Photocopy of typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1979. / Bibliography: leaves 174-182. / Microfiche. / xvi, 182 leaves ill. 29 cm

Thesis (D. Sc.)--University of Sydney, 2003. / Title from title screen (viewed Apr. 28, 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Science to the School of Chemistry, Faculty of Science. Degree awarded 2003; thesis submitted 2002. Includes bibliographical references. Also available in print form.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 224-231).

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Os calcógenos de európio (EuX, onde X representa O, S, Se ou Te) possuem propriedades magneto-ópticas únicas e interessantes, devido ao enorme magnetismo gerado dos elétrons na camada f do átomo pertencente a família dos terras raras, tornando estes materiais atraentes para aplicações na spintrônica (eletrônica baseada nos transporte de spins e não de carga). Neste trabalho investigamos em baixa temperatura o espectro de absorção utilizando luz circularmente polarizada na região próxima do limiar da banda para o telureto de európio EuTe e o seleneto de európio EuSe em alto campo magnético no ordenamento ferromagnético dos spins de Eu^{2+} da rede cristalina. As amostras crescidas por epitaxia por feixe molecular apresentaram um dicroísmo circular magnético intenso no espectro de absorção para a configuração de Faraday. O par de linhas estreitas observadas estão separadas de aproximadamente 200 meV para o EuTe e 300 meV para o EuSe. Em seguida, formulamos um modelo teórico para a interpretação deste espectro de absorção no arcabouço do modelo de transições eletrônicas entre o estado fundamental 4f^{7}({8}^S_{7/2}) e o estado excitado formado dos estados do caroço remanescente 4f^{6}({7}^F_{J=0...6}) mais o estado em que o elétron se encontra na banda de condução 5d(t_{2g}), resultando em uma excelente concordancia qualitativa e quantitativa com o experimento. / Europium chalcogenides (EuX, where X stands for O, S, Se or Te) have very interesting and unique magneto-optical properties, due to the huge magnetism that arises from the electrons in the f?shell of the rare earth element and which makes them attractive for spintronics applications ( spin transport electronics or spin basedelectronics) In this work we investigate the band-edge optical absorption in high magnetic fields in the Faraday geometry for EuTe and EuSe in the ferromagnetic order attained at low temperatures. In thin layers grown by molecular beam epitaxy, an intense magnetic circular dichroism were observed. The doublet of absorption lines showed a separation by about 200meV in EuTe and 300meV in EuSe. Next, we developed a theoretical model for the interpretation of the absorption spectrum, based in the framework of the model of an electronic transition from a localized ground state 4f^{7}({8}^S_{7/2)) to an excited state formed by the core states 4f^{6}({7}^F_{J=0...6}) and the electron extended state in the 5d(t_{2g}) conduction band, yielding an excellent qualitative and quantitavie agreement with experiment.

Neste trabalho, utilizando técnicas magneto-ópticas, estudamos tempos de relaxação spin-rede (T1) do estado fundamental de centros \'F\' e, cristais de halogenetos alcalinos (KCl-KBr). Descrevemos um sistema semi-automático para medidas ópticas de T1, capaz de medir tempos de relação curtos (~1mS), baseado na medida do Dicroísmo Circular magnético (DCM) que apresentam aqueles sais quando portadores de centros paramagnéticos. Obtivemos a dependência de T1 com o campo magnético H (até 65 Kgauss), bem como os espectros de DCM para diferentes concentrações nas matrizes mistas. Uma teoria desenvolvida por Panepucci e Mollenauer (1) para matrizes puras, foi adaptadas para explicar a relaxação spin-rede nos cristais mistos. Os resultados obtidos para o processo direto (T~2.0 K), confrontados com auqela teoria, mostram que o mecanismo de relaxação dominante até 25 KGauss continua sendo a modulação por fônons da interação hiperfins entre o elétron \'F\' e os núcleos vizinhos. / Using magneto-optic techniques we have studied the ground state spin- lattice relaxation times (T1) of \'F\' centers in mixed Alkali Halide cristals (KCl-KBr). We describe a computer assisted system to optically measure short relaxation times (~1mS). The technique is based on the measurement of the Magnetic Circular Dicroism (MCD) presented by F centers. We obtained the T1 magnetic field dependency at 2 K up to 65 kGauss), as well as the MCD spectra for different relative concentration at the mixed matrices. The theory developed by Panepucci and Mollenauer for F centers spin-lattice relaxation in pure matrices was modified to explain the behavior of T1 in mixed cristais. The Direct Process results (T~2.0 K) compared against that theory shows that the main relaxation mecanism, Up to 25 kGauss, continues to be phonon modulation of the hyperfine interaction between \'F\' electrons and surrounding nuclei.

PEGylation of protein side-chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs, and has been enabled by the recent development of many chemoselective reactions for protein side-chain modification. However, there are no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal loss to biological activity. Chapter 1 is a brief introduction to protein PEGylation. In chapter 2 we use the WW domain of the human protein Pin 1 (WW) as a model system to probe the impact of PEG on protein conformational stability. Using a combination of experimental and theoretical approaches, we develop a structure-based method for predicting which sites within WW are most likely to experience PEG-based stabilization, and show that this method correctly predicts the location of a stabilizing PEGylation site within the chicken Src SH3 domain. PEG-based stabilization in WW is associated with enhanced resistance to proteolysis, is entropic in origin, and likely involves disruption by PEG of the network of hydrogen-bound solvent molecules that surround the protein. Chapter 3 shows that PEG-based stabilization of the WW domain depends strongly on the identity of the PEG-protein linker, with the most stabilizing linkers involving conjugation of PEG to an Asn side-chain amide nitrogen. Chapter 4 investigates the interplay between structure-based guidelines for PEG-base stabilization developed in chapter 2 and the different chemistries explored in chapter 3.

Orientador: Raghuvir Krishnaswamy Arni / Coorientador: José Osmar Gaspar / Banca: Marcelo Andrés Fossey / Banca: Priscila Belintani / Resumo: As atividades realizadas compreenderam a produção de cDNA utilizando primer anti-senso e RNA purificado, amplificação do gene codificador da proteína 25K do Cole latent virus (CoLV25K), purificação do fragmento amplificado, ligação em vetor de multiplicação pGEM-T, transformação em células competentes de Escherichia coli linhagem TOP 10, purificação do vetor, digestão enzimática com as enzimas Bam HI e Hind III, subclonagem no vetor de expressão pET28a, transformação em células competentes de E. coli linhagem BL21-RIL, sequenciamento do gene no vetor de expressão e expressão da proteína a 37 o C . Quando expressa a 37 ºC, a proteína, de 25 kDa, foi encontrada em sua totalidade nos corpos de inclusão. Dessa forma, a proteína foi purificada sob condição desnaturante (utilizando 8 M de uréia) e submetida à diálise para seu reenovelamento. Após o reenovelamento, a proteína foi concentrada para aproximadamente 3 mg/mL e foram realizadas medidas de dicroísmo circular, para verificar o seu conteúdo de estrutura secundária, e o espalhamento dinâmico de luz, para estimar a distribuição de tamanho das populações de partículas que estão presentes na solução. Os dados da deconvolução do experimento de dicroísmo circular indicam um percentual de 40-46% α-hélice, 12-14% folha-β, 15-22% voltas e 24-28% de outras estruturas, indicando que a proteína está estruturada; e os dados do espalhamento dinâmico de luz mostraram que a proteína encontra-se estável, monodispersa, mas apresenta um complexo de partículas que deve ser removido para que fique nas condições ideais de cristalização. Foram utilizadas também outras técnicas para tentar alcançar a solubilidade da proteína expressa: abaixando a temperatura... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The experiments performed were, the production of cDNA using anti-sense primers and purified RNA, amplification of the gene encoding the 25K protein of Cole latent virus (CoLV25K), purification of the amplified fragment, cloning in multiplication vector pGEM-T for cell transformation into competent Escherichia coli strain TOP 10, vector purification, enzymatic digestion using the enzymes Bam HI and Hind III, subcloning in expression vector pET28a, transformation into competent cells of E. coli strain BL21-RIL, sequencing of the gene cloned into the expression vector and protein expression at 37 o C. When expressed at 37 °C, the protein with a molecular mass of 25 kDa was detected in inclusion bodies. Thus, the protein was purified under denaturing conditions (using 8 M urea) and subjected to dialysis to stimulate refolding. After refolding, the protein was concentrated to approximately 3 mg / mL and the circular dichroism assay was performed to verify the content of secondary structure, and dynamic light scattering, to estimate the size distribution of particle populations which are present in solution. The data from the deconvolution of circular dichroism experiments indicate a percentage of 40-46% α-helix, 12-14% β-sheet, 15-22% turns and 24-28% of other structures, indicating a structured protein; and the data of the dynamic light scattering showed that the protein is stable, monodispersive, but forms a large complex of particles which must be separated to before crystallization experiments. Other techniques were used to solubilize the expressed protein: lowering the temperature of expression to 18 °C, using the method... (Complete abstract click electronic access below) / Mestre

Porous cellulose nitrate membranes were impregnated with 8CB and PCH5 LCs (liquid crystals) and separations of solutes dissolved in aqueous phases were performed while monitoring solute concentration via UV-VIS spectrometry. The diffusing organic solutes, which consist of one aromatic ring and various functional groups, were selected to exclude molecular size effects on the diffusion and sorption. We studied the effects on solute transport of solute intra-molecular hydrogen bonding and solute/LC intermolecular hydrogen bonding. Hydrogen-bonding effects are a significant factor in the permeation selectivity of positional isomers. The reduction of available hydrogen-bond donors in aromatic ortho-isomers due to intramolecular H-bonding resulted in significant differences in the diffusion relative to the para-isomers which possessed more available H-bond donors. Solutes possessing multiple H-bonding interactions experienced a higher barrier to diffusion and, consequently, lower diffusivities. Diffusing solutes with a single available H-bond donor exhibited faster diffusion than solutes without H-bond donors. PCH5 embedded membranes showed higher solubility and diffusivity than the 8CB embedded membranes due to less dense molecular packing in PCH5 resulting from the bent cyclohexyl ring. The PCH5 LC membranes demonstrated enhanced permeation selectivity for hydroxybenzoic acid and aminophenol isomers primarily due to increased sorption selectivity. Shape selective absorption of rod-like para-isomers in the nematic phase was observed in both 8CB and PCH5 LCs. A nonchiral based HPLC-CD (High Performance Liquid Chromatography-CD) system was developed for the characterization of enantioselective separations. An enantioselective cholesteric liquid crystal membrane was fabricated and evaluated using the nonchiral HPLC-CD system. The cholesteric LC membrane showed enantioselectivity in the cholesteric phase where activation energies of permeation for 1-phenylethanol enantiomers were significantly increased due to the increased interactions between enantiomer and LC phase. The enantioselectivity increased with decreasing pore size of the membrane and increasing chiral dopant compositions. The selectivity decreases when there are no hydrogen bonding interactions between enantiomer and chiral dopant. / Ph. D.

Secreted phospholipase A2 (PLA2) is an interfacial enzyme that catalyzes the calcium-dependent hydrolysis of glycerophospholipids to free fatty acids and lyso-phospholipid, which are further converted to eicosanoids and platelet activating factor with broad biological activities. PLA2 is inactive in solution, but undergoes interfacial activation upon binding to biological membranes. Despite extensive studies on secreted PLA2s, the structural basis for interfacial activation and the effects of site-directed mutations remain largely uncharacterized. Two mutants of human group IIA PLA2, with tryptophans incorporated at the 3rd or 5th position in the N-terminal helix, display dramatic differences in activity compared to the wildtype enzyme. This project analyzes the distinct structural changes that occur in PLA2 and two Trp-mutants during interfacial activation, which are responsible for the observed disparities in activity. Additionally, the thermal stability of both mutants was determined in order to explore possible correlations between resistance to thermal denaturation and enzymatic activity. The V3W mutant shows enhanced activity and a higher optimal temperature compared to the wildtype, which may be promoted at least partially by the high affinity of tryptophan for the lipid-aqueous interface. Contrastingly, the FSW enzyme, which has a tryptophan within the substrate-binding pocket, displays greatly diminished activity compared to both the wild-type and V3W mutant, suggesting inefficient loading of substrate. Circular dichroism and fluorescence studies reveal that the differences in activity of the mutants result from distinct structural changes upon activation. Furthermore, thermal denaturation of V3W was partially reversible, whereas F5W showed no recovery of secondary structure following decrease of temperature. Thus, tryptophan incorporation at two close positions modulates the activity of PLA2 in strikingly different ways, which are associated with defined changes in the secondary structure and the thermal stability of the enzyme. Our results may find industrial or pharmaceutical applications, such as production of fatty acids or development of antibacterial agents.