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.

Submitted by VITO ANTONIO MASTROCHIRICO FILHO null (vito.oceano@gmail.com) on 2016-04-09T21:47:55Z No. of bitstreams: 1 Dissertação Repositório Vito.pdf: 3114594 bytes, checksum: fe4a528d166d3a59bfac7d3a244a016c (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-04-11T13:38:46Z (GMT) No. of bitstreams: 1 mastrochiricofilho_va_me_jabo.pdf: 3114594 bytes, checksum: fe4a528d166d3a59bfac7d3a244a016c (MD5) / Made available in DSpace on 2016-04-11T13:38:46Z (GMT). No. of bitstreams: 1 mastrochiricofilho_va_me_jabo.pdf: 3114594 bytes, checksum: fe4a528d166d3a59bfac7d3a244a016c (MD5) Previous issue date: 2016-02-29 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Pacu (Piaractus mesopotamicus) é uma espécie de peixe Neotropical amplamente distribuída nas bacias dos rios Paraná e Paraguai, e uma das espécies de peixe neotropicais de maior valor para a aquicultura. Uma melhor compreensão do genoma do pacu é necessária para o manejo genético na conservação dos estoques naturais e cultivados. O principal objetivo foi identificar SNPs (Single Nucleotide Polymorphisms) gene-associados no transcriptoma de fígado do pacu e, em seguida, aplicar em análises de variabilidade genética e de parentesco visando um manejo adequado desta importante espécie não modelo na aquicultura. O sequenciamento do transcriptoma foi realizado por meio da plataforma Roche/454, que resultou na formação de 4.110 contigs não redundantes. Destes, 2.051 genes foram identificados e funcionalmente anotados a fim de revelar genes relacionados às características econômicas interessantes para a aquicultura. Foram encontrados 464 SNPs localizados em 5’UTR (10.0%), 3’UTR (17.2%) e em regiões CDS (71,1%), e classificados como sinônimos (70,6%) e não sinônimos (29,4%). Foram genotipados 32 SNPs por meio da técnica Sequenom MassARRAY, dos quais alguns estavam relacionados com sistema imune. A variabilidade genética foi estimada em populações de indivíduos selvagens (Rio Paraná) e de indivíduos cultivados em sete pisciculturas do estado de São Paulo (FF1, FF2, FF3, FF4, FF5, FF6 e FF7). Não foram observadas diferenças significativas entre heterozigosidade observada (Hobs) e esperada (Hexp) para cada população. Análises de diferenciação genética mostraram baixo nível de estruturação genética entre as populações (Fst = 0.064, AMOVA = 93,59% da variação dentro de populações, P<0,05). Análises de parentesco mostraram que a maioria das estações de piscicultura possuíam pelo menos 40% de indivíduos aparentados, com risco de endogamia e necessidade de realização de um programa de acasalamentos direcionados. Nossos resultados proporcionaram importantes recursos genéticos para o pacu, com aplicabilidade para a aquicultura. / Pacu (Piaractus mesopotamicus) is a Neotropical freshwater fish widely distributed in Parana, Paraguay Basin. Wild populations of pacu are threatened by overfishing and it is one of the fish species of highest commercial value for aquaculture. An understanding of the pacu genome is appropriate to genetic management in the conservation of wild and cultivated stocks. The main objective was identify gene-associated SNPs in liver transcriptome of pacu. We used SNPs (Single Nucleotide Polymorphisms) to perform genetic variability and kinship analysis for suitable management of this important non-model species in aquaculture. Transcriptome sequencing was done with the Roche/454 technology and yielded 4,110 non-redundant contigs. Of these, 2,051 genes were identified and functionally annotated to reveal genes correlated to economical traits in aquaculture. We found 464 SNPs in 5’UTR (10.0%), 3’UTR (17.2%) and CDS (71,1%), classified in synonymous (70,6%) and non-synonymous (29,4%). We genotyped 32 feasible SNPs through Sequenom MassARRAY platform and we obtained some SNPs related to immune system. Genetic diversity was estimated in wild individuals (Parana river) and in seven farm fish populations (FF1, FF2, FF3, FF4, FF5, FF6 and FF7). There were no significant differences between observed heterozygosity (Hobs) and expected (Hexp) for each population; and also between observed heterozygosity, expected heterozygosity and minimum allele frequency (MAF), when the population averages were compared (P <0.05). In addition, genetic differentiation analyzes showed low genetic structure of wild and cultivated populations of pacu (Fst = 0.064; AMOVA = 93.59% of the variation within populations; P<0,05). Kinship analysis showed most hatchery stations had at least 40% of related individuals, at risk of inbreeding and the need to perform a directed mating program. Our results showed unprecedented genomic resources for pacu. / FAPESP: 2014/12412-4

Chez tout organisme, l’évolution et l’acclimatation aux changements du milieu de vie sont orchestrés par de nombreux acteurs moléculaires. Parmi eux, les facteurs de transcription (FTs) jouent un rôle clé en régulant l’expression des gènes. Identifier les FTs impliqués dans la production de composés d’intérêt est donc une étape importante dans un contexte biotechnologique. Le laboratoire dispose d’une souche mutante de la microalgue haptophyte Tisochrysis lutea produisant deux fois plus de lipides de réserve que la souche sauvage en condition de privation azotée. Compte tenu du rôle clé des FTs dans l’établissement du phénotype, cette thèse vise à identifier les FTs impliqués dans la mise en place de ce phénotype mutant.Un pipeline bio-informatique d’identification et classification des FTs présents dans le génome de T. lutea a été élaboré. Le manque de donnée chez les haptophytes constituant un vide dans l’étude de l’histoire évolutive des microalgues, une étude comparative des FTs présents dans le génome d’algues de différentes lignées a été réalisée. Celle-ci révèle que l’étude des FTs aide à comprendre et illustrer l’histoire évolutive des microalgues par la mise en évidence de présences/absences de familles de FTs spécifiques de lignée.Afin de comprendre l’établissement du phénotype de la souche mutante de T. lutea, des données transcriptomiques ont permis la construction de réseaux de co-expression et de régulation des gènes chez les deux souches. Leur analyse croisée a identifié sept FTs candidats potentiellement liés au phénotype mutant. Une approche de p-RT-PCR a confirmé l’implication de deux FTs dans la remobilisation de l’'azote en condition de stress azoté. / In every organism, evolution and acclimation to environmental changes are orchestrated by numerous molecular players. Among them, transcription factors (TFs) play a crucial role by regulating gene expression. Therefore, identify TFs involved in the production of high value products is a significant step in a biotechnological context. The laboratory has at its disposal a mutant strain of the haptophyte microalga Tisochrysis lutea producing twice more storage lipids than the wild type strain when exposed to nitrogen deprivation. Given the key role of TFs in phenotype establishment, this PhD aim at identify the TFs involved in that of the mutant phenotype of T. lutea.A TFs identification and classification pipeline was elaborated and applied to T. lutea’s genome. Since the lack of data in haptophytes constitutes a limit in studies on microalgae evolutionary history, a comparative study of TFs identified in the genome of microalgae belonging to different lineages was carried out. This study reveals that TFs could be used to understand and illustrate microalgae evolutionary history through the highlight of lineage specific presence/absence of TF families.Aiming at understanding T. lutea’s mutant strain phenotype establishment, transcriptomic data were used to build gene co-expression networks and gene regulatory networks for both strains. Their comparative analysis identified seven TFs potentially liked to the mutant phenotype. A q-RT-PCR approach confirmed the involvement of two TFs in nitrogen recycling under nitrogen deprivation.

Orientador: Ney Lemke / Resumo: MicroRNAs (miRNAs) são pequenas moléculas de RNA que regulam pós-transcricionalmente a expressão de genes, modelando o transcriptoma e a produção de proteínas. Em geral, os miRNAs são conservados no genoma de eucariotos, sendo considerados elementos vitais em diversos processos biológicos durante o desenvolvimento, tais como crescimento, diferenciação e morte celular. A grande diversidade de miRNAs identificados está restrita a poucas espécies e apenas uma parte do total de alvos de miRNAs preditos foi caracterizada funcionalmente. Nesse contexto, o uso da tecnologia de sequenciamento de alto rendimento (high throughput sequencing) atrelada à análise de nível transcricional por RT-qPCR possibilitam a identificação do microRNoma. A tilápia do Nilo, Oreochromis niloticus, é considerada um excelente modelo biológico para o estudo de miRNAs em vertebrados devido à sua importância econômica e evolutiva. O presente trabalho teve como objetivos: organizar os dados do sequenciamento dos miRNAs da tilapia do Nilo; disponibilizá-los em forma de uma base de dados para a comunidade científica; integrar as informações dos miRNAs identificados com outros bancos de dados de miRNAs; analisar os dados através de análises de bioinformática para determinação de agrupamentos definidos pelo nível de expressão de cada miRNA em seis tipos de tecido (músculo branco, músculo vermelho, testículo, ovário, fígado, olho, cérebro e coração) com distinção entre os gêneros e nas fases do desenvolvimento (2,... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor