Les accidents vasculaires cérébraux (AVC) constituent la 3e cause de mortalité dans les pays industrialisés, et sont à 80% de type ischémique (AVCi). A l’heure actuelle, le seul traitement disponible est l’activateur tissulaire du plasminogène recombinant (rt-PA), dont l’utilisation est très limitée, en raison d’une fenêtre thérapeutique étroite et de l’augmentation du risque de transformations hémorragiques (TH). Après un AVCi, les cliniciens sont confrontés, entre autres, à 3 objectifs d’ordre vasculaire: (1) reperfuser les tissus ischémiés, (2) éviter les TH, ainsi que (3) les ré-occlusions précoces ou tardives. Les travaux du laboratoire ont précédemment établi qu’après une ischémie cérébrale (IC), l’hyperactivation de la poly(ADP-ribose)polymérase (PARP), une enzyme nucléaire, est (1) neurotoxique et (2) contribue aux TH spontanées ou induites par le rt-PA. Par ailleurs, des études suggèrent que les inhibiteurs de PARP pourraient également réduire les phénomènes de ré-occlusion, en inhibant l’activation/agrégation plaquettaires, et ceci via 2 mécanismes : (1) « PARP-indépendant », lié à une analogie structurale de certains inhibiteurs de PARP avec des agonistes plaquettaires, comme l’ADP, et (2) « PARP-dépendant », lié à leur effet anti-inflammatoire. Cependant, à l’heure actuelle, il n’existe aucune donnée dans l’IC. Dans ce contexte, ce travail a consisté à évaluer les effets de plusieurs inhibiteurs de PARP sur l’activation et l’agrégation plaquettaire. Il nous est notamment apparu nécessaire de rechercher si la réduction des TH par les inhibiteurs de PARP pourrait être liée, au moins en partie, à une activité pro-agrégante, qui compromettrait leur association avec le rt-PA. A l’inverse, une activité anti-agrégante, bien que favorisant les hémorragies, pourrait améliorer la reperfusion ou diminuer les risques de ré-occlusion. Dans la 1ère partie, nos résultats montrent in vitro que deux inhibiteurs de PARP (PJ34 et minocycline) sont anti-agrégants plaquettaires, et que cet effet serait « PARP-indépendant », puisque deux autres inhibiteurs de PARP, le 3-aminobenzamide et l’INO-1001, n’ont pas modifié l’agrégation. De plus, sur du sang humain, mais pas murin, le PJ34 exerce un effet anti-agrégant, qui pourrait être lié à un antagonisme du récepteur à l’ADP, P2Y12. La 2nde partie a été réalisé sur des modèles in vivo chez la souris. L’utilisation de 3 tests d’exploration des fonctions plaquettaires (temps de saignement, modèles de thromboembolie pulmonaire et de thrombose carotidienne par le FeCl3) a mis en évidence l’absence d’effet du PJ34 et de la minocycline sur les fonctions plaquettaires, et notamment, pas d’effet pro-agrégant pouvant expliquer la réduction des TH. Dans un modèle de thrombose de l’artère cérébrale moyenne par le FeCl3, le PJ34 n’entrave pas la thrombolyse par le rt-PA, mais au contraire, pourrait tendre à l’améliorer. Parallèlement, dans un modèle d’IC chez la souris, nos travaux ont mis en évidence une augmentation cérébrale de l’adhésion des plaquettes et de l’expression d’ICAM-1. La suite de cette étude sera d’étudier si les inhibiteurs de PARP, en protégeant la paroi vasculaire, pourraient réduire les phénomènes de ré-occlusion. L’ensemble de ce travail s’inscrit dans une thématique plus globale de notre laboratoire qui vise à identifier l’intérêt d’associer un inhibiteur de PARP au rt-PA pour une meilleure prise en charge de la thrombolyse post-AVCi. / Stroke is the 3rd leading cause of death in industrialized countries and 80% are ischemic. The recombinant tissue-plasminogen activator (rt-PA) is currently the only available treatment but its use remains very limited due to a narrow therapeutic window and an increased risk of hemorrhagic transformation (HT). After ischemic stroke, the key vascular objectives of clinicians are : (1) to reperfuse ischemic tissue, (2) to avoid both HT and (3) early or late reocclusions. Our laboratory previously established that after cerebral ischemia (CI), the overactivation of poly(ADP-ribose)polymerase (PARP), a nuclear enzyme, is (1) neurotoxic and (2) contributes to spontaneous or rt-PA-induced HT. Moreover, studies suggest that PARP inhibitors could also reduce the risk of reocclusion by inhibiting platelet activation/aggregation via two mechanisms : (1) one is "PARP-independent" and linked to a structural analogy of certain PARP inhibitors with platelet agonists such as ADP, and (2) the second one is "PARP-dependent" and due to their anti-inflammatory effect. However, so far, there is no data in CI.In this context, the aim of this work was to evaluate the effects of several PARP inhibitors on platelet activation and aggregation. In particular, it appeared necessary to examine whether the reduction of HT by PARP inhibitors could be related, at least in part, to a pro-aggregatory activity, which would then compromise their association with rt-PA. By contrast, an anti-aggregatory activity could improve reperfusion or reduce the risk of reocclusion, although it would also contribute to hemorrhage. In the 1st part, our results show that, in vitro, two PARP inhibitors (PJ34 and minocycline) are antiplatelet agents and that this effect is "PARP-independent" since two other PARP inhibitors, 3-aminobenzamide and INO-1001 did not alter the aggregation. Moreover, in human blood but not in murine one, PJ34 exerts an anti-aggregatory effect which may be related to the antagonism of the ADP receptor P2Y12. The 2nd part was performed on in vivo models in mice. The use of three tests of platelet function exploration (bleeding time and models of pulmonary thromboembolism and FeCl3-induced carotid thrombosis) showed no effect of minocycline and PJ34 on platelet function and in particular, no pro-aggregatory effect which may explain the reduction of HT. In a thrombosis model of the middle cerebral artery by FeCl3, PJ34 does not impede the thrombolysis induced by rt-PA, but even tends to improve it. Meanwhile, in a CI model in mice, our work shows an increase of platelet adhesion and ICAM-1 expression in the brain. The next step will be to investigate whether PARP inhibitors could reduce reocclusions by protecting the vascular wall. All this work is part of a broader topic of our laboratory aims to identify the interest of combining a PARP inhibitor with rt-PA for a better management of post-ischemic thrombolysis.

Les modifications post-traductionnelles des protéines par des polymères d’ADP-ribose (PAR) ou par phosphorylation permet l’assemblage des complexes de la réparation de l’ADN à la chromatine endommagée dont les fonctions sont essentielles pour assurer le maintien de la stabilité du génome. En réponse aux lésions de l’ADN, l’activité de synthèse de PAR des protéines PARP1 et PARP2 est fortement stimulée. Les PAR servent de signalisation pour le recrutement de multiples protéines, dont la protéine plateforme XRCC1.Les études menées au cours de cette thèse ont porté sur l’étude de la régulation des fonctions des protéines PARP1, PARP2 dans la réparation des cassures double brins (CDB) et l’étude des modifications de XRCC1 par phosphorylation en réponse à des dommages de l’ADN. En utilisant des substrats permettant de mesurer l’efficacité des différentes voies de réparation des CDB, nous avons démontré que PARP2, et non PARP1, est impliqué dans la régulation du choix des voies de la réparation des CDB. Plus spécifiquement, nous avons montré que PARP2 stimule l’initiation de la résection des extrémités des CDB dépendante de CtIP, indépendamment de son activité catalytique. Par des approches de vidéo-microscopie, nous avons pu déterminer que PARP2 limite l’accumulation de 53BP1 aux sites de dommages induits par micro-irradiation laser. Nous proposons que la protéine PARP2, en limitant le recrutement de la protéine 53BP1 aux sites de dommages, favorise la réparation des CDB dépendante de la résection des extrémités d’ADN, au détriment de la voie canonique de jonction des extrémités. Ces résultats sont les premiers démontrant un rôle de PARP2 dans le choix des voies de réparation des CDB.En parallèle, nous avons analysé comment la phosphorylation régule les fonctions de la protéine XRCC1. Par des approches in vitro et in vivo, nous avons pu déterminer que l’interdomaine 1 de XRCC1 est phosphorylé par la kinase CDK5. En réponse aux dommages induits par un agent alkylant, XRCC1 est activement déphosphorylé in vivo. De plus, nous avons observé que lorsque l’interdomaine 1 ne peut pas être phosphorylé in vitro, l’interaction de XRCC1 avec les PAR synthétisés par PARP1 et PARP2 augmente, et le recrutement de XRCC1 aux sites de dommages de l’ADN est accru. Ces résultats indiquent pour la première fois que la déphosphorylation de XRCC1 en réponse à un stress génotoxique participe activement à son recrutement aux sites de dommages.Dans leur ensemble, ces travaux ont contribué à améliorer nos connaissances fondamentales des réseaux de protéines impliquées dans la prise en charge des dommages de l’ADN. La compréhension de ces mécanismes est essentielle non seulement car ils participent au maintien de la stabilité du génome mais aussi du fait du développement exponentiel de nouvelles stratégies anti-tumorales qui visent à inhiber les voies de la réparation dans la but de cibler spécifiquement les cellules cancéreuses. / Post-translational modifications of proteins by polymers of ADP-ribose (PAR) or by phosphorylation allow the assembly of DNA repair protein complexes at damaged chromatin and are crucial to ensure genome stability. In response to DNA insults, the synthesis of PAR by the PARP1 and PARP2 proteins is strongly induced. PAR act as a signaling platform for the recruitment of multiples proteins at the sites of DNA damages, including the scaffold protein XRCC1. Research conducted during this PhD have been focused on studying the regulation of PARP1 and PARP2 functions in double-strands break repair (DSBR), and in investigating the role of XRCC1 modifications by phosphorylation in response to DNA damage.Using DNA repair assay allowing us to assess the accuracy of the different DSBR pathways, we demonstrated that PARP2, and not PARP1, is involved in the regulation of DNA double-strands break repair pathway choice. More precisely, we showed that PARP2 stimulates CtIP dependent initiation of end-resection at DSB, independently of its catalytic activity. By live cell imaging, we were able to determine that PARP2 limit 53BP1 accumulation at DNA damage sites induced by laser-microirradiation. We propose that by limiting 53BP1 accumulation at DNA damage sites, PARP2 stimulate DSB repair pathway that depend on DNA end-resection, thus counteracting the canonical end-joining pathway. These results are the first demonstrating a role for PARP2 in DNA DBSR pathway choice.In addition, we analyzed how the functions of XRCC1 are regulated by phosphorylation. Using in vitro and in vivo approaches, we were able to demonstrate that the linker 1 region of XRCC1 is phosphorylated by the CDK5 kinase. XRCC1 is actively dephosphorylated in response to DNA damage induced by an alkylating agent in vivo. We also observed that when the linker 1 cannot be phosphorylated, the XRCC1 interaction between the PAR synthetized by PARP1 and PARP2 is stimulated, and XRCC1 recruitement at the sites of DNA damage is far more efficient. These evidences indicate for the first time that the dephosphorylation of XRCC1 actively participate in its recruitment at the site of DNA damage. Put together, this work contributed to strengthen our fundamental knowledge of the protein network involved in the DNA damage response. Knowledge of those mechanisms is crucial since they participate in maintaining genome stability, and because new antitumoral drugs targeting DNA repair pathways in the attempt to specifically killed tumor cells are exponentially released.

The metabolism of ADP-ribose polymers (PAR) is involved in several cellular processes with a primary focus on maintaining genomic integrity. PAR metabolism following genotoxic stress is transient due to a close coordination between poly(ADP-ribose) polymerases (PARPs) which synthesize PAR and poly(ADP-ribose) glycohydrolase (PARG) which degrades PAR. PARP-1 inhibitors have emerged as promising anticancer therapeutics by increasing chemotherapy sensitivity and selectively target tumors harboring DNA repair defects. Several pharmaceutical companies have PARP-1 inhibitors in clinical trials for treatment of cancer. PARP-1 inhibitors are generally well tolerated, although they typically have poor selectivity among PARPs, and potentially other NAD binding enzymes. The promise of PARP-1 inhibitors as cancer therapeutics has led this dissertation research towards developing alternative tools and approaches to target PAR metabolism.One approach described is an evaluation of high-throughput PARP-1 screening assays as potential tools to discover new classes of PARP-1 inhibitors. These assays were compared to a widely used radiolabeling PARP-1 assay. They were found to offer several advantages that include simplicity, sensitivity, reproducibility, accuracy and eliminating the need for radioactive materials.The primary focus of this dissertation research was to develop PARG inhibitors as an alternative way of targeting PAR metabolism. Lack of viable genetically engineered animals, effective siRNA, and useful pharmacological 20 inhibitors has prevented PARG from being evaluated as a therapeutic target. This dissertation describes the first systematic approach, using Target related Affinity Profiling (TRAP) technology, for the discovery of PARG inhibitors. Identification of several hits led to the first detailed structural activity relationship (SAR) studies defining a pharmacophore for PARG inhibition. Interestingly, these molecules show varying degrees of PARP-1 inhibition, providing the first direct evidence for homology in the active sites of PARP-1 and PARG. Evaluation of a lead inhibitor has provided the first evidence for PARG inhibition in intact cells. Further optimization resulted in a cell permeable inhibitor with reduced toxicity and poor selectivity, providing evidence for a new class of inhibitors that disrupt PAR metabolism by inhibiting both enzymes. The use of dual PARG/PARP-1 inhibitors represents a new approach for therapeutic development of anticancer agents. Finally, directions aimed to overcome remaining challenges are discussed.

No description available.

Previous studies have shown extensive cellular damage can activate poly(ADP-ribose) polymerase-1 (PARP-1) and cause a rapid decrease in the levels of NAD+ and ATP, thereby preventing apoptosis and promoting necrosis and inflammation. The purpose of this study was to extend previous observations that inhibitors of PARP-1 could alter acetaminophen and carbon tetrachloride-induced hepatotoxicity. Bromobenzene (BB) a glutathione dependent hepatotoxicant was tested. Groups of male mice were treated with a single dosage of 112mg/kg (0.075 ml/kg) BB by the intraperitoneal (ip) route. All animals were maintained in a controlled environment and provided food and water ad libitum. This dosage of BB resulted in hepatotoxicity as measured by an increase in serum alanine transferase (ALT). BB treatment resulted in a 5-fold increase in ALT. Moderate hepatotoxicity was detected with this treatment regime. Subsequently, another group of mice were treated with three treatments of nicotinamide at 0.5, 1 and 2 hours following BB treatment. Serum ALT elevations were reduced by 90% at 24 hours following BB and nicotinamide treatments. BB-induced liver pathology was also blocked by nicotinamide. Mortality among BB treated animals was also significantly reduced by nicotinamide treatment. Mortality among mice treated with BB and nicotinamide was near control. The model was verified with a more potent and specific inhibitor, Phen. BB treatment was keep at the same level as in the previous study, and Phen was administered concomitantly. Serum ALT elevations were reduced by 75%. Phen also blocked BB-induced liver pathology. Mortality among mice treated with BB and Phen was reduced 75%. PARP-1 inhibitors appear to alter chemical-induced hepatotoxicity that has either a glutathione dependent or independent mechanism.

The specific interactions of macromolecules along with the activity of enzymes are central to all aspects of biology. It is well recognized that when the relative concentration or activity of macromolecules is perturbed, it can lead to human diseases. Thus, the development of simple methods for the detection of macromolecules and the activity of enzymes in complex environments is important for understanding biology. Moreover, the development of methods for measuring interactions allows for the testing of inhibitors that can be used as tools or drugs for improving human health. Towards this goal, a promising new method has been developed, which is the focus of this thesis, called split-protein reassembly or protein fragment complementation. In this method, a protein reporter, such as the green fluorescent protein or firefly luciferase, is dissected into two fragments, which are attached to designed adaptor proteins. The designed split-protein systems only produce a measurable signal, either fluorescence or luminescence, when a specific macromolecular interaction or activity is present. In this thesis, I have extended previous research on the direct detection of DNA using split-protein sensors utilizing a red fluorescent protein, dsRED from Discosoma that allows for multiplexed DNA detection. I have designed a new split-luciferase based sensor for detection of poly (ADP-ribose) or PAR, which plays a key role in the response to DNA damage and have applied it for monitoring the activity of poly (ADP-ribose) glycohydrolase that controls PAR levels in the cell. Furthermore, I have significantly expanded upon a three-hybrid split-luciferase system for identifying protein kinase inhibitors. I have designed and tested two orthogonal peptide based chemical inducers of dimerization based on BAD and p53mt conjugates. I have studied these chemically induced dimerization systems in detail in order to begin to provide a theoretical basis for the observed experimental results. Finally, in a less related area, I have developed methods for producing water soluble semiconductor nanoparticles called Quantum Dots (QDs), with potential application in biological imaging. I have developed methods for functionalizing the QDs with orthogonal peptides, which can be potentially used for the assembly of high affinity non-covalent QD targeted proteins.

Thèse (Ph. D.)--Université Laval, 2008. / Bibliogr.: f. 250-258. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

ADP-ribose polymer metabolism has been studied in human cells derived from a patient with Glutamyl Ribose Phosphate Storage Disease (GRPSD) and in mouse C3H1OT1/2 cells following oxidative stress induced by hydrogen peroxide (H202 ). It has been postulated that GRPSD resulted from an abnormality in ADP-ribose polymer metabolism. This study has shown that these cells exhibit reduced poly(ADP ribose) polymerase activity which is proposed to result from modification of the enzyme with ribose phosphate groups. The modification in the polymerase is proposed to be secondary to a defect in either ADP-ribosyl proteinlyase or an overproduction of a cellular phosphodiesterase. The metabolism of ADP-ribose polymers was rapidly altered by H202 and there were independent effects on adenine nucleotide pools. The results suggest that ADP-ribose polymer metabolism is involved in cellular defenses to oxidative stress.

A method for assaying poly (ADP-ribose) polymerase (PADPRP) activity in lymphocytes of systemic lupus erythematosus (SLE) patients has been developed. Using this method, PADPRP activity has been studied in lymphocytes from 15 patients and 13 controls. The mean activity in SLE lymphocytes was significantly lower than that in controls and 60% of the SLE patients demonstrated activities below the minimum of the control population. Possible mechanisms for this altered metabolism were investigated. The Km app of PADPRP for NAD; size distribution, branch frequency, and rates of turnover of polymers; competition for substrate; and number of PADPRP molecules were studied. The data demonstrated that SLE lymphocytes have a decreased synthetic capacity rather than alterations in the substrate or in turnover of the product.