Made available in DSpace on 2016-08-17T18:39:30Z (GMT). No. of bitstreams: 1 2590.pdf: 1752867 bytes, checksum: 7dd2196f0a9d489b35a0759a6cc018c6 (MD5) Previous issue date: 2009-09-04 / Pipeline systems for genomic and transcriptomic analysis aim to create communication bridges among the existing analysis tools, therefore reducing researchers efforts. Most of the pipelines found in the literature lack important features which would be useful to the development of genome or transcriptome sequencing projects. Among them, the capacity of tracking the project results along its development, including the generation of partial reports; the presence of a collaborative environment where the involved laboratories can contribute with new data and chromatograms; the possibility to configure analysis parameters; multiple pipeline support and the possibility to include new tools and modules. In this work, a pipeline prototype was developed to overcome these shortcomings. Sequencing projects progresses are tracked along all over their developments. Chromatograms are progressively received along the development of the project and partial reports over newly received data are generated. The communication with the processing server is done via Web service, which offers a universal language interface, allowing client applications in heterogeneous platforms to submit data and execute operations and queries. Pipelines are configured in XML documents written in a predefined format, through which the researchers choose the tools and parameters to be used. The prototype offers support to multiple pipelines executed simultaneously in the same project. Pipelines are executed in parallel by the means of thread pools, what increases efficiency by distributing the workload in multiprocessed systems. Another feature of the prototype is the extensibility as each pipeline step is wrapped in a module. New modules can be easily inserted in the system through the implementation of a programming interface, therefore without the needing of recompilation. Module insertions are done in a declarative way through XML documents. A client application was also developed in the collaborative platform Sakai, allowing different research groups involved in a sequencing project to create pipelines, view results and exchange information on the project current status. To evaluate the efficiency of the prototype, a case study was carried out. Sequences generated from sequencing of Sphenophorus levis transcriptome were submitted and a pipeline was configured to analyze the data. The case study has pointed out that the prototype is efficient and produces good results. / Sistemas de pipeline para análise de genomas e transcriptomas têm o objetivo de criar pontes de comunicação entre as diferentes ferramentas no intuito de reduzir os esforços do pesquisador no processo de análise. A maioria dos pipelines descritos na literatura carece de funcionalidades importantes para o desenvolvimento de projetos de sequenciamento. Entre elas, a capacidade de acompanhar e gerar resultados parciais das análises ao longo do desenvolvimento do projeto; a presença de um ambiente colaborativo onde os diferentes laboratórios envolvidos possam contribuir com novos dados e cromatogramas; a possibilidade da configuração dos parâmetros da análise; o suporte a múltiplos pipelines com diferentes configurações; e o suporte à inclusão de novos programas e módulos. Neste trabalho, foi desenvolvido um protótipo que supre essas deficiências. O progresso dos projetos é acompanhado ao longo de todo o seu desenvolvimento. Para isso, recebe dados brutos de cromatogramas, realiza análises dos dados parciais e emite relatórios com os resultados. A comunicação com o servidor de processamento é realizada via Web service, oferecendo uma interface na linguagem universal XML que permite que aplicações cliente em plataformas heterogêneas submetam dados e realizem operações e consultas. Os pipelines são configurados através de arquivos XML em formato específico, no qual o pesquisador define os programas a parâmetros a utilizar. O protótipo dá suporte a múltiplos pipelines com execução simultânea em um mesmo projeto. A execução dos pipelines é realizada em paralelo por meio de um pool de threads, o que aumenta a eficiência dividindo a carga de processamento em servidores com mais de um núcleo. Uma aplicação cliente foi desenvolvida na plataforma colaborativa, permitindo que os diferentes grupos de pesquisa envolvidos no sequenciamento criem pipelines, visualizem resultados e troquem informações sobre o andamento do projeto. Outro diferencial do protótipo desenvolvido é a extensibilidade. Cada etapa do pipeline é encapsulada em um módulo. Novos módulos podem ser facilmente inseridos sem a necessidade de recompilação de todo o sistema, bastando para isso que o mesmo implemente uma interface específica. A inserção no sistema é realizada declarativamente em arquivos XML. Um estudo de caso foi realizado com a submissão de cromatogramas a partir do sequenciamento de ESTs (Expressed Sequence Tags) de Sphenophorus Levis. Um pipeline foi configurado para o estudo, e sua execução mostrou que o sistema é eficiente e apresenta bons resultados.

Fusarium verticillioides est une moisissure phytopathogène et mycotoxinogène dont l’occurrence est fréquente dans le sol et dans de nombreux céréales et végétaux et plus particulièrement dans le maïs et le blé. La contamination des cultures par F. verticillioides conduit à des maladies variées et est souvent associée avec une accumulation de mycotoxines. Les fumonisines B1 (FB1) and B2 (FB2) sont les toxines les plus dangereuses produites par F. verticillioides. Des conséquences létales de la consommation de produits alimentaires contaminés par les fumonisines ont été reportées chez les animaux et l’homme. Différentes démarches ont été utilisées pour lutter contre cette moisissure et ses toxines mais souvent liées à l’utilisation de fongicides chimiques par ailleurs reconnus pour leurs effets néfastes pour l’homme, les animaux et l’environnement. Des pratiques alternatives ont été développées pour préserver les récoltes en respectant les écosystèmes et la santé humaine et animale. L’utilisation d’amendements organiques est apparue comme une stratégie intéressante de contrôle des maladies des plantes en assurant un apport de PGPM (Plant Growth Promoting Microorganisms) et agents de biocontrôle. Notre étude a donc eu pour but de caractériser les communautés microbiennes, et plus particulièrement celles d’intérêt pour le biocontrôle, d’amendements organiques et sols amendés ainsi que de tester leur potentiel antifongique envers F. verticillioides. Les mécanismes de biocontrôle ont été étudiés en utilisant des approches métabolomiques et transcriptomiques par séquençage haut débit. L’étude des communautés microbiennes dans les amendements et les sols amendés a été réalisée par pyroséquençage de marqueurs qui sont des portions du gène ribosomal 16S et de la séquence ITS2. Le microbiote des amendements est essentiellement constitué par les familles des Pseudonocardiaceae, Bacillaceae et Trichocomaceae qui incluent différents PGPM et agents antifongiques. Par ailleurs les amendements semblent favoriser l’installation des familles d’intérêt dans les sols tout en limitant celle des pathogènes tels les Nectriaceae qui comprennent des Fusarium pathogènes. Les tests d’antagonisme ont montré que les sols amendés réduisaient la croissance de F. verticillioides de façon plus importante que le sol de référence en inhibant la production de microconidies. La production de fumonisines a également été fortement réduite en présence des métabolites produits par le microbiote des sols amendés (plus de 68 % et 92 % pour FB1 and FB2 respectivement). Des souches d’actinomycètes isolées des amendements, et identifiées comme appartenant au genre Streptomyces, ont montré une activité antifongique envers F. verticillioides. Parmi celles-ci, Streptomyces AV05 a été sélectionnée pour les tests supplémentaires en raison de son fort potentiel d’inhibition vis-à-vis de F. verticillioides. Des comparaisons des endométabolomes et transcriptomes des 2 souches cultivées seules ou en confrontation ont été réalisées et de nombreuses différences ont été remarquées pour chacune entre les 2 modalités. Ainsi, 29 métabolites impliqués dans les modifications du métabolome de F. verticillioides en co-culture avec Streptomyces AV05 ont été identifiés et différentes voies métaboliques semblent avoir été affectées. L’étude du transcriptome des 2 souches a donné des résultats allant dans le même sens que celle du métabolome. En effet des changements significatifs de niveau d’expression ont été observés au niveau transcriptomique pour 800 gènes de F. verticillioides et 115 gènes de Streptomyces AV05 en situation de confrontation. L’identification de ces gènes est en cours et plusieurs d’entre eux semblent impliqués dans les modifications des profils métaboliques observées chez les 2 souches. L’intégration des données métabolomiques et transcriptomiques devrait permettre d’améliorer a compréhension des mécanismes mis en jeu au cours de la confrontation. / Fusarium verticillioides is a phytopathogenic and mycotoxigenic filamentous fungus that can be found with high occurrence in soils and in a wide range of cereals and vegetables, particularly in corn and wheat. The F. verticillioides contamination in crops leads to various diseases and is usually associated with an accumulation of mycotoxins. Fumonisin B1 (FB1) and fumonisin B2 (FB2) are the most dangerous mycotoxins produced by Fusarium verticillioides. Lethal consequences caused by fumonisins have been reported in animals and in human due to the consumption of contaminated food products. To deal with this pathogenic fungus and its mycotoxins, several approaches have been applied but they are usually based on the use of chemical agents that are reported to be unsafe for humans, animals and ecosystems. Alternative practices that maintain the quality and the abundance of crops while preserving the ecosystems and human and animal health have been developed. The application of organic amendments has been reported as an interesting strategy for controlling diseases by providing an abundant source of PGPM (Plant Growth Promoting Microorganisms) and biocontrol agents. The aim of our study was to characterize the microbial communities of organic amendments and amended soils focusing on the microbial families of interest for biocontrol and to assess the antifungal potential of amended soils and their microbiota towards F. verticillioides. Mechanisms of biocontrol were studied using metabolomics and transcriptomics approaches. Evaluation of microbial communities in amendments and amended soils using pyrosequencing of the 16S rDNA and the ITS genes showed that the amendments contained mainly the families of Pseudonocardiaceae, Bacillaceae and Trichocomaceae that were believed to include various PGPM and antifungal agents. Furthermore, the amendments were expected to promote the families of interest in soil and also to limit those of pathogens such as Nectriaceae that might contain many pathogenic Fusarium. Antifungal assays showed that the amended soils reduced the F. verticillioides growth better than the reference soil by inhibiting the microconidia production. The fumonisin production was strongly reduced by the metabolites produced by the amended soils’ microbiota (up to 68 % and 92 % for FB1 and FB2 respectively). Some actinomycete isolates from these amendments were identified as Streptomyces and they demonstrated antifungal activity against F. verticillioides. Among the Streptomyces strains, Streptomyces AV05 was selected for further studies because of its strong inhibition towards F. verticillioides. The interaction between the Streptomyces strain AV05 as antagonist agent and F. verticillioides was investigated. The study of the endometabolome and the transcriptome of the two microorganisms was carried out in two different conditions: strains cultivated alone or in confrontation. Many modifications have been noticed into the endometabolome of the 2 microorganisms during the direct confrontation and 29 metabolites involved in the endometabolome alteration of F. verticillioides in co-culture with Streptomyces AV05 were identified. Many fungal metabolic pathways were suggested to have been affected. The results of the study of the mRNA of the 2 strains appeared to be in accordance with the metabolomics results. A change in the transcriptomic level was observed: 800 genes of F. verticillioides and 115 genes of Streptomyces AV05 were found to be expressed differently when the strains were cultivated alone or in confrontation. The identification of these genes is in progress. Many of them are expected to be responsible for the change in the metabolic profiles observed in the bacterial-fungal interaction. The integration of the metabolomic and transcriptomic informations could improve the understanding of the mechanisms of biocontrol during direct confrontation.

Orientador: Stephen Hyslop / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T08:32:10Z (GMT). No. of bitstreams: 1 Cardoso_KiaraCarolina_D.pdf: 27750375 bytes, checksum: a1ca027909bfd58421b986e75bfcd515 (MD5) Previous issue date: 2011 / Resumo: O estudo do transcriptoma de bibliotecas de cDNA da glândula venenífera de serpentes, realizado a partir da análise de ESTs (expressed sequence tags), tem se mostrado útil na identificação de genes expressos neste tecido, inclusive no gênero Bothrops, responsável pela maioria dos acidentes ofídicos no Brasil. Neste trabalho utilizamos uma abordagem transcriptômica para analisar a composição gênica da glândula venenífera da serpente Bothrops alternatus, uma espécie encontrada no sudeste e sul do Brasil, Uruguai, norte da Argentina e leste do Paraguai. Também clonamos e caracterizamos parcialmente a enzima dipeptidilpeptidase IV (DPP IV), uma enzima que cliva peptídeos com prolina ou alanina na penúltima posição em sua porção N-terminal e que tem sido detectada em diversas peçonhas ofídicas. A construção de bibliotecas de cDNA usando métodos convencionais de clonagem, sequenciamento e análise bioinformática resultou em 5,350 ESTs que foram reunidas em 838 contigs and 4512 singletons. Pesquisas a partir de bancos de dados relevantes (BLAST) mostraram 30% de hits e 70% no-hits. Os transcritos relacionados a toxinas correspondem a 23% do total de transcritos e 78% dos hits, respectivamente. A análise por ontologia gênica (GO) detectou genes relacionados ao metabolismo geral, transcrição, tradução, processamento, degradação de polipeptídeos, funções estruturais, e regulação celular. Os principais grupos de toxinas identificados foram metaloproteinases (81%), peptídeos potenciadores da bradicinina/peptídeos natriuréticos do tipo C (8,8%), fosfolipases 'A IND. 2' ('PLA IND. 2'; 5,6%), serinoproteinases (1.9%) e lectinas do tipo C (1,5%). As metaloproteinases eram quase queexclusivamente da classe PIII, com poucas da classe PII e nenhuma da classe PI. As 'PLA IND. 2' eram todas ácidas; nenhuma 'PLA IND. 2' básica foi detectada. Outras toxinas encontradas incluíram a L-aminoácido oxidase, proteínas secretadas ricas em cisteína, DPP IV, hialuronidase, toxinas three-finger e ohanina. Foram identificadas duas proteínas não-tóxicas, a tioredoxina e a Dusp6 (fosfatase de dupla especificidade) que mostraram alto grau de similaridade a proteínas semelhantes de outras serpentes. Também foram observados polimorfismos de nucleotídeos únicos (SNPs - single-nucleotide polymorphisms), microssatélites, transposons e repetições invertidas, todos os quais podem contribuir de alguma forma para a multiplicidade de toxinas na glândula. Estes resultados mostram que a glândula venenífera de B. alternatus possui as principais classes de toxinas encontradas em estudos transcriptômicos e proteômicos de outras espécies botrópicas. ... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Transcriptomic studies of snake venom gland cDNA based on the analysis of expressed sequence tags (ESTs) have been useful in identifying the genes expressed in this organ in a variety of species, including the genus Bothrops, which is responsible for most venomous snakebites in Brazil. In this work, we used a transcriptomic approach to analyze the gene composition of the venom gland of Bothrops alternatus (urutu), a species found in southeastern and southern Brazil, Uruguay, northern Argentina e eastern Paraguay. We also cloned and partially characterized dipeptidylpeptidase IV (DPP IV), an enzyme that cleaves peptides with proline or alanine as the penultimate residue in the N-terminal region and has been identified in several snake venoms. A cDNA library constructed using conventional methods of cloning, sequencing and bioinformatic analysis yielded 5,350 ESTs that formed 838 contigs and 4512 singletons. Databank BLAST searches yielded 30% hits and 70% no-hits. Toxin-related transcripts accounted for 23% of the total transcripts and 78% of the hits. Gene ontology analysis detected genes related to general metabolism, transcription, translation, processing, polypeptide degradation, structural functions and cellular regulation. The main toxin groups identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases 'A IND. 2' ('PLA IND. 2'; 5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively class PIII, with few class PII and no class PI enzymes. The 'PLA IND. 2' were all acidic; no basic 'PLA IND. 2' were detected. Other toxins identified included L-amino acid oxidase, cysteine-rich secretory proteins, DPP IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and a dual specificity phosphatase (Dusp6), shared high sequence homology with similar proteins from other snakes. Single-nucleotide polymorphisms (SNPs), microsatellites, transposons and inverted repeats were also observed and may contribute to the toxin diversity of the gland. These results show that the venom gland of B. alternatus contains the major toxin classes identified in transcriptomic and proteomic studies of other Bothrops species. The predominance of class PIII metalloproteinases agrees with the hemorrhagic activity of this venom, while the low content of serine proteinases and C-type lectins could account for the less intense coagulopathy observed after envenoming by this species. The lack of basic 'PLA IND. 2' agrees with the lower myotoxicity of this venom compared to other Bothrops species. ... Note: The complete abstract is available with the full electronic digital thesis or dissertations / Doutorado / Farmacologia / Doutor em Farmacologia

Over the past few years one of the major concerns in the Atlantic salmon (Salmo salar) farming industry has been the increasing incidence and severity of inflammatory viral diseases. Heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS) are currently two of the most prevalent viral diseases in commercial Atlantic salmon farms in Norway. Mortality levels in both diseases are generally low but morbidity can be very high with the associated chronic inflammatory response lasting for several months. The consequent reduced growth performance is causing considerable financial impact as HSMI has become increasingly widespread in recent years. The impact of CMS is further exacerbated as it generally affects large fish close to harvest. HSMI lesions occur in the atrium and ventricle in the heart including inflammation and necrosis in epi- endo- and myocardium along with myositis of red skeletal muscle. CMS lesions are commonly observed in the spongy myocardium in the atrium and ventricle of the heart with severe mononuclear inflammation and necrosis. Furthermore, circulatory disturbances associated with reduced cardiac function cause multifocal liver steatosis and necrosis in both diseases. Currently there are no vaccines or any other effective treatments for these diseases and so alternative therapies that could potentially modulate the intensity of the inflammatory response could be crucial to improve the clinical manifestation of the diseases. Therefore, the overall aim of the present study was to evaluate the concept of “clinical nutrition” to improve the clinical symptoms of both viral diseases, HSMI and CMS, through the use of functional feeds formulated with reduced lipid content and increased proportions of anti-inflammatory fatty acids to moderate the apparently uncontrolled inflammatory response in the heart tissue associated with both diseases and also alleviate the secondary hepatic lesions. The experimental work consisted of three major dietary trials in Atlantic salmon in seawater. Two large trials investigated the effects of functional feeds in Atlantic salmon challenged with Atlantic salmon piscine reovirus (ASRV) and piscine myocarditis virus (PMCV), the causal agents of HSMI and CMS, respectively. In both trials, heart transcriptome, heart and liver histopathology and tissue lipid and fatty acid compositions and metabolism were determined post-infection in fish fed with the functional feeds in comparison with fish fed with a standard commercial feed formulation considered as a reference diet. All the functional feeds were formulated to have reduced digestible energy through lower dietary lipid and higher protein contents, and increased levels and proportions of anti-inflammatory long-chain polyunsaturated fatty acids (LC-PUFA), particularly eicosapentaenoic acid (EPA) compared with the reference diets. Histopathology, fatty acid composition and gene expression of heart were assessed over a long time-period of 16 weeks and 14 weeks post-challenge with ASRV and PMCV, respectively. Viral load in heart tissue, hepatic histopathology and fatty acid composition of liver and head kidney along with expression of the genes involved in the eicosanoid and LC-PUFA and eicosanoid biosynthesis pathways were also determined in the HSMI trial. The third trial was a nutritional trial evaluating the effects of dietary digestible energy content on lipid and fatty acid metabolism in salmon fed diets containing graded amounts of lipid. Fatty acid composition of liver and heart were assessed over 12 weeks, along with the hepatic expression of genes of lipid and fatty acid metabolism. The results of this research are presented in four chapters (Chapters 2-5) as four paper manuscripts. The manuscripts/Papers are either published (Chapter 2), in review (Chapter 3 and 4) or drafted for submission (Chapter 5) in appropriate peer-reviewed international journals. Chapter 2 and 3 correspond to the HSMI trial, Chapter 4 to the nutritional trial, and Chapter 5 to the CMS trial. Chapter 2 showed that viral load and histopathology scores were lower in fish fed the functional feeds, especially diet FF1, which displayed better performance. Diet strongly influenced the expression of genes related with the immune and inflammatory responses, with delayed expression in fish fed the functional feeds. Up-regulation of pro-inflammatory genes was correlated with the higher viral load observed at early-mid stages of the disease in fish fed the reference diet (ST). Expression of genes related with the immune response at 16-weeks post challenge reflected the differences in immunomodulation between the functional feeds, with fish fed diet FF1 showing lower expression. Therefore, severity of the heart lesions was correlated with the intensity of the immune response and could be associated with tissue anti-inflammatory LC-PUFA levels. Chapter 3 was focused on liver histopathology, fatty acid composition and LC-PUFA biosynthesis, along with phospholipid fatty acid composition and eicosanoid production in head kidney and heart tissue at early and late stages of ASRV infection. Liver was severely affected by the virus at the beginning of the infection in fish fed the reference ST diet, but the level of lesions were similar in all dietary groups at the end of the trial. Hepatic expression of fatty acyl desaturases was significantly depressed in fish fed the ST diet compare with fish fed the functional feeds despite the lower levels of dietary LC-PUFA in that feed. Thus endogenous production and bioavailability of anti-inflammatory LC-PUFA was potentially enhanced in fish fed the functional feeds. Changes in tissue lipid content, mobilization of fatty acids involved in inflammatory responses and changes in expression of transcription factors and genes involved in eicosanoid biosynthesis were more prominent in head kidney, confirming the important role of this organ in dietary immunomodulation after viral infection. To a lesser extent similar changes were observed in heart tissue, suggesting in situ production of eicosanoids could also be important. The unexpected effects of diet on expression of genes of LC-PUFA biosynthesis were specifically investigated in the trial described in Chapter 4. One aim of this study was to clarify whether dietary lipid content or viral infection was the cause of altered expression of desaturase genes between the different diets. Hepatic expression of other genes of lipid and fatty acid metabolism were also determined to evaluate metabolic changes associated with dietary lipid/energy level. In general, reduction of dietary energy and lipid contents while maintaining similar proportions of dietary fatty acids, led to a general up-regulation of genes involved in lipid biosynthetic pathways. Thus salmon fed lower energy diet showed increased liver expression of fatty acyl desaturases in comparison with fish fed higher energy levels. Heart transcriptomic data in Chapter 5 showed a similar delay in the inflammatory response in fish fed the functional feeds after PCMV infection as observed in the HSMI study. Modulation of inflammatory responses, similar to that previously described after ASRV infection, was also observed in fish fed the functional feeds. However, the differences in the expression of immune related genes and the level of heart lesions were not as prominent at mid-late stages of the disease as in fish fed FF1 in the HSMI trial. The present study demonstrated the beneficial effects of a clinical nutrition approach via functional feeds in two viral inflammatory diseases, HSMI and CMS, currently affecting farmed Atlantic salmon.

La Flavescence Dorée (FD) est une maladie épidémique infectant les vignes européennes causée par un phytoplasme (pFD) qui est transmis de vigne à vigne par l’insecte vecteur Scaphoideus titanus. Aucun cépage actuellement cultivé n’est totalement résistant, mais des différences de sensibilité existent. En France, les méthodes de lutte réglementaires se concentrent sur la plantation de pieds certifiés sains, l’arrachage des plants contaminés et des traitements insecticides contre le vecteur dans les zones touchées par la FD. En plus du coût économique de ces moyens de lutte, l’usage des produits phytosanitaires impacte l’environnement et la santé humaine. Une meilleure compréhension des relations entre les différents acteurs du pathosystème vigne-pFD-S. titanus est donc essentielle pour pouvoir appliquer d’autres stratégies de lutte. Dans ce contexte, les principaux objectifs de ma thèse étaient (1) d’identifier et de caractériser au niveau fonctionnel des gènes codant des protéases de surface, les FtsH, potentiellement impliquées dans la virulence du pFD, (2) de caractériser la multiplication et la diffusion du pFD dans la vigne en s’affranchissant des interactions vigne-S. titanus, (3) d’initier la caractérisation du déterminisme génétique de la résistance par analyse d’un pool de descendants issus du croisement entre un cépage sensible (CF) et un cépage peu sensible (Mag) et (4) d’identifier des différences de dérégulations géniques entre le cépage très sensible CS et le cépage peu sensible M par comparaison des transcriptomes. Les résultats indiquent que (1) huit gènes FtsH sont codés par le pFD et s’expriment de manière différentielle selon l’hôte, (2) les différences de sensibilité à la FD chez la vigne sont dues au moins en partie aux interactions vigne-pFD, (3) une ségrégation des caractères « infection des plantes » et « multiplication du pFD » dans cette descendance est obtenue et (4) le M active des voies métaboliques différentes par rapport au CS lors de l’infection. A terme, la compréhension puis l’exploitation des différences de sensibilités des cépages pourront contribuer à l’élaboration de pistes alternatives à la lutte actuelle contre la FD dans le cadre d’une réduction des intrants. / Flavescence Dorée (FD) is a severe epidemic disease of grapevine caused by a wall-less bacteria, a phytoplasma (pFD), transmitted by the insect vector Scaphoideus titanus. There is no resistant variety, but differences in susceptibility between Vitis exist. In France, the actual control consists in insecticide treatments against the vector, up-rooting of diseased plants and sanitary control of plants. It has high economic and environmental impacts. A better understanding of the pathosystem FDp-grapevine-S. titanus is essential to develop alternative measures. In this context, the main objectives of my thesis were (1) to identify and characterize the expression of genes encoding surface proteases, FtsH, potentially involved in the virulence of the pFD, (2) to characterize the FDp multiplication and diffusion in Vitis without taking into account grapevine-S. titanus interactions, (3) to initiate the characterization of genetic determinism by phenotyping a pool of progeny from the cross between the susceptible variety CF and the poorly susceptible variety Mag and, (4) to identify differences in gene expression between the very susceptible variety CS and the poorly susceptible variety M by transcriptomes comparison. Results indicate that (1) eight FtsH genes are encoded by the FDp and are differentially expressed between plant and insect hosts, (2) differences in FD susceptibility in Vitis are caused, at least in part, by Vitis-FDp interactions, (3) a segregation of the characters "plant infection" and "pFD multiplication" is obtained in the progeny and, (4) M activates different metabolic pathways than CS to respond to FDp infection. Such knowledge can contribute to the development of alternative methods for limiting phytosanitary inputs.

Les cellules du cumulus (CCs) forment avec l'ovocyte le complexe ovocyte-cumulus (COC). Au cours de la folliculogénèse, un dialogue interdépendant régi par des jonctions communicantes se crée entre l'ovocyte et les CCs adjacentes. L'ovocyte en sécrétant certains facteurs, permettrait la différentiation et la prolifération des CCs qui, parallèlement, fournissent à l'ovocyte les nutriments nécessaires à sa maturation et son développement. La maturation nucléaire de l'ovocyte est définie par l'expulsion du 1er globule polaire au stade métaphase II (MII). Notre équipe a préalablement démontré que certains gènes exprimés dans les CCs chez l'humain, pouvaient prédire indirectement le potentiel implantatoire embryonnaire et la survenue d'une grossesse. Dans l'objectif d'identifier des marqueurs fiables de la maturité des ovocytes, nous avons analysé le profil transcriptomique des CCs en fonction du stade de maturité des ovocytes (VG, MI et MII). Dans un deuxième temps, nous avons étudié l'impact des conditions de maturation in vivo versus in vitro, sur le profil d'expression de gènes des CCs en fonction du stade de maturation ovocytaire. Nous avons mis en évidence, pour la première fois, une signature spécifique dans les CCs associée à la maturité nucléaire des ovocytes in vivo et in vitro. Nous avons également observé une sous-expression des gènes impliqués dans la maturation ovocytaire et l'expansion des CCs, et une sur-expression des gènes associés au cycle cellulaire dans les CCs dérivées d'ovocytes maturés in vitro, comparées aux CCs issus d'ovocytes in vivo. En comparant l'expression des gènes dans les CCs selon la condition de maturation et le stade de maturité de l'ovocyte, nous avons identifié deux voies de signalisation dominantes : la voie des lipides (transport du cholestérol et des triglycérides) fortement activée en condition in vivo, et le processus de réplication, recombinaison et réparation d'ADN, spécifique aux CCs in vitro. Nos résultats montrent que les conditions de maturation des COCs ont un impact sur la signature moléculaire des CCs. De plus, La signature moléculaire identifiée dans les CCs à différents stades de maturation ovocytaire nous a permis de définir la compétence des CCs. En considérant ce critère, nous avons observé que les ovocytes matures associés à des cellules du cumulus compétents (sur-expression de la signature des CCs au stade MII) présentent un potentiel de formation de blastocyste supérieur aux ovocytes MII entourés des cellules du cumulus incompétents (sur-expression de la signature des CCs au stade VG et/ou MI). Ces résultats ouvrent ainsi de nouvelles perspectives en application clinique. Des études supplémentaires sont toutefois nécessaires afin d'identifier, dans un premier temps, les facteurs qui influencent l'expression des gènes au cours de la maturation ovocytaire in vivo et comprendre ainsi les voies de signalisation altérées par les conditions de culture in vitro. / Cumulus cells (CCs) associated with the oocyte form the cumulus-oocyte complex (COC). During folliculogenesis, interdependent dialogue governed by gap junctions is created between the oocyte and adjacent CCs. The oocyte, by secreting certain factors allows the differentiation and proliferation of CCs which, at the same time, provide nutrients to the oocyte for its maturation and development. Nuclear maturation of oocytes is defined by its transition from germinal vesicle (GV) to metaphase I (MI) up to metaphase II (MII) phase. Our team previously shown that certain genes expressed in the human CCs could predict embryo and the pregnancy outcomes. We analyzed the transcriptomic profile of CCs according to oocyte nuclear maturation stages (GV, MI and MII). The aim of this study was to identify the CCs molecular signature according to nuclear maturation oocyte under in vivo and in vitro conditions. In addition, we studied the impact of culture conditions of the COCs under in vivo and in vitro on the gene expression profile of CCs. We have demonstrated that there is a specific signature in the human CCs associated with the nuclear maturity of human oocytes whatever the culture condition. We have also observed the under-expression of genes involved in oocyte maturation and CCs expansion, and the over-expression of genes associated with cell cycle function in the CCS derived from in vitro versus in vivo oocytes. By comparing gene expression in the CCs according to oocyte nuclear maturation stages, we have identified two dominant signaling pathways: the lipids pathway (cholesterol transport and triglyceride) strongly activated in in vivo conditions, and the process of replication, recombination and DNA repair, which appear to be specific to in vitro CCs. Our results suggest that the maturation conditions of COCs have an impact on the molecular signature of CCs.Moreover, our data showed that matures oocytes can be surrounded by competent (sur-expression of the identified molecular signature of CCs derived from oocyte at MII stage) or incompetent CCs (sur-expression of the identified molecular signature of CCs derived from oocyte at GV or/and MI stages). These results open new perspectives in clinical application.Further studies are needed to identify factors influencing gene expression during oocyte maturation in vivo. These data should help to better understand how/why signaling pathways are altered by culture conditions in vitro.

Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / C. Michael Smith / The tools for arthropods with sucking-mouth parts to attack hosts are mainly in the saliva. For plant-sucking insects, these salivary secretions are primarily produced in the salivary glands. Secreted proteins (also referred to as salivary gland secretomes) are among the important components in the saliva of sucking insects. Gall midges (Cecidomyiidae), a large family of plant-sucking insects, apparently secrete proteins (some of them are effector proteins) into host tissues, inducing various forms of plant outgrowth (galls). Three major insect pest species in the genera Mayetiola, the stem gall midges, are known to produce saliva that can reprogram plant cells and manipulate the host plant growth, causing serious damage to the plants of small grains. The three pest species are the Hessian fly (Mayetiola destructor), the barley midge (Mayetiola hordei), and the oat midge (Mayetiola avenae). Another economically important species of this gall midge family is the wheat midge (Sitodiplosis mosellana). It is a major insect pest of spring wheat and feeds on wheat heads, causing damage to the developing wheat seeds. A global analysis of the salivary gland secretome of first instar larvae of the Hessian fly, (a member of Mayetiola and) a model species for studying insect-plant interactions, has previously revealed a large number of genes encoding Secreted Salivary Gland Proteins, so called SSGPs. For comparison, we conducted analyses on transcripts encoding SSGPs from salivary glands of the first instar larvae of the wheat midge, barley midge, and oat midge. In the first chapter, a transcriptomic analysis of wheat midge has been conducted. In this analysis, a total of 3,500 cDNA clones were sequenced, and 1,301 high quality sequences were obtained and approximately 25% of the cDNAs (with high quality sequences) encoded SSGPs. The SSGPs were grouped into 97 groups based on sequence homology. Among the SSGP-encoding transcripts, 206 encoded unique proteins with no sequence similarity to any known protein and 29 encoded proteins similar to known proteins including proteases, serpines, thioesterases, ankryins, and feritins. The compositions of SSGP transcripts from the wheat midge were then compared with that of Hessian fly. The analyses have identified many common characteristics between the species. Despite these commonalities, no sequence similarity was found between SSGPs from wheat midge and those from Hessian fly, suggesting that SSGPs from these two insect species perform different functions to manipulate host plants. The second chapter contains results of comparative transcriptomic analyses on the barley and oat midges. A total of 2570 cDNA clones were sequenced from the barley midge, and 743 were high quality cDNA sequences, and the analysis identified 458 cDNA clones encoding SSGPs, of these, 178 encoded unique proteins (also called unigenes). Transcripts encoding SSGPs were grouped into 51 groups based on sequence homology. A total of 3226 cDNA clones were sequenced from oat midge, and 718 cDNA sequences were high quality and used for further analysis. The analysis identified 450 cDNA clones encoding SSGPs. Among the SSGP-encoding transcripts, 194 are unigenes, which were placed into 50 groups. The compositions of SSGP transcripts from the barley and oat midges were then compared with that of Hessian fly. The analysis identified five groups containing 102 (57.3%) unigenes from barley midges and seven groups containing 107 (55.1%) unigenes from oat midges which encode SSGPs that are conserved among the three species. The SSGPs conserved among the three midges are from family one (SSGP-1), family 4 (SSGP-4), family 11 (SSGP-11), and family 71 (SSGP-71). The SSGPs conserved among the three species indicate conserved functions such as a role in plant manipulation. Some SSGP unigenes were found to be conserved between only two species. Specifically, there were eight gene groups which are conserved between two species. Within these eight groups 19 (10.7%) unigenes from the barley midge and 25 (12.9%) unigenes from the oat midge were found to be conserved between only the barley and oat midges, whereas no homologues have been found in the Hessian fly. The remaining unigenes encode SSGPs that are unique to different midge species. The highly divergent SSGP groups that have been identified with no homology among the three midges indicate potential roles of these SSGPs in host specification. Due to the important roles of effector proteins in insect-plant interactions for gall midge species and since no insect effector protein have been identified directly from infested plant tissues so far, I have chosen one of the SSGP family, SSGP-1, which are conserved among all three gall midge species, for further analysis in chapter 4. Members in family SSGP-1 are also the most abundantly expressed at the transcript level. Based on Hessian fly data, family 1 contains seven genes and are named SSGP-1A1, SSGP-1A2, SSGP-1B1, SSGP-1C1, SSGP-1C2, SSGP-1D1, and SSGP-1E1. To detect the presence of these proteins in the infested wheat tissues, and to identify probable targets from wheat that interact with the SSGPs in the feeding site, we have generated and purified recombinant proteins for five of the seven proteins, namely SSGP-1A2, SSGP-1B1, SSGP-1C1, SSGP-1D1, and SSGP-1E1 (since SSGP-1A1 and SSGP-1C2 are very similar to SSGP-1A2 and SSGP-1C1, respectively). Antibodies were produced for the recombinant proteins for western blot analyses and indirect immunostaining. Immunostaining on dissected tissues including salivary glands, guts, and Malpighian tubules from 3-day old larvae, was conducted with antibodies against the five SSGPs, and detected a specific localization of all proteins in salivary glands except SSGP-1E1, which exhibited a weak signal in the foregut, in addition to localization in salivary glands. Western blot analyses demonstrated that these five proteins were expressed in larvae at all stages. The continuous production of these proteins suggests that they play roles in initiation and maintenance in Hessian fly infestation. Consistent with their effector functions, these five proteins were detected for the first time in infested wheat tissues based on western blot analyses. To identify possible target proteins from host plants that interact with SSGP-1 family proteins, in vitro pull-down assays were performed. Putative interacting targets for SSGP-1A2, SSGP-1B1, and SSGP-1C1 have been identified by LC-MS/MS. These putative interaction target proteins included uncharacterized proteins, ribosomal proteins, a lipoxygenase, and a tubulin. Identification of these putative targets provided a base for further confirmation of their interaction with Hessian fly effectors in the future.

Les écosystèmes côtiers sont parmi les plus vulnérables aux changements globaux actuels, qui entraînent notamment une augmentation de la température de l'eau, ainsi que de la fréquence des épisodes hypoxiques. La coquille Saint-Jacques, Pecten maximus, est une espèce subtidale évoluant à des profondeurs de 2 à 210 m. Malgré son intérêt commercial et un intérêt écologique majeur, cette espèce n'a fait l'objet que de peu d'études au niveau moléculaire. L'objectif de cette thèse était de caractériser les mécanismes moléculaires régissant l'acclimatation de cette espèce aux contraintes thermique et hypoxique. Nous avons dans un premier temps caractérisé les modifications d'expression des gènes/protéines, par des approches transcriptomiques (RNAseq) et protéomiques (2-DE), dans un tissu, le manteau, d'animaux exposés à une contrainte thermique prolongée (56 jours). Nous avons ainsi pu identifier les voies majeures de régulation (eg., AP-1), les grandes fonctions (eg., cytosquelette) et processus (eg., apoptose) impliqués dans la réponse, mais également d'observer les grandes orientations du métabolisme (eg., dégradation des lipides de réserve). La réponse des organismes à l'hypoxie dépend de leur manière de gérer les faibles teneurs en oxygène. Nous avons d'abord, par une approche comparative avec une espèce intertidale, la moule (Mytilus spp.), caractérisée la réponse physiologique de la coquille Saint-Jacques à l'hypoxie. Nous avons pu ainsi déterminer ses paramètres d'oxyregulation, plus particulièrement son Point critique en 02 (Pc02). Le développement d'une approche protéomique, couplant l'effet de la température et de l'hypoxie, nous a ensuite permis d'identifier plusieurs protéines (CK2, GLN, etc.) potentiellement impliquées dans la réponse au niveau moléculaire. Enfin, dans l'optique de mieux comprendre la physiologie particulière de ces mollusques dans leur environnement naturel, nous avons comparé les signatures protéomiques de deux populations de P. maximus évoluant dans des écosystèmes contrastés, i.e. en limite nord- (Norvège) et au centre- (Brest) de l'aire de répartition de l'espèce. Les résultats suggèrent des différences majeures entre les deux populations au niveau du cytosquelette. En conclusion, ce travail ouvre des perspectives nouvelles pour la compréhension des mécanismes moléculaires régissant l'adaptation des mollusques aux contraintes thermiques et hypoxiques, deux stress particulièrement importants pour les organismes marins dans le contexte du changement global. / Coasts are among the most vulnerable ecosystems to the ongoing global changes, which result in increased water temperatures and frequencies of hypoxic episodes. The great scallop, Pecten maximus, is a subtidal species living at depths of 2-210 m. In spite of its commercial and major ecological values, only few studies at the molecular level were performed on this species. This thesis aimed at characterizing the molecular mechanisms implied in acclimation of this species to thermal and hypoxia stresses. We first characterized the changes of expression of the genes / proteins in response to a long-term thermal stress (56 days), by using both a transcriptomic- (RNAseq) and a proteomic- (2-DE based) approaches, in the mantle tissue of scallops. This allowed us to identify key regulatory pathways (eg., AP-1), the major functions (eg., cytoskeleton) and processes (eg., apoptosis) involved in the response, but also to observe the main orientations of metabolism (eg., degradation of lipid reserves). The response of organisms to hypoxia depends on how they cope with low oxygen availability. Therefore, we first carried out a comparative approach with an intertidal species, the mussel (Mytilus spp.) to characterize the physiological response of P. maximus to hypoxia. Of note, we could determine its oxyregulatory parameters, particularly its critical point in 02 (Pc02). Then, coupling the effects of temperature and of hypoxia, we developed a proteomic approach that allowed us to identify several proteins (CK2, GLN, etc.) potentially involved in the response at the molecular level. Finally, in an effort to better understand the particular physiology of these mollusks in their natural environment, we compared the proteomic signatures of two populations of P. maximus living in highly contrasted ecosystems, ie in the northern limit- (Norway) and the center- (Brest) of the biogeographical distribution of this species. The results suggest major differences between the two populations, especially at the cytoskeleton level. In all, this work opens new avenues for understanding the molecular mechanisms governing the adaptation of mollusks to heat and hypoxia, two stresses that will most probably greatly influence the lifestyle of marine organisms and populations in future years.

Les analyses génomiques ont permis d’identifier des gènes et des protéinesimpliqués dans la maladie d’Alzheimer (MA). Au laboratoire, des approchesdifférentes ont mis en évidence deux gènes différentiellement exprimés dans lamaladie : le gène IL-33, retrouvé comme étant sous-exprimé chez les patientsatteints de MA, et le gène BIN1, identifié par des approches de GWAS et retrouvécomme étant sur-exprimés dans le cerveau des patients.Nous avons observé que la protéine IL-33 avait un impact sur le métabolismedu précurseur du peptide amyloïde (APP) de par sa fonction de facteur de régulationtranscriptionnelle. Nous avons alors cherché à identifier les gènes modulés par IL-33ainsi que des sites potentiels de fixation de la protéine sur l’ADN par des analyses àhaut débit. Il a été observé qu’IL-33 avait une forte implication dans la transcriptiondes gènes et pouvait agir directement sur l’ADN de par son impact sur les histones.De plus, IL-33 augmenterait l’expression de la préséniline 2, ce qui expliquerait alorsson impact sur le métabolisme de l’APP.Nous avons identifié un polymorphisme fonctionnel dans la région régulatricedu gène BIN1 associé à la maladie et pouvant expliquer la variation de sonexpression retrouvée dans le cerveau des patients. Nous avons également retrouvéune interaction de la protéine BIN1 avec Tau. BIN1 est alors le premier déterminantgénétique de la MA retrouvé comme étant associé à Tau et pourrait expliquer le lienentre la pathologie amyloïde et la pathologie Tau.Nos analyses nous ont donc permis, à partir des résultats d’analysesgénomiques, de mieux comprendre les mécanismes impliqués dans laphysiopathologie de la MA. / Genomic analyses allowed to identify genes and proteins involved inAlzheimer’s Disease (AD). In the laboratory, genetic and transcriptomic approachesrevealed two genes differentially expressed in AD: IL-33 gene, found to be underexpressedin AD cases brain, and BIN1 gene, found by GWAS analyses and overexpressedin brains of AD cases.As regards to IL-33, we observed that this protein have an impact on amyloidprecursor protein (APP) metabolism by its transcriptional regulation properties. So,we tried to identify the genes modulated by IL-33 using transcriptomic high-troughputanalyses and we identified IL-33 DNA binding sites by ChIP-on-chip approaches. Weobserved that IL-33 was involved in gene transcription and could act directly on DNAby interaction with histones. We also observed that IL-33 increase the expression ofpresenilin 2, which can explain its effect on APP metabolism.As regards to BIN1, we identified one functional polymorphism in regulatoryregion of this gene associated with AD and allowed to explain the expressionvariation of BIN1 in AD brains. We also found an interaction of BIN1 with Tau. So,BIN1 would be the first genetic risk factor for AD linked to the “Tau pathway” andcould explain the link between amyloid pathology and Tau pathology.The analyses performed in the laboratory allowed to, from genomic analysesresults, a better understanding of mechanisms involved in AD physiopathology.

Les imidazoquinoxalines (imiqualines), molécules bioactives originales analogues chimiques de l’imiquimod et présentant un important potentiel anticancéreux, ont été explorées afin d’élucider leurs mécanismes d'action. Les molécules EAPB0203 et EAPB0503, identifiées lors d’études antérieures comme étant les « têtes de séries », ont montré un effet cytotoxique puissant in vitro sur des lignées cellulaires cancéreuses humaines de mélanome et de lymphomes T. Nous avons étudié l’effet cytotoxique de 13 imiqualines nouvellement synthétisées sur une lignée cellulaire de mélanome humain (A375). Tous les composés ont montré un effet cytotoxique important. L’effet cytotoxique a été démontré sur d’autres lignées cellulaires cancéreuses humaines (côlon, sein et lymphome T de l’adulte).Un blocage du cycle cellulaire en phase G2/M a été mis en évidence par cytométrie en flux sur des cellules A375 traitées par EAPB0203 et EAPB0503. Cet arrêt du cycle cellulaire semble être en relation avec un effet inhibiteur de la polymérisation de la tubuline. En effet, nos résultats ont montré que l’EAPB0503 et 3 autres imiqualines inhibent la polymérisation de la tubuline. L’étude de modélisation moléculaire de la liaison à de la tubuline a montré que ces composés se fixent sur le site de la colchicine.Une analyse transcriptomique sur EAPB0503 a été effectuée pour élucider le mécanisme d'action des imiqualines. Cette étude a été faite sur la lignée A375 en comparaison avec 13 anticancéreux de référence. L’étude transcriptomique a montré que EAPB0503 a une composante antitubuline tout en révélant un mécanisme d’action original. Deux hypothèses mécanistiques pour EAPB0503 ont ainsi été identifiées : 1/ altération de la voie de signalisation liée aux intégrines, 2/ altération de la voie de signalisation du récepteur TNFR et de FasL.Des analyses fonctionnelles in vitro nous ont permis d’explorer ces hypothèses. Ainsi, une altération uniquement des voies de signalisation PI3K/AKT et RAS/MAPK, toutes deux liées aux intégrines, a été observée. La première hypothèse semble donc validée. De plus, ce résultat a été confirmé par l’étude de l’expression et de la phosphorylation de ERK.Finalement, nous avons développé une formulation injectable par voie intraveineuse de EAPB0503 à base de nanocapsules. Cette formulation a d’abord été testée in vitro et in vivo sur un modèle de lymphome. Ces études ont pu mettre en évidence l’absence de toxicité des nanoparticules vides et le maintien de l’activité cytotoxique de EAPB0503 encapsulé in vitro, avec un effet immunomodulateur in vivo qui demande à être exploré. / The imidazoquinoxalines (imiqualines), original bioactive molecules and chemical analogues of imiquimod and with significant anti-cancer potential, were explored in order to elucidate their mechanisms of action. The molecules EAPB0203 and EAPB0503 identified in previous studies as leader, showed potent in vitro cytotoxic effect on human cancer cell lines of melanoma and T lymphoma. We studied the cytotoxic effect 13 newly synthesized imiqualines on a cell line of human melanoma (A375). All compounds showed a significant cytotoxic effect. The cytotoxic effect was shown on other human cancer cell lines (colon, breast and adult T lymphoma).A cell cycle block in G2 / M phase was demonstrated by flow cytometry on A375 cells treated with EAPB0203 and EAPB0503. This cell cycle arrest seems to be related with an inhibitory effect on the polymerization of tubulin. Indeed, our results showed that EAPB0503 and three other imiqualines inhibit tubulin polymerization. The molecular modeling study of binding to tubulin showed that these compounds bind at the colchicine site.A transcriptomic analysis on EAPB0503 was performed to elucidate the mechanism of action of imiqualines. This study was made on the A375 line compared with 13 approuved anticancer molecules. This transcriptomic study showed that EAPB0503 has an antitubulin effect while revealing a novel mechanism of action. Two mechanistic hypotheses for EAPB0503 were identified: 1/ alteration of the signaling pathway linked to integrin, 2/ alteration of the signaling pathway TNFR receptor and FasL.In vitro functional analyses have allowed us to explore these hypotheses. Thus, alteration only PI3K / AKT and RAS / MAPK signaling pathways, both related to integrins, was observed. The first hypothesis seems confirmed. Moreover, this result was confirmed by the study of the expression and phosphorylation of ERK.Finally, we developed an intravenously injectable formulation of EAPB0503 based on nanocapsules. This formulation was first tested in vitro and in vivo on lymphoma model. These studies could highlight the lack of toxicity of empty nanoparticles and retention of the cytotoxic activity of encapsulated EAPB0503 in vitro, with an in vivo immunomodulatory effect which needs to be explored.