In vitro liver models that closely mimic the in vivo microenvironment are central for understanding hepatic functions and intercellular communication processes. Bottom-up shotgun proteomic analysis of the hepatic cells can lend insight into such processes. This technique employs liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of protein abundances by measuring intensities of their corresponding peptides. Organotypic 3D liver models have been developed in our laboratory that consist of hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM), which serves as a mimic for the Space of Disse. Each component within these models is easily separable allowing for systematic evaluation of the cells and PEMs. In this study, proteomes of hepatocytes from PEM containing models, cultured with and without LSECs, were compared to those from monolayers. Changes in core metabolism were evaluated among all culture conditions. Overall, all cultures were ketogenic and performed gluconeogenesis. The presence of the PEM led to increases in proteins associated with mitochondrial-based β-oxidation and peroxisomal proteins. The PEMs also limited production of structural proteins, which are linked to dedifferentiation of hepatocytes, suggesting that cell-ECM interactions are essential for maintenance of their liver-like state. The presence of LSECs increased levels of carboxylesterases and other phase I and phase II detoxification enzymes suggesting that intercellular signaling mediates enzyme abundance. Taken together, these results suggest that the cell-cell (from the LSECs) and cell-ECM (from the PEMs) interactions exert different, yet crucial effects, and both are required for the preservation of metabolic liver functions and differentiated phenotypes. Changes in the PEMs as a result of cell culture were also evaluated but exhibited minimal differences at this time point. Proteomes of LSECs monolayers were also characterized. Enzymes related to the metabolism of amino acids, lipids, oxidative phosphorylation and phase I and phase II detoxification processes were all identified in LSECs monolayers highlighting their role in these processes. Characterization of 3DHL LSECs was not possible due to ion suppression resulting from the presence of excess contaminant proteins. Nonetheless, this study provides a foundation in which LSECs from 3D liver models can be compared against in future studies. / Ph. D.

O crescimento, desenvolvimento e manutenção do tecido ósseo são processos altamente regulados. Diversas proteínas como hormônios, fatores de crescimento e citocinas estão envolvidas nestes processos e exercem atividade direta sobre células osteoblástica e osteoclástica, atuando em sua diferenciação e ativação metabólica. O processo de regeneração óssea é iniciado por fatores estimuladores locais como as proteínas morfogenética óssea (BMP Bone Morphogenetic Proteins). As BMPs são um produto do metabolismo dos osteoblastos, odontoblastos e de várias células tumorais, sendo armazenadas na forma de concentrados no osso, dentina e em células neoplásicas do osteossarcoma e de certos tumores odontogênicos, tais como: fibroma cementificante, cementoblastoma benigno, dentinoma, fibroma odontogênico e odontoma. Esclarecer os mecanismos que controlam a remodelação óssea é uma questão bastante relevante. Nesse sentido, as células-tronco mesenquimais têm despertado grande interesse devido ao seu potencial envolvimento no processo de reparo tissular. A obtenção de osteoblastos funcionais a partir de células-tronco mesenquimais tem sido utilizada na engenharia de tecidos e terapia celular. Desse modo, no presente trabalho foi realizada uma análise proteômica das proteínas envolvidas nas diversas fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea de rato Wistar e humana, no sentido de obter maiores informações sobre a diferenciação celular e a biologia do tecido ósseo. Células-tronco mesenquimais obtidas de medula óssea foram cultivadas em meio osteogênico por diferentes períodos para obter células em diversas fases da diferenciação osteoblástica. Para análise proteômica foram utilizadas ferramentas como a estratégia de shotgun proteomics e quantificação relativa (iTRAQ - Isobaric Tag for Relative and Absolute Quantitation) para separação de proteínas e a espectrometria de massas para a identificação e quantificação relativa de proteínas e peptídeos. Neste contexto, os nossos resultados nos levam a concluir que: as CTMs de medula óssea de rato Wistar expressam genes que estão envolvidos na diferenciação osteogênica quando estimuladas in vitro formando matriz óssea no período de 14 dias, ou seja, o fator estimulante no microambiente é de fundamental importância; as CTMs de medula óssea humana apresentaram resultados semelhantes com as CTMs de ratos em nível genômico durante a diferenciação osteogênica, entretanto quando estimuladas in vitro formaram a matriz óssea no período de 21 dias; utilizando duas abordagens proteômicas, foi possível identificar proteínas importantes que estão envolvidas no processo de diferenciação. Mas cabe salientar que, embora tenham sido detectados genes que parecem envolvidos no processo de diferenciação, isso não teve reflexo no proteoma dessas células nos períodos de 7 e 14 dias da indução de diferenciação à osteogênese, o que indica que a maior parte da funcionalidade dessas células quanto aos outros processos biológicos estão preservados, como por exemplo a proliferação celular permaneceu sem grandes alterações. Isso indica que manipulações de isolamento, cultivo e indução da diferenciação dessas células não afetaram o proteoma, com aspectos positivos para a utilização de células-tronco mesenquimais em terapia celular. Do ponto de vista metodológico, esse trabalho abre perspectivas da utilização de estratégias proteômicas baseadas na marcação por isóbaros em combinação com separação de proteínas por eletroforese unidimensional SDS-PAGE para a análise de amostras biologicamente complexas e de quantidades limitadas de obtenção como células-tronco mesenquimais. O estudo da expressão de proteínas durante as fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea deve refletir seu estado funcional e contribuir para o entendimento das diversas vias envolvidas no processo de diferenciação. / The growth, development and maintenance of bone tissue are highly regulated processes. Several proteins such as hormones, growth factors and cytokines are actively involved in these processes and exert direct activity on osteoblastic and osteoclastic cells, acting in their differentiation and metabolic activation. The process of bone regeneration is initiated by local stimulating factors as bone morphogenetic proteins (BMP). BMPs are a product of the metabolism of osteoblasts, odontoblasts and various tumor cells and is stored in the form of concentrates in bone, dentin and neoplastic cells of osteosarcoma and certain odontogenic tumors such as fibroma cementifying, cementoblastoma benign dentinoma, odontogenic fibroma and odontoma. Clarify the mechanisms that control bone remodeling is a very relevant issue. Accordingly, the mesenchymal stem cells have attracted great interest because of its potential involvement in the process of tissue repair. Obtaining functional osteoblasts from mesenchymal stem cells has been used in tissue engineering and cell therapy. Thus, this present work performed a proteomic analysis of proteins involved in various stages of osteoblast differentiation of mesenchymal stem cells from bone marrow of Wistar rat and human, in order to obtain more information on the biology of cell differentiation and bone tissue. Mesenchymal stem cells obtained from bone marrow were cultured in osteogenic medium for different periods to obtain cells at different stages of osteoblast differentiation. For proteomics analysis tools were used as the strategy of shotgun proteomics and relative quantification (iTRAQ - isobaric Tag for Relative and Absolute quantitation) for protein separation and mass spectrometry to identify proteins. In this context, our results take us to conclude that the MSCs of Wistar rat bone marrow express genes that are involved in osteogenic differentiation in vitro when stimulated to form bone matrix during the 14 days, ie stimulating factor in the microenvironment is of fundamental importance, the MSCs from human bone marrow showed similar results with rat MSCs at the genomic level during osteogenic differentiation, however, when stimulated in vitro formed bone matrix within 21 days, using two proteomic approaches, we could identify proteins important that are involved in the process of differentiation. But it should be noted that although it has been identified genes that seem involved in the process of differentiation, it was not reflected in the proteome of these cells at 7 and 14 days after induction of the osteogenic differentiation, indicating that most of the functionality of these cells and other biological processes are preserved, such as cell proliferation remained without major changes. This indicates that manipulations of isolation, culture and induction of differentiation of these cells did not affect the proteome, with positive aspects to the use of mesenchymal stem cells in cell therapy. From the methodological point of view, this work opens up the use of proteomic strategies based on the score for isobars in combination with protein separation by electrophoresis, one-dimensional SDS-PAGE for the analysis of complex biological samples and limited quantities of production as mesenchymal stem cells. The study of protein expression during stages of osteoblast differentiation of mesenchymal stem cells from bone marrow should reflect their functional status and contribute to the understanding of pathways involved in the process of differentiation.

O crescimento, desenvolvimento e manutenção do tecido ósseo são processos altamente regulados. Diversas proteínas como hormônios, fatores de crescimento e citocinas estão envolvidas nestes processos e exercem atividade direta sobre células osteoblástica e osteoclástica, atuando em sua diferenciação e ativação metabólica. O processo de regeneração óssea é iniciado por fatores estimuladores locais como as proteínas morfogenética óssea (BMP Bone Morphogenetic Proteins). As BMPs são um produto do metabolismo dos osteoblastos, odontoblastos e de várias células tumorais, sendo armazenadas na forma de concentrados no osso, dentina e em células neoplásicas do osteossarcoma e de certos tumores odontogênicos, tais como: fibroma cementificante, cementoblastoma benigno, dentinoma, fibroma odontogênico e odontoma. Esclarecer os mecanismos que controlam a remodelação óssea é uma questão bastante relevante. Nesse sentido, as células-tronco mesenquimais têm despertado grande interesse devido ao seu potencial envolvimento no processo de reparo tissular. A obtenção de osteoblastos funcionais a partir de células-tronco mesenquimais tem sido utilizada na engenharia de tecidos e terapia celular. Desse modo, no presente trabalho foi realizada uma análise proteômica das proteínas envolvidas nas diversas fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea de rato Wistar e humana, no sentido de obter maiores informações sobre a diferenciação celular e a biologia do tecido ósseo. Células-tronco mesenquimais obtidas de medula óssea foram cultivadas em meio osteogênico por diferentes períodos para obter células em diversas fases da diferenciação osteoblástica. Para análise proteômica foram utilizadas ferramentas como a estratégia de shotgun proteomics e quantificação relativa (iTRAQ - Isobaric Tag for Relative and Absolute Quantitation) para separação de proteínas e a espectrometria de massas para a identificação e quantificação relativa de proteínas e peptídeos. Neste contexto, os nossos resultados nos levam a concluir que: as CTMs de medula óssea de rato Wistar expressam genes que estão envolvidos na diferenciação osteogênica quando estimuladas in vitro formando matriz óssea no período de 14 dias, ou seja, o fator estimulante no microambiente é de fundamental importância; as CTMs de medula óssea humana apresentaram resultados semelhantes com as CTMs de ratos em nível genômico durante a diferenciação osteogênica, entretanto quando estimuladas in vitro formaram a matriz óssea no período de 21 dias; utilizando duas abordagens proteômicas, foi possível identificar proteínas importantes que estão envolvidas no processo de diferenciação. Mas cabe salientar que, embora tenham sido detectados genes que parecem envolvidos no processo de diferenciação, isso não teve reflexo no proteoma dessas células nos períodos de 7 e 14 dias da indução de diferenciação à osteogênese, o que indica que a maior parte da funcionalidade dessas células quanto aos outros processos biológicos estão preservados, como por exemplo a proliferação celular permaneceu sem grandes alterações. Isso indica que manipulações de isolamento, cultivo e indução da diferenciação dessas células não afetaram o proteoma, com aspectos positivos para a utilização de células-tronco mesenquimais em terapia celular. Do ponto de vista metodológico, esse trabalho abre perspectivas da utilização de estratégias proteômicas baseadas na marcação por isóbaros em combinação com separação de proteínas por eletroforese unidimensional SDS-PAGE para a análise de amostras biologicamente complexas e de quantidades limitadas de obtenção como células-tronco mesenquimais. O estudo da expressão de proteínas durante as fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea deve refletir seu estado funcional e contribuir para o entendimento das diversas vias envolvidas no processo de diferenciação. / The growth, development and maintenance of bone tissue are highly regulated processes. Several proteins such as hormones, growth factors and cytokines are actively involved in these processes and exert direct activity on osteoblastic and osteoclastic cells, acting in their differentiation and metabolic activation. The process of bone regeneration is initiated by local stimulating factors as bone morphogenetic proteins (BMP). BMPs are a product of the metabolism of osteoblasts, odontoblasts and various tumor cells and is stored in the form of concentrates in bone, dentin and neoplastic cells of osteosarcoma and certain odontogenic tumors such as fibroma cementifying, cementoblastoma benign dentinoma, odontogenic fibroma and odontoma. Clarify the mechanisms that control bone remodeling is a very relevant issue. Accordingly, the mesenchymal stem cells have attracted great interest because of its potential involvement in the process of tissue repair. Obtaining functional osteoblasts from mesenchymal stem cells has been used in tissue engineering and cell therapy. Thus, this present work performed a proteomic analysis of proteins involved in various stages of osteoblast differentiation of mesenchymal stem cells from bone marrow of Wistar rat and human, in order to obtain more information on the biology of cell differentiation and bone tissue. Mesenchymal stem cells obtained from bone marrow were cultured in osteogenic medium for different periods to obtain cells at different stages of osteoblast differentiation. For proteomics analysis tools were used as the strategy of shotgun proteomics and relative quantification (iTRAQ - isobaric Tag for Relative and Absolute quantitation) for protein separation and mass spectrometry to identify proteins. In this context, our results take us to conclude that the MSCs of Wistar rat bone marrow express genes that are involved in osteogenic differentiation in vitro when stimulated to form bone matrix during the 14 days, ie stimulating factor in the microenvironment is of fundamental importance, the MSCs from human bone marrow showed similar results with rat MSCs at the genomic level during osteogenic differentiation, however, when stimulated in vitro formed bone matrix within 21 days, using two proteomic approaches, we could identify proteins important that are involved in the process of differentiation. But it should be noted that although it has been identified genes that seem involved in the process of differentiation, it was not reflected in the proteome of these cells at 7 and 14 days after induction of the osteogenic differentiation, indicating that most of the functionality of these cells and other biological processes are preserved, such as cell proliferation remained without major changes. This indicates that manipulations of isolation, culture and induction of differentiation of these cells did not affect the proteome, with positive aspects to the use of mesenchymal stem cells in cell therapy. From the methodological point of view, this work opens up the use of proteomic strategies based on the score for isobars in combination with protein separation by electrophoresis, one-dimensional SDS-PAGE for the analysis of complex biological samples and limited quantities of production as mesenchymal stem cells. The study of protein expression during stages of osteoblast differentiation of mesenchymal stem cells from bone marrow should reflect their functional status and contribute to the understanding of pathways involved in the process of differentiation.

Submitted by DHARA CARLESSO ZAMPIVA (dhara.zampiva@univates.br) on 2018-08-20T17:17:13Z No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2018EdinaAparecidadosReisBlasi.pdf: 4920714 bytes, checksum: 936b932d7a6e49e5bca50678cab5e1d9 (MD5) 2018Edina_artigo.pdf: 1100636 bytes, checksum: a4919dc64d214daf7c76333e0b77f3a0 (MD5) / Rejected by Ana Paula Lisboa Monteiro (monteiro@univates.br), reason: Inserir o Lattes do autor. on 2018-09-11T18:28:07Z (GMT) / Submitted by DHARA CARLESSO ZAMPIVA (dhara.zampiva@univates.br) on 2018-09-17T17:18:08Z No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2018EdinaAparecidadosReisBlasi.pdf: 4920714 bytes, checksum: 936b932d7a6e49e5bca50678cab5e1d9 (MD5) 2018Edina_artigo.pdf: 1100636 bytes, checksum: a4919dc64d214daf7c76333e0b77f3a0 (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2018-10-03T16:31:01Z (GMT) No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2018EdinaAparecidadosReisBlasi.pdf: 4920714 bytes, checksum: 936b932d7a6e49e5bca50678cab5e1d9 (MD5) 2018Edina_artigo.pdf: 1100636 bytes, checksum: a4919dc64d214daf7c76333e0b77f3a0 (MD5) / Made available in DSpace on 2018-10-03T16:31:01Z (GMT). No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2018EdinaAparecidadosReisBlasi.pdf: 4920714 bytes, checksum: 936b932d7a6e49e5bca50678cab5e1d9 (MD5) 2018Edina_artigo.pdf: 1100636 bytes, checksum: a4919dc64d214daf7c76333e0b77f3a0 (MD5) Previous issue date: 2018-08-20 / High levels of Schizotetranychus oryzae phytophagous mite infestation on rice leaves can severely affect pro- ductivity. Physiological characterization showed that S. oryzae promotes a decrease in chlorophyll concentration and the establishment of a senescence process in rice leaves. Late-infested leaves also present high levels of superoxide radical and hydrogen peroxide accumulation, along with high levels of membrane integrity loss, which is indicative of cell death. To better understand the rice molecular responses to high levels of mite in-festation, we employed the Multidimensional Protein Identification Technology (MudPIT) approach to identify differentially expressed proteins. We identified 83 and 88 proteins uniquely present in control and late-infested leaves, respectively, along with 11 and one proteins more abundant in control and late-infested leaves, re-spectively. S. oryzae infestation induces a decreased abundance of proteins related to translation, protease in-hibition, and photosynthesis. On the other hand, infestation caused increased abundance of proteins involved in protein modification and degradation. Our results also suggest that S. oryzae infestation interferes with in-tracellular transport, DNA structure maintenance, and amino acid and lipid metabolism in rice leaves. Proteomic data were positively correlated with enzymatic assays and RT-qPCR analysis. Our findings describe the protein expression patterns of late-infested rice leaves and suggest several targets which could be tested in future bio-technological approaches aiming to avoid the population increase of phytophagous mite in rice plants.

The new field of ‘omics’ has spawned the development of metaproteomics, an approach that has the ability to identify and decipher the metabolic functions of a proteome derived from a microbial community that is largely uncultivable. With the development and availabilities of high throughput proteomics, high performance liquid chromatography coupled to mass spectrometry (MS) has been leading the field for metaproteomics. MS-based metaproteomics has been successful in its’ investigations of complex microbial communities from soils to the human body. Like the environment, the human body is host to a multitude of microorganisms that reside within the skin, oral cavity, vagina, and gastrointestinal tract, referred to as the human microbiome. The human microbiome is made up of trillions of bacteria that outnumber human genes by several orders of magnitude. These microbes are essential for human survival with a significant dependence on the microbes to encode and carryout metabolic functions that humans have not evolved on their own. Recently, metaproteomics has emerged as the primary technology to understand the metabolic functional signature of the human microbiome. Using a newly developed integrated approach that combines metagenomics and metaproteomics, we attempted to address the following questions: i) do humans share a core functional microbiome and ii) how do microbial communities change in response to disease. This resulted in a comprehensive identification and characterization of the metaproteome from two healthy human gut microbiomes. These analyses have resulted in an extended application to characterize how Crohn’s disease affects the functional signature of the microbiota. Contrary to measuring highly complex and representative gut metaproteomes is a less complex, controlled human-derived microbial community present in the gut of gnotobiotic mice. This human gut model system enhanced the capability to directly monitor fundamental interactions between two dominant phyla, Bacteroides and Firmicutes, in gut microbiomes colonized with two or more phylotypes. These analyses revealed membership abundance and functional differences between phylotypes when present in either a binary or 12-member consortia. This dissertation aims to characterize host microbial interactions and develop MS-based methods that can provide a better understanding of the human gut microbiota composition and function using both approaches.

nÃo hà / O presente estudo avaliou os efeitos da inclusÃo de 13% de farelo de castanha de caju (CNM) na dieta de carneiros Morada Nova nos parÃmetros seminais e proteÃnas do plasma seminal. Vinte carneiros foram distribuÃdos em dois grupos iguais: grupo castanha de caju (CNG) e grupo controle (COG). O CNG e COG receberam 13% e 0% de CNM na dieta durante 90 dias. Os grupos foram comparados para peso corporal, circunferÃncia escrotal, parÃmetros seminais e proteÃnas do plasma seminal, utilizando o MÃtodo de Shotgun Proteomics. Peso corporal e circunferÃncia escrotal aumentaram durante os 90 dias do perÃodo experimental em ambos grupos, porÃm nÃo existiu diferenÃa significativa entre CNG e COG. Contudo, apÃs 60 dias, o consumo de matÃria seca foi inferior no CNG (2,9  0,1; aos 90 dias) em relaÃÃo ao COG (3,4  0,1; aos 90 dias). Os parÃmetros seminais nÃo diferiram entre CNG e COG. A anÃlise shotgun proteomics identificou isoaspartil peptidase L asparaginase, gliceraldeÃdo 3 fosfato desidrogenase e prostaglandina H2 D isomerase com alta expressÃo no CNG quando comparado COG. Ao mesmo tempo, 12 proteÃnas do plasma seminal apresentaram baixa expressÃo no CNG, identificadas como beta galactosidase, caltrina, beta mannosidase, glutationa peroxidase, sorbitol desidrogenase, clusterina, albumina and serotransferrina. AlÃm disso, 20 proteÃnas detectadas no plasma seminal do COG foram ausentes no CNG e cinco proteÃnas foram presentes somente nos carneiros que receberam dieta hiperlipÃdica. Em conclusÃo, o presente estudo descreveu, pela primeira vez, os efeitos de uma dieta hiperlipÃdica nos parÃmetros do sÃmen fresco e do plasma seminal de carneiros. Enquanto os critÃrios seminais nÃo foram afetados, proteÃnas especÃficas do fluido seminal foram alteradas em decorrÃncia da dieta com castanha de caju. Visto os aspectos multifuncionais dessas proteÃnas, nÃs sugerimos que certos aspectos da fertilidades de carneiros podem ser alteradas se a castanha de caju for fornecida. / The present study was carried out to evaluate the effects of 13% of cashew nut meal (CNM) inclusion in the diet of Morada Nova rams on semen parameters and seminal plasma proteins. Twenty rams were distributed in two equal groups: cashew nut group (CNG) and control group (COG). The CNG and COG received 13% and 0% of CNM in the diet for 90 days. The groups were compared for live weight, scrotal circumference, seminal parameters and seminal plasma proteins, using shotgun proteomics. Body weight and scrotal circumference increased during the 90-day experimental period in both cashew nut-fed and control groups but with no differences between CNG and COG. However, after 60 days until 90 days, percentage of dry matter intake (% live weight) was inferior in CNG (2,9  0,1; at 90 days) group than COG (3,4  0,1; at 90 days). The sperm quality parameters were not different between CNG and COG.The shotgun proteomics analysis identified isoaspartyl peptidase L asparaginase, glyceraldehyde 3 phosphate dehydrogenase and prostaglandin H2 D isomerase with higher expression in the cashew nut group as compared to animals receiving the control diet. Conversely, 12 seminal plasma proteins had lower expression in the cashew nut group, identified as beta galactosidase, caltrin, beta mannosidase, glutathione peroxidase, sorbitol dehydrogenase, clusterin, albumin and serotransferrin. Moreover, 20 proteins detected in the seminal plasma of the control animals were absent in the cashew nut fed animals and five were present only in the rams receiving the high fat diet. In conclusion, the present study describes, for the first time, the effects of a high fat diet on parameters of the fresh semen and seminal plasma proteins of rams. While sperm criteria were not affected, specific seminal fluid proteins did change as the result of cashew nut feeding. Given the multifunctional aspects of such proteins, we suggest that certain aspects of the ram fertility can be altered if cashew nut is provided.

In shotgun proteomics, the liquid chromatography step is used to separate peptides in order to analyze as few as possible at the same time in the mass spectrometry step. Each peptide has a retention time, that is how long it takes to pass through the chromatography column. Prediction of the retention time can be used to gain increased identification of peptides or in order to create targeted proteomics experiments. Using machine learning methods such as support vector machines has given a high prediction accuracy, but such methods require known features that the retention time depends on. In this thesis we let a convolutional network, learn to rank the retention times instead of predicting the retention times themselves. We also tested how the prediction accuracy depends on the size of the training set. We found that pairwise ranking of peptides outperforms pointwise ranking and that adding more training data increased accuracy until the end without an increase in training time. This implies that accuracy can be further increased by training on even greater training sets.

No description available.

No description available.

La bactérie à Gram-négatif Pseudomonas aeruginosa est un pathogène humain opportuniste qui peut causer des infections chroniques pouvant conduire à la mort des patients, et plus particulièrement ceux atteints de la mucoviscidose. Il a été montré qu‘un de ses trois systèmes de sécrétion de type VI (SST6) est actif durant les infections chroniques, le SST6-H1. P. aeruginosa est capable d'injecter des toxines de type bactériolytique directement dans le périplasme des autres bactéries à Gram-négatif grâce au SST6-H1, ce qui laisse penser que cette nanomachine pourrait être capitale dans la compétitivité de P. aeruginosa dans les niches polymicrobiennes, comme par exemple un poumon infecté. Cette nanomachine insérée dans l'enveloppe bactérienne est régulée au niveau post-traductionnel par une voie de phosphorylation ressemblant à celles des eucaryotes. Cette voie est constituée par une kinase, PpkA, et une phosphatase, PppA, qui modulent ensemble le niveau de phosphorylation de la protéine Fha1. Nous avons démontré que quatre protéines spécifiques de Pseudomonas appelées TagT, TagS, TagR et TagQ, agissent en amont du couple PpkA/PppA, et sont indispensables pour l'activation du SST6-H1. De plus, elles sont aussi nécessaires lors de compétitions entre P. aeruginosa et d'autres bactéries. Nous avons montré que TagR, connue comme étant une protéine périplasmique, est en fait associée à la membrane externe et cette localisation dépend de TagQ, une lipoprotéine ancrée dans le feuillet interne de la membrane externe. TagT et TagS forment un transporteur de type ABC qui a une activité d'ATPase. L'association de TagR à la membrane externe a été mise en évidence par des études de protéomique à haut débit qui avaient pour but la caractérisation des membranes externe et interne de P. aeruginosa. Grâce à l'analyse des résultats, unmodèle de l'assemblage du SST6-H1 au sein de l'enveloppe a pu être proposé. Ce travail a permis l'identification de plus de 1700 protéines, parmi elles un complexe multi-protéique incluant MagD, une protéine homologue à la macroglobuline humaine. Les résultats obtenus lors de la caractérisation de ce complexe sont aussi présentés dans ce manuscrit. / Pseudomonas aeruginosa is a human opportunistic pathogen that can cause severe infections and death in chronically infected cystic fibrosis (CF) patients. It has been shown that one of its three Type VI Secretion Systems (T6SS), the H1-T6SS, is active during chronic infections in CF patients. P. aeruginosa injects bacteriolytic toxins directly into other Gram-negative bacteria by means of its H1-T6SS, which could be of high importance in its outcome in complex niches such as an infected lung. This trans-envelope nanomachine is posttranslationally regulated by a eukaryotic-like phosphorylation pathway, which includes a kinase-phosphatase pair, PpkA and PppA, respectively. In this work, TagT, TagS, TagR and TagQ, Pseudomonas specific T6SS proteins that are encoded in the same operon as Ppka, PppA and Fha1, were analysed functionally and biochemically. We found that these four proteins are indispensable for the activation of H1-T6SS, by acting upstream of the phosphorylation checkpoint. Moreover, they were also needed for intra- and inter-species fitness mediated by H1-T6SS. We discovered that TagR, a periplasmic protein, associates with the outer membrane (OM) of P. aeruginosa in a TagQ-dependent manner. TagQ is an OM lipoprotein that faces the periplasm. TagT and TagS form a membrane-bound complex, an ABC transporter, with ATPase activity. TagR association with the OM was discovered by shotgun mass spectrometry analyses of the OM and the inner membrane (IM) of P. aeruginosa. In this work, the IM and OM sub-proteomes of P. aeruginosa are also presented, with highlights on T6SS global assembly. Moreover, these two sub-proteomes allowed the identification of a novel envelope-associated complex with macroglobulin-like protein, MagD. The studies concerning this protein and its partners in P. aeruginosa are also presented in this manuscript.