Le seul traitement actuellement disponible pour les accidents vasculaires cérébraux d’origine ischémique est la thrombolyse par l’activateur tissulaire du plasminogène recombinant (rt-PA). Cependant, l’efficacité du rt-PA est souvent partielle ou absente, et des phénomènes de réocclusion du vaisseau peuvent être observés. Par ailleurs, l’administration de rt-PA est associée à un risque hémorragique. Il apparaît donc indispensable de rechercher les mécanismes à l’origine de la toxicité vasculaire du rt-PA, afin de pouvoir développer des stratégies capables de protéger le lit vasculaire. Parmi ces stratégies, notre équipe a montré dans des modèles expérimentaux que l’inhibition d’une enzyme nucléaire, la poly(ADP-ribose) polymérase ou PARP, permet de protéger la barrière hémato-encéphalique, de réduire les hémorragies et d’améliorer la reperfusion cérébrale suite à l’administration post-ischémique de rt-PA. Dans ce contexte, mon travail a consisté à étudier les mécanismes impliqués dans les altérations vasculaires associées à l’administration de rt-PA à la suite de l’ischémie. Mes travaux de recherche ont comporté un volet in vivo et un volet in vitro. Les études réalisées in vivo ont été menées dans un modèle murin d’ischémie cérébrale thrombo-embolique. Nos résultats indiquent que ni l’ischémie, ni le rt-PA, ni l’association au rt-PA d’un puissant inhibiteur de PARP, le PJ34, ne modifient à 24 heures la présence de dépôts de fibrine, marqueur d’hypoperfusion et de réocclusion. Nous nous sommes ensuite intéressés à deux marqueurs endothéliaux d’inflammation : VCAM-1 et ICAM-1, et avons montré que leur expression, qui augmente 24 heures après l’ischémie, n’est pas modifiée par le rt-PA. Enfin, l’association du PJ34 au rt-PA réduit significativement l’expression post-ischémique de VCAM-1, ce qui suggère le rôle de la PARP dans l’expression de cette molécule d’adhésion. La seconde partie de mon travail a été réalisée in vitro sur une lignée de cellules endothéliales cérébrales murines (bEnd.3). Le rt-PA est à l’origine de changements caractéristiques au niveau de l’organisation et de la morphologie de ces cellules. Ces changements ne sont pourtant associés ni à une dégradation de l’expression des molécules de jonctions inter-endothéliales (occludine, VE-cadhérine), ni à une augmentation de l’expression des marqueurs endothéliaux pro-inflammatoires (VCAM-1, ICAM-1). Nous nous sommes également intéressés à d’autres marqueurs de dysfonction endothéliale, les microparticules endothéliales (MPE). Nos résultats montrent que le rt-PA est à l’origine d’une augmentation importante de la libération des MPE. L’utilisation d’un inhibiteur de la protéine p38, le SB203580, et d’un inhibiteur de PARP, le PJ34, permet de réduire cette augmentation, ce qui suggère que p38 et la PARP pourraient être impliquées dans la production de MPE induite par le rt-PA. En conclusion, l’ensemble de ce travail contribue à préciser les effets vasculaires du rt-PA. Parmi ces effets, la mise en évidence de la production de MPE, via la PARP, est particulièrement novatrice. / Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is currently the only approved pharmacological strategy for acute ischemic stroke. However, the efficacy of rt-PA is rarely complete, and arterial reocclusion can be observed. Furthermore, administration of rt-PA increases the risk of hemorrhagic transformations. Therefore, it is essential to seek mechanisms underlying the vascular toxicity of rt-PA in order to develop strategies protecting the vascular bed. Among these strategies, our laboratory has previously shown that inhibition of poly (ADP-ribose) polymerase (PARP), a nuclear enzyme, protects the blood-brain barrier, reduces hemorrhagic transformations and improves cerebral reperfusion following the post-ischemic administration of rt-PA. In this context, the aim of the present work was to establish the post-ischemic mechanisms of rt-PA-induced vascular alterations. The research was divided into (1) in vivo experiments and (2) in vitro studies to examine the effect of rt-PA on the endothelium. The in vivo studies were performed in a mouse model of thrombo-embolic stroke induced by thrombin injection in the middle cerebral artery. Our results showed that neither ischemia, nor rt-PA, nor the association to rt-PA of the potent inhibitor of PARP PJ34 alter cerebral fibrin deposits, a marker of hypoperfusion and reocclusion, at 24 hours after ischemia. We then evaluated the expression of two endothelial markers of inflammation : VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1). Our results showed that their expressions increase 24 hours after ischemia and are not modified by rt-PA. Finally, the association of PJ34 to rt-PA significantly reduced the post-ischemic expression of VCAM-1, suggesting a role for PARP in the expression of this adhesion molecule. The second part of my work was carried out in vitro in cultures of mouse brain-derived endothelial cells bEnd.3. In the presence of rt-PA, the organization and the morphology of the endothelial cells radically changed. However, these changes were associated neither to a degradation of endothelial junction proteins (occludin, VE-cadherin (vascular endothelial-cadherin)), nor to an increase in the expression of pro-inflammatory endothelial markers (VCAM-1, ICAM-1). We were also interested in a recently identified marker of endothelial dysfunction : endothelial microparticles (EMP). Our results showed that rt-PA induces a significant increase in the EMP released by bEnd.3 cells. The use of a p38 inhibitor, SB203580, and the PARP inhibitor, PJ34, reduced this increase, suggesting that p38 and PARP could be involved in the EMP production induced by rt-PA. In conclusion, this work helps to clarify the vascular effects of rt-PA. Among these effects, the highlight of EMP production, through PARP pathway, is particularly original.

Pseudomonas aeruginosa é um importante agente de pneumonia, particularmente em pacientes submetidos à ventilação mecânica, que pode evoluir para sepse, com elevadas taxas de letalidade. Na sepse, o processo inflamatório sistêmico exacerbado favorece o desequilíbrio entre as vias de coagulação e fibrinólise e a instalação de um estado pró-coagulante, com o aparecimento de trombose microvascular, coagulação intravascular disseminada e falência de múltiplos órgãos. Conhecendo a potente atividade pró-inflamatória da toxina ExoU produzida por P. aeruginosa, decorrente de sua atividade fosfolipásica A2, o objetivo desta tese foi investigar seu potencial de indução de alterações hemostáticas relacionadas à patogênese da sepse. Utilizando modelo de sepse em camundongos inoculados, por via intratraqueal, com suspensões de P. aeruginosa produtora de ExoU (PA103) ou de cepa com deleção do gene exoU, não produtora da toxina, foi mostrado que ExoU determinou maior gravidade da infecção, maior taxa de letalidade, leucopenia, trombocitose, hiperpermeabilidade vascular e transudação plasmática, evidenciadas, respectivamente, pela maior concentração de proteínas nos lavados broncoalveolares (LBAs) e acúmulo do corante Azul de Evans, previamente inoculado nos animais, por via endovenosa, no parênquima renal. ExoU favoreceu, também, a ativação plaquetária, confirmada pela maior concentração de plaquetas expressando P-selectina em sua superfície, maior número de micropartículas derivadas de plaquetas e maior concentração plasmática de tromboxano A2. A histopatologia dos pulmões e rins dos animais infectados com PA103 confirmou a formação de microtrombos, que não foram detectados nos animais controles ou infectados com a cepa mutante. Nos pulmões, a produção de ExoU determinou intensa resposta inflamatória com maior concentração de leucócitos totais e polimorfonucleados, interleucina-6 e fator de necrose tumoral-α nos LBAs. A análise imunohistoquímica mostrou intensa deposição de fibrina nos alvéolos e septos interalveolares. A atividade pró-coagulante dependente do fator tissular detectada nos LBAs dos camundongos infectados com PA103 foi independente da produção do inibidor da via de ativação do fator tissular (TFPI), mas associada ao aumento da produção do inibidor do ativador do plasminogênio-1 (PAI-1). Para investigar a participação do fator de ativação plaquetária (PAF) na liberação de PAI-1, foi pesquisada a atividade da enzima PAF-acetil-hidrolase (PAF-AH) nos LBAs dos camundongos. A atividade de PAF-AH apresentou-se significativamente elevada nos LBA dos camundongos infectados com PA103. O tratamento dos animais com um inibidor do PAF, antes da infecção, resultou na diminuição significativa das concentrações de PAI-1 e de leucócitos totais, bem como da atividade pró-coagulante dos LBAs. In vitro, ExoU induziu maior expressão do RNA mensageiro de PAI-1 e maior liberação da proteína PAI-1 nos sobrenadantes de células epiteliais respiratórias da linhagem A549. O tratamento das células A549 com um anticorpo anti-receptor de PAF, antes da infecção, reduziu significativamente a concentração de PAI-1 nos sobrenadantes de células infectadas com a cepa selvagem. Estes resultados demonstraram um novo mecanismo de virulência de P. aeruginosa através da atividade pró-trombótica de ExoU e a possibilidade de utilização da identificação de ExoU em isolados clínicos de pacientes graves como um marcador prognóstico para estes pacientes. / Pseudomonas aeruginosa is an important agent of pneumonia, mainly in patients undergoing mechanical ventilation, which can progress to sepsis with high mortality rates. In sepsis, the systemic inflammatory process favors exacerbated imbalance between the coagulation and fibrinolysis pathways and the installation of a procoagulant state, leading to microvascular thrombosis, disseminated intravascular coagulation and multiple organ failure. Knowing the powerful proinflammatory activity of the P. aeruginosa toxin ExoU, secondary to its phospholipase A2 activity, the goal of this study was to investigate the ExoU potential to induce hemostatic changes related to sepsis pathogenesis. By using a murine model of pneumosepsis, obtained by the intratracheal injection of suspensions of the ExoU-producing PA103 P. aeruginosa strain or of its isogenic mutant PA103ΔexoU, defective in the toxin synthesis, ExoU was shown to enhance the severity of the infection and to induce higher mice mortality rate as well as leukopenia, thrombocytosis, vascular hyperpermeability and plasma transudation, evidenced, respectively, by the higher protein concentration in the bronchoalveolar lavage fluids (BALF) and accumulation of Evans blue dye, previously intravenous injectioned, in mice renal parenchyma. ExoU also favored platelet activation, evidenced by the higher concentration of platelets expressing P-selectin on their surface, greater number of platelet-derived microparticles and increased plasma concentration of thromboxane A2. Histopathology of the lungs and kidneys of PA103-infected animals confirmed the formation of microthrombi, which were not detected in controls or in animals infected with the bacterial mutant. In lungs, ExoU induced an intense inflammatory response with high concentrations of total and polymorphonuclear leukocytes, interleukin-6 and tumor necrosis factor-α in mice BALF. Immunohistochemical analysis showed intense fibrin deposition in the alveoli and interalveolar septa. The tissue factor-dependent procoagulant activity detected in BALF from PA103-infected mice did not depend on decreased production of tissue factor pathway inhibitor (TFPI) production, but was associated with increased concentration of plasminogen activator inhibitor-1 (PAI-1). To investigate the role of platelet activating factor (PAF) in PAI-1 release, we compared the activity of the enzyme PAF-acetylhydrolase (PAF-AH) in BALF from control and infected mice, and we observed that PAF-AH activity was significantly elevated in BALF from PA103-infected animals. Mice treatment with a PAF inhibitor prior to infection resulted in a significant reduction of PAI-1 concentration, as well as of BALF total leukocytes and procoagulant activity. In vitro, ExoU induced higher expression of PAI-1 m RNA and increased release of PAI-1 protein in supernatants from A549 epithelial respiratory cell cultures. A549 cell treatment with an anti-PAF receptor prior to infection significantly reduced PAI-1 concentration in supernatants from cells infected with the wild type strain. These results show a novel mechanism by which a baterial product can favor a prothrombotic activity and ExoU production by infecting P. aeruginosa isolates may have prognostic implications for patients.

Pseudomonas aeruginosa é um importante agente de pneumonia, particularmente em pacientes submetidos à ventilação mecânica, que pode evoluir para sepse, com elevadas taxas de letalidade. Na sepse, o processo inflamatório sistêmico exacerbado favorece o desequilíbrio entre as vias de coagulação e fibrinólise e a instalação de um estado pró-coagulante, com o aparecimento de trombose microvascular, coagulação intravascular disseminada e falência de múltiplos órgãos. Conhecendo a potente atividade pró-inflamatória da toxina ExoU produzida por P. aeruginosa, decorrente de sua atividade fosfolipásica A2, o objetivo desta tese foi investigar seu potencial de indução de alterações hemostáticas relacionadas à patogênese da sepse. Utilizando modelo de sepse em camundongos inoculados, por via intratraqueal, com suspensões de P. aeruginosa produtora de ExoU (PA103) ou de cepa com deleção do gene exoU, não produtora da toxina, foi mostrado que ExoU determinou maior gravidade da infecção, maior taxa de letalidade, leucopenia, trombocitose, hiperpermeabilidade vascular e transudação plasmática, evidenciadas, respectivamente, pela maior concentração de proteínas nos lavados broncoalveolares (LBAs) e acúmulo do corante Azul de Evans, previamente inoculado nos animais, por via endovenosa, no parênquima renal. ExoU favoreceu, também, a ativação plaquetária, confirmada pela maior concentração de plaquetas expressando P-selectina em sua superfície, maior número de micropartículas derivadas de plaquetas e maior concentração plasmática de tromboxano A2. A histopatologia dos pulmões e rins dos animais infectados com PA103 confirmou a formação de microtrombos, que não foram detectados nos animais controles ou infectados com a cepa mutante. Nos pulmões, a produção de ExoU determinou intensa resposta inflamatória com maior concentração de leucócitos totais e polimorfonucleados, interleucina-6 e fator de necrose tumoral-α nos LBAs. A análise imunohistoquímica mostrou intensa deposição de fibrina nos alvéolos e septos interalveolares. A atividade pró-coagulante dependente do fator tissular detectada nos LBAs dos camundongos infectados com PA103 foi independente da produção do inibidor da via de ativação do fator tissular (TFPI), mas associada ao aumento da produção do inibidor do ativador do plasminogênio-1 (PAI-1). Para investigar a participação do fator de ativação plaquetária (PAF) na liberação de PAI-1, foi pesquisada a atividade da enzima PAF-acetil-hidrolase (PAF-AH) nos LBAs dos camundongos. A atividade de PAF-AH apresentou-se significativamente elevada nos LBA dos camundongos infectados com PA103. O tratamento dos animais com um inibidor do PAF, antes da infecção, resultou na diminuição significativa das concentrações de PAI-1 e de leucócitos totais, bem como da atividade pró-coagulante dos LBAs. In vitro, ExoU induziu maior expressão do RNA mensageiro de PAI-1 e maior liberação da proteína PAI-1 nos sobrenadantes de células epiteliais respiratórias da linhagem A549. O tratamento das células A549 com um anticorpo anti-receptor de PAF, antes da infecção, reduziu significativamente a concentração de PAI-1 nos sobrenadantes de células infectadas com a cepa selvagem. Estes resultados demonstraram um novo mecanismo de virulência de P. aeruginosa através da atividade pró-trombótica de ExoU e a possibilidade de utilização da identificação de ExoU em isolados clínicos de pacientes graves como um marcador prognóstico para estes pacientes. / Pseudomonas aeruginosa is an important agent of pneumonia, mainly in patients undergoing mechanical ventilation, which can progress to sepsis with high mortality rates. In sepsis, the systemic inflammatory process favors exacerbated imbalance between the coagulation and fibrinolysis pathways and the installation of a procoagulant state, leading to microvascular thrombosis, disseminated intravascular coagulation and multiple organ failure. Knowing the powerful proinflammatory activity of the P. aeruginosa toxin ExoU, secondary to its phospholipase A2 activity, the goal of this study was to investigate the ExoU potential to induce hemostatic changes related to sepsis pathogenesis. By using a murine model of pneumosepsis, obtained by the intratracheal injection of suspensions of the ExoU-producing PA103 P. aeruginosa strain or of its isogenic mutant PA103ΔexoU, defective in the toxin synthesis, ExoU was shown to enhance the severity of the infection and to induce higher mice mortality rate as well as leukopenia, thrombocytosis, vascular hyperpermeability and plasma transudation, evidenced, respectively, by the higher protein concentration in the bronchoalveolar lavage fluids (BALF) and accumulation of Evans blue dye, previously intravenous injectioned, in mice renal parenchyma. ExoU also favored platelet activation, evidenced by the higher concentration of platelets expressing P-selectin on their surface, greater number of platelet-derived microparticles and increased plasma concentration of thromboxane A2. Histopathology of the lungs and kidneys of PA103-infected animals confirmed the formation of microthrombi, which were not detected in controls or in animals infected with the bacterial mutant. In lungs, ExoU induced an intense inflammatory response with high concentrations of total and polymorphonuclear leukocytes, interleukin-6 and tumor necrosis factor-α in mice BALF. Immunohistochemical analysis showed intense fibrin deposition in the alveoli and interalveolar septa. The tissue factor-dependent procoagulant activity detected in BALF from PA103-infected mice did not depend on decreased production of tissue factor pathway inhibitor (TFPI) production, but was associated with increased concentration of plasminogen activator inhibitor-1 (PAI-1). To investigate the role of platelet activating factor (PAF) in PAI-1 release, we compared the activity of the enzyme PAF-acetylhydrolase (PAF-AH) in BALF from control and infected mice, and we observed that PAF-AH activity was significantly elevated in BALF from PA103-infected animals. Mice treatment with a PAF inhibitor prior to infection resulted in a significant reduction of PAI-1 concentration, as well as of BALF total leukocytes and procoagulant activity. In vitro, ExoU induced higher expression of PAI-1 m RNA and increased release of PAI-1 protein in supernatants from A549 epithelial respiratory cell cultures. A549 cell treatment with an anti-PAF receptor prior to infection significantly reduced PAI-1 concentration in supernatants from cells infected with the wild type strain. These results show a novel mechanism by which a baterial product can favor a prothrombotic activity and ExoU production by infecting P. aeruginosa isolates may have prognostic implications for patients.

Made available in DSpace on 2014-07-29T15:07:55Z (GMT). No. of bitstreams: 1 dissertacao mariana faleiro part 1.pdf: 60728 bytes, checksum: 82e5bf30cd60c4752f1de660f88797ed (MD5) Previous issue date: 2010-03-02 / Humans and dogs show dysplastic lesions in the prostate, such as prostatic intraepithelial neoplasms (PIN) and proliferative inflammatory atrophy (PIA), which are studied due to their malignance potential. The matrix metalloproteinases (MMP) are a family of proteolytic enzymes thought to play an important role in tumor invasion and metastasis in face of their ability to degrade the extracellular matrix (ECM) and basement membrane. The plasminogen activator (PA) system has been suggested to play a central role in cell adhesion, migration, wound healing, angiogenesis, inflammation, regulation of growth factors and tumor invasion. The receptor of plasminogen activator type activator (uPAR) is a component of the PA, with a range of expression in tumor cell and stromal cells. So, this study was aimed to evaluated the expression and correlation between MMP-2 (gelatinase A) and MMP-9 (gelatinase B) as well as the expression of uPAR in normal canine prostate tissue and also in tissue with proliferative disorders, including benign prostatic hyperplasia (HPB), PIA, PIN and carcinoma. And therefore establish relation among the role of these enzymes in the remodeling of the extracellular matrix (ECM) and in the process of tumor invasion and metastasis. For this, it was performed immunohistochemical staining in tissue microarray of 149 paraffin-embedded fragments of prostate tissue selected from 57 prostates of non-castrated adult dogs with or without prostatic diseases. A total of 298 cores were analyzed and it was made 363 diagnoses: 36 (9.9%) normal, 49 (13.5%) BPH, 132 (36.3%) PIA, 75 (20.7%) PIN and 71 (19.6%) carcinomas. It was observed differences in cytoplasmatic immunohistochemical staining by MMP-2 and MMP-9 antibodies in relation to the cell number and intensity of labeling of the acinar epithelial and stromal perilobular cells between normal tissue and in those with proliferative disorders. A correlation between MMP-2 and MMP-9 antibodies occurred just in canine prostates with PIA in relation to the number of labeled cells in acinar epithelium and perilobular stroma, as well as, the staining intensity in the perilobular stromal cells. In relation to uPAR, it was observed differences of immunohistochemical staining of uPAR antibodies in canine prostate. Likewise, there was over expression in dysplastic and neoplasic specimens, but not in normal and benign prostate tissue. A number of epithelial cells labeled for uPAR showed variation among the diagnoses, except between PIN and carcinoma. Less intensity of labeling was observed in acinar epithelial cells of normal prostates compared with PIA, PIN and carcinoma. However, in the normal cells and in those with PIA, there was a difference in the number of cells, as well as in the intensity of stromal labeling. The intensity of labeling of stromal perilobular cells was higher in the PIA. PIA-A (accentuated) and PIA-M (moderated) cells showed greater intensity staining stroma and stromal cells labeled for uPAR, respectively. Thus, this study concludes that there was variation in gelatinases and uPAR expression in canine prostate according to the lesion. Also, there was Less labeling in normal and BPH and higher in PIA, PIN and carcinoma prostate tissues. The correlation between MMP-2 and MMP-9 in canine prostates with PIA indicates that the inflammation likely influenced the activity of these enzymes with simultaneous increase in their expression. The uPAR high expression in inflammatory and neoplasic tissues suggests high ECM proteolytic activity in these situations / Nas espécies humana e canina lesões displásicas da próstata, como a neoplasia intra-epitelial prostática (PIN) e a atrofia inflamatória proliferativa (PIA), são estudadas quanto ao potencial de malignidade. As metaloproteinases (MMP) são enzimas proteolíticas envolvidas no processo de invasão tumoral e metástase, causando destruição de barreiras biológicas como a matriz extracelular (MEC) e a membrana basal (MB). O sistema ativador de plasminogênio (PA) compreende proteínas com ação na adesão celular, regulação da migração, cicatrização, angiogênese, inflamação, regulação de fatores de crescimento e invasão tumoral. O receptor de ativador de plasminogênio tipo uroquinase (uPAR) é um dos componentes do PA, com variação de expressão em células neoplásicas e estromais. Este trabalho teve por objetivo verificar a expressão e a correlação entre MMP-2 e MMP-9, assim como a expressão do uPAR no tecido prostático canino normal e com alterações proliferativas, incluindo a hiperplasia prostática benigna (HPB), a PIA, a PIN e o carcinoma, buscando avaliar o papel dessas proteínas no remodelamento da MEC e no processo de invasão tumoral e metástase. Para isso, foi realizada a imunoistoquímica em lâminas de microarranjo tecidual (TMA), com 149 cores selecionadas de 57 próstatas de cães adultos, não castrados, com ou sem histórico de afecções prostáticas. Foram analisados, para cada anticorpo, 298 cores, perfazendo 363 diagnósticos, sendo 36 (9,9%) normais, 49 (13,5%) HPB, 132 (36,3%) PIA, 75 (20,7%) PIN e 71 (19,6%) carcinomas. Foi possível observar diferença de imunomarcação citoplasmática de MMP-2 e MMP-9 em relação ao número de células e intensidade de imunomarcação nas células epiteliais acinares e estromais periacinares em relação aos diagnósticos. A correlação entre os anticorpos MMP-2 e MMP-9 ocorreu em próstatas caninas com PIA quanto ao número de células imunomarcadas no epitélio acinar e no estroma periacinar, bem como quanto à intensidade de imunomarcação nas células estromais periacinares. Quanto ao uPAR, houve diferença na imunomarcação em relação ao diagnóstico, com maior expressão nas displásicas e neoplásicas em relação ás normais e com HPB. O número de células epiteliais imunomarcadas para uPAR variou entre os diagnósticos, exceto entre PIN e carcinoma. Menor intensidade de imunomarcação epitelial foi constatada nas próstatas normais em relação às com PIA, PIN e carcinoma. Entre as normais e com PIA houve diferença no número de células e intensidade de imunomarcação estromal. A intensidade de imunomarcação estromal foi maior nas com PIA. As PIA-M (inflamação moderada) e PIA-A (inflamação acentuada) apresentaram maior intensidade de imunomarcação estromal e células estromais imunomarcadas para uPAR, respectivamente. Concluiu-se que há variação na expressão das gelatinases e do uPAR na próstata canina, de acordo com a lesão, com menor expressão nas normais e com HPB e maior naquelas com PIA, PIN e carcinoma. A correlação entre MMP-2 e MMP-9 em próstatas caninas com PIA indica que a inflamação influencia a atividade dessas enzimas, com aumento simultâneo na expressão de ambas no microambiente inflamatório. Ainda, o aumento na expressão do uPAR nos microambientes inflamatório e neoplásico sugere maior atividade proteolítica na MEC nesses casos

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-10-21T13:25:36Z No. of bitstreams: 2 Dissertação - Lidiana Candida Piveta - 2016.pdf: 1349571 bytes, checksum: a3430d01588aec9260e619f74991bcd1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-11-08T17:41:10Z (GMT) No. of bitstreams: 2 Dissertação - Lidiana Candida Piveta - 2016.pdf: 1349571 bytes, checksum: a3430d01588aec9260e619f74991bcd1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-11-08T17:41:10Z (GMT). No. of bitstreams: 2 Dissertação - Lidiana Candida Piveta - 2016.pdf: 1349571 bytes, checksum: a3430d01588aec9260e619f74991bcd1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-07-22 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Cataracts is the treatable eye diseases that cause most blindness in the world. The effective treatment is surgical and phacoemulsification the most applied technique. Some complications are associated with the procedure as corneal opacities, uveitis and fibrin deposits. The presence of fibrin in the anterior chamber is associated with the formation of synechia and secondary glaucoma low visual recovery of patients. Some medications, as TPA (Tissue Plasminogen Activator) which acts in the degradation of fibrin, seeing being used to soften these deposits. Tenecteplase is a third generation of the synthetic TPA and have a longer half-life than the others with application in ophthalmology without damage to the cornea and retina of rabbits and humans. The study was conducted on 15 rabbits of New Zealand race, share in three groups GC, GT and GNT. The GNT and GT were surgery by phacoemulsification technics, GT was treated with 0.1 ml intracameral tenecteplase (50 μg) transoperative. The rabbits were evaluated in M0 moments - when selected, underwent phacoemulsification surgery and reassessed in M1d – 1th day M3d – 3th day M7d – 7th day M15d - 15th day and M21d – 21th days. In the clinical evaluation of the use of intracameral tenecteplase in transoperative and postoperative complications were observed, emphasizing the change in IOP, the corneal edema, fibrin deposits, hyphema, aqueous flare and synechia incidences. In the M21d animals were sacrificed, and samples of aqueous humor were collected for physical-chemical analysis (pH, density, concentration of chloride ions and total proteins). No statistical differences were observed in the clinical evaluation of the GC and GNT within the recommended parameters. In physical-chemical analysis of aqueous humor showed no statistical difference between the three groups in terms of pH and concentration of chloride ion. The density values and total protein concentration between the GC and the other groups. / A catarata está entre as oftalmopatias tratáveis que mais causam cegueira no mundo. O único tratamento efetivo é cirúrgico, sendo a facoemulsificação a técnica mais aplicada. Algumas complicações estão associadas ao procedimento como opacidades corneanas, uveítes e depósitos de fibrinas. A presença de fibrina na câmara anterior esta associada à formação de sinéquias, glaucoma secundário e baixa recuperação visual dos pacientes. Algumas medicações vêm sendo usadas para amenizar esses depósitos como os TPA, que atua na degradação da fibrina. A tenecteplase é um TPA sintético de terceira geração que apresenta um tempo de meia vida maior que as outras gerações, com aplicação na oftalmologia sem danos à córnea e retina de coelhos e humanos. O estudo foi realizado com 15 coelhos da raça Nova Zelândia, divididos em três grupos GC, GNT e GT. Os grupos GNT e GT foram operados pela técnica de facoemulsificação, GT recebeu tratamento com 0,1 ml de tenecteplase intracameral (50μg) no transoperatório. Os coelhos foram avaliados nos momentos M0 - quando selecionados, submetidos ao procedimento cirúrgico de facoemulsificação e reavaliados em M1d - 1° dia, M3d - 3°dia, M7d - 7 °dia, M15d - 15° dia e M21d - 21°dia. Na avaliação clínica do uso da tenecteplase intracameral no transoperatório foram observadas as complicações pós-operatórias, dando ênfase à variação da pressão intraocular (PIO), ao edema de córnea, depósito de fibrina, hifema, flare aquoso e incidências de sinéquias. No M5 os animais foram eutanasiados, e coletado amostras do humor aquoso para avaliação físico-química (pH, densidade, concentração de íons de cloreto e proteínas totais). Não foram observadas diferenças estatísticas na avaliação clínica entre o GNT e GT dentro dos parâmetros preconizados. Na avaliação físico-química do humor aquoso não apresentou diferença estatística entre os três grupos quanto aos valores de pH e concentração do íon cloreto. Os valores de densidade e concentração de proteínas totais entre o GC e os demais grupos.

Les accidents vasculaires cérébraux (AVC) constituent un problème majeur de santé publique. Ils sont en majorité de type ischémique, c’est-à-dire liés à l’occlusion d’une artère cérébrale. Le seul traitement actuel de ces AVC ischémiques est la thrombolyse par l’activateur tissulaire du plasminogène recombinant (rt-PA). Cependant, ce traitement est associé à un risque élevé d’hémorragies intracérébrales post-ischémiques, encore appelées transformations hémorragiques (TH), qui contribuent à la dégradation neurologique des patients. Il apparaît donc indispensable de développer des stratégies à associer au rt-PA, afin de protéger le lit vasculaire et de réduire les TH. L’objectif de ce travail de thèse était d’étudier l’implication d’une enzyme, la poly(ADP-ribose)polymérase ou PARP, dans les effets délétères du rt-PA, et plus particulièrement au niveau de la barrière hémato-encéphalique (BHE). Nos travaux ont été menés dans un modèle d’ischémie cérébrale réalisé chez la souris. Dans ce modèle, nous avons mis en évidence le rôle de la PARP dans les TH induites par le rt-PA, grâce à deux techniques : le Western blot d’hémoglobine, permettant d’évaluer la quantité de sang présente dans le parenchyme cérébral, et l’Imagerie par Résonnance Magnétique. Afin de préciser les cibles de la PARP sous-tendant sa contribution aux TH post-thrombolyse, nous nous sommes intéressés à différents constituants de la BHE : la claudine-5, l’occludine et ZO-1 (zonula occludens-1), protéines des jonctions serrées, la VE-cadhérine des jonctions adhérentes et le collagène IV et la laminine, constituants de la lame basale. Nous avons montré que l’ischémie s’accompagne d’une dégradation de la claudine-5, de ZO-1, et de la VE-cadhérine qui est aggravée par le rt-PA ; l’administration d’un puissant inhibiteur de PARP, le PJ34, permet de s’opposer à la dégradation de ces protéines par le rt-PA. Une réduction de la dégradation de la laminine par le rt-PA a également été observée avec le PJ34. Grâce à une collaboration avec le Pr Bérézowski de Lens, nous avons pu montrer dans un modèle in vitro que le PJ34 est capable de traverser la BHE, à la fois dans des conditions « physiologiques » et dans des conditions mimant l’ischémie cérébrale (oxygen/glucose deprivation). Afin de déterminer les voies de signalisation modulées par la PARP conduisant à la dégradation de la BHE et aux TH, nous avons travaillé sur un modèle in vitro de cultures de cellules endothéliales (lignée bEnd.3). Sur ce modèle, nous avons d’ores et déjà pu mettre en évidence une mort cellulaire après un stress excitotoxique et le rôle de la PARP dans cette mort. L’ensemble de ces travaux a permis de démontrer le rôle de la PARP dans la dégradation de différents constituants de la BHE par le rt-PA à la suite de l’ischémie cérébrale. Les futures études in vitro sur cultures cellulaires devraient nous permettre d’explorer les mécanismes mis en jeu dans cette situation pathologique. Une meilleure connaissance de ces mécanismes renforcera l’intérêt des inhibiteurs de PARP pour la prévention des TH post-thrombolyse chez les patients victimes d’AVC ischémiques. / Stroke is a leading public health problem, the majority of which is ischemic, i.e. caused by the occlusion of a cerebral artery. The only pharmacological approved treatment for acute ischemic stroke is thrombolysis by recombinant tissue plasminogen activator (rt-PA). However, this treatment increases the risk of intracerebral hemorrhages, also called hemorrhagic transformations (HT), which contribute to the neurologic aggravation of the patients. It therefore appears essential to develop strategies protecting the vascular bed after cerebral ischemia in order to reduce these HT. The aim of the present work was therefore to study the implication of a nuclear enzyme, the poly(ADP-ribose)polymerase (PARP) in the vascular effects of rt-PA , with special concern for the blood-brain barrier (BBB). Focal cerebral ischemia was performed in mice by permanent endovascular occlusion of the left middle cerebral artery. In this model, we demonstrated the role of PARP in the rt-PA induced HT by two methods: the Western blot of hemoglobin to evaluate the quantity of blood in the cerebral parenchyma, and magnetic resonance imaging. In order to clarify the targets of PARP underlying its contribution to post-thrombolysis HT, we studied several components of the BBB by Western blot: proteins of tight junctions [claudin-5, occludin and zonula occludens-1 (ZO-1)], protein of adherens junction (VE-cadherin) and proteins of basal membrane (collagen IV and laminin). We demonstrated that ischemia induced a marked decrease of claudin-5, ZO-1 and VE-cadherin, which was aggravated by rt-PA. Administration of a potent PARP inhibitor, PJ34, counteracted the degradation of these proteins by rt-PA. A reduction of the degradation of the laminin by rt-PA was also shown with PJ34. Thanks to a collaboration with Pr Berezowski from Lens, we showed in an in vitro BBB model that PJ34 is able to cross the BBB in physiological condition and during oxygen and glucose deprivation, a condition that mimicks cerebral ischemia. In order to determine the molecular pathways modulated by PARP leading to the degradation of the BBB and to HT, we developed an in vitro model of endothelial cell culture (cell line bEnd.3). In this model, we have already shown a cell death after an excitotoxic stress and the role of PARP in this cell death. This work thus demonstrated the role of PARP in the degradation of different components of the BBB induced by rt-PA after cerebral ischemia. The future in vitro studies on cell culture will enable us to further understand the mechanisms implicated in this pathologic situation. A better knowledge of these mechanisms will increase the interest of the use of PARP inhibitors in the prevention of post-thrombolysis HT in patients suffering from ischemic stroke.

Le cancer du sein est la principale cause de mortalité par cancer chez la femme. Deux des facteurs de transcription de la famille Fos, Fra-1 et Fra-2, sont surexprimés dans les cancers du sein agressifs et contribuent au phénotype tumoral en favorisant entre autres, la prolifération, la motilité et l'invasivité. De façon surprenante, les mécanismes moléculaires via lesquels Fra-1 et Fra-2 (et plus généralement le complexe transcriptionnel AP-1 dont ils sont des constituants) gouvernent la transcription de leurs gènes cibles sont quasi-inconnus. Dans ce contexte, en combinant diverses approches (immunoprécipitation de chromatine, interférence à l'ARN…), j'ai étudié les mécanismes moléculaires par lesquels Fra-1 et Fra-2 contrôlent la transcription dérégulée du gène de l'urokinase ou uPA (sérine protéase cruciale dans la progression tumorale et l'établissement de métastases) qui est l'un des nouveaux marqueurs utilisés en clinique pour la mise en place des choix thérapeutiques. Mes travaux montrent de façon originale que (i) Fra-1 et Fra-2 agissent de façon non redondante et coopèrent pour réguler l'expression d'uPA via leur fixation sur un enhancer AP-1 localisé à -1,9 kb du site d'initiation de la transcription (TSS), (ii) Fra-2 est nécessaire au recrutement de RNA Pol II au niveau de l'enhancer, tandis que Fra-1 stimule le passage de RNA Pol II de sa forme initiatrice à sa forme élongatrice et (iii) que la polymérase recrutée à l'enhancer rejoint le TSS par un mécanisme de « tracking », très rarement décrit dans la littérature, en produisant de petits ARNs non codants, bidirectionnels et instables. / Breast cancer is the most frequent malignant disease among women. Two transcription factors, Fra-1 and Fra-2, belonging to the Fos family members, are overexpressed in aggressive breast cancers and contribute to the tumorigenic phenotype by favoring proliferation, motility and invasion. Surprisingly, the molecular mechanisms governed by Fra-1 and Fra-2 (and more generally by the AP-1 transcriptional complex, which they are components of) for the transcription of their target genes are still largely unknown. In this context, by combining different approaches (chromatin immunoprecipitation, RNA interference…), I studied the molecular mechanisms orchestrated by Fra-1 and Fra-2 for the expression of the urokinase (or uPA) gene (encoding a serine protease crucial for tumor progression and metastasis), which is one of the new diagnostic markers now taken into consideration for deciding therapeutic strategies. Interestingly, my results show that (i) Fra-1 and Fra-2 have non redundant functions and cooperate for the transcriptional regulation of uPA through their binding to AP-1 enhancer located 1.9 kb upstream of the transcriptional start site (TSS), (ii) Fra-2 is required for the recruitment of RNA Pol II on this enhancer while Fra-1 allows the conversion of RNA Pol II initiating form into its elongating form and (iii) enhancer-recruited RNA Pol II reaches the TSS by a tracking mechanism, mechanism very rarely described in the literature, during which it synthetizes small, unstable bidirectional, non coding RNAs.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Vascular remodelling due to excessive proliferation and apoptosis resistance of pulmonary arterial smooth muscle (PASMCs) and endothelial cells (ECs) has been attributed to the pathogenesis of pulmonary arterial hypertension (PAH). It is an incurable cardiovascular disorder, which leads to right heart failure and death, if left untreated. Heterozygous germline mutations in the bone morphogenetic protein receptor type II (BMPR2) have been linked with the majority (~75%) of the familial form of the disease (HPAH). Mutations in the BMPR2 gene impinge upon the BMP signalling which perturbs the balance between BMP and TGF-β pathways leading to the clinical course of the disease. Current therapies were discovered prior to the knowledge that PAH has substantial genetic components. Hence, this study aims to identify novel therapeutic intervention and provide novel insights into how the dysfunctional BMPRII signalling contributes to the pathogenesis of PAH. This work demonstrates that cryptolepines and FDA approved drugs (doxorubicin, taxol, digitoxin and podophyllotoxin) inhibit the excessive proliferation and induce apoptosis in BMPR2 mutant PASMCs by modulating the BMP and TGF-β pathways. Moreover, established drug PTC124 has also been tested but has failed to promote translational readthrough. I have also shown that dysregulated apoptosis of PASMCs and HPAECs is mediated through the BMPRII-ALK1-BclxL axis. Finally, the siRNA screen targeting approximately 1000 genes has identified novel proteins including PPP1CA, IGF-1R, MPP1, MCM5 and SRC each capable of modulating the BMPRII signalling. Taken together, this study for the very first time has identified novel compounds with pro-BMP and anti-TGFβ activities which may provide therapeutic intervention prior to or after the onset of PAH. / Commonwealth Scholarship Commission in the UK

Introdução: O seguimento de pacientes com câncer de bexiga superficial apresenta embargos financeiros e psicológicos ao paciente, devido à realização frequente de exames invasivos. Com fim de substituir ou diminuir os exames invasivos, busca-se biomarcadores urinários acurados, que permitam diagnosticar a recidiva tumoral e estratificar pacientes com maior risco de recidiva futura. O objetivo deste estudo é avaliar se expressão na urina de PAI-1, DJ-1, ApoA-1, MMP-9 e IL-8 permite identificar e antecipar a recidiva de câncer de bexiga. Método: A expressão da PAI-1, DJ-1, ApoA-1, MMP-9 e IL-8 foi mensurada por ELISA na urina de 152 pacientes tratados previamente de carcinoma urotelial superficial de bexiga e em seguimento. Os níveis das proteínas foram comparados entre pacientes com e sem recidiva de câncer de bexiga (1) no momento da coleta de urina e (2) durante o seguimento. A ocorrência de recidiva tumoral foi confirmada por análise histopatológica da biópsia de lesões suspeitas, investigadas quando havia alterações na cistoscopia, ultrassom ou citologia oncótica. Pacientes com recidiva diagnosticada no momento da coleta de urina foram excluídos da análise para avaliar o papel antecipatório das cinco proteínas. Foi avaliado se o uso prévio de BCG intra-vesical exercia influência no nível das cinco proteínas estudadas. Resultados: Entre os pacientes avaliados, 16 (10,5%) apresentaram recidiva de carcinoma urotelial no momento da coleta de urina e 21 (15,4%) apresentaram recidiva de carcinoma urotelial durante o seguimento. O seguimento mediano foi de 47 meses (interquartis de 39 e 50 meses). Um painel para o diagnóstico de recidiva tumoral incluindo três biomarcadores (ApoA-1, MMP-9 e IL-8) apresentou razão de risco de 12,9 (IC 95% =3,5-47,4) e um painel para prever pacientes que desenvolverão recidiva durante o seguimento incluindo dois biomarcadores (PAI-1 e IL-8) apresentou razão de risco de 4,1 (IC 95% =1,4-11,4). Os resultados dos painéis não foram influenciados pelo uso prévio de BCG intra-vesical. Conclusão: Os painéis apresentados permitem identificar pacientes com recidiva de carcinoma urotelial de bexiga e prever quais pacientes terão maior risco de desenvolver recidiva no futuro. O uso prévio de BCG intra-vesical não alterou a expressão dos biomarcadores / Purpose: To evaluate if the urinary levels of PAI-1, DJ-1, ApoA-1, MMP-9 and IL-8 can identify and predict tumor recurrence in patients on follow-up of superficial bladder cancer. Methods: We prospectively analyzed the urine of 152 patients previously treated of superficial bladder cancer on follow-up regimen. Five biomarkers (PAI-1, DJ-1, ApoA-1, MMP-9 and IL-8) were assessed by ELISA and compared among patients with and without bladder cancer recurrence (1) in the moment of urine collection and (2) during follow-up. Tumor recurrence was evaluated with cystoscopy, ultrasound and urine oncotic cytology and confirmed by pathological analysis. Patients with recurrence at urine collection were excluded from prediction analysis. A correlation between the level of the biomarkers and previous use of intravesical BCG was investigated. Results: Median follow-up was 47 months (IQR =39-50 months). Among patients included, 16 (10,5%; N =152) and 21 (15,4%; N =136) had bladder cancer recurrence diagnosed in the moment of urine collection and during follow-up, respectively. The panel to diagnose recurrence including 3 biomarkers (ApoA-1, MMP-9 and IL-8) presented OR =12,9 (CI =3,5-47,4), while the panel to predict patients who will have a recurrence during follow-up including 2 biomarkers (PAI-1 and IL-8) presented OR =4,1 (CI =1,4- 11,4). Previous use of intra-vesical BCG didn\'t influence urine biomarkers expression. Conclusions: The 3-biomarker panel can be used to identify patients with bladder cancer recurrence. The 2-biomarker panel can be used to predict patients at greater risk of bladder cancer recurrence during follow-up. Both are reliable in patients with previous use of intravesical BCG