Characterising and understanding factors that affect the rate of molecular evolution in proteins has played a major part in the development of evolutionary theory. The early analyses of amino acid substitutions stimulated the development of the neutral theory of molecular evolution, which later evolved into the nearly neutral theory. More recent work has lead to a better understanding of the role selection plays at the molecular level, but there is still limited understanding of how higher levels of protein organisation affect the way natural selection acts. The investigation of this question is the central aim of this thesis, which is addressed via the analysis of selective pressures in secondary protein structures in insects, mammals and fungi. The analyses for the first two groups were conducted using publically available datasets. To conduct the analyses in fungi, genome sequence data from the fungal genus Microbotryum (sequenced in our laboratory) was assembled and annotated, resulting in the development of a number of bioinformatics tools which are described here. The fungal, insect and mammalian datasets were interrogated with regard to a number of structural features, such as protein secondary structure, position of a site with regard to adaptively evolving sites, hydropathy and solvent-accessibility. These features were correlated with the signals of positive and purifying selection detected using phylogenetic maximum likelihood and Bayesian approaches. I conclude that all of the factors examined can have an effect on the rate of molecular evolution. In particular, disordered and hydrophilic regions of the protein are found to experience fewer physiochemical constraints and contain a higher proportion of adaptively evolving sites. It is also revealed that positively selected residues are ‘clustered’ together spatially, and these trends persist in the three taxa. Finally, I show that this variation in adaptive evolution is a result of both selective events and physiochemical constraint.

Introduction: RNA viruses are economically important pathogens of fish, and among these viruses, infectious pancreatic necrosis virus (IPNV) is of particular concern for the aquaculture industry, especially for farmed rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). This non-enveloped aquatic virus, which was first isolated in the UK in 1971, belongs to the family of Birnaviridae and has a bi-segmented dsRNA genome of about 6kb. IPNV is classified in 6 genogroups with correspondence to 10 known serotypes and an additional proposed genogroup of marine aquabirnaviruses (MABV). IPNV causes high mortality in fry and a reduced mortality in adult fish, respectively. Fish, which survive, can become carriers and this can lead to a clinical outbreak by releasing infective material into water or by vertical transmission via oocytes, milt and seminal fluids. Methods: This project aimed at determining the phylogeny and genomic changes of IPNV in Scotland by whole genome sequence analysis of IPNV isolates (diagnostic TCID50 supernatants) spanning 3 decades since 1982, using next generation sequencing technology. Viral RNA of IPNV culture supernatant (CHSE-214 and TO cell culture) was processed for next generation sequencing on an Illumina MiSeq platform. Library preparation was performed using the Nextera XT DNA Library Kit, prior to sequencing according to the manufacturer's MiSeq Reagent Kit v3 (150cycles) protocol. To optimize whole genome next generation sequencing for IPNV, we compared two RNA processing protocols, the Glasgow (GLAP) and the Goettingen protocol (GOEP) with focus on missing terminal nucleotides after a de novo genome assembly. Sequences were used to determine the phylogeny and selection pressure on the genome as well as a possible virus-host adaptation. Results: The results showed that both protocols were able to give full length genomes as well as genomes with missing terminal nucleotides. The phylogenetic analysis of 57 sequenced IPVN isolates shows that 78.95 % of the isolates group within genogroup V, which includes serogroup Sp and 5.26 % within genogroup I which includes serogroup Ja. Segment A of 15.79 % of the isolate grouped within genogroup III, which includes serotype Ca1 and Te but only 7.02 % of the segment B isolates grouped in the genogroup III. The remaining 8.77 % of segment B groups within genogroup II, containing the Ab serotype. Previous research has shown that residue substitutions at positions 217 and 221 in the major capsid protein VP2 have an impact on the virulence of the virus, leading to different virulence types: virulent (T217, A221), low virulence (P217, A221), avirulent (T217, T221) and persistent (P217, T221). Whole genome sequence results show that 58.93 % of the sequenced isolates belong to the persistent, 32.14 % to the low virulent type, only one isolate was of a virulent type and 7.15 % had not virulence assigned amino acid compositions in positions 217 and 221. The selection pressure analysis showed that especially VP2 is experiencing selection pressure in the variable region. In the VP1 protein we see two sites under positive selection pressure within specific motifs. VP5 showed positive selected sites mostly within the truncated region of the protein. Other proteins showed no particular interesting sites of selection. The codon adaptation analysis showed highest adaptation index for VP2. Besides VP5, which had an CAI index below one, therefore showing negative adaptation, other IPNV proteins had an CAI of barely above the value of 1. The dinucleotide abundance, focussing on CpG, showed that CpG is underrepresented in segment A and B. Discussion Phylogenetic analysis of the sequenced IPNV strains shows separate clustering of different genogroups. Genetic reassortment is observed in segment B showing a grouping within genogroup III and II although the segment A of these isolates was grouping exclusively within III. We found that over 50 % of the isolates belong to the persistent and over 30 % to the low virulent type, assuming that due to not sterilising vaccination these types were selected in the vaccinated population. The results from the CAI calculations indicate an adaptation of IPNV to its host. Together with the findings that CpG is underrepresented in IPNV it suggests that this leads to an immune escape. Especially since the selection pressure analysis showed positive selection in VP2 within the virulence determination sites of the protein, indicating that IPNV "tries" to downregulate immune recognition. The prevalence of mostly persistent type of isolates indicates together with the assumption of adaptation and immune escape that IPNV is evolving with the host in order to ensure survival.

Orientador: Semíramis Guimarães Ferraz Viana / Resumo: Giardia duodenalis é um dos principais agentes etiológicos de diarreia infecciosa, sobretudo em crianças em idade pré-escolar que vivem em comunidades de baixa renda. Estudos da diversidade genética de G. duodenalis ampliaram o conhecimento da epidemiologia nas infecções humanas, entretanto um dos temas mais interessantes e menos conhecidos é a possível interação de Giardia com o microbioma do hospedeiro e com patógenos concomitantes. No presente estudo, avaliou-se a composição e a diversidade da comunidade bacteriana de crianças saudáveis e crianças com diarreia, parasitadas por Giardia e outros protozoários intestinais. Os isolados de Giardia obtidos nessa população foram caracterizados geneticamente. Amostras de fezes foram obtidas de 181 crianças de 0 a 6 anos de idade, das quais 156 crianças hígidas atendidas em centros de educação infantil e 25 crianças com diarreia atendidas no PS Infantil Municipal. Cada amostra de fezes foi processada para o exame microscópico e submetida à extração de DNA a ser empregado em duas etapas distintas: (1) amplificação e sequenciamento Sanger para a caracterização genética de Giardia e o diagnóstico de Blastocystis sp, Dientamoeba fragilis, Enterocytozoon bieneusi e Cryptosporidium spp. e (2) amplificação do gene 16s RNA ribossomal e sequenciamento de nova geração (plataforma Illumina MiSeq) para a caracterização da microbiota intestinal. Giardia (36,5%) e Blastocystis (41,7%) foram os parasitas mais prevalentes. A caracterização genética... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Giardia duodenalis is one of the major etiological agents of infectious diarrhea, especially in preschool children living in low-income settings. Studies focused on genetic diversity of G. duodenalis have provided insights for a better understanding of epidemiology in human infections. However, one of the most interesting and least known issues is the possible interplay between Giardia and the host microbiome and concomitant pathogens. In this work, we evaluated the diversity and composition of bacterial community of healthy children and children presenting with diarrhea infected by Giardia and/or other intestinal protozoa. In addition, Giardia isolates infecting this population were genotyping. A total of 181 stool samples from children aged 0 to 6 years old (156 from daycare children and 25 from diarrheic children attending in an emergence pediatric center) were tested by microscopic examination and submitted to DNA extraction for the following steps: (1) conventional PCR/sequencing for Giardia genotyping and the diagnosis of Blastocystis sp, Dientamoeba fragilis, Enterocytozoon bieneusi and Cryptosporidium spp and (2) next-generation sequencing (Illumina MiSeq) based analysis of intestinal microbiota. Giardia (36.5%) and Blastocystis (41.7%) were the most prevalent parasites. Analysis of Giardia sequences retrieved from 61 isolates revealed infections by assemblages A (31%), B (69%) and mixed infections A+B (3%). Metagenomic analyzes revealed similarity of bacterial microb... (Complete abstract click electronic access below) / Doutor

Obsessive-compulsive disorder (OCD) and the spectrum of associated conditions, affect 2-4% of the population worldwide. Although heritability studies in OCD have shown a 3 - 12 times increased risk for first degree relatives, the identification of the underlying risk-conferring genetic variation using classic genetic association studies has proven to be difficult. The possibility of a larger contribution of rare genetic variants to the risk of psychiatric disorder has been suggested by several successful studies. We expect that a spectrum of risk allele frequencies exists, which includes not only common variation but also a substantial amount of rare genetic variants that contribute to OCD. This thesis is aimed at identifying and functionally characterizing rare genetic variation in the OCD spectrum. Identified statistically significant variants were scrutinized for changes related to synaptic function using high content screening and subsequent functional analyses. Identifying the genetic profile of rare variants found in the OCD spectrum cohort combined with the functional impact that these variants have has provided insight into the etiology of the OCD spectrum. With these approaches a foundation can be laid for the development of a predictive model of the OCD spectrum.

Les cellules cancéreuses sont caractérisées par des altérations de l'ADN causées par des facteurs exogènes, comme l'exposition à des agents environnementaux tels que le tabac ou les UV, ou par des mécanismes endogènes tels que les erreurs de polymérase lors de la réplication de l'ADN. L'analyse des causes et des conséquences de ces altérations permet de mieux comprendre les facteurs et mécanismes à l'origine du développement d'un cancer. Les technologies de séquençages à haut débit offrent l'opportunité d'étudier la nature précise de ces altérations à l'échelle du génome et permettent de révéler des signatures mutationnelles distinctes et spécifiques de cancérigènes, fournissant ainsi des hypothèses sur l'étiologie des cancers.L'objectif de ma thèse a consisté à développer des méthodes et des outils bioinformatiques accessibles et conviviaux permettant de faciliter l'analyse et l'interprétation des signatures mutationnelles à partir de données de séquençage à haut débit. L'application de ces outils et méthodes à des séries originales de tumeurs humaines et de systèmes expérimentaux de mutagénèse et carcinogénèse a permis de mieux caractériser la signature mutationnelle de l'acide aristolochique (AA) ainsi que d'autres cancérigènes d'intérêt / Cellular genomes accumulate alterations following exposures to exogenous factors, like environmental agents such as tobacco smoking or UV, or to endogenous mechanisms such as DNA replication errors. Analysing the causes and consequences of these changes allows a better understanding of the mechanisms underlying cancer development and progression. Next-generation sequencing (NGS) technologies provide the opportunity tostudy the nature of the resulting alterations on a genome-wide scale and started to reveal distinct mutational signatures specific to past carcinogenic exposures providing clues on cancer aetiology.The aim of my thesis was to develop user-friendly bioinformatic tools and methods for facilitating the analysis and interpretation of carcinogen-specific mutational signatures from NGS data. Applying these tools and methods to human tumours and experimental models of mutagenesis led to a better characterisation the mutational signature of aristolochic acid (AA), as well as other carcinogens of interest

Tesis por compendio / [ES] El melanoma es el tipo de cáncer de piel más mortífero y peligroso, ya que tumores de pequeño tamaño pueden generar metástasis. Hasta la fecha, se ha tratado de clasificar desde el punto de vista clínico, epidemiológico y molecular, empleándose actualmente el nivel de exposición solar y la localización del tumor como criterios principales para dividir en distintos grupos a los pacientes de melanoma. En 1998, David Whiteman y colaboradores propusieron un "modelo de vías divergentes" para el desarrollo del melanoma. Este presentaba dos vías: una vinculada a la proliferación melanocítica (nevogénica) y otra relacionada con la exposición solar crónica (CSD). Corroborado desde el punto de vista clínico y epidemiológico, todavía no se ha aportado una caracterización molecular en profundidad. A nivel general se habían identificado genes cuyas mutaciones eran relevantes para el desarrollo del melanoma, como por ejemplo KIT. Sin embargo, todavía se había de estudiar con más detalle la distribución de estas mutaciones entre los distintos subgrupos de melanoma, así como su posible valor pronóstico. En esta tesis se han empleado técnicas de secuenciación - masiva y tradicional - para caracterizar los perfiles mutacionales de las poblaciones del modelo de vías divergentes. Encontramos diferencias tanto en el número de mutaciones como en los genes afectados. También hemos visto cómo los melanomas con mutaciones en KIT parecen desarrollarse por una vía independiente de la etiopatogenia conocida, careciendo el estatus mutacional de este gen de valor pronóstico para la supervivencia de los pacientes. / [CA] El melanoma és el tipus de càncer de pell més mortífer i perillós, ja que fins els tumors de menor mida poden acabar generant metàstasi. Al llarg dels anys, s'ha tractat de classificar des del punt de vista clínic, epidemiològic i molecular. Les classificacions actuals utilitzen el nivell d'exposició solar i la localització tumoral per dividir en diferents grups als pacients de melanoma. Al 1998, David Whiteman i col·laboradors proposaren un model de desenvolupament del melanoma que anomenaren "model de vies divergents". Aquest presentava dos vies per la melanomagènesi: una vinculada a la proliferació melanocítica (nevogènica) i l'altra relacionada amb l'exposició solar crònica (CSD). Malgrat aquest model fou corroborat des del punt de vista clínic i epidemiològic, encara no s'ha aportat una caracterització molecular en profunditat. A nivell general s'havien identificat gens les mutacions dels quals eren rellevants per al desenvolupament del melanoma, com el gen KIT. Però, encara s'havia d'estudiar amb més cura la distribució d'estes mutacions entre els distints subgrups de melanoma, així com el seu possible valor pronòstic. En aquesta tesi s'han emprat tècniques de seqüenciació -massiva i tradicional- per caracteritzar els perfils mutacionals de les dues poblacions proposades pel model de vies divergents, trobant diferències tant al nombre de mutacions com als gens afectats. També hem vist com els melanomes mutats en KIT semblen desenvolupar-se per una via independent de l'etiopatogènia coneguda, mancant l'estatus mutacional d'aquest gen de valor pronòstic per la supervivència dels pacients. / [EN] Melanoma is the deadliest and most dangerous type of skin cancer, given that a small tumor can spread and result in metastasis. Over the years, classifications have been made either from a clinical, epidemiological or molecular point of view. Current classifications use the degree of solar exposure and tumoral location to divide into different melanoma groups. In 1998, David Whiteman and collaborators proposed the divergent pathway model for melanoma development. This presented two pathways to melanomagenesis: one related to melanocytic proliferation (nevogenic) and the other related to chronic sun exposure (CSD). Despite corroborations of this model from the clinic and epidemiology, it is yet to be molecularly characterized in depth. At a general level, different genes had been identified with relevant mutations for the development of melanoma, as is the gene KIT. However, there was a lack of knowledge on how these mutations were distributed among different melanoma subgroups, as well as the potential prognostic value. In this thesis we have implemented sequencing techniques - both massive and traditional - to characterize the mutational profile of the two populations proposed by the divergent pathways model. We found differences both in the number of mutations and in the genes carrying the mutations. We have also seen how melanomas harboring KIT mutations seem to develop in a way which is independent from the known etiology, and how the mutational status of this gene lacks prognostic value on the outcome of the patients. / Millán Esteban, D. (2022). Next-Generation Sequencing in the Identification of Biomarkers in Cutaneous Melanoma According to the Etiopathogenic Development Pathway and their Potential Clinical Relevance [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182977 / Compendio

L'objectif de mon travail de thèse était de développer des outils bio informatiques permettant d'améliorer la traditionnelle gestion de l'information scientifique au sein d'un grand centre de recherche et en particulier au sein d'une plateforme de génomique. Trois outils ont été développés: un cahier de laboratoire électronique, un système de gestion de l'information de laboratoire pour des applications de génomique dont le séquençage de nouvelle génération, ainsi qu'un système de gestion des échantillons pour de grandes bio-banques. Ce travail a été réalisé en étroite collaboration avec des biologistes, épidémiologistes et informaticiens. Il a également inclus la mise en place d'interactions entre les différents outils pour former un système informatique intégré. Les trois outils ont été rapidement adoptés par l'ensemble des scientifiques du centre de recherche et sont désormais utilisés au quotidien pour le suivi de toutes les activités de laboratoire mais aussi plus globalement pour les autres activités scientifiques du centre de recherche. Ces outils sont transposables dans d'autres instituts de recherche / The aim of my thesis work was to develop bioinformatics tools to improve the traditional scientific information management within a large research centre and especially within a genomics platform. Three tools have been developed: an electronic laboratory notebook, a laboratory information management system for genomics applications including next generation sequencing, as well as a sample management system for large biobanks. This work has been conducted in close collaboration with biologists, epidemiologists and IT specialists. It has also included the setup of interactions between the different tools to make an integrated IT system. The three tools have been rapidly adopted by all the scientists of the research centre and are now daily used for the tracking of all the laboratory’s activities but also more globally for the research centre’s other scientific activities. These tools are transposable in other research institutes

8 Summary The thesis "The elucidation of the causes of neurogenetic diseases by the MPS data analysis using advanced algorithms" is focused on processing the massively parallel sequencing (MPS) data from a gene panel, whole-exome sequencing (WES) and whole-genome sequencing (WGS). The aim of the study was to develop a suitable pipeline to evaluate at least 250 MPS gene panel data, 150 WES data and 20 WGS data in order to improve molecular genetic testing of rare neurogenetic disorders. Associated data management and database implementation is also described. Targeted gene panel sequencing A custom-designed gene panel consisting of ge- nes previously associated with the disease was used. In the Epileptic Encephalopathy (EE) panel, two prerequisites need to be met for inclusion into the panel: the gene has to have been published in at least two independent publications OR at least in one publication but in multiple independent families. In the case of the EE panel, 112 genes were included. The targeted gene panel sequencing was then performed on 257 patients with EE. Pathogenic or likely pathogenic (according to ACMG criteria) variants have been found in 28% of patients (72 out of 257). Further analysis of the pathogenic or likely pathogenic variants was performed (76 in total); the variants were grouped by...

La digoxine est un cardiotonique largement employé pour contrôler les symptômes de l'insuffisance cardiaque et de la fibrillation auriculaire. Il est connu depuis les années 1980 que le métabolite principal de la digoxine, la dihydrodigoxine, est produit exclusivement par le microbiome intestinal (métabolisme de premier passage) et plus précisément la bactérie Eggerthella lenta. Aux États-Unis, c'est 14% des participants à une étude qui excrétaient 40% et plus de la dose sous la forme de ce métabolite rapidement éliminable et ayant perdu son affinité pour sa cible. De plus, chaque année, la digoxine est le médicament qui engendre le plus d'hospitalisations pour effets secondaires toxiques. Les effets secondaires très problématiques de la digoxine sont souvent déclenchés par l'ajout d'antibiotiques (surtout les macrolides) à la prescription de digoxine. La théorie explorée ici explique les évènements de toxicité chez les patients métabolisateurs. Ces derniers ont une dose quotidienne de maintien de digoxine plus élevée pour compenser l'action de la bactérie et, lorsque ces patients reçoivent un antibiotique pour une infection non reliée à leur condition cardiaque, l'arrêt du métabolisme par le microbiome engendre une augmentation de la biodisponibilité de la digoxine. Si la concentration plasmatique du médicament augmente trop, les effets secondaires peuvent aller jusqu'à causer la mort. Dans le présent projet, nous avons vérifié la sensibilité de E. lenta à deux antibiotiques de la famille des céphalosporines de seconde génération, in vivo et in vitro. Pour les 18 volontaires qui ont été exposés à 2x500mg de céfprozil durant une semaine, il y a une tendance à la baisse de l'abondance de la bactérie d'intérêt (par 58,3% par rapport au niveau initial), mais pas de significativité au niveau des tests statistiques. Pour les échantillons complets de microbiome fécal, mis en culture avec et sans antibiotiques, il y a une différence statistiquement significative avec une valeur-p de 0,0457, alors que la croissance de E. lenta a été impactée négativement par l'ajout de céfprozil au milieu de culture. Les résultats valident une prémisse importante pour la démonstration du rôle du microbiome dans la pharmacocinétique de la digoxine et la gestion clinique du médicament cardiotonique. / Digoxin is a widely used cardiotonic drug in the management of heart failure and atrial fibrillation. It has been known since the early 1980's that the main metabolite of digoxin, dihydrodigoxin, is synthesized by the gut microbiome during first pass metabolism and is exclusively produced by the bacteria Eggerthella lenta. In a clinical study done in the U.S.A., there were 14% of high metabolizers, for whom over 40% of the oral digoxin dose is transformed to the inactive metabolite and rapidly eliminated. Digoxin toxicity is the leading cause of hospitalization from medication's secondary effects. The toxicity events are often associated with the addition of an antibiotic (mostly from the macrolides class) to the patient's drugs regiments. The theory explored in this project could help explain the toxicity events in metabolizers. These patients have a higher daily digoxin maintenance dose to counteract the effects of the microbiome and are then prescribed antibiotics for an infection unrelated to their heart condition. The antibiotic alters E. lenta negatively, which cannot metabolize digoxin anymore and therefore augments the bioavailability of the cardiotonic. If the plasmatic concentration reaches dangerous levels (over 2ng/ml of plasma), the patients face adverse effects that include death. In the present project, we evaluated the susceptibility of E. lenta to two second generation cephalosporins, in vivo and in vitro. With the 18 healthy volunteers that were exposed to 2x500mg of cefprozil daily for 7 days, we observed a diminution of the abundance of the bacteria of interest by 58,3% from the initial levels. This change did not however produce statistically significant tests results. For the complete fecal microbiome that were cultivated in vitro, with or without cefprozil, the difference between the two conditions resulted in a statistically significant p-value of 0.0457, confirming the sensitivity of E. lenta to this cephalosporin. These results validate an important premise for the demonstration of the importance of the gut microbiome in the pharmacokinetics of digoxin and the clinical management of the drug to avoid toxicity events in clinical practice.

Le succès évolutif de la polyploïdie, notamment de l’allopolyploïdie (où la duplication de génome complet est associée à une hybridation entre génomes différenciés) est en partie lié au fait que cet événement s’accompagne de nombreux changements dans l'organisation du génome et la régulation de l'expression des gènes. On parle du « choc génomique » de l’hybridation interspécifique et de l’allopolyploïdie. Ces sources de diversité génétique, à la fois structurale et fonctionnelle, apparaissent utiles et nécessaires à l'adaptation et l’évolution des espèces. Alors que de nombreuses études portant sur la compréhension des mécanismes moléculaires à l’origine du succès des allopolyploïdes ont concerné les modifications de l’expression des gènes, mes travaux de thèse ont porté sur les agents non codants du génome que sont les éléments transposables et les petits ARN non codants. Le modèle d'étude est le colza (Brassica napus, AACC), espèce allotétraploïde issue de l'hybridation entre les espèces diploïdes navette (B. rapa, AA) et chou (B. oleracea, CC). Nous disposions de colzas néo-synthétisés, étudiés à différentes générations d’autofécondation, permettant de caractériser les changements génomiques accompagnant la formation puis l’évolution du génome néo-allopolyploïde. Une étude a tout d’abord été menée sur un élément transposable (ET) spécifique du génome C, Bot1, en vue d’identifier de nouvelles transpositions survenant chez les colzas néo-synthétisés par rapport aux parents diploïdes, par une approche SSAP. Quelques rares événements de transposition ont été identifiés. Ces résultats, confrontés à ceux obtenus sur deux autres ET, ont permis de mettre en évidence un impact modéré de l’allopolyploïdie sur la transposition de ces différents ET. Par contre, il est apparu que des changements de méthylation auraient accompagné cette allopolyploïdisation, sans doute à l’origine de la réactivation et la transposition de quelques copies de Bot1. Les petits ARN non codants ont été suggérés comme impliqués dans les différents événements génomiques accompagnant la formation d’un génome allopolyploïde. Pour étudier la dynamique d’expression des petits ARN chez des colzas néo-synthétisés pris à deux générations d’autofécondation (S1, S5) en comparaison de leurs parents diploïdes, j’ai exploité des données de séquençage haut débit obtenues pour 11 banques construites à partir des tiges de ces différents génotypes. J’ai ainsi démontré, qu’à une échelle globale, les petits ARN présentaient une réponse immédiate mais transitoire à l’événement d’allopolyploïdie. Les fractions particulièrement affectées par l’allopolyploïdie se sont révélées correspondre (1) à des petits ARN interférents dérivés d’éléments transposables avec une baisse de leur abondance en génération précoce S1, et (2) à des populations de petits ARN de 21 nucléotides exprimées uniquement de manière très précoce, de l’hybride F1 à la génération S1. Nous avons notamment identifié des transcrits de type viral correspondant à ces petits ARN de 21-nt, et présentant les mêmes profils d’expression (de l’hybride F1 à la génération S1), suggérant une réactivation d’éléments viraux endogènes (EVE) en réponse à l’hybridation et l’allopolyploïdie. L’ensemble de mon étude a démontré la mise en place d’une succession des voies de régulation par petits ARN où ET et EVE, réactivés au niveau transcriptionnel, sont immédiatement soumis à une répression post-transcriptionnelle (PTGS), renforcée ensuite par une répression de leur transcription (TGS). L’hypothèse d’une absence de cette régulation par petits ARN lors des phénomènes de nécrose et létalité hybride, amène à envisager ces populations de petits ARN comme les clés de la réussite de la formation d’un génome hybride, où la répression immédiate et efficace des ET et autres endovirus, réactivés suite au choc génomique, se révèle être une nécessité. / The evolutionary success of polyploid species is partly due to the dynamic changes in genome organization and gene expression patterns that occur at the onset of the polyploid formation. These changes are promoted by the merging of divergent genomes into a single nucleus (i.e. allopolyploidy) that causes a “genomic shock”; they are thought to provide a rich source of new genetic material upon which selection can act to promote adaptation and evolution. Many studies have thus aimed to uncover molecular mechanisms that are responsible for the evolutionary success of allopolyploid species, most of them focusing on gene expression changes. In the present PhD thesis, my interest has been concentrated on the non-coding components of the genome: transposable elements and small non-coding RNAs. My study involves oilseed rape (Brassica napus, AACC), a relatively young allopolyploid species that originated from hybridizations between B. rapa (AA) and B. oleracea (CC). Specifically, I have used resynthesized B. napus polyploids advanced by self-pollination of single plants for several generations; I have analyzed these plants at different generations for genomic changes accompanying polyploid formation and subsequent evolution. In a first part, sequence-specific amplification polymorphism (SSAP) targeting the C genome-specific transposable element Bot1, was used to evaluate transposition rate of Bot1 in resynthesized B. napus in comparison with the diploid parents. Only a few transposition events were identified. When combined with the results obtained for two other TEs, this work suggests that allopolyploidy has only a moderate impact on TE transposition and restructuring. The changes observed in SSAP profiles led us to hypothesize that some of them resulted from changes in DNA methylation, resulting in rare but highly specific TE activation and transposition. In a second part, I have concentrated on small non-coding RNAs (sRNAs), which are thought to mediate different aspects of the response to the “genomic shock” induced by allopolyploid formation. Comprehensive analyses of sRNA expression in resynthesized B. napus allopolyploids have been carried out by deep sequencing sRNAs from 11 libraries prepared from stems of three allotetraploids (surveyed at the two generations S1 and S5) and the two diploid parents. Characterization of sRNA distributions in these plants indicates that sRNAs show an immediate but transient response to allopolyploidy. The sRNAs derived from transposable elements (down-regulated in the S1) or targeting unknown sequences (no Blast hit against any available public database) were particularly affected. The use of B. napus mRNAseq data revealed that these latest unknown candidates, which are 21-nt long and over-expressed in the earliest generations (F1, S0, S1) were derived from endogenous viral elements (EVE). We confirmed that these EVEs showed the same expression patterns as the 21-nt long sRNAs that specifically target them (over-expression in the F1, S0 and S1). These results suggest that (at least) some EVEs might be reactivated as a response to the merging of divergent genomes (in interspecific hybrids and newly formed allopolyploids). Altogether, our results have demonstrated a succession of sRNA pathways that counteract the reactivation of some specific TEs and/or EVEs at the onset of polyploid formation; reactivated TEs and/or EVEs being immediately repressed at the post-transcriptional level (PTGS), and then fully repressed by transcriptional gene silencing (TGS) in the subsequent generations. Such data lead to hypothesize that sRNAs are essential to overcome interspecific hybrid incompatibilities due to the uncontrolled and deleterious reactivation of TEs / EVEs. Therefore, sRNAs should be considered as the guardians of genome integrity even in newly-formed allopolyploids.