EHV-1 is a double-stranded DNA virus whereas EAV is a positive sense, single-stranded RNA virus. Therefore, genetically, they are very different from one another. However, both these viruses are endotheliotropic and thus, infect and replicates in equine endothelial cells resulting in vasculitis. Vasculitis is central to the pathogenesis of these two viruses. Thus, the main objective of this thesis was to investigate the inflammatory and innate immune responses of EECs that contribute towards the development of vasculitis following infection with EHV-1 and EAV in-vitro. Since proinflammatory cytokines and chemokines produced by endothelial cells play a significant role in the development of vasculitis, we investigated their gene expression as well as secretion. Results from this study showed that the proinflammatory response of EECs induced by EAV is relatively less when compared with the corresponding results from EHV-1 infected EECs. Furthermore, EAV elicits a lower type I interferon response in EECs when compared with EHV-1. Further investigations revealed an active role played by TLR 3 in inducing the proinflammatory response in EHV-1 infected EECs during the first 6 hours of infection but not in EAV infected EECs. Analyzing the whole transcriptome of EHV-1 and EAV infected EECs revealed a complex pattern of gene regulation and cellular pathways related to cellular immune, inflammatory and apoptotic responses. Finally, we investigated host genetic factors associated with EHV-1 induced myeloencephalopathy but found no evidence for a recessive allele influencing the development of EHM following EHV-1 infection for any genetic locus was identified. However, more complex host-pathogen interactions are possible.

The key to the complexity of higher organisms lies not in the number of protein coding genes they carry, but rather in the intrinsic complexity of the gene regulatory networks. The major effectors of transcriptional regulation are proteins called transcription factors, and in this thesis four papers describing genome-wide studies of seven such factors are presented, together with studies on components of the chromatin and transcriptome. In Paper I, we optimized a large-scale in vivo method, ChIP-chip, to study protein – DNA interactions using microarrays. The metabolic-disease related transcription factors USF1, HNF4a and FOXA2 were studied in 1 % of the genome, and a surprising number of binding sites were found, mostly far from annotated genes. In Paper II, a novel sequencing based method, ChIP-seq, was applied to FOXA2, HNF4a and GABPa, allowing a true genome-wide view of binding sites. A large overlap between the datasets were seen, and molecular interactions were verified in vivo. Using a ChIP-seq specific motif discovery method, we identified both the expected motifs and several for co-localized transcription factors. In Paper III, we identified and studied a novel transcription factor, ZBED6, using the ChIP-seq method. Here, we went from one known binding site to several hundred sites throughout the mouse genome. Finally, in Paper IV, we studied the chromatin landscape by deep sequencing of nucleosomal DNA, and further used RNA-sequencing to quantify expression levels, and extended the knowledge about the binding profiles for the transcription factors NFY and TCF7L2.

Domestic animals have become very different from their wild ancestors during domestication and animal breeding. This provides a good model to unravel the molecular mechanisms underlying phenotypic variation. In my thesis I have studied genes affecting two important traits, leanness in pigs and hair greying-associated melanoma in horses. In the first part of the thesis, I focused on an intronic mutation leading to more muscle growth and less fat deposition in domestic pigs to identify a transcription factor (TF) that binds to the regulatory element overlapping with the mutation. The aim has been to further study the function of the previously unknown TF in mouse myoblast cells and in insulin-producing cells (Paper I-III). We discovered a new TF ZBED6 binding to intron 3 of the IGF2 gene, in which a single nucleotide substitution in pigs abrogates the binding and causes increased leanness in domestic pigs. Silencing of ZBED6 expression in mouse myoblasts increased Igf2 expression, cell proliferation and migration, and myotube formation. This result is in line with the increased leanness phenotype in mutant pigs. Chromatin Immunoprecipitation-sequencing (ChIP-seq) using an anti-ZBED6 antibody identified 1200 ZBED6 target genes besides IGF2 and many are TFs controlling fundamental biological processes. In the first follow-up study we found ZBED6 mainly affected the expression of muscle protein genes by directly regulating Igf2 and Twist2 expression, in agreement with our previous observation of faster myotube formation in ZBED6-silenced cells. ChIP-seq with antibodies against six different histone modifications revealed that ZBED6 preferentially binds to active promoters and modulates transcriptional activity by a novel mechanism rather than by recruiting repressive histone modifications. The second follow-up study revealed that ZBED6 affects the morphology and insulin content and release in pancreatic ß cells. In the second part (Paper IV), we investigate the functional significance of an intronic duplication in the Syntaxin 17 (STX17) gene causing hair greying and melanoma in horses. We found two Microphtalmia-associated transcription factor (MITF) binding sites within the duplication and showed that the duplicated sequence up-regulates reporter gene expression in a melanocyte-specific manner both by reporter assays in mouse melanocytes and in transgenic zebrafish. These results established that the intronic duplication acts as a melanocyte-specific enhancer that becomes much stronger when it is duplicated.

Change in gene expression is a powerful tool for evolution, because seemingly small expression changes can contribute important steps towards adaptation without necessarily affecting the whole organism. There is still much to learn about how gene expression evolves on genome- and population-wide levels, especially in non-model organisms. This thesis addresses some important questions in gene expression evolution via the quantitative measurement of RNA and protein levels in birds. First, I confirmed the state of incomplete dosage compensation in birds by sequencing the transcriptome of collared flycatchers (Ficedula albicollis). I showed that pleiotropy governs the evolution of expression male-bias from the Z chromosome. Sex-linked genes in females were more highly expressed than half the male expression level, indicative of a partial up-regulation. A comparison with data from ostrich (Struthio camelus), a bird with non-degenerated sex chromosomes, showed that sex-linked expression male-bias evolved following sex chromosome degradation. Second, using a combination of RNA sequencing and proteome mass spectrometry in chicken (Gallus gallus), I asked whether complete dosage compensation was achieved through regulation at translation. I showed that this was not the case and that incomplete dosage compensation extends to the protein level in birds. In addition, sex-linked genes showed more often an increased amount of regulation at translational level than autosomal genes. Third, I investigated gene expression divergence between collared and pied flycatchers (Ficedula hypoleuca) using RNA sequencing in multiple tissues and individuals. Tissues differed in the degree of expression variance and in the number of divergent genes, which I identified using expression QST. Variance within species was negatively correlated with expression breadth and protein interactivity, indicating that evolutionary constraints act predominantly within interbreeding populations. Among genes unique to one of the species, I identified one gene, DPP7, falling into a large genomic deletion fixed in pied flycatchers. Fourth, I investigated allele-specific expression (ASE) in the two flycatcher populations. ASE was identified from genetic variants within transcripts using RNA sequencing reads. We developed a Bayesian negative binomial approach that gained statistical power by estimating expression variance from combined SNPs within a transcript and overdispersion from the whole dataset.

Alternative splicing (AS) is a major source of biological diversity and a crucial determinant of cell fate and identity. Characterizing the role of AS regulatory networks in physiological and pathological processes remains challenging. The work presented here addresses this challenge using systems biology analyses of AS regulatory networks in programmed cell death and neurological disorders. The first study describes a genome-wide screen based on splicing-sensitive reporters to identify factors that affect the AS of apoptosis regulators Bclx and Mcl1. The screen identified over 150 factors that affect apoptosis through modulating the pro- and anti-apoptotic splicing variants of these apoptosis regulators. This screen revealed a new functional connection between apoptosis regulation and cell-cycle control through an AS network. It also unearthed many disease-associated factors as AS effectors. The second study describes the functions of the Polyglutamine-binding protein 1 (PQBP1)-mediated AS regulatory network in neurological disorders. PQBP1 is a factor linked to intellectual disability and was unexpectedly identified as an AS effector from the screen described above. We found that PQBP1 influences the splicing of many mRNAs and is associated with a wide range of splicing factors. Depletion of PQBP1 in mouse primary cortical neurons caused defects in neurite outgrowth and altered AS of mRNAs enriched for functions in neuron projection regulation. Disease-mutants of PQBP1 lose associations with splicing factors and cannot complement the aberrant AS patterns and neuron morphology defects in PQBP1 depleted-neurons. This study revealed a novel function of PQBP1 in AS regulation associated with neurite outgrowth and indicated that aberrant AS underlies the pathology of PQBP1-related neurological disorders. A final study examines the dynamics of the Drosophila Sex-lethal AS regulation network using a combination of experimental tools and mathematical modeling. This study demonstrates that the features of Sxl AS regulation have great potentials in building synthetic memory circuits in mammalian cells to track cell fate. Collectively, this work describes the landscape of three diverse AS regulatory networks in various biological processes. The results and methods presented here contribute to our rapidly advancing knowledge of AS regulation in biology and human disease.

Gene-fusions are a prevalent class of genetic variants that are often employed as cancer biomarkers and therapeutic targets. In recent years, high-throughput sequencing of the cellular genome and transcriptome have emerged as a promising approach for the investigation of gene-fusions at the DNA and RNA level. Although, large volumes of sequencing data and complexity of gene-fusion structures presents unique computational challenges. This dissertation describes research that first addresses the bioinformatics challenges associated with the analysis of the massive volumes of sequencing data by developing bioinformatics pipeline and more applied integrated computational workflows. Application of high-throughput sequencing and the proposed bioinformatics approaches for the breast and ovarian cancer study reveals unexpected complex structures of gene-fusions and their functional significance in the onset and progression of cancer. Integrative analysis of gene-fusions at DNA and RNA level shows the key importance of the regulation of gene-fusion at the transcription level in cancer.

This is a two-part project. Proton pump inhibitors (PPIs) have been associated with increased risk of C. difficile infections and increased toxin production when combined with antimicrobial therapy. The first part of this project involved characterization of a hypervirulent NAP1 C. difficile strain, including genome sequencing and assembly, and the development of methods to study its transcriptomics using RNA-Seq, which will enable future researchers to study different expression patterns when toxigenic C. difficile is challenged with PPIs and/or antimicrobials in vitro. The second part of this project involved characterizing a clinical isolate of a NAP1 C. difficile displaying a markedly elevated MIC to metronidazole (MIC = 16 mg/mL), which initially exhibited MIC of 32 mg/mL. A method of obtaining a metronidazole-susceptible revertant from this isolate was developed and a revertant was obtained. The genomes of both isolates were sequenced, assembled, and aligned, then compared to each other for polymorphisms.

Nous proposons de modéliser les données issues des technologies de séquençage du transcriptome (RNA-Seq) à l'aide de la loi binomiale négative, et nous construisons des modèles de segmentation adaptés à leur étude à différentes échelles biologiques, dans le contexte où ces technologies sont devenues un outil précieux pour l'annotation de génome, l'analyse de l'expression des gènes, et la détection de nouveaux transcrits. Nous développons un algorithme de segmentation rapide pour analyser des séries à l'échelle du chromosome, et nous proposons deux méthodes pour l'estimation du nombre de segments, directement lié au nombre de gènes exprimés dans la cellule, qu'ils soient précédemment annotés ou détectés à cette même occasion. L'objectif d'annotation précise des gènes, et plus particulièrement de comparaison des sites de début et fin de transcription entre individus, nous amène naturellement à nous intéresser à la comparaison des localisations de ruptures dans des séries indépendantes. Nous construisons ainsi dans un cadre de segmentation bayésienne des outils de réponse à nos questions pour lesquels nous sommes capable de fournir des mesures d'incertitude. Nous illustrons nos modèles, tous implémentés dans des packages R, sur des données RNA-Seq provenant d'expériences sur la levure, et montrons par exemple que les frontières des introns sont conservées entre conditions tandis que les débuts et fin de transcriptions sont soumis à l'épissage différentiel.

This is a two-part project. Proton pump inhibitors (PPIs) have been associated with increased risk of C. difficile infections and increased toxin production when combined with antimicrobial therapy. The first part of this project involved characterization of a hypervirulent NAP1 C. difficile strain, including genome sequencing and assembly, and the development of methods to study its transcriptomics using RNA-Seq, which will enable future researchers to study different expression patterns when toxigenic C. difficile is challenged with PPIs and/or antimicrobials in vitro. The second part of this project involved characterizing a clinical isolate of a NAP1 C. difficile displaying a markedly elevated MIC to metronidazole (MIC = 16 mg/mL), which initially exhibited MIC of 32 mg/mL. A method of obtaining a metronidazole-susceptible revertant from this isolate was developed and a revertant was obtained. The genomes of both isolates were sequenced, assembled, and aligned, then compared to each other for polymorphisms.

The aim of this study was to gain insight into the nature of resistance genes and mechanisms of resistance present in different ascochyta blight (AB) resistant genotypes of lentil to efficiently select non-allelic AB resistance genes mediating different mechanisms of resistance for gene pyramiding. Recombinant inbred lines (RILs) from all possible crosses among AB resistant Lens culinaris genotypes CDC Robin, 964a-46, ILL 7537 and ILL 1704 were subjected to allelism tests. Efforts were also made to understand the genetics of resistance in the L. ervoides accession L-01-827A. LR-18, a RIL population from the cross CDC Robin × 964a-46 was subjected to quantitative trait loci (QTL) mapping using a comprehensive genetic linkage map previously developed from polymorphic SNPs, SSRs and phenotypic markers. Results of allelism tests suggested that genes conditioning resistance to ascochyta blight in all lentil genotypes were non-allelic. Two complementary recessive resistance genes in L-01-827A were detected. QTL analysis indicated that CDC Robin and 964a-46 were different at two AB resistance QTLs. Histological tests suggested that cell death inhibition in CDC Robin, and reduced colonization of epidermal cells in 964a-46 might be the mechanisms of resistance in these genotypes. Comparing the expression of key genes in the salicylic acid (SA) and jasmonic acid (JA) signaling pathways of CDC Robin and 964a-46 suggested that the SA pathway was strongly triggered in 964a-46. However, the JA pathway was triggered in both, but at a lower expression level in 964a-46 than in CDC Robin. RNA-seq analysis revealed a number of candidate defense genes differentially expressed among genotypes with hypothetical actions in different layers of the plant defense machinery. The expression levels of the six candidate defense genes measured by quantitative real-time PCR analysis was correlated with those of RNA-seq. In conclusion, 964a-46 and CDC Robin mediated resistance to ascochyta blight through different resistant mechanisms, making them ideal candidates for resistance gene pyramiding. Gene pyramiding can be accelerated using closely linked markers to CDC Robin and 964a-46 resistance genes identified through QTL analysis.