Thesis (M.S.)--Indiana University, 2007. / Title from screen (viewed on Apr. 27, 2007) Department of Pharmacology & Toxicology, Indiana University-Purdue University Indianapolis (IUPUI) Includes vita. Includes bibliographical references (leaves 71-84)

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Made available in DSpace on 2014-10-09T12:55:22Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:05:55Z (GMT). No. of bitstreams: 0 / A variabilidade na composição e nas atividades biológicas dos venenos de serpentes vem sendo documentada por diversos autores e pode ser observada em diversos níveis. As diferenças na composição dos venenos apresentam relevância na terapêutica dos envenenamentos ofídicos, tornando o estudo da variação dos venenos de extrema importância para a confecção de antivenenos mais específicos e de maior eficácia nos tratamentos de envenenamentos ofídicos em humanos. Os estudos das variações do veneno de serpentes em populações isoladas são raros, sendo no Brasil os casos mais conhecidos os da Bothrops insularis e Bothrops alcatraz. Diversos estudos sugerem que as oscilações do nível marinho, ocorridas há 11.000 anos, teriam isolado populações de serpentes nas ilhas recentemente formadas, resultando em duas diferentes rotas evolutivas que deram origem a estas espécies. Tendo em vista que a formação da Ilha de São Sebastião-SP ocorreu no mesmo período, isolando populações de animais ali existentes, nos propusemos a avaliar a variação nas atividades do veneno de 80 exemplares de serpentes Bothrops jararaca provenientes da ilha e da sua área de distribuição continental, comparando-as com o Veneno Referência Nacional, para as atividades proteolítica sobre caseína, fosfolipásica indireta, hemorrágica e amidolítica, assim como de seus perfis eletroforéticos (SDS-PAGE) e seu reconhecimento pelo soro antibotrópico comercial através da técnica de Western Blot. A análise eletroforética (SDS-PAGE 12,5%) através de densitometria óptica evidenciou, em sua maioria bandas protéicas de baixo peso molecular e a presença de duas áreas majoritárias com grande variação no número, disposição e intensidade das bandas nas faixas de pesos moleculares de 45 kDa e 25 kDa. A variação da disposição das bandas apresentadas nos géis correlacionou-se com a variação nas atividades. Amostras que apresentaram baixa atividade fosfolipásica foram exatamente aquelas que apresentaram poucas e tênues bandas com aproximadamente 14 kDa. A análise das atividades proteolítica sobre caseína, fosfolipásica e esterásica apresentou um padrão de similaridade que nos permitiu estabelecer duas grandes subpopulações de venenos; uma ao norte da distribuição geográfica amostrada, apresentando altas atividades proteolítica sobre caseína, fosfolipásica e amidolítica e outra ao sul apresentando menores valores para tais variedades. Não foi possível estabelecer uma relação direta entre o padrão de distribuição geográfica com a variação nas atividades hemorrágicas dos venenos amostrados. A técnica de Western Blot permitiu concluir que o soro antibotrópico comercial não apresenta um bom reconhecimento para proteínas de baixo peso molecular. A análise das atividades dos venenos dos exemplares insulares apresentou similaridade com as atividades das amostras de veneno de serpentes coletadas próximas à ilha, sugerindo que o intercâmbio gênico não tenha sido de todo interrompido, provavelmente devido à sua proximidade para com o continente. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / A graviola (Annona muricata L.) à uma fruta bastante utilizada na alimentaÃÃo, pois fornece vitaminas, carboidratos, proteÃnas, lipÃdios, fibras e outras molÃculas para nutriÃÃo humana. Nos Ãltimos anos a graviola (fruto tropical exÃtico) tem apresentado uma posiÃÃo de destaque na economia brasileira, principalmente para Estados do Nordeste como CearÃ, Alagoas, Bahia e Pernambuco. A alta produÃÃo do fruto de graviola no Nordeste se dà principalmente pela floraÃÃo ocorrer durante o ano todo, contudo estima-se que a perda pÃs-colheita chega a 50% da produÃÃo, como ocorre com outros frutos nativos ou exÃticos. Tal perda pÃs-colheita do produto se deve à acelerada taxa de desenvolvimento e senescÃncia do fruto. Apesar de haver alguns estudos sobre graviola, o entendimento bioquÃmico e molecular do processo de amadurecimento à muito limitado tornando-se importante avanÃar nesses conhecimentos a fim de desenvolver estratÃgias para o aumento da vida de prateleira desse fruto. No presente trabalho, buscou-se por um melhor protocolo de extraÃÃo de proteÃnas da polpa de graviola para gerar mapas bidimensionais e identificaÃÃo de possÃveis proteÃnas envolvidas no amadurecimento pÃs-colheita do fruto. Os frutos de graviola no estÃdio prÃ-climatÃrico foram colhidos e deixados amadurecer a temperatura de 25 C por atà 8 dias. Amostras da polpa do fruto foram coletadas nos tempos 0, 2, 4, 6 e 8 dias pÃs-colheita e usadas para a extraÃÃo de proteÃnas e estabelecimento de gÃis bidimensionais (2D). Dois diferentes mÃtodos de extraÃÃo de proteÃnas foram testados e o que apresentou melhor rendimento e spots com boa resoluÃÃo em gÃis bidimensionais foi escolhido para anÃlise de proteÃnas. Os gÃis bidimensionais foram analisados atravÃs do programa Image Master para a identificaÃÃo de spots especÃficos e ou diferenciais, bem como determinaÃÃo de seus pontos isoelÃtricos (pIs) e de respectivas massas moleculares (MM). Os dados de pI e MM foram usados para rastrear proteÃnas homÃlogas em angiospermas atravÃs de buscas nos bancos de dados UniProtKB/Swiss-Prot e UniProtKB/TrEMBL dispondo de mais de 16 milhÃes de informaÃÃes depositadas. GÃis 2D inicialmente produzidos na faixa de pH de 3 a 10 revelaram proteÃnas concentradas na regiÃo de pHs mais Ãcidos. Tal fato determinou que a faixa de pH de 4 a 7 fosse usada para melhor separaÃÃo dos spots em anÃlises seguintes. ApÃs anÃlise dos gÃis bidimensionais 21 spots especÃficos e 6 spots diferenciais foram selecionados. As anÃlises em bancos de dados possibilitaram inferir sobre 26 proteÃnas. Entre essas proteÃnas, vÃrias estavam relacionadas com o amadurecimento de frutos em outras espÃcies tais como: proteÃnas com expressÃo regulada pelo etileno, proteÃnas relacionadas com o estabelecimento das caracterÃsticas organolÃpticas do fruto e proteÃnas do metabolismo energÃtico. Com relaÃÃo ao metabolismo enegÃtico proteÃnas da via glicolÃtica, ciclo de Krebs e CTE foram inferidas. Estudos prÃvios sobre a participaÃÃo da via alternativa de elÃtrons no amadurecimento climatÃrico da graviola foram reforÃados. Entretanto, nesse trabalho foi observado que alÃm da oxidase alternativa (AOX), outros componentes podem participar da regulaÃÃo dessa via. Os resultados obtidos revelam possÃveis proteÃnas alvo para o desenvolvimento de estratÃgias bioquÃmicas e ou moleculares no controle do amadurecimento pÃs-colheita da graviola. / The soursop (Annona muricata L.) fruit is widely used in food, because it provides vitamins, carbohydrates, proteins, lipids and other molecules for human nutrition. In recent years, soursop (exotic tropical fruit) has had a prominent position in the Brazilian economy, especially for northeastern states such as, Ceara, Alagoas, Bahia and Pernambuco. The high production of soursop fruit is mainly in the Northeast by flowering occurs throughout the year, however it is estimated that the post-harvest losses reach 50% of production, as with other native or exotic fruits. Such post-harvest losses of the product are due to the accelerated rate of development and senescence of the fruit. Although some studies on Graviola, biochemical and molecular understanding of the maturation process is very limited making it important to move forward on this knowledge to develop strategies for increasing the shelf life of fruit. In this study, we sought a better protocol for protein extraction from soursop pulp to generate two-dimensional maps and identification of potential proteins involved in post-harvest ripening of the fruit. The soursop fruit in pre-climacteric stage were collected and allowed to ripen at 25  C for up to 8 days. Samples of the fruit pulp were collected at 0, 2, 4, 6 and 8 days post-harvest and used for protein extraction and establishment of two-dimensional gels (2D). Two different protein extraction methods were tested and the best one concerning performance and spots with good resolution in two-dimensional gel was chosen for protein analysis. The two-dimensional gels were analyzed using the Image Master for the identification of specific or differential spots, as well as determination of their isoelectric points (IPs) and their molecular masses (MM). The pI and MM data were used to track homologous proteins in angiosperms through searches on databases UniProtKB / Swiss-Prot and UniProtKB / Tremble featuring more than 16 million deposited information. 2D gels initially produced in the pH range 3 to 10 revealed the protein concentrated in the more acidic pHs. This fact determined the pH range 4-7 was used for better separation of spots in the following analysis. After two-dimensional gel analysis of 21 specific spots and 6 differential spots were selected. The analysis in databases allowed inferences about 26 proteins. Among these proteins, several were related to fruit ripening in other species such as proteins with expression regulated by ethylene, protein related to the establishment of the organoleptic characteristics of the fruit and proteins of energy metabolism. With respect to the metabolism of proteins glycolytic pathway, Krebs cycle and ETC were inferred. Previous studies on the participation of the alternative pathway of electrons in the ripening of climacteric graviola have been strengthened. However, this study also showed that in addition to the alternative oxidase (AOX), other components may participate in the regulation of this pathway. The results reveal possible protein targets for the development of strategies and biochemical and molecular control of postharvest ripening of soursop.

Capillary Electrophoresis (CE) is a high resolution analytical technique that may be employed in the separation and quantification of a wide range of analytes. The enormous efficiency obtained in CE are well suited for complex mixtures in which resolution of a large number of peaks in a short time is desirable. Therefore, CE has a promising future in pharmaC-eutical analysis. The separation mechanism of CE is based on the differential electrophoretic mobility of the solutes inside a buffer filled capillary upon the application of a voltage. Capillary electrophoresis is especially suitable for ionic species. The full potential of this technique can only be realised through the manipulation of numerous experimental parameters. In the present study, a CE method has been developed for the analysis of the macrolide antibiotics: erythromycin, oleandomycin, troleandomycin and josamycin. The selection of initial analysis conditions and optimisation of selectivity are reviewed. A systematic approach to method development was used to maximise analyte differential electrophoretic mobilities, by adjusting the pH. Thereafter, the influences of electrolyte molarity and electrolyte additives were investigated. In addition, some instrumental parameters, such as capillary length emf diameter, applied voltage and injection conditions were varied. The effect of the sample solvent and oncapillary concentration techniques such as FASI, were investigated. Also, the influence of injecting a water plug on the quantity of sample injected was demonstrated. Full resolution was achieved with the addition of methanol to the electrolyte. The applicability of CE for the assay of erythromycin and its related substances was investigated. Two methods were developed and successfully validated using CE: one for the quantitative determination of erythromycin alone and another for erythromycin related substances in the presence of large quantities of erythromycin A. Several related substances and impurities that result from the fermentation process used to produce erythromycin as well as degradation products are known to be present in commercial sa~ples. These impurities include erythromycin B, C, D, E, F, erythromycin enol ether, anhydroerythromycin and N-demethylerythromycin. Currently both the USP and BP official assays for the analysis of erythromycin involve the use of microbiological assays. These methods are limited as they are unable to differentiate between erythromycin and its related substances and degradation products. Furthermore, the microbiological assays are time-consuming and tedious to perform. 11 The CE methods developed for the analysis of erythromycin and for its related substances were fully validated in terms of precision, linearity, accuracy, sensitivity and stability. In addition, erythromycin was subjected to six stress modes and the stressed samples were analysed. An intemal standard was employed to provide acceptable precision for the migration time « 1.80 % RSD) and peak area « 4.44 % RSD). Optimum sensitivity was obtained using low UV wavelengths, with LOO values of less than 10 % for the related substances. The developed method was accurate for erythromycin C, anhydroerythromycin and N-demethylerythromycin, even in the presence of large concentrations of the parent. The method for~ erythromycin related substances was applied to the determination of impurities in three commercial erythromycin bases. The CE methods developed were rapid, precise, specific and stability-indicating and may be used to provide additional information to augment that attained by HPLC for purity assessment and in stability studies of erythromycin. Capillary electrophoresis is a simple, cost-effective technique that is capable of generating high quality data. This technique will become firmly established within pharmaceutical analysis for main peak and related impurity determination assays as familiarity becomes more widespread across the pharmaceutical industry and improvements in instrumentation are performed.

Heparin is an acid mixture of glycosaminoglycans with high negative charge density which naturally occurs in human body. Due to its ability to bind antithrombin III and thus accelerate inhibition of thrombin it has anticoagulant effect. This is abundantly used in clinical practice for operations, in case of embolia or heart-attacks. Protamine is a mixture of small basic peptides, which is used in clinical practice as a heparin antidote. The interaction between heparin and protamine is electrostatic and is also used for determination of heparin in human plasma or blood using affinity methods. In my study it was found that if protamine and heparin are mixed in one vial, a complex is formed. Its resulting charge depends on concentration ratio of protamine and heparin. On the other hand, in case the protamine is injected as a sample and heparin is added to background electrolyte as a protein-binding ligand, it is possible to determine heparin from decreasing protamine peak area. Because of the complexity of protamine-heparin interaction, tetraarginine was used as structurally close model of protamine to increase repeatability of measurements. The method for determination of heparin was optimalised. It uses 20 mM or 60 mM ortho-phosphoric acid as background electrolyte, 1 mg/mL solution of tetraarginine...