Foram examinadas as propriedades conformacionais de um peptídeo (YRWPFGNHL-NH2) presente na primeira alça extra-celular do receptor AT1 do hormônio angiotensina II. Espectros de dicroísmo circular (CD) e fluorescência foram obtidos em solução aquosa (em função do pH e da temperatura), na presença de um solvente indutor de estrutura secundária (trifluoroetanol, TFE), e na presença de micelas carregadas negativamente (dodecil sulfato de sódio, SDS) ou zwitteriônicas (N-hexadecil-N,N-dimetil-3-amônio-1-propano sulfonato, HPS e lisofosfatidilcolina, liso-PC), e de bicamadas contendo um fosfolipídio zwitteriônico (1-palmitoil-2-oleoil fosfatidilcolina, POPC) ou uma mistura de POPC e um fosfolipídio carregado negativamente (ácido l-palmitoil-2-oleoil fosfatídico, POPA). O estudo por calorimetria de titulação permitiu analisar a termodinâmica da ligação. Espectros de CD indicaram uma estrutura flexível em solução aquosa, modulada pelo pH e pela temperatura. Na presença de TFE, de micelas, e de vesículas de POPC:POPA o peptídeo adquire estrutura secundária, sugerindo a presença de uma dobra beta. Espectros de fluorescência intrínseca (W3) mostraram um deslocamento do comprimento de onda máximo de emissão (λmax) para o azul na presença de micelas e de vesículas de POPC:POPA. Observou-se também um aumento da intensidade de fluorescência, exceto no caso de SDS. Esses resultados indicaram que o peptídeo interagiu com os agregados. Não se observou alteração da fluorescência na presença de POPC. Medidas de anisotropia de fluorescência mostraram que, quando ligado, o peptídeo toma-se mais imobilizado. Estudos de supressão de fluorescência empregando agentes supressores aquossolúveis e de membrana sugeriram que o W3 localiza-se próximo à interface bicamada-água. Os resultados mostraram que o peptídeo se liga a micelas zwitteriônicas e negativas, enquanto que, no caso de bicamadas (mais empacotadas), a ligação depende da presença de cargas negativas. Experimentos de calorimetria mostraram que o ΔH de ligação do peptídeo a vesículas é negativo, compatível com a ocorrência de interações eletrostáticas. Foram observadas diferenças qualitativas e quantitativas na ligação do peptídeo às micelas de HPS e liso-PC. A fim de examinar se essas diferenças eram devidas ao empacotamento molecular, foram obtidos espectros de ressonância paramagnética eletrônica de marcadores de spin intercalados nos dois sistemas. Diferenças foram observadas principalmente na região da cabeça polar, as quais poderiam ser responsáveis pela diferença de comportamento do peptídeo em ambos os agregados. Os resultados obtidos para a primeira alça extra-celular do receptor AT1 indicam que a sua conformação pode ser modulada pelo pH e pela polaridade do ambiente, que ela pode interagir com a membrana através de interações hidrofóbicas e eletrostáticas, e ainda que essa interação depende do grau de empacotamento da fase lipídica. Esses resultados estão de acordo com a visão de que domínios extra-membranares de GPCRs situam-se na interface membrana-água. As alterações conformacionais induzidas pelo meio, bem como pela interação entre domínios extra-membranares de GPCRs e a fase lipídica ou pela ligação do agonista, poderiam ter um papel no mecanismo molecular de transdução de sinal. / In this work the conformational properties of a peptide (YRWPFGNHL-NH2) whose sequence is present in the first extra-cellular loop of the angiotensin II AT1 receptor were examined. Circular dichroism (CD) and fluorescence spectra were obtained in aqueous solution (as a function of pH and temperature), in the presence of a secondary structure inducing solvent (trifluoroethanol, TFE), and in the presence of negatively charged (sodium dodecyl sulfate, SDS) or zwitterionic (N-hexadecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, HPS and lysophosphatidylcholine, liso-PC) micelles, and of bilayers containing a zwitterionic phospholipid (l-palmitoyl-2-oleoyl phosphatidylcholine, POPC) or a mixture of POPC and a negatively charged phospholipid (l-palmitoyl-2-oleoyl phosphatidic acid, POPA). Studies of titration calorimetry allowed the analysis of the binding thermodynamics. CD spectra indicated a flexible structure in aqueous solution, which was modulated by pH and temperature. In the presence of TFE, micelles and of POPC:POPA vesicles, the peptide acquired secondary structure, which is suggestive of a β turn. The intrinsic fluorescence spectra (W3) showed a blue-shift of the maximum emission wavelenght (λmax) in the presence of micelles and of POPC:POPA vesicles. An increase in fluorescence intensity was also observed, except in the case of SDS. These results indicated that the peptide interacted with the aggregates. No fluorescence changes were observed in the presence of POPC. Fluorescence anisotropy measurements showed that, when bound to the aggregates, the peptide becomes more immobilized. Fluorescence quenching studies using water soluble and membrane-bound quenchers suggested that W3 is located close to the water-bilayer interface. The results showed that the peptide binds to negativelly charged and zwitterionic micelles, while in the case of bilayers (which are more tightly packed) the binding depends on the presence of negative charges. Titration calorimetry showed that ΔH of peptide binding to the vesicles is negative, which is compatible with the ocurrence of eletrostatic interactions. Qualitative and quantitative differences in binding of the peptide to HPS and liso-PC micelles were observed. In order to examine whether these differences were due to molecular packing, electron paramagnetic resonance spectra of spin labels intercalated in both systems were obtained. Differences were observed, mainly in the polar head group region, that could be responsible for the different behaviour displayed by the peptide in both aggregates. The results obtained for the first extra-cellular loop of the AT1 receptor indicated that its conformation can be modulated by pH and by the polarity of the medium, and that it can interact with the membrane through hydrophobic and electrostatic interactions, and also that this interaction depends on the molecular packing of the lipid phase. These results are in aggrement with the idea that the GPCRs extra-membrane domains are located at the water-membrane interface. Conformational changes induced by the medium, as well as by the interaction between extra-cellular segments and the membrane or by ligand binding, could play a role in the molecular mechanism of signal transduction.

Neste trabalho foi estudada a degradação anaeróbia do alquilbenzeno linear sulfonado (LAS), um surfactante largamente utilizado na fabricação de detergentes e presente em águas residuárias domésticas e industriais. Para isso, foram utilizados dois reatores anaeróbios horizontais de leito fixo (RAHLF) preenchidos com diferentes materiais suporte para imobilização da biomassa: carvão vegetal (RAHLF1) e leito misto de argila expandida e espuma de poliuretano (RAHLF2). O inóculo usado foi lodo proveniente de reator anaeróbio de fluxo ascendente e manta de lodo (UASB) utilizado no tratamento de águas residuárias de suinocultura. Os reatores foram alimentados, numa primeira etapa, com esgoto sintético e, posteriormente, suplementados com 14 mg/L de LAS. O tempo de detenção hidráulica (TDH) utilizado foi de 12 horas. Foi possível constatar que a presença do surfactante na alimentação não afetou a remoção de matéria orgânica (DQO), próxima a 90% em ambos os reatores para afluente com DQO de 550 mg/L. Com o balanço de massa constatou-se que os reatores foram aptos a remover LAS em 30% no RAHLF1 e 35% no RAHLF2. Desse total, 28% e 27%, respectivamente, foram degradados biologicamente, após 343 dias de operação. O restante manteve-se adsorvido nos materiais suportes. O pH no efluente dos RAHLFs manteve-se constante e próximo a 7,0. Alcalinidade foi gerada, assim como ácidos voláteis, apresentando concentrações médias de 342 e 353 mgCaCO3/L e 27 e 24 mgHAc/L no RAHLF1 e RAHLF2, respectivamente, durante operação com 14 mg/L de LAS no afluente. Ácidos acético e propiônico foram observados nos reatores em concentrações de até 50 e 12 mg/L, respectivamente. Em ambos os reatores estiveram presentes microrganismos com morfologias semelhantes a Methanosarcina sp., Methanosaeta sp., bacilos, cocos e filamentos, entre outros. Utilizando-se técnicas de Biologia Molecular (PCR/DGGE) verificou-se, em ambos os reatores, ampla variedade populacional de microrganismos pertencentes aos Domínios Bacteria, Archaea e ao Grupo BRS. Os materiais suportes selecionaram o crescimento de diferentes populações microbianas. No entanto, para o RAHLF2, praticamente não ocorreu estratificação populacional para os dois suportes. No RAHLF1, algumas populações foram semelhantes às observadas no reator de leito misto e outras cresceram mais favoravelmente / The aim of this work was to study the anaerobic degradation of LAS (linear alquilbenzene sulphonate), which is a surfactant widely used in manufacture of detergents and commonly found in domestic and industrial wastewaters. For the experimental purpose it was used two horizontal anaerobic immobilized biomass (HAIB) reactors filled with different support materials for biomass immobilization, such as: vegetal coal (HAIB1) and a mix of expanded clay and polyurethane foam (HAIB2). The biological material used as inoculum was obtained with the sludge of an upflow anaerobic sludge blanket reactor (UASB) treating suine wastewater. The reactors were fed, in a first stage, with synthetic sewage and, in a second, with a synthetic sewage supplemented with 14 mg/L of LAS. The applied hydraulic detention time was of 12 hours. With the chemical routine analyses it was possible to ascertain the evidence that the presence of surfactant in the feeding influent did not affect organic matter removal efficiencies (COD), which were close to 90% for both reactors treating 550 mg/L COD. The mass balance results suggested that the reactors removal efficiency of LAS were of 30% in RAHLF1 and 35% in RAHLF2. Of the total concentration of used LAS in the experiments, 28% and 27% were degraded biologically after 343 days of operating regime. The remaining LAS concentrations were adsorbed on the support materials. The pH values in the systems remained unaltered and close to 7.0. Alkalinity was generated (342 and 353 mgCaCO3/L, respectively), and volatile acids were recorded at concentrations of 24 and 27 mgHAc/L in RAHLF1 and RAHLF2, respectively, both treating effluent containing 14 mg/L of LAS. Acetic and propionic acids were detected in the reactors at concentrations up to 50 and 12 mg/L, for reactors 1 and 2, respectively. In both reactors it was observed microorganisms with morphologies resembling Methanosarcina sp., Methanosaeta sp., rods, cocus, filaments and others. Techniques of Molecular Biology (PCR/DGGE) showed high diversity of Bacteria, Archaea and BRS Group in both reactors. The support material selected different microbial populations. For RAHLF2, however, microbial stratification when using the distinct support material was not observed. In RAHLF1, it was observed that some populations were similar within the reactor of mixing bed where others grew more favorably

Este trabalho teve como objetivo a reprodução, avaliação e caracterização de manchamentos provocados por desodorante antitranspirante, suor, produtos do processo de lavagem como detergente em pó, amaciante, temperatura de passadoria em camisetas de malhas de algodão. Buscando uma melhor caracterização destes manchamentos, foram realizados três grupos de estudo com indivíduos selecionados, propensos à obtenção de manchas na região das axilas. Os indivíduos fizeram uso de desodorante com e sem perfume e utilizaram camisetas por um período de onze dias, sendo que um dos indivíduos fez uso apenas da camiseta sem desodorante e, uma das camisetas foi submetida ao desodorante sem uso por indivíduos. Ao final de cada dia, as camisetas foram lavadas com detergente em pó, amaciante, secas em secadora e passadas, simulando uma condição de uso doméstico. As camisetas manchadas foram submetidas às avaliações instrumentais de espectrofotometria (colorímetro), Microscopia Eletrônica de Varredura (MEV), análise por Dispersão de Energia (EDS) e medição da efusividade e condutividade térmica e Espectrometria de Emissão Atômica por Plasma Acoplado Indutivamente (ICP-OES). Essas camisetas também foram submetidas a dois processos de remoção: por lavagem a seco (LS) e por agentes químicos (RAQ). Após o processo de remoção, as camisetas foram submetidas à avaliação colorimétrica instrumental, condutividade e efusividade térmica, MEV, resistência à ruptura. As análises colorimétricas para todas as camisetas utilizadas pelos participantes dos grupos de estudo mostraram valores de E* de (0,87 a 32,43), indicando uma alteração de cor perceptível visualmente na região das axilas. As microscopias das amostras também mostram um acúmulo de alguns materiais nessa região que pode ter influenciado a troca de calor do material com o ambiente, como mostraram os resultados de efusividade e condutividade. Este material acumulado, analisado pela técnica de ICP-OES, mostrou que a camiseta manchada apresentou 99,7% de alumínio a mais do que na camiseta original na região da axila, o que pode ser oriundo do contato com o desodorante que também apresenta elevados teores de alumínio em sua formulação. Os métodos utilizados para remoção apresentaram resultados satisfatórios, promovendo a remoção dos manchamentos nas camisetas, observado através da diminuição dos valores de E* e nos índices da Escala Cinza de Alteração da Cor, na avaliação colorimétrica instrumental. Entretanto, o processo de remoção por agentes químicos apresentou melhores resultados, tendo cerca de 70% de eficácia quando comparados ao método de lavagem a seco, podendo ser um método mais efetivo na remoção deste tipo de manchamento. / This work was carried out to reproduction, evaluation and characterization of staining caused by antiperspirant deodorant, sweat, products of the washing process and detergent powder, fabric softener, temperature ironing shirts in cotton knits. Searching for a better characterization of these staining, three groups of studies selected, prone to getting stains on the underarm area individuals, were performed. Individuals made use deodorant and fragrance with shirts and used for a period of eleven days, and one of the subjects only used the shirt without deodorant and one of the shirts was submitted to without deodorant use by individuals. At the end of each day the shirts were washed with detergent powder, fabric softener, dryer dried and pressed, simulating a condition of household. The stained shirts were subjected to instrumental evaluations spectrophotometry (colorimeter), Scanning Electron Microscopy (SEM), Energy Dispersive analysis (EDS) and measuring the effusivity and thermal conductivity and Atomic Emission Spectrometry by Inductively Coupled Plasma (ICP-OES). These shirts were also subjected to two processes: removal by cleaning (LS) and chemicals (RAQ). After the removal process tees instrumental colorimetric evaluation, conductivity and thermal effusivity, SEM, tensile strength were submitted. colorimetric analyzes for all t used by the participants of the study groups showed values of E * (0.87 to 32.43), indicating a change of color visually noticeable in the underarm area. The microscopy of the samples also showed an accumulation of some materials in this region that may have influenced the heat exchange material with the environment as showed the results of effusivity and conductivity. This accumulated material, analyzed by ICP-OES technique, showed that the stained shirt showed 99.7% more aluminum than the original shirt, which can be derived from contact with the deodorant that also has a high concentration of aluminum in its formulation. The methods used for removal showed satisfactory results, promoting the removal of staining tees, observed by decreasing the values of E * and indexes of Gray Scale Change Colors, instrumental colorimetric evaluation. However, the process of removal by chemical agents showed better results, about 70% efficiency when compared to the method of cleaning, may be a more effective method in removing this type of stain.

Técnicas analíticas empregadas para a quantificação do teor de lignina em plantas forrageiras, atualmente em uso, são questionáveis quanto às suas acurácias. O método lignina detergente ácido (LDA), que é um dos métodos mais utilizado em Ciência Animal e Agronomia, apresenta algumas falhas, particularmente devido à parcial solubilização da lignina durante a preparação da fibra em detergente ácido (FDA). A lignina Klason (LK), outro método muito usado, apresenta o inconveniente de mensurar a proteína da parede celular como sendo lignina. Em ambos os procedimentos recomenda-se também mensurar cinzas nos resíduos de lignina. A quantificação da concentração de lignina pelo método espectrofotométrico lignina brometo de acetila (LBA) vem ganhando interesse de pesquisadores no Brasil e no exterior. Nesta metodologia, a lignina da planta contida na preparação parede celular (PC) é solubilizada numa solução a 25% de brometo de acetila em ácido acético e a absorbância mensurada é com luz UV a 280 nm. O valor da absorbância é inserido numa equação de regressão e a concentração de lignina é obtida. Para que esta técnica analítica seja mais aceita pelos pesquisadores, ela deve ser, obviamente, convincente e atrativa. O presente trabalho analisou alguns parâmetros relacionados à LBA em 7 gramíneas e 6 leguminosas, em dois estádios de maturidade. Dentre as diferentes temperaturas de pré-secagem, os resultados indicaram que os procedimentos de 55°C com ventilação e liofilização podem ser utilizados com a mesma eficácia. As temperaturas de 55°C sem ventilação e 80°C sem ventilação não são recomendadas, pois aumentaram os valores de FDA e LDA, possivelmente devido ao surgimento de artefatos de técnica como os compostos de Maillard. No método LBA os valores menores das amostras de leguminosas chamaram a atenção e colocaram em questão se a lignina destas plantas seria menos solúvel no reagente brometo de acetila. Dentre algumas alterações na metodologia da técnica LBA, a utilização do moinho de bolas (para diminuir o tamanho particular) nas amostras de PC não mostrou efeito; a hipótese era melhorar a solubilização da lignina usando partículas menores. O uso de um ultrasonicador, que aumenta a vibração das moléculas e assim, facilitaria a solubilização da lignina no reagente brometo de acetila, melhorou a solubilização da lignina em cerca de 10%, tanto nas gramíneas como nas leguminosas. Foi acoplado um ensaio biológico como referência, a degradabilidade in vitro da matéria seca (DIVMS); e como a lignina está intimamente associada à estrutura fibrosa da parede celular, também foi feito um ensaio de degradabilidade in vitro da fibra em detergente neutro (DIVFDN). Os resultados confirmaram o efeito da maturidade, reduzindo a degradabilidade nas plantas mais maduras, e que o teor de lignina de leguminosas é realmente inferior ao de gramíneas. Os resultados de degradabilidade apresentaram coeficientes de correlação mais elevados com o método LBA, quando foi empregada a técnica do ultrasom; o método LK mostrou os menores coeficientes. Também testou-se, com sucesso, a utilização da FDN, como preparação fibrosa, ao invés de PC. A razão é simples: enquanto que a FDN é amplamente conhecida, a preparação PC não o é. Inquestionável que esta manobra facilitará substancialmente a divulgação desse método, tornando-a mais aceitável pela comunidade científica / Analytical techniques employed to quantify lignin content in forages, currently in use, are questionable as to their accuracies. The method acid detergent lignin (ADL), which is one of the most used method in Animal Science and Agronomy, has some flaws, due to the partial lignin solubilization during the preparation of acid detergent fiber (ADF). The Klason lignin method (KL), another analytical procedure commonly used, has the drawback of measuring the cell wall protein as lignin. In both procedures also are recommended to measure ash content in the lignin residues. Quantification of lignin concentration by the spectrophotometric acetyl bromide lignin method (ABL) has been gaining interest from researchers in Brazil and abroad. In this methodology, the lignin contained in the plant cell wall preparation (CW) is solubilized in a 25% acetyl bromide in acetic acid solution and the absorbance is measured with UV light at 280 nm. The absorbance value is inserted in a regression equation and the concentration of lignin is obtained. For this analytical technique be better accepted by researchers, it must be, obviously, convincing and attractive. This study analyzed some LBA-related parameters in 7 grasses and 6 legumes in two stages of maturity. Among the different temperatures of pre-drying, the results indicated that the procedures at 55°C with ventilation and lyophilization can be used just as effectively. Temperatures of 55 and 80 both without ventilation are not recommended because they increased ADF and ADL values, possibly due to the emergence of technical artifacts such as the compounds of Maillard. The ABL method yielded lower values for legume samples which called into question if the lignin of these plants is less soluble in the acetyl bromide reagent. Among some changes studied in the ABL technique it was the utilization of ball milling in the PC samples, which showed no effect; the hypothesis was to reduce the particle size and thus improve the solubilization of lignin. The use of an ultrasonicator, which increases the vibration of molecules and possibly allowing better solubilization of lignin in the acetyl bromide reagent improved lignin solubilization by about 10%, both in grasses. As a reference, an in vitro dry matter degradability (IVDMD) was conducted. Because lignin is closely linked to the fibrous structure of the cell wall, an in vitro neutral detergent fiber degradability (IVNDFD) test of. Results confirmed the effect of maturity, reducing degradability as the plants matured, and that concentration of lignin is lower in legumes than in grasses. The degradability results showed higher correlation coefficients with the ABL method when the ultrasonicator was used; the KL method showed the lowest coefficients. We also tested, successfully, the use of NDF as a fiber preparation, instead of crude CW. While NDF is widely known, CW preparation is not. This maneuver will substantially facilitate the dissemination of this method, making it more acceptable to the scientific community

Quatro bubalinos e quatro bovinos adultos com fístulas ruminais foram utilizados com o objetivo de se estudar os efeitos de diferentes níveis de fibra em detergente neutro na dieta sobre as características de fermentação e digestão ruminal. Foram avaliadas os seguintes parâmetros: degradabilidades da matéria seca, proteína bruta, fibra em detergente neutro do feno de Coast-cross e do farelo de trigo; matéria seca e, proteína bruta do milho em grãos moídos e do farelo de soja; digestibilidade com marcador (Cr2O3); concentração de amônia, produção de ácidos graxos voláteis (acético, propiônico e butírico) e pH do líquido ruminal. Os animais foram delineados em dois Quadrados Latinos (4x4) com arranjo fatorial 4x2 sendo quatro níveis crescentes de FDN na MS (54, 60, 66 e 72%) e duas espécies (bubalinos e bovinos). Cada subperíodo compreendeu 29 dias, sendo 13 de adaptação. Observou-se um efeito significativo (P<0,01) da espécie sobre o pH ruminal, onde os bubalinos apresentaram valor médio (6,78) mais elevado que os bovinos (6,58). Houve efeito da interação tempo x espécie, na concentração de amônia do líquido ruminal somente após 2 horas da alimentação da manhã, onde os bubalinos obtiveram média de 31,76 mg% e os bovinos de 27,74 mg%. Os bubalinos mostraram valores de concentração média de ácidos graxos voláteis, ácido acético e ácido butírico (69,94 mM, 51,31 mM e 6,12 mM) menor (P<0,05) que a dos bovinos (77,96 mM, 56,72 mM e 8,01 mM). Não houve diferença significativa (P>0,05) na concentração de ácido propiônico e na relação acético:propiônico, não sofrendo influência de nenhum dos parâmetros principais analisados. Os bubalinos apresentaram maior desaparecimento ruminal da MS e FDN do feno de Coast-cross e da MS e PB do farelo de soja do que os bovinos, na maioria dos tempos de incubação. O desaparecimento ruminal da PB do feno de Coast-cross foi maior para os bubalinos somente nas últimas horas de incubação. Por outro lado, o desaparecimento ruminal da FDN do farelo de trigo foi mais elevado para os bovinos, somente nas primeiras horas. Para o coeficiente de digestibilidade da MS, houve interação de espécies com níveis de FDN. O coeficiente de digestibilidade da FDN foi influenciada pelos níveis de FDN e para o coeficiente de digestibilidade da PB, não foram notados efeitos de espécies ou níveis de FDN. / Four buffaloes and four cattle rumen fistulated, were used to study the effects of different levels of neutral detergent fiber in the diet on the rumen fermentation and digestion. In situ degradability assay was make of dry matter (DM), crude protein (CP), neutral detergent fiber (NDF) of the Coast-Cross hay (Cynodon dactylon) and the concentrate mixture; digestibility with marker (Cr2O3); ammonia concentration, production of volatile fatty acids (acetic, propionic and butyric) and pH in the ruminal liquid. The animals were designed in two Latin Square experiment (4x4). Treatments were applied in factorial 4 x 2 with four rations with increasing levels of NDF (54, 60, 66 and 72%) and two animal species (buffaloes and cattle). Twenty-nine days subperiods were used, the first thirteen for diet's adaptation. A significant effect was observed (P<0,01) in the species on the ruminal pH, with average of 6.78 in buffaloes and of 6.58 in cattle. There was effect of the interaction time x species, in the rumen ammonia concentration only at 2 hours after feeding with buffaloes having mean of 31.76 mg% and cattle 27.74 mg%. Buffaloes had lower (P<0,05) concentration of volatile fatty acids, acid acetic and acid butyric (69.94 mM, 51.31 mM and 6.12 mM) than cattle (77.96 mM, 56.72 mM and 8.01 mM). There was no significant difference (P>0,05) acid propionic concentration and acetic:propionic relation. The buffaloes showed higher ruminal disappearance of DM and NDF of the Coast-cross hay and DM and CP of the soybean meal than the cattle, in most of the times of incubation. The ruminal disappearance of CP of Coast-cross hay went higher for the buffaloes only in the last hours of incubation. On the other hand, the ruminal disappearance of NDF of wheat bran went higher for the cattle only in the first hours. There was interaction of species with levels of NDF for digestibility coefficient of the DM. The digestibility coefficient of NDF was only influenced by the levels of NDF. The digestibility coefficient of CP wasn't noticed effects of species or levels of NDF.

The Niemann-Pick type C (NPC) disease is a lysosomal lipid storage disorder caused by mutations in one of the two genes NPC1 or NPC2 encoding proteins of the late endosome/lysosome compartment (LE/LY). Defect in these proteins alters vesicular transport and leads to abnormal accumulation of cholesterol (Chol) in LE/LY. There are some lines of evidence suggesting that annexin A6 (AnxA6) participates in vesicular transport of Chol and may interact with membrane domains enriched in Chol and bind Chol. In this work we characterized the membrane microdomains resistant to Triton X-100, i.e., detergent-resistant membranes (DRMs) isolated from NPC patient-derived fibroblasts and from control cells. NPC cells contain a significantly higher amount of DRMs than the control cells that is consistent with the defect in Chol turnover in NPC cells. We also studied the mechanism of AnxA6 involvement in the NPC-induced changes in the membrane organization and showed that in the presence of calcium some AnxA6 molecules associate with the DRMs. This suggests that AnxA6 may play a role in the membrane lateral organization, contributing thus to the etiology of NPC disease. We then focused on the interaction of AnxA6-1 with Chol-rich membranes and on the involvement of its flexible region and VAAEIL sequence in these interactions. For this purpose, kinetics of the interfacial adsorption of human recombinant AnxA6 to Langmuir monolayers containing phosphatidylcholine, Chol and/or cholesteryl acetate were measured. Our data suggest that AnxA6 exhibits the highest affinity to Chol-containing monolayers and that the hydroxyl group of Chol plays a pivotal role in the AnxA6-lipid interactions in vitro.

EPDM (Ethylene-Propylene-Diene Monomer)/PP (Polypropylene) based TPV (Thermoplastic Vulcanizate) was aged in a closed system at stabilized temperature and pressure at 80, 100 and 120oC in distilled water, detergent solution and shiner solution. The variation in properties of TPV upon aging were followed by using DSC (Differential Scanning Calorimetry), TGA (Thermogravimetric Analysis), tensile testing, Shore A rubber hardness testing. DSC analysis indicated that percent crystallinity of PP component in TPV increased at 100oC whereas crystal structure was deformed at 120oC. In addition to this, hardness test showed that the hardness of TPV remained almost constant in distilled water aging except 120oC water aging but TPV became softer in detergent and shiner solution upon all aging temperatures used in this study. Tensile testing confirmed the hardness analysis that the loss in mechanical properties of TPV was observed except 100oC water aging. TGA analysis showed that percent crystallinity increase causes enhancement in degradation temperature of EPDM/PP blend in air. It is detected that TPV is quite resistant to 80oC aging, but TPV loses its resistance to preserve its characteristics at 120oC aging. It is also determined that aging media is as important as temperature to evaluate the performance of TPV. Moreover, it is decided that the rate of aging directly proportional to detrimental rate is arranged in an order from the slowest to the fastest as distilled water, detergent and shiner solution media.

Abstract 3D crystallisation of membrane proteins presents a bottleneck for the determination of the structures of membrane proteins. Obtaining 3D crystals of membrane proteins is made difficult by a number of factors including the poor solubility and instability of membrane proteins outside of their native membrane environment. 2D crystallisation of membrane proteins offers an alternative to preserve the conformational structure and functional activities of membrane proteins within their native bilayer membranes in 2D arrays from which the structure of membrane proteins can be determined. Different techniques exist for obtaining 2D crystals of membrane proteins including surface crystallisation or more commonly 2D crystallisation by detergent removal (using either dilution, dialysis, hydrophobic resin adsorption or cyclodextrin complexation) to promote reconstitution of the protein molecules within bilayer-forming lipids. Another method which has been emerged and is being used increasingly is the lipid monolayer technique for 2D crystallisation of proteins. The use of lipid monolayers to bind and adsorb proteins is an attractive and increasingly important method for generating high localised concentrations of oriented proteins and protein complexes. These bound proteins can be imaged directly, or they may form 2D crystalline arrays that are amenable to structure determination by single particle analysis or 2D electron crystallography. 2D crystals grown by this technique can also be used to initiate the growth of 3D crystals for X-ray diffraction analysis. Many derivatised lipids have been prepared for use with this technique, incorporating a diverse range of ligands to enable binding to specific proteins. Synthetic lipids containing functionalised head groups that chelate Ni2+ or Cu2+ have also been prepared to bind and orient expressed proteins that contain His-tags. Protein-binding monolayer-forming lipids generally consist of two distinct components: (1) a branched hydrocarbon tail to confer fluidity to the monolayer and (2) a functionalised hydrophilic head group to facilitate binding of protein molecules at the air-water interface. Newer examples of these compounds also incorporate perfluorinated hydrocarbon moieties to confer detergent resistance to these lipids. The present work discusses the chemistry of all these functionalised lipids and their contributions to monolayer 2D protein crystallisation. This thesis focuses on the synthesis of novel nickel-chelating fluorinated lipids to be used as a template for 2D crystallisation of His-tagged membrane proteins at the air/water interface. These monolayer-forming lipids have been designed with three distinct components: (i) a branched hydrocarbon tail to confer fluidity of the monolayer, (ii) a perfluorinated central core for detergent resistance, and (iii) a nickel-chelating hydrophilic head group to facilitate binding of recombinant, polyhistidine-tagged fusion proteins. Alkylations of fluorinated alcohols used in these syntheses proceed in good yields only with the application of prolonged sonication and, in some cases, in the presence of phase-transfer catalysts. Biophysical properties of Langmuir monolayers formed by our target synthetic fluorinated lipids were studied, comparing the results obtained with those of DOPC and DOGS Ni-NTA as examples of non fluorinated lipids. The Langmuir films were characterised by surface pressure-area isotherms and X-ray reflectometry to show their fluidity, thickness and packing density. The stability of fluorinated lipid monolayers and their ability to resist the solubilisation effects of a number of detergents were investigated using monolayer and affinity grid techniques. Results showed that fluorinated lipids offer an improved resistance to the solubilisation effects of detergents compared with their non-fluorinated counterparts. A number of trials for 2D crystallisation of both soluble and membrane proteins have been performed using fluorinated lipid monolayers. These new synthetic fluorinated lipids were successfully used to obtain 2D crystals of the His-tagged membrane protein BmrA from Bacillus subtillis by the monolayer technique.

Abstract 3D crystallisation of membrane proteins presents a bottleneck for the determination of the structures of membrane proteins. Obtaining 3D crystals of membrane proteins is made difficult by a number of factors including the poor solubility and instability of membrane proteins outside of their native membrane environment. 2D crystallisation of membrane proteins offers an alternative to preserve the conformational structure and functional activities of membrane proteins within their native bilayer membranes in 2D arrays from which the structure of membrane proteins can be determined. Different techniques exist for obtaining 2D crystals of membrane proteins including surface crystallisation or more commonly 2D crystallisation by detergent removal (using either dilution, dialysis, hydrophobic resin adsorption or cyclodextrin complexation) to promote reconstitution of the protein molecules within bilayer-forming lipids. Another method which has been emerged and is being used increasingly is the lipid monolayer technique for 2D crystallisation of proteins. The use of lipid monolayers to bind and adsorb proteins is an attractive and increasingly important method for generating high localised concentrations of oriented proteins and protein complexes. These bound proteins can be imaged directly, or they may form 2D crystalline arrays that are amenable to structure determination by single particle analysis or 2D electron crystallography. 2D crystals grown by this technique can also be used to initiate the growth of 3D crystals for X-ray diffraction analysis. Many derivatised lipids have been prepared for use with this technique, incorporating a diverse range of ligands to enable binding to specific proteins. Synthetic lipids containing functionalised head groups that chelate Ni2+ or Cu2+ have also been prepared to bind and orient expressed proteins that contain His-tags. Protein-binding monolayer-forming lipids generally consist of two distinct components: (1) a branched hydrocarbon tail to confer fluidity to the monolayer and (2) a functionalised hydrophilic head group to facilitate binding of protein molecules at the air-water interface. Newer examples of these compounds also incorporate perfluorinated hydrocarbon moieties to confer detergent resistance to these lipids. The present work discusses the chemistry of all these functionalised lipids and their contributions to monolayer 2D protein crystallisation. This thesis focuses on the synthesis of novel nickel-chelating fluorinated lipids to be used as a template for 2D crystallisation of His-tagged membrane proteins at the air/water interface. These monolayer-forming lipids have been designed with three distinct components: (i) a branched hydrocarbon tail to confer fluidity of the monolayer, (ii) a perfluorinated central core for detergent resistance, and (iii) a nickel-chelating hydrophilic head group to facilitate binding of recombinant, polyhistidine-tagged fusion proteins. Alkylations of fluorinated alcohols used in these syntheses proceed in good yields only with the application of prolonged sonication and, in some cases, in the presence of phase-transfer catalysts. Biophysical properties of Langmuir monolayers formed by our target synthetic fluorinated lipids were studied, comparing the results obtained with those of DOPC and DOGS Ni-NTA as examples of non fluorinated lipids. The Langmuir films were characterised by surface pressure-area isotherms and X-ray reflectometry to show their fluidity, thickness and packing density. The stability of fluorinated lipid monolayers and their ability to resist the solubilisation effects of a number of detergents were investigated using monolayer and affinity grid techniques. Results showed that fluorinated lipids offer an improved resistance to the solubilisation effects of detergents compared with their non-fluorinated counterparts. A number of trials for 2D crystallisation of both soluble and membrane proteins have been performed using fluorinated lipid monolayers. These new synthetic fluorinated lipids were successfully used to obtain 2D crystals of the His-tagged membrane protein BmrA from Bacillus subtillis by the monolayer technique.

Made available in DSpace on 2014-06-11T19:27:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-06Bitstream added on 2014-06-13T20:36:08Z : No. of bitstreams: 1 ribeiro_ms_me_botfmvz.pdf: 174069 bytes, checksum: 2dd52fa6a3e9b9232005ba7edbfded45 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / The objective of this paper was to compare dried corn plant (DCP) as a roughage (R) source for lactating goats to roughages traditionally used for feeding ruminants: alfalfa hay (AH) and coastcross hay (CCH). Twenty-one crossbred goats weighing an average of 66.48 kg were used. After reaching their lactation peak, they were distributed among seven 3 x 3 latin squares. Each experimental diet consisted of one of the roughages fed ad lib plus a commercial concentrate (C) for lactating goats, fed on a basis of 1 kg for every 2.5 kg of milk. Each group, composed by seven animals kept in a collective pen with slatted floor, stayed in each treatment for two weeks; the first week was an adaptation period, whereas milk yield measurements and milk samples were collected during the second week. Dry or as-fed matter intake was higher in AH than in DCP or CCH. No treatment effect on C intake was detected. The higher dry matter intake in AH led to higher intakes of crude protein, ether extract, minerals, nitrogen-free extract, total digestible nutrients and acid detergent fiber, both in R and in the total diet (R + C). No treatment effect was observed for either crude fiber or neutral detergent fiber intakes in R and in R+C. Milk yield and milk PB content were higher in AH than in DCP or CCH; however, milk yield corrected for 3.5% fat and body weight loss were higher in AH than in CCH, but DCP did not differ from the other two treatments. Production cost per kg of milk was lower for DCP than for AH or CCH. The results show that DCP is a valid alternative roughage for dairy goats.