Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 107-130).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1975. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

In neurons, modification by the lipid palmitate regulates trafficking and function of signaling molecules, neurotransmitter receptors and associated synaptic scaffolding proteins. HIP14 (huntingtin interacting protein 14) is the first identified and characterized mammalian palmitoyl transferase that regulates this process. I have shown that HIP14 has striking effects on modulating trafficking and function of many proteins important for synapse formation and plasticity such as PSD-95, a postsynaptic scaffolding molecule. The importance of the finding that HIP14 is a neuronal palmitoyl transferase is further emphasized by our recent discovery that huntingtin protein folding, trafficking and function are regulated by the enzyme HIP14. Expansion of the polyglutamine tract in huntingtin as seen in Huntington Disease (HD) results in reduced association with HIP14 and decreased palmitoylation of huntingtin, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity. By manipulating HIP14 levels through expression or knockdown, we can manipulate the number of huntingtin inclusion bodies and neuronal cell viability. Overall, these discoveries offer novel mechanism for HD pathogenesis and provide new approaches to therapy for HD. The tight association of HIP14 with wild-type huntingtin, which differs from other known enzyme-substrate interactions, indicates that huntingtin serves other functions beyond being a substrate of HIP14. I have discovered that, in vitro, wild-type huntingtin may facilitate activity of HIP14 to palmitoylate other neuronal substrates such as SNAP25, PSD95 and GAD65. By contrast, mutant htt does not act this way, probably due to lack of interaction with HIP14. Furthermore, immunoprecipitated HIP14 from huntingtin+/- mice also exhibits less enzyme activity in palmitoylating GST-SNAP25 in vitro, suggesting that decreased huntingtin expression compromises HIP14 activity. In vivo, using Acyl Biotin Exchange assay, I have also found that palmitoylation of a number of presynaptic and postsynaptic proteins that are involved in neurotransmission are reduced in huntingtin+/- mice. This study not only ascribes an important biochemical function to wild-type huntingtin, but also suggests that defects in protein palmitoylation in general due to mutant huntingtin lack of ability to facilitate HIP14 activity may contribute to the pathogenesis of HD. / Medicine, Faculty of / Graduate

Rats fed diets which contained 15% of casein and 0.620% of methionine with 0.0002, 0.02 and 0.42% of dietary inorganic sulfate had a dietary sulfate-related change in methionine metabolism. Rats fed the diets low in sulfate (0.0002%) had a 35% increase in methionine metabolism compared to rats fed the diets high in sulfate (0.42%). In contrast, rats fed the diet low in sulfate (0.0002%) had the lowest level of tissue S-adenosyl-methionine and the highest activity of phenylethanolamine-N-methyltransferase activity. Those animals fed the diet normal with respect to sulfate (0.02%) had intermediate levels of S-adenosylmethionine and phenylethanolamine-N-methyl transferase activity. Rats fed the diet high in sulfate (0.42%) had the highest level of tissue S-adenosyl-methionine and the lowest phenylethanol-amine-N-methyl transferase activity. Due to the inverse relationship between S-adenosylmethionine and phenylethanolamine-N-methyl transferase activity, it appears that the catecholamines may function as a methyl sink for the increase in the metabolism of methionine required to provide sulfate for rats fed diets low in sulfate.

<p> The ansamycin antibiotics are unique antibacterial agents that inhibit bacterial DNAdependent RNA polymerase II. Clinical use of this class of antibiotics has primarily been focused on the treatment oftuberculosis using the semi-synthetic rifamycin derivative, rifampin. As drug resistance among different classes of antibiotics continues to rise, there is increased interest in new applications ofrifamycins for diseases other than tuberculosis. Clinical resistance to rifampin has largely been the result of point mutations in the target, RpoB, however chromosomal and transposon mediated enzyme-associated resistance is well documented. As rifamycin antibiotic use becomes more widespread, enzymatic resistance will inevitably become more prevalent. Here we describe the characterization of one of the principle enzymes associated with rifampin inactivation, the rifampin ADP-ribosyl transferase enzyme (ARR). Two chromosomally encoded ARR enzymes from MYcobacterium smegmatis, and Streptomyces coelicolor, and the Tn-encoded ARR-2, widely distributed in Gram negative pathogens, were overexpressed and characterized. These enzymes exhibit comparable, substrate specific steady state kinetic features, and substrate-induced conformational changes that suggest ARR enzymes may demonstrate a preferred order of substrate binding. To gain further insight into the interaction between ARR enzymes and rifampin and NAD+, the three-dimensional crystal structure of ARR from M smegmatis was solved in complex with rifampin. Based on the threedimensional structure of ARRm, an SNl type reaction has been predicted for rifampin ADPribosyl transferase enzymes. This is the first detailed examination of these novel antibioticmodifying enzymes, relevant to their increased use in the clinic. </p> / Thesis / Master of Science (MSc)

Made available in DSpace on 2016-08-10T10:38:34Z (GMT). No. of bitstreams: 1 CONSTANZA THAISE XAVIER SILVA.pdf: 856711 bytes, checksum: aa9a8d8094cd18595af6092fc0d17683 (MD5) Previous issue date: 2012-03-12 / Glaucoma refers to a group of eye diseases that gradually evolve being characterized by typical damage to the optic nerve with consequent changes in vision. The most common type is the primary open angle glaucoma (POAG) corresponding to approximately 60% of cases. The ocular epithelia expressed genes encoding the enzymes Glutathione STransferase (GST). The GST is present in various ocular structures, including aqueous humor, ciliary body, and lens. This study aimed to evaluate the genotypic profile of the gene GSTT1 and GSTM1 polymorphisms in patients with Primary Open Angle Glaucoma and control group in the city of Goiânia. Peripheral blood samples were analyzed by up to 100 samples of patients diagnosed as carriers of glaucoma, proven and 53 specimens from patients with examination within the customary standards, representing the control group. The polymorphism was assessed by PCR and analyzed in 2% agarose gel, stained with etídeo bromide. The frequencies of polymorphisms of GSTM1 and GSTT1 genes were compared with the &#967;2 test (p < 0.05%) and rate risk was assessed by the test of Odds Ratio with 95% confidence interval (&#945; = 0.05). In case it was noted that this was of genotype GSTM1 40% (n = 40) and in controls was 71.6% (n = 38). The null genotype was 60% (n = 60) and 28.3% (n = 15), respectively. The GSTT1 genotype present in case group was 52% (n = 52) and in the control group was 66% (n = 35); already the null genotype was 48% (n = 48) in case group and 34% (n = 18) in the control group. GSTM1 null genotype in the case group was more frequent than in the control group, and this difference statistically significant (p = 0. 0004). The same was not found with the GSTT1 genotype (p = 0.13). Checked also the Association of genotypes GSTM1 null/GSTT1 this the risk of glaucoma to 3.1 times more the chance of disease occurrence, suggesting that individuals who have the GSTM1 null genotype/GSTT1 this can be regarded as one of the risk factors for the development of the POAG. Already for the genotypes GSTM1 null was verified the GSTT1\/risk of 6.7 times more chance of occurrence of disease (p = 0.0004; OR: 6.7; 95% CI:-2.7 20.3), suggesting that individuals who have the GSTM1 null genotype/GSTT1 can be regarded as one of the risk factors for the development of the POAG. / O Glaucoma designa um grupo de doenças oculares que evoluem progressivamente sendo caracterizadas por danos típicos no nervo óptico com consequentes alterações de visão. O tipo mais frequente é o glaucoma primário de ângulo aberto (GPAA) que correspondente aproximadamente 60% dos casos. O epitélio ocular expressa genes que codificam as enzimas Glutationa S-Transferase (GST). A GST está presente em várias estruturas oculares, incluindo humor aquoso, corpo ciliar e cristalino. Este estudo teve como objetivo avaliar o perfil genotípico dos polimorfismos dos genes GSTM1 e GSTT1 em pacientes portadores de Glaucoma Primário de Ângulo Aberto e grupo controle na cidade de Goiânia. Foram analisadas amostras de sangue periférico de 100 amostras de pacientes comprovadamente diagnosticados como portadores de glaucoma e 53 amostras de pacientes com exame dentro dos padrões considerados normais, representando o grupo controle. O polimorfismo foi avaliado por PCR e analisados em gel de agarose a 2% e corado com brometo de etídeo. As frequências dos polimorfismos dos genes GSTM1 e GSTT1 foram comparadas com o teste &#967;2 (p<0,05%) e a taxa de risco foi avaliada pelo teste de Odds Ratio com intervalo de confiança de 95% (&#945;=0,05). No grupo caso observou-se que o genótipo GSTM1 presente foi de 40% (n=40) e nos controles foi de 71,6% (n=38). O genótipo nulo foi 60% (n=60) e 28,3% (n=15), respectivamente. O genótipo GSTT1 presente no grupo caso foi de 52% (n=52) e no grupo controle foi de 66% (n=35); já o genótipo nulo foi de 48% (n=48) no grupo caso e 34% (n=18) no grupo controle. O genótipo GSTM1 nulo no grupo caso foi mais frequente do que no grupo controle, sendo esta diferença estatisticamente significativa (p=0, 0004). O mesmo não foi encontrado com o genótipo GSTT1 (p=0,13). Foi verificado também a associação dos genótipos GSTM1 nulo/GSTT1 presente ao risco de glaucoma para 3,1 vezes mais a chance de ocorrência da doença, sugerindo que indivíduos que apresentam os genótipos GSTM1 nulo/GSTT1 presente pode ser considerado como um dos fatores de risco para o desenvolvimento do GPAA. Já para os genótipos GSTM1/GSTT1 nulos foi verificado o risco de 6,7 vezes mais a chance de ocorrência da doença (p=0,0004; OR: 6,7; IC 95%: 2,7 20,3), sugerindo que indivíduos que apresentam os genótipos GSTM1/GSTT1 nulos pode ser considerado como um dos fatores de risco para o desenvolvimento do GPAA.

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.

Breast cancer is the most frequent malignancy among women, especially in Western societies. Highly penetrant genes such as BRCA1 and BRCA2, together with the reproductive history can constitute only 30% of the cause, so there should be other common genes, which may play a role in breast carcinogenesis according to one&#039 / s lifestyle. In our case, the effect of N-acetyl transferases (NAT1, NAT2) and glutathione-S transferases (GSTM1&amp / GSTT1) were investigated, since variations in these genes may alter their enzymatic activity and therefore their capacity to biotransform xenobiotic compounds. To evaluate the potential association between NAT1, NAT2, GSTM1 and GSTT1 genotypes and development of breast cancer, a hospital based case-control study was conducted in a Turkish study population consisting of 37 histologically confirmed incident breast cancer cases and 34 control subjects with no present or previous history of cancer. The only recognizable difference between case and control groups is the percentage of GSTM1 deletion, 67.6% and 44.1% respectively (p=0.047). The frequency of rapid NAT2 acetylator genotype is 44.4% in cases and 23.5% in controls. Especially, women with NAT2 rapid acetylator and GSTM1 null genotypes were at the elevated risk (OR, 3.8 / CI, 0.9-15.4). NAT1 rapid acetylator genotype showed no association with breast cancer. These results suggest that GSTM1 null genotype is a susceptibility factor for breast cancer, particularly in the presence of NAT2 rapid acetylator genotype.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains xi, 109 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 92-109).

Made available in DSpace on 2015-12-06T23:47:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2007 / Objetivo: Analisar os polimorfismos da proteína p53 no códom 72, as famílias das enzimas metabolizantes GSTM1 e GSTT1 e o Papilomavírus humano (HPV) na carcinogênese do adenocarcinoma do colo uterino. Casuística e Métodos: O estudo caso-controle envolve 43 amostras de adenocarcinoma do colo uterino fixados e blocados em parafina pertencentes ao grupo estudo e 86 amostras de células endocervicais, coletadas com escova endocervical, de mulheres sem tumor cervical pertencentes ao grupo controle. A demonstração da presença do HPV, polimorfismo da p53, da enzima GSTM1, de seu alelo nulo GSTM1*0, da GSTT1 e de seu alelo nulo GSTT1*0 foi realizada utilizando-se a reação em cadeia da polimerase. Resultados: A média de idade do grupo estudo foi 52,49 anos e a do grupo controle foi 48,07 anos. O teste t de Student (p=0,1042) (IC de 95%) mostrou homogeneidade entre os dois grupos. O HPV esteve presente em 42(97,67%) dos casos do grupo estudo e em 27(31,40%) dos controles. A análise estatística confirmou diferença significante (p=0,001) (IC de 95%) e razão de chances foi de 113,79 vezes (IC de 95%:13,671; 945,145) (p<0,001). As análises da presença das isoformas da p53, da associação do HPV e alelos da p53 realizados entre os dois grupos não apresentaram significância estatística Da mesma forma a presença das GSTM1 e GSTM1*0, suas associações com HPV e isoformas da p53 não foram estatisticamente significantes. Por sua vez a análise de GSTT1 e GSTT1*0 pelo teste do χ2, foi significante p=0,001 (IC de 95%) e a razão de chances foi de 4,58 vezes (IC de 95%:2,04; 10,28) (p<0,001). As associações de GSTT1 e GSTT1*0 com HPV evidenciaram p<0,001(IC de 95%). A razão de chances da GSTT1*0 foi de 6,67 vezes (IC de 95%: 1,99; 22,17) (p<0,001). Por outro lado, a razão de chances de GSTT1 foi de 0,15(IC de 95%:0,045; 0,502) (p<0,0021). A associação de GSTT1 e GSTT1*0 com as isoformas da p53 não foram estatisticamente significantes (p=0,056) (IC de 95%). A análise da associação das famílias GSTM1 e GSTT1, por modelo de regressão logística, mostrou que a família GSTM1 não era um fator prognóstico no adenocarcinoma cervical nem isolada, nem associada à família GSTT1. Conclusões: O HPV estava presente em 97,67% dos adenocarcinomas, e associou-se com aumento do risco de 113,79 vezes para a carcinogênese do adenocarcinoma, enquanto a presença de GSTT1*0 mostrou risco para adenocarcinoma de 4,58 vezes. A sua associação com HPV aumentou o risco em adquirir adenocarcinoma para 6,67 vezes. A presença de GSTT1 ofereceu proteção contra a carcinogênese do adenocarcinoma. As isoformas da p53 e a família GSTM1 não participaram da carcinogênese do adenocarcinoma do colo uterino na população estudada. / Purpose: To analyze the codon 72 p53 protein and the metabolizing enzymes GSTM1 and GSTT1 families’ polymorphisms in the uterine cervix adenocarcinoma carcinogenesis. Methods: This case-control study concerned 43 adenocarcinoma samples from formalin-fixed and paraffin- embedded tissues belonging study group and 86 endocervical cell samples, collected by cytobrush, from women without cervical tumor belonged to the control group. Demonstration of HPV presence, p53 polymorphisms, the GSTM1enzyme and its null allele (GSTM1*0), the GSTT1 enzyme and its null allele (GSTT1*0) were performed using the polymerase chain reaction. Results: The study group mean age was 52.49 years and in the control group was 48.07 years. The groups were homogeneous by the t Student test (p=0.1042) (95% CI). The HPV was present in 42(97.67%) cases from study group and in 27(31.40%) from the control group and the χ2 test was significant (p=0.001) (95% CI), the odds ratio was 113.79 (95% CI: 13.671; 947.145) (p<0,001). The p53 isoforms analysis by the Fisher exact test of the two groups wasn’t significant p = 0.397(95% CI). The analysis of HPV and p53 alleles association in the two groups by Fisher exact test was p=0,653 (95% CI).The GSTM1and GSTM1*0 analysis in the two groups didn´t show difference by the χ2 test was p= 0.374 (CI = 95%). The GSTM1and GSTM1*0 associations with HPV and p53 isoforms by Fisher exact test weren’t significant p=0.256 (95% CI) and p = 1.000 (95% CI) respectively. The GSTT1and GSTT1*0 analysis by the χ2 test was p= 0.001 (95% CI) and the odds ratio was 4.58 (95% CI: 2.04; 10.28) (p<0,001). The GSTT1and GSTT1*0 associations with HPV by Fisher exact test was p<0.001(95% CI). The GSTT1*0 odds ratio was 6.67(95% CI: 1.99; 22.17) (p<0.001). The GSTT1 odds ratio was 0.15 (95% CI: 0.045; 0.502) (p<0.0021). The association of GSTT1 and GSTT1*0 with p53 by Fisher exact test wasn’t significant p=0.056 (95% CI). The GSTM1 and GSTT1 families’ association analysis by a regression logistic model shows that the GSTM1 family wasn’t a prognostic factor in cervical adenocarcinoma either alone or with GSTT1 family. Conclusions: HPV was present in 97.67% of the adenocarcinomas. The HPV status showed a risk of 113.79 times for adenocarcinoma carcinogenesis. GSTT1*0 showed a risk for adenocarcinoma of 4.6 times, and in its association with HPV increased the risk in acquiring uterine cervix adenocarcinoma to 6.67 times. The presence of GSTT1 offered protection against adenocarcinoma carcinogenesis. The p53 isoforms and the GSTM1 family didn’t share the adenocarcinoma carcinogenesis in this studied population. / BV UNIFESP: Teses e dissertações