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High Throughput Screening of Gene Expression for the Investigation of Multifactorial Dermatological Disorders

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High Throughput Screening of Gene Expression for the Investigation of Multifactorial Dermatological Disorders High Throughput Screening of Gene Expression for the Investigation of Multifactorial Dermatological Disorders by Máté Manczinger MD. Supervisors: Lajos Kemény MD, PhD, DSc and Lóránt Lakatos PhD PhD Thesis Department of Dermatology and Allergology University of Szeged, Hungary 2016 1 Publications directly related to the subject of the thesis I. Manczinger M, Bocsik A, Kocsis GF, Voros A, Hegedus Z, et al. (2015) The Absence of N-Acetyl-D-glucosamine Causes Attenuation of Virulence of Candida albicans upon Interaction with Vaginal Epithelial Cells In Vitro. Biomed Res Int 2015: 398045. IF: 1.579 II. Manczinger M, Kemeny L (2013) Novel factors in the pathogenesis of psoriasis and potential drug candidates are found with systems biology approach. PLoS One 8: e80751. IF: 3.23 Other publications I. Guban B, Vas K, Balog Z, Manczinger M, Bebes A, et al. (2015) Abnormal regulation of fibronectin production by fibroblasts in psoriasis. Br J Dermatol. IF: 4.225 II. Palotai M, Bagosi Z, Jaszberenyi M, Csabafi K, Dochnal R, Manczinger M, et al. (2013) Ghrelin and nicotine stimulate equally the dopamine release in the rat amygdala. Neurochem Res 38: 1989-1995. IF: 2.551 III. Palotai M, Bagosi Z, Jaszberenyi M, Csabafi K, Dochnal R, Manczinger M, et al. (2013) Ghrelin amplifies the nicotine-induced dopamine release in the rat striatum. Neurochem Int 63: 239-243. IF: 2.650 IV. Heinzlmann A, Kiss G, Toth ZE, Dochnal R, Pal A, Sipos I, Manczinger M, et al. (2012) Intranasal application of secretin, similarly to intracerebroventricular administration, influences the motor behavior of mice probably through specific receptors. J Mol Neurosci 48: 558-564. IF: 2.891 V. Manczinger M, Szabo EZ, Goblos A, Kemeny L, Lakatos L (2012) Switching on RNA silencing suppressor activity by restoring argonaute binding to a viral protein. J Virol 86: 8324-8327. IF: 5.076 VI. Koves K, Kiss G, Heinzlmann A, Dochnal R, Manczinger M, et al. (2011) Secretin attenuates the hereditary repetitive hyperactive movements in a mouse model. J Mol Neurosci 43: 109-114. IF: 2.504 2 Table of contents Publications directly related to the subject of the thesis ............................................................ 1 Other publications ...................................................................................................................... 1 Table of contents ........................................................................................................................ 2 List of abbreviations ................................................................................................................... 4 1. Introduction ............................................................................................................................ 5 1.1. The role of systems biology in the research of multifactorial diseases ........................... 5 1.2. Vulvovaginal candidiasis ................................................................................................. 6 1.2.1. Epidemiology ............................................................................................................ 6 1.2.2. Clinical characteristics .............................................................................................. 6 1.2.3. Pathogenesis and immune defense ............................................................................ 6 1.3. Psoriasis ........................................................................................................................... 8 1.3.1. Epidemiology and clinical characteristics................................................................. 8 1.3.2. Pathophysiology ........................................................................................................ 8 1.3.3. Treatment .................................................................................................................. 8 1.4. High throughput screening of gene expression ............................................................... 9 1.4.1. Microarray ................................................................................................................. 9 1.4.2. Next generation sequencing ...................................................................................... 9 1.5. Network analysis ........................................................................................................... 10 1.5.1. Introduction ............................................................................................................. 10 1.5.2. Scale-free networks, centralities and motifs ........................................................... 10 1.5.3. Network analysis in biomedical research and drug development ........................... 11 2. Aims I ................................................................................................................................... 12 3. Aims II .................................................................................................................................. 12 4. Methods I .............................................................................................................................. 13 4.1. Strains, growth conditions and cell culturing ................................................................ 13 4.2. Viability test .................................................................................................................. 13 4.3. C. albicans adherence assay ........................................................................................... 14 4.4. Total RNA isolation and high throughput sequencing .................................................. 14 4.5. Bioinformatical and statistical analysis ......................................................................... 15 4.6. Quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) .................. 15 5. Methods II ............................................................................................................................ 17 5.1. Microarray meta-analysis .............................................................................................. 17 5.2. Construction of protein-protein, protein-DNA and chemical-protein interaction networks ................................................................................................................................ 18 3 5.3. General network analysis, identification of central nodes and motif detection ............. 18 6. Results I ................................................................................................................................ 21 6.1. Vaginal epithelial cell - C. albicans co-culture as a model of vulvovaginal infection .. 21 6.2. Primary analysis of transcriptome data ......................................................................... 21 6.3. Validation of RNA-Seq data by quantitative real-time PCR (QRT-PCR) .................... 23 6.4. Functional analysis of RNA-Seq data ........................................................................... 24 6.4.1. Analysis of signal transduction pathways involved in hyphal morphogenesis ....... 24 6.4.2. Expression analysis of genes involved in GlcNAc metabolism ............................. 26 6.4.3. Virulence of HXK1 mutant Candida albicans is decreased .................................... 27 6.4.4. GlcNAc is involved in the adherence of C. albicans to vaginal epithelial cells ..... 29 7. Results II .............................................................................................................................. 31 7.1. Detection of DEGs with microarray meta-analysis ....................................................... 31 7.2. General Network analysis .............................................................................................. 32 7.3. Determination of hubs in DEG-derived networks ......................................................... 33 7.4. Motif analysis in DEG-derived networks ...................................................................... 34 7.5. Controller sub-network construction ............................................................................. 35 7.6. Analysis of chemical-protein interaction networks ....................................................... 38 7.7. Effective drugs predominantly act on proteins of the controller sub-network .............. 42 8. Discussion ............................................................................................................................ 43 8.1. In vitro modelling vs. biopsy specimens ....................................................................... 43 8.1.1. Vulvovaginal Candidiasis ....................................................................................... 43 8.1.2. Psoriasis .................................................................................................................. 44 8.2. Filtering data and identification of important genes ...................................................... 44 8.2.1. Vulvovaginal candidiasis ........................................................................................ 44 8.2.2. Psoriasis .................................................................................................................. 44 8.3. Putting genes in context ................................................................................................. 45 8.3.1. Vulvovaginal candidiasis ........................................................................................ 45 8.3.2. Psoriasis .................................................................................................................
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