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.

Asthma is the most common long-term condition in children in the United Kingdom (UK). Asthma-related hospitalisations and mortality are disproportionally higher in the UK, compared with other European countries, however the reasons for this disparity remain unclear. A putative explanation is that that prevalence of suboptimal asthma control in children in the UK is higher than in continental Europe. If this is indeed correct, then the drivers of suboptimal control, such as poor adherence to therapy resulting from poor understanding of the role of preventer medication (inhaled corticosteroids (ICS)) in UK children would be of significant clinical interest. Therefore, in this thesis, I sought to first identify the levels of asthma control and medication adherence in a non-random sample of London secondary school children. Then, I used focus groups to further highlight the barriers to good medication adherence, and generate insights into potential solutions. To achieve these aims, I developed and implemented an online questionnaire to be delivered in schools, which included the validated Asthma Control Test (ACT). Methods: This thesis is divided into three main sections. The first and second sections include original data from an observational research study, which collected data about asthma control, from 24 London secondary schools between December 2014 and March 2016. The aim of the first section was to assess current levels of asthma control and medication adherence among children with asthma in London secondary schools. Data were collected using an online questionnaire, which included the validated ACT to measure asthma control, as well as additional questions about knowledge, healthcare use, medication use, school attendance, lifestyle and emotion and behaviour, using the validated Me and My School (M&MS) questionnaire. The second section of this thesis includes data generated from six focus groups, conducted in four London secondary schools with 56 students. In order to generate data to inform future interventions, discussions focused on the barriers to medication adherence among teenagers, and how these barriers could be addressed. The third section comprises a systematic review of school-based self-management interventions for children with asthma. The review uses a mixed-methods approach, and includes both quantitative and qualitative study data. A process evaluation is also included, to identify intervention elements that are associated with implementation success. Results: 766 children with asthma from 24 schools were surveyed. Almost half of the students (45.7%; n = 350) had poor asthma control by ACT score. Adherence with asthma medication was low, regardless of asthma control (56.2% self-reported forgetting to use their ICS "preventer" inhaler; 29% self-reported not using their SABA "reliever" inhaler when they needed it, at least some of the time). Health care involvement was relatively high, with at least one unplanned GP visit, due to asthma in the previous four weeks, reported by 28.1% of students; at least one unplanned hospital visit was self-reported by 15.7% of students; and at least one unplanned school nurse visit due to asthma was self-reported by 16% of students. At least one whole school absence was reported by 20.9% of students. Unplanned medical care and school absences were higher among children with poor asthma control, according to the ACT. Themes from focus groups suggested that social stigma, fear of embarrassment, forgetfulness, and incorrect attitudes towards medication were all contributory factors to poor medication adherence. Communications with healthcare professionals were also identified as key unmet needs of teenagers with asthma. The findings from the meta-analyses, included in the systematic review of school-based self-management interventions, showed that such interventions were effective in improving several outcomes, largely related to healthcare use. These included hospitalisations, emergency department (ED) visits, and health-related quality of life. There was no evidence that school-based interventions improved school absences, experiences of day and night time symptoms, or the use of medication. The findings from the analysis of the process evaluation studies showed that a theoretical framework is important in the development of a successful intervention. Conclusions: First, in a large non-random sample of secondary school children with asthma, the proportion of children with suboptimal control is worryingly high, and this is associated with general poor adherence to prescribed therapy asthma. Second, focus groups identified practical and social barriers to good adherence, that should be addressed in future studies. Third, previous studies suggest that school based interventions are effective in reducing incidences of unplanned and urgent healthcare use. The systematic review included studies that included relatively hard-to-reach populations, suggesting that such interventions may be effective across diverse populations, including those considered hard-to-reach. The findings in this thesis informed the development of a school-based self-management intervention, to be piloted in London secondary schools, and an NIHR-funded global research group award on improving asthma control in African children.

Orientador: Henrique Nunes de Oliveira / Coorientador: Márcia Cristina de Sena Oliveira / Banca: Simone Cristina Méo Niciura / Banca: Rodrigo Giglioti / Resumo: A bovinocultura é um setor com importante destaque no agronegócio brasileiro. O carrapato Ripicephalus (Boophilus) microplus é responsável por perdas econômicas significativas aos pecuaristas e é vetor de hemoparasitoses como Anaplasma spp e Babesia spp. Sabe-se que os bovinos Bos taurus taurus são mais susceptíveis à infestação por carrapatos do que Bos taurus indicus. Acredita-se que o mesmo ocorra para a infecção por Babesia bovis. Neste trabalho, foram avaliados, em duas colheitas, 355 bovinos da raça Angus, pertencentes a uma fazenda de Uruguaiana-RS, nos quais foram realizadas contagens de carrapatos e colheitas de amostras de sangue para quantificação de B. bovis, pela técnica de qPCR, e genotipagem com chip de 150.000 marcadores SNP. Para qPCR utilizaram-se sequências iniciadoras que flanqueiam um fragmento do gene do citocromo B (mt-cytB), como oligonucleotídeos iniciadores. Após genotipagem dos bovinos com o chip Gene Seek Genomic Profiler™ (GGP-HD) da Illumina Infinium®, foi realizado imputação de genótipos, para recuperação de genótipos faltantes, e controle de qualidade. Foi realizada análise de associação genômica ampla (GWAS), para cada uma das características, infecção por B. bovis e resistência a carrapatos, através do método denominado "Single Step Genomic BLUP" (ssGBLUP). Todos os animais apresentaram infestação por carrapatos e infecção por B. bovis, determinada pela qPCR, e altos valores médios para ambas as características. Algumas regiões cromossômicas ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The cattle industry is a sector with importance in the Brazilian agribusiness. The Ripicephalus (Boophilus) microplus is responsible for economic losses and is a vector for hemoparasitoses, such as Anaplasma spp and Babesia spp. It is known that the Bos tauros animals are more susceptible to infestation by ticks when compared with infestation in Bos indicus animals. It is believed that the same behavior keeps for infection by Babesia bovis. They were evaluated, in two collections, 355 Angus cattle, from a farm in Uruguaiana city, estate of Rio Grande do Sul, where were performed tick counts, quantification of B. bovis by qPCR and genotyping with a 150K chip. Were used as primers, in the qPCR, sequences that flanking the fragment of the cytochrome b gene. The technique was standardized and optimized using specimens of isolates of B. bovis. After genotyping, imputation was carried out, for recovery of missing genotypes, and quality control. Genome association analysis was performed (GWAS), to each of the characteristics, through the method called "Single Step Genomic BLUP" (ssGBLUP). All animals showed tick infestation and infection by B. bovis and high average values for both characteristics. Some regions on chromosomes were identified as significant to the characteristics tick infestation and infection by B. bovis, and seven chromosomes, identified in the present study, were already described in other studies. The present study agrees with other results indicating that the qP... (Complete abstract click electronic access below) / Mestre

Les bactéries du genre Flavobacterium sont retrouvées dans des types d’habitats très divers. Ce genre contient trois espèces ichtyopathogènes : columnare, branchiophilum et psychrophilum qui est responsable de pertes économiques importantes pour l’élevage des salmonidés. Un projet de séquençage et de comparaison des génomes de plusieurs flavobactéries pathogènes de poissons ainsi qu’isolées de différents environnements a été mis en place pour améliorer les connaissances sur ce genre. Les objectifs étaient l’identification des déterminants de virulence et la caractérisation de différents marqueurs moléculaires des traits phénotypiques associés à leur mode de vie. L’analyse des génomes de F. psychrophilum a permis de mettre en évidence une diversité des structures chromosomiques au sein de l’espèce et d’identifier des cibles moléculaires prometteuses pour le développement de tests de diagnostic ainsi que des cibles vaccinales potentielles. Le génome de F. branchiophilum a permis d’identifier des mécanismes moléculaires de virulence originaux. Les caractéristiques du génome de F. indicum révèlent un mode de vie environnemental : sa petite taille et ses faibles capacités de dégradation des bio-polymères suggèrent que F. indicum est adapté à une niche écologique restreinte. Ces nouvelles données ont permis de caractériser in silico des marqueurs moléculaires de caractères phénotypiques. En particulier, un groupe de gènes (dnd) rare et responsable d’une modification étonnante de la molécule d’ADN a été décrit pour la première fois chez les Flavobacteriaceae. Ce projet a permis d’enrichir les connaissances sur les bactéries du genre Flavobacterium et a contribué au développement d’outils pour la santé animale. / Flavobacterium species occur in a wide range of habitats. This genus includes three fish-pathogenic species, namely F. columnare, F. branchiophilum and F. psychrophilum. The latter is responsible for serious economic losses for salmonids farming in France and worlwide. A comparative genomics project including several fish-pathogenic flavobacteria as well as various environmental species has been set up in order to improve the knowledge on this poorly studied genus. Our aims were the identification of virulence determinants associated with pathogenicity and the characterization of various molecular elements reflecting phenotypes associated with their life-style. Analysis of the genomes of several F. psychrophilum isolates revealed the diversity of chromosomal structures within the species and identified in silico promising molecular targets for the development of diagnostic tests as well as potential vaccines targets. Analysis of the F. branchiophilum genome enabled to identify particular molecular virulence mechanisms. The features of the F. indicum genome reflected its environmental lifestyle : its small size and its limited bio-polymers degrading abilities suggested that F. indicum is adapted to a quite narrow ecological niche. These new data have allowed the in silico identification of many molecular elements reflecting phenotypic traits. In particular, a rare gene cluster (dnd) responsible for an unusual DNA structure modification was described for the first time within members of the family Flavobacteriaceae. This project enriched the knowledge on Flavobacterium species and contributed to the development of tools for animal health.

Many problems in the biological sciences reduce to questions of genetic evolution. Entire classes of medical pathology, such as malignant neoplasia or infectious disease, can be viewed in the light of Darwinian competition of genomes. With the benefit of today's maturing sequencing technologies we can observe and quantify genetic evolution with nucleotide resolution. This provides a molecular view of genetic material that has adapted, or is in the process of adapting, to its local selection pressures. A series of problems will be discussed in this thesis, all involving the mathematical modeling of genomic data derived from clonally evolving populations. We use a variety of computational approaches to characterize over-represented features in the data, with the underlying hypothesis that we may be detecting fitness-conferring features of the biology. In Part I we consider the cross-sectional sampling of human tumors via RNA-sequencing, and devise computational pipelines for detecting oncogenic gene fusions and oncovirus infections. Genomic translocation and oncovirus infection can each be a highly penetrant alteration in a tumor's evolutionary history, with famous examples of both populating the cancer biology literature. In order to exert a transforming influence over the host cell, gene fusions and viral genetic programs need to be expressed and thus can be detected via whole transcriptome sequencing of a malignant cell population. We describe our approaches to predicting oncogenic gene fusions (Chapter 2) and quantifying host-viral interactions (Chapter 3) in large panels of human tumor tissue. The alterations that we characterize prompt the larger question of how the genetics of tumors and viruses might vary in time, leading us to the study of serially sampled populations. In Part II we consider longitudinal sampling of a clonally evolving population. Phylogenetic trees are the standard representation of a clonal process, an evolutionary picture as old as Darwin's voyages on the Beagle. Chapter 4 first reviews phylogenetic inference and then introduces a certain phylogenetic tree space that forms the starting point of our work on the topic. Specifically, Chapter 4 describes the construction of our projective tree space along with an explicit implementation for visualizing point clouds of rescaled trees. The Chapter finishes by defining a method for stable dimensionality reduction of large phylogenies, which is useful for analyzing long genomic time series. In Chapter 5 we consider medically relevant instances of clonal evolution and the longitudinal genetic data sets to which they give rise. We analyze data from (i) the sequencing of cancers along their therapeutic course, (ii) the passaging of a xenografted tumor through a mouse model, and (iii) the seasonal surveillance of H3N2 influenza's hemagglutinin segment. A novel approach to predicting influenza vaccine effectiveness is demonstrated using statistics of point clouds in tree spaces. Our investigations into clonal processes may be extended beyond naturally occurring genomes. In Part III we focus on the directed clonal evolution of populations of synthetic RNAs in vitro. Analogous to the selection pressures exerted upon malignant cells or viral particles, these synthetic RNA genomes can be evolved against a desired fitness objective. We investigate fitness objectives related to reprogramming ribosomal translation. Chapter 6 identifies high fitness RNA pseudoknot geometries capable of inducing ribosomal frameshift, while Chapter 7 takes an unbiased approach to evolving sequence and structural elements that promote stop codon readthrough.