The main theme of this thesis research is concerned with developing a computational pipeline for processing Next-generation RNA sequencing (RNA-seq) data. RNA-seq experiments generate tens of millions of short reads for each DNA/RNA sample. The alignment of a large volume of short reads to a reference genome is a key step in NGS data analysis. Although storing alignment information in the Sequence Alignment/Map (SAM) or Binary SAM (BAM) format is now standard, biomedical researchers still have difficulty accessing useful information. In order to assist biomedical researchers to conveniently access essential information from NGS data files in SAM/BAM format, we have developed a Graphical User Interface (GUI) software tool named SAMMate to pipeline human transcriptome quantification. SAMMate allows researchers to easily process NGS data files in SAM/BAM format and is compatible with both single-end and paired-end sequencing technologies. It also allows researchers to accurately calculate gene expression abundance scores.

The goal of this thesis research was to develop software to be used with RNA-Seq data for transcriptome quantification that was capable of handling multireads and quantifying isoforms on a more global level. Current software available for these purposes uses various forms of parameter alteration in order to work with multireads. Many still analyze isoforms per gene or per researcher determined clusters as well. By doing so, the effects of multireads are diminished or possibly wrongly represented. To address this issue, two programs, GWIE and ChromIE, were developed based on a simple iterative EM-like algorithm with no parameter manipulation. These programs are used to produce accurate isoform expression levels.

Orientador: Fernando Sebastián Baldi Rey / Resumo: A compreensão de processos regulatórios e organização molecular dos organismos vivos progrediram consideravelmente na última década. As metodologias também evoluíram com o sequenciamento de DNA e RNA e de ferramentas genômicas permitindo a análise de centenas ou milhares de genes, proteínas ou metabólitos. O uso simultâneo dessas informações auxilia na obteção de informações relevantes sobre as variáveis que envolvem as variações fenotípicas de características de interesse. O objetivo do presente estudo foi integrar dados fenotípicos, genotípicos e transcriptômicos em busca de aprimoramento sobre os mecanismos genéticos e metabólicos que determinam o perfil de ácidos graxos na carne de bovinos Nelore, a fim de contribuir para o melhoramento da composição de ácidos graxos da carne. Foram utilizados machos da raça Nelore terminados em confinamento, abatidos com média de idade 24 meses. Amostras do músculo L. thoracis, entre a 12ª a 13ª costela foram coletadas para as análises de perfil de ácidos graxos, extração de RNA e de DNA. Os resultados foram apresentados nos capítulos 2 e 3. No capítulo 2, o objetivo foi identificar genes diferencialmente expressos pelo método RNA-seq e perfil de ácidos graxos no músculo L. thoracis com uso de componentes principais (principal components: PC). Foram selecionados dois grupos de 10 animais, os quais possuíam valores de PC1 e PC2 extremos (Alto x Baixo) para os grupos somatórios de ácidos graxos (AG): ácidos graxos saturados (AGS), ácidos g... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The understanding of regulatory processes and molecular organization of organisms has progressed considerably in the last 10 years.The methodologies also evolved with the sequencing of DNA, RNA and genomic tools allowing the analysis a lot of genes, proteins or metabolites. The simultaneous use of this information should help to obtain relevant information about the variables that result the phenotypic variations of traits of interest. The objective of the present study was to integrate phenotypic, genotypic and transcriptomic studies in order to clarify the genetic and metabolic mechanisms that determine the fatty acid profile in Nelore beef, in order to contribute to the improvement of the fatty acid composition of the meat. Nelore males were used in feedlot, coming from farms that integrate three breeding programs and slaughtered with an average of 24 months. Samples of the L. thoracis muscle between the 12th to 13th rib were collected for analysis of fatty acid profile, RNA and DNA extraction. The results were presented in chapters 2 and 3. In chapter 2, the objective was to identify genes differentially expressed by RNA-seq method and fatty acid profile in the L. thoracis muscle with the use of Principal Components (PC). Two groups of 10 animals were selected, which had PC1 and PC2 extreme values (High x Low) for the fatty acids (FA) groups: saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), omega 3 (n-3) and omega 6 (n-6... (Complete abstract click electronic access below) / Doutor

Le développement du séquençage ARN, ou RNAseq, a permis l'essor de la recherche intensive de biomarqueurs dans de nombreux domaines de la biologie. L’information complète du transcriptome contenue dans les données de sorties, permet à un bioinformaticien assidu de dépasser les connaissances actuelles et d’accéder, grâce à des pipelines informatiques avancés, à d’innombrables signatures d’intérêts inédites. Dans cette thèse nous mettons en avant que ces marqueurs potentiels, essentiellement explorés pour répondre à des problématiques clinique en conditions pathologiques, peuvent être utilisés pour affiner la caractérisation de types de cellules sans marqueurs strictement spécifiques. Nous nous sommes intéressés aux cellules souches mésenchymateuses (MSCs), un type de cellules souches adultes multipotentes, fortement utilisées en clinique mais ne possédant pas de marqueurs positifs strictement spécifiques.Notre étude se concentre sur la recherche des ARN longs non-codants non annotés. Ces ARNs, aussi nommés "lncRNA", constituent une classe émergente de transcrits encore peu explorée à ce jour. De plus, cette catégorie démontre une spécificité conditionnelle et tissulaire élevée. Nous avons élaboré un pipeline d’analyse RNAseq optimisé pour la reconstruction et la quantification de lncRNAs non annotés.En utilisant les données publiques de RNAseq, venant de différentes sources de MSCs et d'autres types de cellules, nous avons identifié de nouveaux lncRNA non annotés exprimés spécifiquement dans les MSCs.Nous avons développé pour ce projet Kmerator.jl, un outil qui permet de décomposer un transcrit en sous séquences spécifiques (k-mers) afin de chercher et quantifier plus rapidement la signature de nos candidats dans un grand nombre de données RNAseq. Kmerator a également été utilisé dans d'autres applications pour tester la qualité des données RNA-seq disponibles en accés public.Après validation de ces nouveaux biomarqueurs de MSCs par qPCR, nous avons eu recours à plusieurs outils informatiques pour prédire leurs fonctions potentielles. Enfin, nous avons analysé des données RNAseq « single-cell » pour aborder l’hétérogénéité d’expression au sein des populations MSCs. / The development of RNA sequencing, or RNAseq, have opened the path of intensive biomarkers research in many areas of biology. The complete information of the transcriptome contained in the output data, allows a bioinformatician to surpass the current knowledge and to access, thanks to advanced computer pipelines, to signatures of new interest. In this thesis, we are showing that these potential markers, classically used in clinical and pathological conditions, can be used to characterize cell types without extensive markers profile. We have studied mesenchymal stem cells, a type of adult multipotent stem cells, strongly used in clinics but without strickly specific positive markers. Our study mainly focuses on the search for non-annotated, long non-coding RNAs. These RNAs, also called "lncRNA", constitute an emerging class of transcripts and are still lightly explored.In addition, this category presents a highly tissue-related specificity. We have developed an optimized RNAseq pipeline for the reconstruction and quantification of non-annotated lncRNAs.Using public data from RNAseq, coming from different sources of MSC and other cell types, we have identified new non-annotated lncRNAs clearly and specifically expressed in MSCs. to complete this project, we developed Kmerator.jl, a bioinformatical tool that allows to decompose a transcript in k-mer, and select specific sub-sequences, in order to search and quantify at a faster rate the signature of our candidates in a large number of RNAseq dataset. After validation of these new biomarkers of MSCs by qPCR, we used several computer tools to predict their potential functions. Finally, we analyzed single-cell RNAseq data to address the heterogeneity of expression within MSC populations.

Beef quality in Brazil is important for both consumers and the food industry due to high demand and competitiveness in the domestic and international markets. Therefore, it is necessary to develop research to improve beef quality of Nellore cattle (Bos indicus), mainly tenderness, one of the main features to add value to meat. New-generation technologies provide accurate, rapid and inexpensive information on the entire genome, showing great advantage over conventional methods for sequencing and gene expression. However, these new technologies generate large database, which require the use of bioinformatics tools for data analyses of sequencing and for a better understanding of biological regulation mechanisms , cellular control, gene interactions, among other applications. In a previous study, samples were collected from the Longissimus dorsi muscle of 790 animals from Nellore cattle and shear force assessments were made 24 hours after slaughter, with seven and 14 days of aging. Aiming to identify differentially expressed (DE) genes, 34 samples from Nellore animals with extreme levels of estimated breeding value (EBV) for shear force (SF) were selected, sequenced by the method of RNA sequencing (RNA-Seq) (Illumina HiScanSQ). This study performed the processing of data generated by RNA-Seq using software QuasiSeq and Cuffdiff. In the QuasiSeq analysis, 22 DE genes were found, while in the Cuffdiff analysis, 113 DE genes were found. To better understand the biological process involved in meat tenderness, integrative analysis identified possible regulators that can explain the activity of transcriptional regulation in this process using partial correlation coefficient with information theory (PCIT), phenotypic impact factor (PIF) and regulatory impact factor (RIF) methods. The genes found in the PCIT analysis USP2, GBR10, ANO1 and TMBIM4; microRNAs found in RIF analysis bta-mir-133a-2 and bta-mir-22, and the genes with high PIF value MB, ENO3, CA3 could be fundamental to unravel the complex molecular mechanisms that control the meat tenderness in Nellore cattle. / A qualidade da carne bovina no Brasil é importante tanto para o consumidor, como para a indústria alimentícia devido à alta competitividade e exigência do mercado nacional e internacional. Portanto, é necessário o desenvolvimento de pesquisas para melhorar a qualidade da carne bovina da raça Nelore (Bos indicus), principalmente a maciez, que é considerada uma das principais responsáveis por agregar valor à carne. Tecnologias de nova geração proporcionam informações precisas, rápidas e baratas de todo genoma, mostrando grande vantagem em relação aos métodos convencionais de sequenciamento e de estudos de expressão gênica. Essas novas tecnologias geram um grande volume de dados, sendo necessário o uso de ferramentas de bioinformática para realizar as análises de sequenciamento e ter uma maior compreensão de mecanismos biológicos de regulação, controle celular, interações gênicas, entre outras aplicações. Em um estudo prévio, foram coletadas amostras do músculo Longissimus dorsi de 790 animais da raça Nelore e foram realizadas avaliações da força de cisalhamento 24 horas após abate, e com sete e 14 dias de maturação. Com o objetivo de identificar genes diferencialmente expressos (DE), foram selecionadas no total 34 amostras de animais da raça Nelore com valores extremos de valor genético estimado (EBV) para força de cisalhamento (SF), e sequenciados pelo método de sequenciamento de RNA (RNA-Seq) (Illumina HiScanSQ). Neste estudo foi realizado o processamento dos dados gerados pelo RNA-Seq através dos softwares QuasiSeq e Cuffdiff. Foram encontrados 22 genes DE para as análises do QuasiSeq e 113 genes DE para as análises do Cuffdiff. Para melhor compreensão dos processos biológicos envolvidos na maciez da carne, análises integrativas identificaram possíveis reguladores que podem explicar a atividade de regulação transcricional neste processo utilizando os métodos do Coeficiente de Correlação Parcial com Teoria da Informação (PCIT), Fator de Impacto Fenotípico (PIF) e Fator de Impacto Regulatório (RIF). Os genes encontrados nas análises análises do PCIT USP2, GBR10, ANO1 e TMBIM4, assim como os microRNAs encontrados nas análises do RIF, bta-mir-133a-2 e bta-mir-22 e os genes de maior valor de PIF MB, ENO3, CA3 podem ser fundamentais para desvendar os complexos mecanismos moleculares que controlam a maciez da carne na raça Nelore.

Les facteurs environnementaux, tels que les pesticides, peuvent induire des changements phénotypiques dans une variété d'organisme incluant les mammifères. Nous avons étudié chez la souris les effets d'un pesticide largement utilisé, l'atrazine (ATZ), sur la méiose, une étape clé du processus de spermatogenèse. L'utilisation des méthodes de puces à ADN (Gene-Chip) et de séquençage de chromatine immunoprécipité (ChIP-seq) nous a permis de mettre en évidence l'effet de l'ATZ sur une variété de fonctions cellulaires, incluant l'activité GTPase, la fonction mitochondriale et le métabolisme des hormones stéroïdes. De plus, les souris traitées présentent un enrichissement des marques d'histone H3K4me3 au niveau des régions de forte recombinaison (sites de cassures double brin) de gènes très long et une réduction de ces mêmes marques au niveau des régions pseudo-autosomal du chromosome X. Nos données démontrent que l'exposition à l'ATZ interfère avec le déroulement normal de la méiose, ceci affectant la production des spermatozoïdes. Nous avons trouvé que les marques H3K4me3, chez la souris mâle, sont largement affectées par l'ATZ grâce à l'utilisation de technique de séquençage du génome entier. La reprogrammation embryonnaire nécessite l'action coordonnée d'un grand nombre de gène et de facteurs épigénétiques afin de permettre la transition de cellules somatique en cellules germinales. Les modifications épigénétiques imposées pendant la transition des cellules somatiques en cellules germinales et affectées par des expositions nocives, peuvent être héritées et transmises aux générations suivantes via les gamètes. Dans cette étude, nous avons examiné l'héritage des histones modifié aux générations suivantes. Nous avons exposés des femelles gestantes CD1 non consanguines à l'ATZ et les mâles issus de ces femelles ont été croisés pendant trois générations avec des femelles non traitées. Nous avons démontré ici que l'exposition à l'ATZ réduit le nombre de spermatozoïdes sans affecter la morphologie cellulaire ou la proportion des différents types cellulaires constituant l'épithélium séminifère chez les individus issus de la 3ème génération après traitement. Beaucoup de gènes associés avec la réparation de l'ADN, la reproduction et les fonctions mitochondriales sont dérégulés chez les mâles issus de la 3ème génération après traitement. De façon importante, l'exposition à l'ATZ change dramatiquement l'initiation de la transcription, l'épissage et la polyadénylation alternative des ARN. Nous avons aussi observé chez les mâles F3 issus de souris traitées à l'ATZ une altération de la localisation des marques H3K4me3 dans le promoteur de gène associé à la régulation de processus métaboliques cellulaires, à la régulation de la transcription et à la mitose. Les changements de localisation des marques H3K4me3 chez les mâles F3 issus de souris traitées à l'ATZ correspondent à des changements de la localisation de ces marques au niveau de gènes impliqués dans la différenciation des cellules de type souche de la génération F1.Nos données suggèrent que l'héritage transgénérationnel est permis grâce à de multiples voies et repose sur le statut épigénétique de gènes impliqués dans la différenciation des cellules de type souches tels que Pou5f1 et Sox2, l'action des facteurs de transcription et la rétention d'histones dans le sperme. / Environmental factors such as pesticides can cause phenotypic changes in various organisms, including mammals. We studied the effects of the widely used herbicide atrazine (ATZ) on meiosis, a key step of gametogenesis, in male mice. We demonstrate that exposure to ATZ reduces testosterone levels and the number of spermatozoa in the epididymis and delays meiosis. Using Gene-Chip and ChIP-Seq analysis of H3K4me3 marks, we found that a broad range of cellular functions, including GTPase activity, mitochondrial function and steroid-hormone metabolism, are affected by ATZ. Furthermore, treated mice display enriched histone H3K4me3 marks in regions of strong recombination (double-strand break sites), within very large genes and reduced marks in the pseudoautosomal region of X chromosome. Our data demonstrate that atrazine exposure interferes with normal meiosis, which affects spermatozoa production.We found that the H3K4me3 marks in male mice are broadly affected by the widely used herbicide atrazine with genome wide ChIP-sequencing. Embryonic reprogramming requires the coordinated action of many genes and epigenetic factors to perform somatic to germline transition. The epigenetic modifications imposed during somatic to germline transition and affected by harmful exposure can be inherited and transferred to subsequent generations via the gametes. In this study, we examine the inheritance of altered histone modifications by subsequent generations. We exposed pregnant outbred CD1 female mice to the widely used herbicide atrazine (ATZ), and the male progeny were crossed for three generations with untreated females. We demonstrate here that exposure to ATZ reduces the number of spermatozoa without changing the cell morphology or types in testis tissue in the third generation after treatment. Many genes associated with DNA repair, reproduction and mitochondrial function became dysregulated in the third generation (F3) of males after treatment. Importantly, exposure to ATZ dramatically changes the transcription initiation, splicing and alternative polyadenylation of RNA. We also observed altered occupancy of H3K4me3 markers in the F3 generation of ATZ-derived males in gene promoters associated with the regulation of cellular metabolic processes, transcriptional regulation and mitosis. The changes in H3K4me3 occupancy in F3 ATZ-derived males correspond to changes in the H3K4me3 occupancy of stem cell differentiation genes in the F1 generation. Our data suggest that transgenerational inheritance is accomplished through multiple pathways and relies on the epigenetic state of stem cell differentiation genes such as Pou5f1 and Sox2, transcription factor action and sperm histone retention.

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.