Membrane proteins are essential cellular components, responsible for a wide variety of biological functions. In order to better understand such aspects of cell activity, researchers have pursued detailed structural analysis of this class of proteins. Because of the complexities in isolating and studying membrane proteins in their native environment, detergents are often employed as a membrane mimetic media. This thesis examines several features of transmembrane (TM) protein structure and folding in detergents through which we are able to gain insights into membrane protein folding, as well as explore the suitability of detergents as membrane-mimetic environments. We first compare the helix-helix association of a series of model TM sequences in a native bilayer to the corresponding association in a detergent environment. We find that while various classes of helix-helix interaction motifs are preserved in detergents, alterations in detergent solvation may, in turn, lead to altered association affinity. We further explore this phenomenon through investigation of the consequences of the insertion of a strongly polar residue into a TM segment. In these studies we find a correlation between sequence-dependent alterations in detergent solvation and predicted in vivo folding. We also extend such analyses to a variety of detergents and native TM segments, finding that native secondary structure, as it occurs in the context of a full-length protein, is generally well preserved in a variety of detergents. Finally, we assess the determinants of membrane protein folding using two-transmembrane segment constructs, in the process optimizing expression, production and characterization techniques for a diverse range of transmembrane protein sequences. Overall this thesis finds that, detergents are capable of solubilizing membrane proteins in a form suitable for in-depth structural characterization that may not be feasible in other environments. Thus, as an approximation of a native membrane, detergents are able to preserve certain features of membrane proteins such as helix-helix association and native secondary structure.

Membrane proteins are essential cellular components, responsible for a wide variety of biological functions. In order to better understand such aspects of cell activity, researchers have pursued detailed structural analysis of this class of proteins. Because of the complexities in isolating and studying membrane proteins in their native environment, detergents are often employed as a membrane mimetic media. This thesis examines several features of transmembrane (TM) protein structure and folding in detergents through which we are able to gain insights into membrane protein folding, as well as explore the suitability of detergents as membrane-mimetic environments. We first compare the helix-helix association of a series of model TM sequences in a native bilayer to the corresponding association in a detergent environment. We find that while various classes of helix-helix interaction motifs are preserved in detergents, alterations in detergent solvation may, in turn, lead to altered association affinity. We further explore this phenomenon through investigation of the consequences of the insertion of a strongly polar residue into a TM segment. In these studies we find a correlation between sequence-dependent alterations in detergent solvation and predicted in vivo folding. We also extend such analyses to a variety of detergents and native TM segments, finding that native secondary structure, as it occurs in the context of a full-length protein, is generally well preserved in a variety of detergents. Finally, we assess the determinants of membrane protein folding using two-transmembrane segment constructs, in the process optimizing expression, production and characterization techniques for a diverse range of transmembrane protein sequences. Overall this thesis finds that, detergents are capable of solubilizing membrane proteins in a form suitable for in-depth structural characterization that may not be feasible in other environments. Thus, as an approximation of a native membrane, detergents are able to preserve certain features of membrane proteins such as helix-helix association and native secondary structure.

The monotopic membrane protein alMGS, a glycosyltransferase catalyzing glucolipid synthesis in Acholeplasma laidlawii, was overexpressed in Escherichia coli. Optimization of basic growth parameters was performed, and a novel method for detergent and buffer screening using a small size-exclusion chromatography was developed. This resulted in a tremendous increase in protein yields, as well as the unexpected discovery that the protein induces intracellular vesicle formation in E. coli. This was confirmed by sucrose density separation and Cryo-TEM of membranes, and the properties of the vesicles were analyzed using SDS-PAGE, western blot and lipid composition analysis. It is concluded that both alMGS and alDGS, the next enzyme in glucolipid pathway, have the ability to make the membrane bend and eventually form vesicles. This is likely due to structural and electrostatic properties, such as the way the proteins penetrate the membrane interface and thereby expand one monolayer. The highly positively charged binding surfaces of the glycosyltransferases may bind negatively charged lipids, such as Phosphatidylglycerol (PG), in the membrane and withdraw it from the general pool of lipids. This would increase the overall lipid synthesis, since PG is a pace-keeper, and the local concentration of nonbilayer prone lipids, such as Phosphatidylethanolamine, can increase and also induce bending of the membrane. The formation of surplus membrane inside the E. coli cell was used to develop a generic method for overexpression of membrane proteins. A proof-of-principle experiment with a test set of twenty membrane proteins from E. coli resulted in elevated expression levels for about half of the set. Thus, we believe that this method will be a useful tool for overexpression of many membrane proteins. By engineering E. coli mutants with different lipid compositions, fine-tuning membrane properties for different proteins is also possible. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 3: Manuscript.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1990. / Vita. Abstract. Includes bibliographical references (leaves 150-155). Also available via the Internet.

Thesis (Ph. D.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

In the recherche part of our thesis we familiarize ourselves with the human odor, theories of its origin, definition, anatomy of human skin and odors, composition of individual human odors and with odor secretion. We also look closer on the topics of odor absorbent and its use in the world, the transmission of odors to odor sensors and securing of scents. This section of our thesis discusses the resistance and survival abilities of odor, scent identification method and its history, organic acids, and defines the application of laundry detergents and ultrasonic washers. Aim of our thesis was to verify the relevance of the use of chemical detergents in the purification process when working with scents. We tried to verify the ability of detergents to degrade the human scent on odor absorbents to such an extent, that specially trained dogs would not be able to identify it. Our experiment was following a precise determination methodology. In the first phase samples were collected from the hands of targeted persons to a scent carrier - a metal cylinder (extirpate odour), by one researcher. In the second phase samples were collected from the body to a textile carrier, by another researcher. The metal scent carriers were processed. Some of them were left as they were (control sample) and some were exposed to chemical detergents with and without usage of an ultrasonic washer. In the experiment where we tried to identify the odour samples, six bitches of German shepherd were used. They were specially trained for odour identification. Each dog was let three times to identify the target scent exposed to a detergent and three times to identify the target scent not exposed to a detergent. Target smell was randomly deposited between other samples and its position was changed, so that the handler did not know its position and thus was unable to affect the work of his dog. The indicator of positive identification was a sign the dogs were taught - to sit or lay in front of a sample. Any dog was unable to identify the scent which was exposed to chemical detergent and all dogs identify the scent unexposed to a chemical detergent. Our experiment has shown that the use of chemical detergents in the purification process when working with scents is relevant to the degradation of individual human scent.

Made available in DSpace on 2015-07-22T20:50:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-31 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / As leishmanioses constituem um importante problema de saúde pública no Brasil, em razão da incidência da doença associada à alta taxa de morbidade. Devido ao fato da Leishmania ser um parasita intracelular no hospedeiro vertebrado, estudos de moléculas do parasita, que atuam na interação do parasita com a célula do hospedeiro vertebrado, são de alta importância para uma melhor compreensão dos mecanismos de invasão e sobrevivência do parasita. Seguindo este racional, nesta tese foram identificados, purificados e caracterizados os proteofosfoglicanos secretados por formas promastigotas (pPPG) e amastigotas (aPPG) de L. (L.) amazonensis, respectivamente de meios de cultura e de macrófagos infectados com esses parasitas. A purificação dos PPGs foi realizada por combinações de cromatografias em DEAE-Sephadex, Octyl-Sepharose e Bio-Gel A- 0.5 e ultracentrifugações. Por "Western blotting" e radioimunoensaio em fase sólida utilizando diferentes anticorpos monoclonais (mAbs), observou-se que o pPPG apresenta alto peso molecular, contem cadeias fosfoglicanas e é reconhecido por mAbs anti-glicoesfingolipídeos (GSLs) ST-3 e ST-5, e mAb anti-lipofosfoglicano (LPG) VST-1. Em estudos imunohistoquímicos de cortes de lesão de hamsters infectados com L. (L.) amazonensis, observou-se a presença de material reativo com os mAbs ST-3, ST-4 e ST-5 na superfície do parasita e em torno do vacúolo parasitóforo. Após delipidação de cortes da lesão (processo em que são extraídos os glicolipídeos), a reatividade com os mAbs limitou-se a algumas regiões do vacúolo, sugerindo a possível localização dos aPPGs na célula infectada. O aPPG apresentou padrão de migração eletroforética similar ao pPPG, e foi reconhecido pelos mAbs ST-3, ST-4 e ST-5, mas não pelo mAb VST-1. A análise da composição monossacarídica dos PPGs de L. (L.) amazonensis por cromatografia gasosa acoplada a espectrometria de massa mostrou que o pPPG é composto por manose:galactose:glucose, na proporção molar de 1:1,2:1,7. Por outro lado, o aPPG apresenta em sua estrutura manose:galactose:glucose na proporção de 1:2:9. Em experimentos paralelos foram avaliados os níveis de produção de óxido nítrico e do fator de necrose tumoral por macrófagos peritoneais de camundongos BALB/c expostos a diferentes estímulos com antígenos parasitários de L. (L.) amazonensis. Observamos que LPG, pPPG e GSLs per se não estimularam os macrófagos na produção de NO. Por outro lado o aPPG per se mostrou-se capaz de estimular a produção de NO. LPGs e pPPGs foram capazes de estimular a produção de óxido nítrico quando incubados na presença de interferon gama. Por outro lado, GSLs e interferon gama não foram capazes de estimular a produção de NO em macrófagos. Nenhum dos antígenos de Leishmania utilizados estimulou a produção de fator de necrose tumoral. O perfil glicolipídico de amastigotas axênicos de L. (L.) amazonensis em comparação com o de amastigota isolado de lesão foi analisado por HPTLC, tendo sido verificadas diferenças significativas no padrão cromatográfico dos glicolipídeos. Os glicolipídeos de formas axênicas não são reconhecidos por mAbs anti-GSLs. A organização dos glico(esfingo)lipídeos e glicoinositolfosfolipídeos (GIPLs) de formas amastigotas e promastigotas de L. (L.) amazonensis foi analisada. Para isso, membranas resistentes ao detergente não-iônico Triton X-100 foram isoladas a 4ºC. Em formas amastigotas foi verificado que os GSLs, esfingomielina e esteróis estão predominantemente localizados em frações de membranas resistentes ao tratamento com Triton X-100. Em formas promastigotas foi verificado que inositol fosforilceramida, GIPLs e esteróis estão localizados preferencialmente nas membranas de baixa densidade insolúveis em detergente nãoiônico a 4ºC. Em um outro conjunto de experimentos foi analisado o efeito de inibidor de síntese de esfingolipídeo, Aureobadisina A (AbA), em formas promastigotas e amastigotas de L. (L.) amazonensis. Em formas promastigotas foi observado que a AbA inibe o crescimento dos parasitas, entretanto esse efeito é reversível. Em ensaios de infectividade in vivo observou-se que camundongos BALB/c infectados com parasitas tratados com AbA apresentavam um desenvolvimento tardio das lesões em relação aos infectados com parasitas sem tratamento. Por outro lado, em culturas axênicas de amastigotas isoladas de lesão foi observado que a AbA é tóxica aos parasitas. Após a adição da AbA em cultura de macrófagos infectados com formas amastigotas, verificou-se uma diminuição significativa da infecção de macrófagos. Todos estes resultados sugerem que os glicoconjugados de L. (L.) amazonensis analisados nesta tese podem atuar na interação entre o parasita e o hospedeiro, exercendo papéis importantes na sobrevivência e progressão da leishmaniose. / Leishmaniasis is an important public health problem in Brazil. Since Leishmania is an obligatory intracellular parasite in the host, studies of parasite antigens responsible for interaction with the host cell during the infection are important to understand the mechanism of parasite invasion and survival. Following this rationale, in this work it was purified and characterized proteophosphoglycans secreted by promastigote (pPPG) and amastigote (aPPG) forms in culture supernatant and into parasitophorus vacuole of infected macrophages, respectively. The PPGs were purified by combination of chromatography (DEAE-Sephadex, Octyl-Sepharose and Bio-Gel A-0.5) and ultracentrifugation. By Western blotting and solid phase radioimmunoassay using different monoclonal antibodies (mAbs), it was observed that pPPG presents high molecular weight, contains phosphoglycan chains and it is recognized by anti-glycosphingolipids (GSLs) mAbs ST-3, ST-5 and by anti-lipophosphoglycan (LPG) mAb VST-1. Immunohistological analysis of hamster footpad lesions infected with L. (L.) amazonensis using different mAbs showed a strong labeling on amastigote forms and inside/around macrophage parasitophorus vacuole. After delipidation of lesion sections with a mixture of isopropanol/hexane/water (condition in which GSLs are removed) it was observed that the mAbs reactivity localized inside the vacuole remained in infected tissue, thus indicating the possible localization of aPPGs secreted by L. (L.) amazonensis recognized by the mAbs. The aPPG showed similar electrophoretic migration of pPPG, and it was recognized by mAbs anti-GSLs ST-3, ST-4 and ST-5, but not by mAb VST-1. The analysis of monosaccharide compositon of PPGs, determined by gas chromatography - mass spectrometry, showed that pPPG is constituted of mannose:galactose:glucose at molar proportion of 1:1.2:1.7. On the other hand, aPPGs showed in their strucuture mannose:galactose:glucose at proportion of 1:2:9. In parallel assays, nitric oxide (NO) and tumor necrose factor (TNF-α) production by BALB/c macrophages exposed to different L. (L.) amazonensis antigen stimuli were also analyzed. The pPPG, LPG and GSLs by themselves were not able to induce nitric oxide production by macrophages. On the hand, aPPG per se was able to induce nitric oxide production. The LPG and pPPGs were able to synergize with INF-γ the production of NO. Furthermore, GSLs were not able to stimulate NO production in presence of INF-γ. None of the antigens did induce the pro-inflammatory cytokines TNF-α secretion. The glycolipid profile of axenic and lesion amastigotes of L. (L.) amazonensis was analyzed by HPTLC. The results showed a significative difference in glycolipid chromatographic profile among the parasites, and axenic amastigote glycolipids were not recognized by anti-GSL mAbs. The membrane organization of glyco(sphingo)lipids and glycoinositolphospholipids (GIPLs) of L. (L.) amazonensis amastigote and promastigote forms were studied after Triton X-100 extraction at 4ºC. In amastigote forms it was observed that GSLs, sterol and sphingomyelin are present in the Triton X-100 resistant membrane domains. In promastigote parasites it was observed that inositol phosphorylceramide, GIPLs, sterol and a small amount of phosphatidylethanolamine are localized in the low-density membranes insoluble in non-ionic detergent at 4ºC. In another set of experiments, it was analyzed the effect of Aureobasidin A (AbA) in L. (L.) amazonensis promastigote and amastigote forms. AbA inhibited completely promastigotes growth, however, it should be noted that this effect was reversible. Infectivity assays in BALB/c mice using promastigote forms treated with AbA showed a delay in lesion development. On the other hand, the effect of AbA in L. (L.) amazonensis amastigotes showed that this drug is toxic to amastigote forms. AbA effect was also analyzed during the infection of macrophages by amastigote forms of L. (L.) amazonensis. After adition of AbA in the culture, it was observed a significative reduction of phagocytic index. The present study shows that Leishmania glycoconjugates may be crucial in parasite-host cell interaction, playing important role in the parasite survival and in the disease progress. / TEDE / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2014-06-11T19:32:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-06-21Bitstream added on 2014-06-13T18:44:45Z : No. of bitstreams: 1 teixeira_pss_dr_botfmvz.pdf: 156053 bytes, checksum: 9778975fa3550f64bc7f9462a546407e (MD5) / Visando avaliar o efeito da densidade nutricional sobre o desempenho produtivo das coelhas jovens, usaram-se 96 fêmeas do Grupo Genético Botucatu, com 70 dias de idade inicial, alojadas em lotes de quatro por gaiola até 119 dias e individualmente dos 119 aos 140 dias idade. Testaram-se duas rações com densidades diferentes, à base de farelo de soja, milho, farelo de trigo, feno de aveia preta, polpa de citros, calcário cálcitico, fosfato bicálcico, caulim, L-treonina, óleo de soja, premix vitamínico e mineral e sal. A ração de alta densidade foi formulada para conter, na matéria original, 18,4% de proteína bruta, 16,5% de fibra em detergente ácido e 2.500 kcal/kg de energia digestível; e a de baixa densidade, para conter 14,7% de proteína bruta, 24% de fibra em detergente ácido e 2.000 kcal/kg de energia digestível. O experimento foi realizado num esquema fatorial 2x2 (dois períodos, verão e inverno x duas rações), em delineamento inteiramente casualizado. A dieta de alta densidade proporcionou maior ganho de peso, maior ganho diário ajustado para o mesmo consumo e menor consumo de ração, tanto dos 70 aos 119 como dos 119 aos 140 dias de idade. / The objective was to evaluate the effect of nutritional density on the productive performance of young rabbit does. Ninety-six females from the Genetic Group Botucatu were used, starting at the age of 70 days and housed in lots of four per cage up to 119 days and then reared individually from 119 to 140 days of age. We tested two rations having different densities, based on soybean meal, corn grain, wheat bran, black oat hay, citrus pulp, limestone, dibasic calcium phosphate, kaolin, L-threonine, vitamin and mineral premix, soybean oil and salt. The high-density ration was formulated to contain, on an as-fed basis, 18.4% crude protein, 16.5% acid detergent fiber and 2,500 kcal/kg of digestible energy; whereas the low-density ration was formulated to contain 14.7% crude protein, 24% acid detergent fiber and 2,000 kcal/kg of digestible energy. The experiment was conducted according to a 2x2 factorial (two periods, summer and winter x two rations). The high-density diet caused higher weight gain, higher daily gain adjusted for intake and lower food intake, from 70 to 119 days and from 119 to 140 days of age.

O desempenho animal pode ser melhorado pelo incremento na digestibilidade dos alimentos. Um dos elementos neste processo é a acurada caracterização da composição química. Objetivando avaliar quatro métodos para determinar o teor de lignina, foram estudadas cinco gramíneas: Brachiaria brizantha cv. Marandú, Brachiaria brizantha cv. Xaraés (MG-5), Panicum maximum cv. Mombaça, Pennisetum purpureum cv. Cameroon e Pennisetum purpureum cv. Napier. As frações fibrosas da parede celular (PC), fibra em detergente neutro (FDN) e fibra em detergente ácido (FDA) aumentaram conforme as plantas amadureceram, refletindo as mudanças na composição dos componentes da parede celular (celulose, hemicelulose e lignina). Os valores de PC foram superiores aos da FDN indicando solubilização da pectina e outros oligossacarídeos da parede celular na solução de detergente neutro. O método LDA apresentou os menores teores de lignina, evidenciando a solubilização de parte da lignina na solução de detergente ácido. Os resultados de LPer foram maiores que os de LDA, que pode ser devido à oxidação da celulose e pectina pelo permanganato de potássio. Os teores de LK foram maiores que os de LDA possivelmente por contaminação protéica, mas, menores que os de LPer. Os teores de LBA foram maiores que os outros três métodos. A digestibilidade acompanhou, de forma inversa, o estádio de maturidade das plantas. A digestibilidade in vitro apresentou forte correlação negativa com os teores de lignina para todos os métodos, menos para LPer. Encontrou-se um valor de relação de 2,23, entre os métodos LDA e LBA, que, ao ser aplicado, nos teores de LDA, resultou em reta similar ao da LBA. Chama a atenção como este valor de 2,23 é muito próximo ao 2,4 utilizado nas equações B2 e C das frações de carboidratos do \"Cornell Net Carbohydrate & Protein System\" e nas equações do National Research Counsil de 1996, para corrigir o teor de lignina. O método LBA é um método fácil e conveniente para determinar a concentração de lignina em forrageiras e uma boa opção para uso rotineiro nas análises de laboratório. / Animal performance can be improved by enhancing feed digestibility. One of the elements for this process is an accurate characterization of feedstuff chemical composition. With the objective of evaluating four methods used today for lignin determination, five grasses were used: Brachiaria brizantha cv. Marandú, Brachiaria brizantha cv. Xaraés (MG-5), Panicum maximum cv. Mombaça, Pennisetum purpureum cv. Cameroon e Pennisetum purpureum cv. Napier, All fibrous fractions, neutral detergent fiber (NDF), acid detergent fiber (ADF) and cell wall (CW), increased as the plants matured, reflecting the changes in the CW composition (cellulose, hemicellulose and lignin). The values obtained for CW were higher than those obtained for NDF, indicating solubilization of pectin and other cell wall oligosaccharides in the neutral detergent solution. The ADL method produced the lowest lignin values, reflecting lignin solubilization by the acid detergent solution. PerL results were higher than those of ADL, possibly due to hemicellulose and pectin oxidation by potassium permanganate. The values for KL were higher than those of ADL, possibly due to protein contamination, but were lower than PerL values. ABL values were the highest among all methods. Digestibility inversely followed plant maturity throughout the study. In vitro dry matter digestibility showed high negative correlation with lignin contents. A 2,23 ratio between ADL and ABL methods was found, which when applied to ADL values, resulted in a curve similar to ABL method curve. It is interesting to note that, this value of 2,23 is very close to the 2,4 used in carbohydrate fractions B2 and C of the \"Cornell Net Carbohydrate & Protein System\", for the correction of lignin content. The ABL method is easy and convenient for total lignin content determination in forages.

Doctor of Philosophy / Department of Animal Sciences and Industry / Michael D. Tokach / Eleven experiments using 5,434 growing-finishing pigs were performed in addition to the development of a model to predict dietary NE that yields the greatest economic benefit. Two experiments were conducted to determine the effect of dietary phytogenics on growth and carcass performance of growing-finishing pigs. The addition of the combination of two phytogenics products (EOM 1+2) to diets improved ADFI, HCW, and carcass ADG. However, there was no evidence for treatment differences for growth or carcass performance in a second study. Two experiments were conducted to determine the effects of feeding high SID Trp:Lys ratios with and without Ractopamine HCl (RAC) on growth and carcass characteristics of finishing pigs. In Exp. 1, whereas increasing SID Trp:Lys ratio above 20% improved growth and carcass performance when diets contained RAC, pigs fed SID Trp:Lys ratios above 20% in diets without RAC had reduced growth and carcass performance. Contrary in Exp. 2, pigs fed increasing SID Trp:Lys in diet containing RAC did not provide further performance benefits. Three experiments were conducted to determine the optimum dietary SID Lys and CP concentrations in finishing pigs over 100 kg. The SID Lys requirement to obtain 100% of maximum response was 0.55 to 0.63% depending on the response variable. Growth and carcass performance was maximized in diets containing at least 12% dietary CP. Four experiments were conducted to determine the effects of SBM concentration and whether dEB, choline, or K are the reasons that performance is reduced when pigs over 100 kg BW are fed low CP diets. Performance was reduced as SBM concentration was reduced in the diet. Choline, K, and dEB do not appear to be the reason that performance is reduced when SBM concentration is decreased in low CP diets fed to pigs over 100 kg BW. A Microsoft Excel®-based model to predict the value of dietary NE that yields the greatest economic return to the production system was developed. Furthermore, a meta-analysis was conducted to incorporate the impact of NDF on carcass yield in the model.