La cryptographie ADN est un domaine nouveau et prometteur pour la sécurité de l'information. C'est une combinaison des solutions classiques de cryptographie avec les avantages du matériel génétique. En effet, il est possible de bénéficier des avantages des systèmes cryptographiques classiques et de les rendre plus efficaces sur certaines méthodes grâce à l’utilisation de l'ADN. Il y a différentes façons d'utiliser l'ADN pour sécuriser le contenu de l'information. Cette thèse propose deux solutions différentes pour utiliser l'ADN dans la cryptographie : sous sa forme biologique ou alors sous forme numérique. D ‘une part, l'ADN biologique peut être utilisé pour le stockage et pour cacher des données à l'intérieur de celui-ci. L'information secrète est placée dans une molécule de l'ADN et caché parmi d'autres molécules d'ADN. D’autre part, les nombres aléatoires peuvent être générés à partir de séquences numériques d'ADN. Ils représentent une solution pour la génération et la transmission des clés OTP (One-Time-Pad) symétriques. La transmission d'une très longue clé de cryptage n'est pas nécessaire, car chaque séquence possède un numéro d'identification unique dans la base de données. Ce numéro, ou une combinaison de ces numéros, peut alors être transmis. Enfin, la sécurité et la compression sont très importantes lors de la transmission et du stockage des données informatiques. Cependant, la plupart des systèmes de cryptage peuvent augmenter la taille des données, ou encore augmenter la complexité calcul. Ces inconvénients peuvent être résolus en combinant la compression de données avec le cryptage dans un seul processus ou en effectuant le cryptage sélectif des données. / DNA cryptography is a new and promising field in information security. It combines classical solutions in cryptography with the strength of the genetic material. By introducing DNA into the common symmetric key cryptography, it is possible to benefit from the advantages of the classical cryptosystems and solve some of its limitations. There are different ways how DNA can be used to secure information content. It is about using the biological medium of DNA for storing and hiding data. Secret information can be placed in microscopic size of DNA and hidden among a great amount of other DNA structures. Biomolecular computation is possible with specially designed DNA structures. Random numbers can be generated from DNA sequences which can be found in genetic databases in digital form. Genetic databases represent a feasible solution to the One-Time-Pad (OTP) symmetric key generation and transmission problem. The one-time use is ensured due to the great variety of the publicly available, very long (thousands of bases) sequences. Transmission of a very long key is not required because each sequence has a unique identification number in the database and this number can be sent instead. Compression along with information security have always been topics of interest because, as technology advances, the amount of data that is desired to be transmitted, stored, or used in real time applications is becoming greater. Some of the encryption schemes can increase the size of the data, or bring unwanted additional computations. These drawbacks can be solved by several techniques to combine compression with encryption in one process or by performing a selective encryption of the data.

Les génomes des cellules cancéreuses subissent de profonds changements tant au niveau de leur structure, qu'au niveau épigénétique. Les tumeurs de sein présentent en particulier des profils d'anomalies génétiques et épigénétiques complexes et hétérogènes. Alors qu'une meilleure compréhension de la dynamique d'apparition des anomalies permettrait de mieux appréhender la complexité des tumeurs, peu d'informations sont disponibles à ce sujet. En effet la plupart des données, ont été, et sont produites à partir de tumeurs primitives ou de lignées cancéreuses établies et ne renseignent pas sur la séquence d'événements qui accompagnent le passage de l'état normal à cancéreux. De ce fait, nous nous sommes intéressés aux étapes précoces de la cancérogenèse. Nos travaux se basent sur l'utilisation d'un modèle de transformation progressive in vitro de cellules épithéliales mammaires primaires (HMEC). Les cellules, transduites de façon séquentielle à l'aide de constructions rétrovirales portant des shARN et différentes combinaisons d'oncogènes, ont été caractérisées à chaque étape au niveau cellulaire et moléculaire (CFH, MeDIP, Micro-array) afin de répondre aux questions suivantes : (1) Quelle est la séquence d'apparition des modifications épigénétiques et structurales au cours de la transformation tumorale ? (2) Les profils d'anomalies génétiques et épigénétiques sont-ils modulés en fonction de la voie oncogénique initialement activée dans la tumeur ? Contrairement aux données de la littérature, nous avons obtenu des cellules transformées grâce à l'expression de seulement deux éléments génétiques définis et non trois. Nos résultats indiquent que l'inactivation de p53 provoque la mise en place d'un terrain favorable à l'acquisition de nouvelles anomalies génomiques mais induit surtout d'importantes modifications du méthylome. De plus nous avons montré que les profils de remaniements génétiques et épigénétiques dépendent de l'oncogène initialement activé. Pour finir, nos résultats indiquent que la nature de l'oncogène initialement activé et responsable de la transformation, conditionne la dynamique de production et de sélection des anomalies et supportent l'hypothèse que l'hétérogénéité du cancer du sein peut être à l'origine de l'activation de voies oncogéniques distinctes. / The genome of cancer cells undergoes profound changes at genetic and epigenetic level. Breast tumors exhibit in particular complex and heterogeneous genetic and epigenetic profiles. While a better understanding of the dynamics of these changes could allow a better understanding of tumor complexity, little information is available on this subject. In fact, most data have been, and are produced from primary tumors or established cancer cell lines and do not provide information on the sequence of events that accompany the transition from normal to cancerous state. Therefore, we have been interested in the early stages of carcinogenesis. To this aim, we have developed a stepwise transformation model of HMECs (human mammary epithelial cell) by sequential transduction of oncogenes and/or shRNA. Each cellular variant have been characterized at the cellular and molecular level (CGH, MeDIP, and Micro-array) in order to answer the following questions (1) what is the sequence of structural and epigenetic changes during malignant transformation? (2) The patterns of genetic and epigenetic abnormalities are they modulated according to the oncogenic pathway initially activated in the tumor? Contrary to the literature data, we have obtained transformed cells with the expression of only two defined genetic elements. Our results indicate that p53 inactivation promotes the acquisition of genomic alteration but mainly induces significant changes at the DNA methylation level. In addition, we have shown that the remodeling of genetic and epigenetic profiles depends on the oncogene initially activated. Finally, our results suggest that the nature of the oncogene initially activated and responible for the transformation affects the dynamics of production and selection of anomalies, and supports the hypothesis that the heterogeneity of breast cancer may be due to activation of different oncogenic pathways.

Les virus sont omniprésents dans la biosphère et infectent vraisemblablement l'ensemble des êtres vivants. Au sein des écosystèmes, ils ont ainsi un impact sur la diversité des populations microbiennes, l'évolution des génomes de ces populations, et directement ou indirectement sur les cycles biogéochimiques majeurs. Leur caractère protéiforme et l'absence de marqueur unique (tant génétique que physique) font toutefois de l'exploration de la diversité virale une tâche complexe, de telle sorte que nos connaissances sur ces communautés virales environnementales sont encore très limitées. La métagénomique, ou séquençage massif et aléatoire de fragments nucléotidiques extraits d'un prélèvement, offre un point de vue unique sur les génomes viraux. Ce type d'approche, récemment développé, a ainsi mis en évidence la richesse extraordinaire des populations virales environnementales, tant du point de vue des gènes que des génotypes. C'est dans ce cadre de l'étude des communautés virales de l'environnement par métagénomique que se sont inscrits les travaux de cette thèse, organisée autour de quatre axes principaux : • Le développement de nouvelles méthodes d'analyses adaptées aux spécificités des génomes et métagénomes viraux par la mise en place du serveur web Metavir, premier serveur dédié à l'analyse des viromes. Proposant aujourd'hui un ensemble cohérent d'outils pour différents types de viromes, Metavir compte plus de 300 utilisateurs pour plus de 2000 viromes analysés. • Le potentiel fonctionnel des génomes viraux a pu être approché par l'étude conjointe d'un ensemble de viromes. Après une analyse rigoureuse des contaminations potentielles, nous avons pu confirmer que les génomes viraux comprenaient un ensemble limité mais non négligeable de gènes associés au métabolisme cellulaire. La plupart des virus agissent ainsi certainement directement sur le métabolisme de la cellule hôte durant l'infection. • La prépondérance des paramètres environnementaux, et particulièrement de la salinité, en tant que facteurs structurant les communautés virales aquatiques a également pu être mise en avant. La distance géographique entre prélèvements semble n'avoir qu'une influence secondaire, confirmant la capacité importante de dispersion des capsides virales. Une adaptation locale semble toutefois exister dans certains cas, notamment en cas de compétition importante entre les résistances développées par les hôtes et les capacités d'infection des virus. • Enfin, différentes familles de petits virus à ADN simple brin ont pu être caractérisées par une méta-analyse de viromes. Leur apparente simplicité a ainsi révélé des mécanismes d'évolution plus complexes que prévus, impliquant différents cycles et capacités de transfert de gènes jusqu’ici plutôt considérés comme l'apanage des virus à ADN double brin, et remettant en cause les séparations admises entre les différents groupes de virus sur la base de la nature de leur génome. En permettant une étude depuis l'échelle de la communauté jusqu'à des génotypes spécifiques, les viromes constituent des outils de choix pour caractériser la diversité virale, appréhender les différents facteurs régulant ces communautés, et ainsi mieux comprendre la place des virus dans la biosphère. De plus, ces études ont confirmé l'existence d'interactions étroites entre virus et organismes cellulaires, ces interactions semblant nombreuses, multiples dans leurs natures et conséquences, et présentes tout au long de l'histoire du vivant. Ces nouvelles connaissances apportées par l'analyse de viromes permettent donc d'aborder certaines questions fondamentales concernant l'origine des grandes innovations évolutives ou le fonctionnement global des écosystèmes. / Viruses likely infect every organism on Earth (in some cases even other viruses!), and represent vast morphological and genetic diversity. Not surprisingly given their numerical dominance, viruses significantly impact ecosystems through regulating microbial populations, driving major biogeochemical cycles, and shaping the evolution of hosts genomes. However, our understanding of viruses in nature is primitive, especially because the majority of environmental viral genomes remains uncharacterized. Metagenomics (i.e. random and massive sequencing of genomic fragments isolated from a sample) applied to encapsidated genetic templates provides a unique perspective on the viral pangenome. The first viral metagenomes (or viromes) generated entire sets of new questions about viral diversity, especially concerning their genetic and species richness. This work was set within this frame of viral diversity study through metagenomics, and organized into four main themes : • The development of bioinformatics tools adapted to the specific features of viral genomes and metagenomes led to the release of Metavir, the first web server dedicated to virome analysis. Providing a comprehensive set of connected tools, Metavir has now been used by more than 300 users in the analysis of more than 2000 viromes. • The functions encoded within viral genomes were for the first time thoroughly examined, following a rigorous examination of a set of published viromes toward contamination by cellular DNA. A new picture of the viral functional potential could thus be drawn, which confirmed that the range of cellular functions encoded in viral genomes is wider than the one retrieved from the complete genomes currently available, though not as great as previously estimated. • The study of the aquatic viral metagenomes also revealed the importance of salinity in the distribution of viral communities across the globe. The ubiquitous distribution of most viral genotypes confirmed that viral particles seem to be able to move across any distance on Earth. Viruses are thus likely selected based on factors such as the presence of their host in the samples and the competition with other parasites, which can still drive local adaptations. • Finally, viromes were used to better characterize the diversity of different ssDNA viral families. Despite their small size and relative simplicity, these viruses were found to harbor unexpectedly complex cycles and evolutionary mechanisms, in particular a great potential of recombination and gene transfer. Overall, the new genomes assembled from viromes notably challenge the separation between viruses based on the nature of their genome. Eventually, as illustrated by these different works and analyses, viromes are unique and extremely powerful tool to assess and characterize viral genetic diversity. Moreover, considering the tight links between viral and cellular worlds, insights into the viral communities provided by metagenomics make it possible to address fundamental questions such as the origin of important evolutive innovations or the functioning of ecosystems, so that these results are of interest for the whole field of biology.

Submitted by Francine Silva (francine.silva@unipampa.edu.br) on 2016-09-28T20:54:58Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Análise Transcricional de Physcomitrium Acutifolium Broth. Por meio da Técnica de RNA-SEG um enfoque sobre o estresse por frio em plantas.pdf: 2456403 bytes, checksum: f125855c91a08aa6e84cb53239871aa3 (MD5) / Made available in DSpace on 2016-09-28T20:54:58Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Análise Transcricional de Physcomitrium Acutifolium Broth. Por meio da Técnica de RNA-SEG um enfoque sobre o estresse por frio em plantas.pdf: 2456403 bytes, checksum: f125855c91a08aa6e84cb53239871aa3 (MD5) Previous issue date: 2015-05-29 / Os estresses abióticos são responsáveis pela indução de adaptações em plantas. Estas quando submetidas ao estresse, respondem através de mecanismos de sinalização nas rotas fisiológicas, desencadeando um processo de aclimatação. Entretanto, nem sempre este potencial de adaptação é expresso, mas se persistir ao longo do desenvolvimento da planta, torna-se uma adaptação oriunda de mudança genética. Consequentemente, essas alterações incipientes possam ser identificadas em nível transcricional, os precursores de algumas alterações genéticas importantes tais como splicing alternativo. Em ambientes polares a expressão de genes permitiu a adaptação das plantas a temperaturas de congelamento. Entre estas plantas estão os musgos, presentes nos ambientes de climas contrastantes, isto sugere que estes organismos tenham plasticidade fenotípica e genotípica. Ainda que haja poucos estudos destes organismos em relação a diferentes agentes estressores, é amplamente difundido que estes possuem potenciais de resistência aos estresses ambientais. Para descobrir estes potenciais de resistências é necessário estudar os genes relacionados especificamente com o fator estressor em questão, neste caso o estresse ao frio. Para tanto é necessário um processo de sequenciamento dos genes expressos quando a planta é submetida ao estresse. Neste estudo foram realizados testes com explantes cultivados in vitro do musgo Physcomitrium acutifolium Broth. em diferentes temperaturas, com 6 tratamentos variando de 0 a 25 ºC, seguidos das análises fenotípicas e posteriormente genômicas, incluindo processo de sequenciamento e identificação de genes expressos. Os resultados sugerem relação do estresse por baixas temperaturas e o potencial de expressão de genes relacionados ao estresse por frio neste musgo, principalmente por uma identificação de maiores ocorrências de splicing alternativo nas plantas cultivadas a temperatura mais baixa testada. Assim, o uso potencial de espécies de musgo em estudos relacionados à resistência ao congelamento em plantas, torna-se como uma alternativa interessante em processos de biotecnologia vegetal. / The abiotic stresses are responsible for inducing adaptations in plants. When subjected to stress, plants respond by signaling mechanisms in physiological pathways, starting a process of acclimatization. However, not every adaptation potential is identified in phenotypic level, but if the selection pressure led by the stress persist over plant development, there may be a change in genotypic level. Consequently, these incipient changes can be identified in transcriptional level, the precursors of some important genetic changes such as alternative splicing. In polar environments are noted that the plants were adapted to survive at low temperatures, this adjustment is related to the expression of genes, which confer them resistance to freezing. Among these plants are mosses, present in the environments of contrasting climates, this suggests that these organisms have phenotypic and genotypic plasticity. Even if there are few studies of these organisms in relation to different stressors, we know that these have the potential for resistance to environmental stresses. To discover these potential resistance is necessary to study the genes specifically related to the stressor in question, this study is the cold stress. Then you need a sequencing process is necessary genes expressed when the plant is subjected to stress. In this study were performed tests with cultured explants in vitro moss Physcomitrium acutifolium Broth. at different temperatures, with 6 treatments ranged from 0°C to 25°C, followed by phenotypic analysis and subsequently transcriptome analysis based in RNA sequencing process aiming to identify a differential gene expression. The results suggest stress relationship for low temperatures and the potential for expression of genes related to cold stress in this moss, mainly by an identification of a higher occurrences of alternative splicing in plants growing at lowest temperature tested. Thus, the potential use of moss species in studies related to plant resistance to freeze temperatures becomes as an interesting alternative in plant biotechnology processes.

G-quadruplexes are non-canonical nucleic acid secondary structures of increasing biological and medicinal interest due to their proposed physiological functions in transcription, replication, translation and telomere biology. Aberrant G4 formation and stabilisation have been linked to genome instability, cancer and other diseases. However, the specific genes and pathways involved are largely unknown, and the work within this thesis aims to investigate this. Stabilisation of G4s by small molecules can perturb G4-mediated processes and initial studies suggest that this approach has chemotherapeutic potential. I therefore also aimed to identify cell genotypes sensitive to G4-ligand treatment that may offer further therapeutic opportunities. To address these aims, I present the first unbiased genome-wide genetic screen in cells where genes were silenced via short-hairpin RNAs (shRNAs) whilst being treated with either PDS or PhenDC3, two independent G4-stabilising small molecules. I explored gene deficiencies that enhance cell death (sensitisation) or provide a growth advantage (resistance) in the presence of these G4-ligands. Additionally, I present a validation screen, comprising hits uncovered via genome-wide screening, and also the use of this in another cell line of different origin. Sensitivities were enriched in DNA replication, cell cycle, DNA damage repair, splicing and ubiquitin-mediated proteolysis proteins and pathways. Ultimately, I uncovered four synthetic lethalities BRCA1, TOP1, DDX42, GAR1, independent of cell line and ligand. These were validated with three G4-stabilising ligands (PDS, PhenDC3 and CX-5461) using an independent siRNA approach. The latter siRNA methodology was used to screen 12 PDS derivatives with improved medicinal chemistry properties and ultimately identified SA-100-128, as a lead compound. The mechanism behind synthetic lethality with G4-stabilising ligands was explored further for DDX42, which I show has in vitro affinity for both RNA- and DNA-G4s and may represent a previously unknown G4-helicase. Also within this thesis, gene deficiencies that provided a growth advantage to PDS and/or PhenDC3 as uncovered by genome-wide and focused screening were explored. These showed enrichment in transcription, chromatin and lysosome-associated genes. The resistance phenotype of three gene deficiencies, TAF1, DDX39A and ZNF217 was further supported by additional siRNA experiments. Overall, I satisfied the primary aims and established many novel synthetic lethal and resistance interactions that may represent new therapeutic possibilities. Additionally, the results expand our knowledge of G4-biology by identifying genes, functions and subcellular locations previously not known to involve or regulate G4s.

Atualmente, o Brasil é considerado o maior exportador mundial e terceiro maior produtor mundial de carne de frango. Este destaque é resultado, principalmente, do melhoramento animal baseado na estimação do valor genético a partir da mensuração de fenótipos e informações de pedigree. Entretanto, é comum que a seleção não seja feita para cada característica isoladamente devido à correlação genética entre elas. Esta correlação tem como causas a pleiotropia ou a ligação genética. Com este trabalho objetivou-se detectar associações entre características fenotípicas de interesse para a avicultura e SNPs em uma região do cromossomo 1 (168 - 208 cM e 57 - 71 Mbp), onde possíveis QTL ligados foram previamente mapeados. Utilizou-se o Beadchip de SNPs de 60k para genotipar 14 animais da geração Parental (machos TT e fêmeas CC) e 28 F1 da população TCTC desenvolvida pela Embrapa Suínos e Aves. A linhagem TT apresentou maior variabilidade genotípica que CC, porém, os F1 foram superiores às linhagens Parentais com base no número de heterozigotos e MAF. O polimorfismo com maior ocorrência em ambas as gerações foram as transições com 84,3%. Foram selecionados 144 SNPs mais informativos com base na heterozigosidade dos cinco casais F1 que geraram os 453 F2. Houve redução de heterozigotos e MAF em F2, em função da média de F1, decorrente de certo grau de parentesco e endogamia entre os animais que compuseram esta geração. Os blocos de haplótipos construídos demonstraram que os machos TT apresentaram 25 blocos, fêmeas CC (17), F1 (32) e F2 (23) com tamanho médio de 278, 467, 242 e 160 kpb, respectivamente. Foi evidenciado que 236 (42,7%) correlações fenotípicas foram significativas, das quais o maior número constatado foi entre PB_MS e outras 17 características e, o maior valor estimado foi entre PB_MS e EE_MS (-0,90). Do total esperado de 3.456 testes de associação, 609 (17,6%) foram considerados significativos (p < 0,05), sendo 424 (69,6%) com efeito aditivo e 185 com efeito de dominância (30,4%). PV41 apresentou maior número de associações (123), enquanto DOR não foi associado a nenhum SNP. Proporcionalmente, o maior número de SNPs foi associado próximo ao QTL pleiotrópico 2 para 17 características. Já os maiores níveis de significância (p < 9,59 x 10-8) para o efeito aditivo foram evidenciados para SNPs localizados próximos ao QTL pleiotrópico 1 e associados somente com PV41, a saber: Gga_rs13869715 (A < C), Gga_rs13870613 (T < C), Gga_rs14827719 (A < G), GGaluGA019336 (T < C) e GGaluGA019533 (A < C). Foram detectadas associações ainda não descritas na literatura para GP3541, CA3541, INT, PES, CAB, FIG, COR, MOE, PUL, HEM, COL, TRI, TC, PB_MS, EE_MS, CZ_MS. Finalmente, foram indicados possíveis genes candidatos posicionais e funcionais, tais como, IGF1, MYBPC1, MTPN, SOX-5, FGFR1OP2 e TTLL12 que poderão ser empregados na análise de expressão gênica. / Actually, Brazil is considered the world\'s first- and third-biggest exporter and producer of poultry meat, respectively. These performances are mainly consequence of animal breeding based on the estimation of breeding value combining phenotypes and pedigree information. However, usually the selection is not carried out for each trait separately due to genetic correlation between them. This correlation is caused by pleiotropy or linkage. We aimed to detect associations between phenotypic traits of interest to poultry industry and SNPs on a region of chromosome 1 (168 - 208 cM and 57 - 71 Mbp), where putative linked-QTL were previously mapped. A chicken 60k SNP BeadChip was used to genotype 14 animals from Parental generation (TT males and CC females) and 28 F1 of the TCTC population that was developed by Embrapa Swine and Poultry. The TT line showed greater genotypic variability than CC, however, F1 were higher than Parental generation based on the number of heterozygotes and MAF. The polymorphism more frequent in both generations was the transitions with 84.3%. The 144 most informative SNPs were selected based on heterozygosity of the five F1 couples which generated the 453 F2. There was a reduction of heterozygotes and MAF in F2, based on the F1 mean value, as consequence of some degree of relationship and inbreeding between animals that formed this generation. Haplotype blocks demonstrated that the TT males showed 25 blocks, CC female (17) F1 (32) and F2 (23) with an average size of 278, 467, 242 and 160 kbp, respectively. It was observed that 236 (42.7%) phenotypic correlations were significant. Out of these, the highest number was found between PB_MS and other 17 traits and the highest estimated value was between PB_MS and EE_MS (-0.90). Out of 3,456 expected association tests, 609 (17.6 %) were considered significant (p < 0.05), being 424 (69.6%) with additive effect and 185 with dominance effect (30.4%). PV41 presented the highest number of associations (123), while DOR was not associated to any SNP. Proportionally, the highest number of SNPs was associated close to the pleiotropic QTL 2 with 17 traits. On the other hand, the highest significance levels (p < 9.59 x 10-8) for the additive effect were evidenced for SNPs located close to the pleiotropic QTL 1 and associated only with PV41 (Gga_rs13869715 (A < C), Gga_rs13870613 (T < C), Gga_rs14827719 (A < G), GGaluGA019336 (T < C) and GGaluGA019533 (A < C)). Novel associations were detected for GP3541, CA3541, INT, PES, CAB, FIG, COR, MOE, PUL, HEM, COL, TRI, TC, PB_MS, EE_MS, CZ_MS when we compared our results with literature. Finally, putative positional and functional candidate genes were indicated such as IGF1, MYBPC1, MTPN, SOX-5, FGFR1OP2 and TTLL12, which may be used in gene expression analysis.

Aujourd'hui, l'émergence ou la réémergence de maladies transmises par les tiques (TBDs) devient un problème majeur. En raison des problèmes générés par l'utilisation des acaricides (pollution, résistance), il est donc urgent d'identifier de nouvelles approches pour contrôler les populations de tiques. Parmi ces stratégies, la vaccination visant des molécules conservées chez les tiques et impliquées dans leur capacité vectorielle, sont devenues particulièrement attractives. En conséquence, l'identification de cibles antigéniques appropriées est un défi majeur pour la mise en œuvre de ces stratégies de contrôle des tiques et des TBDs. Dans le présent travail, l'objectif principal est d'élucider les interactions moléculaires entre I. ricinus et B. henselae, afin d'identifier des molécules qui pourraient représenter des cibles vaccinales contre les tiques et les agents pathogènes qu'elles transmettent. Dans ce but, nous avons identifié, par séquençage à haut débit, des transcrits d'Ixodes ricinus différentiellement exprimés au niveau des glandes salivaires de la tique en réponse à une infection par B. henselae. Dans un second temps, l'implication d'un de ces transcrits surexprimés lors de l'infection dans la transmission de B. henselae, a été évaluée. Enfin, et en premier lieu, nous avons validé l'utilisation de la technique de gorgement artificiel sur membrane pour infecter I. ricinus par B. henselae et évalué l'impact de différents paramètres sur le gorgement des tiques. Les résultats ont montré que la technique de gorgement sur membrane est bien adaptée à l'infection d'I. ricinus par B. henselae en laboratoire, et que la proportion et le poids des tiques gorgées sont diminués lors de l'infection du sang par la bactérie Le séquençage en 454 des glandes salivaires de tiques a généré une banque de référence contenant 24, 539 transcrits, et la comparaison des glandes salivaires d'I. ricinus infectés et non-infectés par B. henselae a montré que 839 et 517 transcrits étaient respectivement significativement surexprimés et sous-exprimés en réponse à l'infection par des bactéries. Parmi les gènes de fonction connue, 161 transcrits correspondent à 9 familles déjà identifiées, quand les autres correspondent à des gènes de fonction inconnue. L'extinction par RNA interférence du gène le plus surexprimé, IrSPI qui appartient à la famille des inhibiteurs de sérine protéase BPTI/Kunitz, a entraîné une réduction de la taille du repas sanguin prit par les tiques (et donc sa descendance) ainsi que du niveau d'infection au niveau des glandes salivaires. En conclusion, cette étude a démontré que la technique de gorgement artificiel des tiques sur membrane est un outil puissant pour étudier les interactions entre les tiques et les agents pathogènes qu'elles transmettent comme B. henselae. Ce travail apporte aussi une nette avancée en termes de données génétiques sur I. ricinus (dont le génome n'est pas séquencé) et sur les interactions moléculaires entre une bactérie et son vecteur. Enfin, ce travail a permis la mise en évidence d'une molécule représentant un candidat vaccinal très prometteur à la fois pour diminuer la population de tiques et lutter contre les agents pathogènes qu'elles transmettent. Dans le futur, et en fonction de la confirmation du rôle des gènes identifiés ici dans la transmission bactérienne, de nombreux candidats vaccins pourront ainsi être évalués, ouvrant alors de nouvelles perspectives dans la lutte contre les tiques et les maladies dues aux agents qu'elles transmettent / Ticks are obligate blood-feeding ectoparasites of many hosts including mammals, birds and reptiles. After mosquitoes, they are the most important vectors worldwide, and are able to transmit the highest variety of pathogens including virus, bacteria and parasites. Ixodes ricinus (Acari: Ixodidae), the most common tick species in Europe, is a three-life stage hard tick. It is frequently associated with bites in humans, and transmits several pathogens, including Tick-Borne Encephalitis, Babesia spp., Borrellia spp., Anaplasma spp., and to a lesser extent Bartonella spp. Bartonella spp. are facultative intracellular bacteria associated with a number of emerging diseases in humans and animals. It has been demonstrated that I. ricinus is a competent vector for B. henselae that causes cat scratch disease as well as being increasingly associated with a number of other syndromes, particularly ocular infections and endocarditis. Recently, emergence or re-emergence of tick-borne diseases (TBDs) is increasingly becoming a problem. Indeed, and because of the limited success and disadvantages of controlling TBDs via acaricides, new approaches are urgently needed. Therefore, vaccine strategies that target conserved components of ticks that play roles in vector infestation and vector capacity have become particularly attractive. Accordingly, the identification of suitable antigenic targets is a major challenge for the implementation of tick and TBDs control strategies. In the present work, the main objective is to elucidate molecular interactions between I. ricinus and B. henselae in order to identify some targets that may be used as vaccines against ticks and tick-borne pathogens. Two principal points are focused on: primarily, to identify I. ricinus salivary gland differentially expressed transcripts in response to B. henselae infection with next generation sequencing techniques (454 pyrosequencing and HiSeq 2000); secondly, to validate the implication of one of these transcripts in the transmission of B. henselae. For that purpose, and at first, we validated artificial membrane feeding technique for ticks infection by B. henselae and evaluated the impact of several parameters on tick feeding. Results showed that membrane feeding technique is a suitable method to infect I. ricinus with B. henselae and that the proportion and weight of engorged ticks are decreased by B. henselae infection of the blood meal. Transcriptional analysis of the tick salivary glands generated a reference databank containing 24,539 transcripts, and the comparison of B. henselae-infected and non-infected I. ricinus female salivary glands showed that 839 and 517 transcripts were significantly up- and down-regulated in response to bacteria infection, respectively. Among them, 161 transcripts corresponded to 9 groups of ticks salivary gland gene families already described, when the other ones corresponded to genes of unknown function. Silencing the most up-regulated gene IrSPI, which belongs to BPTI/Kunitz family of serine protease inhibitor, resulted in reduction of tick feeding and bacteria load in tick salivary gland. In conclusion, this work demonstrated that artificial-membrane feeding technique is a powerful tool for investigating the interactions between tick and tick-borne pathogens as B. henselae. It also increases the available genomic information for I. ricinus and the knowledge to improve our understanding of the molecular interaction between tick and tick-borne pathogens. At last, it provides a potential vaccine candidate to control tick-borne diseases. In the future, and depending of differentially expressed genes' role confirmation, more and more vaccine candidate will be provided by this work, and the strategy of controlling tick and tick-borne disease will come to a new stage

Asf1 est une protéine chaperon d’histone, qui participe à l’assemblage et au désassemblage des histones H3/H4 sur l’ADN. Asf1 n’est pas essentiel pour la viabilité cellulaire chez S. cerevisiae, mais les voies de surveillance des dommages à l’ADN sont activées de façon constitutive dans les cellules dépourvues d’Asf1 et celles-ci sont hypersensibles à plusieurs types de stress génotoxiques. Chez S. cerevisiae, Asf1 forme un complexe stable avec Rad53 en absence de stress génotoxique. Nos résultats suggèrent qu’au moins trois surfaces d’interaction sont impliquées dans le complexe Asf1-Rad53. Le domaine FHA1 de Rad53 fixe Asf1 phosphorylé sur T270, l’extrémité C-terminale de Rad53 fixe la même surface d’Asf1 impliquée dans la fixation des co-chaperones HirA/CAF-1, et un troisième site putative est constituée de la surface d’Asf1 impliquée dans la fixation de l’histone H3 avec le domaine kinase de Rad53. Lors des stress génotoxiques, Rad53 est phosphorylée et activée. Mes résultats montrent une dissociation totale du complexe Rad53-Asf1 après traitement HU, mais la préservation du complexe après traitement des cellules avec une gamme de concentration de MMS. Nous pensons que la régulation du complexe traduisent des réponses cellulaires distinctes adaptées à des stress génotoxiques spécifiques. Par ailleurs, grâce à la structure du complexe formé par un peptide C-terminal de Rad53 et le domaine N-terminal d’Asf1, nous avons isolé une mutation rad53_A806R-L808R. Nous avons constaté que cette mutation déstabilise l’interaction entre Asf1 et Rad53 et augmente la viabilité des mutants rad9 et rad24 aux stress génotoxiquex. Ce mutant rad53_A806R-L808R semble retourne plus vite dans le cycle cellulaire et/ou traverse plus vite la phase S par rapport à Rad53-WT, et augmente la réparation de l’ADN ou l’adaptation aux dommages du simple mutant rad24Δ. / Asf1 is a histone chaperone, which participates in the assembly and disassembly of histones H3/H4 on DNA. Asf1 is not essential for cell viability in yeast, but the DNA damage checkpoints are constitutively activated in cells lacking Asf1 and they are hypersensitive to several types of genotoxic stress. In yeast, Asf1 forms a stable complex with Rad53 in the absence of genotoxic stress. Our results suggest that this complex involves at Ieast three interaction surfaces. One site involves the H3-binding surface of Asf1 with an as yet undefined surface of Rad53, probably reside in the kinase domain of Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-T270. The third site involves the C-terminal 21 aa of Rad53 bound to the conserved Asf1 N-terminal domain, where Rad53 competes with histone H3/H4 and co-chaperones HirA/CAF-1 for binding to the same surface of Asf1. Rad53 is phosphorylated and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when cells were treated with hydroxyurea but not methyl methane sulfonate, suggesting a regulation of the complex as a function of the stress.In addition to these results, we also found that the rad53-A806R+L808R mutation at the C-terminus of Rad53 destabilized the Asf1-Rad53 interaction and increased the viability of rad9 and rad24 mutants to genotoxic stress. The rad53-ALRR mutant also appeared to re-enter the cell cycle and/or traverse S-phase more rapidly than wild type and increased repair or adaptation when combined with the rad24 mutant.

Convergence of quantitative genetics and genomics is becoming the way that fundamental genetics and applied breeding will be carried out in the next decades. This study bridges the quantitative genetics of complex growth and wood properties traits with genomic technologies towards a more innovative approach to tree breeding. Planted forests play a major role to fulfill the growing world demand for wood products and energy. Eucalypts stand out for their high productivity and versatile wood resulting from the advanced breeding programs associated to clonal propagation and modern silviculture. Despite their fast growth, breeding cycles still take several years and wood properties assessment is limited to a sample of trees in the late stages of selection due to the costs involved in wood phenotyping, not exploitingthe range of genetic variation in wood properties. In this study, we examined fifteen traits including growth and wood chemical and physical properties in 1,000 individuals sampled from an elite Eucalyptus breeding population. Near-infrared spectroscopy (NIRS) models were developed and used for high-throughput phenotyping of wood traits.Highdensity data for 29,090 SNPs was used to obtain accurate pedigree-record-free estimates of trait variance components, heritabilities, genetic and phenotypic correlations, based on a realized relationship matrix, comparing them to pedigree-based estimates. To the best of our knowledge, this is the first study to do this in plants. NIRS predictions were accurate for wood chemical traits and wood density, and variably successful for physical traits. Heritabilities were medium for growth (0.34 to 0.44), high for wood chemical traits (0.56 to 0.85) and variable for wood physical traits (0.11 to 0.63). High positive correlations among growth traits and negative between cellulose and lignin content were observed, while correlations between wood chemical and physical traits and between growth and wood quality traits were low although significant. Phenotypes and SNP markers were then used to build genomic predictive models using a marker density higher than any previous genomic selection study in trees (1 SNP/21 kbp). Two models (RR-BLUP and Bayesian LASSO) that differ regarding the assumed distribution of marker effects were used for genomic predictions. Predictions were compared to those obtained by phenotypic BLUP. Predictive abilities very similar by the two models and strongly correlated to the heritabilities. Accurate genomic-enabled predictions were obtained for wood chemical traits related to lignin, wood density and growth, although generally 15 to 25% lower than those achieved by phenotypic BLUP prediction. Nevertheless, genomic predictions yielded a coincidence above 70% in selecting the top 30 trees ranked by phenotypic selection for growth, wood density and S:G ratio, and 60% when tandem selection was applied. The results of this study open opportunities for an increased use of highthroughput NIRS phenotyping and genome-wide SNP genotyping in Eucalyptus breeding, allowing accurate pedigree-record-free estimation of genetic parameters and prediction of genomic breeding values for yet to be phenotyped trees. These applications should become routine in tree breeding programs for the years to come, significantly reducing the length of breeding cycles while optimizing resource allocation and sustainability of the breeding endeavor. / A convergência da genética quantitativa com a genômica está se tornando a maneira pela qual a genética fundamental e aplicada serão conduzidas nas próximas décadas. Este estudo buscou conectar a genética de fenótipos complexos de crescimento e propriedades de madeira às tecnologias genômicas, em uma abordagem inovadora para o melhoramento florestal. Florestas plantadas têm papel fundamental para satisfazer a crescente demanda mundial por produtos madeireiros e energia. O eucalipto,com sua alta produtividade e madeira versátil, é resultado de programas avançados de melhoramento associados à propagação clonal e silvicultura moderna. Apesar de seu rápido crescimento, ciclos de melhoramento ainda levam muitos anos e a avaliação detalhada de propriedades da madeira é limitada a apenas uma amostra das árvores em estágios avançados de seleção, devido aos altos custos de fenotipagem, não explorando assim toda a variação genética disponível. Neste estudo, examinamos quinze caracteres, incluindo crescimento e propriedades químicas e físicas da madeira, em 1000 indivíduos amostrados de uma população elite de melhoramento. Modelos de espectroscopia de infravermelho próximo (NIRS) foram desenvolvidos e utilizados para fenotipagem de alto desempenho de propriedades de madeira. Genotipagem de alta densidade com 29.090 SNPs foi utilizada para obter estimativas acuradas de componentes de variância, herdabilidades e correlações genéticas baseadas em uma matriz de parentesco realizado, ou seja,sem o uso de pedigree. Este é o primeiro estudo de que temos conhecimento a fazer isso em plantas. Predições NIRS foram precisas para caracteres químicos da madeira e densidade, e apresentaram sucesso variável para caracteres físicos. As herdabilidades foram médias para crescimento (0,34 a 0,44), altas para caracteres químicos de madeira (0,56 a 0,85) e variáveis para caracteres físicos da madeira (0,11 a 0,63). Altas correlações positivas entre caracteres de crescimento e negativas entre celulose e lignina foram observadas, enquanto correlações entre caracteres químicos e físicos da madeira foram baixas, porém significativas. Fenótipos e marcadores SNP foram em seguida utilizados na construção de modelos preditivos com a maior densidade de marcadores já utilizada em estudos de seleção genômica em espécies florestais (1 SNP/21 kpb). Dois modelos de predição (RR-BLUP e LASSO Bayesiano)foram usados nas predições genômicas e comparados ao BLUP fenotípico. Os modelos apresentaram capacidades preditivas similares, fortemente correlacionadas às herdabilidades. Predições genômicas precisas foram obtidas para caracteres relacionados à lignina, densidade e crescimento, embora geralmente 15 a 25% menores do que as predições obtidas por BLUP fenotípico. Contudo, predições genômicas alcançaram coincidências acima de 70% na seleção das melhores 30 árvores ranqueadas pela seleção fenotípica para crescimento, densidade e relação S:G, e de 60% quando seleção em tandem foi aplicada. Os resultados deste estudo abrem enormes oportunidades para o uso combinado de fenotipagem NIRS e genotipagem com SNPs no melhoramento do eucalipto, permitindo estimativas acuradas de parâmetros genéticos e a predição de valores genéticos genômicos para plantas jovens ainda não fenotipadas. Estas aplicações deverão se tornar rotineiras nos programas de melhoramento florestal nos próximos anos, reduzindo significativamente a duração dos ciclos de seleção e, consequentemente, otimizando a alocação de recursos e a sustentabilidade do melhoramento.

Endometriosis is one of the most common gynecological diseases as it is thought to affect up to 15% of the female population. Characterized by the growth and proliferation of endometrial tissue outside of the uterine cavity, it is a complex condition with varying degrees of severity and can affect multiple regions of the body with symptoms ranging from a total lack of symptoms to debilitating pain and infertility. The most accepted theory of how endometriosis initiates is that of retrograde menstruation; however, approximately 90% of women with unobstructed fallopian tubes are thought to have some menstrual debris in the peritoneal cavity. Therefore, this theory does not explain in full why endometriosis occurs in some but not all women who experience retrograde bleeding. Genetic factors are thought to play a major role in the pathogenesis of endometriosis as women with a family history are 5 to 10 times more likely to develop the disease. The goal of this study was to determine if common chromosomal aberrations in the form of additions, deletions, or regions of loss of heterozygosity that may contribute to the establishment or progression of the disease are present in a population of endometriosis patients. DNA was isolated from the peripheral blood of endometriosis patients and endometriosis tissue biopsies, and it was analyzed using oligonucleotide based array comparative genomic hybridization. The results suggest that an addition on chromosome 17p13.3 may play a role in the biological mechanisms involved in endometriosis as it was identified in 75% of the DNA samples obtained from the peripheral blood and 100% of the DNA samples obtained from the tissue biopsies. This chromosomal imbalance is of particular interest as it is located in a region that harbors the tumor suppressor gene, hypermethylated in cancer-1 (HIC-1), whose aberrant expression has been reported in multiple cancers. Endometriosis has long been thought of as a benign disease despite its malignant characteristics, and individuals with endometriosis have been demonstrated to have an increased chance of developing ovarian cancer. This was the first study to examine the DNA from endometriosis patients using oligonucleotide based array comparative genomic hybridization to investigate genetic abnormalities in endometriosis. The findings may provide a novel target for future therapeutic options as well as indicate a link between endometriosis and cancer that has not been previously reported.