Orientador: Célia Regina Nogueira / Resumo: O sistema esquelético é um sistema complexo com intenso metabolismo composto de células, proteínas e minerais. Os osteoblastos, são células fundamentais para o tecido, desempenhando duas funções principais: formação óssea e regulação da reabsorção por meio da modulação da osteoclastogênese. Sendo assim, essas células desempenham funções primordiais para o desenvolvimento e manutenção óssea. Diversas moléculas sistêmicas atuam no tecido, sendo os hormônios tireoidianos um destes. Eles são fundamentais para o metabolismo ósseo já que alterações hormonais culminam em desordens ósseas. Osteoblastos possuem receptores nucleares para T3 e apesar de pouco compreendido, ele afeta diversos aspectos do desenvolvimento da célula, assim como vias modulatórias da remodelação óssea. Diversas linhagens celulares têm sido utilizadas para estudos de osteoblastos, sendo as células-tronco mesenquimais derivadas do tecido adiposo humano (hA-CTMs) um modelo promissor para osteoindução. Desta forma, o objetivo deste trabalho foi verificar a influência do T3 suprafisiológico (T3S) na expressão gênica diferencial em osteoblastos diferenciados a partir de hA-CTMs. As células obtidas de 3 doadores foram submetidas a osteoindução por 16 dias com coquetel de diferenciação (dexametasona, ácido ascórbico e β-glicerofosfato) e caracterizadas pela presença de osteocalcina, fosfatase alcalina e matriz mineralizada. O tratamento com T3 (10-8M) foi realizado por 72h e o RNA foi extraído para preparação das bib... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The skeletal system is complex and have an intense metabolism compound with cells, proteins and minerals. The osteoblasts are fundamental cells to the tissue, executing two mainly functions: bone formation and regulation of reabsorption through osteoclastogenesis modulation. Therefore, these cells perform primordial functions to the bone development and maintenance. Several systemic molecules act in the tissue and the thyroid hormones are one of them. They are fundamental to the bone metabolism, once hormone alterations result in bone disorders. Osteoblasts have T3 nuclear receptors, and although not well elucidated it affects many aspects of the cell development so as pathways that modulates the bone remodeling. Several cells lineages have been used in osteoblasts studies, and human adipose-derived Mesenchymal stem-cells (hA-MSCs) are a promising model to osteoinduction. Thus, the aim of this study was to investigate the supraphysiological T3 (T3S) influence in the gene differential expression in osteoblasts differentiated from hA-MSCs. The cells obtained from three donators were submitted to osteoinduction for 16 days with a differentiation cocktail (dexamethasone, ascorbic acid, β-glycerophosphate) and characterized by the presence of osteocalcin, alkaline phosphate and mineralized matrix. The cells were treated with T3 (10-8M) for 72h and the RNA was extracted to mRNA library prepare and sequenced by Illumina platform. The bioinformatic analysis included the software: Fas... (Complete abstract click electronic access below) / Mestre

Bacterial fruit blotch (BFB) of cucurbits is caused by Acidovorax citrulli, a Gram-negative seedborne bacterium that can cause up to 100% fruit yield losses in the field. Currently, BFB is a major problem for the cucurbits industry worldwide. Thus far, attempts to identify resistance in cucurbit germplasm for controlling BFB have been unsuccessful. Despite the importance of the disease, little is known about the molecular mechanisms of A. citrulli pathogenicity, due to a lack of molecular tools for studying the A. citrulli/cucurbit interaction. The genomic sequence of A. citrulli strain AAC00-1 has been determined, and the components of type III secretion system have been identified. The goal of this research was to develop molecular tools for studying the BFB disease. Nineteen putative type III effector genes were cloned from two representative A. citrulli strains (AAC00-1 and M6). The distribution of 19 type III effectors among A. citrulli strains, collected worldwide, was studied. A novel Gateway-compatible binary vector was developed for transient expression of A. citrulli type III effectors genes in planta. A set of modified vectors for marker-exchange mutagenesis in A. citrulli were constructed. The model plant species Nicotiana benthamiana was found to be susceptible to A. citrulli, while Nicotiana tabacum was resistance to A. citrulli, so therefore could carry nonhost resistance genes. Two T3S effectors, Aave1548 and Aave2166, triggered water soaking-like cell death in N. benthamiana, but HR-like cell death in N. tabacum. Bacterial mutagenesis and in planta disease assay confirmed that both Aave1548 and Aave2166 have significant virulence contributions to A. citrulli in N. benthamiana plant and melon seeds. Aave2166 encodes a putative acetyltransferase that belongs to the YopJ super family, which is conserved in both animal and plant pathogenic bacteria. Wild type but not the putative catalytic mutant (C232A) of Aave2166 can trigger cell death phenotype in N. benthamiana and N. tabacum. N. benthamiana yeast two-hybrid cDNA library screening using Aave2166 identified six N. benthamiana proteins/peptides which specifically interacted with Aave2166. Further characterization of these Aave2166 interactors may allow us to understand the virulence mechanism provided by Aave2166. The identification of nonhost resistance genes that can recognize Aave2166 and other type III effectors may help to develop novel strategies to control BFB disease of cucurbit. / Ph. D.

Chlamydia trachomatis est une bactérie intracellulaire obligatoire. Ce pathogène de l’Homme est la première cause infectieuse de cécité ainsi que de maladies sexuellement transmissible d’origine bacterienne.Utilisant une souche de C. trachomatis L2 exprimant une protéine fluorescente, nous avons développé des méthodes de microscopie et de cytométrie en flux permettant de suivre les différentes étapes du développement de la bactérie. Ces méthodes faciliteront les futures études de l’infection par Chlamydia.Chlamydia interagit avec différents processus cellulaires, et plus particulièrement via la sécrétion d’effecteurs par le système de sécrétion de type 3 (ST3). Nous avons identifié une famille de protéines possédant un signal de ST3 qui partagent un domaine, le DUF582, présent uniquement chez les Chlamydia pathogènes.Nous avons montré que les 5 protéines DUF582 de C. trachomatis sont exprimées à partir du milieu du cycle infectieux. Nous avons démontré que la protéine Hrs interagit avec le DUF582 et que la protéine DUF582 CT619 interagit avec Tsg101. Hrs et Tsg101 sont d’importants composants de la machinerie ESCRT impliquée dans de nombreux processus de fission membranaire.Utilisant l’interférence ARN, nous avons montré que Hrs et Tsg101 ne sont requis ni pour l’entrée, ni pour le développement de la bactérie. Ceci suggère que les protéines DUF582 bloquent des processus dépendant de Hrs/Tsg101. A l’inverse, la bactérie pourrait utiliser la machinerie ESCRT mais l’existence de mécanismes redondants expliquerait l’absence de phénotype dans les expériences d’interférence. Nous discutons trois hypothèses concernant le rôle des protéines DUF582 dans l’infection. / Chlamydia trachomatis is an obligate intracellular human pathogen. It is the first infectious cause of blindness and the most common cause of sexually transmitted diseases of bacterial origin. Using a strain of C. trachomatis serovar L2 expressing a fluorescent protein we developed microscopy and flow cytometry based methods to quantify several steps of its developmental cycle. These methods will facilitate future studies aimed at testing anti-bacterial compounds or various culture conditions. Chlamydiae interfere with many cellular processes, in particular via the secretion of bacterial proteins through a type 3 secretion (T3S) system. We identified a family of proteins that possess T3S signals. They share a domain designated as DUF582, which is only found in pathogenic chlamydiae. We showed that the five DUF582 proteins of C. trachomatis are expressed from the mid phase of infection. We demonstrated that the protein Hrs is a common interactor for the DUF582. In addition the N-terminal part of the DUF582 protein CT619 interacts with Tsg101. Hrs and Tsg101 are both important components of the ESCRT machinery, which is an ancient machinery required for several processes involving membrane fission.Using RNA interference we showed that Hrs and Tsg101 are dispensable for bacterial entry and growth. This last result suggest that DUF582 proteins actually prevent Hrs and/or Tsg101 driven processes. Alternatively, the bacteria might highjack the ESCRT machinery but redundant mechanisms would explain the absence of phenotype on bacterial development observed in the silencing experiments. We discuss three hypotheses as to the possible role of the DUF582 proteins in infection.