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14th CFGBC Symposium

14th CFGBC Scientific Symposium taking place on June 11th 2019 at the Faculty of Medicine, University of Ljubljana, Slovenia

Symposium - Systems medicine in clinical practice

Symposium "Systems medicine in clinical practice" will take place in June 10th-11th 2019 at University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia.

2010-09-27

Posters

Why are classification results for high-dimensional class-imbalanced data biased, and how to address this problem in practice?

Rok Blagus,1 Lara Lusa1

1Faculty of Medicine, University of Ljubljana, Slovenia

Introduction: One of the goals of high-throughput experiments is to develop a multivariate classifier to predict the class of the samples from the measurements derived from the experiment. Frequently the classifiers are developed using class-imbalanced data, i.e., data sets where the number of subjects in each class is not equal. Standard classification methods used on class-imbalanced data produce classifiers that do not accurately predict the smaller class. We investigate if high-dimensionality poses additional challenges and evaluate the effectiveness of some strategies that are available to overcome the effect of class-imbalance.

Results: Our results show that variable selection introduces an additional bias towards classification into the majority class; most new samples are assigned to the majority class from the training set, and as a consequence the class-specific predictive accuracies (PA) differ considerably. This is a consequence of the larger sampling variability of the smaller class. When the class imbalance is not too severe, down-sampling, asymmetric bagging embedding variable selection and threshold adjustment work well in removing the bias, while over-sampling does not. Asymmetric bagging significantly reduced variability of PA compared to simple down-sizing. These results were obtained by a simulation study and confirmed by the analysis of a published data set from breast cancer microarray study.

Conclusions: Our results suggest that using a balanced training set is a good choice for the design of high-dimensional class prediction studies, also in situations where the proportion of samples from each class is not equal in the population. Researchers using class-imbalanced data should be careful in assessing the predictive accuracy of the classifiers and, unless the class-imbalance is mild, they should always use an appropriate method for dealing with the class-imbalance problem.

Affymetrix microarray expression study in congenic mouse line F2 cross to uncover positional candidates for obesity quantitative trait loci

Katarina Cirnski,1 Peter Juvan,2 Rok Košir,2 Damjana Rozman,2 Simon Horvat1

1Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Slovenia, 2Center for Functional Genomics and Bio-Chips, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia

Introduction: Obesity is a complex trait controlled by several genetic and environmental factors. It is a major risk factor for number of diseases, including diabetes, cardiovascular diseases, and cancer. To elucidate the mechanisms of obesity/leanness, additional genes from the common polygenic form of obesity need to be identified. The objective of this study was to identify differentially expressed candidates for mouse obesity quantitative trait loci Fob3b1 and Fob3b2 (Prevoršek et al, 2010) using microarrays.

Materials And Methods: Brain, gonadal fat and liver were used for RNA extraction of FF (homozygous for Fat line alleles at Fob3b) and LL (homozygous for Lean line alleles at Fob3b) F2 females from a cross between the congenic line G and Fat line. Affymetrix GeneChip Mouse Gene 1.0 ST arrays were employed. Data analysis included analysis of variance using modified t-test (R/Bioconductor/Limma) of individual genes, gene enrichment analysis (R/Bioconductor/PGSEA) of gene sets from GO terms and KEGG pathways, and Venn analysis of differentially expressed genes/gene sets. Additionally, genes from the known QTL-containing segment on chromosome 15 were tested separately from the rest of the genes.

Results: In a genome–wide analysis 43 differentially expressed genes in liver and 1 in gonadal fat were identified. A focused analysis on chromosome 15 revealed 1 candidate in brain, 2 in gonadal fat and 9 in liver. These genes are involved in obesity-related processes such as biosynthesis of fatty acids, steroid hormone metabolism, glycerophospholipid metabolism, PPAR signalling pathway and Wnt signalling pathway. This study helped us to significantly narrow down the list of candidates for the obesity Fob3b1 and Fob3b2 QTL loci.

Acknowledgements: We are grateful to the CFGBC (http://cfgbc.mf.uni-lj.si/) for collaboration in microarray analysis and to Zala Prevoršek for help in tissue collections.

Reference:

  1. Prevoršek Z, et al. (2010) Mammalian Genome 21:172–85.

Granulosa cell gene expression in controlled ovarian stimulation after gonadotrophin and GnRH analogue administration

Rok Devjak,1 Klementina Fon Tacer,2 Peter Juvan,2 Irma Virant Klun,1 Damjana Rozman,2 Eda Vrtačnik Bokal1

1Departmen of Obstetrics and Gynecology, University Medical Centre Ljubljana, Slovenia, 2Centre for Functional Genomics and Bio-Chips, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia

Background: For controlled ovarian stimulation (COH) in in vitro fertilization (IVF) program the use of gonadotrophins is combined with GnRH agonists or antagonists to prevent LH surge. The efficiency of both protocols in terms of pregnancy and delivery rate is despite much research still disputable. Since fertilization ability of the oocyte depends on oocyte quality, cumulus and granulosa cells forming a functional unit with the oocyte have been much investigated. To date no research has been made for biomarker identification, which would predict embryo development to the blastocyst stage regarding the GnRH analogue used for prevention of LH surge.

Methods: In this prospective randomized study, biomarkers for blastocyst development in IVF cycles will be identified. Comparison of cumulus cells from unfertilized oocytes and cumulus cells from oocytes that develop to the blastocyst stage will result in differences in gene expression that will serve as biomarkers for blastocyst development. Additionally, it will be tested whether the type of GnRH analogue used (cetrorelix acetate or buserelin acetate) in controlled ovarian stimulation has an impact on the blastocyst development.

Results: Based on cumulus cells gene expression, biomarkers for fertilized oocytes that will develop to the blastocyst stage will be identified. These biomarkers will be useful in cases requiring selective fertilization of a definite number of oocytes or in the decision on the oocytes or embryos appropriate for freezing. It is expected that biomarkers for an individual GnRH analogue treatment will be different, but not in the group of biomarkers related to the development of the oocyte to the blastocyst stage. This implies that GnRH antagonists, which are more women friendly, will not differ in efficiency from GnRH analogues at the bio-molecular level.

Project presentation: Microarray studies of mechanisms in development of necrosis on potato tubers due to potato virus Y infection

Barbara Gerič Stare,1 Irena Mavrič Pleško,1 Jelka Šuštar-Vozlič,1 Mojca Viršček Marn,1 Peter Dolničar,1 Katarina Rudolf Pilih,1 Vladimir Meglič1

1Agricultural institute of Slovenia, Ljubljana, Slovenia

Potato virus Y (PVY) is one of the most important potato pathogens causing up to 90% yield losses. Among various PVY strains, tuber necrotic strain (PVYNTN) is the most aggressive one. It appeared in central European countries in 1980’s and spread across the Europe by the end of the 20th century, causing large crop losses in several countries, including Slovenia. The virus is responsible for development of necrotic symptoms on tubers and other parts of potato plants in sensitive potato cultivars, making the tubers unmarketable. Studies of virus genome and its complete sequencing could not explain the enigma of induction of tuber necroses and also the conditions responsible for tuber necrosis development are not yet fully defined. So far high temperature was pointed out as one of the environmental factors which might influence their development.

In the project effect of different storage temperatures on tuber necrosis development in potato cultivar Igor is studied. To investigate the genes involved in necrotic reaction of potato tubers induced by PVYNTN, gene expression analysis using microarray technology is performed on healthy tubers and infected tubers with and without necrotic symptoms, incubated on different temperature regimes. Potato genome array, a 44K 60-mer array on Agilent technology (AMADID# 015425) designed by Potato Oligo Chip Initiative (POCI) is used in a one colour protocol.

With gene expression analysis we would like to gather new information about physiological and biochemical pathways of hypersensitive reaction of potato genome in the susceptible potato-PVYNTN interaction. We expect that these investigations will contribute to identification of novel candidate genes that can advance potato breeding for virus tolerance and/or resistance.

DairyVis: Web-based application for visualization of cattle candidate genes and genetic markers for milk production and mastitis

Črtomir Gorup,1 Jernej Ogorevc,2 Tanja Kunej,2 Eva Čeh,2 Blaž Zupan,1,3 Peter Dovč2

1Faculty of Computer and Information Science, University of Ljubljana, Slovenia, 2Biotechnical Faculty, University of Ljubljana, Slovenia, 3Baylor College of Medicine, Houston, USA

Introduction: Biological data related to the mammary gland and mastitis is stored in several databases which are poorly interlinked. Data exploration can be therefore laborious and not always up-to-date.

Results: In this poster we present DairyVis, a web-based application which provides up-to-date genome map of cattle candidate genes and genetic markers for milk production and mastitis. DairyVis is based on the existing literature [1] and on on-line bioinformatics resources.

Conclusions: Developed application is easy to use and has a potential to become a valuable tool to scientists interested in milk production and mastitis.

Reference:

  1. J Ogorevc, T Kunej, A Razpet, and P Dovč: (2009) Database of cattle candidate genes and genetic markers for milk production and mastitis. Anim. Genet. 40, pp. 832-51.

 

Figure 1: DairyVis showing the detailed map of the chromosome BTA10.

Structural modeling of transcriptomics data using creative knowledge discovery

Kristina Gruden,1 Petra Kralj Novak,2 Igor Mozetič,2 Vid Podpečan,2 Matjaž Hren,1 Helena Motaln,1 Marko Petek,1 Nada Lavrač2

1National Institute of Biology, Ljubljana, Slovenia, 2Jožef Stefan Institute, Ljubljana, Slovenia

The overall aim of systems biology is to bring a novel perspective into understanding of complex interactions in biological systems. We present a top down approach for modeling of transcriptomics data through information fusion and creative knowledge discovery. By using ontology information as background knowledge for semantic subgroup discovery, rules are constructed that allow recognition of gene groups that are differentially expressed in different types of tissues. This information is further linked with the Biomine engine to visualize gene groups and uncover potential unexpected characteristics of the observed system. In Biomine, data from several publicly available databases were merged into a large graph and a method for link discovery between entities in queries was developed. Obtained models can thus serve as generators of research hypothesis that can be further on experimentally validated. Results of two case studies are presented to illustrate the applicability of the approach.

Polymorphisms in TTK and BUB1B genes in Slovenian patients with gastric cancer

Petra Hudler,1 Mateja Šimic,1 Snježana Frković-Grazio,2 Radovan Komel1

1Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia, 2Department of pathology, Institute of Oncology, Ljubljana, Slovenia

Gastric cancer is a major health problem worldwide because is it still often diagnosed at an advanced stage when the prognosis is poor. It is a fourth most common cancer and the second leading cause of cancer-related death in the world. Molecular mechanisms of its development are complex and still largely unclear. Advances in genomics are showing an important role of genomic instability in gastric carcinogenesis. Genomic instability is probably caused by aberrations in genes, involved in the maintenance of genome integrity. Polymorphisms - minor sequence variations that only subtly change protein expression, activity, or function -rarely cause disease, but can predispose individuals to develop diseases. Recent research indicates that variations in mitotic genes could be responsible for gradual accumulation of other genetic aberrations leading to cancer development.

In our study we analyzed distribution of functionally important SNPs in TTK and BUB1B genes, which are implicated in chromosome segregation. The samples were obtained in cooperation with Institute of Oncology Ljubljana. Genotyping was carried out using real-time PCR, sequencing and high-throughput high resolution DNA melting (Roche). We evaluated the association between these coding SNPs in gastric cancer patients by comparing them with healthy controls. We also determined the association between polymorphisms and clinicopathological features of the patients.

The analyses of candidate genes and determination of their possible effect on gastric cancer development could contribute to better clinical diagnostics and better prediction of the disease progression.

Transcriptome response of Escherichia coli to colicin M treatment

Simona Kamenšek,1 Darja Žgur-Bertok1

1Biotechnical Faculty, University of Ljubljana, Slovenia

Colicins are antibacterial cytotoxins active against strains of the same or related species and are a significant factor in maintaining microbial diversity. They are released into the environment to reduce competition from other bacterial strains. The mode of action of colicins is diverse: pore formation in membranes, degradation of nucleic acids and in case of colicin M inhibition of cell envelope biosynthesis. Studies focused on colicins help to reveal more about their biology and their potential use as probiotics, antibiotic alternatives and even in cancer treatment, etc. Colicin M is a unique colicin that exerts its lethal activity in the periplasm of sensitive cells and its bacteriolytic effect appears to be the consequence of an arrest of peptidoglycan polimerization steps provoked by enzymatic degradation of the undecaprenyl phosphate-linked peptidoglycan precursors. In our study we focused on transcriptome level activities of E. coli strain RW118 (derived from strain K12) treated with purified colicin M for 1h. The untreated culture served as a control. For expression profiling GeneChip E. coli Genome 2.0 Array was used. While the data is still being processed, it is evident that most of the differentially expressed genes are involved in cell wall metabolism.

Possible role of pectin methylesterase inhibitor in spread of Potato virus YNTN in potato plants

Polona Kogovšek,1 Špela Baebler,1 Maruša Pompe Novak,1 Ana Rotter,1 Kristina Gruden,1 Maja Ravnikar1

1Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia

Introduction: Plant viruses employ different strategies for entering plant cells and spreading from cell to cell. One of the mechanisms plant viruses apply is binding to pectin methylesterase (PME) which enables transport of the virus through the cell wall. The PME inhibitor (PMEI) was speculated to be involved in limiting virus movement through the formation of a PME-PMEI complex [1]. The antibacterial and antifungal role of PMEI was already described.

Results: Potato TIGR 10K microarrays and real-time PCR were used for observation of differential expression of genes early after inoculation with two virus isolates, aggressive PVYNTN or mild PVYN. Response was observed in various potato cultivars with different reaction to PVY infection. Data were statistically analyzed as previously described in [2], where analysis with Wilcoxon rank sum test revealed differential expression of genes classified to PMEI family. In potato plants of sensitive cultivar Igor, inoculated with PVYN PMEI genes were up-regulated while in plants inoculated with PVYNTN the expression of those genes did not change. At later stages of infection, we detected higher amounts of isolate PVYNTN then PVYN in systemically infected leaves. Up-regulation of PMEI genes was detected also in resistant potato cultivar Sante inoculated with PVYNTN.

Conclusions: Direct connection of PMEI in plant response to virus infection has not been shown yet. However, our results indicate that PMEI has a role in the response of potato plants to inoculation with PVY virus isolates. We could conclude that elevated expression of PMEI genes in response to inoculation with PVY contributes to the restriction of virus movement and spread. Latter was concluded on the basis of the results obtained in two sensitive potato cultivars, Igor and Nadine, and in resistant cultivar Sante.

References:

  1. Giovane A, et al. (2004) Biochim Biophys Acta 1696, pp. 245-52.
  2. Baebler Š, et al. (2009) Mol Plant Pathol 10, pp. 263-75.

Evaluation of qPCR reference gene stability for circadian studies in peripheral organs of different mouse strains

Rok Košir,1 Jure Ačimovič,1 Anja Korenčič,1 Marko Goličnik,1 Martina Perše,2 Martina Fink,3 Damjana Rozman1

1Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia, 2Insitute of Pathology, Faculty of Medicine, University of Ljubljana, Slovenia, 3University Medical Center Ljubljana, Slovenia

Selection of reference genes is a crucial step of qRT-PCR that ensures accurate data analysis. Recent studies discuss differences in reference gene stability between species and tissues, but none has taken into consideration circadian experiments and potential differences between mouse strains. The aim of this work is to evaluate the circadian stability of candidate reference genes in two frequently used mouse strains, the imbred C57BL6 and mixed strain C57BL6x129Pas.

33 C57BL6 mice and 96 mice with mixed background (51 wild type and 45 Crem-/-) have been included in our study. Livers and adrenals were taken from 4-5 animals per time point every 4 h in a 24 h period. Expression of 10 candidate reference genes was measured by qPCR and their stability evaluated by geNorm, BestKeeper and NormFinder. In all tested cases, identical ranking of genes was obtained irrespective of the algorithm used.

Important differences in reference gene expression were detected when comparing livers of C57BL6 and C57BL6x129Pas mice, but not livers of mixed background w.t. and Crem-/-, nor the adrenals. C57BL6x129Pas generally exhibits a higher stability of reference genes compared to C57BL6. Ppib, 18sRNA and Rplp0 are the best reference genes for circadian studies in the adrenal irrespective of the mouse strain. Rplp0, Hprt1 and Utp6c are most stable in C57BL6x129Pas but, surprisingly, the least stable in C57BL6. Best normalization genes for liver circadian experiments in C57BL6 are Hmbs, Ppib and Eif2a.

Our data show for the first time quantitative differences in expression of potential liver and adrenal reference genes in circadian performed experiments in the two mouse backgrounds. We show that the best normalization genes for liver circadian studies in C57BL6x129Pas mice are indeed the worst selection for C57BL6. This should be taken into consideration to avoid false conclusions in interpreting the circadian qPCR expression data.

Development and validation of new functional cell models

Tomaž Langerholc,1 Lidija Gradišnik,2 Avrelija Cencič1,2

1Department of Microbiology, Biochemistry, Molecular Biology and Biotechnology, Faculty of Agriculture and Life Science, University of Maribor, Slovenia, 2Department of Biochemistry, Faculty of Medicine, University of Maribor, Slovenia

Introduction: Our laboratory is specialized in establishment of new human and animal cell lines to be used as functional cell models for various applications, where normal tissue is required. Our established cell lines are continuous and stable without recombinant technology or cancerous origin. These cell lines mimic the in vivo situation more significantly and represent better models for research than the widely used cell lines in the market.

Results: We described here the human and animal (pig, calf goat, sheep and chicken) small intestinal epithelial and macrophage cell lines. These cell lines were used to build functional cell models of the small intestine, which consist of differentiated epithelial cell layer at the apical side of microporous membrane underlied with macrophages. Using this experimental layout we were able to study intestinal pathogens, bioavailability, selection of probiotic bacterial strains, toxicity of pharmaceutical compounds, etc. Additionally, we are developing new cell lines from human prostate, kidney, liver and urogenital tract that are still in the process of characterization.

Conclusions: Our new cell lines proved to be a better in vitro model for studying cellular processes. Choice of appropriate cell and tissue models is important in experiments with DNA microarray chips; accessibility of primary cells is limited and they are hard to use in experiments, but commercial cancerogenic cell lines are often inappropriate models. Our cell lines circumvent these problems being non-transformed permanent cell cultures, with broad applicability and reliability.

Association and expression studies confirm CTLA4 gene as candidate gene for inflammatory bowel disease susceptibility

Katja Repnik,1 Uroš Potočnik1,2

1Center for Human Molecular Genetics and Pharmacogenomics, Faculty of Medicine, University of Maribor, Slovenia, 2Laboratory for Biochemistry, Molecular Biology and Genomics, Faculty for Chemistry and Chemical Engineering, University of Maribor, Slovenia

Cytotoxic T lymphocyte antigen 4 (CTLA4) gene has been previously associated with risk for a number of chronic autoimmune diseases however the lack of association and expression studies made for CTLA4 gene and its single nucleotide polymorphism (SNP) rs3087243 (CT60) which was shown to influence the CTLA4 functional expression, were inconclusive about the role of CTLA4 gene and its CT60 SNP in inflammatory bowel disease (IBD) susceptibility.

In our study we evaluated functional polymorphism CT60 in CTLA4 gene for potential association with both major subtypes of IBD in Slovenian population. In addition we investigated correlations between CTLA4 CT60 polymorphism and CTLA4 gene expression in peripheral blood lymphocytes and colon biopsies from IBD patients. We genotyped CTLA4 CT60 polymorphism in 266 healthy controls and 481 inflammatory bowel diseases (IBD) patients and found statistically lower frequency of CTLA4 CT60 AA genotype in IBD patients (13.72%) compared to controls (23.31%; p=0.001, OR=0.504) as well as lower allele frequency of minor A allele in IBD patients (0.346) compared to controls (0.461, p<0.001, OR=0.623). The association was confirmed with both major forms of IBD. We also found lower expression of CTLA4 gene in blood lymphocytes from IBD patients compared to controls (p<0.001) and higher CTLA4 expression in biopsies taken from inflamed part of the colon compared to noninflamed part of the colon (p=0.021). We found lower expression of soluble CTLA4 isoform than membrane bound full length isoform in peripheral blood lymphocytes from IBD patients compared to controls (p=0.010) and in lymphocytes from IBD patients with CTLA4 CT60 GG genotype compared to IBD patients with AA genotype (p=0.034).

Our genotype and gene expression data suggest CTLA4 plays role in IBD pathogenesis. Polymorphism CTLA4 CT60 contributes to genetic susceptibility to IBD in Slovenian population and regulates expression of CTLA4 isoforms.

Searching for normalization genes in PC12 and SH-SY5Y cells treated with recombinant human erythropoietin

Klemen Španinger,1 Arthur Sytkowski,2 Nataša Debeljak1

1Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia, 2Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA

Introduction: Erythropoietin (Epo) is a glycoprotein synthesized in kidney cells and is the main regulator of erythropoieses. Epo also has protective role in non-haematopoietic organs like brain, heart and endothelium. Epo activates several signalling pathways (Jak-STAT, PI3K-Akt, RAS-MAPK) and has influence on expression of different genes involved in proliferation and apoptosis.

Aim: Aim of the study is to determine the neuroprotective characteristic of rHuEpo. Neural cell lines SH-SY5Y (human) and PC12 (rat) were grown in different conditions; chronically or acutely treated with recombinant human erythropoietin. To study proliferation of cells the growth characterization assay and RNA isolation was performed. Genes involved in proliferation, differentiation, cell cycle and apoptosis were analysed with RT-qPCR. First step was to determine genes adequate for normalization of genes of interest.

Results: Most stable genes were determined among several house keeping genes and genes of interest. All three available programs NormFinder, BestKeeper and GeNorm gave similar results. In SH-SY5Y cell line Rplp, Stat5b and EpoR genes were most stable and in PC12 cell line Akt, Stat5a and CycA genes. Interesting fact is that among three most stably expressed genes only one is the house keeping gene. Next step was to compare normalization performed with GeNorm (3 most stable genes) to normalization with GAPDH, as the most commonly used normalization factor. Results show that expression level can differ.

Conclusions: Most important step when doing QPCR analysis is to determine normalization genes. This step must carefully be performed specially when analyzing effects of molecules that alternate expression of several gene families (like Epo). In our study, when cells were treated with recombinant human erythropoietin, we can conclude that most commonly used normalization genes are not appropriate for doing normalization analysis.

Studying potato - PVY interaction using system biology approaches

Katja Stare,1 Špela Baebler,1 Jenny Renaut,2 Kamil Witek,3 Jacek Henning,3 Kristina Gruden1

1National Institute of Biology, Ljubljana, Slovenia, 2CRP - Gabriel Lippmann, Belvaux, Luxembourg, 3Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland

Understanding of the plant-pathogen interaction enables selection of efficient strategies for plant protection. Potato cultivars differentially susceptible to the virus Potato virus YNTN are representing a good system to study the plant-pathogen interaction. In our previous research we studied response of different potato genotypes to PVY infection on gene expression level (DNA microarrays). The data were complemented on protein level using 2-D difference Gel Electrophoresis in combination with MS identification. Genotype Rywal with hypersensitive-type resistance to virus was studied in 1 and 3 days post inoculation. After extraction of proteins with buffer (TCA, DTT, acetone) samples and internal standard were labeled with the CyDyes. The isoelectric focusing was performed on the IPGphor (GE Healthcare) using 24 cm pH 3–10 immobilized pH gradient strips (Biorad). The second dimension was carried out on the Ettan Dalt twelve System (GE Healthcare), subsequently gels were scanned. Images were analyzed using the Decyder v6.05.11 software (GE Healthcare). Different statistical tests were applied to search for differentially expressed proteins between healthy and inoculated plants and during the time after inoculation. Spots of interest (16) were excised from preparative gels and digested with trypsin. Mass determinations were performed using the 4800 MALDI TOF/TOF Analyzer (Applied Biosystems). Both peptide mass fingerprinting and tandem mass spectrometry were carried out. Resulting spectra were subjected to a database search through the MASCOT interface (NCBI nr database and potato EST database). Some of identified proteins were RuBisCO, Endochitinase 2, FeSOD, etc. No exact correlations were found between results on proteomic and transcriptional level. However if looking at the process level, proteins involved in processes identified through DNA microarray analysis were among the differentially expressed also in proteomic profiling study.

Downfall of the Erythropoietin

Nina Trošt,1 Klementina Fon Tacer,1 Nataša Debeljak1

1Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia

Introduction: Erythropoietin (Epo), a primer regulator of erythroid progenitor proliferation and differentiation, was shown to have some impact in the biology of cancer. Administration of recombinant human Epo (rHuEpo) and its analogous to anaemic cancer patients receiving chemotherapy showed beneficial for the improved quality of life. Questions have also arisen if Epo may promote tumor cell survival, stimulate tumor growth and contribute to the development of more aggressive cancer phenotypes. Evidence suggest that biologically functional erythropoietin receptor (EpoR) is present in several non-hematopoietic tissue but also in solid tumors, where it may change phosphorylation/dephosphorylation patterns of specific signalling pathways like Jak2/STAT5, PI3K/Akt and Ras/MAPK and consequently influences the gene expression profiles.

Aims: The aim of the study is to investigate the effect of Epo in breast cancer cell lines on the level of gene transcription and cell growth.

Methods: Breast cancer cell lines Hs578T, SKBR3, MDAMB231 and MCF-7 were chronically treated with Epo for 10 weeks and assessed for growth characteristics using Growth Characterization Assays (GCA), such as MTT and cell counting. Quantitative real time PCR (qRT-PCR) was used to analyse gene expression profiles in cell lines acutely treated with Epo (5 U/mL). Genes involved in cell proliferation, cell cycle and apoptosis were analysed.

Results: GCA results showed a prominent change in cell growth characteristics between Epo treated and control samples. On the level of expression, acute Epo treatment induces early gene response in genes involved in cell proliferation and exerts anti-apoptotic effect.

Prospective: Analysis of gene expression profile (qRT-PCR) and protein composition (western blot) in cells after acute and chronic Epo treatment will be performed to explore the mechanisms of Epo effect in breast cancer cells.

The association of brain-derived neurotrophic factor (BDNF) polymorphism Val66Met and suicide in Slovenian population

Alja Videtič Paska,1 Peter Pregelj,2 Gordana Nedić,3 Tomaž Zupanc,4 Matea Nikolac,3 Radovan Komel,1 Dorotea Muck Seler,3 Nela Pivac3

1Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia, 2University Psychiatric Clinic Ljubljana, Slovenia, 3Laboratory for Molecular Neuropsychiatry, Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia, 4Institute of Forensic Medicine, Faculty of Medicine, University of Ljubljana, Slovenia

Introduction: Brain-derived neurotrophic factor (BDNF) mediates neural plasticity, mood, different behaviours, and stress response. BDNF polymorphism (Val66Met) influences the effects of stressful life events or childhood adversity on depression and suicidal behaviour in various psychopathologies. The study evaluated the association between the BDNF Val66Met variants and suicide, committed with violent or nonviolent methods, in victims with or without the stressful childhood experience.

Methods: BDNF Val66Met polymorphism was genotyped on 560 DNA samples from 359 suicide victims and 201 control subjects, collected from autopsies from unrelated Caucasian subjects, subdivided according to the gender, method of suicide, and influence of the childhood adversity.

Results: A similar frequency of BDNF Val66Met variants was found between suicide victims and control groups. A significant difference in the frequency of the combined Met/Met and Met/Val genotypes compared to the homozygous Val/Val genotype was detected between female suicide victims and female controls, between female controls and female suicide victims who used violent suicide methods, and between all suicide victims with or without stressful life events. The combined Met/Met and Met/Val genotypes contributed to this significance.

Conclusions: The combined Met/Met and Met/Val genotypes of the BDNF Val66Met are possible risk factors for completed suicide in female subjects, related to violent method of suicide, and for suicide victims exposed to childhood trauma. These results confirm a major role of BDNF in the increased vulnerability to suicide.

Acknowledgements: Funding for this study was provided by Croatian Ministry of Science, Education and Sport, grants numbers 098-0982522-2455 and 098-0982522-2457, and Croatian-Slovenian bilateral project. In Slovenia the study supported the Slovenian Research Agency, program grant No. P1-0104-0381 and postdoctoral project No. Z3-2180.

Searching for therapeutic targets and diagnostic biomarkers in ovarian endometriosis

Katja Vouk,1 Neli Hevir,1 Nataša Beranič,1 Martina Ribič-Pucelj,2 Cornelia Prehn,3 Jerzy Adamski,3 Tea Lanišnik-Rižner1

1Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia, 2Department of Obstetrics and Gynaecology, University Medical Centre Ljubljana, Slovenia, 3Genome Analysis Centre, Institute of Experimental Genetics, Helmholtz Zentrum Muenchen, Neuherberg, Germany

Introduction: To identify therapeutic targets, prognostic and diagnostic biomarkers for ovarian endometriosis, we are exploring its pathogenesis, with targeted metabolomics and RNA profiling. This is our network reconstruction basis, for understanding molecular interactions involved in cell proliferation. The choice of the disease is based on the facts that it is frequent, lacks effective therapy and specific biomarkers for early diagnosis as well as produces high costs.

Results: In a case-control study we select a well defined group of patients with ovarian endometriosis and a control group of healthy women from Slovenia. We collect plasma, peritoneal fluid and tissue samples of study participants and analyze their expression and metabolic profiles. Over 160 target analytes include amino acids and sugars, however focus on the lipidome, including acylcarnitines, glycerophospholipids and sphingolipids, which is involved in proliferative cell signalling pathways such as angiogenesis, apoptosis, differentiation, cell cycle arrest, oxidative stress, inflammation and immune system response. Analytes are quantified using internal stable isotope-labelled standards by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Bivariate analysis of measurement results shows significant differences in concentrations of several glycerophospholipids and acylcarnitines between the case and the healthy control group.

Conclusions: For testing the validity of candidate biomarkers and establishing their sensitivity and specificity, we need to extend the present groups as well as include an additional control group of patients with benign non-endometriotic ovarian cysts.

Acknowledgements: We are grateful to the staff of the Department of Obstetrics and Gynaecology, UMC Ljubljana, for participating in epidemiological data and sample collection, especially to Ms. M. Andonova, Ms. M. Puklavec and Ms. M. Kavčič for supervising the procedure.

2006 - University of Ljubljana, Faculty of Medicine, Center for Functional Genomics and Bio-chips.