BioMediTech Research Day 2013 Abstract Book

Transcription

BioMediTech Research Day 2013
Abstract Book
December 17, 2013
Finn-Medi 5, auditorium
Biokatu 12
Research Day 2013
Poster presentations
Group 1
Poster session 1/1st coffee break
(10.15-10.45)
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1. Baran Aydogan
4. Marja Pajunen
7. Riikka Nurminen
33. Ana María Soto de la Cruz
13. Katri Leinonen
16. Leena-Maija Vanha-aho
19. Sari Vanhatupa
22. Jenita Pärssinen
25. Johanna Ketolainen/Riikka
Jokimäki
28. Rami Pääkkönen
31. Kirsi Tuppurainen
34. Sina Saari
37. Sanni Virjula
40. Priit Joers
43. Tommi Rantapero
46. Satu Luhtala
49. Ville-Pekka Seppä
52. Laura Airaksinen
55. Hanna Rauhala/Elisa Vuorinen
58. Venkatesh Mallikarjun
61. Minna Hietikko
64. Virpi H. Laitinen
67. Jack George
70. Birgitta Lehtinen
73. Tomas Cervinka
76. Ilmari Tamminen
79. Annika Kohvakka
82. Matti Annala
85. Ana Andjelkovic
88. Fikret Emre Kapucu
91. Anssi Nurminen
94. Jari Väliaho
97. Nathaniel Narra
100. Sanna Turunen
103. Heidi Halonen
106. Joonas Tuominen
109. Joose Kreutzer
Group 2
Poster session 2/last
half an hour of the
lunch break
( 13.00-13.30)
2. Matti Annala
5. Liisa Sjöblom
8. Shanjun Chen
11. Aapo Tervonen
14. Tomi Ryynänen
10. Michelangelo Paci
20. Henrik Hammarén
23. Dmitriy Fayuk
26. Anni Saralahti
Group 3
Poster session 3/2nd coffee break
(15.25-15.55)
29. Tiina Joki
32. Liisa-Ida Sorsa
35. Mostafa Kiamehr
38. Toni Grönroos
41. Johanna Salonen
44. Juha Määttä
47. Cristina A. Nadalutti
50. Suvi Kalliokoski
53. Kalle Lehto
56. Jouni Väliaho
59. Maria Laaksonen
62. Leena Latonen
65. Zsuzsanna Ortutay
68. Nina Gubina
71. Kerstin Lenk
74. Ville Kytölä
77. Päivi Lillsunde
80. Mike Gerards
30. Mauro Scaravilli
17. Danilo Jesus
36. Sanna Auer
39. Susanna Teppo
42. Anni Sorkio
45. Hannu Turpeinen
48. Sanna Huttunen
51. Saara Aittomäki
54. Heidi Kontro
57. Thomas Liuksiala
60. Alejandra Rodríguez Martínez
63. Rolle Rahikainen
66. Jyrki Sivula
69. Giuseppe Cannino
72. Sampo Kukkurainen
75. Jani Sarin
78. Ashwin Sriram
81. Narayan Puthanmadam
Subramaniyam
84. Meeri Mäkinen
87. Sergei Häyrynen
90. Sini Eerola
93. Mirva Järvelä-Stölting
96. Miina Ojansivu
83. Esko Kemppainen
86. Keijo Viiri
89. Elli Käpylä
92. Markus Hannula
95. Magdaléna von
Essen
98. Timo Salpavaara
101. Maiju Hiltunen
104. Kaisa Vuornos
107. Inkeri Vornanen
110. Ashok Aspatwar
3. Antti Ylipää
6. Zuzet Martinez Cordova
9. Francesco Tabaro
12. Iina Vainio
15. Sanna-Kaisa Harjula
18. Kaisa Teittinen
21. Heini Kallio
24. Janne Koivisto
27. Milka Hammarén
99. Shokoufeh Teymouri
102. Laura Johansson
105. Jarno M. A. Tanskanen
108. Kaisa Oksanen
Programme
8.30 Opening words by Susanna Miettinen & Eric Dufour
8:35 Keynote Lecture 1. Bob Lightowlers
First Session: Cancer - from models to patients
9: 15 Juha Saarikettu: Tudor-SN effect on tumour development in the PTEN+/mouse model of cancer
9:30 Kati Kivinummi: SKIL-gene fusions define a new subtype of prostate cancer
9:45 Minna Ampuja: BMP4 inhibits proliferation and induces migration of breast
cancer cells in 3D environment
10:00 Kirsi Kuusisto: Genetic predisposition to breast cancer – in search of novel
variants
13:30 Keynote Lecture 2. Zofia Chrzanowska-Lightowlers
Third Session: in vivo models for health & diseases
14:10 Marteen Szibor: Expression of Ciona Intestinalis alternative oxidase (AOX) in
mouse
14:25 Filippo Scialo: Mitochondrial ROS regulate lifespan by two different mechanisms
14:40 Ines Anderl: The application of flow cytometry to study the cellular immune
response in Drosophila melanogaster larvae
14:55 Markus Ojanen: The role of the proprotein convertase FurinA in the zebrafish
immunity against mycobacteria
14:10 Anantha-Barathi Muthukrishnan: spatial distribution of tsr-venus clusters in
E. coli
15:25-15:55 Coffee Break/ Commercial Exhibition/Poster session 3
10:15-10:45 Coffee Break/Commercial Exhibition/Poster session 1
Fourth Session: enabling techniques
Second Session: in vitro models for health & diseases
10:45 Alexandra Mikhailova: Directed differentiation of corneal epithelial cells from
human induced pluripotent stem cells.
11:00 Anu Hyysalo: Nanofibers for human pluripotent stem cells -derived neural cells
15:55 Javier Gracia: Combining impulse oscillometry and multi-lead impedance
pneumography for regional analysis of dynamic lung function.
15:10 Edite Figueiras: Optical Projection Tomography for 3D live tissue engineering
imaging.
11:15 Marisa Ojala: hiPSC model for hypertrophic cardiomyopathy
16:25 Dhanesh Rajan: Optical non-contact pH measurements with an automatic two
wavelength system.
11:30 Mikael Virta: The capabilities of the Ultimaker 3D printer for biomedical
research purposes
16:40 Barbara Taskinen: Neutral avidin variant for the reversible formation of biotinavidin bridges.
11:45 Florentino Santos: Ex vivo study of atherosclerotic plaque using
microCT12:00-13:30
16:55 IBT Best PhD Thesis 2013
Lunch/Poster session 2
17:25 Hannu Hanhijärvi: Closing remarks
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Juha Saarikettu
2. Cancer Research
Author: Juha Saarikettu(1) (juha.saarikettu@uta.fi)
Group: Olli Silvennoinen
Co-authors: Tekele Fashe(1), Olli Silvennoinen(1,2)
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All affiliations: (1) Institute of Biomedical Technology, Biomeditech, FI-33014
University of Tampere, Finland. (2) School of Medicine, FI-33014 University of
Tampere, Finland
Tudor-SN effect on tumor development in the PTEN+/- mouse model of cancer
The transcriptional coactivator and RNA binding protein Tudor-SN (SND1/p100) is
overexpressed in some cancers. We have recently generated a Tudor-SN knockout
mouse and shown that the body weight and the weight of some organs is decreased
in the knockout. In order to determine if the Tudor-SN expression level can influence
tumorigenesis or tumor growth, we have bred the Tudor-SN knockout to the background
of heterozygous PTEN (+/-) tumor suppressor that develops tumors in various organs
including uterus, prostate, thyroid and colon. Our pilot experiment demonstrates
that tumor development by loss of PTEN allele is partially reverted in the Tudor-SN
homozygous knockout background. The result could be explained by deregulated
expression of insulin-like growth factor-binding proteins and ribosomal proteins in
the Tudor-SN knockout that can result in decreased growth signaling and translational
activity and thereby attenuated tumor growth.
Joonas Tuominen and Kati Kivinummi
Minna Ampuja
2. Cancer Research
Keywords: SKIL, prostate cancer
2. Cancer Research
Keywords: 3D culture, Matrigel, breast cancer, BMP4,
proliferation, migration
Author: Joonas Tuominen (joonas.i.tuominen@uta.fi)
Author: Minna Ampuja (minna.ampuja@uta.fi)
Group: Tapio VIsakorpi
Group: Anne Kallioniemi
Co-authors: Kati Kivinummi, Matti Annala, Matti Nykter, Tapio Visakorpi
All affiliations: Institute of Biomedical Technology
SKIL-genefusions define a new subtype of prostate cancer
We have identified a novel fusion gene TMPRSS2-SKIL. This fusion is mutually
exclusive with ETS/SPINK1 alterations. The fusion merges three first exons of
TMPRSS2 and the full length SKIL and this leads to the overexpression of SKIL due
to the androgen regulated TMPRSS2 promoter. SKIL codes for SKI-like oncogenic
protein which can bind and disturb the heteromeric SMAD complex and this inhibits
the TGF-β signaling pathway. We then further analyzed a transcriptome sequencing
dada from the Cancer Genome Atlas (TCGA) prostate adenocarsinoma project and
that revealed additional SKIL fusion with MIPOL1 and ACPP. Also the analysis of 76
tumors and 22 LuCaP xenografts with qRT-PCR identified SKIL overexpression in the
LuCaP-77 and one clinical sample. Then the whole transcriptome of LuCaP-77 was
sequenced and that revealed a SLC45A3-SKIL fusion. All four fusion positive samples
were negative for ETS or SPINK1 alterations. To determine if SKIL plays significant
role in prostate cancer we created PC-3 cell line with the knockdown of SKIL with two
different siRNA resulted in reduced cell growth, invasiveness and colony formation.
Reduced cell growth was confirmed with LNCaP cells. Then DU145 cell line with
stable overexpression of SKIL was also created. However this didn’t have any effect in
the growth of cells. Invasiveness of DU145 cells was increased in some of the clones
but remained same in others. Also EP156T and RWEP1 cell lines with overexpression
of SKIL were also created and the functional studies of those are currently on goinig.
Co-authors: Riikka Jokimäki, Kati Juuti-Uusitalo, Alejandra Rodrigues-Martinez ,
Emma-Leena Alarmo, Anne Kallioniemi
All affiliations: Institute of Biomedical Technology, University of Tampere and
BioMediTech, Tampere, Finland. Fimlab Laboratories, Tampere, Finland
BMP4 inhibits the proliferation of breast cancer cells and induces an MMPdependent migratory phenotype in MDA-MB-231 cells in 3D environment
Bone morphogenetic protein 4 (BMP4) belongs to the transforming growth factor
β (TGF-β) family of proteins. BMPs have been reported to be involved in cancer
pathogenesis. We have previously shown that BMP4 reduces breast cancer cell
proliferation and simultaneously induces migration in a subset of these cell lines. Here
we examined the effects of BMP4 in a more physiological environment using two
different 3D culture systems; Matrigel, a basement membrane extract, and a synthetic
polyethylene glycol (PEG) gel. The MCF-10A normal breast epithelial cells formed
round acini with correct apicobasal localization of α6 integrin in Matrigel whereas
irregular structures were seen in PEG gel. In PEG gel, BMP4 inhibited the growth of
MCF-10A and all five breast cancer cell lines examined, thus closely resembling the 2D
culture conditions, but in Matrigel, no growth inhibition was observed in MDA-MB-231
and MDA-MB-361 cells. Furthermore, BMP4 induced the expression of the cell cycle
inhibitor p21 both in 2D and 3D culture, thereby partly explaining the growth arrest.
Interestingly, MDA-MB-231 cells formed large stellate structures in response to BMP4
treatment in Matrigel, suggestive of increased cell migration. This effect was reversed by
Batimastat, a broad-spectrum MMP inhibitor, and subsequent analyses showed BMP4
to induce the expression of MMP3 and MMP14. Taken together, our results show that
Matrigel provides a more physiological environment for breast epithelial cells than PEG
gel. Moreover, BMP4 partly recapitulates in 3D culture the growth suppressive abilities
previously seen in 2D culture and induces an MMP-dependent migratory phenotype in
MDA-MB-231 cells.
Kirsi Kuusisto
Alexandra Mikhailova
2. Cancer Research
Keywords: Breast cancer, ovarian cancer, genetic variant
6. Regenerative Medicine
Keywords: Human pluripotent stem cells; differentiation;
corneal epithelium; serum-free
Author: Kirsi Kuusisto (1) (Kirsi.M.Kuusisto@uta.fi)
Group: Johanna Schleutker
Co-authors: Satu-Leena Laasanen (2), Johanna Schleutker (1, 3)
All affiliations: 1) Institute of Biomedical Technology / BiomediTech, University of Tampere and
Fimlab Laboratories, Tampere, Finland. 2) Department of Pediatrics, Genetics Outpatient Clinic,
and Department of Dermatology, Tampere University Hospital, Tampere, Finland. 3)Department
of Medical Biochemistry and Genetics, Institute of Biomedicine, University of Turku, Turku,
Finland
Genetic predisposition to breast cancer – in search of novel variants
Introduction Inherited genetic defects that predispose individuals to breast and ovarian
cancer are for the most part unknown in a majority of families with hereditary breast
and ovarian cancer (HBOC). We aim to identify novel HBOC predisposing genetic
variants in a cohort of high-risk BRCA1/2-negative HBOC families using three different
approach: 1) known candidate gene screening, 2) genome-wide copy number variation
analysis, and 3) whole exome sequencing in selected families.
Methods Seven candidate genes were screened for index individuals from 82 high-risk
BRCA1/2-negative HBOC Finnish families using direct sequencing, MLPA and HRM
method. Statistical analyses were done using Fisher’s exact test and in silico predictions
were performed using PON-P. Genome-wide copy number variation analysis was done
for index individuals from 84 HBOC families using Illumina HumanCytoSNP-12
beadchip. Copy number-affected genes were further studied by Gene Ontology
term enrichment, pathway analyses, and database searches. Copy number validation
experiments were carried out by qPCR. Whole exome sequencing will be carried out for
selected HBOC families and sample collection is currently ongoing.
Results Candidate gene screening identified three previously known breast cancerassociated variants (one in BRCA1 and two in CHEK2) in 11/82 (13.4%) HBOC
families and one novel BRCA2 variant predicted to be pathogenic in 1/82 (1.2%)
HBOC families. Copy number analysis identified potential variants that were enriched
in HBOC cohort compared to controls in EPHA3 and CSMD1 intronic regions and in
5q15 intergenic region.
Conclusion We have identified several potential genetic variants that likely contribute to
HBOC susceptibility in Finnish families.
Author: Alexandra Mikhailova (alexandra.mikhailova@uta.fi)
Group: Heli Skottman
Co-authors: Tanja Ilmarinen, Hannu Uusitalo, Heli Skottman
All affiliations: Institute of Biomedical Technology, University of Tampere, Finland.
BioMediTech, Tampere, Finland. SILK, Department of Ophthalmology, University of
Tampere, Finland. Tauh Eye Center, Tampere University Hospital, Finland
Directed differentiation of corneal epithelial cells from human induced pluripotent
stem cells.
Corneal epithelium is maintained by limbal stem cells, and their transplantation can
be used to treat limbal stem cell deficiency (LSCD). However, this is only possible if
enough healthy limbal tissue is available, making it necessary to search for novel cell
sources which could be used for treating LSCD. Human pluripotent stem cells (hPSCs)
provide unique opportunities for differentiation of limbal and corneal epithelial cells for
cell transplantations.
In order to improve the efficiency and reproducibility of hPSC differentiation towards
limbal epithelial-like cells, signaling cues active during ocular surface ectoderm
development were replicated with two small-molecule inhibitors in combination with
fibroblast growth factor. These early precursor cells were then further differentiated
towards limbal epithelial-like cells. The extent of differentiation was evaluated by
following the expression of key markers using immunocytochemistry and qPCR at
several time-points.
Small molecule induction down-regulated pluripotency marker expression, while upregulating the transcription factors active during ocular development. Protein expression
of the limbal epithelial marker p63 was greatly enhanced, with up to 95% of cells being
p63-positive after five weeks of differentiation. Finally, after a total of six weeks in
differentiation culture, the two markers specific to terminally-differentiated corneal
epithelium, cytokeratins 3 and 12, were expressed in an average of 35% and 71% of
cells, respectively.
In contrast to all earlier studies, corneal epithelial cells were differentiated in serum-free
culture conditions without the use of amniotic membrane or other undefined culture
substrates. This highly efficient differentiation method could potentially be used for
treating LSCD in the future.
Anu Hyysalo
Marisa Ojala
Keywords: Nanofiber, human pluripotent stem cell,
neuron, astrocyte, oligodendrocyte
Keywords: induced pluripotent stem cells, cardiomyocytes,
hypertrophic cardiomyopathy
Author: Anu Hyysalo (anu.hyysalo@uta.fi)
Author: Marisa Ojala (1,2) (marisa.ojala@uta.fi)
Group: Susanna Narkilahti
Group: Katriina Aalto-Setälä (1,2,3)
Co-authors: Mervi Ristola, Tiina Joki, Susanna Narkilahti
Co-authors: Kristiina Rajala (1,2), Risto-Pekka Pölönen (1,2), Kim Larsson (1,2)
All affiliations: Institute of Biomedical Technology, University of Tampere, Tampere,
Finland. BioMediTech, Tampere, Finland
All affiliations: 1. Institute of Biomedical Technology, University of Tampere, Tampere,
Finland. 2. BioMediTech, University of Tampere, Tampere, Finland. 3. Heart Center,
Tampere University Hospital, Tampere, Finland.
NANOFIBERS FOR HUMAN PLURIPOTENT STEM CELL -DERIVED
NEURAL CELLS
hiPSC model for hypertrophic cardiomyopathy
Modeling central nervous system (CNS) processes and deficits in vitro is currently based
on the wide use of 2D cell culture models. However, 2D models lack the structural
architecture that mimic natural environment of cells. Thus, 3D culture conditions are
needed to provide biologically more relevant environment for cells. Synthetic nanofibers
are one option for 3D cell culture models. The benefits of nanofibers include their
assembly into a variety of architectures by manipulating their alignment, stacking and
folding. In addition, they are easy to functionalize through encapsulation or attachment
of bioactive components such as extracellular matrix proteins and growth factors.
Hypertrophic cardiomyopathy (HCM) is a complex autosomal-dominant disease
associated with significant genotypic and phenotypic heterogeneity. HCM is the most
common inherited cardiovascular disorder and the leading cause of sudden cardiac death
in young adults. Typically hypertrophy affects the left ventricle and interventricular
septum and may eventually lead to left ventricular outflow tract obstruction, arrhythmias,
diastolic dysfunction, and sudden death. Other hallmark features are myocyte disarray
and fibrosis. No specific therapy is available to prevent the onset or regression of
hypertrophy.
The aim of this study is to test the suitability of nanofibers for in vitro modeling of
cellular interactions in CNS using human pluripotent stem cell (hPSC) –derived neural
cells. Commercially available electrospun polycaprolactone (PCL) nanofibers were
tested. hPSC-derived neurons, oligodendrocytes and astrocytes were cultured on the
fibers, and the cytocompatibility and the attachment, growth and differentiation of
cells were studied. Nanofibers were non-cytotoxic and immunocytochemical staining
confirmed the presence of the neurons, oligodendrocytes and astrocytes on the
nanofibers. Oligodendrocytes seemed to contact the nanofibers most prominently.
The two most predominant founder mutations for HCM in Finland are in cMYBPC
(Q1061X) accounting for 11,4% and in α-tropomyosin (TPM1, D175N) accounting for
6,5% of the HCM cases. Mutation of each sarcomeric protein is likely to result in a
distinct set of clinical characteristics. The functional consequences and the mechanisms
by which mutations cause diverse phenotypes in HCM are still only partly understood.
Another important question is why some members of the family carrying the same gene
mutation develop different severity of clinical symptoms while some remain completely
asymptomatic.
We have reprogrammed human induced pluripotent stem cells (hiPSCs) from patients
carrying Finnish founder mutations and differentiated hiPSCs into cardiomyocytes. We
have studied the morphology and functionality of HCM hiPSCs by immunocytochemistry,
Ca2+ imaging and patch clamp. With our HCM hiPSC model we can demonstrate
common and new pathophysiological mechanisms of the HCM disease in humans
regarding the two founder mutations in TPM1 and cMYBPC.
Mikael Virta
Florentino Santos
1. Biotechnology
7. Medical Imaging
Keywords: Atherosclerosis, microCT, imaging, phase contrast, iodine staining
Author: Mikael Virta (mikael.virta@tut.fi)
Author: Florentino Santos (florentino.caetanodossantos@tut.fi)
Group: Minna Kellomäki
Group: Professor Hannu Eskola
Co-authors: Atte Joutsen, Hannu Eskola, Juha Salenius
The capabilities of the Ultimaker 3D printer for biomedical research purposes
The Ultimaker 3D printer utilizes fused deposition modeling technique, which is one of
the additive manufacturing processes. The Ultimaker is capable of translating a virtual
solid model into a physical part made of polymer using an additive approach.
The objectives were to study the effects of the printing parameters on the various
properties of printed samples made of different polymer materials, to utilize self made
materials and to determine wheter the Ultimaker can be applied for biomedical research
purposes. The Ultimaker 3D printer was used to fabricate samples for mechanical
testing purposes. The studied polymers were commercial polylactide and medical
grade poly(L/DL-lactide) 70/30. The part properties were determined by tensile testing,
dimensional accuracy measurements and inherent viscosity measurements.
Results showed that the tensile strengths of polylactide parts increased due to higher
printing temperatures. However, the inherent viscosities decreased at the same time.
The melting and following cooling of the polymer material resulted in minor part
shrinkage. Self made materials can be fabricated for printing as long as restrictions of
the printer are taken account of.
In conclusion, the Ultimaker 3D printer has a potential to be utilized for several
research purposes. It is possible to fabricate complex parts for at least non load-bearing
applications and also for biomedical purposes.
All affiliations: FS, AJ and HE:Department of Electronics and Communications
Engineering, Tampere University of Technology, Tampere, Finland. HE: Department
of Radiology, Regional Imaging Centre, Tampere University Hospital. JS: Division of
Vascular Surgery, Department of Surgery, Tampere University Hospital and Medical
School, Tampere, Finland
Ex vivo study of atherosclerotic plaque using microCT
the carotid arteries. Ex vivo microCT studies are crucial to understand the morphology
of the plaque. This work is an initial approach to the use of different pre-imaging and
imaging techniques in microCT for the improvement of tissue separation.
Two patient’s plaques were imaged using different imaging principles. We imaged the
plaques using an Xradia MicroXCT-400 with parameters: 40-80 keV, slice thickness
2.3-4.4 μm and voxel size 2-4 μm. Native images of the plaques were acquired. Plaque
1 was immersed for 48 hours in an I2E (2% iodine in 70% ethanol) staining solution
before imaging. Plaque 2 was imaged using phase contrast for soft tissue enhancement.
Comparisons were done using visual inspection and whole stack histogram spread (HS).
For plaque 1 the comparison between unstained and stained microCT revealed that
staining allows a better separation between soft-hard tissues and between lipidicfibrotic tissues. The HS was six fold higher in the stained than in the native image (0.28
vs. 1.96). Phase contrast enhanced HS for plaque 2 only in 35% (1.06 vs. 1.42) when
compared with the native imaging.
As already confirmed, simple staining protocols are able to improve the separation of
tissues in microCT. Phase contrast didn’t present enough contrast improvement to be
viable. A weakness of I2E staining is that is irreversible. Further work has to be done
to correlate the microCT attenuation of each tissue type with the in vivo CT imaging
attenuations.
Marten Szibor
Filippo Scialó
Lab: Howy
Topic: Mitochondria
4. Mitochondrial Genetics
Keywords: aging, mitochondria, ros,
Marten Szibor1,2, J. Heidler3, Z. Gizatullina4, I.
Salwig2, P.K. Dhandapani1, I. Wittig5, P. Rustin6,7,
E. Dufour8, F.N. Gellerich4, H.T. Jacobs1,8, T. Braun2
Author: Filippo Scialó (filippo.scialo@uta.fi)
1Research Program of Molecular Neurology, University of Helsinki, Biomedicum Helsinki 1,
Helsinki, Finland. 2Dept. I - Cardiac Development and Remodeling, Max Planck Institute for
Heart and Lung Research, Bad Nauheim, Germany. 3Department of Molecular Hematology,
Goethe University, Frankfurt, Germany. 4University Department of Neurology, Otto-vonGuericke University, Magdeburg, Germany. 5Functional Proteomics, SFB 815 core unit, Faculty
of Medicine, Goethe University, Frankfurt, Germany. 6Inserm, UMR 676, Hôpital Robert Debré,
Paris, France. 7Faculté de Médecine Denis Diderot, Université Paris 7, Paris, France. 8Institute
of Biomedical Technology, University of Tampere, Tampere, Finland
Expression of Ciona intestinalis alternative oxidase (AOX) in mouse
The primary objective of this study was to examine the consequences of expressing alternative
mitochondrial respiratory chain enzymes under conditions of mitochondrial dysfunction in vivo.
Multiple evidence indicates that Ciona intestinalis alternative oxidase (AOX) can safely be
expressed in mammalian cells, where it is efficiently targeted to mitochondria and enzymatically
active under conditions where the final enzyme of the electron transport chain the cytochrome c
oxidase (COX) is inhibited (1-3).
AOX was targeted to the ROSA26 locus where it is expressed under the control of the strong
and ubiquitous CAG promoter. ROSA26AOX mice are viable and have no obvious phenotype
or alteration in physiological parameters. AOX is expressed in most tissues with highest level in
heart, skeletal muscle, lung and liver. Functionally, AOX expression allows azide- and antimycin
A-resistant respiration. However, the degree of mitochondrial coupling in AOX-supported
respiration depends on substrate usage and inhibitors of the respiratory chain complexes.
AOX expressing mice represent a valuable and versatile tool to study mitochondrial dysfunction in
vivo. The ROSA26AOX mouse will help to rescue disease models of mitochondrial dysfunction
thereby deciphering molecular mechanisms and aiding in the eventual development of therapies.
In addition, exploiting alternative respiratory chain enzymes is a novel approach to studying
mitochondrial respiration and its control mechanisms.
1. Dassa EP, Dufour E, Goncalves S, Jacobs HT, and Rustin P. 2009. The alternative oxidase, a tool for
compensating cytochrome c oxidase deficiency in human cells. Physiol Plant 137(4):427–434.
2. El-Khoury R, Dufour E, Rak M, Ramanantsoa N, Grandchamp N, Csaba Z, Duvillié B, et al. 2013.
Alternative oxidase expression in the mouse enables bypassing cytochrome c oxidase blockade and limits
mitochondrial ROS overproduction. PLoS Genet 9(1):e1003182.
3. Hakkaart GAJ, Dassa EP, Jacobs HT, and Rustin P. 2006. Allotopic expression of a mitochondrial alternative
oxidase confers cyanide resistance to human cell respiration. EMBO Rep 7(3):341–345.
Group: Alberto Sanz
Co-authors: Ashwin Sriram, Venk Mallikarjun, Mike Murphy *, Alberto Sanz
All affiliations: Institute of biomedical technology
* Medical Research Council (MRC)-Dunn Human Nutrition Unit Wellcome, Trust/
MRC Building Hills Road, Cambridge CB2 2XY, United Kingdom
Mitochondrial ROS regulate lifespan by two different mechanisms
Aging is an intricate biological process by which cells undergo a progressive loss of
cellular surveillance mechanisms. There is a strong connection between lifespan and
mitochondrial function. However, it is clear that aging cannot be solely explained
by the Mitochondrial Free Radical Theory of Aging (MFRTA). The physiological
production of ROS by mitochondria regulates homeostasis by selective activation of
specific pathways. By using the power of Drosophila genetics we demonstrate that ROS
are able to regulate longevity by two different mechanisms. One is the production of
mitochondrial ROS that activate pro-survival pathways involved in protein quality control
(proteostasis) and mitochondrial quality control (mitophagy). According to that, the
reduction of mitochondrial ROS by expressing AOX decreases mitochondrial turnover,
and shortens lifespan under stress. On the other hand, the increase in ROS production
by expressing the NADH alternative dehydrogenase 1 (NDI1) extends lifespan and
increase proteostasis capacity of the flies. Interestingly, the simultaneous expression
of NDI1 and AOX decreases lifespan below normal controls. We hypothesized that
this is caused by the increased pass of electrons through complex I, which damage the
complex by over-producing ROS. This will be the second mechanism by which ROS
regulate lifespan. In order to confirm our hypothesis we reduce the levels of complex
I (by using RNAi against one of the subunits of complex I) in flies expressing NDI1.
As expected, we observed that flies with low levels of respiratory complex I live longer
than flies expressing NDI1.
Ines Anderl & Laura Vesala
Markus Ojanen
5. Molecular Immunology
Keywords: Drosophila larvae, blood cells,
immune defense
Keywords: Proprotein convertase, FURIN, zebrafish,
tuberculosis, M. marinum
Author: Ines Anderl & Laura Vesala
(ines.anderl@uta.fi, laura.vesala@uta.fi)
Author: Markus Ojanen (markus.ojanen@uta.fi)
Group: Dan Hultmark
Co-authors: Dan Hultmark
All affiliations: Ines Anderl & Dan Hultmark: Department of Molecular Biology, Umeå
University, Sweden
Ines Anderl, Laura Vesala & Dan Hultmark: Institute of Biomedical Technology,
University of Tampere, Finland
Group: Marko Pesu
Co-authors: Hannu Turpeinen, Milka Hammarén, Mataleena Parikka, Mika Rämet, Marko Pesu
All affiliations: MO, HT, MH, MPa, MR, MPe: Institute of Biomedical Technology,
BioMediTech, University of Tampere, Tampere, Finland
MPe: Fimlab Laboratories, Pirkanmaa Hospital District, Tampere, Finland
MR: Department of Pediatrics, Tampere University Hospital, Tampere, Finland
MR: Department of Children and Adolescents, Oulu University Hospital, Oulu, Finland
MR: Department of Pediatrics, Medical Research Center Oulu, University of Oulu, Oulu,
Finland
The appllication of flow cytometry to study the celluar immuno response in
Drosophila melanogaster larvae.
The role of the proprotein convertase FurinA in the zebrafish immunity against
mycobacteria
During the cellular immune response of Drosophila melanogaster to endoparasitoid
wasps, lamellocytes are activated to encapsulate and subsequently kill wasp larvae
and eggs. Recent studies revealed that plasmatocytes transform into lamellocytes.
However, the mechanisms and dynamics underlying this transformation are poorly
understood. We used a fly line containing both eater-GFP and MSNF9mo-mcherry and
challenged L1 larvae with endoparasitoid wasps at different time points. We recorded
hemocyte populations and hemocyte numbers with an AccuriC6 flow cytometer. During
the course of infection two eater-GFP positive plasmatocyte populations developed.
One of these started to express MSNF9mo-mcherry yielding two double positive cell
populations that were characterized by high GFP, low mcherry and high mcherry, low
GFP expression, respectively. MSNF9mo-mcherry only cells were mature lamellocytes.
Neither of the double positive populations, nor the MSNF9mo-mcherry only hemocytes
occurred in non-challenged larvae. The hemograms of Drosophila larvae having
successfully combated the parasite were different from those that would succumb to
the infection. We confirmed the flow cytometry results with in vivo microscopy and
immunochemistry. We concluded that flow cytometry is a powerful tool to dissect the
cellular immune response.
Tuberculosis is an epidemic disease caused by Mycobacterium tuberculosis. Not only
the disease leads to over one million deaths annually, but an estimate suggests that one
third of the world population is infected with M. tuberculosis. M. marinum is the closest
relative of M. tuberculosis. The zebrafish - M. marinum model is well established and
it shares comparable pathogenic characteristics to human tuberculosis. FURIN is a
proprotein convertase and cleaves proproteins into their biologically active form at basic
cleavage sites. Among other things, FURIN is involved in immune regulation, bacterial
toxin activation and in virus maturation. Interestingly, zebrafish have two FURIN gene
orthologs: furinA and furinB. In our study, we used heterozygous furinAtd204e mutant
(furinAtd204e/+) zebrafish and the adult zebrafish - M. marinum model to study the role
of FurinA in a mycobacterial infection. In practice, flow cytometry was used to assess
blood cell composition, qRT-PCR in the relative quantification of mRNA amounts and
DNA qPCR in M. marinum copy number quantification Analyses in steady state zebrafish
revealed that FurinA deficiency causes reduced granulocyte amounts and affects T
cell polarization. Experimental mycobacterial infections revealed a fast induction of
the early immune response (1 day post infection) in furinAtd204e/+ zebrafish with
significantly higher tnfa and lta expression compared to WT controls. Furthermore,
in a latent infection the average bacterial burden in furinAtd204e/+ zebrafish was
approximately 40% of that in WT fish. Our results indicate a substantial role for FurinA
in the zebrafish immune system as well as in immunity against mycobacteria.
Barathi Muthukrishnan
Javier Gracia
Lab: Ribeiro, TUT
1. Biotechnology
Keywords: impulse oscillometry, multi-lead impedance
pneumography
Topic: intracellular organization
Authors: Anantha-Barathi Muthukrishnan,
Teppo Annila, Abhishekh Gupta,
Ramakanth Neeli Venkata, and Andre S Ribeiro
Author: Javier Gracia (gracia.javier@tut.fi)
Affiliation: Laboratory of Biosystem Dynamics, Department of Signal Processing,
Tampere University of Technology, FI-33101 Tampere, Finland
Co-authors: Ville-Pekka Seppä, Jari Viik
Spatial distribution of Tsr clusters in Escherichia coli
Escherichia coli possess a chemotaxis network to respond to changes in the
environmental conditions. Tsr is one of the proteins of the family of chemoreceptors.
We used the Tsr tagged with Venus (YFP) construct to quantitatively study the spatiotemporal organization of Tsr-Venus clusters as a function of the amount of these proteins
in the cells and of temperature. We show that they distribute symmetrically relative
to the major cell axis in all conditions. Also, even though the clusters’ numbers, size
and fraction at poles change with induction and with temperature, in all conditions the
correlation between cluster size and distance to midcell is positive and increases in
time. Finally, we show that the spatial distribution of the clusters is anti-correlated with
the nucleoid positioning. Their behavior is thus strikingly similar to that of unwanted
protein aggregates and other large, inert complexes, suggesting that the nucleoid plays
a central role in the internal organization of these organisms.
Group: IP group
Combining impulse oscillometry and multi-lead impedance pneumography for
regional analysis of dynamic lung function
As presented in European Respiratory Society annual congress 2013:
Studies suggest that dynamic lung mechanic properties are not homogeneously
distributed within the lungs [1].
We hypothesise that pressure pulses induced at the mouth by impulse oscillometry (IOS)
produce different volume responses in different lung areas depending on the regional
lung mechanic properties. We developed a novel multi-lead impedance pneumography
measurement method, that unlike earlier impedance methods, has sufficiently high time
resolution to track IOS volume changes in multiple locations [2].
In an initial experiment, three IP signals were recorded at different levels of the thorax
while IOS was being performed. Ensemble averaging of the recorded IP signal gated to
IOS pulses was used to remove respiratory and cardiac impedance changes.
Results show that IOS pressure pulses can be seen in IP measurement. Furthermore, it
was observed that IP signal responses showed consistent differences in shape between
the three measured locations.
We believe that, with the appropriated processing this method will allow for the first
time to assess lung distribution of dynamic air-flow resistance and lung compliance,
thus leading to new insights in pulmonary diseases such as asthma and COPD.
[1] R. Pikkemaat et al. 2012.
[2] J. Gracia, et al. 2012.
Edite Figueiras
Dhanesh Rajan
7. Biomedical Imaging
Keywords: Optical projection tomography,
tissue engineering, 3D imaging
7. Sensor Technology
Keywords: Optical pH measurements,
Non-contact pH measurements
Author: Edite Figueiras (edite.areiasfigueiras@tut.fi)
Author: Dhanesh Rajan (dhanesh.kr@tut.fi)
Group: Jari Hyttinen
Group: Prof. Jukka Lekkala
Co-authors: Ana M. Soto, Danilo Jesus, Jari Hyttinen
Co-authors: Heimo Ihalainen, Jyrki Sivula, Jukka Lekkala
All affiliations: Department of Electronics and Communications Engineering, Tampere
University of Technology
All affiliations: Authors 1,2,4: Department of Automation Science and
Engineering,Tampere University of Technology, Author 3: Adult Stem Cell Group,
Institute of Biomedical Technology, University of Tampere
Optical Projection Tomography for 3D live tissue engineering imaging
Tissue engineering (TE) constructs for regenerative medicine and as in vitro pre-clinical
models, provide new therapies for patients with tissue dysfunctions improving patient’s
health care and addressing major concerns shared by citizens worldwide. Although there
are many imaging techniques, they do not provide the specific information required in
TE research. Imaging techniques for characterization prior to application 3D structure
of scaffolds, as X-ray micro-computed tomography, is relatively well-developed,
whereas there are no methods to investigate the dynamics of the cell behaviors and
interactions with biomaterial scaffolds in 3D engineered tissues over time. In order to
meet the requirements for 3D TE constructs imaging over time, our group is building
and optimizing an optical projection tomography (OPT) system.
OPT is the optical equivalent of X-ray µCT, where a suspended specimen, in an indexmatching liquid, is rotated through a series of angular positions, and an image is captured
at each orientation with a camera imaging sensor. Images collected in each orientation
can be reconstructed employing, e.g., back-projection algorithms. Two imaging modes
can be implemented: transmission imaging (bright-field OPT) and emission tomography
(fuorescence OPT).
The OPT system is now being tested in phantoms based in hydrogels. Simple hydrogels,
as well as hydrogels combined with fibres, micro-particles or cells were imaged. The
first results obtained look promising, and they allow to predict the capabilities of the
system to monitor cell migration, proliferation, differentiation and interaction inside TE
constructs based in hydrogels.
Optical non-contact pH measurements with an automatic two wavelength system.
In cell and tissue cultures, pH is a critical control parameter for maintaining cell viability
and tissue functions. Monitoring pH during culture provides valuable information on
metabolic processes and the overall environment. Electrochemical sensors are used for
pH sensing because they are reliable, fast and easy to use. However, two noticeable
limitations are need for relatively large sample volumes and inevitable physical contact
of sensor to sample. Optical methods can be used to overcome these difficulties in
pH monitoring. A few optical pH sensors are commercially available but those are
not strictly non-contact sensors. But if a pH indicator dye, which could be colour,
fluorescence, protein or DNA based, is suspended in the medium of interest, a noncontact spectroscopic measurement can be employed for measuring the pH dependent
optical changes. We have attempted to measure the pH of cell culture medium with
an automatic two wavelength absorbance measurement method. The medium
(DMEM/F-12, Life Technologies) supplemented with glutamine, serum and antibiotics
contains the pH dye phenol red (8.1mg/L, range pH 6.8 to 8.2) that show strong pH
dependent absorbances at around 558nm and 434nm. In our measurement setup, the
light originating from microcontroller controlled LEDs at these sensitive wavelengths
transmit through the medium. The absorbance ratios computed from transmittance
ratios from detector signals are then converted into corresponding pH using a linear
regression model or a second order exponential model. Preliminary results from
prototype-1 indicate that the technique can measure pH with a resolution 0.1 pH units
in mammalian cell culture environment.
Barbara Taskinen
1. Biotechnology
Keywords: avidin-biotin technology, protein engineering,
biosensor applications, protein-ligand binding
Research Day 2013
Poster presentations
Group 1
Poster session 1/1st coffee break
(10.15-10.45)
Author: Barbara Taskinen (barbara.taskinen@uta.fi)
Group: Vesa P. Hytönen
Co-authors: Dominik Zauner(3), Soili I. Lehtonen(1,4), Masi Koskinen(1,2), Niklas
Kähkönen(1,2), Sampo Kukkurainen(1,2), Juha A.E. Määttä(1,2), Markku S. Kulomaa(1,4),
Andreas Ebner(3), Hermann J. Gruber(3), Vesa P. Hytönen(1,2)
All affiliations: (1) Institute of Biomedical Technology, University of Tampere and Tampere
University Hospital and BioMediTech, Tampere, Finland. (2) Fimlab laboratories, Pirkanmaa
hospital district, Tampere, Finland. (3) Institute of Biophysics, Johannes Kepler University,
Gruberstrasse 40, 4020 Linz, Austria. (4) Tampere University Hospital, Tampere, Finland
Neutral avidin variant for the reversible formation of biotin-avidin bridges
Avidinylated surfaces are a popular tool in biosensor applications. They enable the easy
immobilization of biotinylated analyte molecules because of the stability of the biotin(strept)avidin interaction and the simplicity of biotinylating the analyte (Morgan, H.,
and Taylor, D.M. (1992), Yang, N. et al. (2007)). However, the major drawback of
these surfaces is that the (strept)avidin-biotin binding is irreversible and a new surface
is needed for every new analyte molecule. We previously showed that the chicken
avidin mutant avidin M96H can be used to create reusable biotin-binding biosensor
surfaces (Nordlund, H.R. et al. (2003), Pollheimer, P. et al. (2013)). Avidin M96H has
a histidine mutation in the subunit interface. Protonation of this histidine residue at low
pH causes electrostatic repulsion amongst monomers that leads to an unstable tetramer,
which is easily disrupted in the presence of SDS. The only drawback with this approach
is the high isoelectric point of avidin (9.6) that causes non-specific interactions with
analyte molecules. This interaction makes streptavidin, with an isoelectric point of
5-6, the preferred biotin-binding protein in most applications. To reduce non-specific
binding, we introduced acidifying mutations on the surface of avidin M96H to lower
the isoelectric point close to neutral. Additionally, we introduced mutation R114L in
the biotin-binding pocket, which increased the affinity to conjugated biotin. These new
mutations did not compromise the structure or stability of the protein and resulted in an
avidin that was superior in affinity towards conjugated biotin and in prevention of nonspecific binding towards analyte proteins.
1. Baran Aydogan
4. Marja Pajunen
7. Riikka Nurminen
33. Ana María Soto de la Cruz
13. Katri Leinonen
16. Leena-Maija Vanha-aho
19. Sari Vanhatupa
22. Jenita Pärssinen
25. Johanna Ketolainen/Riikka
Jokimäki
28. Rami Pääkkönen
31. Kirsi Tuppurainen
34. Sina Saari
37. Sanni Virjula
40. Priit Joers
43. Tommi Rantapero
46. Satu Luhtala
49. Ville-Pekka Seppä
52. Laura Airaksinen
55. Hanna Rauhala/Elisa Vuorinen
58. Venkatesh Mallikarjun
61. Minna Hietikko
64. Virpi H. Laitinen
67. Jack George
70. Birgitta Lehtinen
73. Tomas Cervinka
76. Ilmari Tamminen
79. Annika Kohvakka
82. Matti Annala
85. Ana Andjelkovic
88. Fikret Emre Kapucu
91. Anssi Nurminen
94. Jari Väliaho
97. Nathaniel Narra
100. Sanna Turunen
103. Heidi Halonen
106. Joonas Tuominen
109. Joose Kreutzer
Group 2
Poster session 2/last
half an hour of the
lunch break
( 13.00-13.30)
2. Matti Annala
5. Liisa Sjöblom
8. Shanjun Chen
11. Aapo Tervonen
14. Tomi Ryynänen
10. Michelangelo Paci
20. Henrik Hammarén
23. Dmitriy Fayuk
26. Anni Saralahti
Group 3
Poster session 3/2nd coffee break
(15.25-15.55)
29. Tiina Joki
32. Liisa-Ida Sorsa
35. Mostafa Kiamehr
38. Toni Grönroos
41. Johanna Salonen
44. Juha Määttä
47. Cristina A. Nadalutti
50. Suvi Kalliokoski
53. Kalle Lehto
56. Jouni Väliaho
59. Maria Laaksonen
62. Leena Latonen
65. Zsuzsanna Ortutay
68. Nina Gubina
71. Kerstin Lenk
74. Ville Kytölä
77. Päivi Lillsunde
80. Mike Gerards
30. Mauro Scaravilli
17. Danilo Jesus
36. Sanna Auer
39. Susanna Teppo
42. Anni Sorkio
45. Hannu Turpeinen
48. Sanna Huttunen
51. Saara Aittomäki
54. Heidi Kontro
57. Thomas Liuksiala
60. Alejandra Rodríguez Martínez
63. Rolle Rahikainen
66. Jyrki Sivula
69. Giuseppe Cannino
72. Sampo Kukkurainen
75. Jani Sarin
78. Ashwin Sriram
81. Narayan Puthanmadam
Subramaniyam
84. Meeri Mäkinen
87. Sergei Häyrynen
90. Sini Eerola
93. Mirva Järvelä-Stölting
96. Miina Ojansivu
83. Esko Kemppainen
86. Keijo Viiri
89. Elli Käpylä
92. Markus Hannula
95. Magdaléna von
Essen
98. Timo Salpavaara
101. Maiju Hiltunen
104. Kaisa Vuornos
107. Inkeri Vornanen
110. Ashok Aspatwar
3. Antti Ylipää
6. Zuzet Martinez Cordova
9. Francesco Tabaro
12. Iina Vainio
15. Sanna-Kaisa Harjula
18. Kaisa Teittinen
21. Heini Kallio
24. Janne Koivisto
27. Milka Hammarén
99. Shokoufeh Teymouri
102. Laura Johansson
105. Jarno M. A. Tanskanen
108. Kaisa Oksanen
Dogu Baran Aydogan
1. Biotechnology
Keywords: trabecular bone, microstructure, binary
morphology, CT imaging, connectivity, contour tree,
algebraic graph theory
R
E
T
S
POS
T
C
A
R
T
S
B
A
Author: Dogu Baran Aydogan (baran.aydogan@tut.fi)
Co-authors: Dogu Baran Aydogan, Niko Moritz, Hannu Aro and Jari Hyttinen
All affiliations: Tampere University of Technology, Biomeditech, University of Turku
and Turku University Hospital
Analysis of trabecular bone microstructure using contour tree connectivity
Millions of people worldwide suffer from fragility fractures, which cause significant
morbidity, financial costs and even mortality. The gold standard to quantify structural
properties of trabecular bone is based on the morphometric parameters obtained from
μCT images of clinical bone biopsy specimens. The currently used image processing
approaches are not able to fully explain the variation in bone strength. In this study,
we introduce the contour tree connectivity (CTC) as a novel morphometric parameter
to study trabecular bone quality. With CTC, we calculate a new connectivity measure
for trabecular bone by using contour tree representation of binary images and algebraic
graph theory.
To test our approach, we use trabecular bone biopsies obtained from 55 female patients.
We study the correlation of CTC with biomechanical test results as well as other
morphometric parameters obtained from μCT. The results based on our dataset show
that CTC is the 3rd best predictive feature of ultimate bone strength after bone volume
fraction and degree of anisotropy.
Matti Annala [1,2]
Antti Ylipää
2. Cancer Research
Keywords: osteosarcoma, fusion gene, transcriptome
sequencing
2. Cancer Research
Keywords: prostate cancer, RNA-seq, transcriptome
assembly, novel transcript, ERG
Author: Matti Annala [1,2] (matti.annala@uta.fi)
Author: Antti Ylipää (antti.ylipaa@tut.fi)
Group: Matti Nykter
Group: Nykter
Co-authors: Jilong Yang [3,4], Ping Ji [5], Guowen Wang [3,4], Hong Zheng [4,6], David
Cogdell [5], Linru Zhao [4,6], Wei Tian [3,4], Xiaoling Du [7], Zhiwei Fang [8], Baocun
Sun [4,9], Matti Nykter [1], Kexin Chen [4,6], Wei Zhang [5]
Co-authors:Kati Kivinummi1, Matti J Annala1, Kimmo Kartasalo1, Leena Latonen1,
Simo-Pekka Leppänen1, Mauro Scaravilli1, Wei Zhang2, Tapio Visakorpi1, Matti
Nykter1
All affiliations: [1] Institute of Biomedical Technology, University of Tampere, Tampere,
Finland [2] Department of Signal Processing, Tampere University of Technology, Tampere,
Finland [3] Department of Bone and Soft Tissue Tumor, Tianjin Medical University
Cancer Hospital and Institute, Tianjin, PR China [4] National Clinical Research Center
of Cancer, Tianjin Medical University Cancer Hospital and Institute, Tianjin, PR China
[5] Department of Pathology, University of Texas MD Anderson Cancer Center, Houston,
USA [6] Department of Epidemiology and Biostatistics, Tianjin Medical University Cancer
Hospital and Institute, Tianjin, PR China [7] Department of Diagnostics, Tianjin Medical
University, Tianjin, PR China [8] Department of Bone and Soft Tissue Tumors, Beijing
University Cancer Hospital, Beijing, China [9] Department of Pathology, Tianjin Medical
University Cancer Hospital and Institute, Tianjin, PR China
Recurrent LRP1-SNRNP25 and KCNMB4-CCND3 fusion genes promote tumor cell
migration and invasion in human osteosarcoma
The identification of fusion genes such as SYT-SSX1/SSX2, PAX3-FOXO1, TPM3/4ALK, BCOR-CCNB3 and EWS-FLI1 in human sarcomas has provided important
insight into the diagnosis, prognosis, and targeted therapy of sarcomas. No such fusions
have been reported in human osteosarcoma. Here we used transcriptome sequencing to
characterize gene fusions and mutations in 11 human osteosarcomas. We identified two
selectively rearranged genomic loci that gave rise to fusion genes in 5 of 11 samples. When
disrupting rearrangements were taken into account, all 11 samples were found to harbor
inactivating alterations in the TP53 pathway. We also identified frequent gene fusions and
rearrangements associated with 12q amplification. Two fusion genes associated with the
12q locus, LRP1-SNRNP25 and KCNMB4-CCND3, were validated by RT-PCR, Sanger
sequencing and fluorescence in situ hybridization, and were found to be recurrent and
osteosarcoma specific. Expression of LRP1-SNRNP25 and KCNMB4-CCND3 fusion
genes in SAOS-2 osteosarcoma cells promoted cellular migration and invasion, suggesting
an oncogenic contribution independent of 12q amplification.
All affiliations: 1University of Tampere, 2University of Texas M. D. Anderson Cancer
Center
Novel prostate cancer specific transcripts identified using RNA-seq
Prostate cancer is the third most common cause of male cancer deaths in developed
countries, with castration resistance being the most challenging clinical problem. Here
we report an investigation into novel transcripts in primary prostate cancers (PCs),
and castration resistant prostate cancers (CRPCs) in particular. We characterized 28
PCs, 13 CRPCs, and 12 benign prostatic hyperplasias (BPH) using deep transcriptome
sequencing (RNA-seq). Reference-based transcriptome assembly uncovered 145
previously unannotated intergenic PC associated transcripts or isoforms. The expression
patterns of the transcripts were confirmed in two previously published independent
cohorts of primary tumors (n=30 and n=34), 21 PC cell lines, and 25 normal tissues
or cell lines. By integrating publicly available ChIP-sequencing data and transcription
factor (TF)-transcript expression correlations, we identified a transcript that positively
correlated with ERG expression and exhibited an ERG binding event in a PC cell line
coinciding with the canonical ETS-family TF binding motif at its proximal promoter
region. Enrichment of histone modification H3K4me3 and PolII at the promoter of
the transcript in the cell line provided further evidence of open chromatin and active
transcription. We downregulated the expression of the transcript with siRNAs in the cell
line and observed a decrease in cell growth and reduced migration, invasion and colony
formation. Annexin V assay indicated increased rate of apoptosis in the cells. Pathway
analysis indicated that cell cycle, mitosis and apoptosis were the most extensively
affected cellular processes. These results suggested that the transcript significantly
affects tumor growth in ETS-positive prostate cancers.
Marja Pajunen
Liisa Sjöblom
Keywords: Human pluripotent stem cells, neural cells,
hydrogel
2. Cancer Research
Keywords: Prostate cancer, beta-microseminoprotein,
biomarker
Author: Marja Pajunen (marja.pajunen@uta.fi)
Author: Liisa Sjöblom (liisa.sjoblom@uta.fi)
Group: Tapio Visakorpi
Co-authors: Marja Pajunen, Tiina Joki, Laura Ylä-Outinen, Mari Varjola, Heli Skottman,
Susanna Narkilahti
Co-authors: Outi Saramäki, Hans Lilja, Tapio Visakorpi
All affiliations: Institute of Biomedical Technology, BioMediTech, University of
Tampere, Tampere, Finland
3D cultures for Human Pluripotent Stem Cell -Derived Neuronal cells
Background. Neurodegenerative disorders are associated with permanent damages
to cells and brain structures. There have been some success with neural transplants,
however, the cell survival after grafting is still a major challenge. To improve the
survival, cells should be incorporated into protective biomaterials. These biomaterials
should be non-toxic, three dimensional, support cell viability and allow nutrition flux.
In addition to the protection of cells, biomaterials offer a more natural, tissue-like
environment for the cells to grow in. For neural applications hydrogels are considered
to be the best option. Here, we have tested the feasibility of the commercially available
hydrogel PuraMatrixTM as an in vivo growth platform for human pluripotent stem cell
(hPSC) -derived neural cells.
Methods. hPSC -derived neural cells were seeded to different hydrogel concentrations
on top of the gelled material, inside the hydrogel, or under the hydrogel. The cells
were characterized using viability analysis, immunocytochemical staining, time-lapse
monitoring, and confocal imaging. In addition, the maturation and electrical activity
of the neuronal networks inside the hydrogel were characterized using microelectrode
array.
Results. The hydrogel was non-cytotoxic and supported the survival of neural cells,
both neurons and glial cells. Moreover, neurons inside the gel developed more branched
neurite structures compared to the traditional 2D culture. Importantly, these neurons
were also able to form spontaneously functional networks.
Conclusions. Even though there are some challenges related to this material, like the
drastic pH changes during gelation, our data encourages to further study this material
for a growth matrix for human-derived neural cells.
All affiliations: Prostate Cancer Research Center, Institute of Biomedical Technology,
University of Tampere
Validation of beta-microseminoprotein as a prostate cancer biomarker
Prostate cancer (PC) is a significant health problem among men. Most PCs grow slowly
but there are also aggressive cases. Prostate-specific antigen (PSA) is widely used
PC biomarker but it distinguishes poorly aggressive cases from less aggressive ones.
Therefore, more specific markers are needed. MSP/MSMB is one of the most abundant
proteins secreted by the prostate. MSP expression has been observed to be higher in
benign than in cancerous prostate tissue and single nucleotide polymorphism (SNP) in
the promoter of the MSMB gene has been associated with the risk of PC. MSMB forms
a read-through fusion gene with an adjacent gene NCOA4. MSMB-NCOA4 fusion
transcripts have been shown to be regulated by the MSMB promoter. The aim of this
study is to validate MSP as a PC biomarker.
Immunohistochemistry was used to study MSP expression of prostatectomy samples and
castration-resistant prostate cancers (CRPCs). mRNA levels of MSMB, NCOA4 and
MSMB-NCOA4 fusion were examined with Q-RT-PCR from prostatectomy samples.
Circulating MSP levels were measured from serum samples of PC patients and controls.
SNP rs10993994 was genotyped from germ-line DNA of the same cohort. Positive
MSP expression was more frequent in prostatectomy samples than in locally recurrent
CRPCs. MSP expression level was not associated with the progression-free survival in
prostatectomy-treated patients. MSMB-NCOA4 expression was positively correlated
with expression of MSMB. The preliminary results show no significant differences of
the circulating MSP level between cases and controls. Serum MSP level was higher in
men with the low-risk genotype of rs10993994. More studies are warranted to evaluate
MSP as PC biomarker.
Zuzet Martinez Cordova
Riikka Nurminen
Keywords: Furin, myeloid cells, NFK-p65, TGF-β1
2. Cancer Research
Keywords: EMSY, Genetic association study, Imputation,
Prostate cancer
Author:
Zuzet Martinez Cordova (zuzet.martinez.cordova@uta.fi)
Author: Riikka Nurminen (riikka.k.nurminen@uta.fi)
Group: Marko Pesu (Immunoregulation Group)
Co-authors: Anna Oksanen(1), Sanna Hämäläinen(1), Valentina Taverniti(2), Ilkka
Junttila(3), Marko Pesu(1)
Co-authors: Rainer Lehtonen(2), Anssi Auvinen(3), Teuvo Tammela(4), Tiina Wahlfors(1),
Johanna Schleutker(1,5)
All affiliations: 1 Institute of Biomedical Technology, Biomeditech, University of Tampere, 2 Department of Food Science and Microbiology, Universita` degli Studi di Milano, Milan, Italy, 3 School of Medicine, University of Tampere
All affiliations: 1) Institute of Biomedical Technology/BioMediTech, University of
Tampere and Fimlab Laboratories, Tampere, Finland; 2) Department of Biosciences,
University of Helsinki, Helsinki, Finland; 3) School of Health Sciences, University
of Tampere, Tampere, Finland; 4) Department of Urology, University of Tampere and
Tampere University Hospital, Tampere, Finland; 5) Department of Medical Biochemistry
and Genetics, University of Turku, Turku, Finland
Myeloid cell expressed proprotein convertase Furin attenuates inflammation
Proprotein convertase Furin converts proteolytically immature proproteins into functional end products. Previously we have shown that Furin is a key regulator of T cell
mediated peripheral immune tolerance and that it is upregulated upon the activation of
monocytes. The aim of this study is to assess the role of Furin in innate immunity using
a mouse model with a conditional deletion for Furin in myeloid cells (LysMcre-furf/f).
LysMcre-furf/f mice remain healthy during the first year of life. Flow cytometry analysis of the immune cells populations in WT and KO mice did not reveal marked differences in the lymphocyte, macrophage and neutrophil populations. To address the
consequence of Furin deficiency in myeloid cells in vivo, mice were intraperitoneally
injected with LPS and survival and serum cytokines were monitored. LPS injection
resulted in significantly higher mortality of KO mice, and elevated levels of TNFa and
IL-6 in serum. In contrast, IL10 cytokine levels were markedly lower. Furthermore, the
in vitro experiments with peritoneal macrophages demonstrated higher production of
pro-inflammatory cytokines and diminished upregulation of IL-10 upon TLR activation. TGF-β1 is a reported target for Furin as well as a downregulator of activation
NFKb-p65 transcription factor. Furin deficient myeloid cells were observed to produce
significantly lower levels of bioactive TGF-β1 and show hyper-activation of NFKb-p65
upon LPS stimulus.
We conclude that deletion of Furin in myeloid cells results in a proinflammatory immune phenotype, which is due at least in part to the upregulation of NFKb-p65 as result
of a deficiency in TGF-β1 production.
Fine-mapping of chromosomal region 11q13.5 in Finnish prostate cancer
Genetic factors are known to increase the risk of prostate cancer (PrCa) but the
identified PrCa associated single-nucleotide polymorphisms (SNPs) explain only a part
of the PrCa heritability. It is especially important to identify variants that predispose to
aggressive PrCa in order to recognize men who are at risk of aggressive disease outcome.
Chromosomal region at 11q13-14 is connected with PrCa in linkage and genome-wide
association studies. Previously we identified a rare intronic mutation at EMSY (11q13.5)
that increases the risk of aggressive PrCa and associates with familial PrCa in the Finnish
population.
The aim of the study was to characterize the genetic structure of 11q13.5 and to identify
PrCa predisposing variants in the region. The study included a total of 4034 PrCa patients
and 908 controls of Finnish origin. We imputed SNPs and structural variants on 0.9 Mb
at 11q13.5 and validated the associations by genotyping. Imputation was performed
with IMPUTE2 using 1000 Genomes reference population. Haplotype analysis was
conducted with Haploview to study haplotype blocks, linkage disequilibria and haplotype
associations.
The study identified two regions that contributed to PrCa risk. A set of correlated, PrCa
associated SNPs and haplotypes were observed both at EMSY and on an intergenic
region between EMSY and LRRC32. In addition, the intergenic SNPs associated strongly
with PrCa death. The findings indicate that two regions at 11q13.5 contribute to PrCa
predisposition with a complex genetic structure. Further studies are needed to determine
the genetic mechanism of susceptibility and the functionality of the regions.
Shanjun Chen
Francesco Tabaro
Keywords: Mitochondria, wolbachia, Drosophila,
tko, spargel.
3. Computational Biology
Keywords: integrated analysis, tissue images, classification,
ovarian cancer
Author: Shanjun Chen (shanjun.chen@uta.fi)
Author: Francesco Tabaro
Group: Howy Jacobs
Group: Matti Nykter
Co-authors: Marcos T. Oliveira*,§, Alberto Sanz*, Esko Kemppainen*, Atsushi
Fukuoh*, Barbara Schlicht*, Laurie S. Kaguni*,§ & Howard T. Jacobs
Co-authors: F. Tabaro (1), P. Ruusuvuori (2), Y. Liu (3) and W. Zhang (3)
All affiliations: group of mitochondrial gene expression and diseases, Institute of
Biomedical Technology
All affiliations: (1) Computational Biology Group, Institute of Biomedical Technology,
University of Tampere, (2) Department of Signal Processing, Tampere University of
Technology, Tampere, (3) Departments of Pathology, The University of Texas MD Anderson
Cancer Center, Houston, Texas, USA
A cytoplasmic suppressor of a nuclear mutation affecting mitochondrial functions
in Drosophila
Automated Prediction of Pharmacological Treatment Sensitivity in Recurrent
Ovarian Cancer
Phenotypes relevant to oxidative phosphorylation (OXPHOS) in eukaryotes are
jointly determined by nuclear and mitochondrial DNA (mtDNA). Thus, in humans,
the variable clinical presentations of mitochondrial disease patients bearing the same
primary mutation, whether in nuclear or mitochondrial DNA, have been attributed
to putative genetic determinants carried in the ‘other’ genome, though their identity
and the molecular mechanism(s) by which they might act remain elusive. Here we
demonstrate cytoplasmic suppression of the mitochondrial disease-like phenotype of
the Drosophila melanogaster nuclear mutant tko25t, which includes developmental
delay, seizure sensitivity and defective male courtship. The tko25t strain carries
a mutation in a mitoribosomal protein gene, causing OXPHOS deficiency due to
defective intramitochondrial protein synthesis. Phenotypic suppression was associated
with increased mtDNA copy number and increased mitochondrial biogenesis, as
measured by the expression levels of porin (VDAC) and Spargel (PGC1α). Ubiquitous
overexpression of Spargel in tko25t flies phenocopied the suppressor, identifying it
as a key mechanistic target thereof. Suppressor-strain mtDNAs differed from related
nonsuppressor strain mtDNAs by several coding-region polymorphisms and by length
and sequence variation in the non-coding region, in which the origin of mtDNA
replication is located. Cytoplasm from four out of five originally Wolbachia-infected
strains showed the same suppressor effect, whereas that from neither of two uninfected
strains did so, suggesting that the stress of chronic Wolbachia infection may provide
evolutionary selection for improved mitochondrial fitness under metabolic stress. Our
findings provide a paradigm for understanding the role of mtDNA genotype in human
disease.
Treatment of recurrent resistant cancer is a central topic of cancer therapy. Standard
treatments are based on platinum-containing drugs, e.g. cisplatin, carboplatin and
oxaliplatin. These drugs crosslink guanines at DNA level, activating DNA repair systems
and subsequent apoptosis segnaling pathway inducing cell death. The molecular basis
of platinum resistance is not clear, even though some hypoteses have been proposed.
Tissue histology is rutinely used for initial diagnosis of cancer and clinical classification
of tumors. However, tissue features that would be predictive of treatment response are
not well characterized. Here, we set up an automated data analysis pipeline to extract
quantitative features from tissue images and build classifiers for prediction of treatment
resistance. In addition, we combined tissue features with RNA-seq data to build up
more extensive feature sets describing the resistance to pharmacological treatments.
We tested our approach on a cohort of 248 Ovarian Cancer patients from The Cancer
Genome Atlas sharing both histological and RNA-seq data.
We tested three different versions of the feature set: image features, RNA-Seq features
and their combination. We reached almost 70% in the prediction accuracy using on the
image-derived feature set alone. Revision of RNA-seq based feature set is ongoing.
These results show that the feature set extracted from images has predictive power
toward a complex outcome such as pharmacological sensitivity. Our ongoing work aims
to integrate more data sources, such as copy number variation and methylation data.
With integration of diverse data types we hope to obtain clinically applicable prediction
accuracy.
Michelangelo Paci
Aapo Tervonen
Keywords: Action potential, Cardiac cell, Stem cell,
Computational modeling
Keywords: Retinal pigment epithelium, Diffusion,
Permeability, Computational modeling
Author: Michelangelo Paci (michelangelo.paci@tut.fi)
Author: Aapo Tervonen (aapo.tervonen@tut.fi)
Co-authors: Jari Hyttinen, Stefano Severi
Co-authors: Iina Vainio, Soile Nymark, Jari Hyttinen
All affiliations: Michelangelo Paci: Tampere University of Technology, BioMediTech,
Jari Hyttinen: Tampere University of Technology, BioMediTech, Stefano Severi:
University of Bologna
University of Technology and BioMediTech
In-silico modeling of cardiomyocytes derived from human induced pluripotent
stem cells: the long QT 1 case study
The long QT (LQT) syndrome is a cardiac pathological condition characterized by
a delayed heart repolarization which can result in arrhythmia, fainting and sudden
cardiac death. The human induced pluripotent stem cell (hiPSC) technology enabled
the production of LQT-specific cardiomyocytes (CMs), to be used as pathological invitro models.
In this work we focus on the LQT1 syndrome, affecting the slow delayed rectifying K+
current (IKs) channel and resulting in a reduction of IKs. We propose a computational
model of the electrophysiology of hiPSC-CMs affected by LQT1, aimed to study the
effects of this syndrome on the action potential (AP) and the importance that IKs has
in hiPSC-CMs.
Experiments show that LQT1 induces reduced step/tail currents, a slower deactivation
time constant and a decreased IKs maximum conductance (-75%). These data were
integrated into our hiPSC-CM control model and result into a marked prolongation
(+28%) of the AP duration (APD). The same IKs reduction affects only slightly (+4%)
the APD both in the O’Hara-Rudy model of adult CM and in a hybrid CM model we
built by replacing into our hiPSC-CM control model the inward (IK1) and the rapid
delayed rectifying (IKr) currents with their adult versions.
Our simulations suggest (i) that the IKs repolarizing effect is more marked in immature
hiPSC-CMs than in adult CMs and (ii) that adult IKr and IK1 can compensate the LQT1reduced IKs. This is explained in the model by a reduced reserve of repolarization in
hiPSC-CMs than in adult CMs.
Prediction of passive permeability across the retinal pigment epithelium
Retinal pigment epithelium (RPE) is an important part of the normal visual function.
Located behind the retina, one of its main functions is to regulate the transport between
the retina and capillaries. The barrier properties have a role in certain retinal diseases,
such as age-related macular degeneration. In this study, for the first time we introduce
an accurate physical structure-based model of passive diffusion across the RPE.
Our model relates the permeability coefficients of RPE structures to the physicochemical
properties of materials forming the RPE. Model is based on a similar corneal model
(Edwards, A. et al. Pharm Res 18: 1497–508, 2001). Diffusion pathways are described
by separate permeability equations based on the material properties of each pathway
and the interactions between each pathway and the diffusing molecule. The structure
of our tight junction (TJ) model has not been utilized in other structure-based epithelial
models of this type.
Our RPE model was able to predict correct magnitude for the permeabilities and its
behavior corresponds to experimental results. Further, the permeability magnitude and
behavior of the TJ model appear similar to the experimental data of molecules mainly
traversing through the TJs. However, due to the inconsistent experimental data of RPE
permeability, rigorous validation cannot be made.
RPE barrier models would facilitate novel drug development against retinal diseases.
Our model forms a good platform for the future development and refinements as it
combines our knowledge of the RPE structure and permeability.
Iina Vainio (1, 2)
Leinonen Katri
Keywords: epithelial Ca2+ signaling; computational
modeling; ARPE-19; retinal pigment epithelium (RPE);
mechanical stimulation; Ca2+ imaging
2. Cancer Research
Author: Iina Vainio (1, 2) (iina.vainio@tut.fi)
Co-authors: Amna Abu Khamidakh (1, 2), Michelangelo Paci (1, 2), Heli Skottman (2,
3), Kati Juuti-Uusitalo (2, 3), Jari Hyttinen (1, 2) and Soile Nymark (1, 2)
All affiliations: 1 Department of Electronics and Communication Engineering, Tampere
University of Technology, Tampere, Finland, 2 BioMediTech, Tampere, Finland, 3 IBT
– Institute of Biomedical Technology, University of Tampere, Tampere, Finland
Computational model of Ca2+ wave propagation in human retinal pigment
epithelium
Ca2+ signaling is relevant to most biological functions. In retinal pigment epithelium
(RPE) a significant Ca2+ wave is produced by mechanical stimulation. To understand
this process in detail, we constructed a computational model of Ca2+ wave propagation
in ARPE-19 cells based on our Ca2+ imaging measurements (published: Abu Khamidakh
et al. 2013 Exp Eye Res). The model assessed Ca2+ wave propagation by assuming that
cells were experiencing different conditions depending on their location with respect to
the stimulation site only by changing the appropriate environmental parameters or drug
target components. The model describes Ca2+ metabolism after stimulation as follows:
1) Cells near the stimulus site are likely to conduct Ca2+ through plasma membrane
stretch-sensitive Ca2+ channels. 2) The ligand diffusion in the extracellular space
mediates the Ca2+ signal in all locations of the monolayer, the ligand concentration
decreasing with distance. 3) The kinase activity targeted to IP3 receptor defines the
sensitivity of the cell to the ligand. Gap junctions allow the diffusion of Ca2+ and IP3
between the adjacent cells. The model enables the analysis of Ca2+ signal propagation
mechanisms, but it also predicts new pathways of suramin drug effects on P2Y2
receptors suggesting that suramin accelerates the phosphorylation rate of the receptors
by enhancing their desensitization. Our model is the first mathematical model of Ca2+
signaling in ARPE-19 cells and one of the first in general to model epithelial Ca2+
dynamics presenting also predictions about the drug modified parameters.
Author: Leinonen Katri (katri.leinonen@uta.fi)
Group: Professor Tapio Visakorpi
Co-authors: Katri A. Leinonen1, 2, 3, Outi R. Saramäki1, 2, 3, Bungo Furusato4,
Takahiro Kimura5, Hiroyuki Takahashi4, Shin Egawa5, Hiroyoshi Suzuki6, Kerri Keiger7, Sung
Ho Hahm7, William B. Isaacs8, Teemu T. Tolonen1, 2, 3, Ulf-Håkan Stenman9, Teuvo L.J.
Tammela2,10, Matti Nykter1,2,3,11, G. Steven Bova1, 2, 3, and Tapio Visakorpi1, 2, 3
All affiliations: 1Institute of Biomedical Technology, 2Prostate Cancer Research Center,
3BioMediTech, University of Tampere and Tampere University Hospital, 4Department of
Pathology and 5Department of Urology, Jikei University School of Medicine, 3-25-8 NishiShimbashi, Minato-ku, Tokyo 105-8461, Japan, 6Department of Urology, Toho University Sakura
Medical Center, 564-1 Shimoshizu, Sakura, Chiba 285-8741, Japan, 7Department of Pathology,
Johns Hopkins University, Baltimore, MD, USA, 8Department of Urology, Johns Hopkins
University, Baltimore, MD, USA, 9Department of Clinical Chemistry, Helsinki University
Central Hospital, Helsinki, Finland, Department of Urology, School of Medicine, University of
Tampere and Tampere University Hospital, 11Institute of Signal Processing,Tampere University
of Technology, Tampere, Finland
Loss of PTEN is associated with aggressive behavior in ERG positive prostate
cancer
Background: The associations of ERG overexpression with clinical behavior and molecular
pathways of prostate cancer is incompletely known. We assessed the association of ERG
expression with AR, PTEN, SPINK1, Ki-67 and EZH2 expression levels, deletion and
mutations of chromosomal region 3p14 and TP53, and clinicopathological variables.
Methods: The material consisted of 326 prostatectomies, 166 needle biopsies from men treated
primarily with endocrine therapy, 177 transurethral resections of castration-resistant prostate
cancers (CRPC), and 114 CRPC metastases obtained from 32 men. Immunohistochemistry,
fluorescence in situ hybridization, and sequencing was used for the measurements.
Results:ERG expression was found in about 45% of all patient cohorts. In a multivariate
analysis, ERG expression showed independent value of favorable prognosis (p=0.019).
ERG positivity was significantly associated with loss of PTEN expression in prostatectomy
(p=0.0348), and locally recurrent CRPCs (p=0.0042). Loss of PTEN expression was associated
(p=0.0085) with shorter progression-free survival in ERG positive, but not in negative cases.
When metastases in each subject were compared, consistent ERG, PTEN and AR expression
as well as TP53 mutations were found in a majority of subjects.
Conclusions: A similar frequency of ERG positivity from early to late stage of the disease
suggests lack of selection of ERG expression during disease progression. The prognostic
significance of PTEN loss solely in ERG positive cases indicates interaction of these pathways.
The finding of consistent genetic alterations in different metastases suggests that the major
genetic alterations takes place in the primary tumor.
Tomi Ryynänen
Sanna-Kaisa Harjula
7. Materials and devices
Keywords: polystyrene, microelectrode array, MEA,
cardiomyocyte, neuronal cell
Keywords: zebrafish, Mycobacterium marinum
Author: Tomi Ryynänen (tomi.ryynanen@tut.fi)
Group: Jukka Lekkala
Co-authors: Ville Kujala, Laura Ylä-Outinen, Erja Kerkelä, Susanna Narkilahti, Jukka
Lekkala
Author: Sanna-Kaisa Harjula (sanna.harjula@uta.fi)
Group: Mika Rämet
Co-authors: Sanna-Kaisa Harjula, Jenna Ilomäki, Heather Mathie, Nicholas Halfpenny,
Anni Saralahti, Olli Lohi, Mataleena Parikka, Mika Rämet
All affiliations:
TR&JL: Department of Automation Science and Engineering, Tampere University of
Technology and BioMediTech
VK&EK: Heart Group, Institute of Biomedical Technology, University of Tampere and
BioMediTech (both currently elsewhere)
LY&SN: Neuro Group, Institute of Biomedical Technology, University of Tampere and
BioMediTech
All affiliations:
SKH, JI, HM, NH, AS, MP, MR: Institute of Biomedical Technology/BioMediTech,
University of Tampere, Tampere, Finland
OL, MR: Department of Pediatrics, Tampere University Hospital, Tampere, Finland
OL: Paediatric Research Centre, University of Tampere Medical School and Tampere
University Hospital, Tampere, Finland
MR: Department of Pediatrics, Medical Research Center Oulu, University of Oulu, Oulu,
Finland
Polystyrene coated MEA
Genes affecting mycobacterial infection in zebrafish
We have fabricated and tested microelectrode arrays (MEAs) that have polystyrene
as insulator layer instead of industry standard silicon nitride (Si3N4). Evidently, the
most common insulator layer used both in commercial and research MEAs is Si3N4.
However, the available thickness range of Si3N4 coating and thus its capability to
reduce parasitic capacitances is limited. Si3N4 coating also suffers from occasional
quality variations and during the time it wears out when MEAs are reused many times.
The highest motivation for introducing polystyrene as an alternative insulator layer,
however, comes from the fact that vessels made of polystyrene, for example well plates,
have been used for decades by cell scientist and thus there exist far more protocols
to grow cells on polystyrene than on Si3N4. One more benefit related to polystyrene
coating is low capital cost needed to fabricate it. The polystyrene coating on MEAs
has been successfully tested with human embryonic stem cell -derived cardiomyocytes
(hESC-CM) and neuronal cells (hESC-N). Cardiomyocytes attached well to the
plasma treated polystyrene surface which was coated with FBS and 0.1 % gelatine.
Neuronal cells on the contrary preferred the non-plasma treated surfaces coated with
polyethyleneimine and laminin. Detected cardiac field potentials and spontaneous
neuronal network activity were comparable to our earlier experiments with Si3N4 coated
MEAs. Some more experiments are needed to ensure repeatable fabrication process and
optimal polystyrene layer thickness as well as to confirm long term biocompatibility
and adhesion properties.
Tuberculosis, caused by Mycobacterium tuberculosis, is a worldwide health issue. In 2012,
1.3 million people died of tuberculosis and 8.6 people developed the disease. Mycobacterium
marinum is a natural zebrafish pathogen genetically similar to M. tuberculosis. Zebrafish
(Danio rerio) is a small teleost fish in which the advantages of invertebrates and vertebrates
as a genetic model organism are combined. The aim of this study is to identify genes
underlying defence mechanisms against M. marinum infection by carrying out a genebreaking transposon (GBT)-based forward genetic screen and studying the progression of
mycobacterial infection in zebrafish mutants with known defects in their immune defence.
For GBT-based mutagenesis, we inject GBT RP2 construct (a generous gift from Professor
Stephen C. Ekker’s laboratory (Mayo Clinic, Rochester, USA)) with synthetic mRNA of
Tol2 transposase into fertilized wild-type zebrafish eggs and raise the mutated fish. We are
currently doing the primary screen for the defence against M. marinum by monitoring the
survival of infected zebrafish.
We have infected 18 adult F3 mutant zebrafish families and eight of them have shown
altered resistance against M. marinum infection compared to wild-type zebrafish. Currently
we are also infecting zebrafish with known immunological defects. Interesting mutants are
characterized further with the methods we have developed to study the progression of M.
marinum infection in zebrafish. We expect this study to provide novel information about
host mechanisms that are involved in resistance against mycobacterial infection.
Leena-Maija Vanha-aho
Danilo Jesus
Keywords: Drosophila, innate immunity,
wasp infection, blood cells
Keywords: Optical Projection Tomography,
Center of Rotation, Variance
Author: Leena-Maija Vanha-aho
(leena-maija.vanha-aho@uta.fi)
Author: Danilo Jesus (danilo.jesus@tut.fi)
Group: Mika Rämet
Co-authors: Ines Anderl, Henna Myllymäki,
Dan Hultmark, Susanna Valanne, Mika Rämet
Co-authors: Edite Figueiras, Ana Soto, Jary Hyttinen
All affiliations: LMV, IA, HM, DH, SV, MR: Institute of Biomedical Technology, University of Tampere, Finland and BioMediTech, Finland
IA, DH: Department of Molecular Biology, Umeå University, Umeå, Sweden
MR: Department of Pediatrics, Tampere University Hospital, Finland
Functional characterization of the infection-inducible gene edin in Drosophila
melanogaster
The fruit fly, Drosophila melanogaster, is a well-established model organism to study
the mechanisms of innate immunity. It his highly resistant to microbial infection and,
unlike mammals, it has no adaptive immunity. Furthermore, the signaling pathways of
innate immunity are evolutionarily highly conserved. Activation of the pathways upon
an immune challenge triggers the production of antimicrobial peptides (AMPs) and
other effector molecules leading to different immune responses such as blood cell activation. Earlier, our research group has carried out a large-scale screen for genes induced
after Escherichia coli infection in Drosophila S2 cells. Among the genes of highest
expression were AMPs but also genes of unknown function. One of the most highly
induced genes was edin (elevated during infection), which codes for a small peptide
that is secreted from cells into the hemolymph. However, our data shows that Edin has
no antimicrobial properties in vitro or in vivo. Our more recent data indicate that edin
is also highly induced in Drosophila larvae and in the fat body (an immune-responsive
organ) after a natural infection by the parasitic wasp, Leptopilina boulardi. In addition,
our results show that expression of edin in the fat body is necessary for a normal encapsulation response against parasitic wasp eggs that is mediated by Drosophila blood
cells. At the moment, we are further investigating the role of Edin in blood cells after
wasp infection and we aim to elucidate the mechanisms and signaling pathways behind
these phenomena.
Center of rotation detection in Optical Projection Tomography
Optical Projection Tomography (OPT) is an optical imaging technique been used to
analyze, non-invasively, small specimens whose dimensions range from a few millimeters
to 1 cm. OPT is the optical equivalent of X-ray micro-computed tomography, where a
suspended specimen, in an index-matching liquid, is rotated through a series of angular
positions, and an image is captured at each orientation with a camera sensor. Images
collected in each orientation can be reconstructed employing, e.g., back-projection
algorithms. The quality of the reconstruction is quite dependent of the correct detection
of the center of rotation.
This work presents an improved method to detect the center of rotation for the 3D
reconstruction considering the image variance. The first step of this procedure consists
of a broad search to detect the closest position of the center of rotation with a precision
of one pixel. Then, a narrow search around the selected pixel is done, allowing the
detection of the position of the center of rotation with a precision of 1/10 pixels. The
obtained results were compared with different types of samples. Finally, the performance
of the algorithms was compared with other methods such as center of mass and simple
variance method.
The application of the improved method evaluated in this work, will certainly increase
the accuracy of the OPT reconstruction algorithms for different types of samples and
will extend its capability to obtain higher resolution 3D reconstructions of the imaged
specimens.
Kaisa Teittinen
Sari Vanhatupa
2. Cancer Research
Keywords: snoRNA, acute leukemia
Keywords: adipose stem cells, tissue engineering,
BMP-2, osteogenesis, adipogenesis, signalling
Author: Kaisa Teittinen (kaisa.teittinen@uta.fi)
Author: Sari Vanhatupa (sari.vanhatupa@uta.fi)
Group: docent Olli Lohi
Group: Susanna Miettinen
Co-authors: Thomas Liuksiala, Matti Nykter, Olli Lohi
Co-authors: Sari Vanhatupa, Miina Ojansivu, Miia Juntunen, Susanna Miettinen
All affiliations: Tampere Center for Child Health Research, University of Tampere and
Tampere University Hospital, Tampere, Finland (KT, OL)
Institute of Biomedical Technology, University of Tampere, Tampere, Finland (TL,
MN)
Department of Signal Processing, Tampere University of Technology, Tampere, Finland
(TL)
BioMediTech, Tampere, Finland (MN)
Small nucleolar RNAs in pediatric acute leukemia
Small nucleolar RNAs (snoRNAs) guide two different modifications of ribosomal RNA
(rRNA): box H/ACA snoRNAs direct pseudouridylation, and box C/D snoRNAs direct
methylation. Each snoRNA associates with particular protein components in a typespecific manner to form the functional ribonucleoprotein complex (snoRNP) which
carries out the modification. Many snoRNAs reside in the introns of protein-coding
genes, but little is known about the regulation of their expression.
Recently, snoRNAs have been linked to several human malignancies such as cancer.
We have shown that snoRNAs are differentially expressed between the main subgroups
of acute leukemia. This suggests a potential applicability of snoRNAs in classification
of leukemia subgroups, and in leukemia diagnostics. Indeed, when the expression of
a limited set of 15 snoRNAs was studied in pediatric leukemia samples compiled in
a hematological dataset, one snoRNA, SNORD114-3, was overexpressed in samples
representing acute promyelocytic leukemia (APL), a subclass of acute myeloid leukemia
(AML). The higher level of expression of SNORD114-3 differentiated APL from other
AML with notable statistical significance.
In order to shed light on the regulation of snoRNA expression, we have analyzed the
expression of several intronic snoRNAs and their host genes in leukemic cells. In
addition, the effects of overexpression of snoRNAs have been studied in cell lines.
All affiliations: Institute of Biomedical Technology, Adult Stem Cell Research Group,
University of Tampere, Finland. BioMediTech, Tampere, Finland. Science Center,
Tampere, University Hospital, Tampere, Finland
The influence of BMP-2 growth factor on signalling mechanisms and differentiation
fate of adipose stem cells
The combination of mesenchymal stem cells (MSC) with biomaterials and growth
inducing factors is a critical step in the development of bone tissue engineering
applications. Bone morphogenetic protein (BMP)-2 has previously been used in clinical
applications to stimulate bone regeneration. However, there are no clear indications
of the functionality and osteogenic impact of BMP-2 on MSCs, including adipose
stem cells (ASCs), because of contradicting results. In this study, we have analysed
the influence of BMP-2 growth factor stimuli to Smad signalling and differentiation
potential of ASC in vitro. BMP-2 related Smad1/5 activation and osteogenic potential
were analysed in different serum conditions (FBS and HS) with several patient cell
lines by Western Blot, immunofluorescence microscopy and osteogenic differentiation
analysis methods.
Our results indicate that the functionality of BMP-2 is strongly dependent on serum
culture conditions. BMP-2 induced Smad1/5 activation and nuclear translocation in all
cell lines studied, but the effect was prominent only in human serum (HS) conditions.
Interestingly, BMP-2 exhibits a dual role in differentiation process of ASCs, and the
effect on differentiation fate is patient cell line dependent. BMP-2 stimulated osteogenic
effect in some patient lines by inducing ALP activity and mineralization. Whereas,
BMP-2 induction clearly promoted adipogenic processes in other patient lines. These
results indicate that although BMP-2 induces Smad signalling mechanisms, the actual
effect of this signalling might have dual function and has to be individually pre-analysed
in clinical applications to confirm the benefit of the usage of BMP-2 in bone healing
process.
Henrik Hammarén
Heini Kallio
Keywords: Janus kinase 2 (JAK2), pseudokinase
domain, nucleotide binding, rational mutagenesis
2. Cancer Research
Keywords: prostate cancer, PAXgene, formalin,
molecular profile
Author: Henrik Hammarén (henrik.hammaren@uta.fi)
Author: Heini Kallio (heini.kallio@uta.fi)
Group: Olli Silvennoinen
Group: Professor Tapio Visakorpi
Co-authors: Hammarén HM (1), Ungureanu D (1), Hilllert E-K (1), Laitinen AU (1),
Hubbard SR (2), Silvennoinen O (1,3)
Co-authors: Heini Kallio (1), G. Steven Bova (1), Teemu Tolonen (1), Hans G. Lilja (1),
Tapio Visakorpi (1)
All affiliations:
(1) School of Medicine and Institute of Biomedical Technology, University of Tampere,
33014 Tampere, Finland; (2) Structural Biology Program, Kimmel Center for Biology
and Medicine of the Skirball Institute, Department of Biochemistry and Molecular
Pharmacology, New York University School of Medicine, New York, NY, USA; (3)
Department of Internal Medicine, Tampere University Hospital, 33520 Tampere,
Finland
All affiliations:(1)Prostate Cancer Research Center and Institute of Biomedical
Technology, University of Tampere, Tampere, Finland
Elucidating the role of nucleotide binding to the JH2 domain of JAK2 in regulation
of pathogenic JAK-signaling
The pseudokinase domain (JH2) of Janus kinases (JAKs) is a critical regulator of
cytokine signaling. Mutations in this domain have been linked to diseases such as
myeloproliferative neoplasms (MPNs; JAK2) and severe-combined immunodeficiency
(SCID; JAK3). Recent advances in solving the crystal structure (Bandaranayake et al.
2012) and elucidation of biochemical properties of the JAK2 JH2 domain (Ungureanu et
al. 2011) have opened up the possibility to study the, still poorly understood, regulatory
function of this domain. The results show that JAK2 JH2 binds ATP with physiological
affinity and suggest that nucleotide binding is important for the proper function of JAK2.
We set out to investigate the role of nucleotide binding by a multidisciplinary approach.
Structural analysis indicated that nucleotide binding to JAK2 JH2 has a stabilizing effect
on the JH2 domain both in wild type JAK2 JH2 as well as in the V617F mutant form.
Using site-directed mutagenesis and mammalian cell culture systems, we demonstrate
that an intact nucleotide binding cleft is needed for the hyperactivating phenotype of
the MPN-mutation JAK2 V617F. By using a defined mutagenesis approach we are
investigating whether full restoration of normal JAK2 regulation and function can be
achieved in V617F context through manipulation of the nucleotide binding site. This
approach has the potential of opening novel strategies for generating JAK inhibitors
targeting the nucleotide binding site of the pseudokinase domain.
Testing PAXgene fixation for molecular analysis of prostate tissue specimens
Formalin, the most widely used fixative, preserves morphology well, but compromises
macromolecular integrity. This is due to the fact that formalin fixation leads to
degradation and cross-linking of macromolecules, especially that of RNA. On the other
hand, freshly frozen samples provide high quality material for molecular analyses,
but preservation of tissue morphology and histology is restricted in frozen sections.
Therefore, novel approaches for sample fixation and storage are required. PAXgene
is a non-crosslinking fixative that has been shown to enable high quality molecular
analyses, immunohistochemistry and sufficient morphological examination. Therefore,
we set out to test PAXgene fixation for molecular analysis of prostate tissue specimens.
Whole prostates from prostate cancer patients are obtained immediately after surgery
and fixed in PAXgene fixative. Formalin-fixed and freshly frozen samples are used as
controls for DNA and RNA quality, respectively.
If initial tests show that PAXgene-fixed prostates yield high-quality DNA and RNA,
next step is to collect lymph node metastases along with the prostates from patients
going through prostatectomy. Molecular profiles of the primary tumor and metastases
could then be compared using DNA and RNA deep sequencing to learn about the
certain patient’s metastatic process and tumor behavior. The analysis of whole prostates
is critical since prostate cancers generally arise from multiple cancer foci. Thus, it is
obligatory to analyze different cancer cell colonies to understand the molecular profile
of the particular cancer. In the future, PAXgene-fixation could even enable the use of
personalized medicine in the treatment of prostate cancer patients.
Jenita Pärssinen
Dmitriy Fayuk
1. Biotechnology
Keywords: Kindlin-3, atherosclerosis
1. Biotechnology
Keywords: stem cells, neurons, differentiation
Author: Jenita Pärssinen (jenita.parssinen@uta.fi)
Author: Dmitriy Fayuk (dmitriy.fayuk@uta.fi)
Group: Vesa Hytönen
Co-authors: Jenita Pärssinen, PhD1, Niku Oksala, MD, PhD, DSc2,3, Ilkka Seppälä,
MSc, BM2, Emma Raitoharju, MSc2, Terho Lehtimäki, MD, PhD2 , Vesa Hytönen, PhD1
Co-authors: M. MÄKINEN, C. PRAJAPATI, S. PENKKI, A. HYYSALO, L. YLÄOUTINEN, S. NARKILAHTI
All affiliations: 1 Institute of Biomedical Technology and BioMediTech, University of
Tampere Fimlab Laboratories, Tampere, Finland. 2 Department of Clinical Chemistry,
Fimlab Laboratories and University of Tampere, School of Medicine, Tampere, Finland.
3 Division of Vascular Surgery, Department of Surgery, Tampere University Hospital,
Tampere, Finland
All affiliations: Neurogroup, IBT Institute of Biomedical Technology, university of
Tampere
Association of Kindlin-3 with atherosclerotic plaques in human(s)
Background: Human embryonic stem cells (hESC) and human induced pluripotent stem
cells (hIPSC) can be differentiated into neurons that are capable of forming functional
networks in vitro (Heikkilä et al. 2009). So far, the neuronal subtype characterization as
well as analysis of functionality of neuronal networks derived from hIPSCs and hESCs
have not been performed in great detail. Thus, the aim of this study was to determine the
properties of these spontaneously formed functional neuronal networks in vitro.
Methods: Human embryonic (Regea 08/023) and human induced pluripotent (A116,
Hel24.3) stem cells were differentiated into neural precursors using published method
(Lappalainen et al., 2010). The neural networks derived from these precursors were
studied with microelectrode array (MEA) dishes (Multichannel systems) while the
single neurons were tested with whole-cell patch-clamp approach. In addition, the cells
were collected for qRT-PCR array or fixed for immunocytochemical analysis.
Results: Immunocytochemical staining revealed presence of the most common neuronal
subtypes in the networks: glutamatergic, GABAergic, cholinergic, dopaminergic
and serotonergic. Functionally networks demonstrated mature electrical activity and
sensitivity to pharmacological agents: agonists and antagonists of synaptic transmission.
Single neurons demonstrated general firing properties and presence of major voltagegated channels. More detailed analyses are still ongoing.
Conclusions: Different neuronal subtypes can be found in hESC- and hiPSC-derived
neuronal networks in vitro. Neuronal networks formed contain functional dopaminergic,
GABAergic, glutamatergic, serotonergic, and cholinergic signaling systems.
This research is supported by 3DNeuroN project in the European Union’s Seventh
Framework Programme Future and Emerging Technologies, grant agreement N296590;
and competitive research funding of Pirkanmaa Hospital District.
Background: Integrins are shown to have an important role in platelet adhesion
and aggregation as well as in magrophage functional maturation into foam cells in
atherosclerotic lesions. The integrin signaling needs to be tightly controlled since
integrin-mediated platelet adhesion and aggregation for example are essential for sealing
injured blood vessels but excessive platelet aggregation can initiate arterial thrombosis. In
contrast to widely expressed Kindlin-1 and Kindlin-2, expression of Kindlin-3 is restricted
to hematopoietic cells and is particularly abundant in megakaryocytes and platelets as
well as in endothelial cells. Thus Kindlin-3 is potentially important in atherosclerosis and
its thrombotic complications. However, characterization of expression of Kindlin-3 and
its association with plaque stability in human(s) is lacking.
Methods and Results: The expression of Kindlin-3 did not differ in either whole blood
or circulating monocytes from patients with coronary artery disease (n=52) compared
with healthy controls (n=46). However, Kindlin-3 was upregulated (4.6-fold, p<0.0001)
in atherosclerotic plaques (n=68) compared with histologically normal controls (n=24).
Increased Kindlin-3 expression associated with histologically more stable plaques.
Kindlin-3 expression correlated positively with M1 magrophage and especially M2
magrophage markers and negatively with SMC markers in the atherosclerotic plaques.
Kindlin-3 protein co-localized with CD68-positive cells of monocytic origin and HHF3positive cells indicative of SMCs in the plaques and only with CD68 positive cells in the
control samples.
Conclusions: Present findings provide support for the hypothesis that dysregulation of
expression of Kindlin-3 could possibly be involved in the development of atherosclerosis.
CHARACTERIZATION OF FUNCTIONAL HUMAN NEURONAL NETWORKS
IN VITRO
Janne Koivisto
Johanna Ketolainen, Riikka Jokimäki
Keywords: iPS cell differentiation, peripheral neuron,
laminin, fibronectin, Matrigel
2. Cancer Research
Keywords: BMP4, breast cancer, microarray, microRNA
Author: Janne Koivisto (janne.t.koivisto@tut.fi)
Author: Johanna Ketolainen, Riikka Jokimäki
(johanna.ketolainen@uta.fi, riikka.jokimaki@uta.fi)
Group: Katriina Aalto-Setälä
Co-authors: Mari Pekkanen-Mattila, Olli Silvennoinen, Katriina Aalto-Setälä, Minna
Kellomäki
Co-authors: Alejandra Rodriguez Martinez, Nirmal Iyer, Anne Kallioniemi
All affiliations: Heart group, IBT, UTA. Biomaterials & tissue engineering research
group, ELT, TUT
Differentiation of human induced pluripotent stem cells into peripheral neural
cells and culture on selected ECM surfaces.
The development and function of human peripheral nervous system is not well known
on a cellular level. The first protocols to differentiate peripheral sensory neural cells
were published only recently. Peripheral neurons could be used in applications e.g. drug
and toxicology screenings or modeling diseases. For example C nerve fibers that are
responsible for sense of pain, are good targets for these applications.
The most important aim of this study was to produce peripheral sensory neurons from
iPS cells. The nature of differentiated cells was studied according to morphological
characteristics, immunocytochemistry and RT-PCR. The functionality of the cells was
studies with calcium-imaging.
The extracellular matrix (ECM) protein coatings used in cell cultures, were also studied.
The aim was to find the optimal coating suitable for peripheral neurons. The coatings
analyzed were laminin, fibronectin, Matrigel™ and gelatin. They were studied by atom
force microscopy, contact angle measurement from liquid-solid interface and antibody
based fluorescence labeling.
The differentiated neural cells had correct calcium signaling responses, correct
morphology and expressed marker proteins for peripheral neurons. However, the
differentiation efficiency varied a lot. More specifically C-fiber-like peripheral
sensory neurons were only detected in very small amounts. Laminin seemed to be
the best coating, but the differences were not major. After starting this work another
differentiation protocol was published and the differences on efficacy and cell subtype
yields are currently being analyzed.
BioMediTech, Tampere, Finland. Fimlab Laboratories, Tampere, Finland
The Target Genes and microRNAs of Bone Morphogenetic Protein 4 in Breast
Cancer Cell lines
Bone morphogenetic protein 4 (BMP4) belongs to the TGF-β superfamily. We and
others have previously shown that BMP4 inhibits breast cancer cell proliferation but at
the same time increases cell migration and invasion. Currently, the mechanisms behind
these phenomena are studied concentrating on the downstream effectors of BMP4.
Using microarray based screening technique we previously identified a number of
potential BMP4 target genes that are relevant in breast cancer. Of these we chose 12
genes showing most prominent and frequent expression change and further validated
with qRT-PCR that their expression is indeed altered after BMP4 treatment in breast
cancer cell lines. Currently we are investigating the effects of BMP4 treatment on
miRNA expression levels in breast cancer cell lines. The miRNA expression levels
were studied after four hour incubation with BMP4 or vehicle in seven different
breast cancer cell lines along with an immortalized breast epithelial cell line. The data
analysis is currently ongoing and is expected to reveal a set a BMP4 regulated miRNAs.
Subsequently, functional studies, including cell proliferation and migration/invasion
assays, will be conducted with the most promising BMP4 target genes and miRNAs to
assess their possible contribution to BMP4 mediated cancer cell specific phenotypes.
Anni Saralahti
Milka Hammarén
Keywords: Zebrafish, Streptococcus pneumoniae,
host defense, genetic screen
Keywords: Mycobacterium marinum; tuberculosis;
zebrafish; T cell; lymphocyte; T helper response
Author: Anni Saralahti (anni.saralahti@uta.fi)
Author: Milka Hammarén (milka.hammaren@uta.fi)
Group: Mika Rämet
Group: Mataleena Parikka/Mika Rämet
Co-authors: Jenni Jouppila, Sanna-Kaisa Harjula, Mataleena Parikka, Samuli Rounioja,
Mika Rämet
Co-authors: Kaisa Ester Oksanen, Hanna Maria Nisula, Bruno Vincent Luukinen,
Marko Pesu, Mika Rämet, Mataleena Parikka
All affiliations: JJ, SKH, MP, MR: Institute of Biomedical Technology, BioMediTech,
University of Tampere, Tampere, Finland
SR: Fimlab Laboratories, Tampere, Finland
MR: Department of Pediatrics, Tampere University Hospital, Tampere, Finland
MR: Department of Pediatrics, Medical Research Center Oulu, University of Oulu,
Oulu, Finland
All affiliations: BioMediTech, FI-33014 University of Tampere, Finland, Fimlab
Laboratories, Pirkanmaa Hospital District, FI-33520 Tampere, Finland, Department of
Pediatrics, Tampere University Hospital, FI-33521 Tampere, Finland
A forward genetic screen for zebrafish genes involved in pneumococcal infection
Streptococcus pneumoniae (pneumococcus) is a major human pathogen causing lifethreatening infections, such as pneumonia, septicemia, and meningitis. The complex
interactions occurring between the pneumococcus and the host immune system are
only partly understood and, therefore, the optimal treatment and prevention methods
are lacking. Previously, zebrafish (danio rerio) were shown to be valuable hosts in the
study of innate immunity functions associated with pneumococcal infection. In the
present study we have employed the zebrafish embryo model for the forward genetic
screen to identify novel innate immunity components that play a role in the defense
against pneumococcus. Low cost, high fecundity, and relatively short generation time
make the zebrafish a practical model for large-scale genetic screens. In this study, the
gene-breaking transposon based mutagenesis is used to introduce random mutations
into the zebrafish genome. The mutants are crossed further to generate the F3 generation
which is tested for the altered susceptibility for pneumococcal infection. So far, we have
screened 35 mutant zebrafish lines and found one line with an interesting phenotype.
This mutant line will be further tested and characterized by inverse-PCR. Eventually,
the screen is likely to expand our understanding of the innate response to pneumococcal
infection, providing new insights into the treatment of pneumococcal diseases.
Adequate Th2 response associates with restricted bacterial growth in latent
mycobacterial infection of zebrafish
Tuberculosis is still a major health problem worldwide. Currently it is not known what
kind of immune responses lead to successful control and clearance of Mycobacterium
tuberculosis. This gap in knowledge is reflected by the inability to develop sufficient
diagnostic and therapeutic tools to fight tuberculosis. We have used the Mycobacterium
marinum infection model in the adult zebrafish and taken advantage of heterogeneity
of zebrafish population to dissect the characteristics of adaptive immune responses
some of which lead to well-controlled latency or bacterial clearance while others to
progressive infection. Differences in T cell responses between subpopulations were
measured at the transcriptional level. It was discovered that a high total T cell number
was usually associated with lower bacterial loads alongside with a T helper 2 (Th2) type
gene expression signature. At late time-points, spontaneous reactivation with apparent
symptoms was characterized by a low Th2/Th1 ratio and a substantial induction of
Foxp3 reflecting the level of regulatory T cells. Characteristic gata3/tbx21 has potential
as a biomarker for the status of mycobacterial disease.
Rami Pääkkönen
Tiina Joki
Keywords: Tissue engineering, Mesenchymal stem
cells, Chondrogenesis, TGF-beta superfamily proteins,
Bone morphogenetic protein 6, Hypertrophy
Keywords: CellTracker, DiD, co-culture, fluorescent probe,
labeling, human stem cell derived neural cells, long term
Author: Rami Pääkkönen(1,2) (rami.paakkonen@uta.fi)
Co-authors: Katja Ahtiainen(1,2,3,4), Susanna Miettinen(1,2,4)
All affiliations:1.Adult Stem Cells, Institute of Biomedical Technology, University of
Tampere, 33014 Tampere, Finland. 2.BioMediTech, Biokatu 10, 33520 Tampere, Finland.
3.Department of Cell Biology, School of Medicine, University of Tampere, 33014 Tampere,
Finland. 4.Science Center, Tampere University Hospital, Tampere, Finland
The effects of bone morphogenetic protein 6, dexamethasone and human serum on
chondrogenic differentiation of human adipose stem cells in vitro
Background and aims: Injured articular cartilage hardly heals and the current treatments have
only proven applicable for a fraction of patients, hence the demand for a novel treatment.
Mesenchymal stem cells from human adipose tissue are self-proliferative and they can
undergo multilineage differentiation. In this study, we aim to improve the chondrogenic
culturing conditions of human adipose stem cells (hASC).
Methods: hASC were chondrogenically differentiated with six media containing various
combinations of putative chondrogenic bioactive factors, namely TGF-β1, BMP-6,
dexamethasone and human serum. After 2 weeks in 3D high cell-density aggregate culture
the expression of osteogenesis- and chondrogenesis related genes was analyzed with Q-RTPCR array. Histological stainings were used to verify the accumulation of cartilaginous
extracellular matrix.
Results: None of the media induced chondrogenesis exclusively. BMP-6 resulted in
mixed chondrogenic and osteogenic differentiation, although a high dose of BMP-6 did
suppress the hypertrophic marker COL10A1. Dexamethasone boosted the expression of a
variety of ECM related genes, namely COL1A2, COL3A1, COL11A1, COL12A1, COMP,
SERPINH1 and ALP. When combined, a high dose of BMP-6 and dexamethasone resulted
in adipogenic differentiation.
Conclusions: The growth factor combinations studied here were not optimal for
chondrogenesis; the cells underwent concomitant osteogenic and/or adipogenic
differentiation. However, with further refinement of the culturing conditions the
differentiation of hASC should be even more controllable, and accordingly cartilage tissue
engineering with hASC would seem feasible.
Author: Tiina Joki (tiina.joki@uta.fi)
Co-authors: Meeri Mäkinen 1, Laura Ylä-Outinen 1, Heli Skottman 1,2, Riikka
Äänismaa 1, Susanna Narkilahti 1,3
All affiliations: 1 NeuroGroup, Institute of Biomedical Technology/BioMediTech,
University of Tampere, Finland. 2 OpthalmologyGroup, Institute of Biomedical
Technology/BioMediTech, University of Tampere, Finland. 3 The Science Center of
Pirkanmaa Hospital District, Tampere, Finland
Living art – method for labeling living human derived neural cells with fluorescent
probes.
Background: Fluorescent probes attaching to living cells can be used e.g. in long term
follow up studies with possibility to identify the labeled cell population or to enhance
cell visibility in opaque cultures. These features are useful in many research areas in
the field of applied sciences e.g. development of controlled neurotoxicity platforms and
biomaterial research for craft development.
The aim of this study was to create a non-invasive tool for cell visualization and cell
population tracking that can be used in long term studies.
Methods: Cells used in this study were human pluripotent stem cell derived neuronal
cells. Fluorescent probes used were 1) 5-chloromethylfluorescein diacetate (CellTracker
Green CMFDA, CT) and 2) 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine
perchlorate (DiD). Cell viability and proliferation was briefly studied. Also, the labeled
cells were characterized using immunocytochemistry and micro electrode array (MEA).
Results: Most suitable parameters for human neural cells were 10 µM CT with 72 hours
incubation time and 10 µM DiD with 2 hours incubation time. With these parameters
the labeling was visible up to 4 weeks and had no statistical effect on cell viability. CT
had no affect to cell proliferation but DiD caused slight decrease in proliferation in long
term culture. Also, the CT or DiD labeled population and their mixed-cultures expressed
normal spontaneous network activity.
Conclusions: This study indicated that fluorescent probes CT and DiD are suitable for
long-term labeling of human derived neural cultures in vitro. Most importantly, the
labeled neural networks were electrically functional.
Mauro Scaravilli
Kirsi Tuppurainen
2. Cancer Research
Keywords: miRNA, differential expression, MYCBP2.
2. Cancer Research
Keywords: Prostate cancer, microRNA
Author: Mauro Scaravilli (mauro.scaravilli@uta.fi)
Author: Kirsi Tuppurainen (kirsi.tuppurainen@uta.fi)
Group: Prof. Tapio Visakorpi
Co-authors: Mauro Scaravilli1, Kati Porkka1, Elena Martens-Uzunova2, Teuvo Tammela3,
Guido Jenster2 and Tapio Visakorpi1.
Co-authors: Hanna E. Rauhala, Mauro Scaravilli and Tapio Visakorpi
All affiliations: 1Institute of Biomedical Technology, Prostate Cancer Research Center,
University of Tampere and Tampere University Hospital, Tampere, Finland. 2Department of
Urology, Josephine Nefkens Institute, Erasmus University Medical Center, Rotterdam, The
Netherlands. 3Department of Urology, Tampere University Hospital, Tampere, Finland.
Differential expression of miR-1247 in prostate cancer.
Recently there has been increasing attention on the role of microRNAs in the molecular
mechanisms of cancer development. Several genome-wide profiling studies have provided
evidence of aberrant expression of miRNAs in prostate cancer and have highlighted the
potential use of specific miRNA expression signatures as prognostic or predictive markers.
In EU-FP7-ProspeR program, deep-sequencing of 11 pooled samples of 4 normal and
malignant prostates has been recently performed, revealing several putatively differentially
expressed miRNAs that were not present in the miRNA microarray platforms previously
used by ProspeR
The aim of the study is to validate the deep-sequencing data and identify novel miRNAs
whose alterations are involved in the development of prostate cancer. A total of 7 miRNAs,
namely miR-1247, miR-1249, miR-1271, miR-1290, miR-1299 miR-1291, and miR-1269
were selected. qRT-PCR (TaqMan assay) was performed in a subset of 58 clinical samples,
including 8 benign prostatic hyperplasias (BPH) and 28 primary tumors (PCa) obtained
by radical prostatectomy, as well as on 7 BPHs and 15 castration resistant prostate cancers
(CRPC) obtained by transurethral resection of the prostate (TURP). RNU6B was selected as
a qRT-PCR reference gene.
We found significant upregulation of miR-1247 and miR-1249 in TURP-CRPC samples
when compared to TURP-BPH. The expression of miR-1247 was then studied in PCa lines,
showing clear upregulation in the androgen-insensitive PC-3 model, when compared to
other lines. Online target prediction analysis for miR-1247 revealed MYCBP2 (myc-binding
protein 2) transcript as a high-score potential target. Functional studies in vitro performed
on PC-3 and LNCaP models confirmed down-regulation of MYCBP2 at both mRNA and
protein level, while induced overexpression of miR-1247 did not affect cell growth.
Currently, we are performing luciferase assay to confirm the specific binding of miR-1247 to
the 3´-UTR of the putative target gene.
All affiliations: Prostate Cancer Research Center, Institute of Biomedical Technology,
University of Tampere and Tampere University Hospital
CCND1 is a putative miR-193b target in prostate cancer
Prostate cancer is the most frequent cancer of men in Western countries and causes
second most of the cancer-related deaths. In addition to genetic changes also epigenetic
alterations and micro-RNAs (miRNAs) are important in prostate cancer development.
Our group has previously shown that micro-RNA 193b is hypermethylated in 22Rv1
prostate cancer cell line and that it is putative tumor suppressor in prostate cancer. In
melanoma and hepatocellular carcinoma, it has been shown that CCND1, Cyclin D1
protein encoding gene is among the target genes of miR-193b. The aim of this project is
to study if CCND1 is a target of miR-193b in prostate cancer.
According to qRT-PCR and microarray analyses, CCND1 and miR-193b had opposite
expression patterns in prostate cancer cell lines and xenografts. Using a luciferase reporter
gene assay in 22Rv1 cells, we showed that miR-193b targets CCND1 3’UTR. In 22Rv1
cells with low endogenous miR-193b expression, transient miR-193b transfection
reduced CCND1 mRNA levels and Cyclin D1 and phospho-RB protein levels. When
prostate cancer cell lines were treated with cdk4/6 inhibitor, 22Rv1 and VCaP cells with
low miR-193b and high CCND1 expression showed significant growth retardation. In
contrast, the inhibitor did not have any effect on the growth of PC-3 and DU145 cells
with high miR-193b and low CCND1 expression. Furthermore, immunohistochemical
analysis of Cyclin D1 expression showed that CRPC samples have significantly higher
expression of Cyclin D1 compared to PC samples. Therefore, we conclude that CCND1
is, at least, one of the miR-193b targets in prostate cancer.
Liisa-Ida Sorsa
Ana María Soto de la Cruz
Keywords: computational annotation, miRNA,
GRO-seq, primary transcript
Keywords: OPT, SPIM, Tissue Engineering, Hydrogels
Author: Ana María Soto de la Cruz (ana.sotodelacruz@tut.fi)
Author: Liisa-Ida Sorsa (liisa-ida.sorsa@tut.fi)
Group: Matti Nykter
Co-authors: Jari Hyttinen, Edite Figueiras
Co-authors: Merja Heinäniemi, Matti Nykter
All affiliations: Tampere University of Technology, Institute of Biomedical Technology,
University of Tampere, A.I. Virtanen Institute, University of Eastern Finland
All affiliations: Tampere University of Technology, Department of Electronics and
Communications Engineering. BioMediTech
Imaging Hydrogels 3D structure for tissue engineering with OPT and SPIM
Primary miRNA annotation from GRO-seq data
MicroRNAs (miRNAs) play important roles in gene regulation networks. These
molecules are transcribed by RNA polymerases analogously to protein coding genes.
However, many miRNA are processed co-transcriptionally making the annotation of
primary transcripts, which can be tens of kilobases in length, problematic. Accurate
annotation of primary transcripts is necessary for example to study transcriptional
regulation of miRNAs. Current annotation efforts are based on infering the putative
transcriptions start sites (TSS) based on various histone modifications from ChIP-seq
experiments. Here we use recently developed Global run-on sequencing (GRO-seq),
which maps the position, amount and orientation of transcriptionally engaged RNA
polymerases genome-wide, to study nascent RNA with goal to identify primary miRNA
transcripts.
We have developed a data-driven method which annotates primary miRNA transcripts
based on GRO-seq data. We apply our method to HUVEC cells to uncover primary
sequence for miRNA that are transcribed in these cells. We also present results on the
comparison of expression levels between GRO-seq and RNA-seq. Comparison with
annotated protein coding transcripts, primary miRNA annotations in the literature and
histone modification data from ENCODE shows that TSS and transcript bodies can
successfully be uncovered from GRO-seq data, enabling accurate identification of
primary miRNA transcripts.
Hydrogels are hydrophilic polymer networks which have applications in Tissue
Engineering (TE) mainly due to its capability to mimic the extracellular matrix.
However, few have investigated the dynamics of cell behaviours and biological
interactions in three dimension (3D) engineered tissues hydrogels, due to inadequate
imaging technology for high-resolution 3D imaging, real-time, non-destructive and
non-invasive imaging.
Optical Projection Tomography (OPT) and Selective Plane Illumination Microscopy
(SPIM) are promising non-destructive 3D imaging techniques. OPT is the optical
equivalent of X-ray micro-computed tomography (X-ray µCT), where a suspended
specimen, in an index-matching liquid, is rotated through a series of angular positions,
and an image is captured at each orientation. SPIM is a technique where specimens
labeled with fluorophores are illuminated by a planar light sheet perpendicular to the
axis of fluorescence detection. Both techniques are used for 3D high-resolution imaging
of samples with dimensions between 1 and 10 mm (known as the mesoscopic scale).
Currently, an integrated system with OPT and SPIM (OPT-SPIM) techniques is being
built and optimized for TE constructs imaging.
In this work, we show that the OPT-SPIM system is a promising tool to image 3D TE
based hydrogels. We predict that with this system we will be able to image the 3D
structure of hydrogels, as well to monitor over time cell differentiation and maturation
processes, cell attachment, growth, viability (with live/dead staining) and morphology
in 3D TE based hydrogels constructs.
Sina Saari
Mostafa Kiamehr
Keywords: Alternative oxidase, Drosophila
melanogaster, oxidative phosphorylation,
spermatogenesis
1. Biotechnology
Keywords: induced pluripotent stem cell, coronary artery
disease, atherosclerosis, hepatocyte, lipid
Author: Mostafa Kiamehr (mostafa.kiamehr@uta.fi)
Author: Sina Saari (sina.saari@uta.fi)
Group: Katriina Aalto-setälä
Group: Howard T. Jacobs
Co-authors: Leena Viiri
Co-authors: Marcos T. Oliveira, Howard T. Jacobs
All affiliations: iPS cell derived hepatocytes to model atherosclerosis
Alternative Oxidase and Spermatogenesis in Drosophila melanogaster
iPS cell derived hepatocytes to model atherosclerosis
The expression of the mitochondrial alternative oxidase (AOX), a respiratory chain
alternative enzyme from the sea squirt Ciona intestinalis, in the fruit fly Drosophila
melanogaster has proven to counteract deleterious effects caused by dysfunctions in
complexes III and/or IV of the oxidative phosphorylation process (OXPHOS). This
suggests that AOX could potentially be used in gene therapies for human mitochondrial
diseases that currently have no treatment. However, the natural lack of alternative
enzymes in arthropods and vertebrates may present as an impediment to such approach.
We set out to investigate possible negative effects of AOX expression on the fitness of
D. melanogaster, by analyzing the male’s ability to compete against wild-type males
and reproduce. In sperm competition assays, AOX-expressing males were clearly
outcompeted by AOX-nonexpressing males. We analyzed the reproductive organs
macro-morphologically and found a decrease in the size of the seminal vesicles in
AOX-expressing males, indicating that production of mature sperm is compromised,
what explains the lack of sperm competitiveness. We have localized the AOX protein in
the mitochondria of the testis wall cells and those of the seminal vesicle wall cells. How
the expression of AOX causes the decrease in sperm production in D. melanogaster is
yet to be determined, but our preliminary work suggests that AOX function disturbs one
or more events during proper spermatogenesis. This work has been funded by grants of
the Marie Curie Actions, the Academy of Finland and the European Research Council.
Coronary artery disease (CAD) is currently the main cause of morbidity and mortality
in modern world and is predicted to be the number one killer globally in 2020. Available
drug therapies are not capable of preventing 70% of clinical events and at least 10%
of coronary events occur in apparently healthy individuals in the absence of traditional
risk factors such as LDL cholesterol. In this study we are aiming at investigating
new biomarkers to diagnose patients with high risk CAD and discovering possible
new treatments based on the results obtained in vitro. To achieve this, three patients
groups have been recruited: 1) patients with acute myocardial infarction 2) stable CAD
patients and 3) healthy controls. Induced pluripotent stem cell (iPSC) lines have been
generated from skin biopsies of the patients and hepatocytes are being successfully
generated. We are investigating three different protocols for generating the most
functional hepatocytes for our purposes. They will be evaluated according to their
albumin secretion, CYP450 activity, and LDL uptake. Lipidomics will be performed
by mass spectrometry and the secreted lipids will be analyzed from the conditioned
culture media. The achieved data will be compared to that obtained from primary
human hepatocytes.
Sanna Auer
Sanni Virjula
1. Biotechnology
Keywords: adipose stem cells, electrospinning, scaffold, osteogenic differentiation
Author: Sanna Auer (sanna.auer@uta.fi)
Co-authors: Sanna Auer1, Vesa Hytönen1, Jussi Toppari2, Kosti Tapio2, Mohsen
Pourmousa3, Ilpo Vattulainen3, Dongkai Shao2, Markus Ahlskog2
All affiliations: 1 Institute of Biomedical Technology, University of Tampere, 2
Nanoscience Center, University of Jyväskylä, 3 Department of Physics, Technical
Programmable bioactuators
We are aiming to control protein function by electric field. The idea is to immobilize the
selected proteins on carbon nanotubes, or graphene, which are conducting materials and
highly utilized in various novel electronic materials and devices.
In this collaboration with groups from the University of Jyväskylä and Tampere
University of Technology our role is in the expression, engineering and modification
of the selected proteins so that they can be immobilized in fully functional form on the
carbon nanomaterials. The ideal protein candidates are enzymes showing conformational
changes within their active cycle. The aim of the study is to analyze the potential of the
electric field to guide these conformational changes. Such a programmable material
would then work as a bioactuator converting electrical signal into biological readout,
i.e. enzymatic activity.
The first protein candidate selected for the project is a photoprotein luciferase that
catalyzes the conversion of luciferin into oxyluciferin, simultaneously producing
light. The active site of the luciferase is in the cleft between the N- and C-terminal
domains. In the reaction cycle the enzyme adapts a conformational change and the
cleft is closed trapping the substrate and ATP and excluding water. Jamaican click
beetle (Pyrophorus plagiophthalamus) and firefly (Photinus pyralis) luciferases with
engineered biotinylation sites and His-tags are currently expressed in Escherichia coli
(or later also in insect cells) to be immobilized on carbon nanomaterials.
Author: Sanni Virjula (sanni.virjula@uta.fi)
Co-authors: Sari Vanhatupa 1,2,3, Xinxin Zhao 4, Yuan Siang Lui 4, Say Chye Joachim
Loo 4, Susanna Miettinen 1,2,3
All affiliations: 1 Adult Stem Cell Group, Institute of Biomedical Technology, University
of Tampere, Finland. 2 BioMediTech, Tampere, Finland. 3 Science Center, Tampere,
University Hospital, Tampere, Finland. 4 School of Materials Science and Engineering,
Nanyang Technological University, Singapore
Characterization and Comparison of Electrospun Polycaprolactone Based
Composite Scaffolds for Osteogenic Differentiation of Human Adipose Stem Cells
Electrospinning is considered as a promising scaffold fabrication method for bone
tissue engineering, because non-woven membranes of electrospun fibers are thought to
mimic the bone extracellular matrix. In the current study, we investigated the addition
of hydroxyapatite (HA), natural component of bone, into the electrospun scaffolds and
the potency of the scaffolds to modulate the osteogenic differentiation of human adipose
stem cells (hASCs). Adipose tissue is an attractive and abundant source of multipotent
stem cells, and hASCs have already shown their clinical relevancy in bone tissue
engineering applications.
Here, HA was combined with polycaprolactone (PCL) either during spinning resulting
inside the fibers, or precipitated onto the PCL scaffold surface after spinning. Also blank
PCL scaffold was used in comparison. The scaffolds were characterized by scanning
electron microscopy (SEM), tensile test, and apatite forming capacity judged by SEM.
The cell viability in the scaffolds was evaluated by Live/Dead staining and proliferation
by CyQuant Assay. The osteogenic differentiation was analysed after 1-3 weeks
culture by quantitative alkaline phosphatase activity assay (qALP) and immunological
stainings by osteogenic markers, osteopontin and osteocalsin. Studies were conducted
in a presence of basal growth medium and osteoinductive medium conditions.
Our results indicated that HA on the scaffold surface supported best the attachment of
cells, whereas the blank PCL scaffold was found to enhance the osteogenic differentiation
most and carried also best mechanical properties. As a conclusion, HA may not improve
the properties of electrospun PCL scaffolds for bone tissue engineering.
Toni Grönroos
Susanna Teppo
2. Cancer Research
Keywords: TEL-AML1, Acute leukemia, ALL
2. Cancer Research
Author: Toni Grönroos (toni.gronroos@uta.fi)
Group: Docent, Olli Lohi
Co-authors: Kaisa Teittinen, Annemari Uusimäki, Mataleena Parikka, Mika Rämet, Olli
Lohi
All affiliations: Tampere Center for Child Health Research, University of Tampere,
School of Medicine, (TG KT OL). Institute of Biomedical Technology, University
of Tampere, BioMediTech, Tampere, Finland, (AU MP MR). Tampere University
Hospital, Tampere, Finland, (MR, OL)
TEL-AML1 leukemia modeling in zebrafish
TEL-AML1 (ETV6-RUNX1) fusion is the most common genetic rearrangement
in paediatric acute leukemia, occurring in approximately 25 % of childhood pre-B
acute lymphoblastic leukemia (ALL). TEL-AML1 fusion is generated by the t(12,21)
(p13;q22) chromosomal translocation which fuses the 5’ region of the ETS transcription
factor, TEL, in-frame to almost entire AML1 gene. TEL-AML1 is regulated by Telpromoter.
Zebrafish, Danio rerio, has a short generation interval and it produces abundantly
offspring. Eggs are fertilized externally and developing embryos can be kept transparent
with chemical treatment. These characteristics combined with the possibility for
manipulation at all developmental stages makes zebrafish an ideal model organism
for studying hematopoiesis. Furthermore, zebrafish and human hematopoiesis are
morphologically and genetically very similar.
Our main goal is to generate a transgenic zebrafish line expressing TEL-AML1 fusion
gene regulated by natural zebrafish Tel-promoter. We identified and cloned the Telpromoter from the zebrafish gDNA and subcloned it in front of the red fluorescent
protein, DsRed. Thereafter, the synthetic TEL-AML1 fusion gene, based on zebrafish
TEL- and AML1-genes, was cloned between the Tel-promoter and DsRed. We have also
used H2AFVpromoter, as more universal promoter to drive the fusion gene expression.
We are currently crossbreeding our fish lines to generate a stable transgenic fish lines
expressing TEL-AML1 fusion gene.
Author: Susanna Teppo (susanna.teppo@uta.fi)
Group: Olli Lohi
Co-authors: Keijo Viiri, Olli Lohi
All affiliations: Tampere center for child health research, University of Tampere
Modelling of pediatric leukemia in tissue culture
Background Each year around 50 children are diagnosed with leukemia in Finland.
Most, over 85 %, are cured but only after long chemotherapy treatment, and some
patients still relapse. Thus, better understanding of the molecular biology behind the
disease is fundamental. The most common (25 % of patients) genetic variant in acute
lymphoblastic leukemia is a translocation resulting in a fusion of two transcription
factors important in hematopoiesis regulation, TEL and AML1. The fusion protein
is suggested to act as a repressor, especially silencing genes in B-cell differentiation
pathway. However, the mechanism of this repressive function remains unclear.
Methods Aiming at solving the biochemistry behind TEL-AML1-leukemia, we have
generated stable cell lines expressing either inducible TEL-AML1 (Nalm-6-TA)
or shRNA against endogenous TEL-AML1 (REH-shTA). For a control, we have
exploited site-directed mutagenesis to generate a mutant version with substantially
diminished DNA-binding capacity. The effects of TEL-AML1 are studied by examining
proliferation, apoptosis, cell cycle, differentiation and subcellular localization. ChIPseq and gro-seq techniques will be utilized in order to identify the bona fide targets of
TEL-AML1 fusion.
Results and conclusion The preliminary results show 20-fold induction of TEL-AML1
in overexpressing cell lines at both mRNA and protein levels. TEL-AML1 reduces
cell proliferation by 30 % compared to both luciferase-control and the DNA-bindingdisrupted fusion. This model system has shown promising in studying TEL-AML1
function in leukemia at the cell and molecular level.
Priit Joers
Johanna Salonen
Keywords: mtDNA, electron transport chain,
redox state
Keywords: B cell antigen receptor, B cell differentiation,
Bioinformatics, B lymphocyte, MALDI-TOF-MS,
two-dimensional differential gel electrophoresis
Author: Priit Joers (priit.joers@uta.fi)
Author: Johanna Salonen1,2,3 (johanna.m.salonen@uta.fi)
Group: Howard Jacobs
Group: Matti Nykter
Co-authors: Sampsa Järvinen, Mike Gerards, Mikael Parhiala
Co-authors: Gunilla Rönnholm4, Nisse Kalkkinen4, Mauno Vihinen1,2,3,5
All affiliations: Institue of Biomedical Technologies, Tampere University
Design and development of a novel mitochondrial mutator system in D.
melanogaster.
Mitochondrial dysfunction caused by mutations/absence of mt genes extends beyond
defects of ATP production, as homeostasis of this organelle has been implicated in
several crucial functions in organisms (metabolic signalling, cellular differentiation,
oncogenesis, ageing etc.). Therefore mutational analysis of mt genome would provide
valuable information relevant to human senescence and several medical disorders. We
are developing a random mutator system in Drosophila by targeting bacterial restrictionmodification complex (EcoBI) to mitochondrial matrix. This tripartite complex has the
unique ability to cut randomly any DNA molecule that contains its unmethylated binding
site, therefore it is theoretically capable to modify Drosophila mt genome (has 3 sites
for EcoBI) at any position. Moreover, cleavage-deficient version of EcoBI interferes
with mitochondrial gene expression through the process of translocating mtDNA (step
that predates DNA cleavage). This depletes available mitochondrial subunit pools and
therefore functional membrane-inserted complexes, leading to dramatic rise of reactive
oxygen species (ROS), by-products of electron transport along ETC. Increased ROS
trigger several changes, e.g. forcing cell-autonomous differentiation of one type of
immunological cell lineage to another, which resembles the overproliferation phenotype
in human chronic leukaemia. Therefore targeting Type I endonuclease to mitochondria
provides
a. unique mitochondrial mutator system among metazoans coupling random
mitochondrial mutagenesis with possibility of high-throughput screening in Drosophila
model system.
b. convenient way to study alterations in ETC complex ratios and effects caused by
changes in redox state of the cell.
All affiliations: 1Institute of Biomedical Technology, University of Tampere.
2BioMediTech. 3Research Unit, Tampere University Hospital. 4Institute of
Biotechnology, University of Helsinki. 5Department of Experimental Medical Science,
Lund University
Proteomic changes during B cell maturation: 2D-DIGE approach
B cells play a pivotal role in adaptive immune system, since they maintain a delicate
balance between recognition and clearance of foreign pathogens and tolerance to self.
During maturation, B cells progress through a series of developmental stages defined
by specific phenotypic surface markers and the rearrangement and expression of
immunoglobulin (Ig) genes. To get insight into B cell proteome during the maturation
pathway, we studied differential protein expression in eight human cell lines, which
cover four distinctive developmental stages; early pre-B, pre-B, plasma cell and
immature B cell upon anti-IgM stimulation. Our two-dimensional differential gel
electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization-time of
flight-mass spectrometry (MALDI-TOF-MS) based proteomic study indicates the
involvement of large number of proteins with various functions. Notably, cytoskeleton
and ontogeny related proteins were moderately expressed in early pre-B/pre-B cell
stages and silenced in immature B cell (IM-B) stage. The transition from pre-B to
IM-B stage changed significantly the proteome profile, since proteins from a variety
of functional gene ontology (GO) categories appeared. Our long time series analysis
in anti-IgM stimulated Ramos B cells revealed the dynamic regulation of cytoskeleton,
gene expression and metabolic pathways, among other cellular events. In plasma cell
stage, the proteome contained mainly endoplasmic reticulum (ER)/Golgi –system
proteins needed for efficient antibody production. Representative 2D-DIGE maps of
different B cell maturation stages are available online at http://structure.bmc.lu.se/
BcellProteome/.
Anni Sorkio
Tommi Rantapero
Keywords: embryonic stem cell, cell differentiation,
extracellular matrix, retina
Keywords: Prostate cancer,high-throughput sequencing,epigenetics,gene regulatory
networks
Author: Anni Sorkio (anni.sorkio@uta.fi)
Author: Tommi Rantapero (tommi.rantapero@uta.fi)
Group: Heli Skottman
Group: Matti Nykter
Co-authors: Sorkio A.1, 2, Hongisto H.1, 2, Kaarniranta K 3, Uusitalo H.4, Juuti-Uusitalo
K. 1, 2, Skottman H. 1, 2
Co-authors: Simo-Pekka Leppänen, Annika Kohvakka, Kati Waltering, Juha Kesseli,
Tapio Visakorpi, Matti Nykter
All affiliations: 1 Institute of Biomedical Technology, Biokatu 12, 33014 University of
Tampere, Finland. 2 BioMediTech, Tampere, Finland. 3 Department of Ophthalmology,
University of Eastern Finland and Kuopio University Hospital, Kuopio, Finland.
4Department of Ophthalmology, SILK, University of Tampere, Tampere, Finland, TAUH
Eye Center, Tampere University Hospital, Tampere, Finland.
All affiliations: Institute of Biomedical Technology/BioMediTech, University of
Tampere, Tampere, Finland
Structure and barrier properties of human embryonic stem cell derived retinal
pigment epithelial cells are affected by extracellular matrix protein coating
Prostate cancer is a heterogenous disease. Genomic charaterization studies have been
completed to identify genetic aberrations and key oncogenic pathways. Still, key
processes that lead to different clinical subtypes are poorly understood. A number of cell
line model that contain subsets of most recurrent aberrations such as AR or ERG over
expression exists. Experimenting with clinical material is challenging due to steady
state nature and limited amount of the sample. Thus, the key question is how to utilize
information from the models to gain insight into development of clinical subtypes.
Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration,
proliferation and differentiation of cells. We investigated the role of ECM proteins to the
structure and function of human embryonic stem cell derived retinal pigment epithelial
(hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells
on seven ECM proteins and commercially available substrates in adherent differentiation
cultures. Cell pigmentation, expression of RPE specific proteins, fine structure as well
as the production of basal lamina by hESC-RPE on different protein coatings were
evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and
barrier properties on different coatings were investigated by measuring transepithelial
resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated
by early onset of cell pigmentation and further maturation to RPE monolayers after
enrichment. Mature RPE phenotype was verified by RPE specific gene and protein
expression, correct epithelial polarization and phagocytic activity. Significant differences
were found in the degree of RPE cell pigmentation and tightness of epithelial barrier
between different coatings. Furthermore, the thickness of self-assembled basal lamina
and secretion of the key ECM proteins found in the basement membrane of the native
RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion,
this study shows that the cell culture substrate has a major effect on the structure and basal
lamina production during the differentiation and maturation of hESC-RPE potentially
influencing the success of cell integrations and survival after cell transplantation.
Prostate cell lines as models of clinical subtypes
We have generated and collected an extensive set of high throughput measurement data
from prostate cancer cell lines. These include transcriptome, DNA methylation, open
chromatin, transcription factor binding and enhancer RNA expression profiling with
deep sequencing. Using these data we aims to: 1) Identify gene regulatory mechanisms
that are characteristic to cell lines corresponding to different clinical subtypes and to
validate these mechanisms with data from clinical samples. 2) Use these data to build
mathematical models that allow incorporation of enhancer and promoter regulation
information from cell lines for prediction of gene expression in clinical samples.
Epigenetic profiles clearly group the cell lines based on their underlying genomic
aberrations, allowing us to define signatures for clinical subtypes. Ongoing analysis
aims to catalog the gene regulatory elements in each subtype. These catalogs will then
be tested to predict the gene expression profiles in clinical samples. Based on the most
informative features a mathematical model for gene regulatory networks will be built.
Juha Määttä
Hannu Turpeinen
1. Biotechnology
Keywords: protein expression, protein purification,
calorimetry
Author: Juha Määttä (juha.maatta@uta.fi)
Group: Marko Pesu
Co-authors: Zsuzsanna Ortutay1, Marko Pesu1,2
Co-authors: Latifeh Azizi, Niklas Kähkönen, Perrine Pinon, Bernhard Wehrle-Haller,
Vesa Hytönen
All affiliations: 1 Immunoregulation, Institute of Biomedical Technology, University
of Tampere, and BioMediTech, Tampere, Finland, 2 Fimlab laboratories, Pirkanmaa
Hospital District, Finland
All affiliations: Institute of Biomedical Technology, Biokatu 6, FI-33014 University
of Tampere andTampere University Hospital, Finland. Department of Cell Physiology
and Metabolism, Centre Médical Universitaire, 1.Rue Michel-Servet, 1211 Geneva 4,
Switzerland
Biocenter Finland Supported Protein Service
The Protein Service (PS) offers recombinant protein production and follow-up
purification possibilities at a reasonable cost easily affordable for academic clients.
PS offers baculovirus expression system and mammalian cell expression system in
Tampere and Helsinki, respectively. Also E.coli expression is possible in Tampere and
could be scaled up with small scale fermentation. Customers are asked to subclone
inserts to the multiple cloning sites in provided plasmids and to confirm the sequences
by given primers. After small-scale production, final protein is confirmed by western
blot. Optional Scale Up is offered together with protein purification. More information
can be found from core web pages: http://cofa.uta.fi/protein.html
As an example case we produced set of talin head domain proteins in E. Coli (BL21
DE3 Star) cells. After fermentation we purified protein with IMAC and further IEX
chromatography. Yield of one of the proteins after the first purification step was over
40 mg (2.2 mg/ml, 20 ml) and when small patch (500 μL) was further purified with gel
filtration we managed to get approximately 1 mg ultrapure protein. Moreover, protein
stability, hydrodynamic radius and binding properties of the proteins were analyzed
with DSC, DLS and Octet, respectively.
Author: Hannu Turpeinen1 (hannu.turpeinen@uta.fi)
Genetics of the First Seven Proprotein Convertase Enzymes in Health and Disease
Members of the substilisin/kexin like proprotein convertase (PCSK) protease family
cleave and convert immature pro-proteins into their biologically active forms. By
cleaving for example prohormones, cytokines and cell membrane proteins, PCSKs
participate in maintaining the homeostasis in a healthy human body. Conversely, erratic
enzymatic function is thought to contribute to the pathogenesis of a wide variety of
diseases, including obesity and hypercholestrolemia. The first characterized seven
PCSK enzymes (PCSK1-2, FURIN, PCSK4-7) process their substrates at a motif made
up of paired basic amino acid residues. This feature results in a variable degree of
biochemical redundancy in vitro, and consequently, shared substrate molecules between
the different PCSK enzymes, This redundancy has confounded our understanding of the
specific biological functions of PCSKs. The physiological roles of these enzymes have
been best illustrated by the phenotypes of genetically engineered mice and patients that
carry mutations in the PCSK genes. Recent developments in genome-wide methodology
have generated a large amount of novel information on the genetics of the first seven
proprotein convertases. In this review I will summarize the reported genetic alterations
and their associated phenotypes.
Satu Luhtala
Cristina A. Nadalutti
2. Cancer Research
Keywords: HER3, ErbB3, breast cancer,
immunohistochemistry
Keywords: Adhesion, polarization, vascular biology,
celiac disease
Author: Satu Luhtala (satu.luhtala@uta.fi)
Author: Cristina A. Nadalutti (cristina.nadalutti@uta.fi)
Group: Jorma Isola
Group: Prof. Markku Mäki
Co-authors: Synnöve Staff, Minna Tanner, Jorma Isola
Co-authors: Ilma Rita Korponay-Szabo’ (2), Katri Kaukinen (3), Martin Griffin (4), Markku
Mäki (1), Katri Lindfors (1).
All affiliations: Institute of Biosciences and Medical Technology (BioMediTech),
University of Tampere, Tampere, FINLAND
Comparison of HER3 antibodies for determination of HER3 expression in breast
cancer
Background: HER3 (ErbB3) is a transmembrane receptor tyrosine kinase that is
overexpressed in several cancers and is therefore a potential target for modern,
personalized cancer therapies. Currently there is no standardized immunohistochemical
(IHC) method for evaluation of HER3 expression. Variable staining protocols have been
used in the earlier studies clarifying the prognostic significance of HER3 expression. We
aimed to standardize IHC method for reliable evaluation of HER3 expression in breast
carcinoma.
Methods: By using FFPE breast carcinoma samples we compared four commercial
HER3 antibodies: mouse monoclonal antibodies DAK-H3-IC (Dako) and RTJ1 (Leica
Biosystems), rabbit monoclonal antibody SP71 (Spring Bioscience) and rabbit polyclonal
antibody SAB4500793 (Sigma-Aldrich). Membranous and cytoplasmic staining patterns
were analyzed microscopically and were scored as 0, 1+ and 2+ according to the intensity
of the staining and completeness of membranous and cytoplasmic staining. MDA-453
breast cancer cell line and human prostate are well-known of their high HER3 expression
and were used as positive controls. IHC results were confirmed using flow cytometry
analysis.
Results: Feasible HER3 staining protocol was accomplished using HER3 antibody
clone DAK-H3-IC. Other antibodies (RTJ1, SP71, polyclonal SAB4500793) yielded
in nonspecific and non-reproducible stainings. HER3 localized in cell membranes
and cytoplasm, no nuclear staining was seen. Results were comparable also in breast
carcinoma samples fixed with molecular fixative (PAXgene) known to preserve sensitive
epitopes.
Conclusions: Staining method that we describe is technically qualified for routine use.
Assessment of membranous HER3 expression may be clinically useful in selecting
patients that benefit most from pertuzumab or anti-HER3 therapy.
All affiliations: (1)Tampere Center for Child Health Research, University of Tampere
and Tampere University Hospital, Tampere, Finland; (2)Celiac Disease Center, Heim Pal
Children’s Hospital, Budapest and Department of Pediatrics, Medical and Health Science
Center, University of Debrecen, Hungary; (3)School of Medicine, University of Tampere,
Department of Gastroenterology and Alimentary Tract Surgery, Tampere University
Hospital, Tampere, Finland; Department of Medicine, Seinäjoki Central Hospital, Finland;
(4)School of Life and Health Sciences, Aston University, Birmingham, United Kingdom.
Celiac disease patient IgA antibodies induce endothelial adhesion and cell
polarization defects via extracellular transglutaminase 2
We have recently found that celiac disease patient serum-derived autoantibodies targeted
against transglutaminase 2 (TG2) interfere with several steps of angiogenesis, including
endothelial sprouting and migration, though the mechanism involved remained to be fully
characterized. This study now investigated the processes underlying the antiangiogenic
effects exerted by celiac disease patient antibodies (CD IgA) on endothelial cells (HUVECs),
with particular regard to the adhesion, migration and polarization signaling pathway. We
observed that CD IgA reduced endothelial cell numbers by affecting cell adhesion without
increasing apoptosis. HUVECs in the presence of CD IgA showed weak attachment, a
high susceptibility to detach from fibronectin and an disorganized extracellular matrix
(ECM) due to a reduction of protein cross-links. Furthermore, CD IgA led to secretion of
active TG2 from endothelial cells into the culture supernatants. Additionally, cell surface
TG2-mediated integrin clustering in the presence of CD IgA was coupled to augmented
expression of β1-integrin. We also observed that CD IgA-treated HUVECs had migratory
defects and a less polarized phenotype when compared to control groups, and this was
associated with the RhoA signaling pathway. These biological effects mediated by CD IgA
on endothelial cells were partially influenced but not completely abolished by R281, an
irreversible extracellular TG2 enzymatic activity inhibitor.
Taken together our results imply that CD IgA antibodies disturb the extracellular protein
cross-linking function of TG2, thus altering endothelial cell-ECM interactions and thereby
affect endothelial cell adhesion, polarization and motility.
Sanna Huttunen
Ville-Pekka Seppä
Keywords: Adipose stem cells, endothelial
differentiation, vascularization, supercritical carbon
dioxide processing
7. Clinical physiology / Biomedical engineering
Keywords: respiratory loading, respiratory physiology,
lung function measurement
Author: Sanna Huttunen (1,2,3) (sanna.huttunen@uta.fi)
Co-authors: Laura Kyllönen (1,2,3), Kaarlo Paakinaho (1,4), Niina Ahola (1,4), Minna
Kellomäki (1,4), Susanna Miettinen (1,2,3), Suvi Haimi (1,2,3,5)
All affiliations: 1 BioMediTech, Finland. 2 Adult Stem Cells/Institute of Biomedical
Technology, University of Tampere, Finland. 3 Science Centre, Tampere University
Hospital, Finland. 4 Dept. of Electronics and Communications Engineering, Tampere
University of Technology, Finland. 5 Biomaterials Science and Technology Group/MIRA
Institute, University of Twente, The Netherlands
Supercritical Fluid Processed Porous Scaffolds Support the Endothelial
Differentiation of Human Adipose Stem Cells
One of the major challenges in bone tissue engineering is to ensure blood supply for cells
after implantation. Vascularization is essential for the viability of implanted cells especially
in large tissue constructs. Adipose stem cells (ASCs) have been shown to have potential as
a single cell source in the engineering of vascularized bone. In current study, the aim was
to evaluate proliferation and endothelial differentiation of human ASCs (hASCs) on porous
composite scaffolds engineered for bone tissue engineering.
Supercritical carbon dioxide (ScCO2) processing was used to manufacture scaffolds with
the average pore size of 570±220 μm from poly(lactice-co-ε-caprolactone) 70L/30CL and
β-tricalcium phosphate (β-TCP, 40 wt-%, max. particle size 40 μm). hASCs were cultured in
basal medium (BM), endothelial medium (EM) or osteogenic medium (OM) for three weeks.
Cell number was studied by analyzing the total DNA amount. Endothelial differentiation
was evaluated with qRT-PCR technique of endothelial marker genes (PECAM1, VWF and
CDH5). In addition, the protein expression of the same endothelial markers was analyzed
by immunocytochemical staining.
Proliferation of hASCs was the strongest in OM compared to BM and EM. Typical
endothelial marker genes had strong expression in EM according to the qRT-PCR results.
In immunocytochemical staining all studied endothelial markers were positive in EM. As
a conclusion, ScCO2 processed porous PLCL/β-TCP composites together with hASCs are
a promising combination in the engineering of vascularized bone. The hASCs were able to
differentiate towards the endothelial lineage when seeded onto the composites and cultured
in EM.
Author: Ville-Pekka Seppä (ville-pekka.seppa@tut.fi)
Group: Jari Viik
Co-authors: Marko Uitto, Jari Viik
All affiliations: All authors: Department of Electronics and Communications Engineering,
Tampere University of Technology AND Biomeditech
Tidal Breathing Flow-Volume Curves with Impedance Pneumography During
Expiratory Loading
(THIS WORK WAS PREVIOUSLY PRESENTED AS IS IN THE EUROPEAN
RESPIRATORY SOCIETY MEETING 2013)
Introduction: Diagnosis of asthma in the preschool children is difficult due to the lack
of objective lung function tests suitable for this age group. Impedance pneumography
(IP) is a mode of measurement that could enable ambulatory 24h recording of tidal flow
indices. IP can provide accurate flow signal in freely breathing subjects (Seppä et al. Eur
Respir J. 2011;38(55 Suppl):219s), but tidal breathing flow-volume curves (TBFVC) or
difficult respiratory conditions have not been demonstrated.
Objectives: To induce changes in breathing control and mechanics and study the ability
of IP to reproduce the TBFVC and track its changes under difficult conditions.
Methods: TBFVCs were obtained from 17 lung-healthy subjects simultaneously with
a direct mouth pneumotachograph (PNT) and IP during free breathing and expiratory
loading (15 cmH20.l/s). The TBFVC were normalized in flow and volume and the
largest distance between the PNT and IP curves was found for each subject as d max .
Results: The agreement of PNT and IP normalized TBFVCs was found excellent having
d max of 7.2 %±3.1 % and 7.3 %±4.2 % during free and loaded breathing, respectively.
Intense expiratory loading did not degrade the agreement.
Conclusion: We conclude that by using correct electrode placement and proper cardiac
filtering, IP was able to accurately reproduce and track changes in normalized TBFVCs
under free and loaded respiration in healthy subjects.
Suvi Kalliokoski
Saara Aittomäki
Keywords: autoantibodies, passive transfer, transglutaminase 2
Keywords: Drosophila melanogaster, innate immunity,
proprotein convertase
Author: Suvi Kalliokoski (suvi.kalliokoski@uta.fi)
Group: Markku Mäki
Co-authors: Suvi Kalliokoski (1), Sergio Caja (1), Ilma R. Korponay-Szabó (1, 2), Rafael
Frias (3), Outi Koskinen (1), Markku Mäki (1), Katri Kaukinen (4), Katri Lindfors (1)
All affiliations: (1) Tampere Center for Child Health Research, University of Tampere
and Tampere University Hospital, Tampere, Finland; (2) Celiac Disease Center, Heim Pál
Children’s Hospital, Budapest and Department of Paediatrics, Medical and Health Science
Centre, University of Debrecen, Debrecen, Hungary; (3) Central Animal Laboratory,
University of Turku, Turku, Finland; (4) School of Medicine, University of Tampere
and Department of Gastroenterology and Alimentary Tract Surgery, Tampere University
Hospital, Tampere, and Department of Medicine, Seinäjoki Central Hospital, Seinäjoki,
Finland
Injection of celiac patient serum or immunoglobulins to mice reproduces a
condition mimicking early developing celiac disease
Typical features of celiac disease are small bowel villous atrophy, crypt hyperplasia and
inflammation that develop gradually during ingestion of gluten. In addition, patients have
anti-transglutaminase 2 (TG2) autoantibodies in their serum and tissues. In this study we
investigated whether celiac disease can be passively transferred to mice by injecting patient
serum or immunoglobulins (Ig).
Celiac patient sera or purified total IgG were injected to athymic mice daily for 8 days. Nonceliac sera or serum immunoglobulins served as negative controls. A further experiment
with celiac patient or control sera lasting 28 days was performed. Animals were weighted
and observed for diarrhea daily. Tissue samples were harvested at the end of the experiments
and small bowel morphometry, lamina propria infiltration and celiac disease-specific TG2targeted autoantibody deposits were determined.
Interestingly, injection of celiac disease patient serum or total IgG to mice led to growth
failure after eight days and mild diarrhea in a subset of athymic mice. The villus height
crypt depth ratio (Vh/CrD) of the mice injected with celiac patient sera or total IgG was
significantly decreased and lamina propria infiltration was significantly increased in all
experiments in comparison to control mice. The affected mice also had celiac diseasespecific autoantibody deposits targeted to TG2 in several tissues including small intestine.
Taken together, the condition of the mice injected with patient serum or IgG resembles
early developing celiac disease in humans. We conclude that injected patient antibodies
likely cause both deterioration of intestinal mucosa and clinical features of celiac disease
in mice lacking T cells.
Author: Saara Aittomäki (saara.aittomaki@uta.fi)
Group: Marko Pesu
Co-authors: Saara Aittomäki 1) Tapio Lehtinen 1), Susanna Valanne 2), Mika Rämet 2),
Marko Pesu 1,3)
All affiliations: 1)Immunoregulation, Institute of Biomedical Technology, 2)
Experimental Immunology, Institute of Biomedical Technology, 3) Fimlab laboratories,
Pirkanmaa Hospital District
The role of Furin1 in Drosophila immunity
Many important biological processes involve proteolysis of larger protein precursors
by proprotein convertase enzymes. We have investigated the role of the proprotein
convertase family member Furin1 of Drosophila melanogaster in innate immunity.
Furin1 expression was knocked down in the fat body by crossing transgenic UASFurin1-RNAi flies with C564-GAL4 or Fb-GAL4 flies. Adult flies from these crosses
and control crosses were infected with Gram negative Enterobacter cloacae or Gram
positive Micrococcus luteus plus Enterococcus faecalis and their survival was monitored.
Our preliminary results indicate that flies with decreased expression of Furin1 in the fat
body are more sensitive to bacterial infection than control flies. This is possibly due to
the lack of induction of antimicrobial peptide expression via the Toll and Imd signalling
pathways, as analysed by quantitative RT-PCR. The fat body structure of Furin1-RNAi
x Fb-GAL4;UAS-GFP larvae appeared normal when compared to the w1118 x FbGAL4;UAS-GFP control cross larvae. Both RNAi-mediated knockdown of Cactus
and overexpression of IMD rescued the antimicrobial peptide expression from flies
with decreased levels of Furin1 in the fat body. This suggests that Furin1 may function
upstream of the Toll pathway member Cactus and the imd pathway member IMD.
Further studies will be carried out to elucidate the mechanism for Furin1 mediated
regulation of the immune signalling pathways.
Laura Airaksinen
Lehto Kalle
Keywords: Celiac disease, CCR9, CCL25, SNP,
sequencing, genetics
Keywords: X-ray microtomography, retina, dystrophy
Author: Laura Airaksinen (laura.airaksinen@uta.fi)
Group: professor Markku Mäki
Co-authors: Saavalainen P(2), Kaukinen K(3,4), Mäki M(1), Lindfors K(1)
All affiliations: (1) Tampere Center for Child Health Research, University of Tampere
and Tampere University Hospital, Tampere, Finland. (2) Research Programs Unit,
Immunobiology, and Haartman Institute, Department of Molecular Genetics, University of
Helsinki, Helsinki, Finland. (3) School of Medicine, University of Tampere and Department
of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere,
Finland. (4) Department of Medicine, Seinäjoki Central Hospital, Seinäjoki, Finland
Sequencing CCR9 and CCL25 genes in celiac disease
Even though celiac disease is known to be hereditary disease, its genetic background
is not completely understood. HLA genes encoding HLA-DQ2 and HLA-DQ8 proteins
are the only genetic factors predisposing to the disease that are known so far but they
explain only 30-40% of the heredity of celiac disease. HLA-DQ2 or HLA-DQ8 gene
variants are localized in the major histocompatibility complex (MHC) on chromosome
region 6p21.3. However, also other chromosome regions have either shown linkage or
association with celiac disease. Among those is MYO9B locus on chromosome region
19p13. Gene CCL25, coding for a likewise named chemokine, is also situated on this
region. CCL25 plays an essential role in intestinal homing of lymphocytes together with
its receptor CCR9. Interestingly, CCR9 is located on a chromosome region 3p21 and in
a recent genome wide association study this region was associated with celiac disease.
Moreover, CCR9 expression is found to be reduced on epithelial and lamina propria T
cells in untreated CD. All the above presented data makes the chemokine CCL25-CCR9
receptor pair interesting to be studied in celiac disease. The general aim of this study is
to establish the role of CCL25 and its receptor CCR9 in celiac disease and thus reveal
novel information about the genetic background of celiac disease.
DNA was extracted from whole blood samples of 96 HLA-DQ2 positive celiac patients,
96 HLA-DQ2 positive non-celiacs and 96 both HLA-DQ2 and HLA-DQ8 negative
non-celiacs. The exons were sequenced for both CCR9 and CCL25 and SNPs are being
identified. SNP frequencies among celiac disease patients will be compared to those
found in healthy subjects in our study cohort and in SISu (Sequencing Initiative Suomi)
database. If these comparisons reveal different allele frequencies between groups the
identified SNPs will be studied further in larger cohort. In addition, we will study
whether celiac patients carry more coding variants in general.
Author: Lehto Kalle (1,3) (kalle.lehto@tut.fi)
Group: Hyttinen Jari
Co-authors: Tamminen I. (1,3), Skottman H. (2,3), Uusitalo H. (4,5), Nymark S. (1,3),
Aula A. (1,3,6), Hyttinen J. (1,3)
All affiliations: 1. Department of Electronics and Communications Engineering, Tampere University of Technology, Tampere, Finland. 2. Institute of Biomedical Technology, Tampere, Finland. 3. BioMediTech, Tampere, Finland. 4. TAUH Eye Center, Tampere University Hospital, Tampere, Finland. 5. SILK, Department of Ophthalmology,
University of Tampere, Tampere, Finland. 6. Department of Medical Physics, Imaging
Centre, Tampere University Hospital, Tampere, Finland
3D -imaging of Rat Retina Using Contrast Enhanced X-ray Microtomography
Inherited retinal dystrophies such as age-related macula degeneration, retinis pigmentosa, and glaucoma cause irreversible vision loss for millions of people annually. In
dystrophy, the light-sensitive photoreceptor layer degrades as a result of retinal pigment
epithelium malfunction. Present methods for pre-clinical animal retina imaging are restricted, only specific part of the retina can be imaged. Non-destructive high-resolution
X-ray microtomography (µCT) has proven its worth in the context of pre-clinical studies. In this report contrast enhanced µCT is applied to ex-vivo rat eye for anatomical 3D
retina layer analysis.
Control and dystrophic rat eyes were post-mortem harvested and fixed. A huge variety
of imaging pre-sets was tested for optimizing imaging parameters. Both attenuationbased and in-line phase contrast methods were tested, including staining with contrast
agents I2E and IKI. The image packs were post-processed with edge-preserving nonlocal means–filter. Next the retina was segmented by semi-automatic watershed algorithm and corrected manually. Retina thicknesses were calculated by using the specific
surface distance-function.
Optimal imaging parameters were examined over 70 scans. Without staining the image
contrast was poor preventing the thickness analysis of the retina layers. Highest image
quality with 3.1 µm pixel size and decent SNR was achieved by I2E –stained eye still
immersed in the iodine solution. As a result, eight anatomical structures of the retina
were classified clearly. Comparison of µCT images and histological sections verified
that in pre-clinical studies of the eye anatomy the µCT is a promising imaging modality.
Heidi Kontro
Hanna Rauhala and Elisa Vuorinen
7. Experimental diabetes
Keywords: DAPIT, mitochondria, membrane potential,
ROS, ATP synthetase, Hif 1α, β-catenin, glycolysis,
metabolism, EMT
2. Cancer Research
Keywords: karyopherin alpha 7, nuclear transport,
protein-protein interactions, pancreatic cancer
Author: Heidi Kontro (heidi.kontro@uta.fi)
Author: Hanna Rauhala and Elisa Vuorinen
(hanna.e.rauhala@uta.fi)
Group: Heikki Kainulainen
Co-authors: Eric Dufour, Giuseppe Cannino and Heikki Kainulainen
All affiliations: Institute of Biomedical Technology and Pediatric Research Centre, University of
Tampere, Finland. Department of Biology and Physical Activity, University of Jyväskylä, Finland
DAPIT over-expression shifts cellular metabolism to glycolytic direction and
causes EMT-like phenomena in Hek293T cells
Diabetes Associated Protein in Insulin-sensitive Tissues (DAPIT) is a subunit of
mitochondrial ATP synthase and may also be a component of vacuolar ATPase. It is
highly expressed especially in cells with high aerobic metabolism and epithelial cells
related to active transport of nutrients and ions.
The gene expression and/or protein level of DAPIT is up-regulated in various cancers,
cardiomyopathy, metabolic deficiencies and in Parkinson’s disease but the function
of DAPIT has remained unknown.To study the function of DAPIT, we established a
stable transgenic cell-line over-expressing DAPIT constitutively in human embryonal
kidney cells (Hek293T). DAPIT over-expression significantly increased mitochondrial
membrane potential for 16% and reactive oxygen species (ROS) for 26%, as well as the
expression of heat shock protein Hsp60 and voltage dependent anion channel VDAC.
This was accompanied by the significant increase of the transcription factors hypoxia
inducible factor 1α (Hif1α) and β-catenin, as well as down-regulation of Sirtuin 3 (Sirt3),
a mitochondrial deacetylase involved in the modulation of cell metabolism. Accordingly,
cells over-expressing DAPIT consumed more glucose and produced increased amount
of lactate compared to the control cells. At the cell junction level, the excess of DAPIT
caused epithelial to mesenchymal transition (EMT)-like phenomena with shifting
E-cadherin to N-cadherin, and up-regulating other cell junction/adhesion proteins as
vimentin, integrin α2, connexin 43, ZO-1, αSMA and their modulator RhoA GTPase. In
addition, DAPIT over-expression slowed down cell growth and impaired cell migration.
We propose that DAPIT over-expression modulates mitochondrial and transcriptional
functions by shifting the cellular metabolism to more glycolytic direction and leading to
EMT-like cell behavior. DAPIT over-expression couples the changes in mitochondrial
metabolism to physiological and pathophysiological activities in cellular level.
Group: Anna Kallioniemi
Co-authors: Anne Kallioniemi
All affiliations: BioMediTech and Institute of Biomedical Technology, University of
Tampere
Identification of KPNA7 interacting proteins
The correct subcellular localization of proteins is essential for their proper function.
Nuclear import of classical nuclear localization signal containing proteins from the
cytosol into the nucleus takes place by binding to an adaptor protein, karyopherin alpha
(KPNA) and to a transport protein, karyopherin beta 1 (KPNB1). Changes either in the
transport machinery or in the cargo proteins themselves can lead to the mislocalization
of the cargos abolishing their function. Such changes are relatively commonly observed
in numerous diseases, including cancer. Karyopherin alpha 7 (KPNA7) is the newest
member in the KPNA family with no known human cargos. It is known to be expressed
in ovarian and cervical tissues and during embryogenesis, and in a subset of pancreatic
cancer cell lines and pancreatic tumors harboring KPNA7 amplification. We have earlier
shown that silencing KPNA7 in these pancreatic cells causes G1 cell cycle arrest and
reduces their anchorage-independent growth. We hypothesize that this results from the
mislocalization of critical KPNA7-transported proteins. Here, we aimed to identify
KPNA7 interacting proteins in human cells. To this end, we expressed Twin-Strep®tagged KPNA7 in 293T cells. KPNA7-Twin-Strep® containing 293T lysates were
incubated with pancreatic cancer cell line lysates and the formed protein complexes
were purified using Twin-Strep® affinity-based pull-down. Purified protein complexes
were run on SDS-PAGE and individual protein bands were identified by tandem mass
spectrometric analysis. KPNA7-Twin-Strep® interactions with the identified proteins
will be validated using co-immunoprecipitation. For validated KPNA7-interaction
partners, we will address their contribution to the observed KPNA7-silencing induced
phenotype by transiently silencing these interaction partners.
Jouni Väliaho
Thomas Liuksiala
Keywords: Human variation, Mutation database,
Immunodeficiency, Tyrosine kinase
Keywords: snoRNA, leukemia, acute promyelocytic leukemia,
genomic imprinting
Author: Jouni Väliaho (jouni.valiaho@uta.fi)
Group: Matti Nykter
Co-authors: Jouni Väliaho1,2, Imrul Faisal1,2, Csaba Ortutay1,2 and Mauno
Vihinen1,2,3,4
Author: Thomas Liuksiala (thomas.liuksiala@tut.fi)
Group: Prof. Matti Nykter
Co-authors: Kaisa Teittinen, Kirsi Granberg, Merja Heinäniemi, Matti Annala, Markku Mäki,
Matti Nykter and Olli Lohi
All affiliations:
1 Institute of Biomedical Technology, University of Tampere,
Tampere, Finland. 2 BioMediTech, Tampere, Finland. 3 Department of Experimental
Medical Science, Lund University, Lund, Sweden, 4 Research Unit, Tampere University
Hospital, FI-33520 Tampere, Finland
All affiliations: Institute of Biomedical Technology, University of Tampere, Tampere, Finland
(TL, KG, MA, MN). Department of Signal Processing, Tampere University of Technology,
Tampere, Finland (TL, KG, MA). Tampere Center for Child Health Research, University of
Tampere and Tampere University Hospital, Tampere, Finland (KT, MM, OL). A.I. Virtanen
Institute for Molecular Sciences, University of Eastern Finland, Kuopio (MH). BioMediTech,
Tampere, Finland (MN)
Characteristic features of Bruton Tyrosine Kinase Variations
Overexpression of SNORD114-3 marks acute promyelocytic leukemia
The characteristic molecular features of variations, that could be used to distinguish
deleterious mutations from neutral ones is crucial when we try to interpret new variants
found from genome sequences.
Small nucleolar RNAs (snoRNAs) are short non-coding RNA molecules, most of which guide
chemical modifications of other non-coding RNAs, primarily ribosomal RNAs. Some snoRNAs,
however, lack a known target RNA, and are thus called ‘orphan’. Such snoRNAs have been
suggested to have gene regulatory roles and some have been even implicated in tumorigenesis.
Using publicly available microarray gene expression data, our objective was to look for any
peculiarities in the expression of snoRNAs in pediatric leukemias. We compiled a hematological
data set of neoplastic and non-neoplastic samples, all downloaded from the Gene Expression
Omnibus microarray repository. We focused on 883 samples of pediatric leukemias, subdivided
into three categories: early B-ALL, T-ALL and AML. In addition, we considered 35 nonneoplastic samples consisting of hematopoietic stem cells along with naive B- and naive
T-lymphocytes.
Of all the genes on the microarray, 15 were identified as snoRNAs. Looking at the expression
patterns over these snoRNAs, we found two snoRNAs which were expressed consistently
higher than all other snoRNAs in both healthy and leukemic smaples. We also noted that two
other snoRNAs were expressed strongly in naïve B cells, suggesting a B-cell differentiationdependent regulation of expression.
The most striking finding was the high expression of an orphan snoRNA, SNORD114-3, in acute
promyelocytic leukemia (APL), a subclass of AML. This snoRNA has previously been shown to
induce cell growth and profileration of APL cells in vitro. Interestingly, it lies in a genomically
imprinted region, which appears to be deregulated in APL.
These results have been published, see: Liuksiala et al. Overexpression of SNORD114-3 marks
acute promyelocytic leukemia. Leukemia advance online publication, 20 September 2013; doi:
10.1038/leu.2013.250
1495 non-synonymous single nucleotide variations (nsSNV) in Bruton tyrosine kinase
domain (BTK-KD) were analyzed at sequence and structural levels by using a wide
variety of bioinformatic methods. 10% of nsSNVs are known to cause X-linked
agammaglobulinemia (XLA) disorder (dSNV). New pathogenicity prediction method
targeted for BTK-KD variations was developed to maximize the prediction performance
and to address the fundamental question how many of the BTK-KD variants are
pathogenic.
The results suggest 67% of nsSNVs to be harmful, which is more than observed earlier.
Many widely used pathogenicity prediction methods seem to be even more biased
toward pathogenic variations. New structural features and scoring matrices were found
to be useful to characterize disease-causing variations compared to benign cases.
Venkatesh Mallikarjun
Maria Laaksonen
7. Mitochondrial gerontology
Keywords: ROS, mitochondria, complex I
Keywords: lncRNA, RNA-seq, glioblastoma,
non-coding RNA
Author: Venkatesh Mallikarjun
(venkatesh.mallikarjun@uta.fi)
Author: Maria Laaksonen (maria.o.laaksonen@uta.fi)
Group: Alberto Sanz
Group: Matti Nykter
Co-authors: Filippo Scialo, Ashwin Sriram, Nina Gubina
Co-authors: Maria Laaksonen 1, Birgitta Lehtinen 1, Janne Seppälä 1, Tommi Rantapero
1, Kirsi Granberg 1,2, Matti Nykter 1
All affiliations: Institute of Biomedical Technology and Tampere University Hospital,
FI-33014, University of Tampere, Finland.
Mitochondrial complex I self-inflicted damage is a major determinant of longevity
in fruit flies
Aging represents an inexorable decline in the organism’s ability to respond to stress and
is a major risk-factor for many of the most debilitating diseases known to humanity. The
role of mitochondrially derived reactive oxygen species (ROS) in determining longevity
has a long history of research. However, although ROS produced in the mitochondria
can give rise to oxidative damage and disruption of intracellular redox homeostasis,
the relevance of this in aging has been thrown into question by the existence of longlived organisms that have continuously high levels of oxidative damage throughout
their lives, and the fact that antioxidant supplementation has generally negligible or
even deleterious effects on lifespan. Ectopic expression of the yeast alternative NADH
dehydrogenase (NDI1) in Drosophila (where it can bypass mitochondrial complex I)
can extend lifespan. NDI1 can specifically reduce complex I derived-ROS, suggesting
that only these ROS may be detrimental to longevity. Here we show that complex I
activity is specifically decreased in several distinct models of fly aging. Furthermore,
we go on to show that this decline is due to intrinsic damage caused by the complex
itself as exogenous supplementary antioxidants could not protect it. Further experiments
showed that reduction in complex I ROS production, via RNAi-mediated knockdown of
the complex in the presence of NDI1 (to maintain correct electron transport and NAD+/
NADH ratio), conferred an additional increase in lifespan beyond that conferred by
NDI1 alone. Importantly, this increase in lifespan could not be replicated with similar
knockdowns in other oxidative phosphorylation complexes.
All affiliations: 1)Institute of Biomedical Technology, University of Tampere. 2)
Department of Signal Processing, Tampere University of Technology
RNA-sequencing Data Reveals Novel lncRNAs in Glioblastoma
Identification of long non-coding RNAs (lncRNA) has had an impact on cancer
research since they have been recognized as critical components of cancer biology and
linked to oncogenic processes. Certain lncRNAs are known to act as oncogenes, and
lncRNAs could also be used as biomarkers for disease as their expression is disease
specific. In this study, we uncover 53 novel lncRNAs from RNA-seq data of 169
glioblastoma patient primary tumor samples sequenced by The Cancer Genome Atlas
(TCGA) consortium. By association analysis we found that the expression of some of
these lncRNAs correlated with IDH1 mutation status, cancer-related gene expression
signatures and/or patients’ survival rate. Based on associations and gene structure
annotations we selected 23 lncRNAs for further experimentation. To these lncRNAs,
we made gene expression assays in glioblastoma cell lines LN229, SNB19 and T98G.
We found that 15 of them were expressed in at least one of the cell lines. In ongoing
work we are doing quantitative gene expression and RNA interference assays to these
lncRNAs to unveil their functions in glioblastoma cells.
Alejandra Rodríguez Martínez
Minna Hietikko
2. Cancer Research
Keywords: Breast cancer, Bone Morphogenetic
Proteins, SMAD, ChIP-seq, DNase-seq, RNA-seq,
Next Generation Sequencing
Keywords:
Author: Alejandra Rodríguez Martínez (alejandra.rodriguez.martinez@uta.fi)
Group: Markku Mäki
Co-authors: Minna Hietikko1, Pekka Collin2, Timo Reunala3, Markku Mäki1, Katri
Kaukinen2,4, Katri Lindfors1
Co-authors: Minna Ampuja, Tommi Rantapero, Matti Nykter, Anne Kallioniemi
BioMediTech, Tampere, Finland. Fimlab Laboratories, Tampere, Finland. Department
of Signal Processing, Tampere University of Technology, Tampere, Finland.
Author: Minna Hietikko (minna.hietikko@uta.fi)
All affiliations: 1 Tampere Center for Child Health Research, University of Tampere and
Tampere University Hospital, Tampere, Finland. 2 School of Medicine, University of
Tampere and Department of Gastroenterology and Alimentary Tract Surgery, Tampere
University Hospital, Tampere, Finland. 3 Department of Dermatology, Tampere University
Hospital, Tampere, Finland. 4 Department of Medicine, Seinäjoki Central Hospital,
Seinäjoki, Finland.
Deciphering the Mechanisms and Targets of BMP4-induced Transcriptional
Regulation in Breast Cancer Cells by Exploiting State-of-the-art Next-Generation
Sequencing Technologies
Does coeliac patient IgA have similar cellular effects regardless of the disease stage
or type of manifestation?
Determining how gene transcription is regulated is crucial for fully understanding many
biological processes and disease states. Genome-wide landscapes of transcriptional
regulation may be nowadays investigated using next generation sequencing (NGS)
technology. Bone morphogenetic proteins (BMPs) are extracellular ligands that signal
mainly, through the SMAD pathway, resulting in transcriptional activation of target
genes. Furthermore, BMP signaling is highly contextual, hence requiring to be studied
in the specific context of interest. BMPs are well known to play an important role
in cancer development and progression. In breast cancer, BMP4 has emerged as an
inhibitor of proliferation and promoter of cell migration and invasion. Indeed, patterns
of SMAD binding to DNA and other proteins are being characterized with the aid
of NGS methods, but this has not yet been done for BMP4 signaling in any tumor
context. In this project, 3 NGS-based methods (ChIP-seq, DNase-seq and RNA-seq)
will be applied and T-47D and MDA-MB-231 breast cancer cells treated with BMP4
will be compared with vehicle treated controls. This will allow the identification of
SMAD binding sites (ChIP-seq), transcriptionally active sites upon activation of BMP4
signaling (DNase-seq) and differentially expressed genes (RNA-seq). Through the
analysis of these data and comparison of results from different methods we aim to learn
more about mechanisms of transcriptional regulation in general, and understand the
molecular mechanisms of BMP4-elicited transcriptional regulation in breast cancer in
particular. Additionally, identified BMP4 target genes will be functionally studied in 2D
and 3D in vitro models of breast cancer cells.
Background: Coeliac disease is an autoimmune-mediated condition triggered by the
ingestion of gluten containing cereal proteins in genetically susceptible individuals.
Active disease is further characterised by the presence of increased levels of serum
immunoglobulin class A (IgA) antibodies, most notably against transglutaminase 2
(TG2), the major autoantigen of coeliac disease, and gluten-derived gliadin peptides.
Serum IgA antibodies derived from untreated coeliac disease patients with flat small
bowel mucosal morphology have been previously shown to exert a number of biological
functions. However, it is not known whether this applies to IgA derived from patients in
other stages of the disorder or with other types of disease manifestations.
Aim: The aim of the study is to investigate and compare the effects of IgA antibodies
derived from gluten sensitive patients at different stages of coeliac disease or with
different disease manifestations.
Study design: Total IgA fractions are affinity-purified from serum samples from
seven patient groups, including patients with early stage (positive serum antibodies,
normal mucosal structure) and overt (positive serum antibodies, damaged mucosal
structure) coeliac disease, coeliac patients on gluten-free diet, seronegative coeliac
patients, patients with the skin manifestation dermatitis herpetiformis, gluten sensitive
individuals positive for anti-gliadin antibodies, and non-celiac subjects as controls. The
effects of the IgA on membrane ruffling of intestinal epithelial cell line Caco-2, and on
cell migration and tube formation of human umbilical vein endothelial cell line HUVEC
will be tested.
Leena Latonen
Rolle Rahikainen
2. Cancer Research
Keywords: prostate cancer; microRNA; transgenic mice
1. Biotechnology
Keywords: Mechanosensing, Cell-matrix adhesions, talin
Author: Leena Latonen (leena.latonen@uta.fi)
Author: Rolle Rahikainen (rolle.rahikainen@uta.fi)
Co-authors: Melissa Bothe, Anna-Maija Kakkonen, Fuping Zhang, Matti Poutanen and Tapio
Visakorpi
All affiliations: Prostate Cancer Research Center and Institute of Biomedical Technology,
University of Tampere, and Turku Center for Disease Modeling, University of Turku
Expression and role of miR-32 in prostate cancer development in vivo
The androgen receptor (AR) signaling pathway is central to the emergence of castrationresistant prostate cancer (CRPC). Our group has previously searched for androgen-regulated
microRNAs (miRNAs) that may contribute to the development of CRPC, and identified miR-32
to be a putatively important gene in the progression of prostate cancer. Expression of miR-32 is
androgen-regulated and dramatically increased in CRPC. In cultured prostate cancer cells, miR32 increases proliferation and decreases apoptosis. In a neoadjuvant clinical trial material, the
expression of miR-32 is suppressed by androgen deprivation, the current treatment available for
advanced prostate cancer. Thus, miR-32 is a potential marker for aggressive prostate cancer and
is a putative drug target in PC.
To gain insight in miR-32 expression in vivo, we have analyzed a panel of 20 mouse tissues of
male mice for miR-32 expression by qRT-PCR. Expression of miR-32 in most androgen-regulated
tissues tested, including testis, prostate, adrenal glands, and skeletal muscle, is low, with the
exception of epididymis. The highest expression levels were found in kidney, heart muscle, spleen
and lung. To study how increased miR-32 expression contributes to prostate cancer formation
and/or progression in vivo, and to search for in vivo targets of miR-32 in the prostate tissue, we
have established transgenic mice expressing miR-32 specifically in the prostate. FVB/N mouse
strain was used to create transgenic mice with probasin promoter (ARR2PB) driving expression
of miR-32 androgen-responsively in prostate epithelium post-puberty. The mice develop and
breed normally, and express the transgene specifically in the ventral and dorsolateral lobes of the
prostate, with no transgene expression detected in anterior prostate (coagulating gland) or seminal
vesicles up to six months of age. As the frequency of spontaneous prostate cancer is low in mice,
the phenotype is provoked by crossbreeding the ARR2PB-miR32 lines with mice heterozygous
for PTEN tumor suppressor gene and prone to prostatic lesions. Histological analysis of the lesions
in ARR2PB-miR-32 mice in wild-type or heterozygous background for Pten up to 10 months of
age is ongoing. To facilitate RNA expression analysis from tissues processed for histological
analysis, we have set up a tissue processing protocol utilizing PAXgene™ molecular fixative with
sequential paraffin block sections processed to RNA extraction and histological analysis.
With these tools, we aim to test whether miR-32 is an oncomiR for prostate cancer in vivo. In the
future, we will also assess the potency of miR-32 as a therapeutic target and search for the most
important targets of miR-32 in prostate cancer.
Co-authors: Jenita Pärssinen (1), Magdalena Von Essen (1), Bernhard Wehrle-Haller
(2), Vesa Hytönen (1)
All affiliations: (1) Institute of Biomedical Technology, University of Tampere, Finland
(2) University of Geneva, Switzerland
Talin-vinculin interaction in cellular mechanosensing
Mechanosensitive proteins in cell-matrix adhesions allow cells to probe the mechanical
properties of the surrounding tissue and to react to forces exerted on them. This so-called
mechanosensing is known to be essential for many cellular processes, e.g. cell migration,
stem cell differentiation and tissue remodeling, but the molecular mechanisms behind it
are currently only poorly understood. Focal adhesion protein talin directly participates
in the transmission of mechanical forces between the cellular cytoskeleton and the
extracellular matrix. If sufficient force is applied to the rod domain of talin, it partially
unfolds, which allows an interaction between talin and another focal adhesion protein,
vinculin. It has been hypothesized that this mechanically activated interaction between
talin and vinculin may act as the primary mechanosensory switch in many animal cells.
To study the importance of the talin rod domain stability for the talin-vinculin interaction,
we have designed four truncated talin mutants, each containing a defined number of
vinculin binding sites. The vinculin binding helices of these truncated talin proteins
have been further modified by introducing destabilizing point mutations designed by
using steered molecular dynamics simulations. To analyze the effects these mutations
have on the talin vinculin interaction, the mutated talin proteins will be expressed in
talin-1 deficient fibroblasts cultured on hydrogel cell culture substrates with varying
mechanical properties. Immunofluorescence and live cell imaging will be used to
analyze cell morphology and the sizes of focal adhesions in the cells that are cultured
on these substrates and express different talin mutants. The research is in progress.
Virpi H. Laitinen
Zsuzsanna Ortutay
2. Cancer Research
Keywords: hereditary prostate cancer, 2q37, 17q12-q22,
susceptibility alleles, targeted DNA sequencing,
cis-eQTL analysis
Keywords: furin, T cell activation, IL-2, transcription factor
Author: Virpi H. Laitinen (1) (virpi.laitinen@uta.fi)
Group: Marko Pesu
Co-authors: Anna Oksanen, Marko Pesu
Co-authors: Tommi Rantapero (1), Daniel Fischer (2), Elisa M. Vuorinen (1), Teuvo L.J.
Tammela (3), Tiina Wahlfors (1), Johanna Schleutker (1,4)
All affiliations: Institute of Biomedical Technology,University of Tampere.
BioMediTech
All affiliations: (1) Institute of Biomedical Technology/BioMediTech, University of
Tampere and Fimlab Laboratories, Tampere, Finland. (2) School of Health Sciences,
University of Tampere, Tampere, Finland. (3) Department of Urology, Tampere University
Hospital and Medical School, University of Tampere, Tampere, Finland.
(4) Medical
Biochemistry and Genetics, Institute of Biomedicine, University of Turku, Turku, Finland.
Fine mapping of the Finnish hereditary prostate cancer linked loci at 2q37 and
17q12-q22
Linkage and genome-wide association studies have discovered a connection between
hereditary prostate cancer (HPC) and two genomic regions, 2q37 and 17q12-q22. No
candidate gene has been identified at 2q37. The G84E variant in the HOXB13 gene
explains some, but not all of the linkage to 17q12-q22 observed in Finnish HPC families.
To identify novel genes and sequence variants predisposing to HPC, we screened these
two loci by targeted DNA sequencing. The resulting 107,479 unique sequence variants
were filtered using several prioritization criteria. Finally, 58 putative prostate cancer
(PrCa) associated variants co-segregating with the disease in Finnish HPC families
were chosen for validation in 2216 samples. Four novel susceptibility alleles were
identified in the ZNF652, EFCAB13 (17q21) and HDAC4 (2q37) genes.
In addition, we studied the effect of PrCa associated SNPs on the regulation of gene
expression within the two regions. This targeted expression quantitative trait loci
(eQTL) analysis combined the genotype data from targeted DNA sequencing with
transcriptome data obtained from RNA sequencing. The cis-eQTL analysis revealed 24
SNPs possibly regulating the expression of three differentially expressed (DE) genes
at 2q37, and 8 SNPs possibly regulating the expression of five DE genes at 17q12-q22.
The PrCa associated variants identified in this study can be exploited in cancer risk
assessment. In addition, our findings support the role of ZNF652 as a PrCa candidate
gene. The regulatory relationships detected by eQTL mapping provide new insights
into the complex genetic processes contributing to PrCa predisposition.
Author: Zsuzsanna Ortutay (zsuzsanna.ortutay@uta.fi)
Proprotein convertase furin has a dual role in T cell mediated immune responses.
Furin is a member of the proprotein convertase enzyme family. It has a crucial role in
processing physiological proproteins, like growth factors, hormones, plasma proteins
and receptors, and additionally, several pathological proteins, for example bacterial
toxins of B. anthracii and viral envelope glycoproteins of HIV.
T cell activation upregulates the expression of furin, which cleaves and thus activates
several proteins with important roles in T cell functions, including TNFa converting
enzyme (TACE), Notch1, and TGFb family cytokines. T cell specific furin knockout
mice develop age-related autoimmunity; effector T cells from those animals express
high amounts of T cell activation hall mark cytokines, while furin deficient regulatory
T cells have reduced suppressor activity. Notably, the production of interleukin-2 was
decreased after in vitro activation of T cells from the furin deficient model animals.
This study has focused on the effects of furin on TCR mediated signaling. Naïve T cells
from furin deficient animals showed elevated IL-2 production without any stimulation;
however, they respond to activation with a decreased rise in IL-2 expression compared
to wild type counterpart. While the phosphorylation processes were found to be intact,
the TCR signal following gene expression pattern and transcription factor preference
varies in the presence or absence of furin in genome-wide analyses of different helper
T cell subsets.
These results affirm, that furin has an important indispensable role in T cell physiology.
Next to its role in maintenance of immune homeostasis, furin promotes the T cell
mediated immune responses.
Jyrki Sivula
Jack George
Keywords: Adipose stem cells, Immunogenicity,
Immunosuppression, Cytokines, Serum-free,
Cellular therapy
Keywords: Spargel, PGC-1.
Author: Jyrki Sivula (jyrki.sivula@uta.fi)
Author: Jack George (jack.george@uta.fi)
Group: Howy Jacobs
Co-authors: Mimmi Patrikoski 1,2,3, Jyrki Sivula 1,2,3, Heini Huhtala 4, Mika Helminen 4,
Fanny Salo 1,2,3, Bettina Mannerström* 1,2,3, Susanna Miettinen* 1,2,3
All affiliations: 1 Adult stem cell group, Institute of Biomedical Technology, University
of Tampere, Finland. 2 BioMediTech, Tampere, Finland. 3 Science Center, Tampere
University Hospital, Tampere, Finland. 4 Tampere School of Health Sciences, University
of Tampere, Tampere, Finland. * equal contribution
Different culture conditions modulate immunological properties of adipose stem
cells
The potential of human adipose stem cells (ASCs) for regenerative medicine has received
recognition owing to the ease of isolation and the capacity of the cells to differentiate
towards several cell types of mesodermal origin. Additionally, low immunogenicity
and immunosuppressive properties make them a relevant cell source considering
immunomodulation therapies and allogeneic stem cell treatments.
In the current study, immunogenicity and immunosuppression of ASCs were determined
through mixed lymphocyte reactions. The immunogenic response was analyzed after cell
isolation and expansion in fetal bovine serum (FBS), human serum (HS) supplemented
medium, and in xeno-free and serum-free (XF/SF) condition. Additionally, the chemokine
and cytokine secretion (CXCL8, CXCL9, CXCL10, CCL2, CCL5, IL-2, IL-4, IL-6, IL-10,
IL-17A, TNF-α, and IFN-γ) as well as immunophenotype of cells was analyzed.
ASCs were weakly immunogenic when expanded in any of the three conditions. The
strongest suppression was observed with cells expanded in FBS conditions, whereas higher
ASC numbers were required to display suppression in HS or XF/SF conditions. Also,
statistically significant differences in chemokine and cytokine secretion were observed
between direct versus indirect co-cultures, and between different culture conditions. The
characteristic immunophenotype of ASCs was maintained in all conditions. However, in XF/
SF conditions significantly lower expression of CD54 (ICAM-1) and higher expression of
CD45 (LCA) was observed at low passage number.
Although culture conditions have an effect on the immunogenicity, immunosuppression and
cytokine secretion of ASCs, our findings demonstrated that ASCs have low immunogenicity
and immunosuppressive potential whether cultured in FBS, HS or in XF/SF conditions.
The Biology of the PGC-1 Homologue Spargel in Drosophila Melanogaster
BACKGROUND:
The mammalian PGC-1 family members (PGC-1α, PGC-1β and PRC) are a key set
of transcriptional coactivators. They signal environmental and nutrient status to finetune metabolic transcriptional outputs. Spargel is the sole D.Melanogaster PGC-1
homologue. It has been shown to share both structural and functional similarities to
the mammalian PGC-1 members. However, Spargel remains understudied. As such,
further characterisation of Spargel will lead to novel insights into PGC-1 activity and
evolution. Here as an example, a previously unreported link between Spargel activity
and early D.Melanogaster development is presented.
PRINCIPAL FINDINGS
Spargel mRNA expression levels are differentially regulated throughout development.
Particularly, Spargel mRNA is highly expressed in adult Females and early stage embryos. This led us to consider the possibility that Spargel plays an important role during
oogenesis and/or embryogenesis. Interestingly Spargel protein levels don’t correlate
with the ovary mRNA levels suggesting a form of regulated translational repression.
Further evidence of Spargel’s role in development is shown by the infertility of female
Spargel ubiquitous knockdown mutants.
CONCLUSIONS/SIGNIFICANCE:
Spargel is likely an important determinant of D.Melanogaster fecundity. Furthermore
it is potentially a direct link of nutrient availability signals to oogenesis development.
Nina Gubina
Giuseppe Cannino
Keywords: Aging, ROS, Drosophila melanogaster,
RNA sequencing
Keywords: ATP syntase, mtDNA, mtROS.
Author: Nina Gubina (nina.gubina@uta.fi)
Group: Howy Jacobs
Group: Dr. Alberto Sanz
Co-authors: Mike Gerards. Eric Dufour, Atsushi Fukuoh
Co-authors: Nina Gubina, Tommi Rantapero, Csaba Ortutay, Matti Nykter, Alberto
Sanz
All affiliations: Institute of Biosciences and Medical Technology, University of Tampere
All affiliations: IBT
Whole transcriptome analysis of long-lived versus short-lived wild type strains of
Drosophila melanogaster.
Aging is a process of gradual degeneration of cell functions. Nevertheless, it is still
unknown which factors launch aging and determine the difference in lifespan. One of the
possible factors is the generation of by-products of metabolism as for example reactive
oxygen species (ROS). Here we present whole transcriptome data analysis of two wildtype strains – long-lived Oregon R and short-lived Dahomey. Despite to the fact that
Oregon produce less ROS than Dahomey, there was no significant difference between
strains in the expression of well-known antioxidants, stress response genes and ROSproducing factors such as mitochondrial electron transport chain proteins. However,
Oregon R exhibit better tuned transcription and proteostasis, upregulated genes of
immune response, detoxification, nutrient intake, lipid and carbohydrate metabolism,
anti-proliferation and anti-apoptotic factors, whereas Dahomey have upregulated genes
related to enhanced transcription, insulin signaling and cytoskeleton organization, and
G-protein-mediated sensory perception. Taken together, these data link the difference in
longevity between the investigated strains to metabolic misregulation in adult Dahomey
flies which has already been proposed as a marker of aging. The impact of ROS on
aging in the light of this data has still to be clarified.
Author: Giuseppe Cannino (giuseppe.cannino@uta.fi)
A new role of ATP syntase in mitochondrial DNA regulation.
Mitochondria contain their own genetic systems. Mitochondrial DNA (mtDNA) is a
circular double-stranded molecule used for replication, transcription and translation.
The number of copies mtDNA changes as a result of alterations of the cellular content
of mitochondria, as well as variation in the number of mitochondrial genomes per
mitochondrion. In some pathological states, collectively described as ‘mtDNA depletion
syndrome’, mtDNA copy number is drastically decreased, often in a tissue-specific
manner. The genetic defect underlying mtDNA depletion syndrome remains unknown
in many cases, and the associated pathological mechanisms are not well understood.
Previously work in our lab, using an RNAi screening strategy combined with
fluorescence microscopy, has identified otherwise non-essential genes whose products
are involved in the faithful maintenance of mtDNA copy number in Drosophila S2
cells. Interestingly almost all of the nuclear-coded subunits of OXPHOS complex V
(ATP synthase) were revealed as positives, by this screen. I dissected the relationship
between complex V and mtDNA copy number, knocking-down each of three ATP
synthase subunits: subunits delta (CG2968), epsilon (CG9032) and G (CG6105). We
demonstrate that the RNA inhibition of each of these subunits decreases mtDNA copy
number, accompanied by decreased growth, cellular respiration, mitochondrial mass
and mitochondrial membrane potential, plus increased production of mitochondrial
superoxide (ROS). In addition we found that ROS levels can modulate mitochondrial
copy number. In conclusion our results suggest that the depletion of ComplexV can alter
mitochondrial biogenesis via ROS pathway by increasing the mitophagy.
Birgitta Lehtinen
Kerstin Lenk
2. Cancer Research
Keywords: immunohistochemistry, gene fusion,
FGFR, FGFR3, TACC3, fusion protein, IHC,
clinical association, glioblastoma, glioma, low grade
glioma, brain cancer
7. Computational Neuroscience
Keywords: in vitro, multielectrode array, human
pluripotent stem cells, simulation, neural development
Author: Birgitta Lehtinen (birgitta.lehtinen@uta.fi)
Group: Prof. Jari Hyttinen
Group: Matti Nykter
Co-authors: Barbara Priwitzer, Laura Ylä-Outinen, Lukas Tietz, Susanna Narkilahti,
Jari A K Hyttinen
Co-authors: Birgitta Lehtinen1,2, Hannu Haapasalo3, Matti Annala1, Matti Nykter1,
Kirsi Granberg1
Author: Kerstin Lenk (kerstin.lenk@tut.fi)
All affiliations: 1 Institute of Biomedical Technology, University of Tampere. 2
Department of Signal Processing, Tampere University of Technology. 3 Department of
Pathology, Fimlab Laboratories and University of Tampere, Tampere, Finland
All affiliations: Department of Electronics and Communications Engineering,
Tampere University of Technology and BioMediTech, Tampere, Finland. Faculty of
Engineering and Computer Science, Brandenburg University of Technology CottbusSenftenberg, Senftenberg, Germany. NeuroGroup, Institute of Biomedical Technology/
BioMediTech, University of Tampere, Tampere, Finland
Validation and clinical association analysis of the tumorigenic FGFR3-TACC3
gene fusion
Simulation of a developing neuronal cell network derived from human pluripotent
stem cells
Aberrant activation of FGFR3 (fibroblast growth factor receptor 3) has been reported to
be tumorigenic in various cancer types. Escaping miRNA-mediated regulation, FGFR3
is activated when fusing with another gene, most commonly with transforming acidic
coiled coil 3 (TACC3). Among brain cancers, gene fusions involving FGFR3 have been
detected in glioblastoma and certain low grade gliomas. The endogenous FGFR3 protein
levels are extremely low in brain partly due to miRNA-mediated regulation and gene
fusions lead to prominent protein expression. However, FGFR3 fusion recurrence and
fusion partners have been analyzed only in limited patient cohort and there are no reports
about their clinical associations. In this study, we have performed immunohistochemistry
for FGFR3 on tissue microarrays with over a 1’000 clinical glioma samples (including
epedymomas, plexus papillomas, pilocytic astrocytomas, other lower-grade gliomas
and glioblastomas). FGFR3 positive staining has been detected in subpopulations of
all included tumor types. The presence of fusion transcript will be measured with PCR
in positive cases to validate immunohistochemistry-based approach for FGFR3 fusion
detection. Furthermore, we have extensive clinical records for all the cases, and they will
be used for clinical association analysis of FGFR3 fusions. Altogether, this study will
evaluate the suitability of immunohistochemical methods for FGFR3 fusion detection
as well as investigate their recurrence and clinical significance in glioma.
In the past, we developed a simple model called INEX which simulates neuronal activity
as observed in a neuronal network cultivated on a multielectrode array (MEA). In vitro
MEA recordings from neuronal cell network derived from human pluripotent stem cells
(hPSCNNs) were done between 7 and 26 days in vitro following the development of
the neuronal network. The aim of the presented work is the simulation of maturating
hPSCNNs and, thus, to evaluate their functioning and network development in different
developmental stages in silico. The model is based on an inhomogeneous Poisson
process to simulate neurons which are active without external input or stimulus as
observed in MEA experiments. Each neuron has either an inhibitory (negative synaptic
strength) or an excitatory (positive synaptic strength) effect to its neighbors. In the
present work, the model is used to simulate a maturating hPSCNN. These hPSCNNs
exhibit highly variable network structure and time-varying dynamics. To explore and
validate the developing burst and spike activities of such network simulations we
calculated features adapted from spikes and bursts. The features show that the presented
model has potential to simulate maturating neuronal activity of hPSCNNs. We hope
that more detailed models of the developing connectivity in the model can be used to
understand the maturating process of hPSCNNs and other cell types.
Sampo Kukkurainen
Tomas Cervinka
7. Image processing
Keywords: Bone strength; cortical bone; distance
regularized level set evolution; pQCT; segmentation
Author: Tomas Cervinka (tomas.cervinka@tut.fi)
Co-authors: Jari Hyttinen, Harri Sievänen
All affiliations: BioMediTech, Tampere, Finland; Department of Electronics and
Communications Engineering, Tampere University of Technology, Tampere, Finland;
Bone Research Group, UKK Institute, Tampere, Finland; Pirkanmaa Hospital District,
Science Center, Tampere, Finland
Automatic segmentation algorithm for accurate cortical bone detection in
peripheral quantitative computed tomography data.
An accurate assessment of bone strength is an important goal in clinical bone research.
For appropriate information on bone strength, precise segmentation of actual crosssectional bone geometry is needed. This is, however, not always guaranteed by currently
used threshold-based analyses, mainly due to the partial volume effect, relatively low
signal to noise ratio etc. Therefore, in this paper, we introduce an automatic, simple and
fast approach for reliable segmentation of cortical bone cross-sectional area without
density based thresholds. Using 25 in vivo pQCT images of distal tibia we compare
these new segmentation results with those obtained from commonly applied simple
density thresholds and recent state of the art level set method. Manual segmentation
done by three independent evaluators was considered a gold standard in this study. The
new approach showed about 50% less variation in error compared to threshold based
analyses irrespective of used preprocessing. Moreover, in conjunction with a recently
introduced statistical preprocessing of the pQCT image the segmentation results were
similar (difference 2%) to manual segmentation. In conclusion, the proposed cortical
detection algorithm performed well and can enhance the cortical bone analysis of
tomographic images in clinical studies. In broader perspective, more reliable cortical
analysis may result in improved estimation of whole bone strength. Obviously, the
main limitation of present study pertains to manual segmentation of cortical bone
the accuracy of which strongly depends on previous experience of raters. Accurately
digitized true cortical cross-sections at high spatial resolution would have provided a
more appropriate reference method.
Ville Kytölä
Jani Sarin
2. Cancer Research
Keywords: Transcription factor, regulation,
network, prostate cancer
2. Cancer Research
Keywords:
Author: Jani Sarin (jani.sarin@uta.fi)
Author: Ville Kytölä (ville.kytola@tut.fi)
Group: Jorma Isola
Group: Matti Nykter
Co-authors: Satu Luhtala, Jorma Isola
Co-authors: Antti Ylipää, Juha Kesseli, Matti Nykter
All affiliations: University of Tampere. Tampere University of Technology
Computational construction of prostate cell specific regulatory networks
Reliable and thorough characterization of gene regulation is essential in understanding
origin and progression of a disease. As disease related dysregulation includes a large
number of regulatory molecules such as transcription factors and microRNAs, complete
experimental mapping of regulatory networks is not feasible. While computational
analysis can be used to predict regulatory interactions, common approaches such as
genome scanning with transcription factor (TF) binding motifs yield large false positive
rates.
Here we combine traditional TF binding predictions based on position weight matrix
scanning with DNAse-sequencing data from prostate cancer cell line (LNCaP). By
focusing the analysis only on the areas of open chromatin allows us to reduce the
number of false positives and to generate a set of biologically relevant TF target gene
predictions in specific cellular context. These predictions are then further refined by
incorporation of gene expression data and imposing a criteria of expression correlation
between regulator and the target gene in a collection of clinical prostate cancer data.
This approach allows us to construct disease specific networks and thus to propose
hypothesis on oncogenic regulatory mechanisms. Using mathematical modeling with
targeted validation data we can characterize these predicted mechanisms in detail.
Quantitation of HER2-HER3 receptor tyrosine kinase dimers using proximity
ligation assay
HER2 and HER3 (human epidermal growth factor receptors 2 and 3) are membranebound receptor tyrosine kinases. Different members of the HER receptor family can
form dimers, which lead to the activation of growth-promoting signalling. HER2 is a
widely studied target of breast cancer research, and the gene coding for HER2 protein
is estimated to be over-expressed in about 15 % of breast cancers. The role of HER3
in breast cancer is less well-characterized. However, point mutations in the ERBB3
(HER3) gene exist in breast tumors and cell lines, although their role in cancer biology
is not known.
It is believed that the point mutations in ERBB3 gene affect the dimerization of HER2
and HER3 proteins. To test this hypothesis, proximity ligation assay (PLA) was used
to determine the amount of HER2-HER3 dimers in different breast cancer cell lines in
order to correlate the amount of dimers to the previously quantified ERBB3 expression
levels in the corresponding cell lines.
Our preliminary results show that the PLA assay can be applied on cultured cells as
well as paraffin-embedded clinical breast tumor samples. Dimers are visualized as
distinct fluorescent dots (diameter 0.5-1 um), which are localized in the cell membrane.
The semi-confocal Zeiss Apotome microscope (using oil-40X and oil-63X objective
lenses) was found particularly useful for PLA microscopy. ImageJ software was used
to quantitate the dots in image focus stacks. The results are expressed as the average
number of dimers per cancer cell.
Tamminen Ilmari
Päivi Lillsunde
7. Biomedical imaging
Keywords: X-ray microtomography,
regenerative medicine, stem cells, three-dimensional
imaging
Keywords: Mitochondrial DNA, replication,
Drosophila melanogaster
Author: Tamminen Ilmari (ilmari.tamminen@uta.fi)
Author: Päivi Lillsunde (paivi.lillsunde@uta.fi)
Group: Hyttinen Jari
Group: Laurie S. Kaguni
Co-authors: Sivula J. (2), Miettinen S. (2), Aula A. (1,3), Hyttinen J, (1).
Co-authors: Howard T. Jacobs (1), Laurie S. Kaguni (1,2)
All affiliations: 1. Department of Electronics and Communications Engineering, Tampere
University of Technology, Tampere, Finland. 2. Institute of Biomedical Technology,
University of Tampere, Tampere Finland. 3. Department of Medical Physics, Imaging
Centre, Tampere University Hospital, Tampere, Finland.
Specific Antibody Staining for 3D X-ray microtomography (µCT) Imaging
Regenerative medicine rarely relies on flat scaffolds. Instead, three-dimensional fibrous,
gel or foamy biomaterials are preferred for carrying stem cells and for supporting the
healing processes of the impaired tissues. Conventional microscopes are not able to
provide comprehensive insight from the inner structures of the 3D biomaterial-cellhybrid samples. If the biomaterial is opaque, conventional optical microscopy allows
only examination of the surface. X-ray absorption, however, depends on the density of
the medium instead of its optical properties. µCT devices could be called “3D X-ray
microscopes” as they can provide fine structural information from the studied materials,
no matter are they opaque to visible light or not. Many biomaterials consist of low
atomic number elements like hydrogen, carbon, nitrogen and oxygen. Hence, their
X-ray absorption is close to that of water and cells cannot be distinguished from the wet
biomaterials. Antibody guided localization of contrast agents allows visualization of
even molecular details and their 3D-distributions in the biomaterials by µCT imaging.
In this study, we have demonstrated the proof of concept for application of µCT together
with metal particle precipitation based antibody stainings for examining subcellular
structures and organelles. For example, actin cytoskeletons and nuclei of human derived
stem cells were stained with the antibody guided metal particle precipitation and parallel
fluorescence stains. Signals from different stains superimposed almost perfectly with
each other as it was shown with light, fluorescence and X-ray microscopes.
All affiliations: 1) Institute of Biomedical Technology, University of Tampere, 33014
Tampere, Finland, 2) Department of Biochemistry and Molecular Biology and Center
for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI
48824-1319, USA
Analysis of mitochondrial DNA replication intermediates in Drosophila
melanogaster
Replication of the mitochondrial genome has been studied extensively in mammalian
and yeast models. The mechanisms of mitochondrial DNA replication in the widelyused model organism Drosophila melanogaster, however, remain largely unknown.
Although the nuclear-encoded key players of the mitochondrial DNA replication
machinery, DNA polymerase gamma, the mitochondrial DNA helicase and the singlestranded DNA-binding protein, have been described, the precise mode of replication
as well as the factors contributing to its regulation must be defined. Mitochondrial
replication intermediates can be separated and identified by two-dimensional agarose
gel electrophoresis, thus enabling the analysis of the progression of replication. By
analyzing insect cell lines and fly strains with modified expression of the replisome
proteins, this study aims to provide novel aspects in the functional properties of these
proteins in vivo. The data will be complemented with electron microscopy analyses,
which can be used to describe further the architecture of the mitochondrial replisome.
These results will shed light on the molecular mechanisms of mitochondrial DNA
replication, a prerequisite for the expression of proteins of the electron transport chain.
Ashwin Sriram
Annika Kohvakka
Keywords: Alternative Oxidase, Pink, Mitophagy, Lifespan
2. Cancer Research
Keywords: Prostate cancer, long non-coding RNA
Author: Ashwin Sriram (ashwin.sriram@uta.fi)
Author: Annika Kohvakka (annika.kohvakka@uta.fi)
Group: Alberto Sanz
Co-authors: Filippo Scialo1, Venkatesh Mallikarjun, Nina Gubina, Alba Naudi, Victoria
Ayala, Giuseppe Cannino, Eric Dufour, Reinald Pamplona and Alberto Sanz
Co-authors: Kati Kivinummi, Antti Ylipää, Matti Nykter, and Tapio Visakorpi
All affiliations: Institute of Bio-Medical technology
All affiliations: Prostate Cancer Research Centre, Institute of Biomedical Technology,
Mitochondrial ROS production regulates mitophagy in vivo.
Characterization of novel lncRNAs in prostate cancer
The environmental temperature plays a major role in determining the lifespan of the fruit
fly. Here we report that, a H2O2 signal is required for adapting to higher temperatures.
Understanding the transduction of this signal will help us to modulate the lifespan of fruit
flies. We have decreased mitochondrial H2O2 production expressing AOX (Alternative
Oxidase) from Ciona intestinallis and targeting catalase to the mitochondrial matrix.
On the other hand, we have overexpressed Superoxide dismutase 2, which decreases
superoxide levels, but increases H2O2. Intriguingly, we observed a significant reduction
in lifespan when H2O2 levels are decreased, and a modest increased when they are
elevated. The reduction in H2O2 markedly reduced the levels of PINK1 an instrumental
mediator of mitophagy. As a consequence, we observed a decrease in autophagy, and a
notable accumulation of damaged proteins and dysfunctional mitochondria in fly with
lower levels of H2O2. In summary, we showed that a mitochondrial H2O2 signal is
required to correctly regulate mitochondrial turnover under stress conditions.
Long non-coding RNAs (lncRNAs) are a group of regulatory RNAs that have gained
more interest in recent years. Little is still known about the biological roles of these
molecules, but many of them are suggested to regulate gene expression by interacting
with chromatin-modification complexes [1]. Moreover, lncRNAs have been associated
with several cancers [2]. In prostate cancer, overexpression of two lncRNAs has
proposed to be a plausible cause for castrate-resistance in prostatic tumours [3]. In
addition, lncRNAs are shown to be expressed tissue-specifically, which make them
potential biomarkers [4].
In our previous studies RNA sequencing data of prostatectomy samples revealed 145
unannotated transcripts that were highly expressed in several prostate tumour tissues.
Further investigation showed that one transcript, a lncRNA called PCAT-5, had a
significant effect on tumour growth in ERG-positive prostate cancers. To further assess
the potential of these transcripts, we will validate the expression of the transcripts in
150 prostatectomy samples with Fluidigm. To study the effects the transcripts have on
growth of prostate cancer cell lines, siRNA library screening will also be performed.
Later on, the association of the transcripts with functionally interesting proteins is
studied with protein lysate array-siRNA screen.
[1] Rinn and Chang, Annu. Rev. Biochem., 2012, 81:145–166
[2] Presner and Chinnaiyan, Cancer Discovery, 2011, 1(5):391–407
[3] Yang et al., Nature, 2013, 500(7464):598-602
[4] Cabili et al., Genes Dev., 2011, 25(18):1915-27
Mike Gerards
Narayan Puthanmadam Subramaniyam
Keywords: mitochondrial quality control
Keywords: EEG, epilepsy, non-linear, network theory, recurrence.
Author: Mike Gerards (mike.gerards@uta.fi)
Group: Mitochondrial gene expression and disease
Co-authors: H.T. Jacobs
All affiliations: Institute of Biomedical Technology and Tampere University Hospital, University
of Tampere, Tampere, Finland
The search for new mtDNA replication factors: functional characterization of
unusual suspects
Mitochondrial DNA (mtDNA) replication occurs semi-independently of the nuclear DNA
and requires a unique set of proteins. While many different proteins are known to be
involved in nuclear DNA replication, only a relative small number have been identified for
mtDNA replication. It is unlikely that these few proteins account for the complete replication
process of the mtDNA. In the search for other proteins involved in mtDNA replication, a
genome wide RNAi screen in Drosophila melanogaster cell lines was previously performed.
Besides the usual suspects, many interesting and unexpected proteins were identified that
could possibly play a role in mtDNA replication. Among these were CG3539 and CG14084
which are both involved in vesicle transport as SNARE (binding) proteins.
In the current study the function of these proteins and their relation to the mitochondria
were investigated in more detail. Cellular localization studies showed that these proteins
were not localized in the mitochondrion, instead CG14084 co-localized with Golgi and early
endosome markers, whereas CG3539 was mainly localized at the cell membrane and in
some cells exclusively co-localized with Golgi and early endosome markers. Furthermore,
live imaging using Blue Fluorescent Protein tagged CG14084 showed that the CG14084
containing compartments were always in close proximity of the lysosomes indicating there
might be a direct interaction. Knock down of both proteins in S2 cells showed no change in
mtDNA copy number compared to control. However, knock down of CG3539 resulted in a
1.7 fold increase of CG14084 expression and knock down of CG14084 in a 4.3 fold increase
of CG3539 expression. Additionally, knock down of CG3539 but not of CG14084 lead to a
30% decrease in mitochondrial mass.
These results imply that these proteins play an important role in the maturation and/or
functionality of the endosomes and lysosomes. Possibly knock down of these proteins leads
to a defect in mitochondrial quality control due to defective maturation or functionality
of the endosomes and lysosomes. More research needs to be conducted to determine the
exact role of these proteins in the endosomal and/or lysosomal pathway, how they affect
mitochondria and why they were among the positives in the screen for mtDNA replication
factors.
Author: Narayan Puthanmadam Subramaniyam (narayan.ps@tut.fi)
Co-authors: Jari Hyttinen
All affiliations: Department of Electronics and Communications Engineering, Tampere University of Technology. Biomeditech
Analysis of nonlinear dynamics of healthy and epileptic EEG signals using recurrence based complex network approach
Axioms
Epileptic electroencephalographic (EEG) signals are highly non-linear and non-stationary. Different non-linear methods like correlation dimension, Lyapunov exponent, entropy and more recently recurrence quantification analysis (RQA) have been used to characterize the non-linear dynamics underlying interictal (between seizures) and ictal (during seizure) activities. While RQA
is sensitive to embedding parameters other non-linear methods mentioned above require long and
stationary data.
Research aim
1.Develop a non-linear method that is robust to data non-stationarity and embedding parameters.
2.To investigate and characterize the difference in non-linear dynamics of ictal and inter-ictal
signals
Methods and Materials
We propose recurrence network (RN) approach to quantify the non-linear dynamics of the epileptic EEG data. The time series data is transformed into attractors using time delay embedding.
Based on proximity measures, the attractor data is converted into RN. Complex network measures like clustering coefficient (C) and path length (L) are computed on RN. RN constructed from
chaotic attractors show small world property with high C and low L whereas periodic attractors
show regular network property with high C and L. The dataset used to test the method is obtained
from online database and consists of altogether 500 signals from interictal, ictal and healthy (eyes
open and eyes closed) EEG activity.
Results and Conclusion
1.The interictal signals are generated by chaotic system as the RN constructed from their attractors exhibit small world property.
2.The behaviour of RN constructed from ictal attractors is consistent with that of periodic attractors displaying regular network property with high C and L.
3.The attractors of eyes closed EEG exhibit small world property while that of eyes open EEG
also exhibit small world property but relatively to a lesser degree indicating that generation of
alpha rhythm is more chaotic than normal background EEG.
In conclusion, the newly proposed RN based approach for EEG signals is able to characterize
different classes of EEG signals based on complex network measures derived from RN of their
attractors.
Matti Annala
Esko Kemppainen
Keywords: laboratory information management
Keywords: Drosophila, mitochondria, metabolism
Author: Matti Annala (matti.annala@tut.fi)
Author: Esko Kemppainen (esko.kemppainen@uta.fi)
Group: Matti Nykter
Group: Howard Jacobs
Co-authors: Aleksi Kallio, Matti Nykter
Co-authors: Jack George
Finland. Department of Signal Processing, Tampere University of Technology, Tampere,
Finland (for all authors)
Finland
Inzyme: a lightweight laboratory information management system
Biomedical research is a collaborative enterprise that often involves large samples and
thousands of measurements. Keeping track of the samples, experiments and results
involved in a project can pose a formidable challenge, with the necessary information
scattered in spreadsheets and notebooks of involved personnel. Laboratory information
management systems provide a centralized mechanism for tracking these assets, but are
often cumbersome to use and neglected in the frantic pace of research.
We introduce Inzyme, a web-based laboratory information management system designed
with speed and ease of use in mind. Inzyme is based on a flexible database engine that
allows items to be extended with new attributes and maintains a timestamped history of
all modifications. Existing data can be imported directly into Inzyme as spreadsheets.
Items are linked together based on their origin and associations, allowing users to
perform complex queries such as “patients with at least one biopsy of Gleason grade
>= 6 and a prostatectomy sample” across millions of samples. Items are displayed in a
spreadsheet-like view that allows sorting, filtering and multi-item editing. To automate
repetitive tasks, Inzyme allows users to create protocols, or stored rulesets for item
transformation. If a DNA extraction protocol is applied to a set of 50 samples, Inzyme
calculates the reagents needed for each sample, prints out instructions for the experiment,
and automatically adds new samples to the database after the experiment is complete.
NUTRITIONAL MODULATION OF MITOCHONDRIAL DYSFUNCTION IN A
DROSOPHILA MODEL OF HUMAN MITOCHONDRIAL DISORDERS
Mutations in genes encoding the components of the mitochondrial translational apparatus
give rise to various human mitochondrial disorders with a range of clinical phenotypes.
We have used a Drosophila mutant harbouring a missense mutation in the gene technical
knockout (tko) which encodes the highly conserved mitoribosomal protein S12, to
elucidate the pathology and adaptation to such defects in multicellular organisms. The
tko25t mutant flies exhibit a phenotype of developmental delay, sensitivity to paralytic
and epileptic seizures and hearing impairment, features analogous common clinical
manifestations of mitochondrial disease in humans. Transcriptome-wide analysis of the
mutant flies revealed several compensatory changes in the expression of genes required
for the transport and catabolism of fats and proteins, and in anaplerotic pathways. The
mutant flies also exhibit transcriptional changes with the hypothetical physiological
outcome of reduced intestinal absorption and increased renal clearance of sugars. In
agreement with this, the mutant flies respond favorably to low dietary sugar content
during development while high sugar diet exacerbates the developmental defects. Based
on a metabolomics analysis of wild-type and mutant larvae reared on high and low sugar
diets, we believe that the combination of mitochondrial dysfunction and high sugar diet
deprive the tko25t-mutants of NADPH required to drive anabolic and redox reactions
during development. This detrimental metabolic state is at least partially rescued on a
low sugar diet through a mechanism likely to involve dietary modulation of oxidative
stress and a few key enzymes regulating the cellular NADP/NADPH ratio.
Meeri Mäkinen
Ana Andjelkovic
Keywords: human pluripotent stem cells, neural networks
Keywords: Drosophila, cleft thorax, JNK pathway,
alternative oxidase, reactive oxygen species
Author: Meeri Mäkinen (meeri.makinen@uta.fi)
Author: Ana Andjelkovic (ana.andjelkovic@uta.fi)
Group: Howard T. Jacobs
Co-authors: L. YLÄ-OUTINEN, D. FAYUK, S. NARKILAHTI
Co-authors: Kia K.Kemppainen, Howard T. Jacobs
All affiliations: IBT, Univ. of Tampere / Biomeditech, Tampere, Finland
All affiliations: Institute of Biomedical Technology, Tampere, Finland
Development of network connectivity in human pluripotent stem cell derived
neural networks
Neural cells integrate into existing networks using similar mechanisms as newborn
neural cells during fetal development (Jäderstad et al., 2010, Stephens et al 2011). During
embryogenesis, the developing brain expresses a form of spontaneous synchronous
network activity (Voigt et al., 2001). Similar activity develops spontaneously in the
networks formed by human pluripotent stem cell (hPSC) derived neurons in vitro
(Heikkilä et al., 2009). However, the cellular composition and the intercellular connections
in spontaneously active neural networks remain unclear. Here, we studied the cellular
components of network communication underlying the formation of early activity in
hPSC derived neuronal networks.
hPSCs were differentiated to neurons as described earlier (Lappalainen et al., 2010).
The analysis of functional cellular subpopulations and connections requires activity
measurements. The activity of neural cell cultures is commonly measured with
microelectrode arrays (MEAs) or calcium imaging. These two methods were combined
with pharmacological studies to follow the spontaneous network activity and to dissect
the mechanisms mediating activity during the maturation of the networks.
The early network activity was found to be mediated by gap junctions as well as by
glutamatergic and GABAergic signalling. The amount of gap junction and GABA
mediated signaling seemed to change during network maturation. In addition, the
dependency of the network activity from these mechanisms was different between
differently derived networks. Furthermore, networks were found to contain subnetworks
responding differently to pharmacological treatments.
The activity in hPSC derived neuronal networks is mediated by mechanisms similar
to those occurring during the early brain development. The described pharmacological
protocols seemed promising for studying differences in cellular composition and
functional connections. Thus, human pluripotent stem cell derived neuronal cell cultures
are a suitable in vitro platform for studying the integration of neurons into functional
tissue.
AOX and cleft thorax in Drosophila
Most eukaryotes possess an alternative mitochondrial respiratory chain which can
bypass the mitochondrial oxidative phosphorylation system under specific physiological
conditions. One of the alternative respiratory enzymes is the alternative oxidase (AOX).
AOX is found in plants, fungi and some metazoans, although not in vertebrates or
arthropods. We found that the ubiquitously acting, drug-inducible transgene expression
driver, tub-GeneSwitch (tubGS), in combination with high doses of the inducing drug the synthetic steroid mifepristone, but in the absence of any target transgene, produces
detrimental phenotypes in Drosophila melanogaster including pupal death, cleft thorax,
defective bristle morphology, impaired leg development and notched wing phenotype.
However, when the expression of Ciona intestinalis AOX is concomitantly driven by
tubGS or by a driver-independent promoter, it alleviates all mentioned phenotypes
produced by mifepristone-activated tubGS. Morover, AOX rescues cleft thorax
in pannier heterozygous mutant flies, and in pannier driven basket and hemipterous
knockdown flies. These observations are of potential medical relevance, since failure
of midline closure is a common developmental abnormality in humans, recognised in
congenital disorders such as spina bifida, cleft lip and palate.
Some specific mutations in the pannier and ultraspiracle genes also give rise to cleft
thorax in Drosophila melanogaster. The same occurs when the JNK pathway is disrupted
in specific epidermal cells during development. The aim of my project is to elucidate the
molecular mechanism by which AOX rescues this developmental defect. My starting
hypothesis is that the failure of midline closure is due to excessive production of reactive
oxygen species (ROS) in specific cells, leading to the dysregulation of downstream
signaling, and that AOX can correct the defect by decreasing ROS production at complex
III of the mitochondrial respiratory chain, for which it provides a by-pass.
Keijo Viiri
Sergei Häyrynen
7. Molecular Biology & Epigenetics
Keywords: Polycomb proteins, Histone modifications,
non coding RNA, Histone methylation
Keywords: Alternative splicing, prostate cancer, RNA-seq
Author: Sergei Häyrynen (sergei.hayrynen@tut.fi)
Author: Keijo Viiri (keijo.viiri@uta.fi)
Group: Matti Nykter
Group: R. Jenner (London), in Finland affiliated with Markku Mäki’s lab
Co-authors: Matti Nykter.
Co-authors: Aditi Kanhere, Manuel Beltran-Nebot, Richard Jenner, Kalle Kurppa, Katri
Lindfors, Markku Mäki
All affiliations: Institute of Biomedical Technology, University of Tampere.
All affiliations: Univ Tampere, School of Medicine, Tampere, Finland & UCL Cancer
Instite, London, UK.
Computational Analysis of Alternative Splicing Using RNA-seq data
Short non-coding RNA mediated epigenetic gene regulation during cell
differentiation – using differentiation aberrancies in Coeliac Disease as a model
platform
Alternative splicing is a biological mechanism affecting transcription process in
eukaryotes. It enables producing multiple RNA transcripts from a single gene. Occuring
in estimated 95 % of human genes its contribution to transcriptional diversity is
significant. Analyzing alternative splicing computationally has been proven challenging,
but recent publications on the subject have provided tools aiming to solve the problem.
Cell type specific expression programs are orchestrated through regulated access to
chromatin. Polycomb group (PcG) proteins regulate developmental gene expression.
PcG proteins are essential for embryogenesis across Metazoa and this is mirrored by
their requirement for embryonic stem (ES) cell self-renewal and pluripotency. Polycomb
function is also necessary for the maintenance of cell identity and cell differentiation
throughout life and deregulation of polycomb proteins has been implicated in cancer.
We have previously found that polycomb target genes produce short non-coding
RNAs that interact with polycomb repressive complex 2 (PRC2) . These RNAs are
transcribed from CpG islands and other CpG-rich sequences at promoters, introns,
exons and intragenic sites. Our results suggests that short ncRNA expression and
concomitant PRC2 recruitment is dynamic and tightly regulated process during the cell
differentiation. In future we seek to test our hypothesis that recruitment of polycomb
proteins to the target genes via short ncRNAs is a universal mechanism during cell
differentiation in health and disease. Our preliminary results suggest that illegitimate
polycomb activity is contributing to the lack of epithelial cell differentiation in the small
intestine of the coeliacs providing us a good disease model to study ncRNA-polycomb
cross talk in future.
Here we present comparative analysis of three published state of the art methods for
alternative splicing using prostate adenocarcinoma samples from the Cancer Genome
Atlas. These methods are Miso, that implements Bayesian modeling approach,
SpliceSeq, that maps reads to splice graphs, and Alexa-seq which relies on simpler
approach of creating extensive annotation database. All these methods base the analysis
on existing transcript annotations and use RNA-seq data as an input. Using these tools
we explored the splicing patterns in prostate adenocarcinoma and tested consistency of
the variants identified by different methods. Project resulted in valuable information on
how alternative splicing analysis can be performed effectively.
Fikret Emre Kapucu
Elli Käpylä
Keywords: MEA, hESCs, neuronal networks,
burst analysis
7. Biomaterials
Author: Fikret Emre Kapucu (emre.kapucu@tut.fi)
Author: Elli Käpylä 1,2 (elli.kapyla@tut.fi)
Co-authors: Fikret Emre Kapucu 1,2, Jarno Tanskanen 1,2, Laura Ylä-Outinen2,3,
Susanna Narkilahti 2,3, Jari Hyttinen 1,2nen
Co-authors: Elli Käpylä1,2, Tomáš Sedlačík3, Dogu Baran Aydogan1,2, František
Rypáček3, Minna Kellomäki1,2
All affiliations: 1 Department of Electronics and Communications Engineering, Tampere
University of Technology, Tampere, Finland. 2 Institute of Biosciences and Medical
Technology, Tampere, Finland. 3 NeuroGroup, Institute of Biomedical Technology,
University of Tampere and Tampere University Hospital, Tampere, Finland
All affiliations: 1 Department of Electronics and Communications Engineering,
Tampere University of Technology, Tampere, Finland. 2 BioMediTech Tampere,
Finland. 3 Institute of Macromolecular Chemistry of the Academy of Sciences of the
Czech Republic, Prague, Czech Republic
Analyzing network bursts of maturing human embryonic stem cell derived neurons
It is previously shown that human embryonic stem cell (hESC) derived neuronal cells
can form spontaneously functional neuronal networks. There is need for deriving
quantitative metrics for a reliable tracking of cells’ maturing process. We analyzed their
maturing process by means of burst activities.
Deriving parameters from neuronal bursts such as burst durations, burst amplitudes,
bursting frequency etc is a quantitative way of following the network development.
However it is only possible by detecting the bursts without using any fixed parameters
or predefined values. Thus, Cumulative Moving Average (CMA) algorithm which is an
adaptive method for burst analysis is chosen. It is possible to detect different bursts in a
single analysis which have not firing with similar statistics with this algorithm.
We utilized the CMA algorithm for the recordings acquired from maturing hESC
derived neuronal cells on different time points. Cells are plated in micro electrode array
(MEA) wells and kept in an incubator between recordings. All recordings were made
using MEAs purchased from Multi Channel Systems MCS GmbH (MCS, Reutlingen,
Germany).
In this work, it is shown that even though maturing of hESCs is a complex process it is
possible to interpret some of it by means of burst parameters. It is a promising step for
creating a reliable metric for quantifying the maturation. Furthermore the parameters
which are used in this work can be combined with the information acquired from single
spikes for an enhanced connectivity analysis.
In conclusion, neurons derived from hESCs provide us a way of understanding the
development and functioning of human neuronal networks during their maturation.
Thus better tools and quantification methods are crucial to improve our perception
while interpreting their behaviours.
Direct laser writing of synthetic poly(amino acid) hydrogels
Hydrogels are promising matrix candidates for tissue engineering due to their high water
content and biomimetic properties. The combination of hydrogels with the additive
manufacturing technique of direct-laser writing by two-photon polymerization (2PPDLW) holds great potential for recreating complex extracellular microarchitectures.
However, this approach requires a wider selection of hydrogels applicable for
2PP-DLW. In this work, we studied the 2PP-DLW of synthetic hydrogels based on
methacryloylated and acryloylated poly(amino acid)s (poly(AA)s) for the first time.
Poly(AA) hydrogels are biodegradable by enzyme-catalyzed hydrolysis and can be
functionalized by cell-adhesion peptides. We compared the performance of these novel
materials to a standard poly(ethylene glycol) diacrylate (PEGda) hydrogel in terms of
polymerization and damage thresholds, voxel size, line width and post-polymerization
swelling and deformation. We showed that the tested poly(AA)s are applicable to 2PPDLW with 80% water content. The acryloylated poly(AA)s performed better than the
methacryloylated analogs by producing more stable three-dimensional microstructures.
These findings could in the future be used to fabricate biomimetic scaffolds for soft
tissue engineering applications.
Sini Eerola
2. Cancer Research
Keywords: prostate cancer, Pim kinases,
phosphorylation and NFATc transcription factors
Author: Sini Eerola (sini.eerola@uta.fi)
Group: Päivi Koskinen
Co-authors: Sini Eerola, Niina Santio, Petri Kouvonen, Riitta Vahakoski, Garry
Corthals, Eeva Rainio and Päivi Koskinen
All affiliations: Turku Centre for Biotechnology, University of Turku and Åbo
Akademi University, Drug Discovery Graduate School, Finland, Turku Bioimaging and
Department of Biology, University of Turku
Pim kinases promote prostate cancer cell migration by regulating NFATc activity
NFATc (Nuclear Factor of Activated T cells) transcription factors are ubiquitously
expressed in human tissues and regulate various cellular processes including immune
responses and development. NFATc localization and activation are regulated by
phosphorylation. We have previously shown that the oncogenic Pim-1 kinase directly
interacts with NFATc1, phosphorylates it and enhances NFATc-dependent transactivation
in both immune and neuronal cells. Interestingly, knock-out animals lacking all three
pim family genes show defects in T-cell proliferation, suggesting a role for Pim kinases
in NFATc regulation also in vivo. Since both Pim kinases and NFATc proteins have
recently been linked to several functions important for cancer progression such as cell
survival, migration and angiogenesis, we investigated whether NFATc proteins could
mediate the pro-migratory effects of Pim kinases.
Here we show that PC-3 prostate cancer cells exhibit constitutive NFAT-activity.
Moreover, the Pim kinase inhibitor DHPCC-9 as well as mutation of Pim target sites in
NFATc1 significantly decrease the pro-migratory effects of NFATc1 in PC-3 cells. These
results suggest that NFATc proteins are important players in the signalling pathways
through which Pim kinases regulate cancer cell motility.
Anssi Nurminen
Markus Hannula
Mirva Järvelä-Stölting
Keywords: X-ray microtomography, biomaterials
Keywords: Drosophila melanogaster, innate immunity,
G-protein coupled receptor kinase 2, Toll pathway,
Eye Transformer, JAK-STAT pathway
Author: Markus Hannula (markus.hannula@tut.fi)
Co-authors: Markus Hannula(1,2), Kaarlo Paakinaho(1,2), Minna Kellomäki(1,2),
Antti Aula(1,2,3), Jari Hyttinen(1,2)
All affiliations: (1) Department of Electronics and Communications Engineering,
Tampere University of Technology, Tampere, Finland. (2) BioMediTech, Tampere,
Finland. (3) Department of Medical Physics, Imaging Centre, Tampere University
Hospital, Tampere, Finland
Pixel size determination for reliable structure analysis of porous biomaterials
X-ray microtomography (µCT) imaging enables three-dimensional non-destructive
analysis of biomaterial structures. The present µCT imaging devices and methods
allow effective analyses with resolutions down to one micrometer. However, imaging
with high resolution is time-consuming and can cover only a relatively small volume.
Thus, especially for series with large number of samples, the resolution needs to be
optimised in order to achieve accuracy where the results are reliable but the imaging
time stays reasonable. In this study, porous tissue engineering scaffold structures were
imaged with X-radia MicroXCT-400 device. The porous structures were manufactured
by a supercritical carbon dioxide foaming process from melt-extruded poly(lactice-co-ε
caprolactone) - β-tricalcium phosphate composites. Pixel size varied from 2 to 30µm
and the effect of pixel size on morphological parameters of the material was assessed.
Morphological parameters, such as porosity and thickness, were calculated at different
resolutions. The parameter values at the smallest pixel size were used as a reference and
the aim was to find out the largest pixel size which yielded similar parameter values.
This approach will lead to optimized imaging times for similar biomaterials and large
sample series.
Author: Mirva Järvelä-Stölting (mirva.jarvela-stolting@uta.fi)
Group: Mika Rämet
Co-authors: Susanna Valanne, Mika Rämet
All affiliations: Experimental Immunology Research Group, IBT, University of Tampere
Gprk2 and ET - regulators of innate immunity in Drosophila melanogaster
Innate immune response is the primary step of immune defense. In mammals, the major
role of innate immunity lasts through lifetime as a primary mechanism of pathogen
defense, but it also has a significant role as a regulator of adaptive immune response. Invertebrates, like Drosophila melanogaster, do not have the adaptive immune response,
which provides the opportunity to study innate immune system separately in the Drosophila model.
G protein-coupled receptor kinase 2 (Gprk2) and Eye Transformer (ET) have been identified as regulators of innate immunity in large genome-wide in vitro RNAi screens.
Gprk2 is a protein coding gene that has been shown to be an important regulator of Toll
signalling, one of the two Drosophila NFkappaB pathways. It also has been shown to
co-immunoprecipitate with the Drosophila IkappaB factor Cactus. Another Drosophila protein coding gene, ET, has been identified as a negative regulator of JAK/STAT
cascade. The experimental evidence indicates that ET co-immunoprecipitates with the
Drosophila JAK called hopscotch and Dome, the Drosophila JAK/STAT receptor. Both
Gprk2 and ET have been identified in our laboratory as important regulators of innate
immunity, but the exact mechanisms of function are still unknown. In this study the aim
is to recognize proteins that interact with Gprk2 and ET. The purpose is to compare the
differences between identified genes in situations when immune response is activated
or inactive.
Väliaho Jari
Magdaléna von Essen
7. Point-of-care diagnostics
Keywords: Point of care diagnostics, microfluidics,
europium, nanoparticle, thyrotropin, immunoassay, finite
7. Protein Dynamics
Keywords: Mechanobiology, protein dynamics,
mechanotransduction, heart muscle, skeletal muscle,
epidermis, cancer
element method, microstructures
Author: Väliaho Jari (a) (jari.valiaho@tut.fi)
Group: Micro- and nanosystems research group
Co-authors: Välimaa Lasse (b), Kallio Pasi (a)
All affiliations: (a)The Department of Automation Science and Engineering, Tampere University
of Technology,Finland. (b)The Department of Biotechnology, University of Turku, Finland
Improving the performance of a microfluidic immunoassay
Point-of-care devices are more and more based on microfluidics, where small fluid
volumes are handled inside micro scale channels. With microfluidics it has been possible
to miniaturize devices and thus decrease sample and reagent consumption compared to
conventional methods.
The objective of this research was to study how the performance of a microfluidic
immunoassay cartridge would be enhanced. The aim was to study how different
polystyrene grades and reaction chamber geometries affect the immunoassay
performance.
Injection molded microfluidic cartridges were used in the research. Concentration of
thyroid stimulating hormone was measured with fluorescence nanoparticles containing
Europium(III)-chelates. The effect of material and different chamber structures on an
immunoassay was examined. A simplified finite element method (FEM) model was
created to simulate the immunoreactions occurring inside a chamber.
Material comparisons showed that polystyrene grade Empera 124N gave the highest
sensitivity for the immunoassay. FEM models of original reaction chambers showed
that the system was fully diffusion limited. New smaller geometries were designed
using the FEM model. Measurement sensitivity was increased about 1.4-fold and
the limit of detection was decreased from 1 mU/L to 0.52 mU/L. Micro structures on
the reacting surface were also tested and they increased measurement sensitivity by
3.8â€“fold compared to a smooth surface.
In conclusion, already a simple FEM model can be used to describe what happens in a
system and how the system can be optimized. As a smaller reaction chamber was used,
sensitivity and the limit of detection were enhanced and reagent consumption during
fabrication was decreased.
Author: Magdaléna von Essen (magdalena.vonessen@uta.fi)
Group: Vesa P. Hytönen
Co-authors: Vesa P. Hytönen
Mechanobiology and Disease
Introduction: The ability of cells to receive mechanical signals from the extracellular
environment is fundamental to life. The way cells respond to the mechanical cues is shaping
their phenotype in health and in disease. Mechanobiology, formed by merging cell and
molecular biology, is concerned with the impacts of mechanical forces on the cell structure,
its viability and locomotion, proliferation, growth and differentiation. Mechanosensitive
proteins are able to transduce the mechanical impulses from the extracellular space
through cytoskeletal structures towards the nucleus and affect important cell functions.
Hence, a mutation in the force sensitive system may cause changes in the way cells
communicates with their environment, alter cell development and cause pathological
changes demonstrating in a disease. The phenotypes and genotypes of various diseases
have been studied intensively. The role of mechanical force in the cellular development
and protein dynamics with the association to diseases is however not yet understood.
Aim and Benefit: In this work we provide an overview of mechanosensitive protein
structures with their diseases association. The benefit of the work lies in providing a
comprehensive overview of the mechanosensitive proteins and their importance in the cell
survival and proper functioning.
Methods: Public databases and published literature were studied to collect all data of
the review. NCBI (PubMed, Gene, Protein, SNP and OMIM), Uniprot and KEGG were
investigated for gene location, tissue specificity, protein cell location, metabolic pathways
and known disease causing mutations. RCSB Protein Data Bank was further used to collect
the protein structural information and homology patterns.
Results: Extensive literature and database search resulted in a collection of 140 protein
structures (different tissue specific protein variants were included). Majority of the listed
proteins are already associated with a disease. The protein dataset was sorted based on
the protein function (force transduction, force generation), protein localization within
cell (plasmatic membrane, cytoskeleton and cytoplasm, and nuclear lamina and nucleus),
associated disease (cardiovascular, muscular, epidermal, renal, mental, skeletal and cancer
diseases) and based on the similarities in the protein structure (spectrin, immunoglobulin,
fibronectin, calponin, plecstrin repeats).
Miina Ojansivu
Nathaniel Narra
Keywords: adipose stem cells, osteogenesis,
bioactive glass, bone tissue engineering
Keywords: reconstruction plate, finite element analysis,
mandible reconstruction, rapid prototyping, maxillofacial
surgery
Author: Miina Ojansivu (miina.ojansivu@uta.fi)
Co-authors:Sari Vanhatupa (1,2,3), Leena Björkvik (4), Leena Hupa (4), Susanna
Miettinen (1,2,3)
All affiliations: 1 Institute of Biomedical Technology, Adult Stem Cell Research Group,
University of Tampere, Finland. 2 BioMediTech, Tampere, Finland. 3 Science Center,
Tampere, University Hospital, Tampere, Finland. 4 Process chemistry centre, Åbo
Akademi University, Turku, Finland
Bioactive glass ions strongly enhance osteogenic response in adipose stem cells
Adipose stem cells (ASCs) are a promising autologous cell source for the applications
of regenerative medicine. They are multipotent stem cells able to differentiate in vitro
for example towards bone, cartilage and fat cells. Of the various biomaterials used
in bone tissue engineering, bioactive glass (BaG) has been shown to be especially
advantageous due to its osteogenesis-inducing ability. However, the osteogenesisinducing mechanism of BaG is currently unknown. To shed light on this, ASCs were
cultured in BaG ions containing extracts prepared from different BaG compositions.
Bioactive glass extracts were prepared from each glass type using basic medium (BM)
and osteomedium (OM) as an extraction basis in the protocol. To assess osteogenesis,
alkaline phosphatase (ALP) activity was analyzed after 7 and 14 days of culture. In
addition, mineralization was determined by Alizarin red staining at 14 days. At 7 d
time point none of the conditions induced osteogenesis of ASCs. The situation was
changed at 14 d: some of the OM extracts induced higher ALP activities compared to
the OM control. In addition, the mineralization in OM extracts was excessive. Parallel
to the increased mineral formation, the cell amount in OM extracts was decreased as
observed by live/dead staining and CyQuantÂ® proliferation assay (Invitrogen). These
results imply that BaG ions are able to stimulate osteogenesis of ASCs, even though
osteogenic supplements are still required. The OM extracts could potentially provide a
fast and effective way to differentiate ASCs in vitro prior to their utilization in clinical
bone tissue engineering applications.
Author: Nathaniel Narra (nathaniel.narragirish@tut.fi)
Co-authors:Jiří Valášek, Markus Hannula, Petr Marcián George K. Sándor, Jari
Hyttinen, Jan Wolff
Finite element analysis of customized reconstruction plates for mandibular
continuity defect therapy
Large mandibular continuity defects pose a significant challenge in oral maxillofacial
surgery. One solution to this problem is to use computer-guided surgical planning
and additive manufacturing technology to produce patient-specific reconstruction
plates. However, when designing customized plates, it is important to assess potential
biomechanical responses that may vary substantially depending on the size and
geometry of the defect.
The aim of this study was to assess the design of two customized plates using finite
element method (FEM). These plates were designed for the reconstruction of the
lower left mandibles of two ameloblastoma cases (patient1/plate1 and patient2/plate2)
with large bone resections differing in both geometry and size. Simulations revealed
maximum von Mises stresses of 63 MPa and 108 MPa in plates 1 and 2, and 65 MPa
and 190 MPa in the fixation screws of patients 1 and 2. The equivalent strain induced
in the bone at the screw-bone interface reached maximum values of 2739 micro-strain
for patient 1 and 19575 micro-strain for patient 2. The results demonstrate the influence
of design on the stresses induced in the plate and screw bodies. Of particular note,
however, are the differences in the induced strains. Unphysiologically high strains in
bone adjacent to screws can cause micro-damage leading to bone resorption. This can
adversely affect the anchoring capabilities of the screws. Thus, while custom plates offer
optimal anatomical fit, attention should be paid to the expected physiological forces on
the plates and the induced stresses and strains in the plate-screw-bone assembly.
Timo Salpavaara
Shokoufeh teymouri
7. Sensor Technology
Keywords: Biodegradable, Sensor
7. Biomedical Engineering
Keywords: Chemical surface modification; Polyimide;
ARPE-19; Collagen IV
Author: Timo Salpavaara (timo.salpavaara@tut.fi)
Author: Shokoufeh teymouri (shokoufeh.teymouri@tut.fi)
Group: Jukka Lekkala
Co-authors: Ville Ellä, Minna Kellomäki, Jukka Lekkala
All affiliations: TUT
Manufacturing methods for biodegradable sensors
The biodegradable sensors can be used in implantable applications to contemporarily
monitor the recovery and to ensure the functioning of the implants. The biodegradable
sensors have an advantage compared with conventional sensors because there is no need
for removal operation when the sensor has completed its purpose. The manufacturing
methods of sensors of this kind are still under research. The traditional manufacturing
methods have been tested with the biodegradable materials. In the sensor manufacturing,
there is a need for both conductive and non-conductive patterns and layers. Metals
like iron, zinc and magnesium can be considered as conductive materials. There are
also conductive polymers but, at the moment, the resistivities of those polymers are
still too high for most conventional purposes. The biodegradable polymers like PLA
and PCL can be used as insulators even though they absorb a little bit water during
the implantation. Materials like magnesium dioxide, silicon dioxide and silicon nitride
may be used as insulators as well even though the latter two are not really considered
to be biodegradable. In this study, E-beam evaporation has been tested to deploy
magnesium and oxide patterns by using physical masks. 3D printing technology has
been successfully utilized to make easily modifiable PLA masks. Polymers are shaped
ether by using molding or spin coating. The testing of these methods and materials
paves the way for the manufacturing of fully functional biodegradable sensors.
Co-authors: Shokoufeh Teymouri 1,2 , Maiju Hiltunen 1,2 , Kati Juuti-Uusitalo 2,3 ,
Anni Sorkio 2,3 , Heli Skottman 2,3 , Minna Kellomäki 1,2
University of Technology, Tampere, Finland. 2 Institute of Biomedical Technology,
Biokatu 12, 33014 University of Tampere, Tampere, Finland. 3 BioMediTech, Biokatu
10, 33520 Tampere, Finland
Chemical surface modification of polyimide membrane for retinal applications
Different natural and synthetic polymers have been studied as an insulator and carrier for
retinal prosthesis or as a scaffold for cell transplantation. The use of synthetic polymers
outmatches natural polymers in some aspects including degradation, processability and
strength. The surface of synthetic polymers can be modified by proteins to promote the
cell attachment and subsequently the tissue adhesion. Chemical surface modification
is a stable mean of protein immobilization in which proteins can be grafted covalently
onto the surface. The generated covalent coupling protects the protein from shear
stresses and changes in pH of environment.
In this study, the surface of polyimide (PI) membrane was modified by covalent
immobilization of collagen IV through a four-step protocol. The morphology and
hyrophilicity of the surface were studied by atomic force microscopy (AFM) and water
contact angle measurements. The number of carboxyl groups on the membranes was
determined using Toluidine Blue O (TBO) method. The modified PI substrate was
further evaluated by in vitro study of ARPE-19 (spontaneously transformed human
adult RPE cell line) cell interactions.
Collagen-modified PI was found to be more hydrophilic in comparison to control
membranes. The protocol did not significantly affect the final surface roughness of
membranes. On surface modified membrane, the phalloidin was relocated to cell-cell
boundaries and the protein ZO-1 represents the hexagonal ARPE-19 cell morphology.
The surface modified membrane tested in this study show good potential as ARPE-19
cell substrate. However, means to prevent the aberrant cell division are suggested.
Sanna Turunen
Maiju Hiltunen
7. Biomaterials and tissue engineering
Keywords: Direct laser writing, two-photon
polymerization, microstructure, neuronal cell
growth guidance
7. Biomedical Engineering
Keywords: Conducting polymer, coating, electrode,
electrochemical polymerization
Author: Sanna Turunen (1,2) (sanna.turunen@tut.fi)
Author: Maiju Hiltunen (maiju.hiltunen@tut.fi)
Co-authors: Jenni Koskela (1,2), Elli Käpylä (1,2), Minna Lähteenmäki (1,2), Konstantina
Terzaki (3), Costas Fotakis (3), Maria Farsari (3), Jouko Viitanen (4), Laura Ylä-Outinen (2,5,6),
Susanna Narkilahti (2,5,6), Minna Kellomäki (1,2)
All affiliations: (1) Biomaterials and Tissue Engineering Group, Department of Electronics
and Communications Engineering, Tampere University of Technology, Tampere, Finland.
(2) BioMediTech, Tampere, Finland. (3) Institute of Electronic Structure and Laser (IESL),
Foundation for Research and Technology Hellas (FORTH), Heraklion, Crete, Greece. (4) VTT
Technical Research Centre of Finland, Tampere, Finland. (5) NeuroGroup, Institute of Biomedical
Technology, University of Tampere, Tampere, Finland. (6) The Science Center of Pirkanmaa
Hospital District, Tampere, Finland
Direct laser writing of microstructures from proteins and synthetic photopolymers
for neuronal cell growth guidance
As the primary goal of neural tissue engineering is to guide neurite extension in a desired
direction, it is important to study the neuronal response to different microscale topographical
and chemical cues. Direct laser writing by two-photon polymerization offers a feasible
method to fabricate 2D micropatterns and 3D microstructures from various photosensitive
materials. In this study, photopolymerization of three different synthetic photopolymers,
i.e. methacrylated poly(ε-caprolactone)-based oligomer (PCL-o), poly(ethylene glycol)
diacrylate (PEGda) and polymer-ceramic hybrid material Ormocomp®, and two proteins,
i.e. avidin and bovine serum albumin (BSA), was demonstrated. Their suitability as
starting materials for the fabrication of microstructured platforms for directional neurite
outgrowth studies was assessed in terms of optimal processing parameters, overall
processability, achievable feature size, retention of bioactivity and possible cytotoxicity
of the used material-photoinitiator combinations. Due to its superior photocrosslinking
properties and ability to support neuronal cell migration, Ormocomp® was selected as a
starting material instead of PCL-o and PEGda for the fabrication of cell growth guidance
structures. In addition to synthetic Ormocomp®, avidin and biotinylated BSA proteins
were chosen to be further investigated as raw materials for direct laser written cell
guidance micropatterns. As a proof of concept, two-and three-dimensional biochemical
and topographical cues were polymerized with a custom-built laser system and tested for
their ability to guide neurite outgrowth of human pluripotent stem cell (hPSC) derived
neuronal cells.
Co-authors: Virpi Savolainen, Niina Onnela, Soile Nymark, Jari Hyttinen, Minna
Kellomäki
University of Technology. BioMediTech
Improving the electrode properties and performance by conductive polymer
coating
Microelectrode applications to measure or stimulate bioelectric activity of tissues
or cells call for new improved materials with increased effective surface area that
enhances the electrochemical behaviour of the electrodes. In the biomedical field, the
compatibility of the material with living cells/tissue is also essential. There are several
methods available to increase the effective surface area of the electrode but most of them
do not remove the drawback that metals are not optimal substrates for cells. Another
approach is to use conductive polymer coating on the electrodes. The nodular surface
structure of conducting polymers results in large specific surface area. Furthermore,
many conducting polymers are already proved to be compatible with different cell/
tissue types. In this study, the electrodes were coated with electroconductive polypyrrole
layer. Different coating thicknesses were achieved by controlling the charge density
in the electrochemical polymerization process. The properties and performance of the
coated electrodes were monitored by various surface analytical and electrochemical
techniques, such as atomic force and scanning electron microscopes, energy dispersive
x-ray spectroscopy and electrochemical impedance spectroscopy. Although the coating
thickness was only a few microns at the most, the polypyrrole coating improved the
electrodes by increasing the specific surface area of the electrodes, and by clearly
decreasing the low frequency impedance values. The study also revealed that the
coating thickness and the conditions of usage environment have a significant effect on
the electrode behavior and performance. Further experiments are still needed to verify
the function and compatibility of the coated electrodes with cells.
Laura Johansson
Heidi Halonen
7. Biomaterials
Keywords: extrusion, blend, polymer
Author: Laura Johansson (laura.johansson@tut.fi)
Keywords: adipogenic differentiation, adipose stem cells,
in vitro culturing, mechanical stimulation, osteogenic
differentiation, vibration loading
Author: Heidi Halonen (1,2) (heidi.halonen@tut.fi)
Co-authors: Ville Ellä, Minna Kellomäki
All affiliations: Polymer blending in twin-screw extruder. Laura Johansson, Ville
Ellä, Minna Kellomäki. Biomaterials and Tissue Engineering Group. Department of
Electronics and Communications Engineering, Tampere University of Technology,
Tampere, Finland. BioMediTech, Tampere, Finland.
Co-authors: Kyllönen Laura (2,3,4), Beev Nikolai (5), Miettinen Susanna (2,3,4), Haimi
Suvi (2,3,4), Hyttinen Jari (1,2)
Polymer blending in twin-screw extruder
Twin-screw extrusion is an effective method for mixing polymeric blends. Co-rotating
twin-screw extruders are widely used in compounding processes in plastics industry.
Blending is done to alter the properties of the desired end product. In the most cases the
blending of two different polymers result in a material which properties are the average
of the original materials in respect of the mixture ratio.
In this study the materials used were poly(D,L-lactide) (PLA), poly(butylene succinate)
(PBS) and three different molecular weight poly(ethylene glycolide)s (PEG). The
amount of PBS and PEGs used in blends was 5-30 wt-% and 5-15 wt-% respectively.
PLA/PBS blends were immiscible at all tested mixing ratios. The PEGs being low
molecular weight substances decreased the melt viscosity of the blends. In consequence
the processing temperatures could be lowered. PLA and PEGs formed miscible blends
at all tested mixing ratios and therefore the blends had good optical properties while the
PLA/PBS blends were opaque.
Two types of blends with different properties were accomplished. The addition of PBS
had greater effect on the mechanical properties: even with a small addition of PBS
the strain increased, whereas PEG blends showed no significant changes in tensile
properties. Although further testing of the blends is still needed the results indicate that
the blended materials can be used to spin fine fibers.
University of Technology, Tampere. 2 BioMediTech, Tampere. 3 Institute of Biomedical
Technology, University of Tampere, Tampere. 4 Science Center of Pirkanmaa Hospital
District, Tampere. 5 VTT Technical Research Centre of Finland, Helsinki.
Subwoofer as a cell stimulator
Introduction: Tissue engineering provides new methods to treat severe bone defects.
Cells benefit from dynamic mechanical stimulation during culturing. Also we have
observed correspondingly with in vitro bone tissue engineering.
Methods: We constructed a prototype of the cell stimulator that utilized a commercial
subwoofer (Lab12, Eminence) as a vertically vibrating platform. The system was driven
by a home-made controller. The stimulator performed in open-loop and closed-loop
modes. Vertical peak accelerations of the system were measured, when altogether five
cell culture dishes of human adipose stem cells were simultaneously stimulated (3
Gpeak, 50 and 100 Hz) with a modulated square wave.
Results: The working principle of the stimulator is simple and the device is easy to
use. The performance was the most adequate at the 50 Hz vibration frequency of the
closed-loop mode. The stimulation increased both ALP activity and collagen production
significantly when compared to static conditions. Mineralized areas were observed only
in the vibrated samples. Also adipogenesis was inhibited. These responses were higher
with the 100 Hz vibration frequency.
Discussion: Subwoofer produces continuously accelerating linear motion for large
sample sizes, even at high vibration magnitudes.
Kaisa Vuornos
Jarno M. A. Tanskanen
Keywords: adipose stem cells, tendon tissue
engineering, polylactide
Keywords: neuronal cell network, microelectrode array, MEA,
connectivity analysis, phase lock value
Author: Kaisa Vuornos (kaisa.vuornos@uta.fi)
Author: Jarno M. A. Tanskanen (jarno.m.tanskanen@tut.fi)
Co-authors: Miina Björninen [1] [2], Elina Talvitie [2] [3], Minna Kellomäki [2] [3],
Susanna Miettinen [1] [2], Suvi Haimi [1] [2] [4]
Co-authors: Jarno M. A. Tanskanen (1), Ville Raatikainen, Susanna Narkilahti (2), and Jari A. K.
Hyttinen (1)
All affiliations: [1] Adult Stem Cells, Institute of Biomedical Technology (IBT), University of Tampere, Tampere, Finland. [2] BioMediTech, Tampere, Finland. [3] Unit
of Electronics and Communications Engineering, Tampere University of Technology,
Tampere, Finland. [4] Department of Biomaterials Science and Technology, Enschede,
University of Twente, the Netherlands.
All affiliations: (1) Department of Electronics and Communications Engineering, Tampere
University of Technology, and BioMediTech. (2) Institute of Biomedical Technology, University
of Tampere, and BioMediTech.
Human adipose stem cells cultured in tenogenic differentiation medium on braided polylactide scaffold is a potential approach for tendon tissue engineering
Currently, there is a growing demand for an efficient tendon tissue engineering construct due to an increasing amount of tendon injuries in relation to sports activities and
degenerative conditions. This in vitro study proposes a strategy to differentiate multipotent mesenchymal human adipose stem cells (hASCs) towards tenogenic lineage on
braided poly-L-D-lactide (P(L/D)LA) scaffold stimulated with soluble factors.
A suitable tenogenic differentiation medium (TM) containing tenogenesis promoting
growth factor combined with ascorbic acid was chosen based on significantly higher
cell proliferation and total collagen content together with positive tenogenic marker
gene and protein expression profile in phase 1 of study. The braided 8-filament PLA
scaffold was chosen based on higher cell proliferation and total collagen content alongside positive gene and protein expression profile in phase 2 of study. The TM for hASCs
was combined with the braided 8-filament PLA scaffold for phase 3, where the cell
proliferation and total collagen content results were significantly higher compared to
control, in addition to which the hASC tenogenic differentiation gene expression profile
was gained.
The results demonstrated that growth factor and ascorbic acid supplementation significantly increased hASC proliferation and ECM collagen content. This tissue engineering
strategy of pretreating hASCs with TM in braided PLA scaffolds prior to in vivo transplantation might be potential for functional musculoskeletal tissue engineering applications. However, more investigations are needed to confirm the optimal TM composition
and scaffold architecture required to support further the hASC tenogenic differentiation
process and to study the in vivo functionality of the engineered tendon construct.
Phase Lock Analysis of Signals from Neuronal Networks on Multi-well
Microelectrode Array with a Common Ground/Reference
Here, microelectrode arrays (MEAs) [1] were employed to record electrical field
potentials generated by neuronal cells and populations, i.e., neuronal cell networks. To
investigate the properties of neuronal networks, it is traditional to observe the action
potential spikes and compute characteristic figures, such as average number of spikes
per minute or spike burst duration. To gain information on the networks, information
from several channels should be jointly analyzed. In this paper, we investigated phase
lock value (PLV) [2] as a network connectivity measure.
We performed PLV analysis on six-well MEA data. Such MEAs have a common
ground and reference electrode (CGRE) for all the microelectrodes in all the wells.
We computed PLVs over all channel pairs in a single well and over all channels on one
MEA plate with regard to one channel.
In conclusion, PLV may be a useful measure of network connectivity. Due attention
must be paid on the signals, analysis and the results, especially analyzing signals from a
system with CGRE: PLV may sometimes be high even between signals measured from
different wells, which might indicate a common signal component being measured from
the physically, but not fully electrically, separate wells.
Acknowledgment: This research has been supported by the 3DNeuroN project in the
European Union’s Seventh Framework Programme, Future and Emerging Technologies,
grant agreement n°296590.
[1] F. Mormann, K. Lehnertz, P. David, and C. E. Elger. Physica D: Nonlinear
Phenomena, 144:358-369, 2000.
[2] M. Taketani and M. Baudry (Eds.), Advances in Network Electrophysiology.
Springer, 2006.
Joonas Tuominen
Inkeri Vornanen
2. Cancer Research
Keywords: SKIL, prostate cancer
Keywords: neuron network, simulation model, topology,
3D, MEA
Author: Joonas Tuominen
(joonas.i.tuominen@uta.fi)
Group: Tapio VIsakorpi
Co-authors: Kati Kivinummi, Matti Annala,
Matti Nykter, Tapio Visakorpi
All affiliations: Institute of Biomedical Technology
SKIL-genefusions define a new subtype of prostate cancer
We have identified a novel fusion gene TMPRSS2-SKIL. This fusion is mutually
exclusive with ETS/SPINK1 alterations. The fusion merges three first exons of
TMPRSS2 and the full length SKIL and this leads to the overexpression of SKIL due
to the androgen regulated TMPRSS2 promoter. SKIL codes for SKI-like oncogenic
protein which can bind and disturb the heteromeric SMAD complex and this inhibits
the TGF-Î² signaling pathway. We then further analyzed a transcriptome sequencing
dada from the Cancer Genome Atlas (TCGA) prostate adenocarsinoma project and
that revealed additional SKIL fusion with MIPOL1 and ACPP. Also the analysis of 76
tumors and 22 LuCaP xenografts with qRT-PCR identified SKIL overexpression in the
LuCaP-77 and one clinical sample. Then the whole transcriptome of LuCaP-77 was
sequenced and that revealed a SLC45A3-SKIL fusion. All four fusion positive samples
were negative for ETS or SPINK1 alterations. To determine if SKIL plays significant
role in prostate cancer we created PC-3 cell line with the knockdown of SKIL with two
different siRNA resulted in reduced cell growth, invasiveness and colony formation.
Reduced cell growth was confirmed with LNCaP cells. Then DU145 cell line with stable
overexpression of SKIL was also created. However this didnâ€™t have any effect in
the growth of cells. Invasiveness of DU145 cells was increased in some of the clones
but remained same in others. Also EP156T and RWEP1 cell lines with overexpression
of SKIL were also created and the functional studies of those are currently on goinig.
Author: Inkeri Vornanen
(inkeri.vornanen@tut.fi)
Co-authors: I. Vornanen (1,2), J. M. A. Tanskanen (2), J. Hyttinen (2), K. Lenk (2)
All affiliations: (1) Aalto University, School of Science, Department of Biomedical
Engineering and Computational Science, Espoo, (2) Tampere University of Technology
Department of Electronics and Communications Engineering, and BioMediTech,
Tampere
Modelling Neuron Network Dynamics from 2D to 3D
Neurons form in their natural environment 3-dimensional (3D) structures. However,
most of the in vitro cultures of neurons are planar. This study aims to construct a
simulation model of a neuronal network to predict, how the network dynamics change,
as the neuronal network is transformed from 2D to 3D. We will compare the bursting
behavior of our simulation model in 2D and 3D space to neuronal networks consisting of
human embryonic stem cell (hESC) derived neurons measured with 2D microelectrode
array (MEA), which records the electrical activity of the neuron culture at multiple
locations simultaneously.
We based our model on inhibitory-exhibitory probabilistic neuronal network model
called INEX, which is a very simple non-topological neuron network model consisting
of Poisson neurons. We added a simple 2D and 3D topology to the model: first the
neurons are placed randomly in 2D or 3D space and then connected to the nearest
neurons so that the resulting network is roughly 10%-connected.
Our simulations illustrate that adding the topology increases the activity of the network:
the spikes and bursts are more frequent in the both 2D and 3D networks than in networks
without topology. Also, 3D networks are more active than 2D networks. The very simple
topological model used in the study can induce a change in the network behavior, which
suggests that topology has an important role in the neuron network dynamics, and
encourages us to improve the topology model towards a more biologically plausible
one for more realistic results.
Kaisa Oksanen
Joose Kreutzer
7. Infectious disease
Keywords: zebrafish, tuberculosis,
Mycobacterium marinum, vaccination
1. Biotechnology
Keywords: Microfabrication, Normoxia, Cell culture,
Long-term, MEA, PDMS
Author: Kaisa Oksanen (kaisa.oksanen@uta.fi)
Group: Mika Rämet
Co-authors: Eleanor Sherwood, Maarit Ahava, Leena Mäkinen, Mataleena Parikka,
Mika Rämet
All affiliations: Institute of Biomedical Technology, BioMediTech, University of
Tampere, FIN-33014 Tampere, Finland: KO, ES, MA, LM, MP, MR. Department of
Pediatrics, Tampere University Hospital, FIN-33521 Tampere, Finland: MR
Investigating potential novel tuberculosis vaccine antigens using adult zebrafish
model
Tuberculosis remains a major global health challenge despite extensive vaccination
schemes with the current live vaccine, Bacillus Calmette–Guérin (BCG). To relieve
the global burden, research into novel vaccines is needed. The zebrafish (Danio rerio)
displays a disease similar to human tuberculosis, making it an attractive model to
study the disease and its prevention. We have recently shown that zebrafish can be
protected against mycobacterial disease by vaccination. In the present study, we use
adult zebrafish as a model for investigating novel tuberculosis vaccine candidates.
We screen through selected mycobacterial genes by Mycobacterium tuberculosis and
a natural zebrafish pathogen, Mycobacterium marinum, for their antigenic properties
and protective effects against mycobacteriosis. The screening is done using DNA-based
vaccination that represents an alternative for immunizing against tuberculosis and a
tool for vaccine antigen screening. Genes associated with different bacterial metabolic
states are included in the antigen screen to allow specific targeting of certain disease
phases. Our aim is to identify novel vaccine antigens or combinations of antigens that
confer protection to the latent and/or active form of mycobacterial infection. Due to
the remarkable similarities between human and zebrafish mycobacteriosis, the antigens
identified in the zebrafish screen might in the future be utilized for designing a novel,
protective vaccine against tuberculosis.
Author: Joose Kreutzer (joose.kreutzer@tut.fi)
Group: Pasi Kallio
Co-authors: Antti Mäki, Pasi Kallio
All affiliations: Tampere University of Technology (TUT), Micro- and nanosystems
research group (MST-group). BioMediTech (BMT)
Culture Chamber for Long-Term Normoxia Studies of Cells on MEA plate
To mimic normal human body conditions, cells should be cultured in a biomimetic
and controlled environment. Typically, cells are cultured inside an incubator with 5%
CO2 and atmospheric O2 concentration (~20%). However, those concentrations are
not physiologically relevant for most of the human tissues in vivo. Although 5% CO2
concentration is physiologically high, it is required in many culture solutions to maintain
proper pH level of the growth medium. O2 concentration in tissues in vivo (normoxia)
varies. For example for the brain tissue, the normoxia is from 0.5% to 7%. Furthermore,
constant culture conditions are difficult to maintain outside an incubator. For example,
in long-term extracellular recordings with microelectrode arrays (MEA) evaporation
and altering gas concentrations are typical challenges.
We have developed a cell culture chamber that provides controlled environment for
long-term normoxia studies on a MEA plate. The chamber maintains constant gas
concentration (for both 5% CO2 and normoxia) and additionally, relatively fast changes
of O2 concentration can also be executed to study for example cell behaviour in hypoxic
conditions. Chamber material enables gas exchange but keeps water vapour inside the
chamber. Evaporation is typical issue affecting molecular concentrations of the medium.
We have shown that in the studied chamber evaporation is as small as 7% in a threeday experiment that is similar to evaporation inside an incubator. Thus, the chamber
supports the long-term culture with different gas concentrations and maintains the stable
condition. Different chamber designs can also be done to meet the requirements of the
physiological study.
Ashok Aspatwar
7. Neurobiology
Keywords: morphant zebrafish, antisense morpholinos,
apoptosis, ataxia, abnormal development
Author: 1Ashok Aspatwar (ashok.aspatwar@uta.fi)
Group: Prof. Seppo Parkkila
Co-authors: 2Tolvanen MEE, 2Barker H, 2 Ortutay C, 1Kuuslahti M, 1 Pan P, 2 Parikka
M, 2Rämet M, 2 Vihinen M, 4Parkkila S
All affiliations: 1Institute of Biomedical Technology and School of Medicine, University of Tampere and BioMediTech, 33014 Tampere, Finland. 2 Institute of Biomedical
Technology University of Tampere and BioMediTech, 33014 Tampere, Finland. 3 Institute of Biomedical Technology University of Tampere and BioMediTech, 33014 Tampere, Fimlab Laboratories (University of Tampere Hospital), and Department of Experimental Medical Science, Lund University, Lund, Sweden. 4Institute of Biomedical
Technology and School of Medicine, University of Tampere, BioMediTech and Fimlab
Laboratories (Tampere University Hospital), 33014 Tampere, Finland
Abnormal embryonic development and ataxia in CA10 and CA11 morphant zebrafish
Carbonic anhydrase related proteins (CARPs) X and XI are highly conserved across
the species and are predominantly expressed in the human and mouse brain. However,
the biological role of these proteins is still an enigma. In this study, we analyzed the
expression pattern of CA10 and CA11 genes using RT-qPCR during embryonic development and in different adult zebrafish tissues. The CA 10 and CA11 genes were
knocked down using antisense morpholinos (MOs) to get insights into the role of these
genes during embryonic development in 0-5 day post fertilized (dpf) zebrafish embryos.
Expression analysis showed that these genes are predominantly expressed in the brain
and expression in several other tissues. Knockdown of CA10 and CA11 genes using
antisense MOs showed developmental delay with a high rate of mortality in 1-5 dpf
larvae. Morphant zebrafish larvae resulted in a curved body axis, pericardial edema and
abnormalities in the eye. The TUNEL essay showed apoptotic cell death in the brain
region. Histological examination revealed gross morphologic defects in the brain region
and in the muscle. Furthermore, analysis of swim pattern showed ataxic movement in
5 dpf morphant zebrafish larvae. Taken together, our data suggests that CARP X and
CARP XI play important role in embryonic development. The suppression of CA10 and
CA11 expression leads to ataxia in zebrafish and introduces a novel zebrafish model
in which to investigate the mechanisms of CARP X and CARP XI-related ataxia and
mental retardation in humans.

BioMediTech Research Day 2013 Abstract Book

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