La fermentation œnologique et le vieillissement oxydatif des vins sous voile représentent des modes de vie très contrastés qui sont effectués par deux lignées différentes de souches de levures de l'espèce Saccharomyces cerevisiae. Dans cette thèse, nous avons comparé le génome de souches de levures de voile à celui de levures de vin afin de détecter leurs spécificités. Nous tout d'abord sélectionné 16 souches (8 levures de vin et 8 levures de voile) isolées en France, Hongrie, Italie et Espagne, pour séquencer leur génome sur une plateforme Illumina (HiSeq2000). Nous avons également développé un ensemble de souches de vin et de voile haploïdes pour l'évaluation moléculaire de différentes cibles. Nous avons également mis au point un milieu synthétique mimant le vin à cette fin. A partir de la comparaison des séquences du génome nous avons établi une phylogénie qui montre que les levures de voile représentent un groupe spécifique de levure, différentes des levures de vin, puis à partir de différentes méthodes (analyse en composantes principale, diversité nucléotidique et D de Tajima) nous avons identifié des régions divergentes. Ces régions variantes comprennent des gènes remplissant plusieurs fonctions clé associées à la croissance en voile. En particulier, des variations alléliques ont été rencontrées chez les levures de voile pour plusieurs gènes impliqués dans la régulation de l'expression de FLO11 tels que les voies MAP kinase, ou des voies Ras/cAMP/PKA, ainsi que pour plusieurs gènes impliqués dans l'homéostasie des cations divalents de métaux de transition tels que le zinc, le cuivre ou le fer. La comparaison des transcriptomes d'une levure de voile et d'une levure de vin sur notre milieu synthétique a révélé des différences d'expression pour les floculines (FLO1, 5, 8, 11) ainsi que pour le transport des hexoses, mais a également suggéré que la levure de voile P3-D5 était en situation de carence en zinc et en inositol par rapport à la levure de vin, tandis que la levure de vin K1 exprimait certains gènes suggérant des défauts mitochondriaux. L'impact de la variation allélique de plusieurs gènes a été évalué dans le phénotype de voile: le transporteur de zinc à haute affinité Zrt1 ainsi que la pyruvate décarboxylase majeure Pdc1. / Wine fermentation and flor ageing are performed by two groups of the yeast Saccharomyces cerevisiae, with very different lifestyles. In this thesis we have studied the genome of flor yeast in comparison to wine yeast in order to unravel their specificities. We have first selected 16 strains (8 wine and 8 flor) from France, Hungary, Italy and Spain in order to sequence their genome sequence on an Illumina HiSeq2000 platform. Three flor strains and two wine strains were haploidized in order to obtain a set of haploid flor strains for the molecular evaluation of different targets. We developed as well a synthetic media mimicking wine for that purpose. From the genome sequence we have drawn a phylogeny that showed that flor yeasts represent a specific lineage of yeast, different from the wine strains lineage, and identified divergent regions. These regions contain genes involved in key functions and several associated with velum growth. Remarkably, many genes involved in FLO11 regulation such as MAP kinase, or Ras/PKA pathways were mutated among flor strains and many variations were encountered in genes involved in metal homeostasis such as zinc and divalent metal transporters. A transcriptome analysis comparing one flor and one wine yeast on our wine synthetic media revealed expression differences associated to floculins and hexose transport, but also suggested that flor yeast P3-D5 face a zinc and inositol deficiency, whereas wine yeast K1 presented mitochondrial defects. The impact of allelic variation of several genes coding for the high affinity zinc transporter (ZRT1), and the major pyruvate decarboxylase (PDC1) has been evaluated in order to assess their role in the flor phenotype.

Heavy metal pollutants, discharged into the ecosystem as waste by anthropogenic activities, contaminate drinking water for millions of people and animals in many regions of the world. Long term exposure to these metals, leads to several lethal diseases like cancer, keratosis, gangrene, diabetes, cardio- vascular disorders, etc. Therefore, removal of these pollutants from soil, water and environment is of great importance for human welfare. One of the possible eco-friendly solutions to this problem is the use of microorganisms that can accumulate the heavy metals from the contaminated sources, hence reducing the pollutant contents to a safe level. In this thesis an arsenic resistant bacterium Lysinibacillus sphaericus B1-CDA, a chromium resistant bacterium Enterobacter cloacae B2-DHA and a nickel resistant bacterium Lysinibacillus sp. BA2 were isolated and studied. The minimum inhibitory concentration values of these isolates are 500 mM sodium arsenate, 5.5 mM potassium chromate and 9 mM nickel chloride, respectively. The time of flight-secondary ion mass spectrometry and inductively coupled plasma-mass spectroscopy analyses revealed that after 120 h of exposure, the intracellular accumulation of arsenic in B1-CDA and chromium in B2-DHA were 5.0 mg/g dwt and 320 μg/g dwt of cell biomass, respectively. However, the arsenic and chromium contents in the liquid medium were reduced to 50% and 81%, respectively. The adsorption values of BA2 when exposed to nickel for 6 h were 238.04 mg of Ni(II) per gram of dead biomass indicating BA2 can reduce nickel content in the solution to 53.89%. Scanning electron micrograph depicted the effect of these metals on cellular morphology of the isolates. The genetic composition of B1-CDA and B2-DHA were studied in detail by sequencing of whole genomes. All genes of B1-CDA and B2-DHA predicted to be associated with resistance to heavy metals were annotated. The findings in this study accentuate the significance of these bacteria in removing toxic metals from the contaminated sources. The genetic mechanisms of these isolates in absorbing and thus removing toxic metals could be used as vehicles to cope with metal toxicity of the contaminated effluents discharged to the nature by industries and other human activities.

Campylobacter est responsable de la zoonose bactérienne d’origine alimentaire la plus fréquemment reportée en Europe. Cette bactérie étant ubiquitaire, les sources et voies d’infection de l’Homme sont nombreuses. Cependant, afin de diminuer l’incidence de la maladie, il est nécessaire d’identifier les principaux réservoirs impliqués dans les infections humaines. Pour cela, nous avons dans un premier temps investigué la présence de Campylobacter dans trois réservoirs animaux (volaille, bovin, animaux de compagnie), ainsi que la diversité génétique des isolats de C. jejuni, en comparaison à celle d’isolats cliniques, à l’aide des techniques MLST (Multilocus sequence typing) et CGF (Comparative Genomic Fingerprinting). Afin d’identifier l’origine des campylobactérioses avec précision et de compenser notamment les limites techniques de la MLST, 15 marqueurs génétiques ont été sélectionnés comme marqueurs potentiellement indicateurs de l’hôte, après analyse de plus de 800 génomes de C. jejuni. Par la suite, la capacité de la MLST, la CGF40 et des 15 marqueurs à identifier l’origine des campylobactérioses a été étudiée. Ainsi, les 15 marqueurs se sont révélés être particulièrement performants pour l’attribution de sources des campylobactérioses, suivis ensuite par la MLST, tandis que la CGF40 est apparue comme étant peu adaptée. A partir des données MLST et des 15 marqueurs génétiques, une implication majoritaire des volailles et des bovins a été mis en évidence en France, tandis que les animaux de compagnie et l’environnement (comprenant eau et oiseaux sauvages) étaient faiblement impliqués. Ceci permet ainsi de renforcer les efforts de recherche relatifs aux moyens de lutte contre Campylobacter menés dans ces réservoirs. Ce travail a également permis de mettre en évidence de potentielles spécificités nationales dans la dynamique de transmission de C. jejuni à l’Homme. / Campylobacter is the causal agent of the main bacterial foodborne gastroenteritis in Europe. Since Campylobacter is frequently found in animal reservoirs, sources of human infection and transmission routes are various. However, to decrease the human burden of campylobacteriosis, it is essential to quantify the relative importance of the several reservoirs in human infections. For this purpose, we assessed the contamination of chicken, cattle and pets by Campylobacter spp., and further characterized C. jejuni isolates using MLST (Multilocus Sequence Typing) and CGF (Comparative Genomic Fingerprinting) in comparison with French clinical isolates. Then, in order to identify the most likely origin of campylobacteriosis cases in France and overcome MLST limitations in source attribution, about 800 C. jejuni genomes were analyzed which resulted in the identification of 15 genes as promising host segregating markers for source attribution. Subsequently, we assessed the ability of MLST, CGF40 and the 15 host-segregating markers to identify the most likely origin of campylobacteriosis. The 15 host-segregating markers were the most powerful in source attribution, followed by MLST, while CGF40 appeared to be not suitable for source attribution in our study. Based on MLST and the 15 markers, assignments of clinical cases emphasize the significant implication of chicken and ruminant in human infection by Campylobacter, while pets and the environment (including water and wild birds) were slightly involved, reinforcing the interest to focus control strategies on livestock. Finally this work highlights potential national variations in the transmission dynamics of C. jejuni to human.

<p>Humans have altered natural landscape since the agricultural revolution, but it has been most destructive since human globalization and rampant industrialization in the last two centuries. These activities deteriorate and fragments natural habitat of many wild species that creates small isolated populations that lose genetic diversity over time. Loss of genetic diversity reduces the adaptive capacity of a population to respond to future environmental change and increases their extinction risks. Implementing strategies for wildlife conservation is a challenge primarily because of our lack of understanding of the biology of many wild species, the risks they are currently facing, and their evolutionary histories. With the advent of genomic and computational techniques, it is now possible to address these concerns. In my research, I used genomics to study the evolutionary history of the Montezuma Quail (<i>Cyrtonyx montezumae</i>) and created monitoring tools that can be readily applied by wildlife managers for its conservation. Montezuma Quail is a small gamebird found mostly in Mexico with peripheral populations existing in Arizona, New Mexico, and Texas. Montezuma Quail are going through species wide decline in the United States and are listed as vulnerable in the state of Texas due to their small population sizes and geographic isolation from rest of the range. My results show that Texas quail are genetically distinct and significantly less diverse than Arizona quail. Analysis of whole genome sequences from multiple individuals show that due to small population sizes and isolation, Texas quail are significantly more inbred and genetic drift is the major contributor for loss of genetic diversity we see today. Inbreeding is negatively impacting Texas quail as they carry more deleterious alleles within their genome that reduce fitness of the individuals. Demographic models predict that both Arizona and Texas populations were formed via founding bottlenecks around 20,000 years ago. Texas populations have maintained small population sizes since its split from the ancestral populations and are less efficient in purging new deleterious mutations that arise post-bottleneck. The inferences from my research not only carries direct implications for Montezuma Quail conservationists, but also illustrate the power of evolutionary genomics in implementing targeted management strategies for any species that face existential threats in today’s waning world. </p>

A compound-heterozygous variant occurs when a child inherits a variant from each parent, with these variants occurring at a different position within the same gene and on opposite homologous chromosomes. These inherited variants may result in two nonfunctional versions of the same gene. Compound-heterozygous variants cannot be identified unless a patients' DNA sequence data is phased. Phasing is a computationally demanding process that requires the use of multiple software tools in order to determine which nucleotide was inherited from which parent. First, in Chapter 1, we review the literature to better understand what research has been conducted on the role of compound-heterozygous variants in pediatric cancers and what methods are being used to identify them. In Chapter 2, we develop a pipeline to make it easier for us and other researchers to phase and identify compound-heterozygous variants using VCF files from trios or individuals. We then use this pipeline in Chapter 3 to survey the prevalence of compound-heterozygous variants across 7 pediatric disease types. We show the importance of identifying compound heterozygous and what information would be missed if this variant type was not included in study design. In Chapter 4, we develop a software tool to phase trio data using a combination of Mendelian inheritance logic and an existing phasing software program. We show that our software tool increases the total number of variants that can be phased. Finally, in Chapter 5, we use phased data of three nuclear families, each family having one child with pediatric cancer, to evaluate the potential to use inherited genomic variants to inform diagnostic decisions. The work contained within this dissertation shows the importance of not overlooking compound-heterozygous variants when trying to identify potentially causal genes in pediatric disease. In addition, this work provides software tools that are openly available for other researchers to use; these tools make it easier to phase patient DNA sequence data and to identify compound-heterozygous variants.

No description available.

Campylobacteriosis is a leading foodborne illness worldwide, primarily caused by Campylobacter jejuni (C. jejuni) which is associated with poultry consumption. The emergence of antibiotic resistance has emphasized the need for alternative strategies to control C. jejuni colonization in poultry. To assess the prevalence of C. jejuni in poultry, 270 cloacal swab samples were collected from broilers raised under No-Antibiotics Ever system. Among these samples, 16.3% were identified as C. jejuni positive. Notably, these isolates exhibited a diverse range of virulence factors and antimicrobial resistance genes, with 61.36% of isolates showing hyper-motile and 20.45% demonstrating multidrug resistance. Following isolation, whole genome sequencing was conducted on four selected strains using a hybrid sequencing approach. Subsequently, the complete genomes of these C. jejuni strains were analyzed to identify vaccine candidates using reverse vaccinology. Three conserved potential vaccine candidates were identified as suitable targets for vaccine development, namely phospholipase A (PldA), TonB dependent transporter (ChuA), and cytolethal distending toxin (CdtB). Furthermore, the gene expression of these candidates was examined in four C. jejuni strains during host-pathogen interactions using avian macrophage cell line HD11. Significant upregulation of all three candidate genes were observed in the four tested C. jejuni strains during interaction with host cells, indicating their crucial role in C. jejuni infection. Additionally, the expression of immune genes was evaluated in avian macrophage cells to understand the immune responses during C. jejuni infection. The infection resulted in the upregulation of toll-like receptor genes (TLR-4), pro-inflammatory genes (IL-1β, IFN-γ, IL-6, IL-8L1), anti-inflammatory gene (IL-10), and iNOS2 gene expression. The observed immune response demonstrates the potential of C. jejuni to induce host immunity for protection. In conclusion, our study identifies three conserved potential vaccine candidates and provides insights into the immune responses induced by C. jejuni infection in avian macrophage cells. These findings are crucial for the development of an effective vaccine against C. jejuni, aiming to reduce C. jejuni transmission through poultry consumption and the risk of human infection.

No description available.

Understanding the transmission dynamics of infectious diseases is important to well-informed public health policy, responsive infection control and individual patient management. The on-going revolution in whole-genome sequencing provides unprecedented resolution for detecting evidence of recent transmission and characterising population-level transmission dynamics. In this thesis, I develop and apply evolutionary approaches to investigating transmission, focusing on three globally important pathogens. Hepatitis C virus (HCV) is a major cause of liver disease affecting 150 million people and killing 350,000 annually. I conducted a meta-analysis of twentieth-century HCV epidemics, finding that the age of the epidemic can be predicted by genetic diversity. Using the coalescent, I fitted classic susceptible-infected (SI), susceptible-infected-susceptible (SIS) and susceptible-infected-recovered (SIR) epidemiological models. Most epidemics showed signatures of SI dynamics, but three, from Argentina, Hong Kong and Thailand, revealed complex SIR dynamics. Norovirus is the leading viral cause of diarrhoea, estimated to cost the NHS around £115 million annually. I analysed whole norovirus genomes via a stochastic transmission model, finding that up to 86% of hospital infection was attributable to transmission from another patient in the hospital. In contrast, the rate of new introductions to hospital by infected patients was extremely low (<0.0001%), underlining the importance of ward management during outbreaks. Campylobacter is the most commonly identified cause of bacterial gastroenteritis worldwide. I developed a zoonotic transmission model based on phylogeography approaches to test whether three strains previously associated with multiple host species were in fact aggregates of strongly host-restricted sub-strains, or genuine generalists. Members of the same strain isolated from different host species were often more closely related than those isolated from the same host species. I estimated 419, 389 and 31 zoonotic transmissions in ST-21, ST-45 and ST-828 respectively, strongly supporting the hypothesis that these strains are adapted to a generalist lifestyle.

Le gliome infiltrant du tronc cérébral est une tumeur rare, non opérable et inéluctablement fatale. En raison du manque de ressource biologique disponible, aucun progrès dans la compréhension de la biologie de ces tumeurs n’a été fait jusqu’à ces dernières années, laissant la radiothérapie pour seul traitement efficace, et seulement transitoirement. Enfin, grâce à la mise en place de collecte d’échantillons de gliomes infiltrant du tronc cérébral au diagnostic ou à l’autopsie, un nombre sans précédent d’analyses biologiques et génomiques a pu être mené et améliorer la connaissance de ces tumeurs. Si ces études ont montré que ces gliomes pédiatriques étaient bien différents de ceux de l’adulte, elles ont aussi fait apparaître la présence d’anomalies génétiques récurrentes spécifiques de ces tumeurs sous-tentorielles. Ainsi le Platelet-Derived Growth Factor Receptor Alpha (PDGFRα) est apparu comme cible prédominante dans ces tumeurs compte tenu des nombreuses anomalies génétiques constatées. La recherche d’un médicament efficace pouvant inhiber cette voie nous a conduit à évaluer l’effet du dasatinib qui est un inhibiteur multi-ciblé. Nous en rapportons ici l’efficacité in vitro sur de nouvelles lignées cellulaires de gliomes infiltrants du tronc cérébral établies à partir de biopsies stéréotaxiques réalisées au diagnostic. Sachant néanmoins que les thérapies ciblées restent peu efficaces en clinique quand elles sont utilisées seules, nous mettons en évidence l’intérêt de combiner le dasatinib avec un inhibiteur de MET, 2ème oncogène fréquemment amplifié dans ces tumeurs. D’autre part, une stratégie originale de criblage médicamenteux a été mise en œuvre. Celle-ci a permis de définir de manière fonctionnelle de nouveaux médicaments potentiellement efficaces dans les gliomes infiltrants du tronc cérébral, incluant les inhibiteurs d’Histone deacetylases (HDAC), les inhibiteurs des Cyclin-Dependent Kinases (CDK) ou encore les inhibiteurs du protéasome. Enfin par la technique de séquençage génome-entier, de nouvelles anomalies génétiques jamais rencontrées dans aucun autre cancer ont été détectées. Parmi celles-ci se trouvent des mutations d’histone H3K27M dont la fréquence élevée (80%) suggère leur rôle fondamental dans la genèse de ces tumeurs. Des mutations activatrices d’ACVR1/ALK2 ont été également mises en évidence. Celles-ci représentent désormais de nouvelles cibles à explorer.Ce travail de thèse rapporte la recherche de nouvelles cibles thérapeutiques d’une part, via une approche exploratoire par criblage médicamenteux et recherche d’anomalies génétiques par séquençage « génome-entier », et d’autre part, via une approche de validation préclinique sur le plan des thérapies ciblées de type inhibiteurs de tyrosine-kinases. / Diffuse Intrinsic Pontine Glioma (DIPG) is a rare, unresectable and universally fatal tumor. Due to the lack of available material, no improvements have been made in the knowledge of the biology of this tumor until recent years, leaving radiotherapy as the only efficient treatment, and only transiently. Recently, the effort engaged for collecting samples in this disease at the diagnosis or at the autopsy resulted in an unprecedented number of analyses consequently improving our knowledge in DIPG. Those studies bring evidences for their differences with adult gliomas, but also with other pediatric supratentorial glioma showing specific genomic alterations. Thus, Platelet-Derived Growth Factor Receptor Alpha (PDGFRα) appeared to be one of the major target given its frequent aberrations found in those tumors. Investigating an effective drug to inhibit this pathway led us to evaluate the effect of dasatinib, which is known as a multi-targeted inhibitor. We report here the in vitro efficacy of dasatinib on new cell lines of DIPG developed from stereotaxic biopsy at diagnosis. Because therapies are largely inefficient in the clinic when they are used as a monotherapy, we bring out our interest on combining dasatinib with an inhibitor of MET, which is the 2nd most common amplified oncogene in these tumors.Additionally, an innovative strategy of pharmacological screening has been successfully tested. New drugs, potentially efficient in DIPG, have been fonctionnaly-defined, including Histone deacetylase inhibitors (HDACi), Cyclin-Dependent Kinases inhibitors (CDKi) and proteasome inhibitors as well.Finally, by using whole genome sequencing (WGS), we have been able to discover new genetic abnormalities, never encountered before in other cancers. Among those, mutations of histone H3K27M with a high frequency of 80% were found, suggesting that they have a fundamental role in tumors genesis. Moreover, ACVR1/ALK2 activating mutations have been identified as well. And this gene now represents a new target to explore. This work reports the research of new therapeutic targets through an exploratory approach using drug screening and WGS on the one hand, and on the other hand through a preclinical validation approach in terms of targeted therapies with tyrosine-kinases inhibitors.