Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-11-29T18:54:35Z No. of bitstreams: 1 Helder Jacobina Santos As y-glutamil... 2005.pdf: 33243351 bytes, checksum: 707639696a2cd425ea90f9fb81248893 (MD5) / Made available in DSpace on 2012-11-29T18:54:35Z (GMT). No. of bitstreams: 1 Helder Jacobina Santos As y-glutamil... 2005.pdf: 33243351 bytes, checksum: 707639696a2cd425ea90f9fb81248893 (MD5) Previous issue date: 2005 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A carbamazepina é a droga de eleição usada no tratamento de pacientes com epilepsia secundariamente generalizada. Além disso, a carbamazepina tem sido implicada no aumento sérico de algumas enzimas. Alguns autores encontraram prevalência de 13% e os outros, 22% e até 53% de alterações para as fosfatases alcalinas. Objetivo; As metas deste trabalho são determinar a proporção de alterações amostrais nas atividades das gama-glutamil transferase (GGT), fosfatases alcalinas totais (FA), transaminases (AST e ALT) e as concentrações séricas da carbamazepina em pacientes de ambulatórios de epilepsia. Metodologia: 0 desenho do estudo é descritivo, aprovado pelo Comitê de Ética local, no qual uma amostra de conveniência de 52 pacientes epilépticos de acompanhamento ambulatorial foi organizada por faixa etária de 12 a 30 e de 31 a 90 anos e, subdivididos por tempo de uso. As atividades séricas das GGT, FA, AST e ALT foram determinadas, assim como, as concentrações séricas da carbamazepina. As proporções de alterações por variáveis foram descritas. Resultados: 52% dos pacientes apresentaram alteração em pelo menos uma enzima, 42% com alterações nas FA, 18%, nas GGT, 12% nas AST e 2%, nas ALT. A faixa etária de 12 a 30 anos apresentou 56% de alterações nas FA enquanto que aquela de 31 a 90 anos, apenas 18%. Conclusão: a faixa etária pode colaborar com o aumento de prevalência das fosfatases alcalinas alteradas nestes pacientes. Quanto aos baixos valores para GGT e transamin / Carbamazepine is the drug utilized in the treatment of patients who bear epilepsy with secondary generalization. Furthermore, carbamazepine has been implicated with the serum increase of certain enzymes. Some authors have found a prevalence of 13% while others have found 22%, or even 53% of alterations for alkaline phosphatases. Objective: the goals of this study are to determine the ratio of alterations of the serum activities of the enzymes gamma-glutamil transferase (GGT), alkaline phosphatases (AF) and transaminases (AST, aspartate aminotransferase; and ALT, alanine aminotransferase) were determined as well as the serum concentrations of carbamazepine in samples taken from the metropolitan region of Salvador, Bahia. Methodology: the design of this study is descriptive and it has been approved by the local Ethics Committee. A convenience sample of 52 epileptic ward patients was distributed according to age groups of 12 to 30 and 31 to 90 years-old, and was also subdivided according to the period of usage. The serum activities of the enzymes GGT, AF, AST, and ALT were determined as well as the serum concentrations of carbamazepine. The proportions of alterations per variables were described. Results: 52% of the patients showed alteration in at least one enzyme, 42% with alterations in the AF, 18% in the GGT, 2% in the ALT, and 12% in the AST. The age group of 12 to 30 years-old showed 56% of the alterations in AF while the group of 31 to 90 year olds showed only 18% alterations. Conclusion: age group may contribute to the increase of prevalence of altered alkaline phosphatases in these patients. In regard to the low values 70 altered alkaline phosphatases In these patients. In regard to the low values of GGT and transaminases, futher studies would be necessary for better understanding of those variations.

As septinas constituem uma conservada família de proteínas de ligação a nucleotídeos de guanina e formação de heterofilamentos. Em mamíferos, tais proteínas estão envolvidas em uma variedade de processos celulares tais como citocinese, exocitose e tráfego de vesículas. A SEPT4 tem sido identificada em depósitos filamentosos e inclusões citoplasmáticas em Alzheimer e doença de Parkinson, respectivamente. A SEPT4 é a única proteína em associação com proteínas aberrantes em depósitos em ambos os tipos de doenças Assim, estudos adicionais de propriedades bioquímicas estruturais e funções fisiológicas para a SEPT4 podem promover importantes insights em relação ao mecanismo das doenças neurodegenerativas citadas acima. O desenovelamento térmico do domínio GTPase do SEPT4-G revelou um intermediário que agrega rapidamente na forma de fibras tipo amilóide em condições fisiológicas. Neste estudo a análise cinética da agregação do SEPT4-G foi monitorada utilizando fluorescência extrínseca e dicroísmo circular. Com os resultados e análises realizados durante este trabalho de mestrado foi possível compreender com mais detalhes a cinética de formação de agregados tipo amilóide do domínio SEPT4-G. Este trabalho também descreve a cristalização, coleta de dados, resolução e refinamento do modelo cristalográfico para a enzima Glutationa-S-Transferase de Xylella fastidiosa. Tal enzima está associada à patogenicidade da bactéria X. fastidiosa, responsável por várias doenças em plantas economicamente importantes, incluindo a Clorose Variegada dos Citros (CVC) ou Amarelinho. Algumas análises também foram realizadas após a obtenção do modelo cristalográfico demonstrando as diferenças estruturais entre GSTs bacterianas. / The septins are a conserved family of guanine nucleotides-binding and hetero-filament forming. proteins. In mammals they are involved in a variety of cellular processes, such as cytokinesis, exocytosis, and vesicle trafficking. SEPT4 has been reported to accumulate in tau-based filamentous deposits and cytoplasmic inclusions in Alzheimers and Parkinsons diseases respectively. Sept4 is unique in its association with the aberrant protein depositions in both types of diseases. Further studies on the biochemical, structural properties and physiological functions of Sept4 may therefore provide important insights into the common mechanism underlying diverse neurodegenerative disorders. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediary which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, the kinetic analysis of aggregation of SEPT4-G was monitored using extrinsic fluorescence and circular dichroism spectroscopy. The aggregates have the ability to bind specific dyes such as Thioflavin-T (ThT), suggesting that they are amyloid in nature. Fibrils formation was monitored by the increase in ThT emission and electron microscopy as a function of temperature, pH, metal ions and protein concentration. Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (XfGST) was crystallized by the vapour-diffusion method and its crystallography structure was solved for molecular replacement and refined. Afterwards, sequential and structural analyses were carried out for XfGST and others GSTs.

Orientador: Maria Salete Costa Gurgel / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T01:46:42Z (GMT). No. of bitstreams: 1 CardosoFilho_Cassio_M.pdf: 1571004 bytes, checksum: 252da74a9dd5114a82f05ed36b7face1 (MD5) Previous issue date: 2007 / Resumo: Introdução: As deleções dos genes GSTM1 e GSTT1 têm sido associadas ao aumento do risco de várias neoplasias, porém não há consenso sobre suas influências no câncer de mama. Objetivo: Avaliar a ocorrência das deleções homozigóticas dos GSTM1 e GSTT1 em mulheres com câncer de mama esporádico (CME) ¿ casos - e em mulheres sem câncer ¿ controles - e comparar as características clínicas e biológicas dos CME entre mulheres portadoras e não-portadoras das referidas deleções. Casuística e Método: Foram avaliados 177 casos e 169 controles, com determinação das freqüências das referidas deleções pelo PCR. Estas foram correlacionadas às características clínicas e biológicas através do cálculo de odds ratio com seus respectivos intervalos de confiança de 95%. Resultados: Dos casos e controles, respectivamente, 46% e 45% não apresentaram qualquer deleção, 37% e 35% apenas do GSTM1, 11% e 10% apenas do GSTT1, 6% e 9% apresentaram ambos os genes deletados. Observou-se freqüência menor da deleção do GSTM1 em mulheres pardas (p=0,1128), OR=0,48 (0,24 ¿ 0,98). O risco foi menor de ocorrência de tumores grau nuclear 3 em pacientes com deleção do GSTT1 (p=0,04), OR=0,37 (0,15 - 0,90). A deleção homozigótica de pelo menos um dos genes associou-se com mulheres que não amamentaram (p=0,0202), OR=0,41 (0,19 ¿ 0,88), e com a ausência de expressão dos receptores hormonais (p=0,0300), ORadj=2,25 (1,03 ¿ 4,90). Já a deleção de ambos os genes associou-se ao aumento de risco da ocorrência de tipos histológicos diferentes do carcinoma ductal invasivo clássico (p=0,0571), ORadj=12,09 (1,03 ¿ 142,03). Conclusões: mulheres com CME e antecedente de miscigenação com etnia negra tiveram menor freqüência da deleção do GSTM1, enquanto os tumores com grau de diferenciação nuclear mais favorável relacionaram-se à deleção do GSTT1. Já as pacientes com pelo menos um dos genes deletados apresentaram maior risco de tumores que não expressam receptores hormonais, e a deleção combinada de ambos os genes associou-se ao aumento de risco para tipo histológico não ductal clássico / Abstract: Introduction: The GSTM1 and GSTT1 gene deletions have been associated with an increased risk of various neoplasms, although there is a lack of consensus about their influence on breast cancer. Objective: To evaluate the occurrence of homozygous deletions of the GSTM1 and GSTT1 genes in women with sporadic breast cancer (SBC) ¿ cases ¿ and in women without cancer ¿ controls ¿ and compare the clinical and biological characteristics of SBC among women with and without the referred deletions. Case Study and Method: The study evaluated 177 cases and 169 controls, determining the frequency of the abovementioned deletions by PCR. These were correlated with the clinical and biological characteristics by calculating the odds ratios and their 95% confidence intervals. Results: Of the cases and controls, 46% and 45% did not present any deletion, 37% and 35% had only GSTM1 deletion, 11% and 10% had only GSTT1 deletion, 6% and 9% had both genes deleted, respectively. A lower frequency of GSTM1 deletion was observed in mulatto women (p=0.1128), OR=0.48 (0.24 ¿ 0.98). The risk of occurring nuclear grade 3 tumors was lower in patients with GSTT1 deletion (p=0.04), OR=0.37 (0.15 ¿ 0.90). Homozygous deletion of at least one gene was associated with women who had not breastfed (p=0.0202), OR=0.41 (0.19 ¿ 0.88), and with absence of hormone receptor expression (p=0.0300), ORadj=2.25 (1.03 ¿ 4.90). The deletion of both genes was associated with an increased risk of occurring histologic types different from classic invasive ductal carcinoma (p=0.0571), ORadj=12.09 (1.03 ¿ 142.03). Conclusions: women with SBC and a history of miscegination with the black race had a lower frequency of GSTM1 deletion, while tumors with a more favorable degree of nuclear differentiation were related to GSTT1 deletion. Patients with at least one gene deleted had a higher risk for tumors that did not express hormone receptors, and the combined deletion of both genes was associtated with a greater risk for the non-classic ductal histologic type / Mestrado / Tocoginecologia / Mestre em Tocoginecologia

Orientadores: Lair Zambon, Carmen Silvia Passos Lima / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T12:57:06Z (GMT). No. of bitstreams: 1 Honma_HelenNaemi_M.pdf: 6073042 bytes, checksum: 8de4d548610b680b2101397d10fc9e52 (MD5) Previous issue date: 2007 / Resumo: A citocromo P- 450 1A1 (CYP1A1) é uma importante enzima do metabolismo carcinogênico, em virtude da regulação polimórfica, a CYP1A1 é um marcador genético promissor para susceptibilidade de certas malignidades, particularmente o câncer de pulmão (CP). A primeira variação detectada (chamada de m1 ou *2A) foi a transição de T?C no exon 7 com 1194pb, criando um sítio de clivagem para a enzima MspI. Esta alteração foi mais prevalente em Asiáticos do que em Caucasóides. Os genes GSTM1, GSTT1 e GSTP1 vem sendo estudados extensivamente em relação ao CP, pois estão relacionados com a perda completa ou redução da atividade de certos xenobióticos metabolizados por enzimas. O genótipo variante Val/Val do gene GSTP1*B tem uma maior atividade para com o epóxido diol hidrocarbono e BPDE, portanto um menor potencial de detoxificação, resultando em um grande risco para o câncer. O objetivo deste estudo foi analisar a susceptibilidade dos genes polimórficos para o risco em CP e a correlação dos genótipos com as interações das variáveis clínicas. Para cumprir tais objetivos, o DNA genômico de 200 pacientes com CP e 264 doadores de sangue (controles), foram analisados por meio da reação em cadeia da polimerase (PCR) e digestão enzimática. Freqüências similares da deleção homozigótica dos genes polimórficos isolados GSTM1 (45,5% vs 48,1% p= 0,14), GSTT1 (13,5% vs 12,9% p=0,54), GSTM1/T1 ambos nulos (6,0% vs 4,9% p=0,83), da variante Val/Val do gene GSTP1 (12,5% vs 11,4% p=0,87) e da variante CYP1A1*2A (5,5% vs 3,4% p= 0,69) foram observadas em pacientes e controles. Não foram observadas diferenças significativas entre as freqüências de todos estes genótipos. Os resultados sugerem que os genótipos isolados e combinados não influenciaram o risco para CP, mas podemos sugerir que na população Afro-Brasileira estudada, há indícios de uma associação dos genótipos hetero/ variante do gene CYP1A1*2A e GSTs possam levar a uma maior susceptibilidade para o CP / Abstract: Cytochrome P-450 A1 (CYP1A1 ) is an important enzyme of carcinogen metabolism. Due to polymorphic regulation, CYP1A1 is a biomarker of genetic susceptibility to certain malignancies, especially lung cancer (LC). The first detected variation, named m1 or *2A, was a T-to-C transition in exon 7, with 1194bp, which produced a breakage point for the MspI enzyme. Originally, more Asians than Caucasians presented with LC. Intensive studies have shown that GSTM1, GSTT1 and GSTP1 genes are related to LC, which may lead to whole loss or certain hazard reduction. The GSTP1 Val/Val variant genotype has a strong activity, especially along with hydrocarbon diol epoxide and BPDE, leading to detoxification potency. The purpose of this study was to analyze the susceptibility of polymorphism genes to LC risk and the correlation of genotypes with clinical variations. In order to achieve these objectives, the genomic DNA of 200 patients with LC and 264 blood donors (controls) were analyzed through PCR and enzymatic digestion. Similar frequencies of homozygotic deletion of polymorphic isolated genes were observed in patients and controls, as follows: GSTM1 (45.5% vs 48.1%, p= 0.14), GSTT1 (13.5% vs 12.9%, p= 0.54), GSTM1/T1 both null (6.0% vs 4.9%, p= 0.83), GSTP1 gene Val/Val variant (12.5% vs 11.4%, p= 0.87) and the CYP1A1*2A variant (5.5% vs 3.4%, p= 0.69). The differences observed among the frequencies of all combined genotypes were not significant. The final results suggested that isolated and combined genotypes did not influence LC risk; on the other hand, there is a high susceptibility for LC in African-Brazilian people with CYP1A1*2A and GSTs hetero/variant genotype / Mestrado / Clinica Medica / Mestre em Clinica Medica

Orientador: Laura Sterian Ward / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T20:51:35Z (GMT). No. of bitstreams: 1 Bufalo_NatassiaElena_M.pdf: 3282180 bytes, checksum: 0aa23171bc5ca7c18ed18eb22b3a23d2 (MD5) Previous issue date: 2007 / Resumo: Tanto a doença de Graves como o câncer da tiróide são doenças de etiologia multifatorial e envolvem uma interação entre meio ambiente e fatores genéticos de predisposição. O ábito e fumar é um fator de risco reconhecido para o desenvolvimento da doença de Graves, particularmente para a oftalmopatia de Graves. Ao contrário, estudos epidemiológicos têm freqüentemente demonstrado a redução no risco ao carcinoma diferenciado da tiróide entre tabagistas. A herança de polimorfismos de genes relacionados com a metabolização e com a detoxificação de xenobióticos, assim como a herança de genes relacionados com a vida e a morte celular, desempenham um importante papel na suscetibilidade a doenças. Os objetivos foram determinar a influência dos polimorfismos dos genes CYP1A1, GSTM1, GSTT1, GSTP1 e 72TP53 no risco para a doença de Graves e o papel do gene CYP1A1 na tumorigênese tiroidiana. Para avaliar o papel destes genes na doença de Graves foi estudado um total de 400 pacientes com doença de Graves, comparados com 574 indivíduos-controle. Para analisar o papel destes genes no câncer da tiróide foi estudado 248 pacientes com nódulos tiroidianos, comparados com 277 indivíduos-controle, todos pareados para sexo, idade e etnia. As análises genotípicas foram feitas em DNA extraído de sangue periférico, através de amplificação por PCR, seguido de restrição enzimática para os genes CYP1A1, GSTP1 e 72TP53 e PCR-duplex para os genes GSTM1 e GSTT1. Não se encontrou relação entre os genótipos de GSTM1 e GSTT1 e a suscetibilidade à doença de Graves. Contudo, as variantes de GSTP1 (p<0.0001), CYP1A1 m1 (p<0.0033) e Pro/ProTP53 (p<0.0035) foram mais freqüentes em pacientes com doença de Graves do que nos controles. A análise de regressão logística multivariada corrigida parasexo, idade e etnia indicou que o hábito de fumar e a herança das variantes dos genes GSTP1, CYP1A1 e Pro/ProTP53 são importantes fatores de risco para a doença de Graves. Em relação aos nódulos tiroidianos, o genótipo selvagem do gene CYP1A1 foi mais freqüente entre pacientes com carcinoma papilífero (74.26%) do que na população-controle (62.45%) (p= 0.0147), diminuindo o risco para o desenvolvimento deste câncer (OR=0.564; 95% IC= 0.357 - 0.894). A análise de regressão logística multivariada corrigida para sexo, idade e etnia mostrou uma correlação inversa entre o hábito de fumar e a herança do gene CYP1A1 e a suscetibilidade ao carcinoma papilífero. Conclui-se que os polimorfismos de GSTP1, CYP1A1 e TP53 podem estar associados à suscetibilidade relacionada com o tabagismo para a doença de Graves e que o genótipo de CYP1A1 pode estar associado à redução no risco para o carcinoma papilífero entre fumantes / Abstract: Graves's disease and differentiated thyroid cancer are multifactorial diseases with environmental and genetic interactions. Cigarette smoking is a well-recognized risk factor for Graves¿ disease and, particularly, for Graves¿ ophthalmopathy. Conversely, epidemiologic studies have consistently reported a reduced risk of differentiated thyroid cancer in tobacco consumers. Inheritance of germline polymorphisms genes related with metabolizing and detoxification of xenobioticos, besides genes involved in major DNA repair/apoptosis pathways, might have an important role in the susceptibility to these diseases. To assess the influence of the GST, CYP and TP53 gene polymorphisms in the risk of Graves' disease and the CYP1A1 role in thyroid tumorigenesis, we used a PCR strategy to genotype for GSTT1, GSTM1, GSTP1, CYP1A1 and codon 72 of TP53 a group of 400 Graves¿ disease patients compared to 574 control individuals with similar environmental exposure features and 248 patients with thyroid nodules and 277 controls with similar ethnic backgrounds. DNA was extracted from a blood sample and submitted to PCR-RFLP for CYP1A1, GSTP1 and 72TP53 genes and PCR-duplex GSTM1 and GSTT1 genes assays. GSTM1 and GSTT1 genotypes were equally distributed in Graves' disease and controls. However, GSTP1 variants (p<0.0001), CYP1A1 variants (p<0.0033), and Pro/ProTP53 (p<0.0035) appeared more frequently in Graves¿ disease than in controls. A multivariate analysis indicated that cigarette smoking and the inheritance of GSTP1 variants, CYP1A1 variants and Pro/ProTP53 were important risk factors of risk for Graves¿ disease. Among the thyroid nodules, the wild-type CYP1A1 m1 genotype was more frequent in papillary carcinomas patients (74.26%) than in the control population (62.45%) (p= 0.0147) indicating that the variants of these genes reduce the risk for this cancer (OR=0.564; 95% IC= 0.357 to 0.894). Multiple logistic regression analysis showed an inverse correlation between cigarette smoking and CYP1A1 germline inheritance and the susceptibility to papillary carcinomas. We concluded that GSTP1, CYP1A1 and TP53 germline polymorphisms may be associated with smoking-related Graves¿ disease susceptibility and CYP1A1 genotype might be associated to the reported reduced risk to papillary carcinomas among smokers / Mestrado / Clinica Medica / Mestre em Ciências Básicas

Orientador: Maria Salete Costa Gurgel / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T12:25:32Z (GMT). No. of bitstreams: 1 Franco_RicardoLaier_M.pdf: 1204778 bytes, checksum: 4a5d4c7eb9039f14fc0392b4e8d9abb5 (MD5) Previous issue date: 2008 / Resumo: Introdução: A glutationa S-transferase (GST) é um sistema enzimático localizado no citosol celular e é responsável pela eliminação de toxinas. Estudos realizados in vitro mostraram que a presença deste sistema nas células do carcinoma de mama pode promover a eliminação do quimioterápico, levando assim à diminuição da eficácia desta modalidade terapêutica em mulheres com carcinoma de mama que expressam tal sistema enzimático em suas células. Objetivo: O presente estudo teve como objetivo avaliar a associação entre a expressão da enzima GST Pi em células tumorais e a sobrevida global e livre de doença em mulheres com carcinoma de mama submetidas a tratamento quimioterápico. Sujeitos e Métodos: Entre janeiro de 1995 e junho de 1997, 554 pacientes foram submetidas a tratamento cirúrgico para câncer de mama no CAISM-UNICAMP. Destas, 160 tinham entre 18 e 70 anos ao diagnóstico, doença não metastática, foram submetidas à quadrantectomia/mastectomia com margens livres e linfadenectomia axilar, seguida de quimioterapia adjuvante, e foram acompanhadas até agosto de 2006. Os blocos de parafina estavam disponíveis para 95 destas pacientes. Neles foi realizada a pesquisa da GST Pi através da reação de imuno-histoquímica e sua expressão foi correlacionada com os dados clínicos obtidos de seus respectivos prontuários. Resultados: Dos 95 casos, 36 (38%) foram positivos para a expressão da GST Pi e 59 (62%) negativos. A expressão da GST Pi nas células tumorais não mostrou associação com a idade ao diagnóstico, tipo histológico do tumor, estágio da doença e expressão de receptores de estrogênio e progesterona. Os cânceres de mama com positividade para a enzima GST Pi mostraram associação significativa com tumores de grau histológico I e negatividade para a expressão da proteína HER-2. Não se observou associação com a sobrevida livre de doença e sobrevida global, após seguimento médio de 10,68 anos. Conclusões: Os achados deste estudo indicam que pacientes com tumores positivos para a enzima GST Pi têm o mesmo prognóstico do que pacientes com tumores negativos para GST Pi após serem tratados com quimioterapia / Abstract: Introduction: The glutathione S-transferase is an enzymatic system located in the cytosol of the cells and it is responsible for the elimination of toxins. 'In vitro¿ studies showed that the presence of this enzymatic system in breast carcinoma cells can accelerate the elimination of the drug used in the chemotherapy. This would decrease the efficacy of this kind of treatment in women with GST Pi -positive breast cancer cells. Objectives: The scope of the present study was to evaluate the association between GST Pi-positive breast cancer and overall and disease free-survival in women with breast carcinoma submitted to chemotherapy. Methods: Between January, 1995 and July, 1997, 554 patients were undergone to breast cancer surgical treatment at CAISM-UNICAMP. Out of 554 patients, 160 were between 18 and 70 years old in the diagnosis, did not have metastatic disease, were submitted to quadrantectomy/mastectomy with free surgical margins and axillary dissection followed by adjuvant chemotherapy and had complete follow-up until August, 2006. The paraffin blocks were available for 95 of these patients. The immunohistochemical reactions were done and the GST Pi expression was correlated with the clinical data obtained from their respective medical records. Results: Of the 95 cases studied, 36 (38%) were positive for the GST Pi expression and 59 (62%) did not express the enzymatic system. The expression of GST Pi in breast cancer cells showed no relation with age, histological type, stage of disease and estrogen and progesterone receptor status. The GST Pi-positive breast cancers showed a significant relation with lower histological grade tumors and with negativity of the expression of HER-2. Also, the expression of GST Pi had no relation with overall and disease free survival after a median follow-up of 10.68 years. Conclusions: The findings of this study indicate that patients with GST Pi-positive tumors have the same prognosis as patients with GST Pi-negative tumors after chemotherapy / Mestrado / Tocoginecologia / Mestre em Tocoginecologia

Orientador: Carmen Silvia Passos Lima / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T16:24:35Z (GMT). No. of bitstreams: 1 RinckJunior_JoseAugusto_D.pdf: 4925898 bytes, checksum: cf6e78a6e4ad84c7bbe48d34d75fdae6 (MD5) Previous issue date: 2015 / Resumo: As glutationa S-transferases (GSTs) são enzimas detoxificantes. Os genes GSTM1 e GSTT1 são polimórficos e quando deletados perdem a expressão enzimática. As proteínas codificadas pelos genes XPD são responsáveis pelo reparo de lesões do DNA causadas pela luz solar. Polimorfismos nestes genes também podem codificar proteínas com funções comprometidas, em especial o Lys751Gln e o Asp312Asn do gene XPD. Ainda não é claro o papel dos polimorfismos destes genes no risco de melanoma cutâneo (MC) ou se estão associados com os aspectos clínicopatológicos. Foram incluídos 489 indivíduos (231 pacientes, 258 controles). A genotipagem foi realizada por reação em cadeia da polimerase e digestão enzimática. O risco de MC esteve aumentado em 2,00 (IC 95%: 1,05-3,81, P= 0,03) vezes em portadores do genótipo GSTT1 nulo + Asp/Asn + Asn/Asn do XPD Asp312Asn. O GSTT1 nulo elevou o risco de MC metastático em 3,75 (IC 95%: 1,48-9,44, P= 0,006) vezes e se combinado ao GSTM1 nulo em 7,33 (IC 95%: 2,09-25,68, P= 0,003) vezes. O alelo 312Asn elevou o risco de MC no tronco ou membros em 1,80 (IC 95%: 1,19-2,73, P= 0,005) vezes e do subtipo extensivo superficial ou nodular em 1,80 (IC 95%: 1,14-2,84, P= 0,01) vezes. Os genótipos Asn/Asn + Gln/Gln elevou o risco de MC de níveis I, II ou III de Clark em 2,46 (IC 95%: 1,12-5,37, P= 0,02) vezes. Os genótipos GSTM1 nulo + GSTT1 nulo (HR: 3,18; IC95%: 1,21-8,36, P= 0,01) e o genótipo GSTT1nulo + Gln/Gln (HR: 5,93; IC95%: 1,53-22,91, P= 0,01) estiveram associados a maior risco de morte. Concluímos que os referidos polimorfismos em combinações específicas podem aumentar a susceptibilidade ao MC e influenciar suas características clínicopatológicas e sobrevida / Abstract: The glutathione S-transferases (GST) are detoxifying enzymes; the GSTM1 and GSTT1 genes are polymorphic and when deleted lose enzyme expression. The XPD proteins are responsible for DNA damage repair caused by sunlight. Polymorphisms (SNPs) in XPD genes may also result proteins with impaired function, in particular Lys751Gln and Asp312Asn. However It¿s not entirely clear whether the SNPs of these genes influence the risk of cutaneous melanoma (CM) or are associated with clinic pathological aspects of this disease. In the present study 489 individuals were included (231 patients and 258 controls). Genotyping was performed by polymerase chain reaction and enzyme digestion. The risk of MC was increased 2.00-fold (95% CI: 1.05-3.81, P= 0.03) in carriers of the GSTT1 null combined with Asp/Asn + Asn/Asn genotype of XPD Asp312Asn. The GSTT1 null genotype and GSTT1 null + GSTM1 null genotype increased the risk of metastatic MC by 3.75-fold (95% CI: 1.48-9.44, P= 0.006) and 7.33-fold (95% CI: 2.09-25.68, P= 0.003), respectively. The allele 312Asn increased the risk of MC in the trunk or limbs in 1.80-fold (95% CI: 1.19-2.73, P= 0.005) and superficial spreading or nodular subtype in 1.80-fold (95%: 1.14-2.84, P= 0.01). The combined Asn/Asn + Gln/Gln genotype raised the risk of MC in Clark¿s level I, II or III in 2.46-fold (95% CI: 1.12-5.37, P= 0.02). The genotype GSTM1null + GSTT1 null (HR: 3.18; 95% CI: 1.21-8.36, P= 0.01) and GSTT1 null + Gln/Gln (HR: 5.93; 95% CI: 1.53-22.91, P= 0.01) were predictive of lower overall survival. In conclusion, polymorphisms in specific genes combinations may increase susceptibility to MC and influence their clinic pathological features and survival / Doutorado / Clinica Medica / Doutor em Clínica Médica

Orientador: Carmen Silvia Passos Lima / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T18:37:49Z (GMT). No. of bitstreams: 1 Pincinato_EderdeCarvalho_D.pdf: 3229024 bytes, checksum: a92cc669d0c70abe36aab0ff261da986 (MD5) Previous issue date: 2015 / Resumo: A cisplatina (CDDP) associada à radioterapia (RT) é utilizada no tratamento de portadores de carcinoma de células escamosas de cabeça e pescoço (CCECP). A resposta ao tratamento bem como seus efeitos colaterais variam de indivíduo para indivíduo e tal fato pode ser explicado pela variabilidade genética no metabolismo da CDDP. O objetivo deste estudo foi o de verificar se habilidades herdadas na detoxificação da CDDP, mediadas pelas enzimas GSTM1, GSTT1 e GSTP1, alteram os efeitos terapêuticos, colaterais e a concentração de CDDP urinária em pacientes com CCECP. Foram avaliados, de forma prospectiva, 90 pacientes com CCECP tratados com CDDP associada à RT. Os genótipos dos polimorfismos GSTM1, GSTT1 e GSTP1 Ile105Val foram analisados por meio da reação em cadeia da polimerase (PCR) multiplex e PCR seguida de digestão enzimática, respectivamente, em DNA de sangue periférico. A resposta ao tratamento foi avaliada por exame clínico e tomografia computadorizada do pescoço. Os efeitos colaterais ao tratamento foram graduados por questionários e exames laboratoriais. As toxicidades renal e auditiva foram avaliadas por clearance de creatinina estimado (ClCrea), taxa de filtração glomerular com EDTA-51Cr (TFG) e audiometria tonal limiar. As concentrações urinárias da CDDP foram realizadas por cromatografia líquida de alta eficiência (HPLC). Pacientes com a deleção homozigótica do gene GSTT1 estiveram sob risco 0,09 (IC 95%: 0,02-0,41) vezes menor de ocorrência de vômitos, 0,22 (IC 95%: 0,05-0,89) vezes menor de ototoxicidade e menor redução de ClCrea (81,69 ± 21,40 para 84,13 ± 25,69 versus 93,16 ± 28,94 para 77,52 ± 23,96 mL/min/1,73m²) e de TFG (79,56 ± 20,68 para 69,94 ± 21,40 versus 84,38 ± 19,96 para 62,87 ± 20,72 mL/min/1,73m²) do que os portadores do gene. Já os pacientes com o alelo Val do polimorfismo GSTP1 Ile105Val estiveram sob risco 6,32 vezes maior de ocorrência de vômitos acentuados (IC 95%: 2,05-19,51), 3,35 vezes maior de ototoxicidade acentuada (IC 95%: 1,03-10,96) e maior redução de TFG (80,87 ± 21,73 para 66,97 ± 24,96 versus 85,35 ± 18,76 para 62,19 ± 17,40 mL/min/1,73m²) do que os portadores do genótipo Ile/Ile. Maior concentração urinária de CDDP foi observada em pacientes com a deleção homozigótica do gene GSTT1, quando comparados aos pacientes com a presença do gene (429,58 ± 116,24 versus 253,42 ± 95,20 ug CDDP/mg creatinina). Concluímos que estes polimorfismos desempenham papéis importantes na ocorrência de eventos adversos da terapêutica e excreção urinária do CDDP. Acreditamos que estes resultados possam constituir a base preliminar para o tratamento personalizado futuro de pacientes com CCECP / Abstract: Cisplatin (CDDP) associated with radiotherapy (RT) is used in treatment of patients with head and neck squamous cell carcinoma (HNSCC). The response to treatment as well as its side effects vary among individuals, and this fact may be explaned by the genetic variability in metabolism of CDDP. The aim of this study was to access if inherited ability to cellular CDDP detoxification, mediated by GSTM1, GSTT1 and GSTP1 enzymes alters the therapeutic and side effects of CDDP and RT and urinary concentration of CDDP in HNSCC patients. We evaluated, prospectively, 90 consecutive HNSCC patients, who received CDDP associated RT as treatment. Genotypes of GSTM1, GSTT1 and GSTP1 Ile105Val polymorphisms were analyzed by multiplex polymerase chain reaction (PCR) and PCR followed by restriction enzyme digestion, respectively, in peripheral blood DNA. Treatment response was assessed by clinical examination and computed tomography of neck. Treatment side effects were ranked through questionnaire and laboratory tests. Renal and hearing toxicities were assessed using, respectively, estimated creatinine clearance and glomerular filtration 51Cr-EDTA and pure tone threshold audiometry. Urinary doses of CDDP were performed by high performance liquid chromatography (HPLC). Patients with the GSTT1-null genotype had a 0.09 times (95% CI:0.02-0.41) decreased risk for vomiting, 0.22 times (95% CI:0.05-0.89) decreased risk for ototoxicity, less creatinine clearance decreases (81.69 ± 21.40 to 84.13 ± 25.69 versus 93.16 ± 28.94 to 77.52 ± 23.96 mL/min/1.73m²) and glomerular filtration 51Cr-EDTA (79.56 ± 20.68 to 69.94 ± 21.40 versus 84.38 ± 19.96 to 62.87 ± 20.72 mL/min/1.73m²) than GSTT1-present genotype. Patients with Val allele of GSTP1 Ile105Val polymorphism had 6.32 higher risk presenting high grade vomiting (95% CI: 2.05-19.51), 3.35 higher risk to high grade ototoxicity (95% CI: 1.03-10.96) and higher glomerular filtration 51Cr-EDTA reduction (80.87 ± 21.73 to 66.97 ± 24.96 versus 85.35 ± 18.76 to 62.19 ± 17.40 mL/min/1.73m²) when compared to wild genotype. Higher CDDP urinary level was observed in patients with the GSTT1-null genotype, compared to patients with the gene (429.58 ± 116.24 versus 253.42 ± 95.20 ug CDDP/mg creatinine). We concluded that these genetic polymorphisms have important roles in complete response rate, in occurrence of side effects, and in urinary CDDP excretion. We believe that this data may constitute preliminary basis of future personalized treatment of HNSCC patients / Doutorado / Clinica Medica / Doutor em Clínica Médica

Francisella tularensis is a Gram-negative, facultative intracellular coccobacillus and the etiologic agent of tularemia for a wide variety of vertebrate and invertebrate animal species. Several species and subspecies of Francisella are currently recognized. However, the majority of infections are caused by F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B). Given the low infectious dose, multiple transmission routes, severity of illness, and lack of licensed vaccines, F. tularensis has long been considered a potential biological weapon and is now classified as a category A select agent by the National Institutes of Health and the Centers for Disease Control and Prevention. The investigation of the mechanisms of pathogenesis by F. tularensis type A and B strains is hindered by the difficulty and lack of methods to mutate the putative genes that encode for virulence factors. New genetic tools have been developed that have enabled mutagenesis of F. tularensis type A and type B stains. However, site-specific mutations remain difficult to execute or these methods generate random mutations. In this study a novel method was developed to create site-directed mutations in a putative capsule biosynthesis locus to knock out encapsulation of the attenuated F. tularensis live vaccine strain. Two suicide vectors for mutagenesis of F. tularensis were constructed based on the commercial PCR cloning vector pSC-A. These vectors were created by inserting into the cloning site a kanamycin resistance gene boarded upstream by 1.3 kb of N-terminal DNA and downstream by 1.3 kb of C-terminal DNA that flanks the target gene. Cryotransformation was used to introduce the vectors into F. tularensis. Open reading frame (ORF) FTT0793, which may encode for an ABC transporter involved in capsule export, was initially selected for mutagenesis in order to generate a mutant that was nonencapsulated, but could still synthesize capsule and induce a host immune response. Mutagenesis of this gene was successful. However, phenotypic assays could not confirm that the mutant was nonencapsulated compared to the parent. Therefore, adjacent ORFs FTT0798 and FTT0799, which may encode for a galactosyl transferase and mannosyl transferase, respectively, were also deleted to completely knock out capsule synthesis. The resulting mutant appeared to be nonencapsulated as determined by negative staining transmission electron microscopy. In this study, a plasmid and method for generating allelic exchange mutants is reported, which should be useful for generating additional mutants of F. tularensis for use in clarifing the roles of specific genes. This vector is currently being used to make a nonencapsulated mutant of a virulent type A strain to determine the role of capsule in virulence. / Master of Science

Motivation: The different coping styles used to respond to psychosocial stress have been linked to the development of cardiovascular disease (CVD). However, the manner in which the cardiovascular system is influenced differs between the coping styles. Of the different coping styles, defensive active coping (AC) has been shown to be the most detrimental to cardiovascular health. This is worsened by augmented α-adrenergic cardiac responses found in Africans. Furthermore, many studies have found that the prevalence of hypertension and other CVDs is much higher in urban Africans when compared to their Caucasian counterparts. This can be attributed to certain lifestyle changes implemented by Africans in the transition that occurs with urbanization, where they are forced to cope with an urban-dwelling lifestyle. One of these lifestyle factors, which also poses as a cardiovascular risk factor, is increased usage and in some cases abuse of alcohol. Certain discrepancies exist between ethnicities with regard to the metabolism of alcohol, which influences the effect of alcohol on the individual. Alcohol usage as a possible manner of coping has been supported in many instances, but the interdependent effects of alcohol usage and AC as cardiovascular risk factors has only been found in African men. Further investigation is needed to determine if coping and alcohol abuse act in tandem only in African men, or also in other ethnic or sex groups. What also needs to be discussed is whether the inconsistencies between ethnicities regarding alcohol metabolism, plays a part in the development of CVD in a bi-ethnic gender cohort. Objectives: The main aims of this study were to determine 1) receiver operated characteristic (ROC) ethnic specific cut points of alcohol usage in the prediction of ambulatory hypertension, and 2) to assess if these cut points in defensive active groups revealed increased cardiometabolic risk in a bi-ethnic sex cohort, and if so, whether the increased risk will be associated with a specific race or sex group? Methodology: This sub-study forms part of the SABPA (Sympathetic activity and Ambulatory Blood Pressure in Africans) study, conducted from 2008 to 2009. After exclusion criteria were applied, our bi-ethnic sex cohort consisted of 390 individuals. These participants were all from the Kenneth Kaunda Education District of the North-West province in South Africa, and they all signed informed consent prior to participation. The SABPA study was approved by the Ethics Review Board of the North-West University, with additional ethical approval for this sub-study. All procedures in this study complied with the guidelines of the Declaration of Helsinki. Each participant completed a psychosocial battery supervised by registered clinical psychologists, and information regarding their medication use and medical history was obtained. They also completed the Coping Style Indicator questionnaire which was developed by Amirkhan, to identify the coping style habitually used. Ambulatory blood pressure and ECG measurements were recorded for a 24h period with the Cardiotens CE120®. Anthropometric measurements were performed by ISAK (International Society for the Advancement of Kinanthropometry) level 2 accredited anthropometrists using calibrated instruments. Out of this, the body surface area were calculated. The physical activity of each participant was determined by use of the Actical® omnidirectional accelerometer. Resting blood samples were collected by a registered nurse. The following blood serum levels were determined: gamma-glutamyl transferase (γ-GT) as a marker for alcohol usage, C-reactive protein, cholesterol, high density lipoprotein, triglycerides, cotinine, reactive oxygen species and glycated haemoglobin levels. All statistical analyses were done using Statistica version 12.0. Descriptive statistics were conducted to state the baseline characteristics of the entire group, while Chi-square (X2) tests were used to determine prevalence for medications and pathology. ROC analyses were computed to establish a cut point for γ-GT predicting ambulatory hypertension in each ethnicity as well as in the entire group. Independent t-tests identified confounders, after which two-way analysis of covariance (ANCOVA) tests were computed to test a 2 x 2 main effects interaction (race x γ-GT cut points) for all cardiometabolic risk markers and to compare the different ethnic groups. ANCOVAs were then performed in the ethnic groups with high γ-GT as well as in above mean AC for the graphs that followed. Lastly, odds ratios (OR‟s) with 95% confidence intervals (CI‟s) were calculated in several models to highlight the odds of high alcohol intake to predict ambulatory hypertension in the ethnic-sex groups as well as in AC ethnic-sex groups. Significant values were noted as p ≤ 0.05. Results: The Africans revealed higher cardiometabolic risk markers, above mean defensive active coping, seeking social support with less avoidance coping scores. ROC analyses revealed that ambulatory hypertension commences at a much higher level of γ-GT in the Africans [55.7U/l (AUC=0.69; 95% CI: 0.61; 0.76)] with sensitivity /specificity of 47%/83% compared to the Caucasians [19.5U/l (AUC=0.747; 95% CI: 0.68; 0.82)] with sensitivity/specificity of 70%/73%. The Caucasians thus reveal an increased sensitivity for alcohol ingestion at a much lower γ-GT cut point compared to the Africans. When comparing ethnic specific ROC cut point groups, we found that certain levels of cardiometabolic risk factors such as C-reactive protein, systolic blood pressure, waist circumference and silent ischemic events, were significantly higher in the African group, especially in above mean AC groups. Out of the Africans with high γ-GT levels, 73% used the AC style, suggesting hypervigilant AC coping and increased CVD risk in Africans. Clinical significance was determined by OR‟s, which demonstrated that high γ-GT levels in AC African men predicted ambulatory hypertension with an OR of 7.37 (95% CI: 6.71 – 8.05). Higher alcohol intake predicted ambulatory hypertension in AC Caucasians with an OR of 2.77 (95% CI: 2.31 – 3.23) in men and 6.42 (95% CI: 5.85 – 7.0) in women respectively. Conclusion: γ-GT cut-points in defensive active groups revealed increased cardiometabolic risk markers in a bi-ethnic sex cohort. A possible hypermetabolic state in African men may initially protect them against CVD morbidity but if chronically challenged with no forthcoming social support, CVD risk is imminent. / MSc (Physiology), North-West University, Potchefstroom Campus, 2015