O fungo filamentoso Trichoderma reesei é um dos principais fungos utilizados para a produção em larga escala de enzimas devido a sua grande capacidade de produção e secreção de holocelulases para aplicação em processos de sacarificação da biomassa vegetal lignocelulósica. Embora o T. reesei seja utilizado como um dos principais produtores de celulases a nível industrial, diversos processos e estudos são realizados com o objetivo de aprimorar o entendimento de todo o mecanismo de degradação de biomassa vegetal além de prover o aumento da eficiência tanto da produção quanto da atividade das celulases. No presente trabalho foi realizado a construção de uma linhagem mutante para o gene cel74a (codificando para uma xiloglucanase) e a caracterização funcional de CEL74A na regulação gênica de holocelulases durante o cultivo em bagaço de cana-de-açúcar. Os nossos resultados mostraram que deleção de cel74a possivelmente pode estar envolvida no sinergismo da regulação da expressão de holocelulases durante o cultivo em bagaço de cana-de-açúcar. A partir da análise do perfil de expressão gênica, foi possível observar a redução na expressão de todos os genes de celulases testados (cel7a, cel7b e cel6a) embora não tenha afetado a atividade enzimática, ao passo que as hemicelulases (xyn1 e xyn2) apresentaram aumento tanto na expressão quanto na atividade enzimática. Na linhagem ?cel74a, foi observado redução na liberação de glicose, xilose e galactose após a hidrólise de xiloglucano. Além disso, a atividade de CEL74A foi modulada na presença de cálcio e pode ser necessária para a atuação mais eficiente de outras enzimas envolvidas na degradação de xiloglucano. Desta forma, em T. reesei, CEL74A apresenta um papel importante tanto na regulação de genes holocelulolíticos quanto para a degradação eficiente de bagaço de cana-de-açúcar, contribuindo para a elucidação de mecanismos pelos quais este fungo utiliza para a utilização do bagaço de cana-de-açúcar como fonte de carbono / The filamentous fungus Trichoderma reesei is one of the main fungi used for the large-scale production of enzymes due to their great capacity of production and secretion of holocellulases for application in saccharification processes of lignocellulosic plant biomass. Although T. reesei is used as one of the main producers of cellulases at industrial level, several processes and studies are carried out with the aim of improving the understanding of the whole plant biomass degradation mechanism, as well as increasing the efficiency of both the production and cellulase activity. In the present work the construction of a mutant lineage for the cel74a gene (coding for a xyloglucanase) and the functional characterization of CEL74A in the gene regulation of holocellulases during the cultivation of sugarcane bagasse were carried out. Our results showed that deletion of cel74a may be involved in the regulation of holocellulase expression during sugarcane bagasse cultivation. From the analysis of the gene expression profile, it was possible to observe the reduction in the expression of all tested cellulase genes (cel7a, cel7b and cel6a), although it did not affect the enzymatic activity, whereas the hemicellulases (xyn1 and xyn2) presented increase in both expression and enzymatic activity. In the ?cel74a strain, a reduction in glucose, xylose and galactose release was observed after xyloglucan hydrolysis. In addition, the activity of CEL74A was modulated in the presence of calcium and may be required for the more efficient performance of other enzymes involved in the degradation of xyloglucan. Thus, in T. reesei, CEL74A plays an important role both in the regulation of holocellulolytic genes and in the efficient degradation of sugarcane bagasse, contributing to the elucidation of mechanisms by which this fungus uses for the use of sugarcane bagasse as source of carbon

Em estudos de proteção ao genoma contendo lesões de C. crescentus, foi feita uma varredura em uma biblioteca clones mutados pelo transposon Tn5 e sensíveis à luz UV-B e MMS. Dos 249 clones selecionados conseguimos identificar mutações em 102 genes, distribuídos em dez categorias funcionais, incluindo metabolismo de DNA. Além de genes já descritos como atuantes na defesa de genoma a lesões, os dados ainda adicionam funções potenciais para outros genes. Investigação mais detalhada indica que vários dos genes identificados podem afetar o estresse e ciclo celular, podendo estar relacionados a respostas do tipo pontos de checagem. Além disso, verificamos que mutantes para genes envolvidos em reparo excisão de nucleotídeos são mais sensíveis à H2O2, indicando que nessa bactéria, este sistema de reparo atua também em lesões oxidativas no DNA. Os dados obtidos são pioneiros no estabelecimento de funções para vários genes de C. crescentus e de outras alfa-proteobactérias. / This work aimed to identify genes related to DNA damage protection in C. crescentus, by means of screening a Tn5 -mutated library for clones sensitive to UV-B light and MMS. Out of 249 selected clones, we were able to identify mutations in 102 genes, classified in ten different functional categories, including DNA metabolism. In addition to genes already described as playing a role in genome defense to lesions, the results provide new potential functions to several other genes. More detailed investigation indicates also that the mutations in some of these genes may also affect cell stress and cell cycle, being potentially involved in check-point responses. Moreover, mutants for nucleotide excision repair genes were also found to be sensitive to H2O2, indicating that this DNA repair system also acts in DNA oxidative damage. These data are the first establishing a role in genome protection for several genes in C. crescentus, as well as other alpha-proteobacteria.

Le Lupus Erythémateux Systémique (LES) est une maladie autoimmune sévère dont laquelle les lymphocytes B (LB) jouent un rôle central. Afin de rechercher des anomalies génétiques propre à ces cellules, notre laboratoire a étudié l’expression des gènes dans les LB de patients atteints d’un LES, par rapport à des sujets témoins. Ce projet a pour but d’étudier un de ces gènes (FKBP11), surexprimé dans les LB au cours du LES. Pour cela nous avons créé, à l’aide de lentivirus, des souris transgéniques reproduisant de manière ubiquitaire la surexpression de ce gène. Ces souris développent une hyperplasie des organes lymphoïdes, une hyper gamma globulinémie de type IgG3, et présentent une réponse humorale augmentée après immunisation avec un antigène T-indépendant. Notre étude met en évidence que la surexpression de FKBP11 favorise le développement des plasmocytes dans leur phase initiale dépendante de l’expression de Pax5, après induction de la différenciation plasmocytaire in vitro. Enfin, les souris développent des autoanticorps variés. De plus, le rôle d’une surexpression de FKBP11 dans la rupture de tolérance B a été montré chez des souris transgéniques anti-ADN 56R, croisées avec notre modèle lentigénique, qui voient leur production d’autoanticorps augmentée. La surexpression de FKBP11 dans le modèle C57BL/6lpr/lpr accentue le caractère lymphoprolifératif et l’hyper gamma globulinémie présents dans ces souris. Au cours de ce projet de thèse, j’ai également initié l’étude de deux autres gènes surexprimés dans les LB de patients lupiques (PRDX4, TRIB1), par le développement de modèles transgéniques murins. / Systemic Lupus Erythematosus (SLE) is a severe autoimmune disease where B cells play a central role and carry intrinsic genetic abnormalities.Today, only a few SLE genes have been validated in humans. Looking for these intrinsic B cell abnormalities in SLE, we have performed a microarray analysis of the human transcriptoma in B cells from quiescent SLE patients, in comparison to normal subjects. The project proposes to explore the consequences of overexpression of one of these genes: FKBP11. For this purpose, a lentiviral construct allowing the ubiquitous overexpression of FKBP11 was produced, and has been used to generate lentigenic mice. These mice develop a hyperplasia of lymphoid organs, an IgG3 hypergammaglobulinemia and an increase of humoral immune response after immunisation with a T-independent antigen. Our study points out that the overexpression of FKBP11 promotes the plasmocyte development, at the initial stage which is dependent on Pax5 expression, after in vitro induction of the plasmacytic differentiation. Finally, these mice produce various autoantibodies. The role of FKBP11 overexpression in B cell central tolerance breakdown has been demonstrated in anti-DNA 56R transgenic mice crossed with our lentigenic model. These mice have an increase of autoantibody production. The FKBP11 overexpression in C57BL/6lpr/lpr model increases the lymphoproliferative syndrome and the hypergammaglobulinemia in this model. During this thesis, I have also initiated the study of two others genes which were found overexpressed in B cells of lupus patients (PRDX4, TRIB1), by the development of transgenic murine models.

L’objectif de ce travail était de mieux comprendre les mécanismes moléculaires de l’adaptation microbienne à l’environnement fromager par des approches de génomique fonctionnelle via le modèle de Brevibacterium, un genre bactérien largement utilisé en technologie fromagère, mais dont l’implantation est parfois difficile à maîtriser.L’analyse génomique comparative de 23 souches de Brevibacterium, dont 12 issues de fromages, a révélé des différences en déterminants génétiques impliqués dans la capacité à croître à la surface du fromage. Parmi ces différences, plusieurs sont corrélées à la phylogénie des souches, et d’autres résultent de transferts horizontaux, notamment dans le cas des gènes liés à l’acquisition du fer et à la biosynthèse de bactériocines. Nous avons identifié des îlots génomiques correspondant à des transferts de gènes d’acquisition du fer entre des souches fromagères de Brevibacterium et des bactéries d’affinage appartenant à d’autres genres. Nous avons également mis en évidence un transposon conjugatif codant pour la synthèse de bactériocines présent chez des souches de Brevibacterium d'origine fromagère mais aussi chez une souche fromagère du genre Corynebacterium.L’étude fonctionnelle des interactions biotiques entre Brevibacterium et Hafnia alvei, une autre bactérie d’affinage du fromage, a été menée dans un modèle fromager développé au cours de ce travail. En couplant des analyses microbiologiques, biochimiques et transcriptomiques (RNA-seq), nous avons mis en évidence l’existence de différents mécanismes d’interaction entre ces bactéries. Ceux-ci concernent notamment l’acquisition du fer, la protéolyse, la lipolyse, le métabolisme soufré et le catabolisme du D-galactonate. Nos résultats suggèrent que dans la relation mutualiste observée entre certaines souches de Brevibacterium et H. alvei, cette dernière sécrète des sidérophores qui sont utilisés par Brevibacterium pour capter le fer plus efficacement, stimulant ainsi sa croissance. En contrepartie, Brevibacterium sécrète des lipases et des protéases qui dégradent les caséines et triglycérides du fromage en constituants énergétiques favorisant la croissance de H. alvei. Ce type d’interaction est intéressant à considérer pour la formulation des ferments d'affinage car il en résulte une meilleure capacité de tous les partenaires à coloniser le fromage, et ainsi à générer les propriétés technologiques recherchées. / The objective of this study was to better understand the molecular mechanisms of microbial adaptation to the cheese habitat by functional genomic approaches using Brevibacterium as a model microorganism. This bacterium is widely used for the manufacturing of cheese but its growth on the cheese surface is sometimes difficult to control.Comparative genomic analysis of 23 Brevibacterium strains, including 12 strains isolated from cheeses, revealed differences in genetic determinants involved in the growth on the cheese surface. Some of them are correlated to strain phylogeny and others are the result of gene transfers, especially those involved in iron acquisition and bacteriocin biosynthesis. We identified genomic islands corresponding to transfers of genes involved in iron acquisition between cheese-associated Brevibacterium strains and cheese-associated strains belonging to other genera. We also detected a conjugative transposon encoding bacteriocin production, which is present in cheese-associated Brevibacterium strains as well as in a cheese-associated Corynebacterium strain.Functional study of biotic interactions between Brevibacterium and Hafnia alvei, another cheese-ripening bacterium, was performed in a model cheese developed in this study. By coupling microbial, biochemical and transcriptomic (RNA-seq) analyses, we revealed several interaction mechanisms between these bacteria. These concern, in particular, iron acquisition, proteolysis, lipolysis, sulfur metabolism and D-galactonate catabolism. Our findings suggest that in the mutualistic relationship between some Brevibacterium strains and H. alvei, the latter stimulates Brevibacterium growth by the secretion of siderophores, which can be used by Brevibacterium to capture iron more efficiently. In return, Brevibacterium secretes lipases and proteases, which degrade cheese caseins and triglycerides into energetic substrates that stimulate H. alvei growth. This type of interaction is interesting to consider in the formulation of ripening cultures because it results in a better ability of all partners to colonize the cheese, and thus to generate the desired technological properties.