Understanding the genetic basis of biological processes is a fundamental component of modern ecology and evolutionary biology studies. With the recent advent of next generation sequencing (NGS) technologies, it is now possible to perform large genome and transcriptome projects for ecologically important non-model species. In this thesis, I focused on the development and application of genomic resources of two non-model bird species, the black grouse (Tetrao tetrix) and the great snipe (Gallinago media). Using the chicken genome as a reference, I developed a reference guided NGS pipeline to assemble the complete draft genome of black grouse. The draft genome has a good coverage of the main 29 chromosomes of the chicken genome. The genome was used to develop a vast number of genetic markers. Comparing this genome with that of other species, I identified the genomic regions which were important for the lineage specific evolution of black grouse. I also sequenced and characterised the spleen transcriptome of the black grouse. I identified and validated a large number of gene-based microsatellite markers from the transcriptome and identified and confirmed the expression of immune related genes. Using a similar RNA-Seq approach, I also sequenced the blood transcriptomes of 14 great snipe males with different mating success. I identified genes and single nucleotide polymorphisms (SNPs) which might be related to male mating success in this species, both in terms of gene expression levels and genetic variation structure. For the immunologically important major histocompatibility complex (MHC) gene region of black grouse, I constructed a fosmid library and used it to sequence the complete core MHC region of this species. This resource allowed me to perform a comprehensive comparative genomics analysis of the galliform MHC, by which I found that some genes in this region were affected by selective forces. I was also able to develop a single locus genotyping protocol for the duplicated MHC BLB (class IIB) genes and found that the two black grouse BLB loci followed different evolutionary trajectories. This thesis set an example of developing genomic resources in non-model species and applying them in addressing questions relevant to ecology and evolutionary biology.

The study of variation found in DNA is fundamental in human genetic studies. Single nucleotide polymorphisms (SNPs) are simple to document because they can be captured in single DNA sequence reads. Larger structural variation including duplications, insertions, deletions, termed as copy number variation (CNV), inversions and translocations are more challenging to discover. Recent studies using microarray and sequencing technologies have demonstrated the prevalence of structural variation in humans. They can disrupt genic and regulatory sequences, be associated with disease, and fuel evolution. Therefore, it is important to identify and characterize both SNPs and structural variants to fully understand their impact. This thesis presents the analysis of structural variation in the human genome. The primary DNA sample used for my experiments is the DNA of J. Craig Venter, also termed HuRef. It was the first personal human genome sequenced. I combined computational re-analysis of sequence data with microarray-based analysis, and detected 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing study. The results indicated that the genomes of two individuals differed 1.3% by CNV, 0.3% by inversion and 0.1% by SNP. Structural variation discovery is dependent on the strategy used. No single approach can readily capture all types of variation, and a combination of strategies is required. I analyzed the formation mechanisms of all HuRef structural variants. The results showed that the relative proportion of mutational processes changed across size range: the majority of small variants (<1kb) were associated with nonhomologous processes and microsatellite events; median size variants (<10kb) were commonly related to minisatellites and retrotransposons; and large variants were associated with nonallelic homologous recombination. Eight new breakpoint-resolved HuRef inversions were genotyped in populations to elucidate these understudied variants. I discovered that the structures of inversion could be complex, could create conjoined genes, and their frequencies could exhibit population differentiation. The data here contributes to our understanding of structural variation in humans. It shows the need to use multiple strategies to identify variants, and it emphasizes the importance to examine the full complement of variation in all biomedical studies.

No description available.

Herpes simplex virus type 1 (HSV-1) is a double stranded DNA virus that causes epithelial skin infections and persists through the life of the host by infecting neurons, where it can switch to a latent state to evade an immune response. In rare cases during primary infection or after reactivation, instead of undergoing lytic infection at the epithelial surface, it instead travels to the brain and causes herpes simplex virus encephalitis (HSVE) which can have a ≥70% mortality rate if untreated. As the virus takes over its host cell, it gains control of the host cell machinery and manipulates host gene expression in order to evade the immune system and to pool its resources into the replication of the virus. One aspect of the dysregulated gene expression involves microRNAs (miRNAs). MiRNAs are short, non-coding RNAs that bind to the 3' untranslated region (3'UTR) of messenger RNAs (mRNAs), leading to translational repression of the target. Dysregulated miRNAs are often down-regulated during infection as the virus takes over, but many miRNAs have also been found to be up-regulated as well1–5. The aim of this study is to observe the full cellular miRNA changes in the context of an acute viral encephalitic infection using HSV-1, and to further characterize selected up-regulated miRNAs to determine their function in the context of the disease state. Of particular note were miR-141 and miR-200c which showed anti-apoptotic effects on neuronal cell culture and did not impact cell viability during an over-expression of the miRNAs. MiR-141, miR-183 and miR-200a expression was enriched within specific areas of the brain during infection. In addition, the potential for miR-150 to bind to a bioinformatically predicted target site within the shared 3'UTR of the HSV-1 UL18, UL19 and UL20 genes was explored. Examining the changes in expression of this class of regulatory RNAs and investigating their potential functions may yield new insight into the relationship between host and virus during infection.

In human microbiome analysis, sequencing of bacterial 16S rRNA genes has revealed a role for the gut microbiota in maintaining health and contributing to various pathologies. Novel community analysis techniques must be evaluated in terms of bias, sensitivity, and reproducibility and compared to existing techniques to be effectively implemented. Next- generation sequencing technologies offer many advantages over traditional fingerprinting methods, but this extensive evaluation required for the most efficacious use of data has not been performed previously. Illumina libraries were generated from the V3 region of the 16S rRNA gene of samples taken from 12 unique sites within the gastrointestinal tract for each of 4 individuals. Fingerprint data were generated from these samples and prominent bands were sequenced. Sequenced bands were matched with OTUs within their respective libraries. The results demonstrate that denaturing gradient gel electrophoresis (DGGE) represents relatively abundant bacterial taxa (>0.1%) beta-diversity of all samples was compared using Principal Coordinates Analysis (PCoA) of UniFrac distances and Multi-Response Permutation Procedure (MRPP) was applied to measure sample cluster strength and significance; indicator species analysis of fingerprint bands and Illumina OTUs were also compared. The results demonstrate overall similarities between community profiling methods but also indicate that sequence data were not subject to the same limitations observed with the DGGE method (i.e., only abundant taxa bands are resolved, unable to distinguish disparate samples). In addition, the effect of stochastic fluctuations in ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? differ for DGGE and next-generation sequencing. I compared pooled and individual reactions for samples of high and low template concentration for both Illumina and DGGE using the combined V3-V4 region of the 16S rRNA gene, and demonstrated that template concentration has a greater impact on reproducibility than pooling. This research shows congruity between two disparate molecular methods, identifies sources of bias, and establishes new guidelines for minimizing bias in microbial community analyses.

The study of variation found in DNA is fundamental in human genetic studies. Single nucleotide polymorphisms (SNPs) are simple to document because they can be captured in single DNA sequence reads. Larger structural variation including duplications, insertions, deletions, termed as copy number variation (CNV), inversions and translocations are more challenging to discover. Recent studies using microarray and sequencing technologies have demonstrated the prevalence of structural variation in humans. They can disrupt genic and regulatory sequences, be associated with disease, and fuel evolution. Therefore, it is important to identify and characterize both SNPs and structural variants to fully understand their impact. This thesis presents the analysis of structural variation in the human genome. The primary DNA sample used for my experiments is the DNA of J. Craig Venter, also termed HuRef. It was the first personal human genome sequenced. I combined computational re-analysis of sequence data with microarray-based analysis, and detected 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing study. The results indicated that the genomes of two individuals differed 1.3% by CNV, 0.3% by inversion and 0.1% by SNP. Structural variation discovery is dependent on the strategy used. No single approach can readily capture all types of variation, and a combination of strategies is required. I analyzed the formation mechanisms of all HuRef structural variants. The results showed that the relative proportion of mutational processes changed across size range: the majority of small variants (<1kb) were associated with nonhomologous processes and microsatellite events; median size variants (<10kb) were commonly related to minisatellites and retrotransposons; and large variants were associated with nonallelic homologous recombination. Eight new breakpoint-resolved HuRef inversions were genotyped in populations to elucidate these understudied variants. I discovered that the structures of inversion could be complex, could create conjoined genes, and their frequencies could exhibit population differentiation. The data here contributes to our understanding of structural variation in humans. It shows the need to use multiple strategies to identify variants, and it emphasizes the importance to examine the full complement of variation in all biomedical studies.

Orientador: Erick da Cruz Castelli / Resumo: O Complexo Principal de Histocompatibilidade (MHC) é um complexo gênico que está intimamente envolvido com a regulação do sistema imune. Esse complexo comporta o sistema de Antígenos Leucocitários Humano (HLA), cuja principal importância está relacionada com o reconhecimento do que é próprio ou não do organismo. HLA-C é o gene polimórfico menos variável dos genes HLA clássicos e o que tem menor expressão nos tecidos, exceto na interface materno-fetal, em que é o único gene clássico expresso. A molécula codificada por esse gene possui significante função na apresentação antigênica e regulação da atividade de células NK, o que permite uma íntima associação com situações fisiológicas, como gestação, e patológicas, como doenças infecciosas, autoimunes, inflamatórias, neoplasias e rejeições a enxertos transplantados. Sua porção gênica mais estudada é a que codifica a fenda de ligação a peptídeos antigênicos, devido sua destacada importância na apresentação de antígenos a células T citotóxicas. No entanto, outras regiões do gene, que são negligenciadas nos estudos de variabilidade, também merecem destaque por influenciarem na sinalização e modulação da citotoxicidade de células efetoras, na ancoragem e estabilidade da molécula na membrana plasmática e na internalização e reciclagem da molécula HLA-C. Desta maneira, nós exploramos a variabilidade dos segmentos que codificam α3 (éxon 4), transmembrana (éxon 5) and cauda citoplasmática (éxon 6 and éxon 7) da molécula HLA-C em uma popu... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Major Histocompatibility Complex (MHC) is a gene complex closely involved in the regulation of the immune system. This complex includes the Human Leukocyte Antigen (HLA) system, whose main role is related to the recognition of self/non-self structures of humans. HLA-C is the least variable polymorphic gene of classical HLA genes and has the lowest expression in tissues, except at the maternal-fetal interface, where it is the only classical HLA class I expressed gene. The molecule encoded by this gene has a significant role in the antigen presentation and regulation of NK cells activities, which allows an intimate association with physiological conditions, such as pregnancy, and pathological conditions like infectious, autoimmune, and inflammatory diseases, cancer, and transplantation rejection. The most studied HLA-C portion is that encoding the peptide-binding groove, due to its outstanding importance in presentation of antigens to cytotoxic T cells. However, other regions of the gene, which are neglected in the variability studies, are also important in influencing the signaling and modulation of effector cell cytotoxicity, in the anchorage and stability of the molecule on the cell surface, and in the internalization and recycling of the HLA-C molecule. Here, we explore the variability of the segments encoding the α3 (exon 4), transmembrane (exon 5) and cytoplasmic tail (exon 6 and exon 7) domains of the HLA-C molecule in an admixed population sample from Southeastern B... (Complete abstract click electronic access below) / Mestre

No description available.

En ville, les systèmes de récupération et d'infiltration des eaux pluviales dans le sous-sol ont pour conséquence d'augmenter la connectivité hydrologique entre la surface et la nappe phréatique. Ces pratiques d'infiltration produisent de nombreuses perturbations physico-chimiques au niveau de la nappe (ex. augmentation des variations thermiques, baisse des concentrations en oxygène dissous, enrichissement de la nappe en matière organique dissoute) mais les conséquences sur le compartiment microbien restent peu connues. L'objectif principal de la thèse est de déterminer les effets de l'infiltration des eaux pluviales sur les communautés microbiennes des nappes phréatiques, aussi bien en termes d'abondance, d'activités que de diversité génétique bactérienne. En se basant sur les changements environnementaux associés à l'infiltration des eaux pluviales et l'analyse des communautés bactériennes, un objectif fondamental est d'évaluer l'importance des phénomènes de dispersion (ex. transferts) et de sélection par des facteurs abiotiques (ex. disponibilité des ressources nutritives) sur les assemblages bactériens au sein des nappes phréatiques. Ces travaux ont été axés sur des expérimentations de terrain utilisant deux approches d'échantillonnage : une méthode active (prélèvements d'eau) et une méthode passive (incubation de substrats artificiels). La description des communautés a été effectuée par une méthode de séquençage de nouvelle génération (i.e. Illumina MiSeq) en se basant sur le gène rrs. Les résultats de ce travail mettent en avant une influence significative des pratiques d'infiltration sur les bactériomes d'un aquifère. En effet, le développement, les activités et la diversité des micro-organismes retrouvés dans la nappe ont été stimulés significativement par l'enrichissement en carbone organique dissous biodégradable engendré par ces pratiques. Néanmoins, cet impact est fortement réduit dans les systèmes étudiés où la zone non saturée est épaisse (> 10 m) et agit comme un filtre physique, chimique et biologique efficace entre le bassin d'infiltration et l'aquifère. Les faibles similarités entre les structures génétiques des bactériomes des eaux d'infiltration et dans la nappe indiquent que la zone non saturée joue un rôle efficace sur la rétention des bactéries dans les systèmes étudiés. En conclusion, cette thèse constitue la première étude d'envergure visant à quantifier la réponse du compartiment microbien des aquifères à des perturbations engendrées par l'infiltration des eaux pluviales en milieu urbain. Elle ouvre aussi de nouvelles perspectives sur les méthodes et outils d'évaluation de la qualité des nappes phréatiques / In urban area, managed aquifer recharge (MAR) systems raises hydrological connectivity between surface and groundwater. These infiltration practices are the cause of many disturbances in groundwaters (e.g. increase of thermal variations, decrease of dissolved oxygen or enrichment in organic matter) but associated consequences on microbial compartment remains unclear. The main aim of the thesis is to determine the effects of stormwater runoff infiltration on microbial communities of groundwater, in terms of abundance, activities and bacterial diversity. Based on environmental changes associated to MAR practices and bacterial community analyses, a fundamental question is to assess the importance of dispersal (e.g. transfers) and selection by abiotic factors (e.g. nutrients availability) on groundwater communities assemblage. This study is based on field experiments with two complementary strategies of sampling: an active one (i.e. groundwater sampling) and a passive one (incubation of artificial substrate). Communities’ description was made by next-generation sequencing (i.e. Illumina MiSeq) of rrs gene. The results showed a significant influence of MAR practices on microbial communities. Growth, activities and diversity of groundwater micro-organisms were mainly stimulated by biodegradable dissolved organic carbon enrichment associated to MAR practices. Nonetheless, this impact was reduced in systems where the vadose zone is thick (> 10 m) and acts as a physical, chemical and biological filter between the infiltration basin and the aquifer. Low similarities between bacterial communities of infiltration waters and bacterial communities of groundwaters reveal that vadose zone is effective on the retention of bacteria in studied systems. To conclude, this thesis constitutes the first major study that aimed to quantify microbial compartment response to disturbances caused by MAR practices in urban area. It also opens new perspectives on assessment tool for groundwater quality

L'identification de gènes ayant permis à des populations de s'adapter à leur environnement local constitue une des problématiques majeures du domaine de la génétique des populations. Les méthodes statistiques actuelles répondant à cette problématique ne sont plus adaptées aux données de séquençage nouvelle génération (NGS). Nous proposons dans cette thèse de nouvelles statistiques adaptées à ces nouveaux volumes de données, destinées à la détection de gènes sous sélection. Nos méthodes reposent exclusivement sur l'Analyse en Composantes Principales, dont nous justifierons l'utilisation en génétique des populations. Nous expliquerons également les raisons pour lesquelles nos approches généralisent les méthodes statistiques existantes et démontrons l'intérêt d'utiliser une approche basée sur l'Analyse en Composantes Principales en comparant nos méthodes à celles de l'état de l'art. Notre travail a notamment abouti au développement de pcadapt, une librairie R permettant l'utilisation de nos statistiques de détection sur des données génétiques variées. / Identifying genes involved in local adaptation is of major interest in population genetics. Current statistical methods for genome scans are no longer suited to the analysis of Next Generation Sequencing (NGS) data. We propose new statistical methods to perform genome scans on massive datasets. Our methods rely exclusively on Principal Component Analysis which use in population genetics will be discussed extensively. We also explain the reasons why our approaches can be seen as extensions of existing methods and demonstrate how our PCA-based statistics compare with state-of-the-art methods. Our work has led to the development of pcadapt, an R package designed for outlier detection for various genetic data.