Made available in DSpace on 2014-12-17T14:03:36Z (GMT). No. of bitstreams: 1 JullianeTAM_TESE_Parcial.pdf: 7138229 bytes, checksum: a06700fccf67cc2db086f62bf86db506 (MD5) Previous issue date: 2014-02-26 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / studies using UV as a source of DNA damage. However, even though unrepaired UV-induced DNA damages are related to mutagenesis, cell death and tumorigenesis, they do not explain phenotypes such as neurodegeneration and internal tumors observed in patients with syndromes like Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS) that are associated with NER deficiency. Recent evidences point to a role of NER in the repair of 8-oxodG, a typical substrate of Base Excision Repair (BER). Since deficiencies in BER result in genomic instability, neurodegenerative diseases and cancer, it was investigated in this research the impact of XPC deficiency on BER functions in human cells. It was analyzed both the expression and the cellular localization of APE1, OGG1 e PARP-1, the mainly BER enzymes, in different NER-deficient human fibroblasts. The endogenous levels of these enzymes are reduced in XPC deficient cells. Surprisingly, XP-C fibroblasts were more resistant to oxidative agents than the other NER deficient fibroblasts, despite presenting the highest of 8-oxodG. Furthermore, subtle changes in the nuclear and mitochondrial localization of APE1 were detected in XP-C fibroblasts. To confirm the impact of XPC deficiency in the regulation of APE1 and OGG1 expression and activity, we constructed a XPC-complemented cell line. Although the XPC complementation was only partial, we found that XPC-complemented cells presented increased levels of OGG1 than XPC-deficient cells. The extracts from XPC-complemented cells also presented an elevated OGG1 enzimatic activity. However, it was not observed changes in APE1 expression and activity in the XPCcomplemented cells. In addition, we found that full-length APE1 (37 kDa) and OGG1- ? are in the mitochondria of XPC-deficient fibroblasts and XPC-complemented fibroblasts before and after induction of oxidative stress. On the other hand, the expression of APE1 and PARP-1 are not altered in brain and liver of XPC knockout mice. However, XPC deficiency changed the APE1 localization in hypoccampus and hypothalamus. We also observed a physical interaction between XPC and APE1 proteins in human cells. In conclusion, the data suggest that XPC protein has a role in the regulation of OGG1 expression and activity in human cells and is involved mainly in the regulation of APE1 localization in mice. Aditionally, the response of NER deficient cells under oxidative stress may not be only associated to the NER deficiency per se, but it may include the new functions of NER enzymes in regulation of expression and cell localization of BER proteins / A maior parte do nosso conhecimento sobre a via de Reparo de Excis?o Nucleot?deos (NER) vem de estudos usando a luz ultravioleta (UV) como fonte de danos no DNA. Contudo, embora os danos no DNA causados pela luz UV sejam relacionados ? ocorr?ncia de mutag?nese, morte celular e tumorig?nese, eles n?o justificam fen?tipos como neurodegenera??o e tumorig?nese observados em pacientes com s?ndromes como Xeroderma Pigmentosum (XP) e S?ndrome de Cockayne (CS), as quais s?o associadas ? defici?ncia na via NER. Adicionalmente, evid?ncias mais recentes indicam o envolvimento da via NER no reparo de 8-oxodG, um substrato t?pico da via de Reparo por Excis?o de Bases (BER). Uma vez que a defici?ncia na via BER resulta em instabilidade gen?mica, doen?as neurodegenerativas e c?ncer, foi investigado neste trabalho o impacto da defici?ncia em XPC nas fun??es da via BER em c?lulas humanas. Foram realizadas an?lises da express?o e da localiza??o celular de APE1, OGG1 e PARP-1, principais enzimas da via BER, em fibroblastos humanos deficientes na via NER. Os resultados demonstraram que os n?veis end?genos de APE1, PARP-1 e OGG1 s?o reduzidos nos fibroblastos deficientes em XPC, os quais foram mais resistentes a diferentes tipos de agentes oxidantes e apresentaram n?veis elevados de 8-oxodG quando comparados aos demais fibroblastos deficientes na via NER. Adicionalmente, altera??es sutis na localiza??o nuclear e mitocondrial de APE1 foram observadas nos fibroblastos deficientes em XPC. Para confirmar o impacto da defici?ncia de XPC na regula??o da express?o e atividade de APE1 e OGG1, foi constru?da uma linhagem complementada com XPC. Embora a complementa??o tenha sido parcial, foi poss?vel observar que os fibroblastos parcialmente complementados com XPC apresentaram n?veis maiores de express?o de OGG1 quando comparados aos fibroblastos deficientes em XPC. Os extratos dos fibroblastos parcialmente complementados com XPC tamb?m apresentaram uma elevada atividade enzim?tica de OGG1. Contudo, n?o foram observadas mudan?as na express?o e atividade de APE1 nos fibroblastos parcialmente complementados com XPC. Adicionalmente, foi poss?vel verificar a presen?a da forma completa de APE1 (37 kDa) e de OGG1-? na mitoc?ndria dos fibroblastos deficientes em XPC e parcialmente complementados com XPC. Por outro lado, observou-se que a express?o de APE1 e PARP-1 n?o ? alterada no c?rebro e f?gado de camundongos knockouts para XPC. Contudo, a defici?ncia em XPC resultou em mudan?as na localiza??o celular de APE1 no hipocampo e hipot?lamo. Ainda, foi observada a ocorr?ncia de uma intera??o f?sica entre as prote?nas XPC e APE1 em c?lulas humanas. Em conclus?o, os dados sugerem que a prote?na XPC possui um papel na regula??o da express?o e da atividade de OGG1 em c?lulas humanas e est? envolvida na regula??o da localiza??o celular de APE1 principalmente em camundongos. Adicionalmente, as respostas celulares dos fibroblastos deficientes na via NER ao estresse oxidativo podem n?o estar associadas ? defici?ncia na via NER per se, mas podem incluir novas fun??es das enzimas da via NER na regula??o da express?o e localiza??o celular das prote?nas da via BER / 2020-01-01

La stabilité du génome est compromise par les déséquilibres du pool de dNTPs qui affectent la vitesse de progression des fourches de réplication. Par exemple, la déficience en cytidine désaminase (CDA) conduit à un excès de dCTP qui induit un ralentissement des fourches de réplication. Les résultats obtenus au cours de ma thèse ont permis de mettre en évidence un nouveau mécanisme par lequel un déséquilibre du pool de nucléotides compromet la complétion de la réplication et la ségrégation correcte des chromosomes. En utilisant des techniques de peignage moléculaire, de microscopie électronique et d’imagerie cellulaire permettant de quantifier le niveau basal de PAR, nous avons montré que la réplication incomplète de l’ADN lorsque la CDA est absente est due à l’inhibition partielle de PARP-1, et n’est pas liée au ralentissement de la vitesse de progression des fourches de réplication. En effet, l’accumulation intracellulaire de dCTP inhibe l’activité de PARP-1 ce qui réduit l’activation de Chk1 et l’efficacité des points de contrôle situés en aval, favorisant ainsi l’accumulation de séquences d’ADN non répliquées en mitose. Celles-ci conduisent alors à la formation de ponts anaphasiques ultrafins (UFBs) entre les chromatides sœurs au niveau de sites difficiles à répliquer tels que les centromères et les sites fragiles. Ces résultats ont des implications directes dans le syndrome de Bloom (BS), une maladie génétique rare combinant prédisposition aux cancers et instabilité génétique. Ce syndrome est la conséquence de la mutation du gène BLM, codant pour une hélicase RecQ du même nom. La déficience en BLM conduit à une chute de l’expression de la CDA résultant en une augmentation des UFBs entièrement due à l’inhibition de PARP-1 par la dCTP, indépendamment de BLM. Ces travaux décrivent ainsi une conséquence pathologique encore inconnue du déséquilibre du pool de nucléotides et révèlent un rôle inattendu de PARP-1 dans la surveillance des séquences d’ADN non répliquées prévenant leur accumulation en mitose et les défauts de ségrégation des chromosomes associés. / Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that in CDA-deficient cells DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. Indeed, the intracellular accumulation of dCTP inhibits PARP-1 activity compromising the activation of Chk1 and the downstream checkpoints efficiency, allowing the subsequent accumulation of unreplicated DNA in mitosis. This unreplicated DNA leads to the formation of ultrafine anaphase bridges (UFBs) between sister-chromatids at “difficult-to-replicate” sites such as centromeres and fragile sites. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3’-5’ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of UFBs due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our results describe previously unknown pathological consequences of the distortion of dNTP pools and reveal an unexpected role for PARP-1 in preventing unreplicated DNA accumulation in mitosis and in preventing chromosome segregation defects.

Para que ocorra a transição do epitélio normal de laringe para carcinoma escamoso é necessário um processo de múltiplas etapas tal como exposição prolongada ao fumo e álcool e uma possível associação à infecção pelo HPV. Vários tipos de marcadores moleculares vêm sendo estudados na carcinogênese da laringe, entre eles proteínas associadas a apoptose (bcl-2 e PARP-1) assim como proteínas envolvidas em múltiplos processos biológicos como a Galectina-3. Neste estudo foram realizadas análise imunoistoquímica quantitativa e qualitativa para bcl-2, PARP-1 e galectina-3 em 65 pacientes diagnosticados com câncer de laringe subdivididos em: carcinoma de laringe in situ (CLIS), carcinoma de laringe com metástase (CLM), sem metástase (CLS) e linfonodos cervicais (LC). A detecção e tipificação do HPV foram realizadas pela reação em cadeia da polimerase (PCR) e os tipos de HPV avaliados foram HPV 6, 11, 16, 18, 31 e 33. Na avaliação quantitativa de galectina-3 observou-se um significativo aumento de expressão no carcinoma invasivo de laringe (CLS e CLM) quando comparado com carcinoma in situ (CLIS), podendo concluir que essa proteína seria um bom marcador pra progressão de câncer de laringe. Para as proteínas PARP-1 e bcl-2 não houve diferença nos níveis de expressão nos grupos analisados. Na análise qualitativa PARP-1 apresentou uma homogeneidade de marcação tanto alta como baixa entre os grupos. Em relação à Galectina-3 observou-se um predomínio de casos com alta expressão, diferentemente da proteína bcl-2 onde o predomínio foi de baixa expressão em todos os casos de carcinoma de laringe e seus respectivos linfonodos metastáticos. Dos 65 pacientes, 55 (84,6%), foram positivos para beta-globina e 7 (12.7%) dos 55 pacientes foram positivos para HPV. Não foi possível verificar quaisquer correlações entre as proteínas Galectina-3, bcl-2 e PARP-1 e o HPV devido ao baixo índice de casos positivos. / To occur the transition from normal epithelium to squamous cell carcinoma is a necessary a for multiple stages process, such as smoking and alcohol abuse and a possible association with HPV infection. Several types of molecular markers have been studied in cancer of larynx, including proteins associated with apoptosis (bcl-2 and PARP-1) and proteins involved in many biological process such as galectin-3. In this study, analyses of qualitative and quantitative immunohistochemistry was performed for bcl-2, PARP-1 and galectin-3 in 65 patients diagnosed with laryngeal squamous cell carcinoma divided into in situ laryngeal carcinomas(LSCCS), laryngeal squamols cells carcinomas without metastases (LSCCWT) and with metastasis (LSCCW) and cervical lymph nodes (CL). HPV detection and typing was performed by PCR and the HPV types evaluated were HPV 6, 11, 16, 18, 31 and 33. In quantitative of galectin-3 there was observed a significant increase of expression in invasive laryngeal squamous cell carcinoma (LSCCWT and LSCCW) compared with in situ laryngeal carcinomas (LSCCS), may indicating that this protein could be a good marker for progression of laryngeal carcinoma. For PARP-1 and bcl-2 protein there was no difference in the levels of expression in all groups studied. In qualitative analysis PARP-1 showed a homogenous immunolabeling in both high and low among the groups. In relation to Galectin-3, it was observed a predominance of cases with high expression, unlike the protein bcl-2 where the expression prevalence was low in all cases of laryngeal carcinoma and their metastatic lymph nodes. Of the 65 patients, 55 (84.6%) were positive for beta-globin and 7 (12.7%) of 55 patients were positive for HPV. Because of a low incidence of HPV in the cases studied, it was not possible correlate the proteins bcl-2, PARP-1 and Galectin-3 with the presence of HPV.

Para que ocorra a transição do epitélio normal de laringe para carcinoma escamoso é necessário um processo de múltiplas etapas tal como exposição prolongada ao fumo e álcool e uma possível associação à infecção pelo HPV. Vários tipos de marcadores moleculares vêm sendo estudados na carcinogênese da laringe, entre eles proteínas associadas a apoptose (bcl-2 e PARP-1) assim como proteínas envolvidas em múltiplos processos biológicos como a Galectina-3. Neste estudo foram realizadas análise imunoistoquímica quantitativa e qualitativa para bcl-2, PARP-1 e galectina-3 em 65 pacientes diagnosticados com câncer de laringe subdivididos em: carcinoma de laringe in situ (CLIS), carcinoma de laringe com metástase (CLM), sem metástase (CLS) e linfonodos cervicais (LC). A detecção e tipificação do HPV foram realizadas pela reação em cadeia da polimerase (PCR) e os tipos de HPV avaliados foram HPV 6, 11, 16, 18, 31 e 33. Na avaliação quantitativa de galectina-3 observou-se um significativo aumento de expressão no carcinoma invasivo de laringe (CLS e CLM) quando comparado com carcinoma in situ (CLIS), podendo concluir que essa proteína seria um bom marcador pra progressão de câncer de laringe. Para as proteínas PARP-1 e bcl-2 não houve diferença nos níveis de expressão nos grupos analisados. Na análise qualitativa PARP-1 apresentou uma homogeneidade de marcação tanto alta como baixa entre os grupos. Em relação à Galectina-3 observou-se um predomínio de casos com alta expressão, diferentemente da proteína bcl-2 onde o predomínio foi de baixa expressão em todos os casos de carcinoma de laringe e seus respectivos linfonodos metastáticos. Dos 65 pacientes, 55 (84,6%), foram positivos para beta-globina e 7 (12.7%) dos 55 pacientes foram positivos para HPV. Não foi possível verificar quaisquer correlações entre as proteínas Galectina-3, bcl-2 e PARP-1 e o HPV devido ao baixo índice de casos positivos. / To occur the transition from normal epithelium to squamous cell carcinoma is a necessary a for multiple stages process, such as smoking and alcohol abuse and a possible association with HPV infection. Several types of molecular markers have been studied in cancer of larynx, including proteins associated with apoptosis (bcl-2 and PARP-1) and proteins involved in many biological process such as galectin-3. In this study, analyses of qualitative and quantitative immunohistochemistry was performed for bcl-2, PARP-1 and galectin-3 in 65 patients diagnosed with laryngeal squamous cell carcinoma divided into in situ laryngeal carcinomas(LSCCS), laryngeal squamols cells carcinomas without metastases (LSCCWT) and with metastasis (LSCCW) and cervical lymph nodes (CL). HPV detection and typing was performed by PCR and the HPV types evaluated were HPV 6, 11, 16, 18, 31 and 33. In quantitative of galectin-3 there was observed a significant increase of expression in invasive laryngeal squamous cell carcinoma (LSCCWT and LSCCW) compared with in situ laryngeal carcinomas (LSCCS), may indicating that this protein could be a good marker for progression of laryngeal carcinoma. For PARP-1 and bcl-2 protein there was no difference in the levels of expression in all groups studied. In qualitative analysis PARP-1 showed a homogenous immunolabeling in both high and low among the groups. In relation to Galectin-3, it was observed a predominance of cases with high expression, unlike the protein bcl-2 where the expression prevalence was low in all cases of laryngeal carcinoma and their metastatic lymph nodes. Of the 65 patients, 55 (84.6%) were positive for beta-globin and 7 (12.7%) of 55 patients were positive for HPV. Because of a low incidence of HPV in the cases studied, it was not possible correlate the proteins bcl-2, PARP-1 and Galectin-3 with the presence of HPV.

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.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Les modifications post-traductionnelles des protéines de réparation de l'ADN et des facteurs chromatiniens par poly(ADP-ribose)ylation et par phosphorylation sont essentielles pour le maintien de l'intégrité de l'ADN et de la chromatine, en particulier dans la réponse cellulaire aux dommages de l'ADN induits par radiation ionisantes (RI). Parmi les protéines impliquées dans ces deux processus nous trouvons, respectivement, la poly(ADP-ribose) polymérase-1 (PARP-1) et PARP-2, et la kinase dépendante des cyclines Cdk5 : PARP-1 et PARP-2 sont impliqué dans le mécanisme de réparationdes cassures simples brin (CSBs) de l'ADN (Single Strand Break Repair : SSBR ) et la déplétion de Cdk5 a été liée à l'augmentation de la sensibilité des cellules aux inhibiteurs de PARPs. Nous avons montré, en utilisant des cellules HeLa stablement déplété pour Cdk5 ou PARP-2, que ces deux protéines sont impliquées dans les deux sous-voies du SSBR, le short- (SPR) et le long-patch repair (LPR). L'absence de Cdk5 ou PARP-2 entraîne des modifications du fonctionnement du SSBR, notamment en termes de recrutement des protéines de réparation PARP-1 et XRCC1, impliquées dans le SPR, et PCNA, protéine clé du LPR, au site du photo-dommage. PARP-2 et Cdk5 agissent aussi sur la balance du niveau des poly(ADP-ribose) car en absence de Cdk5 une hyper-activation de PARP-1 a été montrée, et en absence de PARP-2 une diminution de l'activité de la protéine poly(ADP-ribose) glycohydrolase (PARG) a été aussi observée. Cependant, malgré ces changements les deux lignées cellulaires dépourvues de Cdk5 (Cdk5KD) ou de PARP-2 (PARP-2KD) réparent de façon normale les CSBs radio-induites, mais, intéressement et contrairement aux cellules PARP-2KD, les cellules Cdk5KDsont sensibles à l'effet létal des RI. De plus nous avons montré que Cdk5, PARP-2 et PARG sont toutes les trois impliquées dans la régulation du recrutement et de dissociation du facteur chromatinien ALC1 suggérant leur implication dans la régulation de la dynamique de la chromatine en réponse aux photo-dommages de l'ADN. Ces résultats avec l'observation de la diminution du recrutement de PARP-1 dans les cellules Cdk5KD et PARP-2KD, montrent l'apparition d'un réseau complexe de phosphorylation et de poly(ADP-ribose)ylation en réponse aux RI qui implique Cdk5 , PARP-1,PARP-2 and PARG et qui est fort probablement initié par l'activité kinase de Cdk5.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

No description available.

No description available.