L'identification des réservoirs cellulaires du VIH ainsi que la compréhension des mécanismes moléculaires impliqués dans le maintien et la réactivation de la latence sont essentiels afin d'élaborer une stratégie efficace pour éliminer le virus de l'organisme. Mon travail de thèse a porté d'une part, sur l'étude de l'infection du tissu adipeux par le VIH et son rôle potentiel comme nouveau tissu cible, et d'autre part, sur la recherche de gènes cellulaires candidats impliqués dans le contrôle de la latence du VIH.<br />Dans un premier temps, nous avons voulu déterminer si la faible production virale obtenue après infection des préadipocytes par le VIH est due à une faible efficacité d'infection ou de réplication. Nous avons montré que l'infection par les virus pseudotypés avec des glycoprotéines d'enveloppe de tropisme X4 et R5 est restreinte en raison d'une co-expression insuffisante des récepteurs viraux à la surface des préadipocytes. Cela suggère que le tissu adipeux n'est pas une cible privilégiée pour le virus in vivo et ne peut donc être considéré comme un nouveau réservoir pour le VIH.<br />Dans un deuxième temps, nous avons analysé le transcriptome différentiel de lignées cellulaires infectées de façon latente par le VIH et stimulées ou non pour la réactivation de la latence par un inhibiteur d'histone déacétylase. Parmi les gènes caractérisés, deux nous ont paru particulièrement intéressants : NCoA3 et IRF8. Après avoir confirmé les modifications de l'expression de ces gènes, nous avons analysé leurs rôles fonctionnels sur la transcription du VIH. Les facteurs NCoA3 et IRF8 pourraient respectivement être impliqués dans la réactivation et le maintien de la latence virale.

The whitefly Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae) is a serious pest of protected vegetable and ornamental crops in most temperate regions of the world. Neonicotinoids, pymetrozine (a feeding blocker), spiromesifen (a tetronic acid derivative), bifenthrin (a pyrethroid), and pyriproxyfen (a juvenile hormone mimic) are among the most important insecticides used to control this species. Bioassays were used to quantify responses of recently-collected strains of T. vaporariorum to three neonicotinoids (imidacloprid, thiamethoxam, and acetamiprid), pymetrozine, spiromesifen, bifenthrin, and pyriproxyfen. 454 pyrosequencing was exploited to generate the first transcriptome for this species. PCR-sequencing was used to identify mutations in the target proteins of spiromesifen and bifenthrin potentially associated with resistance to these compounds. Microarray sequencing technology was employed to investigate differences in gene expression associated with pyriproxyfen resistance. Resistance to neonicotinoids was age-specific in expression and consistently associated with resistance to pymetrozine, supporting a hypothesis of metabolic resistance analogous to that in the tobacco whitefly, Bemisia tabaci. Bioassays also showed moderate to high level resistance to spiromesifen, bifenthrin and pyriproxyfen in some strains. Analysis of the transcriptome identified genes encoding enzymes involved in the detoxification of xenobiotics (cytochrome P450s, carboxyl/cholinesterases, and glutathione-s transferases) and ones encoding insecticide targets: acetyl-coA carboxylase (ACCase), the target of spiromesifen and the voltage-gated sodium channel protein targeted by pyrethroids. PCR-sequencing revealed a single nucleotide polymorphism in the ACCase gene, which was consistently associated with spiromesifen resistance. Three amino-acid substitutions in the sodium channel of pyrethroid-resistant T. vaporariorum were found in positions previously implicated in pyrethroid resistance in B. tabaci. Microarray sequencing disclosed that a cytochrome P450 gene (CYP4G61) was overexpressed in a strain selected for increased pyriproxyfen resistance. The implications of these results and opportunities for further work are discussed.

12 month embargo; Available online 9 November 2016 / SCN8A encephalopathy is a severe, early-onset epilepsy disorder resulting from de novo gain-of-function mutations in the voltage-gated sodium channel Na(v)1.6. To identify the effects of this disorder on mRNA expression, RNA-seq was performed on brain tissue from a knock-in mouse expressing the patient mutation p.Asn1768Asp (N1768D). RNA was isolated from forebrain, cerebellum, and brainstem both before and after seizure onset, and from age-matched wildtype littermates. Altered transcript profiles were observed only in forebrain and only after seizures. The abundance of 50 transcripts increased more than 3-fold and 15 transcripts decreased more than 3 fold after seizures. The elevated transcripts included two anti-convulsant neuropeptides and more than a dozen genes involved in reactive astrocytosis and response to neuronal damage. There was no change in the level of transcripts encoding other voltage-gated sodium, potassium or calcium channels. Reactive astrocytosis was observed in the hippocampus of mutant mice after seizures. There is considerable overlap between the genes affected in this genetic model of epilepsy and those altered by chemically induced seizures, traumatic brain injury, ischemia, and inflammation. The data support the view that gain-of-function mutations of SCN8A lead to pathogenic alterations in brain function contributing to encephalopathy.

<p>This research examines three potential mechanisms by which bacteria can adapt to different temperatures: changes in strain-level population structure, gene regulation and particle colonization. For the first two mechanisms, I utilize bacterial strains from the Vibrionaceae family due to their ease of culturability, ubiquity in coastal environments and status as a model system for marine bacteria. I first examine vibrio seasonal dynamics in temperate, coastal water and compare the thermal performance of strains that occupy different thermal environments. Our results suggest that there are tradeoffs in adaptation to specific temperatures and that thermal specialization can occur at a very fine phylogenetic scale. The observed thermal specialization over relatively short evolutionary time-scales indicates that few genes or cellular processes may limit expansion to a different thermal niche. I then compare the genomic and transcriptional changes associated with thermal adaptation in closely-related vibrio strains under heat and cold stress. The two vibrio strains have very similar genomes and overall exhibit similar transcriptional profiles in response to temperature stress but their temperature preferences are determined by differential transcriptional responses in shared genes as well as temperature-dependent regulation of unique genes. Finally, I investigate the temporal dynamics of particle-attached and free-living bacterial community in coastal seawater and find that microhabitats exert a stronger forcing on microbial communities than environmental variability, suggesting that particle-attachment could buffer the impacts of environmental changes and particle-associated communities likely respond to the presence of distinct eukaryotes rather than commonly-measured environmental parameters. Integrating these results will offer new perspectives on the mechanisms by which bacteria respond to seasonal temperature changes as well as potential adaptations to climate change-driven warming of the surface oceans.</p> / Dissertation

L’objectif de cette thèse a été d’enrichir nos connaissances sur les bactériesintracellulaires strictes et spécialement Coxiella burnetii, agent responsable de la fièvre Q.Pour ce faire, nous avons d’une part amélioré les techniques de préparation de l’ARN pourles études transcriptionnelles et d’autre part utilisé la technologie des puces à ADN pouranalyser le transcriptome ainsi que la diversité génomique de C. burnetii.L’utilisation d’échantillons cliniques dans les études transcriptionnelles est limitéepar la quantité de matériel disponible qui ne permet pas d’analyser simultanément les profilsdu pathogène et de son hôte au cours de l’infection. De ce fait, nous avons développé, sur unmodèle d’escarres obtenus à partir de malades de fièvre boutonneuse méditerranéenne, unestratégie basée sur l’hybridation soustractive pour séparer les ARN eucaryotes etprocaryotes dans le but d’entreprendre des hybridations de puces à ADN.C. burnetii est une bactérie hautement résistante aux stress environnementaux commele changement de pH, la dessiccation, mais aussi le changement de température. Nous avonsparticulièrement étudié la réponse précoce de C. burnetii lors d’une exposition à une hauteet faible température. L’analyse globale du profil transcriptionnel a montré que la réponsede C. burnetii était limitée et similaire pour les différents stress appliqués. Cependant,malgré cette faible réponse, il apparait clairement qu’une accumulation de ppGpp, un arrêtde la croissance et des modifications de la membrane et de la paroi cellulaire permettraient àC. burnetii de résister à ces stress. Toutes ces régulations géniques convergent vers unchangement d’état de la bactérie vers une forme pseudo-sporulée. De plus, nous avonsobservé une organisation spatiale des gènes différentiellement exprimés. Nos analyses bioinformatiquesont montré que ces clusters de régulation ne répondaient ni au paradigmepromoteur - facteur de transcription – opéron, ni à des réseaux biologiques. Ayant retrouvéce phénomène dans plusieurs autres études transcriptionnelles chez d’autres bactériesintracellulaires, nous spéculons que ces clusters de régulations pourraient être dus à unerégulation épigénétique qu’il reste à caractériser.Différentes méthodes de typage ont déjà été mises au point pour classer les isolats deC. burnetii dans le but d'explorer son pouvoir pathogène. Ici, nous présentons une méthodede génomotypage basée sur la présence ou l'absence de gènes à l'aide de puces à ADN.Nous avons testé notre stratégie de génomotypage sur 52 isolats provenant de différenteszones géographiques, de différents hôtes et de patients présentant différentes manifestationscliniques. L'analyse a révélé la présence de 10 génomotypes organisés en 3 groupes avecune topologie congruente à celle observée avec le Multi Spacer Typing. Nous avons aussidécouvert 4 génomotypes particulièrement associés à la fièvre Q aiguë, alors que tous lesgénomotypes étaient associés à la forme chronique. De plus, le génomotypage a révélé queles isolats retrouvés dans les tiques dures, y compris la souche de référence Nine Mileappartiennent au même genomotype.Globalement, les données que nous avons obtenues confirment le fait que les puces àADN sont un outil adapté pour l’analyse de la pathogénicité de C .burnetti mais aussi desautres bactéries intracellulaires strictes. Cependant, de nouvelles technologies plusrésolutives comme le DNA ou RNAseq semblent être plus prometteuses mais restent encoreà optimiser / The objective of this thesis was to increase knowledge of obligate intracellular bacteriaand specifically, the causative agent of Q fever C. burnetii. In this regard, we have improvedstrategies to purify RNA for the transcriptional studies. We also used the technologymicroarrays to analyze the transcriptome and genomic diversity of C. burnetii.The use of clinical samples in the transcriptional studies is limited by the amount ofmaterial available and thus the transcriptional profiles of the pathogen and its host duringinfection can not be simultaneously analyze. We developed, with a model of eschars obtainedfrom Mediterranean spotted fever patients, a strategy based on subtractive hybridization toseparate RNA eukaryotic and prokaryotic cells in order to perform microarray experiments.Analysis of the survival strategies used by this bacterium to adapt to new environmentalconditions is critical for our understanding of C. burnetii pathogenicity. Here, we report theearly transcriptional response of C. burnetii under temperature stresses. Our data show thatC. burnetii exhibited minor changes in gene regulation under short exposure to heat or coldshock. While small differences were observed, C. burnetii seemed to respond similarly to coldand heat shock. The expression profiles obtained using microarrays produced in-house wereconfirmed by quantitative RT-PCR. Under temperature stresses, 190 genes were differentiallyexpressed in at least one condition, with a fold change of up to 4. Globally, the differentiallyexpressed genes in C. burnetii were associated with bacterial division, (p)ppGpp synthesis,wall and membrane biogenesis and, especially, lipopolysaccharide and peptidoglycansynthesis. These findings could be associated with growth arrest and witnessed transformationof the bacteria to a spore-like form. Unexpectedly, clusters of neighboring genes weredifferentially expressed. These clusters do not belong to operons or genetic networks; theyhave no evident associated functions and are not under the control of the same promoters. Wealso found undescribed but comparable clusters of regulation in previously reportedtranscriptomic analyses of intracellular bacteria, including Rickettsia sp. and Listeriamonocytogenes. The transcriptomic patterns of C. burnetii observed under temperature stressespermits the recognition of unpredicted clusters of regulation for which the trigger mechanismremains unidentified but which may be the result of a new mechanism of epigenetic regulation.Different typing methods have been previously developed to classify C. burnetii isolatesin order to explore its pathogenicity. Here, we report a comprehensive genomotyping methodbased on presence or absence of genes using microarray. The genomotyping method was thentested on 52 isolates obtained from different geographic areas, different hosts and isolated frompatient with different clinical manifestations. The analysis reveals the presence of10 genomotypes organized in 3 groups with a topology congruent with that of Multi SpacerTyping. We also found out 4 genomotypes especially associated with acute Q fever whereas allthe genomotypes could be associated to chronic human infection. Serendipity, genomotypingreveals that hard ticks isolates including Nine Mile belong to the same genomotype.Overall, the data we obtained confirm that DNA microarrays are a suitable tool forexploring pathogenicity of C. Burnetti and other obligate intracellular bacteria. However newtechnologies such as DNAseq or RNAseq seem more promising but still need to optimize andalso are still expensive compared to microarray.

<p>RNA binding proteins (RBPs) and non-coding RNAs orchestrate gene expression in part through the recognition specific sites in mRNA. Thus understanding the connection between binding to specific sites and regulation of the whole transcript is essential. Current methods to do this can either identify the binding sites or quantitate binding to whole transcripts, but not both. Furthermore reliance of binding site detection on ultraviolet crosslinking results in inefficient identification of binding sites, and insufficient data to assess binding strength at sites. I have overcome these limitations by combining aspects of current methods to develop a new method called DO-RIP-seq (digestion optimization RNA immunoprecipitations with deep sequencing) that can quantitate the binding strength of RBPs at sites in mRNA, and also relate binding sites to binding of the whole mRNA. DO-RIP-seq was developed using the well-studied RBP ELAVL1/HuR as a test case, and applied to the less well-studied RBP known as RBM38/RNPC1. The quantitative data from DO-RIP-seq out-performed current binding site methods at predicting other features of the binding sites of HuR and RBM38, for example the lack of RNA secondary structure, and preferences in binding to particular sub-motifs. My studies indicate that DO-RIP-seq will be useful in uncovering the determinants of RNA-protein interactions, and studying dynamic biological processes that could modulate these interactions.</p> / Dissertation

Pathogenic Yersinia cause a manifold of diseases in humans ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to pneumonic and bubonic plague (Y. pestis), while all three have a common virulence strategy that relies on a well-studied type III secretion system and its effector proteins to colonize the host and evade immune responses. However, the role of other protein secretion and/or translocation systems in virulence of Yersinia species is not well known. In this thesis, we sought to investigate the contribution of twin-arginine translocation (Tat) pathway and its secreted substrates to the physiology and virulence of Y. pseudotuberculosis. Tat pathway uniquely exports folded proteins including virulence factors across the cytoplasmic membranes of bacteria. The proteins exported by Tat pathway contain a highly conserved twin-arginine motif in the N-terminal signal peptide. We found that the loss of Tat pathway causes a drastic change of the transcriptome of Y. pseudotuberculosis in stationary phase at environmental temperature with differential regulation of genes involved in virulence, carbon metabolism and stress responses. Phenotypic analysis revealed novel phenotypes of the Tat-deficient strain with defects in iron acquisition, acid resistance, copper oxidation and envelope integrity, which we were partly able to associate with the related Tat substrates. Moreover, increased glucose consumption and accumulation of intracellular fumarate were observed in response to inactivation of Tat pathway implicating a generic effect in cellular physiology. We evaluated the direct role of 22 in silico predicted Tat substrate mutants in the mouse infection model and found only one strain, ΔsufI, exhibited a similar degree of attenuation as Tat-deficient strain. Comparative in vivo characterization studies demonstrated a minor defect for ΔsufI in colonization of intestinal tissues compared to the Tat-deficient strain during early infection, whereas both SufI and TatC were required for dissemination from mesenteric lymph nodes and further systemic spread during late infection. This verifies that SufI has a major role in attenuation seen for the Tat deficient strain both during late infection and initial colonization. It is possible that other Tat substrates such as those involved in iron acquisition and copper resistance also has a role in establishing infection. Further phenotypic analysis indicated that SufI function is required for cell division and stress-survival. Transcriptomic analysis revealed that the highest number of differentially regulated genes in response to loss of Tat and SufI were involved in metabolism and transport. Taken together, this thesis presents a thorough analysis of the involvement of Tat pathway in the overall physiology and virulence strategies of Y. pseudotuberculosis. Finally, we propose that strong effects in virulence render TatC and SufI as potential targets for development of novel antimicrobial compounds

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries. Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Dans la première partie j'ai analysé des jeux de données de RNA-seq de transcriptome de petits ARNs disponibles dans les bases de données publiques. J'y ai observé 2 points intrigants : - une grande partie des lectures (bien que courtes) ne peux pas être alignée sur le génome de référence sans discordance et cette fraction non-alignable est parfois majoritaire. - de nombreuses lectures ont des tailles autours de 15-18nt qui ne correspondent à aucun type de petits ARNs connues, cette fraction est également majoritaires dans certains cas. Ces expériences sont souvent conçues pour la détection des miRNAs et l'analyse bioinformatique de ces données passent toujours par un alignement sur le génome de référence ou sur des séquences connues pour donner des petits ARNs. J'ai donc simplement éliminé la contrainte d'alignement dans l'analyse de ces données et effectué un regroupement des lectures par similarité (à la manière des ESTs). Ce regroupement donne une vision différente des données dans laquelle la notion de position génomique n'est plus centrale et ouvre la possibilité d'y découvrir des phénomènes non-standard. La deuxième partie est tirée d'une collaboration avec le laboratoire U675 INSERM. J'ai fait l'analyse bioinformatique des gènes dérégulés par la répression par RNAi du gène REST dans une lignée de neuroblastome de souris (N18). Ce gène est un facteur de transcription qui réprime les gènes neuronaux dans les cellules non neuronales. Ce répertoire de gènes dérégulés est potentiellement constitué de gènes clefs dans la biologie des neurones. / In first part of this thesis I have analysed small RNA-seq transcriptome data. I have noticed : - a large fraction of reads can't be aligned perfectly on reference genome - lot of reads are very short (15-18 nt) and don't match on previously known functionnal small RNAs. These experiments are designed for miRNA discovery and bioinformatics analysis of these data use alignments on genome or on known small RNA precursors sequences. I have eliminated the alignment and I have clustered these sequences. This clustering let me to observe these data with a new view in wich the genomic location is not central and open the gate to discover unconventional events. The second part is the analysis of deregulate genes by the silencing of the gene REST/NRSF in mouse N18 cell line. This gene is a transcription factor and it works as a repressor of neuronal genes in non neuronal cells. This deregulate genes repertoire potentially contains key genes in neuron biology. We found in this repertoire a network of genes centered on SWI/SNF complex including SMARCA2. This gene was associated to schizophrenia (SZ) in association studies and structural variation studies. In this network we found another genes associated to SZ. We show that these genes exhibit positive evolution in primate compare to rodents.