Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 143-169). Available also in a digital version from Dissertation Abstracts.

Thesis (M. Phil.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 103-121).

Thesis (Ph. D.)--Florida State University, 2004. / Advisor: Dr. Michael Chapman, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Sept. 15, 2005). Document formatted into pages; contains vi, 76 pages. Includes bibliographical references.

Thèse (Ph. D.)--Université Laval, 2003. / Bibliogr.: f. [241]-283. Publié aussi en version électronique.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 90-99). Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 90-99). Also available online.

Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 114-124).

Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 121-133).

Orientador: Edwil Aparecida de Lucca Gattás / Banca: Eleonora Cano Carmona / Banca: Rubens Monti / Resumo: A levedura Pichia pastoris vem sendo largamente utilizada como um eficiente sistema de expressão para a produção de proteínas heterólogas, pois é um sistema seguro, fácil e mais barato que sistemas de expressão de outros eucariotos. Neste trabalho, a enzima de interesse é a glicerol quinase (GK), que cataliza a transferência do fosfato terminal do ATP para o glicerol originando glicerol-3-fosfato e ADP. Esta reação pode ser utilizada na determinação da concentração de glicerol, subproduto da fermentação alcoólica. A leitura do consumo de glicerol é realizada pela determinação espectrofotométrica do NADH gerado na reação de oxido-redução catalizada pela enzima glicerol-3-fosfato desidrogenase. Este estudo de indução foi realizado em diferentes condições de crescimento da levedura Pichia pastoris. Os resultados mostraram a seleção do melhor clone da levedura Pichia pastoris para a expressão extracelular da enzima glicerol quinase, e a determinação das melhores condições do meio de cultura para a produção da enzima de interesse foram: concentração do meio de cultura BMMY (20 vezes), densidade inicial de célula (0,1 mg/mL), concentração de metanol na fase de indução (1%), natureza do tampão (fosfato de potássio), pH (6,0), suplementação de glicerol no meio BMMY (1%), peptona (marca Difco), sem adição de sulfato de amônio, caseína e glicina, uso do meio BMMY e liofilização do mesmo. Estudos de parâmetros cinéticos foram realizados e a atividade máxima da GK foi obtida em pH 9,8, a 50ºC e 2,5 μM de substrato, por metodologia clássica, além da presença de sulfato de magnésio e diluição da enzima de 30 vezes. A enzima apresentou alta estabilidade térmica ― a atividade foi completamente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The yeast Pichia pastoris has been widely used as an efficient expression system for production of heterologous proteins because it is a safe, easy and cheaper than expression systems in other eukaryotes.In this studie, the enzyme of interest is glycerol kinase (GK), which catalizes the transfer of terminal phosphate from ATP to glycerol resulting glycerol-3-phosphate and ADP. This reaction can be used in determining the concentration of glycerol, a byproduct of fermentation. The reading of the consumption of glycerol is carried out by spectrophotometric determination of NADH generated in the redox reaction catalyzed by the enzyme glycerol-3-phosphate dehydrogenase. This study of induction was performed in different conditions of growth of the yeast Pichia pastoris. The results show that selecting the best clone of the yeast Pichia pastoris for the expression of extracellular enzyme glycerol kinase, and determining the best conditions of the culture medium for producing the enzyme of interest were: concentration of the culture medium BMMY (20 times), initial cell density (0.1 mg/mL), methanol concentration in the induction phase (1%), nature of buffer (potassium phosphate), pH (6.0), glycerol supplementation in BMMY medium (1%), peptone (Difco), without addition of ammonium sulfate, casein and glycine in BMMY and lyophilized medium. Studies of kinetic parameters were conducted and the GK maximum activity was obtained at pH 9.8 at 50°C and 2.5 μM substrate by conventional method, besides the presence of magnesium sulfate and diluting the enzyme 30 times. The enzyme showed high thermal stability - the activity was fully maintained up to 50°C for one hour - and at pH 7.0 for 7 days and kept under refrigeration, freeze-dried extract showed a decrease in enzymatic activity. Calculated by... (Complete abstract click electronic access below) / Mestre

Alzheimer’s disease (AD) is neuropathologically characterized by excessive beta -amyloid (Abeta)plaques and neurofibrillary tangles composed of hyperphosphorylated tau in the brain. Although the etiology of genetic cases of AD has been attributed to mutations in presenilin and amyloid precursor protein (APP) genes, in most sporadic cases of AD, the etiology is still unknown and various predisposing factors could contribute to the pathology of AD. Predominant among these possible predisposing factors that have been implicated in AD are age, hypertension, traumatic brain injury, diabetes, chronic neuroinflammation, alteration in calcium levels and oxidative stress. Since both inflammation and altered calcium levels are implicated in the pathogenesis of AD, we wanted to study the effect of altered levels of calcium on inflammation and the subsequent effect of selective calcium channel blockers on the production of pro-inflammatory cytokines and chemokines. Our hypothesis is that Abeta depending on it conformation, may contribute to altered levels of intracellular calcium in neurons and glial cells. We wanted to determine which conformation of Abeta was most pathogenic in terms of increasing inflammation and calcium influx and further elucidate the possibility of a link between altered calcium levels and inflammation. In addition, we wanted to test whether calcium channel blockers could inhibit the inflammation mediated by the most pathogenic form of Abeta by antagonizing the calcium influx triggered by Abeta. Our results in human glial and neuronal cells demonstrate that the high molecular weight oligomers are the most potent at stimulating the release of pro-inflammatory cytokines IL-6 and IL-8 as well as increasing intracellular levels of calcium compared to other conformations of Abeta. Further, L-type calcium channel blockers and calmodulin kinase inhibitors are able to significantly reduce the levels of IL-6 and IL-8. These results suggest that Abeta induced alteration of intracellular calcium levels contributes to its pro-inflammatory effect.