published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [115]-133).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves [78]-86).

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 154-163.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 78-93).

Made available in DSpace on 2018-06-27T18:56:01Z (GMT). No. of bitstreams: 2 Amanda Lima de Oliveira.pdf: 513074 bytes, checksum: 393de3a3c8893cb2dad143281cc76ca4 (MD5) Amanda Lima de Oliveira.pdf.jpg: 3435 bytes, checksum: d98e4a9d95435991a70b3e949f124696 (MD5) Previous issue date: 2012-11-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cyclodextrins (CDs) are cyclic oligosaccharides formed by 6 (αCD), 7 (bCD) or 8 (γCD) glucose units with an internal hydrophobic cavity and outside surface hydrophilic. These three derivatives, the b-cyclodextrin (bCD) seems to be the most advantageous for pharmaceutical use for their availability, cavity size and low cost. The CDs have a future quite promising for their properties as greater absorption of drugs through the biological barriers and time of release, however, some types may not be considered non-toxic. The objective of this study was to investigate the intraperitoneal administration of βCD, M-β-CD and HP-ß-CD for 8 weeks with administered dose of 65.65 mg of CDs/kg rats/day, on parameters of biochemical analyzes, enzymes of energy metabolism, enzymes tiolicas sensitive to increase reactive oxygen species and to make this relationship, also evaluate parameters of oxidative stress in cerebral cortex, liver, kidneys and heart of wistar rats. The results showed that for the group treated with βCD there has been a significant increase in serum urea and creatinine levels, indicating nephrotoxicity, however not related to the other parameters. There was also a great reduction in serum levels of iron for the 3 CDs. The heart showed a reduction in the activity of CKmitocondrial and increase for AK by M-β-CD and reduction of CKmit by HP-ß-CD, but showed a reduction in the levels of diclorofluorceina (DCF) to the 3 CDs and protein carbonyl) by βCD. For the rim there was no significant change in comreducao activity of CKmit by HP-β-CD. In liver tissue, the βCD and M-β-CD reduced the activity of PK, but this is not reflected in blood glucose levels. In the cerebral cortex, the βCD reduced the activity of enzymes CK mitochondrial and PK, also reduced TBARS, but increased carbonyl protein. The indices lipidemic reduced reported by other researchers was not observed in this work, because the group of M-β- CD has a significant increase in serum levels of LDL cholesterol, in addition to aspartate aminostransferase AST, albumin, total protein, alkaline phosphatase, sodium, calcium, magnesium and phosphate. Our results indicate that some CDs alter enzymes crucial for energy metabolism, mainly of brain tissue with a reduction in activity and the PK by βCD. If changes in the activity of these enzymes occur in people who use drugs by intraperitoneal route, it is possible that the energy metabolism and brain functioning may be affected causing damage to the tissue. However more studies are needed to elucidate how there was a reduction of serum iron and as the cyclodextrins affect a structure so well protected by blood-brain barrier as the brain. / As ciclodextrinas (CDs) são oligossacarídeos cíclicos formados por 6 (αCD), 7 (bCD) ou 8 (γCD) unidades de glicose com uma cavidade interna hidrofóbica e superfície externa hidrofílica. Destes três derivados, a b-ciclodextrina (bCD) parece ser a mais vantajosa para utilização farmacêutica pela sua disponibilidade, tamanho da cavidade e baixo custo. O interesse pelas CDs se dá pelas suas propriedades como maior absorção dos fármacos através das barreiras biológicas e tempo de liberação, entretanto, alguns tipos não podem ser considerados atóxicas. O objetivo deste estudo foi investigar a administração intraperitoneal de βCD (Beta Ciclodextrina), M-β-CD (Metil Beta Ciclodextrina) e HP-β-CD (Hidroxypropil Beta Ciclodextrina) durante 8 semanas com dose administrada de 65,65 mg das CDs/kg rato/dia, sobre parâmetros de análises bioquímicas, de enzimas do metabolismo energético, enzimas tiólicas sensíveis ao aumento de espécies reativas e para fazer a relação, também avaliar parâmetros de estresse oxidativo em córtex cerebral, fígado, rins e coração de ratos wistar. Os resultados mostraram que para o grupo tratado com βCD houve um aumento significativo nos níveis séricos de uréia e creatinina, indicando nefrotoxidade, porém não relacionada com os demais parâmetros. Também houve uma grande redução nos níveis séricos de ferro para as 3 CDs. O coração apresentou redução na atividade da Creatinaquinase mitocondrial (CKmit) e aumento para Adenilatoquinase (AK) pela M- β-CD e redução da CKmit pela HP-β-CD, porém apresentou uma redução nos níveis de diclorofluoresceína (DCF) para as 3 CDs e carbonilas proteicas pela βCD. Para o rim houve alteração significativa com redução na atividade da CKmit pela HP-β-CD. No tecido hepático, a βCD e M-β-CD reduziram a atividade da Piruvatoquinase (PK), porém isto não refletiu nos níveis glicêmicos. No córtex cerebral, a βCD reduziu a atividade das enzimas CK mitocondrial e PK, também reduziu TBARS (Espécies reativas ao ácido tiobarbitúrico), mas aumentou carbonilas proteicas. Os índices lipidêmicos reduzidos relatados por outros pesquisadores não foi observado neste trabalho, pois o grupo da M-β-CD apresentou um aumento significativo nos níveis séricos de LDL (lipoproteína de baixa densidade), além de AST (aspartato aminostransferase), albumina, proteínas totais, fosfatase alcalina, sódio, cálcio, magnésio e fosfato. Os resultados indicam que algumas CDs alteram enzimas cruciais do metabolismo energético, principalmente do tecido cerebral com redução na atividade da PK pela βCD. Possíveis alterações na atividade destas enzimas podem afetar o metabolismo energético e o funcionamento cerebral causando dano ao tecido. Entretanto mais estudos são necessários para elucidar de que forma ocorreu a redução sérica de ferro e como as ciclodextrinas afetaram uma estrutura tão bem protegida pela barreira hemato-encefálica como a cerebral.

The work in this thesis focuses on the study of three main aspects of proteins, viz, Protein structure, stability, and dynamics. Chapter 1 is a general introduction to the topics studied in this thesis. Chapter 2 deals with the first aspect, i.e., protein structure in which we describe an approach to use saturation mutagenesis phenotypes to guide protein structure prediction. Chapters 3 and 4 discuss how to increase protein stability using surface electrostatics, and Chapter 5 details a method to predict whether a proline substitution in a given protein would be stabilizing or destabilizing. Hence, Chapters 3-5 can be associated with the second aspect, i.e., protein stability. The third aspect, namely protein dynamics, is dealt with in Chapters 6 and 7 which study conformational dynamics of adenylate kinase. Protein structure prediction is a difficult problem with two major bottlenecks, namely, generation of accurate models and the selection of the most appropriate models from a large pool of decoys. In Chapter 2, the problem of model discrimination is addressed using mutant phenotype information derived from saturation mutagenesis library. A library of ~1500 single-site mutants of the E. coli toxin CcdB (Controller of Cell Division or Death B) has been previously constructed in our lab. The pooled library was characterized in terms of individual mutant phenotypes at various expression levels which were derived from the relative populations of mutants at each expression level. The relative populations of mutants were estimated using deep sequencing. Mutational tolerances were derived from the phenotypic data and were used to define an empirical parameter which correlated with a structural parameter, residue depth. We further studied how this new parameter can be used for model discrimination. Increasing protein stability in a rational way is a challenging problem and has been addressed by various approaches. One of the most commonly used approaches is optimization of protein core residues. Recently, optimization of protein surface electrostatics has been shown to be a useful approach for increasing stability of proteins. In Chapter 3, from analyses of a dataset of ~1750 non-homologues proteins, we show that proteins having a pI away from physiological pH, possess a significant fraction of unfavorably placed charged amino acids on their surface. One way to increase protein stability in such cases might be to alter these surface charges. This hypothesis was validated experimentally by making charge reversal mutations at putative unfavorable positions on the surface of maltose binding protein, MBP. The observed stabilization can potentially be increased by combining multiple individually stabilizing mutations. Different combinations of such mutations were made and tested in Chapter 4 to decide which mutants can be combined to achieve net stabilization. Ideas were tested through systematic experimentation which involved generation of two-site, three-site, and four-site mutations. A maximum increase in melting temperature (Tm) of 3-4 °C over wild-type protein was achieved upon combination of individually stabilizing mutants. Proline (Pro) has two special stereo-chemical properties when it is a part of a polypeptide chain. First the φ value of Pro has a very constrained distribution and second, Pro lacks an amide hydrogen. Due to these properties, introduction of Pro might perturb stability/activity of the protein. In Chapter 5 we describe a procedure to accurately predict the effects of Pro introduction on protein stability. Pro scanning mutagenesis was carried out on the model protein CcdB and the in vivo activity of the individual mutants was also examined. A decision tree was constructed, using the special stereo-chemical properties of Pro to maximize correlation of predicted phenotype with the in vivo activity. Binary classification as perturbing or non-perturbing of every Pro substitution was possible using the decision tree. The performance of the decision tree was assessed on various test systems, and the average accuracy was found to be ~75%. The role of conformational dynamics in enzyme catalysis has been explored in great detail in the literature. In Chapter 6, with the help of very long (350 ns), fully atomistic, explicit solvent molecular dynamics simulations, we studied conformational dynamics of adenylate kinase. We found the existence of a relatively stable state which lies intermediate between the open and closed conformations of the enzyme. The finding was further confirmed by computing a two dimensional configurational free energy surface when motions along each of the two movable domains (LID and NMP) are considered as reaction coordinates. We also discussed possible roles of the intermediate state during enzyme catalysis. The role of water in stabilization of intermediate states was also discussed. In Chapter 7, we studied dynamical coupling between LID and NMP domains of adenylate kinase during domain opening. Our observation suggests that the LID domain should start opening prior to the NMP domain. On the domain opening trajectory, the free energy surface of LID domain was found to be very rugged. We discuss a possible role of water in the ruggedness of the domain motions. The Appendix contains 3 supplementary parts of the thesis. Appendix I is a mutant dataset obtained from 454 sequencing analysis. It includes the normalized number of reads per mutation at each expression level along with mutational sensitivity score. Appendix II is parameters used for one of the electrostatic calculations. Appendix III contains a list of PDB ids used for database analysis in surface electrostatics work discussed in Chapter 3.

Bordetella pertussis produces many virulent factors including adenylate cyclase toxin (CyaA) This toxin preferentially invades cells of immune system with integrin receptor CD11b/CD18 and weakens the immune system of the host. CyaA affects invaded cells in two ways. First, CyaA creates a cation-selective pores in the membrane of invaded cell and causes colloidal osmotic lysis. Second, CyaA converts cytosolic ATP into signal molecule cAMP, which causes a loss of physiological function of invaded cell and also leads to cellular death. The aim of my thesis was to test a suitability of a new model system composed from synthetic lipids - diphytanoyls, for a characterization of pore-forming properties of adenylate cyclase toxin. In the past, asolectin model system comprising many different lipid was used for characterization but it was found to be too complex for defining the role of individual lipids in CyaA activity. Further the effect of cholesterol for activity of CyaA was studied in a new model system because it was found recently that translocation of adenylate cyclase domain takes place at lipids rafts with high concentration of cholesterol. The last aim of my thesis was to characterize a newly discovered type of channel with the two conductance levels. Key words: Bordetella pertussis, adenylate...

Right ventricular function (RV) is one of the most important predictors of prognosis in many cardiovascular disease states. Despite the significance of RV function to survival, there are no therapies that directly nor selectively improve RV function. As well, the basis for RV failure is poorly understood. This is particularly relevant for patients with pulmonary arterial hypertension (PAH), where RV failure in the setting of pressure overload is the leading cause of death. PAH will be introduced in the 2nd chapter of this thesis by comparing and refining contemporary mortality risk assessment strategies. I will then explore 1) RV neurohormonal function and, 2) RV energetics, two molecular pathways thought to be involved in the pathogenesis and progression of maladaptive RV failure. I employed small animal molecular imaging using positron emission tomography (PET) to non-invasively investigate these pathways. The PET imaging techniques employed in this thesis have the unique potential for translation to human studies, to further explore disease mechanisms.

Secretin and VIP stimulate cardiac adenylyl cyclase activity and exert a positive inotropic action in several mammalian species. This study examined positive inotropic activity and cAMP levels in rat papillary muscle. Isoproterenol and secretin increased contractions by 150 ± 31% and 129 ± 27%, respectively. VIP increased contraction by 30 ± 21% only at 10 μM. Isoproterenol significantly increased cAMP levels by 82%, whereas increases by secretin (58%) and VIP (56%) were not significant. These results are consistent with reports that secretin and VIP stimulate cardiac adenylyl cyclase in the rat, but suggest that cAMP tissue levels cannot totally explain the positive inotropic responses to secretin and VIP.