To identify novel therapeutic targets for basal-like breast cancer (BLBC) subtype, we investigated several DNA repair mechanisms associated with maintenance of high genomic instability for cell survival in cancer cells. We identified that the mismatch repair proteins, MSH2 and MSH6 (referred to as MSH2/6 hereafter), are highly elevated across BLBC samples. High expression level of MSH2/6 in BLBC is associated with worse prognosis and survivability for patients. Therefore, we knocked out MSH2 in BLBC cell lines and performed in vivo xenograft and syngeneic mice model studies to find significant attenuation of tumor growth in MSH2 KO group. Also, MSH2-deficient BLBC cells have increased rate of new mutations. Additionally, we tested the efficacy of conventional chemotherapeutics and radiation treatment that would further tip the genomic instability in MSH2-deficient BLBC cells towards cell death, but found them to be ineffective. Next, we performed high-throughput screening of 1280 FDA-approved compounds to discover that calcium channel blockers preferentially kill MSH2-deficient BLBC cells. This was likely due to association of significantly mutated pathways that involved calcium ion binding and calmodulin binding sites. Here we provide evidence of an alternative therapeutic strategy targeting DNA repair genes in BLBC patients utilizing bioinformatics analysis, high-throughput drug screening, in vitro,and vivoexperimentalmodels.

Deafness is the most common sensory deficit in humans, affecting 278 million people worldwide. Non-syndromic hearing loss (NSHL), hearing loss not associated with other symptoms, is the most common type of hearing loss and most NSHL in developed countries is due to a genetic cause. The inner ear is a remarkably complex organ, and as such, there are estimated to be hundreds of genes with mutations that can cause hearing loss. To date, 62 of these genes have been identified. This extreme genetic heterogeneity has made comprehensive genetic testing for deafness all but impossible due to low-throughput genetic testing methods that sequence a single gene at a time. The human genome project was completed in 2003. Soon after, genomic technologies, including massively parallel sequencing, were developed. MPS gives the ability to sequence millions or billions of DNA base-pairs of the genome simultaneously. The goal of my thesis work was to use these newly developed genomic technologies to create a comprehensive genetic testing platform for deafness and use this platform to answer key scientific questions about genetic deafness. This platform would need to be relatively inexpensive, highly sensitive, and accurate enough for clinical diagnostics. In order to accomplish this goal we first determined the best methods to use for this platform by comparing available methods for isolation of all exons of all genes implicated in deafness and massively parallel sequencers. We performed this pilot study on a limited number of patient samples, but were able to determine that solution-phase targeted genomic enrichment (TGE) and Illumina sequencing presented the best combination of sensitivity and cost. We decided to call this platform and diagnostic pipeline OtoSCOPE®. Also during this study we identified several weaknesses with the standard method for TGE that we sought to improve. The next aim was to focus on these weaknesses to develop an improved protocol for TGE that was highly reproducible and efficient. We developed a new protocol and tested the limits of sequencer capacity. These findings allowed us to translate OtoSCOPE® to the clinical setting and use it to perform comprehensive genetic testing on a large number of individuals in research studies. Finally, we used the OtoSCOPE® platform to answer crucial questions about genetic deafness that had remained unanswered due to the low-throughput genetic testing methods available previously. By screening 1,000 normal hearing individuals from 6 populations we determined the carrier frequency for non-DFNB1 recessive deafness-causing mutations to be 3.3%. Our findings will also help us to interpret variants uncovered during analysis of deafness genes in affected individuals. When we used OtoSCOPE® to screen 100 individuals with apparent genetic deafness, we were able to provide a genetic diagnosis in 45%, a large increase compared to previous gene-by-gene sequencing methods. Because it provides a pinpointed etiological diagnosis, genetic testing with a comprehensive platform like OtoSCOPE® could provide an attractive alternative to the newborn hearing screen. In addition, this research lays the groundwork for molecular therapies to restore or reverse hearing loss that are tailored to specific genes or genetic mutations. Therefore, a molecular diagnosis with a comprehensive platform like OtoSCOPE® is integral for those affected by hearing loss.

Sorghum (Sorghum bicolor L. Moench spp.) is a bioenergy crop with several appealing biological features to be explored in plant breeding for increasing efficiency in bioenergy production. The possibility to connect the influence of quantitative trait loci over time and between traits highlight the Bayesian networks as a powerful probabilistic framework to design novel genomic prediction models. In this study, we phenotyped a diverse panel of 869 sorghum lines in four different environments (2 locations in 2 years) with biweekly measurements from 30 days after planting (DAP) to 120 DAP for plant height and dry biomass at the end of the season. Genotyping-by-sequencing was performed, resulting in the scoring of 100,435 biallelic SNP markers. We developed and evaluated several genomic pre- diction models: Bayesian Network (BN), Pleiotropic Bayesian Network (PBN), and Dynamic Bayesian Network (DBN). Assumptions for BN, PBN, and DBN were independence, dependence between traits, and dependence between time points, respectively. For benchmarking, we used multivariate GBLUP models that considered only time points for plant height (MTi- GBLUP), and both time points for plant height and dry biomass (MTr-GBLUP) modeling unstructured variance-covariance matrix for genetic effects and residuals. Coincidence indices (CI) were computed for understanding the success in selecting for dry biomass using plant height measurements, as well as a coincidence index based on lines (CIL) using the posterior draws from the Bayesian networks to understand genetic plasticity over time. In the 5-fold cross-validation scheme, prediction accuracies ranged from 0.48 (PBN) to 0.51 (MTr- GBLUP) for dry biomass and from 0.47 (DBN-DAP120) to 0.74 (MTi-GBLUP-DAP60) for plant height. The forward-chaining cross-validation showed a substantial increment in prediction accuracies when using the DBN model, with r = 0.6 (train on slice 30:45 to predict 120 DAP) to 0.94 (train on slice 30:90 to predict 105 DAP) compared to the BN and PBN, and similar to multivariate GBLUP models. Both the CI and CIL indices showed that the ranking of promising inbred lines changed minimally after 45 DAP for plant height. These results suggest that 45 DAP is an optimal developmental stage for imposing the two-level indirect selection framework, where indirect selection for plant height at the end of the season (first-level target trait) can be done based on its ranking with 45 DAP (secondary trait) as well as for dry biomass (second-level target trait). With the advance of robotic technologies for field-based phenotyping, the development of novel approaches such as the two-level indirect selection framework will be imperative to boost genetic gain per unit of time. / O sorgo (Sorghum bicolor L. Moench spp.) é uma cultura bioenergética com várias características atrativas para serem exploradas no melhoramento de plantas para aumentar a eficiência de produção de bioenergia. A possibilidade de conectar informações genômicas em caracteres quantitativos ao longo do tempo, e entre caracteres, destacam as Redes Bayesianas como uma ferramenta probabilística poderosa para delinear novos modelos de predição genômica. Neste estudo, um painel diverso de 869 linhagens de sorgo foi fenotipado em quatro ambientes diferentes (2 locais em 2 anos) com medidas a cada duas semanas de 30 a 120 dias após o plantio (DAP), para altura de plantas e biomassa seca no fim da safra. Um procedimento de Genotipagem por sequenciamento foi executado, resultando na chamada de 100.435 marcadores baseados em Polimorfismos de Nucleotídeos Únicos (SNPs) bialélicos. Neste estudo foram desenvolvidos e avaliados os modelos de predição genômica: Rede Bayesiana (BN), Rede Bayesiana Pleiotrópica (PBN), e Rede Bayesiana Dinâmica (DBN). Os pressupostos para BN, PBN, e DBN foram independência, dependência entre caracteres, e dependência entre pontos no tempo, respectivamente. Para fins comparativos, formulações de modelos multivariados GBLUP foram utilizados considerando dependência entre pontos de tempo para altura de plantas (MTi-GBLUP), e ambos os pontos de tempo para a altura de plantas e biomassa seca (MTr-GBLUP), modelando matriz de variância-covariância não estruturada para efeitos genéticos e residuais. Índices de coincidência (IC) foram calculados para entender o sucesso na seleção indireta de biomassa seca usando medidas de altura de plantas, bem como um índice de coincidência baseado em linhagens (CIL), usando as amostras das posteriores das redes Bayesianas para entender a plasticidade genética ao longo do tempo. No esquema de validação cruzada 5-fold, as acurácias das predições variaram de 0,48 (PBN) a 0,51 (MTr-GBLUP) para biomassa seca e de 0,47 (DBN-DAP120) a 0,74 (MTi-GBLUP-DAP60) para altura de plantas. A validação cruzada forward-chaining mostrou um incremento substancial nas acurácias das predições ao usar o modelo DBN, com r = 0,6 (treinando no intervalo 30:45 para prever 120 DAP) até 0,94 (treinando no intervalo 30:90 para prever 105 DAP) em comparação com o BN e PBN, e semelhante aos modelos multivariados GBLUP. Os índices CI e CIL mostraram que o ranking de linhagens promissoras mudou minimamente após 45 DAP para altura de plantas. Estes resultados sugerem que 45 DAP é um estágio de desenvolvimento ideal para impor a estrutura de seleção indireta em dois níveis, onde a seleção indireta para a altura da planta no final da estação (caractere alvo de primeiro nível) pode ser feita com base na sua classificação com 45 DAP (caractere secundário), bem como para a biomassa seca (caractere alvo de segundo nível). Com o avanço das tecnologias robóticas para a fenotipagem baseada em campo, o desenvolvimento de novas abordagens, como a estrutura de seleção indireta em dois níveis, serão imperativas para aumentar o ganho genético por unidade de tempo.

The upland (Pseudacris feriarum) and midland (P. triseriata) chorus frogs are closely related cryptic species that are best distinguished genetically. The distribution of these species within the Commonwealth of Kentucky has previously been defined by only a handful of genetic samples, making delineation of range limits for each species difficult. Accurate understanding of species distributions, and the genetic structure within them, are vitally important for conservation management of amphibian species. In this study, I have collected genetic samples from across the putative ranges of P. triseriata and P. feriarum in Kentucky and used next-generation sequencing technology to generate more fine-scale estimates of species ranges. The genetic data generated in this study support the delineation of two species in Kentucky, and the species assignments of all individuals and populations are in general concordance with the previously hypothesized species distributions. However, I have identified two previously unrecognized contact zones for these species and revealed areas of hybridization. By delineating species distributions and identifying potentially important regions of genetic admixture, this study will be informative to future conservation management and conservation genetic research of chorus frogs in Kentucky.

La sélection génomique (GS) est un outil puissant pour prédire les phénotypes ou les valeurs génétiques d'individus encore non observés, sur la base d'un panel à la fois phénotypé et génotypé. Les modèles mixtes GBLUP habituellement utilisés prennent en compte tous les marqueurs simultanément, en postulant que leurs effets suivent tous la même distribution gaussienne. Les connaissances des mécanismes biologiques sous-jacent à la variation phénotypique ne sont donc pas pris en compte dans une telle modélisation. Le but de cette thèse est d'intégrer dans des modèles GBLUP des connaissances a priori, comme des régions génomique impliquées dans la variation des caractères d'intérêt ou encore des réseaux de gènes, afin d'évaluer le potentiel d'amélioration de la précision de prédiction. Ces modèles ont été appliqués à l'espèce de tournesol Helianthus annuus L., sur trois caractères (la floraison, le rendement et la sénescence foliaire) dans 13 environnements différents. L'un des principaux défis des études sur les hybrides de tournesol est de modéliser la vigueur hybride, ou hétérosis. Différentes hypothèses, incluant la dominance, la superdominance et l'épistasie ont été proposées pour clarifier les mécanismes génétiques sous-jacents au phénomène de l'hétérosis, mais leur importance n'est pas clairement connue. Dans ce contexte, la première partie de cette étude a eu pour but de tester l'efficacité de la GS dans une population d'hybrides provenant du croisement de 36 lignées femelles avec 36 lignées mâles. Pour cela des modèles prenant en compte des effets non-additifs ont été expérimentés, et les résultats validés expérimentalement en champ sur deux années. La prédiction des valeurs génétiques des hybrides ayant été concluante, nous avons ensuite cherché des informations a priori à intégrer à ces modèles. Des SNPs impliqués dans la variation des trois caractères d'intérêt ont été recherchés à l'aide de plusieurs modèles de GWAS (additifs et non-additifs). De plus, dans la perspective de tester des modèles prenant en compte des interactions épistatiques, des SNPs localisés dans des réseaux de gènes connus ont été recherchés. La dernière partie de cette thèse a eu pour but d'intégrer aux modèles GBLUP ces régions génomiques impliquées dans la variation des caractères. Deux méthodes ont été utilisées pour cela, à savoir la modélisation des informations a priori dans la partie aléatoire (modèle MultiBLUP) ou dans la partie fixe des modèles. Ces méthodes ne montrent pas d'amélioration significative des précisions de prédiction par rapport aux modèles GBLUP sans information a priori. / Genomic selection is a powerful tool for predicting phenotypes or genetic values of non-observed individuals, based on a panel both phenotyped and genotyped. The mixed models GBLUP usually utilized take into account all markers simultaneously, assuming that all their effects all follow the same Gaussian distribution. Knowledge of the biological mechanisms underlying phenotypic variation is therefore not taken into account in such modeling. The aim of this thesis is to integrate in GBLUP models a priori knowledge, such as genomic regions involved in the variation of the traits of interest or networks of genes, in order to evaluate the potential for improvement of accuracies. These models were applied to the Helianthus annuus L. sunflower specie on three traits (flowering time, yield and leaf senescence) in 13 several environments. One of the main challenges of genetic studies on sunflower hybrids is to model hybrid vigor, or heterosis. Different hypotheses, including dominance, over-dominance and epistasis have been proposed to clarify the genetic mechanisms underlying the heterosis phenomenon, but their importance is not clearly known. In this context, the first part of this study aimed to test the efficiency of the GS in an hybrid population from the crossing of 36 female lines with 36 male lines. For this purpose, models taking into account non-additive effects were experimented, and the results validated experimentally in field over two years. The prediction of the genetic values of the hybrids was conclusive, so we looked for a priori information to integrate with these models. SNPs involved in the variation of the three traits of interest were searched using several models of GWAS (additive and non-additive). Moreover, in order to test models taking into account epistatic interactions, SNPs located in known gene networks have been sought. Finally the integration of the genomic regions involved in the variation of the traits, into the GBLUP models, was conducted. Two methods were implemented for this, namely the modeling of a priori information in the random part (MultiBLUP model) or in the fixed part of the models. These methods do not show significant improvement in accuracies compared to GBLUP models without a priori information.

Microorganisms are capable of producing advanced biofuels that can be used as ‘drop-in’ alternatives to conventional liquid fuels. However, vital physiological processes and membrane properties are often disrupted by the presence of biofuel and limit the production yields. In order to make microbial biofuels a competitive fuel source, finding mechanisms for improving resistance to the toxic effects of biofuel production is vital. This investigation aims to identify resistance mechanisms from microorganisms that have evolved to withstand hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters. First, using genomic DNA from Marinobacter aquaeolei, we constructed a transgenic library that we expressed in Escherichia coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy of a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance. Additionally, we used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E. coli host. We exposed cells containing the library to pinene, selecting for genes that improved tolerance. Importantly, we found that expressing the sigma factor RpoD from P. putida greatly expanded the diversity of tolerance genes recovered. With low expression of rpoDP. putida, we isolated a single pinene tolerance gene; with increased expression of the sigma factor our selection experiments returned multiple distinct tolerance mechanisms, including some that have been previously documented and also new mechanisms. Interestingly, high levels of rpoDP. putida induction resulted in decreased diversity. We found that the tolerance levels provided by some genes are highly sensitive to the level of induction of rpoDP. putida, while others provide tolerance across a wide range of rpoDP. putida levels. This method for unlocking diversity in tolerance screening using heterologous sigma factor expression was applicable to both plasmid and fosmid-based transgenic libraries. These results suggest that by controlling the expression of appropriate heterologous sigma factors, we can greatly increase the searchable genomic space within transgenic libraries. This dissertation describes a method of effectively screening genomic DNA from multiple organisms for genes to mitigate biofuel stress and shows how tolerance genes can improve bacterial growth in the presence of toxic biofuel compounds. These identified genes can be targeted in future studies as candidates for use in biofuel production strains to increase biofuel yields.

La différenciation et le développement requièrent une régulation fine de l’expression desgènes, médiée en partie par les modifications épigénétiques. Parmi les modificationsd’histones, la chromatine bivalente, signature chromatinienne atypique associant lesmarques permissive H3K4me2/3 et répressive H3K27me3, est de par sa plasticité, pressentiepour jouer un rôle décisionnel dans l’acquisition d’une identité cellulaire. Pour étudier le rôlede la chromatine bivalente au cours du développement, nous avons choisi d’utiliserl’empreinte parentale. Ce cadre développemental bien caractérisé, conduit à l’expression decertains gènes à partir d’un seul des deux allèles selon son origine parentale. La méthylationdifférentielle de l’ADN d’une région clé, appelée ICR (Imprinting Control Region), bienqu’absolument requise pour l’expression mono-allélique de ces gènes, n’est pas suffisantepour rendre compte de la complexité du profil d’expression de ces gènes suggérantl’implication d’autres mécanismes. Sur 15 ICR méthylés sur l’allèle maternel, nous avonsprécisément mis en évidence que la chromatine bivalente est présente par défaut sur l’allèlenon-méthylé lorsque celui-ci est transcriptionnellement inactif, quel que soit le stadedéveloppemental ou le tissu étudié, participant ainsi à la régulation fine de l’expressiontissu-spécifique à partir de ces régions. Dans leur ensemble, nos données révèlent que lachromatine bivalente joue un rôle moins dynamique que pressentie. Ainsi, au niveau del’empreinte parentale, sa fonction principale serait de protéger l’allèle non-méthylé des ICRcontre l’acquisition de méthylation tout en aidant à le maintenir réprimé dans certainstissus. Nous proposons que la chromatine bivalente joue un rôle similaire sur l’ensemble desîlots CpG du génome, contribuant ainsi à la protection de l’identité cellulaire. Afin decompléter cette première étude, j’ai étudié la régulation de l’expression d’un candidat de larégulation de la dynamique de la chromatine bivalente, l’histone déméthylase pourH3K27me3, JMJD3. Les résultats obtenus suggèrent que l’induction d’expression observéeau cours de la différenciation neurale s’appuie sur une dynamique de la structuretridimensionnelle de la chromatine qui pourrait elle-même être régulée par la transcriptiond’un eARN (enhancer ARN) et l’hydroxyméthylation. Ce modèle souligne un mode derégulation complexe de ce nouvel acteur épigénétique, impliquant des régionsintragéniques, et pourrait notamment permettre de comprendre les mécanismes impliquésdans sa dérégulation dans les cancers. / Fine-tuned regulation of gene expression is required for cell fate determination anddevelopment. Epigenetics modifications are well documented to be instrumental in thisprocess. Among them, bivalent chromatin, an unusual chromatin signature, which associatesthe permissive mark H3K4me2/3 and the repressive mark H3K27me3, is believed to arbitrategene expression during cell commitment. To study its precise role in development, we haveundertaken to study bivalency in the context of genomic imprinting. This well-defineddevelopmental frame is a process restricting expression of some genes to one parental alleleonly. The constitutive differential DNA methylation at the key region called ICR (ImprintingControl Region), is absolutely required but not sufficient to explain the complexity of themono-allelic expression pattern of imprinted genes, indicating that other mechanisms couldbe involved. Specifically, on 15 maternally methylated ICR, we showed that bivalentchromatin is acquired by default on the unmethylated allele of ICR when it istranscriptionally inactive whatever the developmental stage or the tissue studied and thuscontribute to tissue-specific expression from these regions. Altogether, our results revealthat chromatin bivalency is much less dynamic than proposed. In the context of genomicimprinting, it seems to plays more a safeguard function at ICR by protecting theunmethylated allele against DNA methylation acquisition while keeping it silent in a subsetof tissues. To complete this study, I studied the regulation of JMJD3, a histone demethylasefor H3K27me3, candidate to regulate bivalency dynamic. Our results suggest that theinduction of Jmjd3 expression observed during neural differentiation rely on the dynamic ofthe tridimensional architecture at the locus which could be regulated by the transcription ofan eRNA (enhancer RNA) and by hydroxymethylation. This model highlight a complex way ofregulation for this new epigenetics actor, involving intragenic regions and could help tounderstand how Jmjd3 expression is deregulated in a pathological context such as in cancer.

Comprendre l’émergence des maladies dans les agroécosystèmes nécessite d’étudier l’histoire évolutive des populations bactériennes associées aux plantes. L’objectif de ce travail était de déterminer les évènements évolutifsconduisant à l’émergence des lignées pathogènes ou pathovars dans l’espèce Xanthomonas arboricola. Une analyse de génétique des populations a été menée sur un panel de souches phytopathogènes et commensales et complétée par l’inférence des gains et pertes de facteurs de virulence. Cette espèce possède une structure de population épidémique ; les clones épidémiques ont émergé suite à l’acquisition de facteurs de virulence à partir d’un fond recombinant de souches commensales. Une analyse de génomique des populations et la reconstruction de scénarios de divergence entre ces clones et le réseau de souches recombinantes, a montré la persistance d’un flux de gènes asymétrique entre ces deux groupes, dans le sens souches pathogènes vers souches commensales. Enfin, l’histoire évolutive du principal facteur de virulence des Xanthomonas, le système de sécrétion de type 3, a été retracée au sein du genre, et a montré que celui-ci avait été acquis ancestralement puis perdu dans certaines souches commensales. En conclusion, l’ancêtre commun de X. arboricola possédait des facteurs de virulence et au sein des souches commensales, certaines ont perdu ces facteurs, tandis que d’autres ont conservé le répertoire ancestral. Ces dernières diffèrent peu de certains agents pathogènes, et pourraient représenter un risque pour de nouvelles émergences. Des travaux de génomique fonctionnelle permettraient de valider ces hypothèses. / Deciphering the evolutionary history of bacterial populations associated to plants is necessary to understand diseaseemergence in agroecosystems. The aim of this study is to unveil the evolutionary events responsible for pathogeniclineages or pathovar emergences in Xanthomonas arboricola. This species is composed of both plant pathogenic andcommensal strains Population genetics analyses and gain and loss inferences of virulence factors showed that X. arboricola exhibits an epidemic population structure, within which epidemic clones emerged from a recombinogenic background population following virulence factor acquisition. Population genomics and inference of divergence scenarii between epidemic clones and the network of recombinant strains showed persistence of homologous recombination along divergence of these two groups, with an asymmetric gene flux from pathogenic strains to commensal ones. Finally, evolutionary history of the type three secretion system (T3SS), the main virulence factor in Xanthomonas genus, was studied at genus scale and showed that T3SS was ancestrally acquired and lost in commensal strains. Altogether these analyses allowed us to show that the common ancestor of X.arboricola had virulence factors, and that within commensal strains, some lost these virulence factors whereas others kept the ancestral repertoire. These latter strains have a similar repertoire to that of some pathogenic strains, and could represent a risk for new disease emergence. Functional genomics could allow us to validate these hypotheses.

The rapid spread of carbapenem-resistant lineages of Klebsiella pneumoniae, clustered within the clonal group CG258, is a growing public health problem associated with healthcareassociated infections. The objective of this study was to perform a genomic analysis of KPC-2 and/or CTX-M β-lactamase-producing strains of K. pneumoniae belonging to CG258 (ST11, ST258, ST340, ST437) circulating at the human-animal-environment interface, in Brazil and South America. The analysis was conducted to characterize the antimicrobial resistome, virulome, genetic elements of transfer and mobilization associated with the dissemination of the blaKPC-2 gene, and to perform a detailed comparative genomic analysis of the CG258; with subsequent pathogenicity evaluation in an invertebrate (Galleria mellonella) model of infection, aiming to identify biomarkers of virulence. The main results are presented in the format of six manuscripts. Manuscript I: New draft genome sequence of a Klebsiella pneumoniae strain 1194/11, belonging to ST340, showing a wide resisto-me. Manuscript II: The first draft genome sequence of a Klebsiella pneumoniae 606B ST340 carrying blaCTX-M-15 in food-producing animal isolated in Brazil. Manuscript III: The first draft genome sequence of a Klebsiella pneumoniae strain Kp171, recovered from a water sample collected in an urban river in Brazil, demonstrating that anthropogenic activities, including the release of wastewater and sewage from hospitals, may have contributed to the contamination of aquatic environments, raising a concern to public health. Manuscript IV: Identification and complete sequence analysis of an IncX3 plasmid carrying a non-Tn4401 genetic element (NTEKPC-Ic), originating from a hospital associated lineage of K. pneumoniae ST340, showing the spread of blaKPC-2 in new Incompatibility group. Manuscript V: Dissemination of blaKPC-2 in novel non-Tn4401 Element (NTEKPC-IId) carry by new small IncQ1 and Col-Like plasmids in lineages of Klebsiella pneumoniae ST11 and ST340. Manuscript VI: Yersiniabactin, colibactin and wider resistome contribute to enhanced virulence and persistence of KPC-2-producing K. pneumoniae CG258 in South America. The results obtained in the present study allow us to obtain a first genomic landscape of K. pneumoniae lineages of the CG258, circulating at the human-animal-environment interface, in Brazil and South America. In this regard, most likely the interplay of yersiniabactin and/or colibactin, and resistance to clinically significant antibiotics (as carbapenems and polymyxins) are contributing to the emergence of highly virulent and MDR lineages that pose great risk to human health. On the other hand, the wide antimicrobial resistome (antibiotics, disinfectants and heavy metals) could be contributing to adaptation of KPC-2- and/or CTX-M-producing K. pneumoniae CG258 in the human-animal-environment interface, highlighting the urgent need for enhanced control efforts. In conclusion, these findings could contribute to the development of strategies for prevention, diagnosis and treatment of K. pneumoniae infections. / A rápida disseminação de linhagens de Klebsiella pneumoniae resistentes aos carbapenêmicos, agrupadas dentro do grupo clonal GC258, e um crescente problema de saúde pública associado com infecções relacionadas a assistência a saúde. O objetivo deste estudo foi realizar uma análise genômica de cepas de K. pneumoniae produtoras de β-lactamases KPC-2 e/ou CTX-M, pertencentes ao GC258 (ST11, ST258, ST340, ST437), circulando na interface humana-ambiente-animal, no Brasil e na América do Sul. A análise foi direcionada para caracterizar o resistoma e viruloma, elementos genéticos de transferência e mobilização associados com a disseminação de genes blaKPC-2, e realizar uma análise de genômica comparativa detalhada do GC258, com posterior avaliação da patogenicidade em modelo invertebrado (Galleria mellonella) de infecção, visando identificar biomarcadores de virulência. Os principais resultados são apresentados na forma de seis manuscritos. Manuscrito I: Nova sequência \"draft\" do genoma de K. pneumoniae 1194/11isolado de amostra clínica, pertencente ao ST340, mostrando um amplo resistoma. Manuscrito II: O reporte da primeira sequência \"draft\" do genoma de K. pneumoniae 606B (ST340), contendo blaCTX-M-15 em animais de produção isolados no Brasil. Manuscrito III: O primeiro esboço da sequência do genoma de K. pneumoniae Kp171, recuperado de uma amostra de água coletada em um rio urbano no Brasil, demonstrando que atividades antrópicas, incluindo a liberação de esgoto e esgoto de hospitais, podem ter contribuído para a contaminação ambientes aquáticos, levantando uma preocupação para a saúde pública. Manuscripto IV: Identificação e análise de sequencia completa de um plasmídeo IncX3 portador de um elemento genético não Tn4401 (NTEKPC-Ic), originado de uma linhagem hospitalar associada a K. pneumoniae ST340, mostrando a disseminação de blaKPC-2 no novo grupo Incompatibilidade. Manuscrito V: Disseminação de blaKPC-2 no novo elemento non-Tn4401 (NTEKPC-IId) portado por novos pequenos plasmídeos IncQ1 e Col-Like em linhagens de K. pneumoniae ST11 e ST340. Manuscrito VI: Os resultados obtidos no presente estudo permitem gerar um panorama genômico das linhagens de K. pneumoniae do GC258, circulando na interface humana-animal-ambiente, no Brasil e na América do Sul. De principal interesse, a convergência da virulência associada com genes codificando yersiniabactina e/ou a colibactina e a resistência a antibióticos clinicamente significativos (como carbapenemicos e polimixinas), estão contribuindo para o aparecimento de linhagens altamente virulentas e multirresistentes que apresentam um grande risco a saúde humana. Por outro lado, a ampla resistência aos antimicrobiana (antibióticos, desinfetantes e metais pesados) poderia estar contribuindo para a adaptação de estirpes de K. pneumoniae do GC258, produtoras de KPC-2- e/ou CTX-M, na interface humana-ambiente-animal, destacando a necessidade urgente de medidas para o controle de disseminação. Em conclusão, esses achados poderiam contribuir para o desenvolvimento de estratégias de prevenção, diagnóstico e tratamento das infecções por K. pneumoniae.

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