Thesis (Ph. D.)--University of Wisconsin--Madison, 1965. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Pharmacology and Toxicology. Title from title-page of electronic thesis. Bibliography: leaves 219-238.

Growth and deposition of amyloid fibrils, polymers of proteins with a cross beta-sheet structure, are associated with a significant number of human pathologies including Alzheimer’s disease, Parkinson’s disease, prion diseases, type II diabetes, and senile systematic or dialysis-related amyloidoses. The broader objective of my research is to identify the basic mechanisms regulating nucleation and growth of amyloid fibrils. There is increasing evidence that amyloid formation may proceed along at least two distinct assembly pathways for the formation of rigid fibrils. One pathway involves the nucleated polymerization of the characteristic rigid fibrils from partially denatured monomers and the other proceeds via the growth of globular oligomers and their associated curvilinear fibrils (also known as protofibrils) which, in ways yet to be determined, transform into late-stage rigid fibrils. These oligomeric intermediates of fibril assembly, in particular, have been implicated as the predominant aggregate species causing cellular toxicity in amyloid diseases. Yet, amyloid oligomers and curvilinear fibrils are considered transient, metastable aggregates. This raises the question whether and how such transient aggregate species can be responsible for most of the cell/tissue toxicity? In this dissertation, I report on my investigation of several basic questions related to the mechanisms of amyloid formation. Using the model amyloid hen egg-white lysozyme, I participated in research to characterize the distinct kinetics of amyloid formation along distinct assembly pathways, to determine the morphological features of the various aggregate species emerging along either pathway, and to investigate the structural evolution of the monomers from their native state to the amyloid cross- sheet structure (chapter 3). Chapters 4-6 represent the core of my dissertation work. There I investigated whether amyloid aggregates from three different amyloid proteins, formed under denaturing condition, could undergo prion-like proliferation upon return to physiological solution conditions. I was also intimately involved in a project on the conditions inducing amyloid spherulites formation by polyglutamic acid and the mechanisms resulting in the formation of this often-overlooked amyloid aggregate structure (chapter 7). In the appendix I provide a short summary of the various experimental techniques I have used in the above experiments.

Differences have been shown in the steady-state accumulation and half-lives between Brassica FAD3 (BF3) and tung FAD3 (TF3) proteins expressed in yeast cells cultured at 30°C. TF3 has a greater steady-state accumulation and longer half-life than BF3. These differences are attributed to post-translational modification and have been shown to be controlled by an Nterminal element. I attempted to determine specific amino acids important for regulation, and further characterize the mechanism contributing to the differences. Through site-directed mutagenesis, it was shown that replacing lysine residues with asparagines in the BF3 and TF3 Ntermini increased protein stability, while replacing an asparagine with lysine in the TF3 Nterminus decreased its stability. Furthermore, I showed that the TF3 polyglutamic region (six consecutive glutamic acid residues) is primarily responsible for the higher steady-state amount of TF3 in comparison to BF3. This negatively charged region likely acts as an electrostatic shield protecting the protein from degradation.

Submitted by Erik de Lima Andrade null (erik_andrade88@hotmail.com) on 2017-03-06T18:12:25Z No. of bitstreams: 1 Dissertação_Erik de Lima Andrade.pdf: 2096082 bytes, checksum: fdfbde2d75d685f0c1cda5fc47b0b3f1 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-03-10T14:43:15Z (GMT) No. of bitstreams: 1 andrade_el_me_soro.pdf: 2096082 bytes, checksum: fdfbde2d75d685f0c1cda5fc47b0b3f1 (MD5) / Made available in DSpace on 2017-03-10T14:43:15Z (GMT). No. of bitstreams: 1 andrade_el_me_soro.pdf: 2096082 bytes, checksum: fdfbde2d75d685f0c1cda5fc47b0b3f1 (MD5) Previous issue date: 2017-02-09 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A adequação no tratamento da água, para abastecimento público envolve a compreensão da vulnerabilidade de mananciais e de situações de risco à saúde humana. Da mesma forma, o sucesso do tratamento de água de superfície depende, fortemente, da eficácia do desempenho do coagulante. Com a qualidade dos mananciais comprometida por diferentes cenários, tornase indispensável, o processo de desenvolvimento de abordagens eficazes para as questões de caráter econômico e ambiental. O tratamento de água de superfície objetiva, de modo geral, a remoção de impurezas com o propósito de enquadrá-la no padrão de potabilidade, essencialmente, através do uso de produtos químicos. Neste sentido, o presente trabalho analisou a eficiência de três produtos comerciais: sulfato de alumínio, policloreto de alumínio e PGα21Ca. Para tanto, foram realizados ensaios de coagulação-floculação e sedimentação, em água da ETA de Salto de Pirapora, São Paulo, Brasil. Os resultados obtidos do PGα21Ca revelam caráter, essencialmente, inorgânico, com teores elevados de sulfato de cálcio (87%) e 5% de ácido γ-poliglutâmico, entre outros compostos químicos. O PGα21Ca apresentou maior eficiência de remoção de turbidez e cor aparente, com valores de 1,93 NTU de turbidez remanescente e 96,89% de remoção de cor aparente e pouca alteração no pH do meio, demonstrando efetividade para o tratamento de água para abastecimento público. O PAC apresentou resultados inferiores para clarificação da água quando comparado ao PGα21Ca e melhores resultados quando comparado ao sulfato de alumínio. Utilizando-se o PGα21Ca em água de Rio Grande, não obtém-se resultados tão expressivos quando comparado a água da ETA de Salto de Pirapora. / The suitability in the treatment of water for public supply involves understanding the vulnerability of sources and situations of risk to human health. Similarly, the success of surface water treatment depends strongly on the effectiveness of the coagulant performance. With the quality of water sources compromised by different scenarios, it is essential to the effective approaches development process for the economic and environmental character issues. The water treatment lens surface generally the removal of impurities in order to fit it in potable standard, mainly through the use of chemicals. In this sense, the present study examined the effectiveness of three commercial products: aluminum sulphate, polyaluminum chloride and PGα21Ca. Therefore, tests were carried out coagulation-flocculation and sedimentation in water of Salto de Pirapora WTP, São Paulo, Brazil. The results obtained reveal the character PGα21Ca essentially inorganic, with high levels of calcium sulfate (87%) and 5% γ-polyglutamic acid, and other chemical compounds. The PGα21Ca showed higher turbidity and apparent color removal efficiency, with values of 1.93 NTU turbidity and remaining 96.89% removal of apparent color and little change in pH of the medium, demonstrating the effectiveness for water treatment. The PAC presented lower results for clarification of water when compared to PGα21Ca and better results when compared to aluminum sulfate. Using PGα21Ca in Rio Grande water did not obtain significant results when compared to the water of the Salto de Pirapora WTP. / FAPESP: 2015/02650-8

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