No / TCDD-inducible poly-ADP-ribose polymerase (TIPARP; also known as PARP7 and ARTD14) is a mono-ADP- ribosyltransferase that has emerged as an important regulator of innate immunity, stem cell pluripotency, and transcription factor regulation. Characterizing TIPARP’s catalytic activity and identifying its target proteins are critical to understanding its cellular function. Here we describe methods that we use to characterize TIPARP catalytic activity and its mono-ADP-ribosylation of its target proteins.

Yes / The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and member of the basic helix-loop-helix-PAS family. AHR is activated by numerous dietary and endogenous compounds that contribute to its regulation of genes in diverse signaling pathways including xenobiotic metabolism, vascular development, immune responses and cell cycle control. However, it is most widely studied for its role in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. The AHR target gene and mono-ADP-ribosyltransferase, TCDD-inducible poly-ADP-ribose polymerase (TIPARP), was recently shown to be part of a novel negative feedback loop regulating AHR activity through mono-ADP-ribosylation. However, the molecular characterization of how AHR regulates TIPARP remains elusive. Here we show that activated AHR is recruited to the TIPARP promoter, through its binding to two genomic regions that each contain multiple AHR response elements (AHREs), AHR regulates the expression of both TIPARP but also TIPARP-AS1, a long non-coding RNA (lncRNA) which lies upstream of TIPARP exon 1 and is expressed in the opposite orientation. Reporter gene and deletion studies showed that the distal AHRE cluster predominantly regulated TIPARP expression while the proximal cluster regulated TIPARP-AS1. Moreover, time course and promoter activity assays suggest that TIPARP and TIPARP-AS1 work in concert to regulate AHR signaling. Collectively, these data show an added level of complexity in the AHR signaling cascade which involves lncRNAs, whose functions remain poorly understood. / This work was supported by Canadian Institutes of Health Research (CIHR) operating grants (MOP-494265 and MOP-125919), an unrestricted research grant from the Dow Chemical Company, and the Johan Throne Holst Foundation to J.M. G.G. was supported by European Union Seventh Framework Program (FP7-PEOPLE2013-COFUND) under the Grant Agreement n609020 - Scientia Fellows

La PARP-1 est une enzyme nucléaire qui modifie de façon post-traductionnelle plusieurs protéines nucléaires via son activité de poly(ADP-ribosyl)ation, souvent en réponse aux bris de l'ADN. Elle est ainsi impliquée dans plusieurs fonctions cellulaires vitales, incluant la réparation de l'ADN, la prolifération, la différenciation ainsi que la transcription de l'ADN pour n'en nommer que quelques- unes. Les promoteurs humains, de rat, de souris et de Drosophile du gène de la PARP-1 ont été clones et démontrent une structure commune aux gènes constitutifs (housekeeping). La transcription du gène de la PARP-1 est en partie régulée positivement par les facteurs de transcription Sp1 et Sp3 et négativement par le facteur de transcription NFI. Nous avons récemment démontré que les niveaux d'expression de la PARP-1, Sp1 et Sp3 sont fortement modulés par l'état de la densité et de la différenciation cellulaire chez des cultures primaires de cellules épithéliales de cornée de lapin (CECL). Par conséquent, la PARP-1 pourrait jouer un rôle durant la cicatrisation cornéenne puisqu'elle est fortement influencée durant les événements de migration, prolifération et différenciation qui caractérisent ce phénomène. La PARP-1 joue un rôle d'autant plus spécifique durant la cicatrisation cornéenne. En plus d'être influencée par la densité et la différenciation cellulaire, son expression est également fortement influencée par la présence de la fibronectine (FN). La FN, une protéine de la matrice extracellulaire, est sécrétée de façon massive durant la cicatrisation de l'épithélium cornéen. Considérant la relation étroite qui existe entre l'expression de la PARP-1 et de Sp1, nous avons également envisagé la possibilité que Sp1 soit une cible potentielle de la poly(ADP-ribosyl)ation par la PARP-1. PARP-1 pourrait ainsi réguler la transcription de son propre gène. En conclusion, notre étude a démontrée par quels mécanismes moléculaires la PARP joue son rôle durant la cicatrisation de l'épithélium cornéen. La PARP-1 pourrait ainsi constituer un modérateur de l'expression génique durant la phase hautement prolifique qui caractérise la cicatrisation cornéenne. / Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that post-translationally modifies a variety of proteins through its poly(ADP-ribosyl)ation activity in response to DNA strand break. PARP-1 is thus involved in several vital cellular functions such as DNA repair, cell proliferation and differentiation as well as DNA transcription. The promoter from the human, rat, mouse and Drosophila PARP-1 genes have been identified and cloned. Each of them has a structure common to housekeeping genes. PARP-1 gene transcription is positively regulated by the positive transcription factors Sp1 and Sp3 whereas it is repressed by members of the nuclear factor-l (NFI) family of transcription factors. We recently demonstrated that PARP-1, Sp1 and Sp3 expression was similarly influenced by both cell density and cell differentiation in primary cultures of rabbit corneal epithelial cells (RCECs). Because it may influence the migration, proliferation and differentiation properties of the epithelial cells from the cornea, PARP-1 may therefore play a significant function during corneal wound healing as well. We recently demonstrated that fibronectin, a major component from the extracellular matrix that is transitorily expressed during corneal wound healing, positively influenced the expression and DNA binding of Sp1 in RCECs. Similarly, PARP-1 gene expression strongly responded (positively) to the presence of FN in tissue culture plates, a further evidence that link PARP-1 expression to corneal wound healing as FN is massively secreted during that process. Moreover, and considering the close relationship between PARP-1 and Sp1 expression, we hypothesized that Sp1 could represent a potential targetfor poly(ADP-ribosyl)ation by PARP-1 and thereby alter its overall regulatory influence. We indeed demonstrated that Sp1 could be added polymers of ADPribose by PARP-1, a post-translational modification that also resulted in restricting the positive regulatory influence Sp1 might exert on its downstream target genes. In conclusion, PARP-1 indeed seems to be a potent regulator of gene expression during the highly proliferative phase that characterizes corneal wound healing.

Background: Lung dysfunction constitutes a severe complication after major cardiac surgery with cardiopulmonary bypass (CPB), substantially contributing to postoperative morbidity and mortality. The current possibilities of preventive and therapeutic interventions, however, remain insufficient. We, therefore, investigated the effects of intraoperative application of the antioxidant and anti-inflammatory green tea polyphenol epigallocatechin-3-gallate (EGCG) on CPB-associated lung injury. Materials and methods: Thirty piglets (8 - 15 kg) were divided into four groups: sham-operated and saline-treated control group (n = 7); sham-operated and EGCG-treated control group (EGCG-control group; n = 7); CPB group (n = 10); and CPB + EGCG group (n = 6). The CPB groups underwent 120 min of CPB followed by 90 min of recovery time. In the CPB + EGCG group, EGCG (10 mg/kg body weight) was administered intravenously before and after CPB. Hemodynamic monitoring, blood gas analysis, hematoxylin-eosin staining, and immunohistochemistry of lung tissue were performed. Results: Histologic examination revealed thickening of the alveolar wall and enhanced alveolar neutrophil infiltration in the CPB group (P < 0.05) compared with those in the control group, which was prevented by EGCG (P < 0.05). In the CPB group, higher formation of poly(ADP-ribose) and nuclear translocation of apoptosis-inducing factor were detected in comparison with those in the control group (P < 0.001), which were both reduced in the CPB + EGCG group (P < 0.001). Compared with the control group, the EGCG-control group showed thickening of the alveolar wall and increased neutrophil infiltration (P < 0.05). Conclusions: CPB leads to lung edema, pulmonary neutrophil infiltration, and presumably initiation of poly(ADP-ribose) polymerase-dependent cell death signaling in the lung. EGCG appears to attenuate CPB-associated lung injury, suggesting that this may provide a novel pharmacologic approach.

Les ribosomes sont responsables de la traduction des ARNm en protéines. Des modifications de la composition des ribosomes altèrent son activité de traduction et favorisent la tumorigenèse. L’identification des altérations de composition des ribosomes dans les cancers du sein pourrait être un nouveau mécanisme de tumorigenèse mammaire et ouvrir de nouvelles perspectives thérapeutiques. En effet, les cancers du sein restent la première cause de mortalité liés aux cancers chez la femme et leur hétérogénéité induit un problème thérapeutique important. Dans ce contexte, les altérations de composition des ribosomes dans les cancers du sein ont été abordées dans des cohortes humaines et dans des modèles cellulaires de l’EMT (Transition Epithélio-Mésenchymateuse), un processus impliqué dans la tumorigenèse mammaire. Ces travaux ont permis d’identifier : i) deux facteurs impliqués dans la biogenèse des ribosomes, FBL (fibrillarine) et NCL (nucléoline) dont les variations d’expression sont associées à un mauvais pronostic chez les patientes ; et ii) des variations de composition du ribosome et de son activité traductionnelle dans l’EMT. L’ensemble de ces résultats soutient l’existence d’altérations de composition des ribosomes dans les cancers du sein / Ribosomes are responsible of translating mRNAs to proteins. Alterations of ribosome composition modify its translation activity and favour tumourigenesis. Identification of ribosomes composition alterations in breast cancers might correspond to a new mechanism responsible of mammary tumourigenesis and might open up novel therapeutic approaches. Indeed breast cancers represent the first cause of women mortality due to cancers and their heterogeneity induces an important therapeutic problem.In this context, alterations of ribosomes composition were determined in human cohorts and in EMT (Epithelial to Mesenchymal Transition) cellular models, the EMT being a process involved in mammary tumourigenesis. This studies identify : (i) two factors involved in ribosome biogenesis, FBL (fibrillarin) and NCL (nucleolin) whose expression variations are associated with poor prognosis in patients and (ii) variations of ribosome composition and its translational activity in EMT. Altogether, this data support the presence of ribosomes composition alterations in breast cancers

Macrófagos infectados com Leishmania são protegidos de apoptose, entretanto não se conhece o mecanismo de transdução de sinal intracelular que interfere neste processo de morte. Neste trabalho, células J 774 em cultura, com privação de nutrientes, sofrem apoptose, a qual aumenta na presença dos indutores camptotecina (CPT) ou fator de necrose tumoral recombinante (rTNF). Estas células quando infectadas com amastigotas ou promastigotas de Leishmania (L.) chagasi (5 parasitos/uma célula) são protegidas de apoptose. Avaliando as possíveis vias intracelulares envolvidas nesse processo, observamos que a privação de nutrientes altera o potencial de membrana da mitocôndria, havendo reversão com a infecção tanto com promastigotas e amastigotas, entretanto a reversão da alteração do potencial de membrana induzida por rTNF só foi observada com infecção com promastigotas. Tanto a atividade de caspase 3, como a detecção de caspase 3 clivada induzidas por H202 são revertidas com a infecção com promastigotas ou amastigotas. Quando analisamos a expressão de poli (ADP ribose) polimerase (PARP), em relação às células sem indução, a indução por CPT não levou ao aumento da PARP de 116 kDa, mas, aumento da banda de 24 kDA. Por outro lado, a infecção por amastigota de Leishmania (L.) chagasi em células J774 levou à diminuição da expressão de PARP de 116 kDa, mas aumento da de 24 kDa. Nas células infectadas por promastigotas de Leishmania (L.) chagasi, observamos uma diminuição da banda de 116 kDa, aparecimento de uma molécula de 89kDa e diminuição da expressão da de 24 kDa. Nas células sob indução por CPT, a infecção levou a resultados similares, exceto a diminuição da molécula de 24 kDa quando infectado por amastigota. Avaliando-se a influência da proteína do choque térmico de 83 kDa de Leishmania infantum, como possível fator que interferiria no processo de apoptose, observamos que a fagocitose de bactérias Escherichia coli (M15) contendo plasmídio com gene de HSP83 expressando essa proteína, leva a diminuição da apoptose nessas células, mesmo quando induzidas por CPT ou rTNF. Nossos dados mostram que a infecção de macrófagos J774 in vitro por Leishmania (L.) chagasi, interfere no processo de apoptose afetando diversas vias de sinalização intracelular de apoptose, tanto extrínsecas quanto intrínsecas, sendo que promastigota é mais efetiva em inibir apoptose nesta linhagem macrofágica. / Macrophages infected by Leishmania are protected from apoptosis, however the mechanism of intracellular signal transduction that interferes in this death process remains unknown. In this work, J774 cells in culture, under nutrient deprivation undergo apoptosis, which is increased in the presence of inducers: camptothecin (CPT) or recombinant tumoral necrosis factor (rTNF). These cells infected by amastigotes or promastigotes of Leishmania (L.) chagasi (5 parasites per cell) are protected from apoptosis. Evaluating the possible intracellular pathways involved in this process, we observed nutrient deprivation alters the mitochondrial membrane potential, reversed by both amastigote and promastigote infection, in contrast, mitochondrial membrane potential was altered by rTNF and it was reversed only by promastigotes. Both caspase 3 activity and caspase 3 cleavage detection induced by H2O2 are reversed with amastigote or promastigote infection. When we analysed the expression of poly (ADP-ribose) polymerase, related to no induced cells . CPT induction didnLt increase 116 kDa PARP, but increased a 24 kDa fragment. Otherwise, Leishmania (L.) chagasi amastigote infection in J774 cells decreased 116 kDa PARP, but increased a 24 kDa fragment. Incells infected by Leishmania (L.) chagasi , we observed a decrease of 116 kDa fragment, appearance of a 89 kDa fragment and a decreasing of a 24 kDa fragment. In the cells under CPT induction similar results were found, except a decreasing of a 24 kDa when infected by amastigote. Evaluating the Leishmania (L.) infantum Heat Shock Protein of 83 kDa, as a possible factor that interferes in the apoptosis process, we observed that a phagocytosis of Escherichia coli (M15) bacteria with a HSP83 gene within a plasmid expressing this protein induced by isopropyl &#946; - D- tiogalactopiranosideo (IPTG), considerably diminished apoptosis in these cells even when induced by CPT or rTNF. Our data show that Leishmania (L.) chagasi infection in J774 macrophages in vitro notoriously interferes in the apoptosis process affecting several intracellular pathways involved in both extrinsic and intrinsic pathways, more prominently with promastigote in this macrophage cell lineage.

La Poly(ADP-ribosyl)ation est une modification post-traductionnelle de protéines, impliquée dans un grand nombre de processus biologiques, dont la réparation de l’ADN. Alors que la fonction et le mode d’action de la Poly(ADP-ribose) (PAR) Polymérase 1 (PARP1), activée en réponse aux dommages de l’ADN sont bien compris, on en sait beaucoup moins sur la fonction et la régulation de l’enzyme de dégradation du PAR, la Poly(ADP-ribose) glycohydrolase (PARG). Dans le contexte de ce projet de thèse, nous décrivons de nouvelles lignées U2OS stables, déficientes pour toutes les isoformes de PARG, permettant la complémentation inductible avec chacun des isoformes de PARG. Ces modèles nous ont permis d’évaluer les contributions relatives des isoformes à la réparation de dommages à l’ADN. Nous avons identifié un nouveau partenaire cellulaire de PARG : la protéine-kinase dépendante des dommages à l’ADN (DNA-PK). Nous explorons l’interaction fonctionnelle de ces deux protéines dans le contexte de la réponse cellulaire à la camptothécine (CPT), un agent anticancéreux inhibant la topoisomérase I et provoquant l’activation simultanée de PARP1 et DNA-PK. / Poly (ADP-ribosyl) ation is a post-translational modification of proteins involved in a large number of biological processes, including DNA repair. While the function and mode of action of Poly (ADP-ribose) (PAR) Polymerase 1 (PARP1), activated in response to DNA damage, is well understood, much less is known about the function and regulation the PAR degrading enzyme, Poly (ADP-ribose) glycohydrolase (PARG). In the context of this thesis project, we describe new stable U2OS lines, deficient for all PARG isoforms, allowing the inducible complementation with each of the PARG isoforms. These models allowed us to evaluate the relative contributions of the isoforms to DNA damage repair. We have identified a new cellular partner of PARG: the DNA-dependent protein kinase-dependent kinase (DNA-PK). We explore the functional interaction between these two proteins in the context of the cellular response to camptothecin (CPT), an anticancer drug that inhibits topoisomerase I and induces the simultaneous activation of PARP1 and DNA-PK.

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.

Macrófagos infectados com Leishmania são protegidos de apoptose, entretanto não se conhece o mecanismo de transdução de sinal intracelular que interfere neste processo de morte. Neste trabalho, células J 774 em cultura, com privação de nutrientes, sofrem apoptose, a qual aumenta na presença dos indutores camptotecina (CPT) ou fator de necrose tumoral recombinante (rTNF). Estas células quando infectadas com amastigotas ou promastigotas de Leishmania (L.) chagasi (5 parasitos/uma célula) são protegidas de apoptose. Avaliando as possíveis vias intracelulares envolvidas nesse processo, observamos que a privação de nutrientes altera o potencial de membrana da mitocôndria, havendo reversão com a infecção tanto com promastigotas e amastigotas, entretanto a reversão da alteração do potencial de membrana induzida por rTNF só foi observada com infecção com promastigotas. Tanto a atividade de caspase 3, como a detecção de caspase 3 clivada induzidas por H202 são revertidas com a infecção com promastigotas ou amastigotas. Quando analisamos a expressão de poli (ADP ribose) polimerase (PARP), em relação às células sem indução, a indução por CPT não levou ao aumento da PARP de 116 kDa, mas, aumento da banda de 24 kDA. Por outro lado, a infecção por amastigota de Leishmania (L.) chagasi em células J774 levou à diminuição da expressão de PARP de 116 kDa, mas aumento da de 24 kDa. Nas células infectadas por promastigotas de Leishmania (L.) chagasi, observamos uma diminuição da banda de 116 kDa, aparecimento de uma molécula de 89kDa e diminuição da expressão da de 24 kDa. Nas células sob indução por CPT, a infecção levou a resultados similares, exceto a diminuição da molécula de 24 kDa quando infectado por amastigota. Avaliando-se a influência da proteína do choque térmico de 83 kDa de Leishmania infantum, como possível fator que interferiria no processo de apoptose, observamos que a fagocitose de bactérias Escherichia coli (M15) contendo plasmídio com gene de HSP83 expressando essa proteína, leva a diminuição da apoptose nessas células, mesmo quando induzidas por CPT ou rTNF. Nossos dados mostram que a infecção de macrófagos J774 in vitro por Leishmania (L.) chagasi, interfere no processo de apoptose afetando diversas vias de sinalização intracelular de apoptose, tanto extrínsecas quanto intrínsecas, sendo que promastigota é mais efetiva em inibir apoptose nesta linhagem macrofágica. / Macrophages infected by Leishmania are protected from apoptosis, however the mechanism of intracellular signal transduction that interferes in this death process remains unknown. In this work, J774 cells in culture, under nutrient deprivation undergo apoptosis, which is increased in the presence of inducers: camptothecin (CPT) or recombinant tumoral necrosis factor (rTNF). These cells infected by amastigotes or promastigotes of Leishmania (L.) chagasi (5 parasites per cell) are protected from apoptosis. Evaluating the possible intracellular pathways involved in this process, we observed nutrient deprivation alters the mitochondrial membrane potential, reversed by both amastigote and promastigote infection, in contrast, mitochondrial membrane potential was altered by rTNF and it was reversed only by promastigotes. Both caspase 3 activity and caspase 3 cleavage detection induced by H2O2 are reversed with amastigote or promastigote infection. When we analysed the expression of poly (ADP-ribose) polymerase, related to no induced cells . CPT induction didnLt increase 116 kDa PARP, but increased a 24 kDa fragment. Otherwise, Leishmania (L.) chagasi amastigote infection in J774 cells decreased 116 kDa PARP, but increased a 24 kDa fragment. Incells infected by Leishmania (L.) chagasi , we observed a decrease of 116 kDa fragment, appearance of a 89 kDa fragment and a decreasing of a 24 kDa fragment. In the cells under CPT induction similar results were found, except a decreasing of a 24 kDa when infected by amastigote. Evaluating the Leishmania (L.) infantum Heat Shock Protein of 83 kDa, as a possible factor that interferes in the apoptosis process, we observed that a phagocytosis of Escherichia coli (M15) bacteria with a HSP83 gene within a plasmid expressing this protein induced by isopropyl &#946; - D- tiogalactopiranosideo (IPTG), considerably diminished apoptosis in these cells even when induced by CPT or rTNF. Our data show that Leishmania (L.) chagasi infection in J774 macrophages in vitro notoriously interferes in the apoptosis process affecting several intracellular pathways involved in both extrinsic and intrinsic pathways, more prominently with promastigote in this macrophage cell lineage.

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.