched GMOs on the world market are plants that have different characteristics such as resistance to
herbicides or insects. The number of cultivated GMOs has been growing in recent years despite the intense discussion
about the benefits or damage that these organisms may have on humans and ecosystems [2,3]. For this reason, there
is great interest in developing effective methods for the identification of GMOs in different stages of the chain of
cultivation, processing and distribution [4]. This work describes the development of a rapid molecular application to
detect genetically modified (GM) plants at low cost. The new multiplex PCR assay allows the detection of six
transgenic sequences (P-35S, T-nos, bar, ctp2-cp4epsps, P35S-pat and FMV35S) used in most GM plant events and a
chloroplast DNA (cpDNA) trnL gene as internal control. The seven target regions were designed to yield amplicons
with different lengths to be discriminated by electrophoresis. Our method successfully detected the presence of
transgenic elements in 19 samples of reference materials from GM plants. The seven target regions were easily
discriminated both by conventional and capillary electrophoresis. The use of target regions of small length (<270 bp)
allows the analysis of forensic samples with degraded DNA, as the detection of illegal GMO crops or to verify the
labelling of food products.
[1] Parekh SR: The GMO handbook: genetically modified animals, microbes, and plants in biotechnology: Humana Press; 2004.
[2] von Götz F: See what you eat—broad GMO screening with microarrays. Analytical and bioanalytical chemistry 2010, 396(6):1961-1967.
[3] Hamels S, Glouden T, Gillard K, Mazzara M, Debode F, Foti N, Sneyers M, Nuez TE, Pla M, Berben G: A PCR-microarray method for the screening of genetically
modified organisms. European Food Research and Technology 2009, 228(4):531-541.
[4] Hemmer W: Foods derived from genetically modified organisms and detection methods: Agency BATS; 1997.
P48. Inferences regarding gene-flow between autochnous and allochthonous Scots pine
populations in Serra do Gerês based on ISSR and SCoT markers
1
1
1
1
2
2
Ivo Pavia , Maria Lemos , Andreia Delgado , Jani Pires , Maria João Gaspar , Ana Carvalho , José Lima-Brito
1
2
2
University of Trás-os-Montes and Alto Douro, 5001-801 Vila Real, Portugal
Center of Agricultural Genomics and Biotechnology (CGBA), University of Trás-os-Montes and Alto Douro, 5001-801 Vila Real, Portugal
Two native populations of Scots pine (Pinus sylvestris L.) present at Serra do Gerês (NW, Portugal) are possibly the last
remnant nuclei of a glacial refugium present in Portugal. Refugia populations are locally well adapted and constitute
reservoirs of intra-specific genetic diversity. These populations are peripheral and have experienced different
selection regimes relatively to central populations, reinforcing the need of its conservation. However, most Scots pine
populations in Portugal resulted from plantation of allochthonous material with a Central Europe origin performed in
the last century, some of which geographically close to the native populations. In this study we intend to infer the
possibility of pollen introgression in the Scots pine natural populations. We analyzed the data obtained with the
amplification of seven ISSR and eight SCoTs markers on genomic DNA from 142 individuals from two native Scots pine
population (Biduiça and Ribeira das Negras), the geographically closest allochthonous populations (Pedra Bela and
Peneda), and two foreign populations (Germany and Sweden). The sampled individuals from each of the native
populations were divided in two groups: trees with less than 30y.o.,and trees with more than 80y.o. We found that
the younger individuals of the native populations presented genetic similarity with the nearest allochthonous
populations, possibly due to pollen exchange, but differed from the older individuals. These results allowed us to
believe that these autochthons populations are now exchanging pollen with the geographically closest allochthonous
populations planted during the XX century. Therefore, is urgent to define conservation strategies for these native
populations, to avoid jeopardizing the preservation of these genetic resources of Serra do Gerês.
Acknowledgements: Study supported by the FCT project PTDC/AGR-CFL/110988/2009. AC acknowledged the post-doctoral grant SFRH/BPD/68932/2010 attributed by
FCT.
74
P49. Involvement of Arabidopsis SUMO proteases as modulators of Arabidopsis thaliana
development and response to stress
1
1,2
1
1
3
Pedro Humberto Castro ,*, Tiago Lourenço ,*, Miguel Ângelo Santos , Sara Freitas , Javier Ruiz-Albert , Rui Manuel
1
3
2
Tavares , Eduardo Rodrigues Bejarano , Herlander Azevedo
1
Biosystems and Integrative Sciences Institute (BioISI), Plant Functional Biology Center, University of Minho, Campus de Gualtar, 4710-057 Braga,
Portugal
2
CIBIO, InBIO - Research Network in Biodiversity and Evolutionary Biology, Universidade do Porto, Campus Agrário de Vairão, 4485-661 Vairão,
Portugal
3
Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas
(IHSM-UMA-CSIC), Dept. Biología Celular, Genética y Fisiología, Universidad de Málaga, Campus Teatinos, 29071 Málaga, Spain
* equal contribution
One of the major objectives of current plant biology is to understand how plants can overcome environmental
stresses without being significantly affected in growth. These findings can be translated into more stress resistant
crops without significant yield losses. Environmental conditions are constantly changing. Therefore, strategies for
stress tolerance include fast and reversible responses by post-translational modifications (PTMs), that modulate the
activity of key proteins. The Small Ubiquitin-like Modifier (SUMO) is a PTM peptide that modifies various central
regulators of the stress response [1]. SUMO attachment (sumoylation) requires SUMO peptides to first be processed
by SUMO proteases (ULPs), and then conjugated to a target’s lysine via SUMO E1 activase and SUMO E2 conjugase,
aided by SUMO E3 ligases. Deconjugation of the SUMO peptide can subsequently be carried out by the SUMO
proteases. ULPs constitute a relatively larger gene family of SUMO pathway components and may be a source of
specificity in the pathway, yet they are less functionally characterized when compared to the SUMO conjugation
components. We have characterized the developmental and environmental stress responses of previously
uncharacterized Arabidopsis T-DNA insertion mutants disrupting two functionally redundant ULPs, resulting in diverse
developmental defects and constitutively increased SUMO-conjugate levels.
This work is funded by FEDER through the Operational Competitiveness Program - COMPETE - and by national funds through the Foundation for Science and Technology
- FCT - within the scope of project “SUMOdulator” (Refs. FCOMP-01-0124-FEDER-028459 and PTDC/BIA-PLA/3850/2012).
[1] Castro PH, Tavares RM, Bejarano ER, Azevedo H. 2012. SUMO, a heavyweight player in plant abiotic stress responses. Cell Mol Life Sci 69(19):3269-3283.
P50. Identification of protein kinases involved in the phosphoregulation of Bax-dependent cell
death
1
1
1
1
2
Lisandra Castro , Sara Alves , Susana R. Chaves , Maria João Sousa , Stéphen Manon , Manuela Côrte-Real
1
2
1
Department of Biology, Centre of Molecular and Environmental Biology, University of Minho, Braga, Portugal
CNRS, UMR5095, Université de Bordeaux 2, Bordeaux, France
Apoptosis dysfunctions underlie multiple human pathologies, such as degenerative diseases and cancer. Apoptosis is
thus tightly regulated, mainly by Bcl-2 protein family [1]. The major pro-apoptotic member of this family is Bax, which
plays a central role in controlling outer mitochondrial membrane integrity in response to apoptotic stimuli. Bax can be
regulated by phosphorylation/dephosphorylation, though it is not clear how, and only a few Bax residues and kinases
involved in Bax phosphoregulation have been identified [2]. In order to identify novel kinases involved in
phosphorylation of Bax, we heterologously expressed human Bax in yeast cells lacking non-essential kinases and
determined whether there were differences in Bax phosphorylation profile. With this approach, we found putative
kinase candidates, as well as one yeast kinase (Rim11p), which have as human ortholog GSK3β kinase, previously
described as involved in Bax phosporylation. [3]. We also found new putative yeast kinases candidates, not previously
implicated in this regulation. Therefore, the proposed approach will allow assessing the consequences of this posttranslation modification on Bax function, and provide novel insights on phosphoregulation of this key apoptosis
regulator. Since protein kinases are potential drug targets, this study provides a basis for further therapeutic strategies
against diseases involving apoptotic dysfunctions.
This work was supported by FEDER through POFC – COMPETE and by Fundação para a Ciência e Tecnologia through projects PEst-OE/BIA/UI4050/2014 and FCTANR/BEX-BCM/0175/2012, and fellowships to L. Castro (SFRH/BD/93589/2013) and S. Chaves (SFRH/ BPD/89980/2012).
[1] Cory S andAdams JM Nature Reviews Cancer (2002) 2(9):647-656
[2] Renault TT andManon S Biochimie (2011) 93(9):1379-1391
[3] Linseman DA, et al. (2004) J Neurosc 24(44):9993-10002
75
P51. Antibacterial activity of genetically engineered silk-elastin-like films and fibres containing
silver nanoparticles
1,2
1,2
1,2
Raul Machado , Andreia Maria Silva , André da Costa , Andreia Castro Gomes
3,4
5,6
5,6
1,2
, Senentxu Lanceros-Mendez , Vitor Sencadas and Margarida Casal
1,2
, José Carlos Rodríguez-Cabello
1
Departamento de Biologia, Universidade do Minho, Braga, Portugal
3
Bioforge (Group for Advanced Materials and Nanobiotechnology), Centro I+D, Universidad de Valladolid, Valladolid, Spain
4
Networking Research Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), E-47011 Valladolid, Spain
5
Center of Physics of University of Minho, Universidade do Minho, Braga, Portugal
6
Departamento de Física, Universidade do Minho, Braga, Portugal
2
Advances in protein engineering combined with the use of recombinant DNA technology allow the design and
production of recombinant Protein-Based Polymers (rPBPs) with an absolute control over its composition, sequence
and length. The silk-elastin-like proteins (SELPs) are a class of rPBPs which composition is based on silk and elastin
repeating units, combining in the same polypeptide chain the outstanding mechanical and biological properties of
both proteins [1]. As base materials for biomedical purposes, SELP nanofibre mats demonstrate potential to be
applied as wound dressing materials for skin regeneration applications [2]. The increasing antimicrobial resistance
associated with the excessive and inappropriate use of antibiotics demands the research for new pathogen-free
healthcare polymeric materials. In this regard, silver (Ag) is a metal with well-known antimicrobial activity against a
broad spectrum of microorganisms [3]. In this work we report the fabrication of antimicrobial SELP/Ag materials
produced by electrospinning and solvent casting using water or formic acid as solvents and AgNO 3 at different
concentrations (1, 3, 5 wt%). The produced materials were characterized and evaluated for their antimicrobial
performance. The results indicate the formation of silver nanoparticles during the fabrication process and a
distribution throughout the fibres and films. As the produced materials are highly water soluble, water insolubility was
rendered by exposure to methanol-saturated air. FTIR analysis of the methanol-treated samples demonstrated that
water insolubility is mediated through a β-sheet conformation-driven mechanism. The silver containing materials
showed a strong antibacterial activity against Gram+ and Gram– bacteria namely Staphylococcus aureus, Bacillus
subtilis, Escherichia coli and Pseudomonas aeruginosa. Furthermore, the SELP/Ag materials did not reveal a cytotoxic
effect against normal human skin fibroblasts suggesting its potential application as antimicrobial wound dressing
medical devices.
[1] Machado R, Azevedo-Silva J, Correia C, Collins T, Arias FJ, Rodríguez-Cabello JC, Casal M. High level expression and facile purification of recombinant silk-elastin-like
polymers in auto induction shake flask cultures. AMB Express, 2013, 3:11.
[2] Machado R, da Costa A, Sencadas V, Garcia-Arévalo C, Costa CM, Padrão J, Gomes AC, Lanceros-Méndez S, Rodríguez-Cabello JC, Casal M. Electrospun silk-elastin-like
fibre mats for tissue engineering applications. Biomedical Materials, 2013b, 8:065009.
[3] Prabhu S, Poulose EK. Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects. International Nano Letters, 2012,
2: 32.
P52.
Search for Mechanisms Underlying the Anticancer Activity of Lactoferrin
1
1,2
1,2
1
2
3
Cátia Pereira ; Marília Gonçalves. , Luís Loureiro , Lisandra Castro , Hernâni Gerós , Lígia R. Rodrigues and Manuela
1
Côrte-Real
1
Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.
Centro de Investigação e Tecnologias Agroambientais e Biológicas (CITAB), Department of Biology, University of Minho, Braga, Portugal.
3
Centre of Biological Engineer (CEB), Department of Biological Engineer, University of Minho, Braga, Portugal.
2
Lactoferrin (Lf) is an iron-binding glycoprotein with multiple biological functions. Some reports have shown that Lf is
anticarcinogenic, although the molecular mechanism underlying its cytotoxicity to cancer cells remains elusive. In the
scope of a broader project aiming to elucidate the cellular targets of this protein, different analytical and biochemical
methods, including flow cytometry were used to assess the cytotoxicity in three breast cell lines with different
invasive properties. Results will be discussed in terms of molecular mechanisms underlying Lf anticancer activity.
This work was supported by FEDER through POFC – COMPETE and by Fundação para a Ciência e Tecnologia through projects PEst-OE/BIA/UI4050/2014, FCT-ANR/BEXBCM/0175/2012 and PTDC/SAU-BMA/121028/2010.
76
P53. PKA and TOR pathways regulate the negative effects of ammonium during aging of
Saccharomyces cerevisiae that are associated to replicative stress induction
1,2
1,2
3
Fernanda Leitão-Correia , Júlia Santos , Maria João Sousa , and Cecília Leão
1,2
1
Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal
ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal
3
Molecular and Environmental Biology Centre (CBMA)/Department of Biology, University of Minho, Braga, Portugal
2
The yeast Saccharomyces cerevisiae is highly exploited as a eukaryotic model to study mechanisms involved in aging
regulation. Manipulation of several single nutrients from the culture medium is known to extend cell survival in a nondividing state in yeast, known as chronological life span (CLS) and many medium components have been identified as
longevity affecting agents [1]. Here we present results showing that ammonium - a nitrogen source commonly used by
S. cerevisiae - is one of these agents that can modulate yeast longevity. The results we obtained show that
ammonium, when present in the culture medium, induces CLS shortening that was accompanied by the induction of
replicative stress and by an impairment of the essential amino acids consumption. Mep1p and Mep2p transporters
appear to partially mediate ammonium-induced cell death. Furthermore, TOR1 deletion reverted ammonium effects
both in amino acid restricted and non-restricted cultures, whereas, Ras2p and Sch9p seem to mediate cell death in
response to ammonium under amino acid restriction conditions. Our studies also indicate that ammonium could have
a crucial role in the nutritional equilibrium between nitrogen sources and glucose required for yeast longevity, mainly
through the pathways identified as signaling glucose effects on aging.
[1] Santos J, Leão C and Sousa MJ. Growth culture conditions and nutrient signaling modulating yeast chronological longevity. Oxid Med Cell Longev. 2012; 2012:680304.
P54. Strategies for Synthetic Biology: lego-ization of different genetic elements to increase
gene expression
1
1
Patrícia Apura , Sandra Cristina Viegas and Cecília Maria Arraiano
1
1
Instituto de Tecnologia Química e Biológica António Xavier/UNL, Av. da República, Estação Agronómica Nacional, 2780-157 Oeiras, Portugal
The levels of expression of a gene can depend on multiple factors: the efficiency of its transcription, the intracellular
concentration and stability of its mRNA and its capability to be translated into proteins, and the post-translational
regulation of protein yield. Accordingly, steady-state protein levels can be controlled by using combinations of
variable-strength promoters to change transcription rates, by employing different ribosome-binding sites to alter
translation efficiency and by adding degradation tags to adjust rates of protein turnover. The lego-ization of different
genetic elements allows the creation of biological devices with defined properties and ultimately systems that can
perform more complex functionalities. Degradation of mRNA has a major role in post-transcriptional control gene
expression. The steady-state level of a continuously synthesized message is directly proportional to its half-life.
Stability elements in the 5’- and 3’- UTR of the mRNA can change transcripts stability and impact on the amount of
mRNA available for translation. We have designed a set of different constructs using different stability elements
described throughout the literature, which were tested with the help of GFP as the reporter gene. The use of
fluorescent proteins is advantageous since they allow fluorescence measurements directly in living bacterial cells. We
measured the influence of genetic elements in the expression pattern in E. coli MG1655, by fluorescence intensity
measurements, and our results revealed a great variability on protein synthesis arising from the sequence elements
combined in the different constructs. The effect of different 5’ stabilizing elements on the expression of our gene of
interest was also tested. We are currently analysing the decay rate of gfp mRNA in the different constructs to evaluate
the co-relation between mRNA stability and the expression levels obtained.
77
P55. Candida albicans gene mistranslation as a modulator of host-pathogen interactions and
pathogenesis.
1
1
1
Edgar Lopes* ,Carla Oliveira , Ana R. Bezerra , Manuel Santos
1
1
Institute for Biomedicine – iBiMED, Health Sciences, University of Aveiro, Portugal.
Candida albicans is a commensal and a pathogen that occurs in a broad range of human body sites. It has the
remarkable capacity to survive and proliferate in environments with drastic changes conditions. This human pathogen
Ser
decodes the leucine-CUG codon both as leucine and serine using a novel serine tRNA (tRNA CAG ). In contrast to other
tRNAs, this is charged by two aaRSs, SerRS and LeuRS.This genetic code alteration should cause proteome disruption
and be highly detrimental due to the distinct characteristics of the two amino acids: serine is hydrophobic while
1
leucine is hydrophilic . However, C. albicans incorporates about 3% of Leu and 97% of Ser at thousands of CUGs,
producing heterogeneous mixtures of polypeptides from single mRNAs. These values are flexible as leucine
misincorporation fluctuates between 0.6% and 5% in response to environmental stress. Remarkably, misincorporation
of leucine can be artificially increased up to 98% without visible effects on fitness. The aim of this study was to identify
molecules and pathways involved in the regulation of mistranslation, and ultimately contribute to a better knowledge
of this C. albicans unique feature. To accomplish this, levels of the SerRS and LeuRS were determined using a mutant
gene reporter based on the GFP. The GFP opening reading frame of this probe was fused to the promoters of the
SerRS and LeuRS genes (SES1 and CDC60 genes) and its fluorescence was indicative of their activity. Additionally, to
identify putative regulators (kinases and transcription factors) of the SerRS and LeuRS activity, C. albicans strains from
a kinase and transcription factor knockout collections were transformed with the same fluorescent reporter system
and activity of both aaRSs were quantified in several physiological conditions, using epifluorescence microscopy. We
hope that the expression profiles of the two aaRSs will shed light on the mechanism of regulation of genetic code
ambiguity in C. albicans.
1. Santos, M. A., Ueda, T., Watanabe, K., Tuite, M. F. (1997) Mol. Microbiol. 26, 423-431.
2. Moura, G. R., Paredes, J. A, Santos, M. A. (2010) FEBS Letters, 584, 334-341
78
Sponsors
BIOGNÓSTICA
Email: geral@biognostica.com
www.biognostica.pt
NZYTECH
www.nzytech.com
ALFAGENE®
Email: info@alfagene.pt
www.alfagene.pt
SARSTEDT
Email: info.pt@sarstedt.com
www.sarstedt.com
BIOPORTUGAL
Email: bioportugal@bioportugal.pt
www.bioportugal.pt
DUX
http://www.dux.com
COMISSÃO DE VITICULTURA DA
REGIÃO DOS VINHOS VERDES
www.vinhoverde.pt
FUNDAÇÃO PARA A CIÊNCIA E TECNOLOGIA
www.fct.pt
79
CENTRO DE GENÉTICA CLÍNICA
http://www.cgcgenetics.com/pt
CÂMARA MUNICIPAL DE BRAGA
www.cm-braga.pt

Session 3

Transcription

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