Thesis advisor: Kenneth Williams / Proliferating cell nuclear antigen (PCNA) is a DNA polymerase δ auxiliary protein during cell cycle. However, PCNA is also present in quiescent cells undergoing DNA repair. This study investigates the expression of PCNA in CNS macrophages of SIV infected Rhesus macaque and examines the presence of PCNA in monocytes prior to differentiation into CNS macrophages. Accumulation of macrophages in the SIV infected brain, together with the creation of multinucleated giant cells (MNGC), form SIV lesions, which lead to neurological disorders. From the twelve animals used in this study, four were analyzed by flow cytometry to detect PCNA expression in monocytes, but no expression of PCNA was detected. Peripheral blood mononuclear cells (PBMC) of five animals were also analyzed using cytospin preparations to confirm results obtained by flow cytometry. The study did not find induction of PCNA mRNA by qRT-PCR in infected animals. Animals that developed SIVE were analyzed by immunohistochemistry to identify PCNA in recently immigrated macrophages. Together results suggest that 1) PCNA is not expressed in monocytes; 2) PCNA in monocyte/macrophages is induced in SIV infected animals and it is not associated with cell cycle; 3) Recently immigrated monocytes of CNS are PCNA negative and 4) majority PCNA+ macrophages in the SIVE lesions are perivascular macrophages. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

国立情報学研究所で電子化したコンテンツを使用している。

DNA mismatch repair (MMR) is a critical genome-maintenance system. It ensures genome stability by correcting mismatches generated during DNA replication, suppressing homologous recombination, and inducing apoptosis in response to severe DNA damage. As a result, defects in MMR lead to genome-wide mutations and susceptibility to both hereditary and sporadic cancer syndromes. The hallmark of cancer cells defective in MMR is their ability to display frequent instability in simple repetitive DNA sequences, a phenomenon called microsatellite instability (MSI). However, only ~70% of the MSI-positive tumors have identifiable MMR gene mutations, indicating that additional factor(s) are responsible for the MSI phenotype in the remaining 30% MSI-tumors. We demonstrate here that phosphorylation of proliferating cell nuclear antigen (PCNA), an MMR component required for the initiation and resynthesis steps of the repair reactions, blocks in vitro MMR. We found that nuclear extracts derived from colorectal cell lines containing high levels of phosphorylated PCNA are not only defective in MMR, but also inhibitory to MMR activity in HeLa extracts. To determine if PCNA phosphorylation inhibits MMR, several PCNA isoforms that mimic phosphorylated or non-phosphorylated PCNA were examined for their effects on MMR activity. We show that all phosphorylated PCNA mimics block MMR at the initiation step but MMR was not affected by the non-phosphorylated mimetic PCNA. In vitro gap-filling experiments reveal that the phosphorylated PCNA induces a mutational frequency several fold higher than non-phosphorylated PCNA. Since PCNA has been shown to interact with MMR initiation factors MutSα and MutLα, we examined the interactions of phosphorylated PCNA with these two initiation factors. Interestingly, PCNA phosphorylation reduces the PCNA-MutSα interaction, but not the PCNA-MutLα interaction. Since PCNA is proposed to transfer MutSα to the mismatch site, the simplest explanation of the result is that PCNA phosphorylation inhibits MMR by blocking MutSα-mismatch binding activity. Taken together, our results reveal that PCNA phosphorylation induces genetic instability by inhibiting MMR at the initiation step and by promoting DNA polymerase-catalyzed mis-incorporations. This study provides a novel mechanism by which posttranslational modifications inhibit MMR, leading to genome instability and tumorigenesis. A second part of the study is to determine MMR function of several MutLα mutants associated with relapse leukemia patients. One of the mutants contains a phenylalanine99 to leucine substitution in the MLH1 subunit of MutLα. We show that this mutation inhibits MMR by blocking both the ATPase activity and the endonuclease activity associated with MutLα, supporting the importance of the MutLα ATPase and the endonuclease activities in MMR.

One major pathway to overcome DNA damage induced replication blocks is translesion DNA synthesis, which is the replicative bypass of DNA damage by non-classical polymerases. For the cell to utilize translesion synthesis the non-classical DNA polymerase is recruited to sites of DNA damage, and a polymerase switch occurs between the stalled classical polymerase and the incoming non-classical polymerase. This process requires the replication accessory factor proliferating cell nuclear antigen (PCNA) and its monoubiquitination at Lys-164. To better understand the role of PCNA during translesion synthesis, I biochemically and structural characterized two PCNA mutant proteins, G178S and E113G PCNA, which are defective in translesion synthesis. The X-ray crystal structure of both mutant proteins showed a shift in an extended loop, called loop J, compared to the wild type PCNA structure. Steady state kinetic studies determined that in contrast to wild type PCNA which stimulates the non-classical polymerases, the two PCNA mutant proteins fail to stimulate the activity of the non-classical polymerase pol η. These results indicate that loop J in PCNA plays an essential role in facilitating translesion synthesis. During the structural studies of the E113G PCNA mutant protein I observed a unique PCNA structure that failed to form the characteristic PCNA ring shape structure, through traditional intersubunit interactions of domain A and domain B on neighboring subunits. Instead this non-trimeric PCNA structure formed A-A and B-B intersubunit interactions. The B-B interface is structurally similar to the A-B interface observed for the trimeric ring shaped form. In contrast the A-A interface is stabilized by hydrophobic interactions. The location of the E113G substitution is directly within this hydrophobic surface and would not be favorable in the wild type protein. This suggests that the side chain of Glu-113 promotes trimer formation by destabilizing these possible alternate subunit interactions. To biochemically and structurally characterize the impact of monoubiquitinating PCNA (Ub-PCNA), I developed an Ub-PCNA analog by splitting the protein into two self-assembling polypeptides. This analog supports cell growth and translesion synthesis in vivo, and steady state kinetic studies showed that the Ub-PCNA analog stimulates the catalytic activity of pol η in vitro. The X-ray crystal structure of Ub-PCNA showed that the ubiquitin moieties are located on the back face of PCNA. Surprisingly, the attachment of ubiquitin does not change PCNA's conformation. This implies that PCNA ubiquitination does not cause an allosteric change to PCNA, and instead facilitates non-classical polymerase recruitment to the back of PCNA by forming a new binding surface for the non-classical polymerases.

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Proliferating cell nuclear antigen (PCNA) is a key factor involved in Eukaryotic DNA replication and repair, as well as other cellular pathways. Its importance comes mainly from two aspects: the large numbers of interacting partners and the mechanism of facilitated diffusion along the DNA. The large numbers of interacting partners makes PCNA a necessary factor to consider when studying DNA replication, either in vitro or in vivo. The mechanism of facilitated diffusion along the DNA, i.e. sliding along the duplex, reduces the six degrees of freedom of the molecule, three degrees of freedom of translation and three degrees of freedom of rotation, to only two, translation along the duplex and rotational tracking of the helix. Through this mechanism PCNA can recruit its partner proteins and localize them to the right spot on the DNA, maybe in the right spatial orientation, more effectively and in coordination with other proteins. Passive loading of the closed PCNA ring on the DNA without free ends is a topologically forbidden process. Replication factor C (RFC) uses energy of ATP hydrolysis to mechanically open the PCNA ring and load it on the dsDNA. The first half of the introduction gives overview of PCNA and RFC and the loading mechanism of PCNA on dsDNA. The second half is dedicated to a diffusion model and to an algorithm for analyzing PCNA sliding. PCNA and RFC were successfully purified, simulations and a mean squared displacement analysis algorithm were run and showed good stability and experimental PCNA sliding data was analyzed and led to parameters similar to the ones in literature.

O Orlistat é um membro de uma classe de drogas usadas como tratamento para obesidade. No entanto, a segurança de seu uso em longo prazo ainda não é conhecida. O Orlistat exerce sua atividade no lúmen do trato gastrintestinal inibindo a enzima lipase pancreática, responsável pela hidrólise dos triacilglicerídeos normalmente ingeridos com a dieta. Esse medicamento, quimicamente sintetizado, é um derivado da lipstatina, inibidora natural da lipase produzida pelo Streptomyces toxytricini. Assim, a excreção de gordura fecal fica significativamente aumentada com o uso do Orlistat. Estudos epidemiológicos e em modelos experimentais, sugerem que o aumento das dietas hipergordurosas tem efeito promotor para o câncer colorretal. Tal efeito deve ser relacionado, pelo menos parcialmente, às mudanças intra-colônicas causadas pela ação direta da gordura nas células da mucosa do cólon, os colonócitos. O estudo atual tem como objetivo verificar os efeitos do Orlistat na formação colônica de focos de criptas aberrantes (FCA) e na proliferação celular epitelial da mucosa gastrintestinal. Ratos Wistar machos receberam dieta padrão ou dieta com aumento de gordura, suplementada ou não com Orlistat (200 mg/kg), e duas doses semanais do carcinógeno químico Dimetilhidrazina (DMH) (25 mg/kg). Após 30 dias, nos animais tratados com DMH, o Orlistat foi associado a um aumento significativo no número de FCAs colônicos e na proliferação celular epitelial da mucosa do cólon, independentemente da dieta. Os achados obtidos neste trabalho permitem concluir que o aumento do teor de gordura na luz do cólon distal, em decorrência da ação do Orlistat, pode potencializar a ação da DMH na formação de FCAs e no aumento da proliferação celular epitelial da mucosa colônica. / Orlistat is a member of a drug class used as obesity treatment. However, the security of its use in a long period of time is not known yet. Orlistat has its activity in the lumen of the gastrointestinal tract inhibiting the pancreatic lipase enzyme responsible for the hydrolyze of the triglycerides that usually are swallowed with the diet. This drug chemically synthesized, is a derived from lipstatina, that inhibit naturally the lipase produced by Streptomyces Toxytricini. So, the excretion of fat excrement stays significantly increased with the use of Orlistat. Epidemiologic studies and in experimental pattern, suggest that the increase of the high-fat diets facilitate the appearance of the colorectal cancer. Such effect must be related, at least partially to the changes intracolonic caused by the direct action of the fat in the cells of the colon mucous, the colonocytes. The current study has as objective to check the effects of the Orlistat on the formation of rat colonic aberrant crypt foci (ACF) and in the epithelial cell proliferation of the gastrointestinal mucous. Male Wistar rats received either a standard diet or a high fat diet (HFD), supplemented or not with Orlistat (200 mg/kg chow) and two doses of the carcinogen dimethyl-hydrazine (25 mg/Kg). After 30 days, Orlistat was associated to a significant increase in the number of colonic ACFs and cell proliferation in DMH-treated animals, independently of the HFD. The find got in this study permit to conclude that the increase in the level of adiposity inside the distal colon, due to Orlistat action, can potencializar the DMH action in the formation of ACFs and in the increase of epithelial cell proliferation of the colônica mucous.

O estudo das alterações neoplásicas do sistema nervoso (SN) de cães apresenta enorme desafio devido suas particularidades, entre as espécies de animais domésticos o cão Canis familiares é o que apresenta a maior ocorrência destes neoplasmas. O desenvolvimento deste trabalho visa estabelecer o estudo anátomopatológico, molecular e epidemiológico em neurooncologia veterinária, avaliando pela técnica de imuno-histoquímica marcadores da proliferação celular PCNA e Ki-67, expressão da proteína p53 e marcadores de diferenciação celular. Foram utilizados neste estudo 18 animais, da espécie canina, que deram entrada ao Serviço de Patologia vinculado ao Hospital veterinário (HOVET) e ao Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, e que tiveram diagnóstico de neoplasia intracraniana primária confirmado, entre eles (8) meningiomas e (3) astrocitomas. Não houve predileção sexual e a idade média do aparecimento tumoral foi de 9 anos. O valor médio do índice proliferativo (IP) de meningiomas benignos para PCNA é 4,8% e Ki-67 é 2,8%, no astrocitoma fibrilar o valor do IP é para PCNA 1% e Ki-67 1%, no astrocitoma anaplásico o valor do IP é para PCNA 10% e Ki-67 5% e no xantoastrocitoma pleomórfico o valor do IP é para PCNA 20% e Ki-67 4%.Não foi detectada alterações em p53 e o IP foi útil para definir o comportamento tumoral benigno ou maligno e dois tipos tumorais que não haviam sido descritos em cães: xantoastrocitoma pleomórfico e tumor de parênquima pineal de diferenciação intermediária puderam ser diagnosticados graças ao conjunto de informações coletadas, morfologia celular, marcadores de diferenciação celular e índice proliferativo. / The study of neoplastic alterations of dogs nervous system (NS) presents huge challenges due their particularities, between domestic animals species the dog Canis familiars presents the highest of these neoplasms. The development of this research aims to establish the anatomical-pathological, molecular and epidemiology study in veterinary neuro-oncology, evaluating by Immunohistochemistry technique markers of cell proliferation PCNA and Ki-67, expression of p53 protein and markers of cell differentiation. In this study were used 18 animals, dogs, which had entry at the Pathology Service of the Veterinary Hospital (HOVET) and at the Department of pathology from Faculdade de Medicina Veterinária e Zootecnia of University of São Paulo, which had confirmed diagnose of primary intracranial neoplasia, among them (8) meningiomas and (3) astrocytomas. There wasn\'t sexual preference and the mean age of tumor manifestation was 9 years old. The mean value of labelling index (LI) of benign meningiomas for PCNA is 4,8% and Ki-67 is 2,8%, in fibrillary astrocytoma the mean value of LI is 1% for PCNA and 1% for Ki-67, in anaplastic astrocytoma the mean value of LI is 10% for PCNA and 5% for Ki-67 and in pleomorphic xanthoastrocytoma the mean value of LI is 20% for PCNA and 4% for Ki-67. The p53 alterations wasn\'t detected and the LI was useful to set the benign or malign tumor behavior and two tumor types which had not been described in dogs: pleomorphic xanthoastrocytoma and pineal parenchymal tumour of intermediate differentiation could be diagnosed through the set of information collected, cell morphology, markers of cell differentiation and labelling index.

O Orlistat é um membro de uma classe de drogas usadas como tratamento para obesidade. No entanto, a segurança de seu uso em longo prazo ainda não é conhecida. O Orlistat exerce sua atividade no lúmen do trato gastrintestinal inibindo a enzima lipase pancreática, responsável pela hidrólise dos triacilglicerídeos normalmente ingeridos com a dieta. Esse medicamento, quimicamente sintetizado, é um derivado da lipstatina, inibidora natural da lipase produzida pelo Streptomyces toxytricini. Assim, a excreção de gordura fecal fica significativamente aumentada com o uso do Orlistat. Estudos epidemiológicos e em modelos experimentais, sugerem que o aumento das dietas hipergordurosas tem efeito promotor para o câncer colorretal. Tal efeito deve ser relacionado, pelo menos parcialmente, às mudanças intra-colônicas causadas pela ação direta da gordura nas células da mucosa do cólon, os colonócitos. O estudo atual tem como objetivo verificar os efeitos do Orlistat na formação colônica de focos de criptas aberrantes (FCA) e na proliferação celular epitelial da mucosa gastrintestinal. Ratos Wistar machos receberam dieta padrão ou dieta com aumento de gordura, suplementada ou não com Orlistat (200 mg/kg), e duas doses semanais do carcinógeno químico Dimetilhidrazina (DMH) (25 mg/kg). Após 30 dias, nos animais tratados com DMH, o Orlistat foi associado a um aumento significativo no número de FCAs colônicos e na proliferação celular epitelial da mucosa do cólon, independentemente da dieta. Os achados obtidos neste trabalho permitem concluir que o aumento do teor de gordura na luz do cólon distal, em decorrência da ação do Orlistat, pode potencializar a ação da DMH na formação de FCAs e no aumento da proliferação celular epitelial da mucosa colônica. / Orlistat is a member of a drug class used as obesity treatment. However, the security of its use in a long period of time is not known yet. Orlistat has its activity in the lumen of the gastrointestinal tract inhibiting the pancreatic lipase enzyme responsible for the hydrolyze of the triglycerides that usually are swallowed with the diet. This drug chemically synthesized, is a derived from lipstatina, that inhibit naturally the lipase produced by Streptomyces Toxytricini. So, the excretion of fat excrement stays significantly increased with the use of Orlistat. Epidemiologic studies and in experimental pattern, suggest that the increase of the high-fat diets facilitate the appearance of the colorectal cancer. Such effect must be related, at least partially to the changes intracolonic caused by the direct action of the fat in the cells of the colon mucous, the colonocytes. The current study has as objective to check the effects of the Orlistat on the formation of rat colonic aberrant crypt foci (ACF) and in the epithelial cell proliferation of the gastrointestinal mucous. Male Wistar rats received either a standard diet or a high fat diet (HFD), supplemented or not with Orlistat (200 mg/kg chow) and two doses of the carcinogen dimethyl-hydrazine (25 mg/Kg). After 30 days, Orlistat was associated to a significant increase in the number of colonic ACFs and cell proliferation in DMH-treated animals, independently of the HFD. The find got in this study permit to conclude that the increase in the level of adiposity inside the distal colon, due to Orlistat action, can potencializar the DMH action in the formation of ACFs and in the increase of epithelial cell proliferation of the colônica mucous.

O estudo das alterações neoplásicas do sistema nervoso (SN) de cães apresenta enorme desafio devido suas particularidades, entre as espécies de animais domésticos o cão Canis familiares é o que apresenta a maior ocorrência destes neoplasmas. O desenvolvimento deste trabalho visa estabelecer o estudo anátomopatológico, molecular e epidemiológico em neurooncologia veterinária, avaliando pela técnica de imuno-histoquímica marcadores da proliferação celular PCNA e Ki-67, expressão da proteína p53 e marcadores de diferenciação celular. Foram utilizados neste estudo 18 animais, da espécie canina, que deram entrada ao Serviço de Patologia vinculado ao Hospital veterinário (HOVET) e ao Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, e que tiveram diagnóstico de neoplasia intracraniana primária confirmado, entre eles (8) meningiomas e (3) astrocitomas. Não houve predileção sexual e a idade média do aparecimento tumoral foi de 9 anos. O valor médio do índice proliferativo (IP) de meningiomas benignos para PCNA é 4,8% e Ki-67 é 2,8%, no astrocitoma fibrilar o valor do IP é para PCNA 1% e Ki-67 1%, no astrocitoma anaplásico o valor do IP é para PCNA 10% e Ki-67 5% e no xantoastrocitoma pleomórfico o valor do IP é para PCNA 20% e Ki-67 4%.Não foi detectada alterações em p53 e o IP foi útil para definir o comportamento tumoral benigno ou maligno e dois tipos tumorais que não haviam sido descritos em cães: xantoastrocitoma pleomórfico e tumor de parênquima pineal de diferenciação intermediária puderam ser diagnosticados graças ao conjunto de informações coletadas, morfologia celular, marcadores de diferenciação celular e índice proliferativo. / The study of neoplastic alterations of dogs nervous system (NS) presents huge challenges due their particularities, between domestic animals species the dog Canis familiars presents the highest of these neoplasms. The development of this research aims to establish the anatomical-pathological, molecular and epidemiology study in veterinary neuro-oncology, evaluating by Immunohistochemistry technique markers of cell proliferation PCNA and Ki-67, expression of p53 protein and markers of cell differentiation. In this study were used 18 animals, dogs, which had entry at the Pathology Service of the Veterinary Hospital (HOVET) and at the Department of pathology from Faculdade de Medicina Veterinária e Zootecnia of University of São Paulo, which had confirmed diagnose of primary intracranial neoplasia, among them (8) meningiomas and (3) astrocytomas. There wasn\'t sexual preference and the mean age of tumor manifestation was 9 years old. The mean value of labelling index (LI) of benign meningiomas for PCNA is 4,8% and Ki-67 is 2,8%, in fibrillary astrocytoma the mean value of LI is 1% for PCNA and 1% for Ki-67, in anaplastic astrocytoma the mean value of LI is 10% for PCNA and 5% for Ki-67 and in pleomorphic xanthoastrocytoma the mean value of LI is 20% for PCNA and 4% for Ki-67. The p53 alterations wasn\'t detected and the LI was useful to set the benign or malign tumor behavior and two tumor types which had not been described in dogs: pleomorphic xanthoastrocytoma and pineal parenchymal tumour of intermediate differentiation could be diagnosed through the set of information collected, cell morphology, markers of cell differentiation and labelling index.