Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 129-137). Also available on the World Wide Web.

nÃo hà / As taxas de morbimortalidade se elevam quando a diarreia està associada à desnutriÃÃo. Entretanto, os mecanismos pelos quais as deficiÃncias nutricionais afetam o intestino sÃo em grande parte desconhecidos. O objetivo desse trabalho foi avaliar alteraÃÃes morfomÃtricas, nas proteÃnas transportadoras de substratos e no transporte intestinal de eletrÃlitos e Ãgua em modelo de desnutriÃÃo em camundongos. Objetivamos ainda, analisar o efeito da toxina do cÃlera (TC) associada ou nÃo à desnutriÃÃo, sobre as proteÃnas transportadoras de substratos, sobre o transporte hidroeletrolÃtico em camundongos e por fim, avaliamos os efeitos de soluÃÃes de reidrataÃÃo oral (SRO) da OMS (SGli) e modificadas com glutamina (SGln), alanil-glutamina(SAla-Gln) e arginina (SArg) nesse transporte. Camundongos (n=20) receberam por 7 dias uma raÃÃo deficiente em proteÃnas, gorduras e minerais (DBR). Segmentos de Ãleo foram obtidos antes e no 7 dia da dieta, para estudos de morfometria, imunohistoquÃmica para as proteÃnas: SGTL-1, PepT-1, CAT-1 e SN-2 e avaliaÃÃo por RT-qPCR da expressÃo RNAm dessas proteÃnas transportadoras. O modelo de perfusÃo intestinal por 75 min em camundongos (n=6) foi utilizado para avaliar o transporte intestinal de Ãgua e eletrÃlitos e para avaliar o papel de soluÃÃes de reidrataÃÃo oral em camundongos nutridos e desnutridos expostos ou nÃo à toxina do cÃlera (1Âg/ml). Animais desnutridos apresentaram perda ponderal, atrofia dos vilos e reduÃÃo na expressÃo por imunofluorescÃncia da SGTL-1. A desnutriÃÃo causou ainda reduÃÃo na expressÃo do RNAm da SGTL-1 e PEPT-1 e aumento na expressÃo do RNAm para o SN-2 no ileo de camundongos. No modelo de perfusÃo intestinal, a desnutriÃÃo aguda aumentou a secreÃÃo intestinal de eletrÃlitos e Ãgua. A TC aumentou a secreÃÃo de eletrÃlitos e Ãgua em modelo de perfusÃo intestinal de camundongos. A TC aumentou a transcriÃÃo para o RNAm dos transportadores intestinais SGTL-1, PEPT-1 e CAT-1, mas nÃo aumentou a transcriÃÃo para o SN-2. As soluÃÃes de reidrataÃÃo com glicose (SGli), glutamina (SGln), alanil-glutamina (SAla-Gln) e arginina (SArg) diminuÃram a secreÃÃo de eletrÃlitos induzida pela TC. Apenas a SGln nÃo conseguiu diminuir significativamente a secreÃÃo de Ãgua induzida pela TC. Apenas a SGli reduziu a secreÃÃo de Ãgua induzida pela TC. SGli, SAla-Gln e SArg, mas nÃo SGln, diminuÃram a secreÃÃo de sÃdio e cloreto induzida pela TC. A desnutriÃÃo associada à diarreia pela TC causou reduÃÃo na transcriÃÃo para o RNAm dos transportadores intestinais SGTL-1, PEPT-1, CAT-1 e SN-2. A desnutriÃÃo associada à TC aumentou a secreÃÃo de Ãgua quando comparado ao grupo nutrido exposto à TC. SGli, SAla-Gln e SArg, mas nÃo SGln, diminuÃram a secreÃÃo de Ãgua induzida pela desnutriÃÃo associada à TC. Todas as soluÃÃes diminuÃram a secreÃÃo de sÃdio e cloreto induzida pela desnutriÃÃo associada à TC. / The morbidity and mortality rates rise when diarrhea is associated with malnutrition. However, the mechanisms by which nutritional deficiencies affect the gut are largely unknown. The aim of this study was to evaluate morphological changes in transport proteins and substrates in intestinal transport of electrolytes and water in malnutrition model in mice. We aim to further analyze the effect of cholera toxin (CT) with or without malnutrition on the carrier proteins substrates on electrolyte transport in mice and finally, we evaluate the effects of oral rehydration solutions (ORS) of OMS (SGli) e modified with glutamine (SGln), alanyl-glutamine (SAla-Gln) and arginine (SArg) in this transport. Mice (n=20) received for 7 days a diet deficient in protein, fat and minerals (DBR). Segments of ileum were obtained before and on day 7 of the diet, for studies of morphology, immunohistochemistry for proteins: SGTL-1, PEPT-1, CAT-1 and SN-2 and evaluated by RT-qPCR of mRNA expression of these transport proteins. The model of intestinal perfusion for 75 min in mice (n=6) was used to evaluate the intestinal transport of water and electrolytes and to evaluate the role of oral rehydration solutions in mice exposed nourished and malnourished or without cholera toxin (1Âg/ml). Malnourished animals showed weight loss, atrophy of the villi and reduced expression by immunofluorescence of SGTL-1. Malnutrition also caused a reduction in mRNA expression SGTL-1 and PEPT-1 and increased mRNA expression for SN-2 in mice ileum. In the model of intestinal perfusion, acute malnutrition increased intestinal secretion of electrolytes and water. CT increased the secretion of electrolytes and water into the intestinal perfusion model mice. SGli, SGln, SAla-Gln and SArg decreased secretion of electrolytes induced by CT. Just SGln failed to significantly decrease water secretion induced by CT. CT increased mRNA transcription for intestinal transporters SGTL-1, PEPT-1 and CAT-1 but not to increased transcription SN -2. Only SGli reduced water secretion induced by CT. SGli, SAla-Gln and SArg, but not SGln , decreased secretion of sodium and chloride induced by CT. The malnutrition associated with diarrhea caused by TC reduction in the transcription of mRNA for intestinal transporters SGT -1, PEPT-1, CAT-1 and SN-2. Malnutrition associated with CT increased the secretion of water when compared to the group fed exposed to TC. SGli , SAla-Gln and SArg, but not SGln, decreased water secretion induced by malnutrition associated with TC. All solutions decreased secretion of sodium and chlorid induced malnutrition associated with TC.

O principal problema no desenvolvimento de vacinas de segunda geração é a baixa resposta contra os antígenos, geralmente extremamente purificados. Isto tem levado à busca de novos adjuvantes que sejam mais eficientes e menos tóxicos. Este trabalho objetivava o estudo de uma proteína de choque térmico de Mycobacterium leprae - 18kDa-hsp - recombinante, produzida em levedura, veiculada em lipossomos, como modelo de proteína carregadora para antígenos fracos. Além de um estudo de estabilização de lipossomos por liofilização. A 18kDa-hsp apresenta certas características peculiares que levaram ao desenvolvimento de um método de dosagem próprio e extensível a outras proteínas com baixo teor em aminoácido cromóforos. Tendo uso potencial como proteína carregadora, a 18kDa-hsp é estável, suportando bem aquecimento, liofilização, acilação e associação com lipossomos de fosfolípides, sem perda significativa de sua atividade. Estudos paralelos de estabilização de lipossomos unilamelares mostraram que se pode usar trealose como crioprotetora no processo de liofilização-reidratação, sem perdas significativas do conteúdo dos lipossomos e com manutenção das estruturas das vesículas. Finalmente, o sistema lipossomos-18kDa-hsp mostra-se promissor como um sistema estável para veiculação de imunógenos fracos. / The major problem on development of second generation vaccines is the poor response against some immunogens. It has leading an intense research to find other substances non toxic and more efficient on enhancement of the immunological response. The aim of this work was the study of a heat-shock protein from Mycobacterium leprae - 18kDa-hsp - produced in yeast and vehiculated in liposomes, as a protein carrier model for weak immunogens. Furthermore, there was a study on stabilization of liposomes by lyophilization. The peculiar protperties of the 18kDa-hsp lead us to develop a new method for quantification that can be extended to other proteins with low chromophore content. Potentially a carrier protein, the 18kDa-hsp is very stab1e: it has no significant loss of its antigenic reactivity when heated, lyophilized, acylated and associated to liposomes. Parallel studies on liposomes stabilisation show trehalose as a good cryoprotector. The sugar can avoid the leakage of the entrapped marker (protein) and maintain the vesicles structures on the lyophilisation-rehydration processo Finally, the system liposomes-18kDa-hsp is promising for vehiculation of weak immunogens.

No description available.

Pituitary hormones are known to alter sex steroid receptor levels in the liver, and possibly the actions of the steroids as well. Recently, two classes of estrogen binding proteins have been characterized in male rat hepatic cytosol: a high affinity, low capacity estrogen receptor, and a lower affinity, higher capacity sex steroid binding component (moderate affinity component). It is of interest that the moderate affinity component binds both androgens and estrogens. A high affinity, low capacity androgen receptor has not been convincingly demonstrated in rat hepatic cytosol. Therefore, we have investigated the relationship of the moderate affinity component to sex steroid modulation of hepatic aryl hydrocarbon hydroxylase (AHH) activity as a possible control mechanism. Because of the sexual dimorphism for hepatic drug and steroid metabolism known to occur in rat liver, we chose this model to study. We have shown that no sex difference exists for the binding of pH]-estradiol to the estrogen receptor from either immature or adult rats. However, the moderate affinity component does exhibit a sex difference. We did not detect binding to the moderate affinity component in adult female or immature rats of either sex. This site could normally only be measured in the adult male. These findings were consistent with the age and sex dependent elevation of male AHH activity. We have also observed that gonadectomy of the male reduced the levels of AHH activity and the capacity of the moderate affinity component in a testosterone reversible fashion. These results were obtained using either unlabeled estradiol or dihydrotestosterone (DHT) as competitors for [³H]-estradiol binding. Administration of mestranol reduced AHH activity and the capacity of the moderate affinity component in the male. The moderate affinity component was not detected in the pseudoherma-phroditic rat which resembled the female, rather than the male, with respect to control and induced AHH activity. Hypophysectomy of the female resulted in an increase in AHH activity and detection of the moderate affinity component. Hypophysectomy of the male reduced both the capacity of the moderate affinity component and AHH activity. Unlike the gonadectomized male, testosterone had no restorative effect on the levels of AHH activity or the capacity of the moderate affinity component in the hypophy-sectomized rat. Continuous infusion of rat growth hormone (rGH) reversed the effect of hypophysectomy on the increased AHH activity and capacity of the moderate affinity component in the female. Administration of rGH to the hypophysectomized male abolished the detection of the moderate affinity component and reduced AHH activity to control female levels. This suggested rGH may be the pituitary hormone involved in production of the female level of metabolism. The effects of prolactin were not as clear. Therefore, we have demonstrated the modulation of AHH activity by peripheral sex steroids, and the regulation of these parameters by rGH. We have shown, the capacity of the moderate affinity component to vary in a manner that paralleled changes in hepatic AHH activity in different physiological models. Changes in the estrogen receptor were not found to be consistent with changes in AHH activity in these models. We conclude that the moderate affinity component is comparable to the male hepatic cytosolic DHT-binding protein. Furthermore, this component is associated with sex steroid action on hepatic AHH activity in the male rat. Interestingly, we have also shown this component as well as the estrogen receptor, to bind polycyclic aromatic hydrocarbons. Both 3-methylcholanthrene and benzo[a]pyrene competed for [³H]-estradiol binding to the estrogen receptor and moderate affinity component. In addition, dioxin congeners demonstrated specificity for the estrogen receptor in the female. However, this was not observed for the estrogen receptor or moderate affinity component in the male. The significance of this is presently unclear. / Pharmaceutical Sciences, Faculty of / Graduate

O principal problema no desenvolvimento de vacinas de segunda geração é a baixa resposta contra os antígenos, geralmente extremamente purificados. Isto tem levado à busca de novos adjuvantes que sejam mais eficientes e menos tóxicos. Este trabalho objetivava o estudo de uma proteína de choque térmico de Mycobacterium leprae - 18kDa-hsp - recombinante, produzida em levedura, veiculada em lipossomos, como modelo de proteína carregadora para antígenos fracos. Além de um estudo de estabilização de lipossomos por liofilização. A 18kDa-hsp apresenta certas características peculiares que levaram ao desenvolvimento de um método de dosagem próprio e extensível a outras proteínas com baixo teor em aminoácido cromóforos. Tendo uso potencial como proteína carregadora, a 18kDa-hsp é estável, suportando bem aquecimento, liofilização, acilação e associação com lipossomos de fosfolípides, sem perda significativa de sua atividade. Estudos paralelos de estabilização de lipossomos unilamelares mostraram que se pode usar trealose como crioprotetora no processo de liofilização-reidratação, sem perdas significativas do conteúdo dos lipossomos e com manutenção das estruturas das vesículas. Finalmente, o sistema lipossomos-18kDa-hsp mostra-se promissor como um sistema estável para veiculação de imunógenos fracos. / The major problem on development of second generation vaccines is the poor response against some immunogens. It has leading an intense research to find other substances non toxic and more efficient on enhancement of the immunological response. The aim of this work was the study of a heat-shock protein from Mycobacterium leprae - 18kDa-hsp - produced in yeast and vehiculated in liposomes, as a protein carrier model for weak immunogens. Furthermore, there was a study on stabilization of liposomes by lyophilization. The peculiar protperties of the 18kDa-hsp lead us to develop a new method for quantification that can be extended to other proteins with low chromophore content. Potentially a carrier protein, the 18kDa-hsp is very stab1e: it has no significant loss of its antigenic reactivity when heated, lyophilized, acylated and associated to liposomes. Parallel studies on liposomes stabilisation show trehalose as a good cryoprotector. The sugar can avoid the leakage of the entrapped marker (protein) and maintain the vesicles structures on the lyophilisation-rehydration processo Finally, the system liposomes-18kDa-hsp is promising for vehiculation of weak immunogens.

No description available.

No description available.

No description available.

No description available.