No description available.

Submitted by Fabiana Gonçalves Pinto (benf@ndc.uff.br) on 2016-10-24T17:42:22Z No. of bitstreams: 1 Fernanda Soares Pessanha.pdf: 2199380 bytes, checksum: bb078e15b653af8e5dfd1387ea2db7e4 (MD5) / Made available in DSpace on 2016-10-24T17:42:22Z (GMT). No. of bitstreams: 1 Fernanda Soares Pessanha.pdf: 2199380 bytes, checksum: bb078e15b653af8e5dfd1387ea2db7e4 (MD5) Previous issue date: 2015 / Mestrado Acadêmico em Ciências do Cuidado em Saúde / Introdução: Para a correta reparação das feridas, as diversas fases do processo de cicatrização devem ocorrer na sequencia correta, numa intensidade ideal. Vários fatores afetam a cicatrização das feridas ao interferir em uma ou mais fases deste processo, tal como, a presença de infecção. Objetivo geral: Analisar as cepas de Pseudomonas aeruginosa encontradas nas feridas crônicas tratadas com gel de carboximetilcelulose a 2% ou com placa de poliuretano. Método: Pesquisa observacional descritiva, com abordagem quantitativa, realizada através da coleta de material biológico de feridas crônicas de pacientes atendidos em serviços ambulatoriais, empregando swabs. A pesquisa foi aprovada pelo Comitê de Ética em Pesquisa (Hospital Universitário Antônio Pedro – UFF) com número de parecer 815.353. As cepas de P. aeruginosa foram identificadas por MALDI-TOF MS, submetidas a testes de susceptibilidade aos antimicrobianos, identificação de genes de virulência através de PCR e tipagem molecular através de PFGE. Resultados: Das 43 feridas a partir das quais foram coletados swabs, em 31 (72,09%) obteve-se isolamento de P. aeruginosa (foram identificadas 48 cepas). Estas feridas têm 3,6 vezes mais chances de desenvolverem infecção quando comparadas àquelas a partir das quais esse microrganismo não foi isolado. As cepas isoladas dos pacientes em uso de gel de carboximetilcelulose a 2% apresentaram maiores taxas de resistência a gentamicina e ciprofloxacino (ambos com 7,89%). Já as cepas isoladas dos pacientes tratados com placa de poliuretano, destacaram-se pela resistência a ciprofloxacino (90%). Foram identificadas três cepas multirresistentes de duas feridas tratadas com placa de poliuretano impregnada com prata. Foram positivas para presença do gene exoS 26 cepas (54,16%), e 13 (27,08%), para o gene exoU. Observou-se mesmo perfil de PFGE entre as cepas coletadas em diferentes momentos de onze pacientes, enquanto que em seis pacientes as cepas coletadas em diferentes momentos foram distintas. Não houve semelhança de padrões de fragmentação de DNA entre cepas derivadas de pacientes diferentes. Conclusão: A maioria das feridas não apresentava sinais clínicos de infecção. Foram identificadas 48 cepas de P. aeruginosa. O isolamento deste microrganismo é fator de risco para desenvolvimento de infecção. As cepas de P. aeruginosa têm baixos índices de resistência antimicrobiana, com apenas três cepas multiresistentes. Os desbridamentos realizados nas feridas crônicas não têm sido efetivos para descolonização de P. aeruginosa, já que um mesmo clone bacteriano foi identificado na ferida em diferentes momentos, na maioria dos casos / Introduction: For proper wound healing, various stages of the healing process must occur in a correct sequence and an ideal intensity. Several factors affect the wounf healing on one or more phases of this process, such as the presence of infection. General objective: To analyze Pseudomonas aeruginosa strains found in chronic wounds treated with 2% carboxymethylcellulose gel or polyurethane plate. Method: Descriptive observational research with a quantitative approach, carried out through the collection of biological material of chronic wounds of patients attended in outpatient services, using swabs. The study was approved by the Research Ethics Committee (Academic Hospital Antonio Pedro - UFF) with number 815.353. P. aeruginosa strains were identified by MALDI-TOF MS, subjected to antimicrobial susceptibility testing, identification of virulence genes by PCR and molecular typing by PFGE. Results: Of the 43 wounds from which swabs were collected, at 31 (72.09%) was obtained isolation of P. aeruginosa (48 strains have been identified). These wounds are 3.6 times more likely to develop infection when compared to those from which this microorganism was not identified. The strains isolated from patients using 2% carboxymethyl cellulose gel showed more resistance rates to gentamicin and ciprofloxacin (both 7.89%). Already the strains isolated from patients treated with polyurethane plate, highlighted by the resistance to ciprofloxacin (90%). Three multiresistant strains were identified from two wounds treated with polyurethane plate impregnated with silver. 26 strains (54.16%) were positive for the presence of exoS gene and 13 (27.08%), for the exoU gene. It was observed even PFGE profile among strains collected at different times of eleven patients, while in six patients, strains collected at different times were different. There was no resemblance DNA fragmentation patterns among strains derived from different patients. Conclusion: Most of the wounds showed no clinical signs of infection. 48 strains of P. aeruginosa have been identified. The isolation of this microorganism is a risk factor for development of infection in chronic wounds. Strains of P. aeruginosa demonstrated low antimicrobial resistance rates and only three multi-resistant strains were identified. The debridement performed in chronic wounds is not effective for removing colonization by P. aeruginosa, because same bacterials clones was identified in the wound swabs collected in same patients at different times in most cases

Whole cell biosensors have been extensively used for monitoring toxicity and contamination of compounds in environmental biology and microbial ecology. However, their application in the pharmaceutical and cosmetics industries for preservative efficacy testing (PET) has been limited. According to several pharmacopoeias, preservatives should be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. The aim of the study was to construct and develop whole cell microbial biosensors with Pseudomonas aeruginosa ATCC 9027. Constitutive promoters: PlysS, Pspc, Ptat, Plpp and PldcC and the lux-cassette were inserted into plasmid pME4510 and transformed into P. aeruginosa ATCC 9027 cells to produce bioluminescent strains. Plasmids were found to be maintained stably (~50 copies per cell) throughout the growth and death cycle. The novel bioluminescent strains were validated in accordance with the pharmacopoeia using bioluminescence detection and quantification followed by comparison with the traditional plate counting method. The bioluminescent method was found to be accurate, precise and equivalent at a range of 103 – 107 CFU/mL, as compared with plate counting. Recovery of bacterial cells was quantified using bioluminescence; this method proved to be accurate with percentage recoveries between 70-130% for all bioluminescent strains. The method was also more precise (relative standard deviation less than 15%) than the traditional plate counting method or the ATP bioluminescent method. Therefore, the bioluminescent constructs passed/exceeded pharmacopoeial specified criteria for range, limit of detection, accuracy, precision and equivalence. Physiology of the validated bioluminescent strains was studied by assessing the growth and death patterns using constitutive gene expression linked with bacterial replication. Promoter strengths were evaluated at various stages of the growth and death pattern and related to promoter sequences. PlysS, Ptat and Plpp were relatively strong promoters whilst PldcC and Pspc were relatively weak promoters. Relative promoter strength decreased in the order of Plpp>Ptat>PlysS>PldcC>Pspc during the exponential phase whilst Ptat was stronger than Plpp during the stationary phase of growth. Plpp had its highest level of expression during the exponential phase, while Ptat had relatively stable lux expression during the stationary phase. Correlations between relative bioluminescence and CFU at 24 hours were greater than 0.9 indicating a strong relationship for all bioluminescent strains. Reduction in correlation coefficients to approximately 0.6 between relative bioluminescence and CFU and between relative fluorescence and CFU beyond 24 hours indicated that a certain proportion of cells were viable but non-culturable. Tat-pME-lux showed steady bioluminescence compared to CFU count (R>0.9) throughout 28 days of growth. Equivalence analysis showed no significant difference between the bioluminescence and plate count method throughout 28 days of growth for all five bioluminescent strains. Applicability of these novel bioluminescent strains was evaluated for preservative efficacy tests (PET) using bacterial replication and bioluminescence as a measure of constitutive gene expression. PET using benzalkonium chloride and benzyl alcohol showed no significant difference between the bioluminescent method and the plate count method. Good correlations between bioluminescence, CFU count and fluorescence were obtained for benzalkonium chloride (BKC) concentrations (R>0.9) between 0.0003% and 0.0025% against strains lysR25, lppR4 and tatH5. Similarly, good correlations (R>0.9) between the three parameters were obtained for benzyl alcohol (BA) concentrations between 0.125% and 2% against strains lysR25, lppR4 and tatH5. The bioluminescent method and traditional plate counting method were equivalent for concentrations of BKC (0.0003 - 0.02%) and BA (0.25 - 2%) during preservative efficacy tests. These bioluminescent constructs therefore are good candidates for selection for preservative efficacy testing. The bioluminescent method and traditional plate counting method were also found to be equivalent for construct tatH5 at a concentration of 0.125% BA. PET testing with BKC and BA showed that tatH5-pMElux (R>0.9) had consistently high correlation coefficients between CFU and relative bioluminescence. Together with the results from growth and death kinetics, where tatH5 showed the greatest constitutive expression, it can be concluded that P. aeruginosa ATCC 9027 tatH5-pMElux is the best construct for testing various antimicrobial agents. This study has shown that according to the pharmacopoeial requirements, the bioluminescent method is more accurate, precise and equivalent to the traditional plate counting method and therefore can be utilised instead of the traditional plate counting method for the purpose of preservative efficacy testing.

La Fibrose kystique (FK), causée par des mutations du canal Cl- CFTR, entraîne une dysfonction de la sécrétion de Cl- et un débalancement dans la sécrétion des fluides. La diminution de la clairance mucociliaire qui s’en suit occasionne une accumulation du mucus. Cet environnement est alors favorable à l’installation d’infections et d’inflammation chroniques, responsables de lésions au niveau de l’épithélium respiratoire. Le vieillissement de la population FK, suite à la prise en charge plus appropriée de la maladie, est accompagné par l’émergence de pathologies associées, telles que le diabète. Celui-ci, ainsi que plusieurs autres facteurs comme l’infection à Pseudomonas aeruginosa, contribuent au déclin progressif de la fonction pulmonaire, principale cause de mortalité et de morbidité des patients FK. Le maintien de la fonction pulmonaire est dépendant notamment du transport ionique et liquidien régulant la clairance mucociliaire ainsi que de la réparation épithéliale nécessaire à la génération d’un épithélium fonctionnel en réponse aux agressions. Nous avons donc évalué l’impact de l’hyperglycémie et des exoproduits de P. aeruginosa sur ces deux mécanismes. Nos résultats ont tout d’abord montré qu’un niveau de glucose élevé diminue les courants Cl- CFTR et potassique et altère la réparation de l’épithélium bronchique FK et non FK. Nous avons aussi observé que l’hyperglycémie limite l’impact bénéfique de la correction de CFTR sur la réparation épithéliale. Dans un second temps, nous avons évalué l’impact de l’infection à Pseudomonas aeruginosa sur le CFTR, qui tient un rôle important dans la fonctionnalité de l’épithélium des voies aériennes non-FK. Nous avons noté que l’expression du CFTR ainsi que sa fonction sont réduites par l’exposition aux produits bactériens dans les cellules non-FK. De plus, ces exoproduits compromettent la maturation du CFTR muté par les correcteurs ainsi que leur bénéfice sur la réparation de l’épithélium FK. Finalement, nous avons testé différentes combinaisons de composés correcteurs et potentiateurs de CFTR afin de déterminer quelle stratégie serait la plus efficace afin de favoriser la réparation épithéliale bronchique FK malgré la présence d’infection. / Cystic fibrosis (CF), caused by mutations in the CFTR gene, is characterized by dysfunctional Cl- secretion and an imbalance in ion/fluid transport resulting in a decrease in mucociliary clearance. Accumulation of mucus then occurs and this favors bacterial infection in the airways. Chronic infection and inflammation is then responsible for progressive injuries to the lung epithelium. These mechanisms are associated with a decline in lung function, the main cause of morbidity and mortality in CF. The recent improvement in clinical care of patients with CF has led to an increase in median life expectancy, which allows the emergence of comorbidities, such as CF-related diabetes (CFRD). Because Pseudomonas aeruginosa infection and CFRD have been associated with decreased lung function, we investigated their impact on ion transports and epithelial repair, two main functions of airway epithelia. First, our results showed a reduction in Cl- secretion by CFTR and total K+ currents through CF and non-CF airway epithelial cells in hyperglycemic conditions. Moreover, our data indicated a decrease in wound closure rates of airway cell monolayers after exposure to high glucose. We also demonstrated an impairment of the beneficial effect of CFTR correctors on repair rates. The second part of our studies reveals a deleterious impact of Pseudomonas aeruginosa diffusible material (PsaDM) on CFTR expression and function in non-CF airways cells. Importantly, we showed, for the first time, that the presence of PsaDM altered the functional rescue of mutated CFTR by correctors and dampened their beneficial effect on CF wound repair. Finally, we tested several different combinations of corrector and potentiators in order to identify the most efficient compounds to improve the repair rates of CF monolayers despite the presence of PsaDM. Overall, our research demonstrated a deleterious impact of CFRD and PsaDM on ion transports and wound closure. Moreover, the new therapies with correctors may also be impacted by these two components.

Pseudomonas aeruginosa est une bactérie Gram-négative qui présente une grande résistance aux antibiotiques, lui permettant de sévir dans le milieu hospitalier en infectant plus particulièrement les patients immunodéprimés. Cette résistance est principalement due au système d’efflux membranaire MexAB-OprM, capable d’exporter les antibiotiques en dehors de la cellule. Cette pompe à efflux est composée de trois protéines, MexA, MexB et OprM, incorporées dans les membranes internes et externes de la paroi bactérienne. Les structures de MexA, OprM et AcrB -une protéine présente chez E. coli, homologue de MexB- ont été déterminées individuellement par cristallographie des rayons X. Cependant, la structure du complexe entier, regroupant les trois protéines en interaction, ainsi que le mécanisme de cette pompe font toujours défaut. Le renforcement de nos connaissances structurales et fonctionnelles est donc capital pour lutter plus efficacement contre ces bactéries, par de nouvelles stratégies médicamenteuses. Ce travail porte sur l’étude de la structure et de la stœchiométrie de l’assemblage des protéines OprM et MexA au sein d’une membrane lipidique. La caractérisation du complexe OprM/MexA a été réalisée à l’aide de nouvelles techniques de caractérisation physico-chimique des surfaces, telle que la Microbalance à Cristal de Quartz avec Mesure de Dissipation (QCM-D), et par des méthodes d’imagerie, telles que la Cryo-Microscopie Electronique en Transmission (CryoMET) et la Cryo-Tomographie Electronique (CryoTE). En QCM-D, les mesures d’interaction entre OprM et MexA ont été réalisées sur support solide en contrôlant l’orientation d’OprM placée dans un environnement lipidique. Après ajout de la protéine MexA, la formation de complexes OprM/MexA a été mise évidence. Pour comprendre l’organisation de ce complexe, nous avons procédé à une étude comparative de l’organisation des protéines OprM, MexA et du complexe OprM/MexA incorporés dans une membrane lipidique, par CryoMET. Trois types d’organisation, respectivement spécifiques d’OprM, de MexA et du complexe OprM/MexA, ont été mis en évidence. Une analyse structurale de ces trois différents assemblages, pris en sandwich entre deux membranes lipidiques, a été menée par CryoTE. La reconstitution de la protéine OprM conduit à la formation de protéoliposomes, dû à des interactions intervenant entre les protéines OprM au niveau de leurs hélices périplasmiques. La protéine MexA s’organise sous forme d’une structure annulaire de 13 nm de hauteur au sein des membranes lipidiques, et d’une structure plus complexe de 26 nm de hauteur, résultant de l’empilement tête-bêche de deux structures annulaires de 13 nm. Ce travail révèle les dimensions exactes de l’assemblage formé par MexA, et permet de localiser à proximité des membranes les domaines non résolus dans la structure cristallographique. La reconstitution du complexe OprM/MexA révèle une disposition régulière des deux protéines dans les membranes lipidiques. Au sein des complexes, les protéines OprM sont présentes sous forme de trimères. Dans la membrane opposée, à l’aplomb d’une molécule d’OprM, MexA ne forme pas une structure annulaire similaire à celle décrite précédemment, indiquant un état d’oligomérisation différent de celui observé dans les assemblages MexA. Les densités de MexA sont compatibles avec la présence de quelques molécules de MexA. Cependant des structures annulaires de MexA, positionnées à l’aplomb de trois trimères d’OprM sont visibles. Notre étude montre que MexA adopte des structures oligomériques spécifiques en fonction de ses interactions avec les membranes lipidiques ou avec son partenaire OprM. / The structure determination of membrane protein in lipid environment can be carried out using cryo electron microscopy combined with the recent development of data collection and image processing. We describe a protocol to study assemblies or stacks of membrane protein reconstitued into a lipid membrane using both cryo electron tomography and single particle analysis which is an alternative approach to electron crystallography for solving 3D structure. We show the organization of the successive layers of OprM molecules revealing the protein-protein interactions between OprM molecules of two successive lipid bilayers.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-04-25T19:30:35Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_FuncaoPulmonarPacientes.pdf: 366003 bytes, checksum: 42150f32a4cc6c057aadbf811a928a70 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-04-30T12:36:57Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_FuncaoPulmonarPacientes.pdf: 366003 bytes, checksum: 42150f32a4cc6c057aadbf811a928a70 (MD5) / Made available in DSpace on 2013-04-30T12:36:57Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_FuncaoPulmonarPacientes.pdf: 366003 bytes, checksum: 42150f32a4cc6c057aadbf811a928a70 (MD5) Previous issue date: 2008 / A Pseudomonas aeruginosa aparece como um patógeno oportunista na fibrose cística, sendo a maior fonte de infecção pulmonar e é responsável por altas taxas de mortalidade nestes pacientes. O objetivo deste trabalho é de mostrar as características espirométricas e clínicas dos pacientes com fibrose cística atendidos no Hospital Universitário João de Barros Barreto (HUJBB), no Pará. Foi realizado um estudo retrospectivo nos prontuários de 44 pacientes acompanhados no HUJBB, no período de 1997-2007, que atenderam aos critérios de inclusão, sendo 14 colonizados por P. aeruginosa, com a ocorrência de 9 óbitos. Com nível de significância de 5%. No grupo colonizado 10 eram do sexo feminino, a idade média dos sintomas iniciais foi de 0.3±0.6 anos, com diferença significativa quando comparado com pacientes não colonizados (p<0.05). A idade média ao diagnóstico foi de 13.1±10.8 nos colonizados todos apresentavam sintomas respiratórios persistentes ao diagnóstico. A média dos valores percentuais preditos das espirometrias, referentes à avaliação inicial e final, do grupo colonizado foi VEF1(60.0± 25.0%) e (47,82±16.1%) e FEF25-75%(42.5± 22.9%) e (26,5±17.9%) e no grupo não colonizado foi VEF1(79.2± 21.0%) e (79,6±18.0%) e FEF25-75%(69.2± 26.7%) e (68,9±26.8%), respectivamente (p<0.005). A média do escore de Shwachman inicial nos colonizados foi de 42.9±13.5 e nos não colonizados foi de 68.4±15.1(p<0.0001) e na avaliação final foi de 36.6±18.7 e 73.6±12.3 (p<0.0001), respectivamente, com diferença significativa entre os grupos. Os fatores relacionados aos óbitos encontrados foram a colonização por P. aeruginosa, estado nutricional deficiente e VEF1 reduzido. Na casuística estudada foi encontrado maior comprometimento da função pulmonar e idade média mais elevada ao diagnóstico do que em outros estados brasileiros. Estes dados reforçam a necessidade de ações para um diagnóstico precoce no estado do Pará, propiciando uma abordagem terapêutica eficaz e direcionada, buscando aumento de sobrevida e melhoria da qualidade de vida destes indivíduos, objetivo principal das ações dos profissionais envolvidos nesta assistência. / Pseudomonas aeruginosa is an opportunistic pathogen in cystic fibrosis becoming the most frequent cause of lung infection; it is responsible for high mortality rates in patients who present this disease. The goal of this study is to showing spirometric and clinic characteristics of patients who present cystic fibrosis and who are attended at João de Barros Barreto University Hospital (HUJBB), in the State of Pará. A retrospective study was performed with 44 patients´ notes record who were attended at HUJBB during the 1997 to 2007 time frame; these patients fit into inclusion criteria, in which 14 presented P. aeruginosa bacteria colony, nine patients died. The significance rate presented was of 5%. Within the group who was colonized by P. aeruginosa, ten patients were women and the median age for their initial symptoms was of 0.3 ± 0.6 year which was significantly different when compared with patients who didn´t present the bacteria colony (p<0.05). The median age for diagnosis was of 13.1 ± 10.8 in colonized patients and all of them presented respiratory symptoms pertaining to the diagnosis. The median of the predicted percentage values of spirometries for initial and final evaluation of the colonized group was VEF1 (60.0 ± 25.0%) and (47,82 ± 16.1%) and FEF25-75% (42.5 ± 22.9%) and (26.5 ± 17.9%), and in the non-colonized group, the median was VEF1 (79.2 ± 21.0%) and (79,6 ± 18.0%) and FEF25-75% (69.2 ± 26.7%) and (68.9 ± 26.8%), respectively (p<0.005). The median initial Shwachman score in the colonized patients was 42.9 ± 13.5 and in the non-colonized patients it was 68.4 ± 15.1 (p<0.0001), and at the final evaluation the median was 36.6 ± 18.7 and 73.6 ± 12.3 (p<0.0001), respectively, showing important difference between the two groups. Factors related to deaths found in the study were related to P. aeruginosa colonization, inadequate nutritional status and reduced VEF1. In the studied casuistry, a larger damage to respiratory function and older median age for diagnosis were found in the State of Pará when compared to other Brazilian States. These data reinforce the need for action towards precocious diagnosis in the State of Pará so that an efficient and guided therapeutic approach is put into practice guiding to survival increase and quality of life improvement for these individuals, which is the main goal of professionals involved in the assistance practice area.

Sepsis is a leading cause of death among critically ill patients that results in metabolic alterations including hypercatabolism, lipoatrophy, and muscle wasting, contributing to the development of cachexia. Septic cachexia is associated with loss of body weight, fat mass, and lean mass and dysregulated immune function. There are currently no efficacious treatment strategies for septic cachexia, and nutritional interventions have limited success in preventing hypercatabolic wasting. Pyocyanin is a virulence factor produced by sepsis-causing Pseudomonas aeruginosa that has been shown to activate the aryl hydrocarbon receptor (AhR), increase inflammation, and produce reactive oxygen species. Thus, pyocyanin represents a novel mechanistic target in the development of septic cachexia. In Aim 1, we hypothesized that pyocyanin reduces adipocyte differentiation and activates AhR in vitro and in vivo. In vitro, pyocyanin reduced differentiation of 3T3-L1 cells to adipocytes and promoted expression of proinflammatory cytokines. These effects were associated with activation of AhR. We established an in vivo model of pyocyanin-induced cachexia using repeat intraperitoneal exposure to pyocyanin in male and female C57BL/6J mice. Acutely, pyocyanin reduced differentiation of stem cells isolated from adipose stromal vascular tissue and augmented expression of proinflammatory cytokines. Chronically, pyocyanin reduced body weight and fat mass, which was associated with adipose-specific AhR activation. Pyocyanin had sexually dimorphic effects on lipolysis and adipocyte inflammation. These data suggest a role of pyocyanin in adipose cachexia associated with sepsis. In Aim 2, we hypothesized that pyocyanin activates adipocyte AhR to promote adipose tissue wasting and cachexia. To test this hypothesis, we used a mouse model of adipocyte-specific deficiency of AhR and chronically administered pyocyanin to male and female mice. In male mice with adipocyte AhR deficiency, effects of pyocyanin to promote adipose wasting and cachexia were attenuated. In contrast, female adipocyte AhR deficient mice had an augmented response to pyocyanin to decrease body weight. Results suggest divergent mechanisms of pyocyanin to regulate adiposity and body weight through adipocyte AhR between male and female mice. These data support a role for pyocyanin in the development of adipose cachexia associated with Pseudomonas aeruginosa sepsis that is partially regulated by adipocyte AhR. Targeting pyocyanin’s effects on adipocytes represents a potentially novel therapeutic approach for septic cachexia that could mitigate septic cachexia, a condition associated with increased risk of mortality in this population.

L'agression pulmonaire aiguë et le SDRA sont caractérisés par une augmentation de la perméabilité de la barrière alvéolo-capillaire à l'origine de l'exsudation d'un liquide d'oedème riche en protéines conduisant à une altération sévère des échanges gazeux. En dépit des progrès réalisés ces dernières années dans la prise en charge des patients, la mortalité demeure élevée (30-40%). L'agression pulmonaire aiguë est marquée par un état procoagulant et antifibrinolyique à l'origine de dépôts de fibrine et de membranes hyalines dans le poumon distal. La protéine C activée est un important régulateur de la coagulation et exerce des propriétés anti-inflammatoires pulmonaires. Ainsi, dans un premier temps, notre travail a consisté à caractériser l'équilibre pulmonaire au cours de l'administration de protéine C activée in vivo sur un modèle murin de pneumopathie aiguë à P. aeruginosa (Pa). Nos résultats ont montré que l'administration de PCa à la phase précoce de l'agression pulmonaire aiguë induite par Pa tendait à augmenter la formation de l'oedème pulmonaire avec une perte de la compartimentalisation de la réponse inflammatoire. Dans un deuxième temps, nous avons évalué l'équilibre hydrique pulmonaire dépendant du tonus β-adrénergique endogène au cours de la pneumonie aiguë à E. coli. L'inhibition du tonus β-adrénergique endogène induisait une augmentation de l'oedème pulmonaire en diminuant la résorption de l'oedème alvéolaire et en majorant le trouble de perméabilité vasculaire pulmonaire. Enfin, la stimulation β2-adrénergique exogène diminuait l'oedème pulmonaire au décours de la pneumonie à Pa. Ainsi, des approches pharmacologiques différentes peuvent moduler l'équilibre hydrique pulmonaire au cours des pneumonies aiguës. Les connaissances fondamentales acquises au cours de ce travail de thèse seront directement valorisables par la mise au point de nouvelles stratégies thérapeutiques au cours de l'agression pulmonaire aiguë secondaire à la pneumopathie aiguë

Pseudomonas aeruginosa et Yersinia pestis sont responsables d'infections graves chez les individus immunodéprimés et de la peste, respectivement. Leur pathogénicité repose sur de nombreux facteurs de virulence dont le système de sécrétion de type III (SST3) qui a une action prépondérante lors d'infections aiguës. Le SST3 est composé d'une base ancrée dans la double membrane bactérienne, d'une aiguille creuse érigée à la surface et d'un pore de translocation inséré dans la membrane de la cellule hôte permettant à la bactérie d'y injecter des toxines. L'objet de cette thèse est l'étude de l'interactome de l'antigène protecteur V, PcrV chez P. aeruginosa et LcrV chez Y. pestis. Celui-ci est situé au sommet de l'aiguille et est nécessaire au processus de translocation des toxines. L'étude des propriétés biochimiques de la protéine in vitro nous a permis de mettre en évidence sa capacité à former des oligomères présentant une structure en forme d'anneaux. Les multimères ont été observés par chromatographie d'exclusion de taille, gel natif, spectrométrie de masse native et MET. Leur formation est dépendante de la présence de l'hélice α12 C terminale de PcrV et de l'intégrité de ses résidus hydrophobes. Le processus d'assemblage de la protéine est nécessaire à sa fonction in vivo : des mutants qui sont incapables d'oligomériser perdent leur cytotoxicité envers les cellules eucaryotes. <br />Puis, l'interaction directe entre PcrV et la sous unité formant l'aiguille, PscF, a été mise en évidence in vitro par co-purification. De plus, deux mutants ponctuels de PscF dont le phénotype présente un défaut de translocation se sont montrés défectueux pour la liaison avec PcrV. Enfin, l'hélice C terminale de PscF est échangeable avec l'hélice α12 C terminale de PcrV comme l'atteste la capacité de polymérisation d'un hybride créé entre ces deux protéines, suggérant un rôle de celle-ci dans la formation du complexe F-V. L'ensemble de ces études montre que l'assemblage multimérique des antigènes V ainsi que leur position au sommet de l'aiguille sont des éléments essentiels à leur fonction, avec un rôle prépondérant de l'hélice α12 C terminale de PcrV. Ces conclusions pourraient permettre de mieux cibler les développements futurs de nouveaux vaccins ou agents antimicrobiens.

L'exoenzyme S, une toxine de type III, de Pseudomonas aeruginosa possède un domaine GAP (GTPase Activating Protein) (ExoSGAP) inhibant les Rho GTPases (Rho, Rac, Cdc42) et la phagocytose dans les cellules de Mammifères en culture. J'ai utilisé une approche de transgenèse chez Drosophila melanogaster en utilisant un système d'expression tissu-spécifique inductible (UAS-Gal4) afin d'exprimer ExoSGAP. Nous avons montré qu'ExoSGAP cible in vivo les Rho GTPases Rho, Rac1, Rac2 et Cdc42. ExoSGAP affecte la résistance des mouches aux infections en inhibant la phagocytose des bactéries par les plasmatocytes, des cellules de type macrophage, mais n'a pas d'effet sur les voies NF-kB. Une approche génétique a permis d'identifier de nouvelles cibles de la toxine, en recherchant des gènes dont la dérégulation modifie le phénotype d'œil ou d'aile induit par l'expression d'ExoSGAP. Nous avons identifié plusieurs gènes pouvant avoir un rôle dans les voies des JNK et NF-kB Ces résultats valident une stratégie d'étude des toxines de type III par transgenèse chez la drosophile.J'ai parallèlement montré la spécificité de la GTPase Rac2 dans la résistance des mouches aux infections bactériennes. Rac2 participe notamment à la phagocytose.<br />Les travaux du Dr. H. Tricoire ont permis d'identifier 180 gènes dont la dérégulation modifie la réponse des mouches à un stress oxydant. J'ai testé 105 de ces lignées pour leur résistance aux infections, afin d'étudier une corrélation possible entre la réponse aux stress oxydant et infectieux. Ce crible a permis de montrer l'implication d'une protéine à domaine lectine PSLR (Pseudomonas Sensitive Lectin Receptor) dans la réponse immunitaire de la drosophile.