<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.

La leptospirose est une zoonose d’importance médicale majeure dans les îles du Sud-Ouest de l’Océan Indien (SOOI) dont certaines enregistrent des incidences parmi les plus élevées au monde. Durant la dernière décennie, les données épidémiologiques moléculaires obtenues avec une approche « One Health » ont mis en évidence une grande diversité de lignées de leptospires ainsi que différentes chaines de transmission sur les différentes îles de la région. Les données moléculaires montrent la présence de leptospires pathogènes et de réservoirs animaux introduits ou endémiques de cette région. La distribution de ces différentes lignées de leptospires est associée à (i) un contraste épidémiologique incluant des différences dans la sévérité des cas humains et (ii) des niveaux de spécificité d’hôtes différents selon les leptospires considérés. Plus particulièrement, les leptospires endémiques du SOOI semblent être moins pathogènes chez les humains et montrent une plus forte affinité pour leur réservoir que les leptospires cosmopolites. Pour compléter nos connaissances sur l’histoire évolutive des leptospires du SOOI, nous avons produit des données provenant de chauves-souris de l’Afrique de l’Est. Ces données confirment la spécificité de certaines lignées de leptospires envers leurs hôtes chiroptères et suggèrent que les chauves-souris d’Afrique ont colonisé Madagascar tout en étant infectées par leurs leptospires. Afin de mieux comprendre le rôle des différents leptospires dans l’épidémiologie régionale de la leptospirose, nous avons mesuré la pathogénicité de trois souches de leptospires retrouvées dans cette région à l’aide d’un modèle hamster. Des souches de Leptospira mayottensis et Leptospira borgpetersenii ont été isolés respectivement de Tenrec ecaudatus (tenrec) et Triaenops menamena (chauve-souris), deux mammifères endémiques du SOOI. Une souche de Leptospira interrogans, dont le génotype est retrouvé dans la majorité des cas humains graves à la Réunion, a été isolée de Rattus rattus (rat). En cohérence avec les données épidémiologiques humaines de Mayotte et de La Réunion, les leptospires endémiques se sont révélées être significativement moins pathogènes que la souche L. interrogans. La spécificité d’hôte des deux souches isolées de mammifères endémiques a été mise à l’épreuve par des infections expérimentales de Rattus norvegicus, connu comme un réservoir important de leptospires. Les rats ont été infectés avec les trois isolats précédemment utilisés. Les rats infectés par les souches endémiques n’ont pas développé d’infection rénale chronique contrairement à la souche cosmopolite. Ces résultats montrent que la spécificité d’hôte des leptospires endémiques observée in natura est probablement due à des facteurs génétiques plutôt qu’à des facteurs écologiques, comme un manque de contacts physiques entre les réservoirs animaux endémiques et introduits. Enfin, le séquençage complet de souches de leptospires du SOOI a été réalisé afin d’identifier des caractéristiques génétiques pouvant être associées à la pathogénicité et la spécificité d’hôte des leptospires pathogènes. Une classification précise de souches de leptospires du SOOI a pu être réalisée sur la base des génomes complets. La comparaison de ces génomes a permis d’identifier des gènes spécifiques à un groupe ou une espèce de leptospires. Cependant des modifications génomiques complexes rendent difficiles l’identification de caractéristiques génomiques responsables d’un phénotype particulier tel que la virulence ou la spécificité d’hôte. / Leptospirosis is a zoonosis of main medical concern on several islands of southwestern Indian Ocean (SWIO), some of which recording among the highest human incidence worldwide. Over the last decade, molecular epidemiology investigations carried out under a One Health framework have revealed a wide variety of Leptospira lineages and distinct transmission chains throughout the islands of the region. These islands are home to pathogenic Leptospira lineages and animal reservoirs that are either introduced or endemic to the SWIO region. Interestingly, the regional distribution of Leptospira diversity is associated with (i) a contrasted severity of human cases and (ii) distinct levels of specificity of Leptospira towards their mammalian hosts. Specifically, endemic Leptospira appear less pathogenic in humans and display higher specificity towards their animal reservoirs than their cosmopolitan counterparts. To complete the dataset of Leptospira diversity in the SWIO region, we produced data from bats of eastern Africa. Results support the previously observed pattern of host specificity of Leptospira towards their bats hosts and, overlaid upon the biogeographic history of Malagasy bats, suggest that these volant mammals have colonized Madagascar from continental Africa while hosting pathogenic Leptospira. To better understand the role of distinct Leptospira lineages in the contrasted epidemiology observed in the SWIO, we investigated the pathogenicity of three Leptospira isolates from this region using a hamster model. Leptospira mayottensis and Leptospira borgpetersenii isolates were obtained from Tenrec ecaudatus (tenrec) on Mayotte and Triaenops menamena (bat) in Madagascar, respectively, both mammals endemic to the SWIO region. A Leptospira interrogans strain, which genotype has been reported in the majority of human acute cases on La Réunion, was isolated from the introduced Rattus rattus (rat). In keeping with a distinct severity of the disease on Mayotte and La Réunion, endemic bat-borne and tenrec-borne Leptospira were significantly less pathogenic than the control cosmopolitan rat-borne isolate. The host specificity of the isolates obtained from endemic hosts was addressed using experimental infection of Rattus norvegicus, a known reservoir of pathogenic Leptospira. This animal model was challenged with all three isolates and mostly failed in supporting chronic infection with bat-borne and tenrec-borne Leptospira. Hence, the strong host-specificity of endemic Leptospira toward their hosts observed in the wild likely results from genetic determinants shaped by long-term co-evolutionary processes rather than from ecological constraints such as a lack of physical contact between introduced and endemic animal reservoirs. Finally, we undertook full genome sequencing of regional strains in order to highlight genomic features that may be associated with virulence and host specificity. Whole genome sequencing allowed the accurate classification of Leptospira isolates obtained on SWIO islands. Comparative genomics allowed to identify genes specific to a group or species of Leptospira but complex changes in Leptospira genome make difficult the identification of genomic elements responsible for specific traits such as virulence and host specificity.

O gênero Xylella é composto de uma única espécie, Xylella fastidiosa, bactéria Gram-negativa, não flagelada, que coloniza o xilema de uma diversidade de plantas cultivadas e silvestres em várias partes do mundo. Em algumas dessas plantas, a bactéria é considerada agente causal de doenças, como a Clorose Variegada do Citros em laranjeiras, a Doença de Pierce das videiras e escaldadura da folha de cafeeiro. Onze diferentes cepas de X. fastidiosa, isoladas de distintos hospedeiros, já tiveram seus genomas sequenciados, entre essas, as cepas 9a5c, isolada de laranjeira, e Temecula 1, isolada de videira. Análises desses genomas indicam uma razoável variabilidade entre suas respectivas sequências e evidenciam vários genes associados a mecanismos de virulência e patogenicidade desta bactéria. No presente trabalho descrevemos o sequenciamento, a montagem e a anotação dos genomas das cepas U24d e Fb7, isoladas de laranjeiras, e da cepa 3124 isolada de cafeeiro, os quais apresentam, respectivamente 2.681.334 pb, 2.733.974 pb e 2.748.594 pb. Destas, apenas a cepa U24d apresenta um plasmídeo, o qual é idêntico ao pXF51 previamente identificado na cepa 9a5c. O genoma da cepa U24d é praticamente colinear ao genoma da cepa 9a5c enquanto que os genomas das cepas Fb7 e 3124 apresentaram maior colinearidade com a cepa Temecula1. Entre as diversas alterações encontradas nas análises comparativas destes genomas, destacamos a inserção no gene pilQ verificada no genoma da cepa U24d. Essa mutação causa ausência do pilus do tipo IV com consequente deficiência na motilidade twitching, sendo que plantas infectadas com a cepa U24d apresentam sintomas localizados restritos ao ponto de inoculação. Na cepa Fb7, detectamos a ausência de formação de biofilme no cultivo in vitro possivelmente devido ausência da expressão dos transcritos de mrkD e pspA, que codificam respectivamente adesina do pilus curto e adesina similar à hemaglutinina. Postulamos que estes genes não são expressos em decorrência de um defeito na via de sinalização de DSF (Fator de Sinalização Difusível) reflexo de uma mutação em rpfC no genoma de Fb7. Assim como as demais cepas de X. fastidiosa, também os genomas de U24d, Fb7 e 3124 apresentaram elevado conteúdo de Elementos Genéticos Móveis (EGM), que aparecem em maior número nas cepas sul-americanas. Os estudos do pangenoma de X. fastidiosa mostraram que essa espécie tem um genoma aberto e grande parte dos genes de EGMs correspondem a genes acessórios. A grande quantidade de EGMs em X. fastidiosa pode estar relacionada a falta do sistema CRISPR/cas completo, um provável resultado de eventos de erosão do genoma desta espécie. A inferência filogenética por MSLA mostrou uma clara distinção dos grupos de cepas da América do Norte em relação às do Sul, sugerindo a ocorrência de mais eventos de recombinações genéticas nas cepas sul-americanas, provavelmente pela falta de isolamento geográfico. Assim, é possível que as cepas norte e sul-americanas sofreram divergência alopátrica e simpátrica, respectivamente. / The genus Xylella consists of a single species, Xylella fastidiosa, a Gram-negative and non-flagellated bacterium that colonizes the xylem of a diversity of cultivated and wild plants in several parts of the world. In some of these plants, this bacterium is considered causal agent of diseases such as the Citrus Variegated Cholorosis in orange trees, Pierce\'s Disease of grapevines and coffee leaf scald. Eleven different strains of X. fastidiosa isolated from different hosts had their genomes sequenced, including 9a5c and Temecula1 strains, respectively isolated from orange tree and grapevine. Analyses of these genomes indicate a reasonable variability in their sequences and showed several genes associated with pathogenicity and virulence mechanisms of this bacterium. In this work we describe the genome sequencing, assembly and annotation of the strains U24d and Fb7, isolated from orange trees, and 3124 isolated from coffee, which have, respectively, 2,681,334 bp, 2,733,974 bp and 2,748,594 bp. Of these, only strain U24d has a plasmid, identical to pXF51 from strain 9a5c. The genome of U24d strain is almost collinear to the genome of strain 9a5c while the genomes of strains Fb7 and 3124 had higher collinearity to Temecula1 strain. Among many changes found in the comparative analysis of these genomes, we highlight an on insertion in pilQ gene that was found in U24d strain genome. This mutation causes lack of type IV pilus with a consequent deficiency in twitching motility. Moreover orange trees infected with U24d strain showed localized symptoms near to the inoculation point. We verified that Fb7 strain does not form biofilm in vitro possibly due to the absence of expression of mrkD and pspA transcripts, which encode, respectively, a short pilus adhesin and a hemagglutinin-like adhesin. We postulate that these genes are not expressed due to a defect in the signaling pathway of DSF (Diffusible Signal Factor) reflecting a mutation on rpfC in the Fb7 genome. Similarly to other X. fastidiosa strains, the genomes of U24d, Fb7 and 3124 also showed high content of mobile genetic elements (MGE), which appear in larger numbers in South American strains. Pan genome studies of X. fastidiosa showed that this species has a open genome and that most of MGE genes correspond to accessory genes. The large number of MGE in X. fastidiosa may be related to the lack of a complete system CRISPR/cas, likely a result of erosion events of the genome of this species. The phylogenetic reconstruction by MLSA showed a clear distinction between groups of strains from North and South America, suggesting the occurrence of more recombination events in South American strains, probably due to lack of geographical isolation. Thus it is possible that North and South American strains underwent allopatric and sympatric divergence, respectively.

Next-generation sequencing technology provides high-resolution data for epidemiological surveillance of bacterial pathogens on local and global scales. This approach has been used for many species including Methicillin Resistant Staphylococcus aureus (MRSA). In this thesis I demonstrate the utility of these data for understanding the spread of the globally disseminated clone MRSA ST239. I focus both on local and national-level epidemiology through sequence data of 71 isolates recovered from four hospitals representing three cities in Turkey; Istanbul (x2). Ankara and Izmir. I analyse whole genome sequence data from a further 33 ST239 isolates from global sources. These data were combined with previously published data for phylogenetic analysis based only on the core genome. I demonstrate how transmission events can be inferred from this approach on multiple levels; within hospital, between hospitals and between countries. The data pointed to a European origin of ST239, and independent introductions from Europe to Turkey, South America and East Asia. I also demonstrate how whole genome sequence data can be used to develop bespoke PCR assays, based on phage variation, for rapid local epidemiology. Finally, I consider how the sequence data might be used to explain variation in virulence potential, and describe the distribution and transfer of an important phage-borne virulence determinant, sasX, within Europe. Finally, I identified a single isolate with very strong biofilm forming ability likely due to the over-expression of the important adhesion SasG.