A ampla diversidade metabólica das cianobactérias é associada não somente a sua importância nos ciclos biogeoquímicos, mas também a sua distribuição global. Tal característica também é responsável pela capacidade destes organismos em produzir uma ampla variedade de substâncias de estruturas incomuns e atividades de interesse para o homem. Microcystis é um gênero cianobacteriano reconhecido como produtor de mais de duas centenas de produtos naturais, incluindo cianotoxinas. Microcystis aeruginosa é uma espécie frequentemente encontrada em florações, portanto causando preocupações sobre sua influência ecológica, especialmente em corpos d\'água doce utilizados para consumo humano. Desta forma, o objetivo deste trabalho foi o levantamento da diversidade e quantidade de metabólitos secundários que podem ser produzidos pela espécie, através de análises genômicas, além de variáveis que podem potencialmente interferir nas análises computacionais, procurando-se por padrões na espécie, e comparando-se 18 linhagens de todos os continentes. Foi encontrado o total de 235 agrupamentos relacionados ao metabolismo secundário, categorizados em 12 classes segundo as estruturas de seus produtos, nas 18 linhagens, evidenciando a riqueza de agrupamentos relacionados ao metabolismo secundário encontrados nesta espécie. Destes agrupamentos, os mais abundantes pertencem às categorias dos Terpenos, Híbridos, Bacteriocinas e NRPS. Entre as NRPS, nenhuma foi comum a todas as linhagens. Ainda, a quantidade de agrupamentos variou entre 6 e 21, e a quantidade de categorias de produtos variou entre 4 e 10, mostrando uma distribuição heterogênea de agrupamentos e tipos de metabólitos preditos. Esta distribuição heterogênea foi detalhada para melhor compreensão deste padrão encontrado na espécie. Dos agrupamentos de NRPS, os três mais frequentes foram selecionados para uma análise pormenorizada de sua estrutura e sequência: aeruginosina (15 linhagens), microcistina (11 linhagens), e micropeptina (15 linhagens). O agrupamento de micropeptina encontrado nas linhagens SPC777, TAIHU98 e PCC 9806 se mostrou amplamente dissimilar com relação à referência utilizada, potencialmente indicando um erro de identificação causado pela plataforma antiSMASH utilizada para a localização dos agrupamentos. Análises de colinearidade genômica mostram uma baixíssima sintenia entre os genomas das linhagens em análise, sugerindo frequentes eventos de reorganização genômica. Ainda, análises de pangenoma mostram um cenário em que mais genomas desta espécie são necessários para a estimativa da quantidade total de genes diferentes que a espécie pode possuir, o que é interessante para futuros estudos de procura de metabólitos secundários. Análises do genoma cerne apontam para uma estimativa segura de 1.944 genes comuns a todos os genomas desta espécie, o que corresponde entre 35% e 50% dos genes em cada linhagem. Análises estatísticas apontam para diferentes graus de interferência não linear da quantidade de sequências contíguas na observação de diferentes padrões de outras características genômicas, sugerindo precaução nas expectativas com relação ao metabolismo secundário em caso de linhagens em que a montagem gênica ultrapasse o limite superior aproximado de 100 sequências contíguas. / The wide metabolic diversity of cyanobacteria is associated not only with their importance in biogeochemical cycles, but also with their global distribution. Such a feature is also responsible for the ability of these organisms to produce a wide variety of substances with unusual structures and activities of interest to man. Microcystis is a cyanobacterial genus recognized as a producer of more than two hundred natural products, including cyanotoxins. Microcystis aeruginosa is a species frequently found in cyanobacterial blooms, thus causing concerns about its ecological influence, especially in freshwater bodies used for human consumption. In this way, the objective of this work was the survey of the diversity and quantity of secondary metabolites that can be produced by the species, through genomic analyzes, besides variables that can potentially interfere in the computational analyzes, searching for patterns in the species, and comparing 18 strains from all the continents. A total of 235 clusters, categorized in 12 classes according to the structure of their products, were found in the 18 strains, evidencing the richness of clusters related to the secondary metabolism found in this species. Of these clusters, the most abundant belong to the categories of Terpenes, Hybrids, Bacteriocins and NRPS. Among NRPS, none were common to all strains. Also, the number of groups ranged from 6 to 21, and the number of product categories ranged from 4 to 10, showing a heterogeneous distribution of predicted groupings and types of metabolites. Such a heterogeneous distribution was detailed for a better understanding of this pattern found in the species. Of the NRPS clusters, the three most frequent were selected for a detailed analysis of their structure and sequence: aeruginosin (15 strains), microcystin (11 strains), and micropeptin (15 strains). The micropeptide cluster found in the SPC777, TAIHU98 and PCC 9806 strains was widely dissimilar to the reference, só potentially indicating an identification error caused by the antiSMASH platform used to locate the clusters. Genomic collinearity analyzes showed a very low synteny among the genomes of the strains under analysis, suggesting frequent events of genomic reorganization. Also, pangenome analyzes show a scenario in which more genomes of this species are needed for the estimation of the total amount of different genes the species may possess, which is interesting for future studies conserning secondary metabolites. Coregenome analyzes point to a reliable estimate of 1,944 genes common to all genomes of this species, which corresponds to 30% up to 50% of the genes in each strain. Statistical analyzes point to different degrees of non-linear interference of the number of contiguous sequences on the observation of different patterns of other genomic characteristics, suggesting necessary caution about expectations regarding the secondary metabolism in case of strains in which the gene assembly exceeds the approximate upper limit of 100 contiguous sequences.

A ampla diversidade metabólica das cianobactérias é associada não somente a sua importância nos ciclos biogeoquímicos, mas também a sua distribuição global. Tal característica também é responsável pela capacidade destes organismos em produzir uma ampla variedade de substâncias de estruturas incomuns e atividades de interesse para o homem. Microcystis é um gênero cianobacteriano reconhecido como produtor de mais de duas centenas de produtos naturais, incluindo cianotoxinas. Microcystis aeruginosa é uma espécie frequentemente encontrada em florações, portanto causando preocupações sobre sua influência ecológica, especialmente em corpos d\'água doce utilizados para consumo humano. Desta forma, o objetivo deste trabalho foi o levantamento da diversidade e quantidade de metabólitos secundários que podem ser produzidos pela espécie, através de análises genômicas, além de variáveis que podem potencialmente interferir nas análises computacionais, procurando-se por padrões na espécie, e comparando-se 18 linhagens de todos os continentes. Foi encontrado o total de 235 agrupamentos relacionados ao metabolismo secundário, categorizados em 12 classes segundo as estruturas de seus produtos, nas 18 linhagens, evidenciando a riqueza de agrupamentos relacionados ao metabolismo secundário encontrados nesta espécie. Destes agrupamentos, os mais abundantes pertencem às categorias dos Terpenos, Híbridos, Bacteriocinas e NRPS. Entre as NRPS, nenhuma foi comum a todas as linhagens. Ainda, a quantidade de agrupamentos variou entre 6 e 21, e a quantidade de categorias de produtos variou entre 4 e 10, mostrando uma distribuição heterogênea de agrupamentos e tipos de metabólitos preditos. Esta distribuição heterogênea foi detalhada para melhor compreensão deste padrão encontrado na espécie. Dos agrupamentos de NRPS, os três mais frequentes foram selecionados para uma análise pormenorizada de sua estrutura e sequência: aeruginosina (15 linhagens), microcistina (11 linhagens), e micropeptina (15 linhagens). O agrupamento de micropeptina encontrado nas linhagens SPC777, TAIHU98 e PCC 9806 se mostrou amplamente dissimilar com relação à referência utilizada, potencialmente indicando um erro de identificação causado pela plataforma antiSMASH utilizada para a localização dos agrupamentos. Análises de colinearidade genômica mostram uma baixíssima sintenia entre os genomas das linhagens em análise, sugerindo frequentes eventos de reorganização genômica. Ainda, análises de pangenoma mostram um cenário em que mais genomas desta espécie são necessários para a estimativa da quantidade total de genes diferentes que a espécie pode possuir, o que é interessante para futuros estudos de procura de metabólitos secundários. Análises do genoma cerne apontam para uma estimativa segura de 1.944 genes comuns a todos os genomas desta espécie, o que corresponde entre 35% e 50% dos genes em cada linhagem. Análises estatísticas apontam para diferentes graus de interferência não linear da quantidade de sequências contíguas na observação de diferentes padrões de outras características genômicas, sugerindo precaução nas expectativas com relação ao metabolismo secundário em caso de linhagens em que a montagem gênica ultrapasse o limite superior aproximado de 100 sequências contíguas. / The wide metabolic diversity of cyanobacteria is associated not only with their importance in biogeochemical cycles, but also with their global distribution. Such a feature is also responsible for the ability of these organisms to produce a wide variety of substances with unusual structures and activities of interest to man. Microcystis is a cyanobacterial genus recognized as a producer of more than two hundred natural products, including cyanotoxins. Microcystis aeruginosa is a species frequently found in cyanobacterial blooms, thus causing concerns about its ecological influence, especially in freshwater bodies used for human consumption. In this way, the objective of this work was the survey of the diversity and quantity of secondary metabolites that can be produced by the species, through genomic analyzes, besides variables that can potentially interfere in the computational analyzes, searching for patterns in the species, and comparing 18 strains from all the continents. A total of 235 clusters, categorized in 12 classes according to the structure of their products, were found in the 18 strains, evidencing the richness of clusters related to the secondary metabolism found in this species. Of these clusters, the most abundant belong to the categories of Terpenes, Hybrids, Bacteriocins and NRPS. Among NRPS, none were common to all strains. Also, the number of groups ranged from 6 to 21, and the number of product categories ranged from 4 to 10, showing a heterogeneous distribution of predicted groupings and types of metabolites. Such a heterogeneous distribution was detailed for a better understanding of this pattern found in the species. Of the NRPS clusters, the three most frequent were selected for a detailed analysis of their structure and sequence: aeruginosin (15 strains), microcystin (11 strains), and micropeptin (15 strains). The micropeptide cluster found in the SPC777, TAIHU98 and PCC 9806 strains was widely dissimilar to the reference, só potentially indicating an identification error caused by the antiSMASH platform used to locate the clusters. Genomic collinearity analyzes showed a very low synteny among the genomes of the strains under analysis, suggesting frequent events of genomic reorganization. Also, pangenome analyzes show a scenario in which more genomes of this species are needed for the estimation of the total amount of different genes the species may possess, which is interesting for future studies conserning secondary metabolites. Coregenome analyzes point to a reliable estimate of 1,944 genes common to all genomes of this species, which corresponds to 30% up to 50% of the genes in each strain. Statistical analyzes point to different degrees of non-linear interference of the number of contiguous sequences on the observation of different patterns of other genomic characteristics, suggesting necessary caution about expectations regarding the secondary metabolism in case of strains in which the gene assembly exceeds the approximate upper limit of 100 contiguous sequences.

Mycoplasma hyopneumoniae, Mycoplasma flocculare e Mycoplasma hyorhinis são capazes de aderir e colonizar o trato respiratório de suínos. Enquanto a presença de M. flocculare é considerada assintomática, M. hyopneumoniae e M. hyorhynis são relacionados ao desenvolvimento de patologias. M. hyopneumoniae é o agente etiológico da pneumonia enzoótica suína e M. hyorhynis além dos pulmões pode atingir outros sítios e hospedeiros, estando relacionado a artrites, poliserosites e desenvolvimento de vários tipos de câncer em humanos. Apesar dos avanços tecnológicos na área de genômica, raros são os dados quanto ao papel de M. flocculare no trato respiratório suíno. Além do mais, informações relativas à transcrição gênica nessas espécies são escassas, apesar da importância desses microrganismos. Neste estudo são apresentados os dados da sequência do genoma de uma linhagem de M. flocculare, bem como do genoma de um novo isolado de M. hyopneumoniae. Com estas novas sequências foram realizadas análises de genômica comparativa visando a identificação de características que pudessem explicar os diferentes comportamentos quanto à patogenicidade dessas espécies. Além disso, a análise global dos transcritomas de cada uma das espécies foi realizada e o perfil transcricional entre M. hyopneumoniae, M. flocculare e M. hyorhynis foi analisado comparativamente objetivando identificar características peculiares para cada um dos mapas transcricionais, além de compreender a coordenação do modo de transcrição gênica em Mycoplasma. De um modo geral, as três espécies de Mycoplasma que habitam o trato respiratório suíno possuem grandes semelhanças na composição gênica, assim como na abundância de transcritos. A análise do repertório transcricional, mostra que os genomas são transcritos quase que em sua totalidade, incluindo as regiões intergênicas, nas três espécies. M. hyopneumoniae e M. flocculare apresentam conteúdo gênico e perfil transcricional muito semelhantes. Uma importante diferença encontrada entre estas duas espécies refere-se à presença exclusiva de genes e transcritos de adesinas específicas. M. hyorhynis possui genes e transcritos exclusivos, os quais sabidamente estão relacionados à sua capacidade mutacional, de invasividade e infecção de diferentes sítios. Por fim, a análise comparativa dos genomas, e a obtenção dos mapas transcricionais para M. hyopneumoniae, M. flocculare e M. hyorhynis, foram abordagens que resultaram em um grande número de informações, as quais são importantes para embasamento de futuros estudos de caracterização dos mecanismos moleculares, como os eventos de regulação da transcrição gênica, no gênero Mycoplasma. / Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare are able to adhere and to colonize the swine respiratory tract. While M. flocculare presence is virtually assymptomatic, M. hyopneumoniae and M. hyorhynis infections may cause respiratory disease. M. hyopneumoniae is the causative agent of swine enzootic pneumonia and M. hyorhynis may affect the lungs and other sites in a diversity of hosts and has been related to arthritis, poliserosites and to the development of several types of human cancer. Despite genomics technological advances, there are very few data about the possible role of M. flocculare in the swine respiratory tract. Moreover, little information about gene transcription is available in these species, despite the importance of these microorganisms. In this work the genome sequences of M. flocculare and a new isolate of M. hyopneumoniae are presented. A comparative genomic analyzes was performed to identify possible characteristics that may help to explain the different behaviors of these species in the swine respiratory tracts. Furthermore, a transcriptome map of each species was performed and a comparative transcriptional profile analysis between M. hyopneumoniae, M. flocculare and M. hyorhynis was undertaken to identify the exclusive features for each of the transcriptional maps, in addition to understanding the coordination mode of gene transcription in Mycoplasma. In general, the three Mycoplasma species that inhabit the swine respiratory tract have a similar gene composition as well as the abundance of transcripts. The transcriptome maps showed that most of the predicted genes are transcribed from these Mycoplasma genomes, as well as some intergenic regions. M. hyopneumoniae and M. flocculare present very similar gene content and transcriptional profile. However, an important difference between these two species is related to the exclusive presence of genes and transcripts of some specific adhesins. M. hyorhynis presents exclusive genes and transcripts that have been related to its invasiveness, mutation rate and infection of different sites. Finally, the comparative analysis of the genomes and transcriptional maps between M. hyopneumoniae, M. flocculare and M. hyorhynis have resulted in a large amount of information, which are important for future studies of the molecular characterization, as transcriptional regulation in the Mycoplasma spp.

Les phénomènes évolutifs qui perturbent l’organisation des gènes dans les génomes eucaryotes sont de deux types : les changements dans l’ordre des gènes, ou réarrangements, et les modifications du contenu en gènes du génome, par duplications, délétions ou gains de gènes. Ces processus sont mal connus, tant au niveau de leurs mécanismes d’apparition que de leur impact fonctionnel et sélectif. Ce travail de thèse s’articule autour de deux projets. Le premier s’intéresse à la distribution des points de cassure de réarrangements évolutifs entre un génome ancestral et ses descendants modernes. Cette distribution a été modélisée en fonction des caractéristiques locales du génome pour mettre en évidence quels facteurs influencent la probabilité de cassure. Nos résultats montrent que la distribution des cassures peut s’expliquer simplement comme une fonction de la longueur des espaces intergéniques, fonction qui est cependant non-linéaire contrairement aux attentes sous un régime aléatoire classique. La répartition des points de cassure dans les génomes semble principalement liée à des propriétés de structure, et n’est que peu soumise à des contraintes de sélection. Elle pourrait être liée à la structure chromatinienne du génome. Le second projet s’inscrit dans le cadre du séquençage du génome du poisson zèbre, et fournit un aperçu global de l’organisation de ce génome. Les génomes de poissons téléostéens sont anciennement dupliqués : l’analyse est axée sur les conséquences de cette duplication. Les résultats montrent que le génome du poisson zèbre présente une organisation assez typique d’un génome téléostéen. Les gènes retenus en deux copies après la duplication du génome appartiennent à des catégories fonctionnelles particulières, et sont biaisés vers des gènes déjà conservés après les duplications 1R et 2R ayant eu lieu au début de l’histoire des vertébrés. / Evolutionary processes disrupting the gene organisation in eukaryotic genomes belong to two categories: changes in the order of the genes, known as rearrangements, and changes in the content of the genome by gene duplications, deletions and gains. The mechanisms through which these events arise, and their functional and selective impact on genomes, are poorly understood. This thesis covers two different projects. Firstly, we investigated the distribution of rearrangement breakpoints between an ancestral genome and its modern descendants. This distribution was modelled according to local genomic characteristics to highlight factors influencing the breakage process. Our results show that the distribution of breakpoints can be simply explained as a function of intergenic spacers length, although in a non-linear fashion differing from classical random expectations. The repartition of breakpoints in genomes seems to be linked to structural properties, and is only marginally affected by selective constraints. It might in fact reflect local chromatin structure in the genome. The second project is part of the joint sequencing effort for the zebrafish genome, and provides an overview of the organisation of this genome. Teleost fish genomes are anciently duplicated: the analysis focuses on the consequences of this duplication. Results show that the zebrafish genome displays a typical teleost fish genome organisation. Genes retained in two copies after the whole genome duplication belong to specific functional categories, and are biased towards genes already conserved as duplicates after the 1R and 2R duplication events that have taken place early in vertebrate history.

Dermatófitos são fungos filamentosos que infectam substratos queratinizados como pele, unha e cabelo em busca de nutrientes para se desenvolverem e permanecerem no hospedeiro. Pertencem aos gêneros Epidermophyton, Microsporum ou Trichophyton, os quais, dependendo de seu habitat natural, são classificados em espécies geofílicas, zoofílicas ou antropofílicas. O uso indiscriminado de antifúngicos levou à seleção de cepas resistentes, e o comportamento invasivo desses patógenos em pacientes imunodeprimidos aumentou nos últimos anos, dificultando o tratamento das dermatofitoses. Há, portanto, a necessidade de estudos para um melhor entendimento da biologia dos dermatófitos devido as suas importâncias médica e/ou veterinária e o escasso conhecimento da interação destes patógenos com os hospedeiros. No presente trabalho, analisamos oito espécies de dermatófitos: Arthroderma benhamiae, Microsporum canis, Microsporum gypseum, Trichophyton interdigitale, Trichophyton equinum, Trichophyton rubrum, Trichophyton tonsurans e Trichophyton verrucosum. Análises de genômica comparativa e de expressão de genes potencialmente envolvidos na degradação de queratina foram realizadas. Além disso, efetuamos o sequenciamento genômico em larga escala de uma das linhagens. A estrutura dos genes sub3, sub5 e sub7, que codificam serina endopeptidases com atividade queratinolítica, mep3 e mep4, que codificam proteínas pertencentes ao grupo das metaloendopeptidases, dppV, lap1 e lap2, que codificam exopeptidases, foi analisada por meio de ferramentas computacionais. Essas análises revelaram que os genes que codificam proteases possuem alto grau de conservação em suas estruturas, que é menor quando comparadas apenas suas regiões não codificadoras. As análises permitiram também a identificação em regiões promotoras de consensos específicos a gêneros de dermatófitos. Observamos que o acúmulo de transcritos destes genes, avaliados durante o cultivo em queratina, mimetizando o processo infeccioso, não está correlacionado à similaridade das sequências gênicas entre as espécies. Não encontramos correlação entre o nicho preferencial dos dermatófitos e suas sequências gênicas ou níveis transcricionais. Observamos que, na grande maioria das vezes, genes que codificam endo e exopeptidases, possuem acúmulo de transcritos em períodos iniciais de degradação de queratina. Nossos resultados sugerem que diferenças pontuais na sequencia gênica, diferenças em regiões promotoras ou, até mesmo, expressão variável destes genes que codificam um conjunto proteico com funções sinérgicas e provavelmente compensatórias, contribuam para os diferentes graus de reações inflamatórias no hospedeiro, bem como para a especificidade patógeno-hospedeiro. / Dermatophytes are filamentous fungi that infect keratinized substrates such as skin, nail and hair, searching for nutrients for their development and permanence in the host. They belong to the genera Epidermophyton, Microsporum or Trichophyton, and, depending on their natural habitat, are classified into geophilics, zoophilics or anthropophilics species. The indiscriminate use of antifungals has led to the selection of resistant strains, and the invasive behaviour of these pathogens in immunocompromised patients increased in the last years, hampering the treatment of the dermatophytoses. Therefore, there is a need of studies for a better understanding of the biology of the dermatophytes due to their medical and/or veterinary importance and the scarce knowledge about the interaction of these pathogens with their hosts. In this work, we analyzed eight species of dermatophytes: Arthroderma benhamiae, Microsporum canis, Microsporum gypseum, Trichophyton interdigitale, Trichophyton equinum, Trichophyton rubrum, Trichophyton tonsurans, and Trichophyton verrucosum. Comparative genomics and gene expression analyses of genes potentially involved in keratin degradation were performed. Moreover, we performed a large-scale genome sequencing of one of the strains. The structure of the genes sub3, sub5, and sub7, which encode serine endopeptidases with keratinolytic activity, mep3, and mep4, which encode proteins belonging to the group of the metalloendopeptidases, dppV, lap1, and lap2, encoding exopeptidases, were analyzed by computational tools. These analyses revealed that the genes encoding proteases possesses high degree of conservation in their structures, which are lower when their non-coding regions are compared. The analyses also allowed the identification of consensus in promoter regions, specific of dermatophytes genera. We observed that the transcripts accumulation of these genes, evaluated during the cultivation in keratin, mimicking the infection process, is not correlated to the gene sequence similarities among the species. We have not found any correlation between the preferential niche of dermatophytes and their gene sequences or transcription levels. Most of the times, we observed that genes encoding endo and exopeptidases accumulated transcripts at the beginning of keratin degradation. Our results suggest that specific differences in the genic sequencing, differences in promoter regions, or even variable expression of these genes encoding a set of proteins with synergic and probably compensatory functions, contribute to different levels of inflammatory reactions in the host, as well as to the host-pathogen specificity.

Potato cyst nematodes (PCN) are economically relevant plant parasites that infect potato crops. The genomes of three PCN species are available and genome data have been generated for several populations of PCN, to address questions related to the molecular basis of plant parasitism. In this thesis, I employ approaches of comparative genomics to highlight differences and similarities between PCNs and other nematode species. I present two new software solutions to address challenges associated with the field of comparative genomics: BlobTools, a taxonomic interrogation toolkit for quality control of genome assemblies, and KinFin, a solution for the analysis of protein orthology data. I apply both software solutions to genomic datasets of nematodes, platyhelminths, and tardigrades. Based on KinFin analysis of plant parasitic nematodes, I identify protein families in PCNs likely to be involved in host-parasitic interaction, termed effectors, and discuss their functions. I highlight examples of horizontal gene transfer from bacteria to plant parasitic nematodes. Through genomic data of European and South American populations of PCNs, I address variation in populations, infer phylogenetic relationships, and try to estimate the effect of selection on effector genes identified through KinFin. Furthermore, I estimate the rate of variation across the reference genomes of two PCNs.

La vigne (Vitis vinifera) possède un métabolisme secondaire particulièrement riche donnant naissance à une large palette de molécules dont certaines sont impliquées dans les défenses contre les pathogènes et d'autres dans la grande diversité d’arômes qui fait la renommée des vins. L’analyse de la séquence de référence du génome de la vigne a permis de mettre en évidence une remarquable expansion de certaines familles de gènes liés au métabolisme secondaire par rapport aux autres plantes. Dans ce travail, j'ai étudié les familles gènes codant pour les cytochromes P450, dont certains sont impliqués dans la production d’arômes, les gènes codant pour les stilbènes synthases (STS), les endo-β-1,3-glucanases et les gènes de résistance de type NBS impliqués dans les défenses de la vigne. Ma thèse vise à proposer des hypothèses expliquant l’organisation structurale de ces familles de gènes et ainsi à mieux comprendre pourquoi certaines familles présentent une amplification dans le génome de la vigne. Des approches bioinformatiques ont été utilisées afin d’étudier ces différentes familles de gènes. Les gènes cytochromes P450 et gènes R de type NBS ont tout d'abord été annotés de manière manuelle dans le génome de référence de la vigne. L’expression des gènes endo-β-1,3- glucanases, STS et cytochromes P450 a été analysée en utilisant une approche transcriptomique à grande échelle. Pour ce faire, un outil a été développé durant cette thèse pour estimer le niveau d’expression des gènes à partir de données RNA-Seq disponibles dans les banques de données publiques. Parallèlement, des données de reséquençage d’ADN de 56 cépages et espèces de vigne ont été analysées, afin de déterminer les variations structurales de type CNV au sein des familles de gènes à domaine NBS et de gènes STS. Ces différents travaux ont permis de montrer que l’amplification des familles de gènes étudiées n’est pas spécifique du génome de référence mais est retrouvée dans l'ensemble du genre Vitis, mais également de mettre en évidence des variations structurales au sein des différents génomes étudiés. L'analyse de la famille STS a montré que ces gènes sont organisés en blocs de duplication, et que les gènes plus conservés sont aussi les plus exprimés. Nous avons également montré que les gènes à domaine NBS sont organisés en cluster, dont certains sont particulièrement soumis à variation. Ces travaux contribuent à une meilleure connaissance de facteurs de défense efficaces et durables ainsi que des gènes impliqués dans la synthèse d’arômes dans la vigne. Ces connaissances pourront bénéficier aux programmes de création variétale mis en œuvre à l’INRA de Colmar. / Grapevine (Vitis vinifera) has a particularly rich secondary metabolism, giving rise to a wide range of molecules, some of which are involved in defences against pathogens and others in the great diversity of aromas that make wines famous. Analysis of grapevine reference genome has shown a remarkable expansion of certain families of genes linked to secondary metabolism in comparison with the other plants. In this work, I have analysed gene families coding for cytochromes P450, some of them being involved in the production of aromas, genes coding for stilbene synthases (STS), endo-β-1,3-glucanases and NBS type resistance genes involved in grapevine defences. My thesis intends to propose hypothesis to explain the structural organisation of these families and therefore better understand why some of these families are amplified in the grapevine genome. Bioinformatic approaches have been used to study these different genes families. The cytochromes P450 and R genes of NBS type were manually annotated to improve the knowledge of these families of genes. The expression of endo-β-1,3-glucanases, STS and cytochromes P450 genes has been quantified using a large-scale transcriptomic approach. To this purpose, a tool has been developed during this thesis to estimate the level of genes expression from RNA- Seq data available in public databases. In the meantime, DNA resequencing data from 56 cultivars and grapevine species have been analysed to identify structural variations of CNV types within the genes with a NBS domain and the STS genes. These works showed that the amplification of the gene families of interest was not specific to the reference genome but occurred at the scale of the Vitis genus, but also to highlighted structural variations in different genomes. Regarding the STS genes, blocks of duplication and more conserved and expressed genes were identified. For the genes with NBS domain, a clustered organisation has been highlighted with some clusters varying more than others in the studied genotypes. These works contribute to a better knowledge of gene families for efficient and durable defence against pathogens and optimal aromas synthesis in grapevine. This knowledge will benefit to breeding programs currently in progress at INRA Colmar.

The identification of disease genes (genes that when mutated cause human diseases) is an important and challenging problem. Proper diagnosis, prevention, as well as care for patients require an understanding of disease pathophysiology, which is best understood when the underlying causative gene(s) or genetic element(s) are identified. While the availability of the sequenced human genome helped to lead to the discovery of more than 1,900 disease genes, the rate of disease gene discovery is still occurring at a slow pace. The use of genetic linkage methods have successfully led to the identification of numerous disease genes. However, linkage studies are ultimately restricted by available meioses (clinical samples) which result in numerous candidate disease genes. This thesis addresses candidate gene prioritizations in disease gene discovery as applied toward a genetically heterogeneous disease known as Bardet-Biedl Syndrome (BBS). Specifically, the integration of various functional information and the development of a novel comparative genomic approach (Computational Orthologous Prioritization - COP) that led to the identification of BBS3 and BBS11. Functional data integration and application of the COP method may be helpful toward the identification of other disease genes.

Genomic imprinting is responsible for monoallelic gene expression that depends on the sex of the parent from which the alleles (one active, one silent) were inherited. X-chromosome inactivation is also a form of monoallelic gene expression. One of the two X chromosomes is transcriptionally silenced in the somatic cells of females, effectively equalising gene dosage with males who have only one X chromosome that is not complemented by a gene poor Y chromosome. X chromosome inactivation is random in eutherian mammals, but imprinted in marsupials, and in the extraembryonic membranes of some placentals. Imprinting and X inactivation have been studied in great detail in placental mammals (particularly humans and mice), and appear to occur also in marsupial mammals. However, both phenomena appear to have evolved specifically in mammals, since there is no evidence of imprinting or X inactivation in non-mammalian vertebrates, which do not show parent of origin effects and possess different sex chromosomes and dosage compensation mechanisms to mammals.¶ In order to understand how imprinting and X inactivation evolved, I have focused on the mammals most distantly related to human and mouse. I compared the sequence, location and expression of genes from major imprinted domains, and genes that regulate genomic imprinting and X-chromosome inactivation in the three extant mammalian groups and other vertebrates. Specifically, I studied the evolution of an autosomal region that is imprinted in humans and mouse, the evolution of the X-linked region thought to control X inactivation, and the evolution of the genes thought to establish and control differential expression of various imprinted loci. This thesis is presented as a collection of research papers that examines each of these topics, and a review and discussion that synthesizes my findings.¶ The first paper reports a study of the imprinted locus responsible for the human Prader-Willi and Angelman syndromes (PWS and AS). A search for kangaroo and platypus orthologues of PWS-AS genes identified only the putative AS gene UBE3A, and showed it was in a completely different genomic context to that of humans and mice. The only PWS gene found in marsupials (SNRPN) was located in tandem with its ancient paralogue SNRPB, on a different chromosome to UBE3A. Monotremes apparently have no orthologue of SNRPN. The several intronless genes of the PWS-AS domain also have no orthologues in marsupials or monotremes or non-mammal vertebrates, but all have close paralogues scattered about the genome from which they evidently retrotransposed. UBE3A in marsupials and monotremes, and SNRPN in marsupials were found to be expressed from both alleles, so are not imprinted. Thus, the PWA-AS imprinted domain was assembled from many non-imprinted components relatively recently, demonstrating that the evolution of imprinting has been an ongoing process during mammalian radiation.¶ In the second paper, I examine the evolution of the X-inactivation centre, the key regulatory region responsible for X-chromosome inactivation in humans and mice, which is imprinted in mouse extraembryonic membranes. By sequencing and aligning flanking regions across the three mammal groups and non-mammal vertebrates, I discovered that the region homologous to the X-inactivation centre, though intact in birds and frogs, was disrupted independently in marsupial and monotreme mammals. I showed that the key regulatory RNA of this locus (X-inactive specific transcript or XIST) is absent, explaining why a decade-long search for marsupial XIST was unsuccessful. Thus, XIST is eutherian-specific and is therefore not a basic requirement for X-chromosome inactivation in all mammals.¶ The broader significance of the findings reported in these two papers is explored with respect to other current work regarding the evolution and construction of imprinted loci in mammals in the form of a review. This comparison enabled me to conclude that like the PWS-AS domain and the X-inactivation centre, many domains show unexpected construction from disparate genomic elements that correlate with their acquisition of imprinting.¶ The fourth and last paper examines the evolution of CCCTC-binding Factor (CTCF) and its parologue Brother Of Regulator of Imprinted Sites (BORIS) which contribute to the establishment and interpretation of genomic imprinting at the Insulin-Like Growth Factor 2/H19 locus. In this paper I show that the duplication of CTCF giving rise to BORIS occurred much earlier than previously recognised, and demonstrate that a major change in BORIS expression (restriction to the germline) occurred in concert with the evolution of genomic imprinting. The papers that form the bulk of this thesis show that the evolution of epigenetic traits such as genomic imprinting and X-chromosome inactivation is labile and has apparently responded rapidly to different selective pressures during the independent evolution of the three mammal groups. I have introduced these papers, and discussed them generally in terms of current theories of how and why these forms of monoallelic expression have evolved in mammals.

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