Orientador: Danísio Prado Munari / Coorientador: Ricardo Vieira Ventura / Banca: Lenira El Faro Zadra / Banca: Ana Fabrícia Braga Magalhães / Banca: Rodrigo Pelicioni Savegnago / Banca: João Ademir de Oliveira / Resumo: Dentre as informações fornecidas pelas metodologias que utilizam marcadores do tipo polimorfismo de nucleotídeo único (SNPs), as de segmentos de homozigose (ROH) e de desequilíbrio de ligação têm colaborado para estudos de aplicação direta da informação genômica em populações de bovinos de corte, como em estudos de associação com cobertura ampla do genoma, de seleção genômica e de estrutura da população, dentre outros. Atualmente a imputação vem sendo utilizada principalmente para reduzir custos com a genotipagem dos animais e pode ser utilizada combinando informações genômicas de diferentes painéis. Para que dados imputados sejam utilizados de forma eficiente, é necessário que a imputação tenha sido implementada de forma que todos os animais tenham seus genótipos inferidos com elevada acurácia. No entanto, esta é verificada apenas se houver o genótipo real para avaliar a confiabilidade do genótipo imputado. Dessa maneira, os objetivos deste trabalho foram: (1) estudar a imputação de painéis comercial e customizados de baixa densidade para painéis de alta densidade (Illumina e Affymetrix), assim como para um painel combinado (Illumina + Affymetrix) para bovinos da raça Nelore, e estudar o desequilíbrio de ligação e conformação de blocos de haplótipos antes e após a imputação; (2) estudar estratégias para predição da acurácia de imputação, utilizando redes neurais artificiais e regressão linear múltipla; (3) estudar os segmentos de homozigose e, com isso, a endogamia presente ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Among all the information provided by methodologies that use single nucleotide polymorphism (SNPs), the runs of homozygosity (ROH) and linkage disequilibrium have been used for studies that explore genomic information in beef cattle population, as the genome-wide association, genomic selection, the structure of population and others. Nowadays, the imputation is used in these studies to reduce genomic costs and this also can be used combining genomic information from different panels. The animals used to be imputed should present genotypes inferred with high accuracy to allow the use imputed genotypes in other studies. However, the accuracy is verified only if there is a real genotype to evaluate the imputed genotype. Therefore, this study aimed: (1) Evaluate imputation of commercial and customized low density panels to high density panels (Illumina and Affymetrix), as well as to a combined panel (Illumina + Affymetrix) in Nelore beef cattle, and estimating linkage disequilibrium and haplotype blocks conformation to high density panels individually and after imputation; (2) Study a strategy to predict imputation accuracy using artificial neural network and linear regression; (3) Study runs of homozygosity and inbreeding in a populations from Nelore beef cattle, as well as identify genes present in ROH with high frequency in population. For ROH studies were used 34 bulls from different lines and the progeny, totalizing 809 Nelore animals genotyped with information of 509.107 SN... (Complete abstract click electronic access below) / Doutor

Orientador: Roberto Carvalheiro / Coorientador: Haroldo Henrique de Rezende Neves / Coorientador: Herman Arend Mulder / Banca: Ricardo Vieira Ventura / Banca: Lucia Galvão de Albuquerque / Banca: Gerson Antonio de Oliveira Júnior / Banca: Diercles Francisco Cardoso / Resumo: A existência de um controle genético sobre a variância residual torna possível a seleção para uniformidade de produção. Desta forma, os objetivos do presente estudo foram: i) investigar a presença deste componente genético na variância residual do peso ao sobreano (PS) em bovinos da raça Nelore (N = 194.628, touros = 625), usando duas abordagens: dois passos (two-step) e modelo linear generalizado hierárquico duplo (double hierarchical generalized linear model, DHGLM); e ii) identificar, através de estudo de associação genômica ampla (genome-wide association study, GWAS), regiões genômicas associadas com uniformidade do PS usando differentes variáveis respostas: EBV desregredido (dEBVv) obtido a partir de soluções de um DHGLM assumindo correlação genética não-nula entre média e variância residual (rmv ≠ 0); e dEBVv_r0 e variância dos resíduos log-transformada (ln_σ2ê) obtidos a partir de soluções de um DHGLM assumindo rmv = 0. Os resultados confirmam a presença de heterogeneidade genética de variância residual bem como oportunidade de seleção (coeficiente de variação genética da variância residual (GCVE) variando de 0,10 a 0,17). Porém, a seleção pode ser limitada devido à magnitude da variância genética da variância residual e à forte e positiva correlação genética entre a média e a variância (0,20 e 0,76 para a abordagem em dois passos e DHGLM, respectivamente). Além disso, foi possível observar que um grande número de progênies por touro é necessário para obter EBV com mai... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The existence of genetic control on residual variance makes possible the selection for uniformity. Thus, the objectives of the present study were: i) to investigate the presence of this genetic component on the residual variance of yearling weight (YW) in Nellore cattle (N = 194,628, sires = 625) using two approaches: two step approach and double hierarchical generalized linear model (DHGLM); and ii) to identify, through a genome-wide association study (GWAS), genomic regions associated with uniformity of YW using different response variables: deregressed EBV (dEBVv) obtained from solutions of a DHGLM assuming non-null genetic correlation between mean and residual variance (rmv ≠ 0); and dEBVv_r0 and log-transformed variance of estimated residuals (ln_σê 2) obtained from solutions of a DHGLM assuming rmv = 0. The results confirm the presence of genetic heterogeneity of residual variance as well as opportunity of selection (coefficient of genetic variance of residual variance (GCVE) ranging from 0.10 to 0.17). However, selection may be limited by the magnitude of the genetic variance of the residual variance and the strong and positive genetic correlation between mean and variance (0.20 and 0.76 for two-step approach and DHGLM, respectively). In addition, it was observed that large sire families are required to obtain higher EBVv accuracies (heritability of residual variance, hv 2 < 0.007). The mean-variance relationship was reduced by using Box-Cox transformation, which reduc... (Complete abstract click electronic access below) / Doutor

Orientador: Henrique Nunes de [UNESP] Oliveira / Coorientador: Rodrigo Giglioti / Coorientador: Fernando Flores Cardoso / Banca: Danísio Prado Munari / Banca: Ricardo da Fonseca / Banca: Guilherme Jordão de Magalhães Rosa / Banca: Maria Eugênia Zerlotti Mercadante / Resumo: Babesiose bovina é uma doença transmitida pelo carrapato, sendo que a Babesia bovis é considerada a espécie mais patogênica. Ambos são considerados como um entrave na melhoria da produtividade da bovinocultura de corte nos trópicos, especialmente para animais de raças taurinas e suas cruzas. Esse estudo analisou uma população de bovinos Hereford e Braford e foi composto por quatro capítulos com os seguintes objetivos: Capítulo 1) Revisão de literatura; Capítulo 2) Estimação de parâmetros genéticos para contagem de carrapatos (TC) e B. bovis usando modelos lineares e modelos lineares generalizados; Capítulo 3) Avaliar a habilidade de predição e a possibilidade de aplicação da seleção genômica e conduzir estudos de associação genômica ampla (GWAS) para nível de infecção de B. bovis (IB); Capítulo 4) Procurar por estruturas causais entre TC, IB, ganho de peso do nascimento a desmama (WG) e ganho de peso da desmama ao sobreano (YG) usando a abordagem do modelo de equação estrutural (SEM). Os carrapatos foram contados manualmente em um lado do animal. A quantificação de B. bovis foi feita por meio de ensaios de qPCR. No Capítulo 2, os dados de contagem de carrapato e B. bovis estavam em escala logaritímica para as análises usando modelos lineares. O modelo de Poisson foi aplicado para contagem de carrapato sem tranformação logarítimica e para os modelos probit o fenótipo foi considerado como ausência (0) ou presença (1) de B. bovis baseado em três diferentes limiares (BBt1: IB usa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bovine babesiosis is a tick-borne disease, and the Babesia bovis is considered the most pathogenic species. Both parasites constitute major drawbacks for improvement of beef cattle productivity in the tropics, especially when purebred and crossbred taurine animals are used. This study analyzed a population of Hereford and Braford cattle and it was composed of four chapters with the following objectives: Chapter 1) Literature review; Chapter 2) Estimate genetic parameters for tick count (TC) and B. bovis using linear and generalized linear models; Chapter 3) Evaluate predictive ability and application of genomic selection and performed genome wide association studies (GWAS) for B. bovis infection level (IB) using single step GBLUP model; Chapter 4) Search for causal structures to investigate potential functional relationships among TC, IB, weight gain from birth to weaning (WG), and weight gain from weaning to yearling (YG) using structural equation modeling (SEM). Tick counts were performed by manually counting adult female ticks on one side of each animal. The B. bovis quantification was performed using a qPCR assay. In the Chapter 2, the tick count and B. bovis records were in log scale for analysis using linear model. A Poisson model was applied for tick count without log transformation and for probit model the phenotype was assessed by absence or presence of B. bovis based on three different thresholds (BBt1: IB using the threshold observed; BBt2: BB using threshold as a ... (Complete abstract click electronic access below) / Doutor

Os elementos transponíveis (TEs) constituem grande parte do material genético de diversos eucariotos, alcançando entre 50-80% do genoma de gramíneas. Os projetos genoma proporcionaram um aumento das informações disponíveis sobre estes elementos, o que evidenciou sua importância e possibilitou o desenvolvimento de novas abordagens para seu estudo. O sistema Mutator (Mu) de milho é o mais ativo e mutagênico transposon de plantas. Além do elemento autônomo, MuDR, o sistema compreende ainda um conjunto de elementos bastante heterogêneo em sua seqüência e estrutura, chamados MuLEs, que podem conter até mesmo fragmentos de genes do hospedeiro. As seqüências de transposons mais abundantemente expressas no transcriptoma de cana-de-açúcar são relacionadas a MuDR e se agrupam em quatro clados (nomeados Classes I, II, II e IV), existentes antes da divergência entre Mono e Eudicotiledôneas. O trabalho apresentado aqui teve o objetivo de aprofundar o conhecimento sobre o sistema Mutator em cana-de-açúcar a partir da análise comparativa entre seqüências dessa planta e de arroz (cujo genoma está totalmente seqüenciado). Foi possível avaliar a abundância e diversidade do sistema Mu em gramíneas, ficando evidente uma amplificação de elementos clado-específica, tendo a Classe II sofrido uma explosão no número de cópias ao longo da evolução destas plantas. Análises estruturais revelaram que, enquanto as Classes I e II compreendem elementos com características de transposons, as Classes III e IV são, na verdade, transposases domesticadas. Foram completamente seqüenciados dois clones de BAC de cana-de-açúcar, um proveniente de cada parental do híbrido (Saccharum officinarum e Saccharum spontaneum), ambos contendo elementos da Classe III. Estes elementos foram caracterizados e a seqüência genômica de cana foi comparada com sua ortóloga em arroz, revelando um acúmulo de TEs nas regiões intergênicas. / Transposable elements (TEs) constitute great part of eukaryote genetic material, in grasses, they comprise between 50-80% of the genome. Genome projects have significantly increased the amount of information about these elements, revealing their importance and allowing the development of new approaches for their study. The Mutator system (Mu) of maize is the most active and mutagenic plant transposon. Beyond the autonomous element, MuDR, the system comprises a very heterogeneous, in sequence and structure, set of elements, called MuLEs, that can contain even host gene fragments. The most abundant transposon related sequences expressed in sugarcane transcriptoma are the MuDR-like. They group into four clades (called Classes I, II, III and IV) that exist prior to the Mono and Eudicot split. The aim of this work is to gain knowledge about the Mutator system in sugarcane through the comparative analysis against rice (whose genome is completely sequenced). The results described the abundance and diversity of the Mu system in grasses, evidencing a clado-specific amplification with a burst of Class II along the evolution of this plant group. Structural analyses showed that, while Classes I and II comprise elements with transposon characteristics, Classes III and IV are domesticated transposases. One BAC clone from each sugarcane parental genotype (Saccharum officinarum and Saccharum spontaneum) have been completely sequenced, both containing Class III elements. These elements have been characterized and the sugarcane genomic sequences were compared with their orthologues in rice. The comparative analyses showed an accumulation of TEs in the intergenic regions.

O advento do sequenciamento de nova geração (NGS - Next Generation Sequencing) nos últimos anos proporcionou um aumento expressivo no número de projetos genômicos. De maneira simplificada, as máquinas sequenciadoras geram como resultado fragmentos de DNA que são utilizados por programas montadores de genoma. Esses programas tentam juntar os fragmentos de DNA de modo a obter a representação completa da sequência genômica (por exemplo um cromossomo) da espécie sendo sequenciada. Em alguns casos o processo de montagem pode ser executado com maior facilidade para organismos com genomas de tamanhos pequenos (por exemplo bactérias com genoma em torno de 5Mpb), através de pipelines que automatizam a maior parte da tarefa. Um cenário mais complicado surge quando a espécie possui genoma com grande comprimento (acima de 1Gpb) e elementos repetidos, como no caso de alguns eucariotos. Nesses casos o resultado da montagem é geralmente composto por milhares de fragmentos (chamados de contigs), uma ordem de magnitude muito superior ao número de cromossomos estimado para um organismo (comumente da ordem de dois dígitos), dando origem a uma montagem altamente fragmentada. Uma atividade comum nesses projetos é a comparação da montagem com a de outro genoma como forma de validação e também para identificação de regiões conservadas entre os organismos. Embora o problema de alinhamento par-a-par de genomas grandes seja bem contornado por abordagens existentes, o alinhamento múltiplo (AM) de genomas grandes em estado fragmentado ainda é uma tarefa de difícil resolução, por demandar alto custo computacional e grande quantidade de tempo. Este trabalho consiste em uma metologia para fazer alinhamento múltiplo de genomas grandes de eucariotos com montagens altamente fragmentadas. Nossa implementação, baseada em alinhamento estrela, se mostrou capaz de fazer AM de grupos de montagens com diversos níveis de fragmentação. O maior deles, um conjunto de 5 genomas de répteis, levou 14 horas de processamento para fornecer um mapa de regiões conservadas entre as espécies. O algoritmo foi implementado em um software que batizamos de FROG (FRagment Overlap multiple Genome alignment), de código aberto e disponível sob licença GPLv3. / The advent of Next Generation Sequencing (NGS) in recent years has led to an expressive increase in the number of genomic projects. In a simplified way, sequencing machines generate DNA fragments that are used by genome assembler software. These programs try to merge the DNA fragments to obtain the complete representation of the genomic sequence (for example a chromosome) of the species being sequenced. In some cases the assembling process can be performed more easily for organisms with small-sized genomes (e.g. bacteria with a genome length of approximately 5Mpb) through pipelines that automate most of the task. A trickier scenario arises when the species has a very large genome (above 1Gbp) and complex elements, as in the case of some eukaryotes. In those cases the result of the assembly is usually composed of thousands of fragments (called contigs), an order of magnitude much higher than the number of chromosomes estimated for an organism (usually in the order two digits), giving rise to a highly fragmented assembly. A common activity in these projects is the comparison of the assembly with that of another genome as a form of validation and also to identify common elements between organisms. Although the problem of pairwise alignment of large genomes is well circumvented by existing approaches, multiple alignment of large genomes with highly fragmented assemblies remains a difficult task due to its time and computational requirements. This work consists of a methodology for doing multiple alignment of large eukaryotic genomes with highly fragmented assemblies, a problem that few solutions are able to cope with. Our star alignment-based implementation, was able to accomplish a MSA of groups of assemblies with different levels of fragmentation. The largest of them, a set of 5 reptilian genomes where the B. jararaca assembly (800,000 contigs, N50 of 3.1Kbp) was used as anchor, took 14 hours of execution time to provide a map of conserved regions among the participating species. The algorithm was implemented in a software named FROG (FRagment Overlap multiple Genome alignment), available under the General Public License v3 (GPLv3) terms.

Introdução: As arboviroses causam graves problemas de saúde pública no Brasil e em muitos dos países da América Latina. A epidemiologia molecular é um instrumento valioso na compreensão da dispersão, persistência e diversidade desses patógenos virais. Objetivos: Neste projeto, buscamos investigar a dinâmica epidemiológica molecular dos arbovíroses (com especial enfoque aos vírus da dengue-DENV, chikungunya - CHIKV e zika -ZIKV) que circularam no estado do Amapá entre os anos de 2013 e 2016. Métodos: 824 amostras de plasma humano foram coletadas pelos laboratórios de Saúde Publica (LACEN) no estado do Amapá entre os anos de 2013 e 2016; essas amostras foram obtidas de pacientes que apresentavam sintomas consistentes com uma das arboviroses. O material genético viral presente nestas amostras foi extraído e os ensaios de qPCR foram realizados. Todas as amostras foram submetidas inicialmente a um ensaio triplex (ZIKV/DENV/CHIKV), as amostras negativas foram posteriormente submetidas a um ensaio de pan-flavivírus. As amostras positivas para um dos ensaios foram submetidas a NGS (sequenciamento de nova geração). Resultados: Das 824 amostras testadas, 36 foram positivas para DENV, ZIKV ou CHIKV; desses 36 positivos, 24 foram para DENV, 11 para CHIKV e 1 para ZIKV. Foram obtidos 27 genomas completos: 16 de DENV (15 DENV1, genótipo V e 1 DENV2, genótipo III) e 11 de CHIKV (genótipo asiático / caribenho). Das 788 amostras testadas com o ensaio de pan-flavivírus, 22 amostras foram positivas; porem apenas uma amostra produziu genoma completo pela técnica de NGS. Este genoma foi relacionado com um flavivírus com semelhante em 76,81% com o vírus Long Pine Key - LPKV, que anteriormente só tinha sido descrito em mosquitos. Árvores de Maximum likelihood e Maximum clade credibility foram construídas utilizando os genomas do DENV1 obtidos neste estudo. Essas árvores exibiam duas linhagens distintas de DENV1, genótipo V presentes na América Latina. Uma destas linhagens tem um padrão de circulação que inclui países do Caribe, América Central e América do Sul (incluindo Brasil); a outra linhagem distinta circula dentro das fronteiras do Brasil. As árvores também indicam que o DENV1 presente no estado do Amapá é da linhagem que tem o padrão de circulação que inclui o Caribe e as Américas Central e do Sul e que essa linhagem surgiu no Amapá entre 2005 e 2010. Conclusão: Este estudo fornece dados importantes sobre as arboviroses no Amapá e os dados genômicos mais recentes disponíveis para a região, bem como o contexto brasileiro e latino-americano para esses dados. Dados dessa natureza são inestimáveis nos esforços das autoridades de saúde pública para a prevenção e controle de epidemias por estes agentes. / Introduction: Arboviral febrile illnesses plague the nation of Brazil and many of its surrounding Latin America countries. Molecular epidemiology is a growing and increasingly invaluable tool in the field of public health for understanding the dispersal, persistence, and diversity of these impactful viral pathogens. Objectives: In this project, the identities and molecular epidemiological dynamics of arboviruses circulating in the Brazilian state of Amapá between the years 2013 and 2016, with special focus on DENV, CHIKV and ZIKV, were investigated and given Brazilian and Latin American geographical and temporal context via molecular epidemiological analyses. Methods: 824 human blood plasma samples were collected from LACEN laboratories in the state of Amapá between the years 2013 and 2016; these samples originated from patients showing symptoms consistent with any of the common arboviral febrile illnesses. The viral genetic material present in these samples was extracted and qPCR diagnostics assays were performed; all samples first underwent a triplex assay (ZIKV/DENV/CHIKV - ZDC), then the samples yielding negative results for the triplex assay underwent a pan-flavivirus assay. The samples yielding positive results for either assay were submitted for NGS and all whole viral genomes subsequently obtained underwent phylogenetic molecular epidemiological analyses. Results: Of the 824 samples tested, 36 tested positive for the ZDC assay; of those positives, 24 tested positive for DENV, 11 for CHIKV, and 1 for ZIKV. 27 full genomes were obtained from these ZDC positives: 16 of DENV (15 DENV1, genotype V and 1 DENV2, genotype III) and 11 of CHIKV (Asian and Caribbean genotype). Of the 788 samples tested with the pan-flavivirus assay, 22 samples yielded positive results, from only one of which a genome was obtainable. This genome was found to be closely related to a flavivirus previously only found in mosquitoes (76.8% identity with Long Pine Key Virus - LPKV). Maximum likelihood and maximum clade credibility trees were constructed using the DENV1 genomes obtained from this study. These trees displayed two distinct lineages of DENV1, genotype V present in Latin America, one of which has a circulation pattern spanning widely across the Caribbean and Central and South America (including Brazil), while the other circulates within Brazilian borders. The trees also indicate that the DENV1 present in the state of Amapá is of the lineage having the wider circulation pattern and that this lineage emerged in Amapá between 2005 and 2010. Conclusion: This study provides important data concerning the range of the arboviral landscape in Amapá and the most recent genomic data available for the region as well as Brazilian and Latin American context to that data. Data of this nature are invaluable in the efforts of public health officials for the prevention and control of epidemics of these impactful arboviral pathogens.

Studying the microbiome in natural populations could improve our understanding of the biological factors that influence microbiome variation. If host genetic variation is important in microbiota assembly, then understanding genetic divergence among natural populations could be informative. Despite advances in sequencing technology, we have not yet taken full advantage of this technology in natural populations. Here we integrate genome-wide population genomic and microbiome analyses in wild threespine stickleback (Gasterosteus aculeatus) fish distributed throughout western Oregon, USA. We found that gut microbiome varied in diversity and composition more among than within wild host populations. Furthermore, this among population variation was better explained by host population genetic divergence than by environment and geography. We also identified a subset of gut microbial taxa that were most strongly sorted both across environments and across genetically divergent populations. We believe this study contributes generalizable methods and findings in host systems. This thesis includes supplemental materials. / 2021-04-30

A Bothrops jararaca é a serpente de maior importância médica no Brasil. Vários estudos foram realizados com o objetivo de caracterizar os componentes do veneno de serpentes, entretanto, a base molecular dos genomas das serpentes é pouco conhecida. Assim, foi realizado o sequenciamento e montagem do genoma da serpente Bothrops jararaca. Foram construídas bibliotecas tipo shotgun e mate-pair para realização de corridas de sequenciamento usando a tecnologia Illumina e sequências complementares foram obtidas em equipamento PACBIO RS II. Uma biblioteca de BACs também foi construída e 768 pools de 12 BACs foram sequenciados. Um grande conjunto de segmentos genômicos foi obtido e foi possível identificar genes de várias toxinas, entre elas SVMPs, SVSPs, BPPs, CRISPs e VEGF. Ainda foi possível depreender o contexto genômico de muitos destes genes e identificamos os principais elementos repetitivos genômicos. Estes achados são relevantes para o entendimento da função e evolução do sistema venenífero e podem servir de base para outros estudos futuramente. / The pit viper Bothrops jararaca is the most medically important snake in Brazil. Several studies were conducted in order to characterize the components of snake venom. However, the molecular basis of snake genomes is poorly known. Hence, it was carried out the sequencing and assembly of the Bothrops jararaca snake genome. Shotgun and mate-pair libraries were constructed to perform sequencing runs using Illumina technology and complementary sequences were obtained in PACBIO RS II equipment. A BAC library was also constructed and 768 pools of 12 BACs were sequenced. A large number of genomic segments was obtained. It was possible to identify genes of several toxins, including SVMPs, SVSPs, BPPs, CRISPs and VEGF. In addition, it was possible to infer the genomic context related to most of these genes and identify the main genomic repetitive elements. These findings are relevant for understanding the function and evolution of the venom system and it provides the basis for further studies.

Le Pseudomonas aeruginosa est un pathogène nosocomial majeur. Le clone ST235 est le plus prévalent des clones internationaux dits à hautris que. Ce clone est très fréquemment multi résistant aux antibiotiques, ce qui complique la prise en charge des infections dont il est à l’origine.Malgré son importance clinique, la base moléculaire Du succès du clone ST235 n’est pas comprise.Dans ce travail, nous avons cherché à comprendre l’origine spacio temporelle de ce clone et les bases moléculaires de son succès. A l’aide d’outils bio informatiques existants ,nous avons trouvé que le clone ST235 a émergé en Europe en 1984 et que tous les isolates ST235 produisent l’exotoxine ExoU. Nous avons également identifié 22 gènes Contigus spécifiques de ce clone et impliqués dans l’efflux transmembranaire, dans le traitement de l’ADN et dans la transformation bactérienne. Cette combinaison unique de gènes a pu contribuer à la gravité des infections dues à ce clone et à sa capacité à acquérir des gènes de résistance aux antibiotiques. Ainsi, la diffusion mondiale de ce clone a probablement été favorisée par l’utilisation extensive des fluoroquinolones, puis il est de venu localement résistant aux amino glycosides, aux β-lactamines, et aux carbapénèmes par mutation et acquisition d’éléments de résistance. Nous avons majoritairement utilisé des outils existants,mais avons découvert que les programmes de détection des séquences d’insertions (IS, ayant un rôle important dans l’évolution des génomes bactériens) ne sont pas adaptés aux données dont nous disposions. Nous avons ainsi mis au point un outil (appelé panISa) qui détecte de façon précise et sensible les IS à partir de données brutes de séquençage de génomes bactériens. / Pseudomonas aeruginosa is a major nosocomial pathogen with ST235 being the most prevalent of the so-called ‘international’ or ‘high-risk’ clones. This clone is associated with poor clinical outcomes in part due to multi- and high-level antibiotic resistance. Despite its clinical importance, the molecular basis for the success of the ST235 clone is poorly understood. Thus this thesis aimed to understand the origin of ST235 and the molecular basis for its success, including the design of bioinformatics tools for finding insertion sequences (IS) of bacterial genomes.To fulfill these objectives, this thesis was divided into 2 parts.First, the genomes of 79 P. aeruginosa ST235 isolates collected worldwide over a 27-year period were examined. A phylogenetic network was built using Hamming distance-based method, namely the NeighborNet. Then we have found the Time to the Most Recent Common Ancestor (TMRCA) by applying a Bayesian approach. Additionally, we have identified antibiotic resistance determinants, CRISPR-Cas systems, and ST235-specific genes profiles. The results suggested that the ST235 sublineage emerged in Europe around 1984, coinciding with the introduction of fluoroquinolones as an antipseudomonal treatment. The ST235 sublineage seemingly spreads from Europe via two independent clones. ST235 isolates then appeared to acquire resistance determinants to aminoglycosides, β-lactams, and carbapenems locally. Additionally, all the ST235 genomes contained the exoU-encoded exotoxin and identified 22 ST235-specific genes clustering in blocks and implicated in transmembrane efflux, DNA processing and bacterial transformation. These unique genes may have contributed to the poor outcome associated with P. aeruginosa ST235 infections and increased the ability of this international clone to acquire mobile resistance elements.The second part was to design a new Insertion Sequence (IS) searching tool on next-generation sequencing data, named panISa. This tool identifies the IS position, direct target repeats (DR) and inverted repeats (IR) from short read data (.bam/.sam) by investigating only the reference genome (without any IS database). To validate our proposal, we used simulated reads from 5 species: Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, and Vibrio cholerae with 30 random ISs. The experiment set is constituted by reads of various lengths (100, 150, and 300 nucleotides) and coverage of simulated reads at 20x, 40x, 60x, 80x, and 100x. We performed sensitivity and precision analyses to evaluate panISa and found that the sensitivity of IS position is not significantly different when the read length is changed, while the modifications become significant depending on species and read coverage. When focusing on the different read coverage, we found a significant difference only at 20x. For the other situations (40x-100x) we obtained a very good mean of sensitivity equal to 98% (95%CI: 97.9%-98.2%). Similarly, the mean of DR sensitivity of DR identification is high: 99.98% (95%CI: 99.957%-99.998%), but the mean of IR sensitivity is 73.99% (95%CI: 71.162%-76.826%), which should be improved. Focusing on precision instead of sensibility, the precision of IS position is significantly different when changing the species, read coverage, or read length. However, the mean of each precision value is larger than 95%, which is very good.In conclusion, P. aeruginosa ST235 (i) has become prevalent across the globe potentially due to the selective pressure of fluoroquinolones and (ii) readily became resistant to aminoglycosides, β-lactams, and carbapenems through mutation and acquisition of resistance elements among local populations. Concerning the second point, our panISa proposal is a sensitive and highly precise tool for identifying insertion sequences from short reads of bacterial data, which will be useful to study the epidemiology or bacterial evolution.

The persistence of woody plant populations faces numerous environmental challenges, including climate change, hybridisation and population fragmentation. Here I explore the genomic signatures and relative importance of these pressures in Dwarf Birch (Betula nana), which has declined significantly over the last century across the Scottish Highlands. Firstly, I find that future climate is likely to result in a significant range reduction and that relict populations are likely to display reduced fitness. Secondly, I show that combining multiple mutation rate markers yields more accurate estimates of demographic history and the impact of fragmentation. I develop a novel method to derive high mutation rate markers from short sequencing reads, to facilitate more widespread application. Thirdly, I assess the degree of local adaptation, and explore potential for composite provenancing for the restoration of B. nana populations. Surprisingly, the data yields little evidence of adaptive introgression from the related tree B. pubescens, suggesting that this may not be an alternative route to climate tolerance. Finally, I review published literature on the population structure and genetic diversity of genus Betula in Europe and consider options for the conservation and management of B. nana, including assisted gene flow and prioritization of in situ genetic diversity.