Um enfoque analítico, envolvendo centrifugação diferencial de homogeneizados de cecos gástricos de Rhynchosciara americana preparados em condições brandas e vigorosas, suporta a asserção de que existe uma classe principal de aril-α- e β -glicosidases, aril-α- e β-galactosidases e celobiases que é ligada à membrana plasmática, e duas classes de maltases, aminopeptidases, carboxipeptidases e dipeptidases. Maltases ocorrem na membrana plasmática e no interior de lisossomos na forma solúvel, enquanto que as peptidases (amino-carboxi- e dipeptidase) são encontradas na membrana plasmática e citossol. A ocorrência das enzimas citadas acima na membrana plasmática das células cecais, foi confirmada por resultados obtidos em preparações de microvilosidades realizadas com técnica desenvolvida para enterocito de mamíferos. O tratamento de microvilosidades com solução hiperosmótica de Tris, seguido de centrifugação em gradiente de densidade, permitiu a separação de quatro frações de membrana e de uma fração que contém microvilosidades intactas e, provavelmente, microfilamentos isolados. Eletroferogramas do material recolhido do gradiente mostrou diferenças entre as frações de membrana e sugeriu a ocorrência de actina como componente majoritário dos microfilamentos das microvilosidades. Ensaios enzimáticos nas diferentes frações do gradiente confirmaram a ocorrência de celobiase e carboxipeptidase ligadas à membrana plasmática. Os resultados de distribuição intracelular de hidrolases nos cecos de R. americana permitiram propor que a digestão terminal de proteínas ocorre na membrana plasmática e no citossol, por ação de amino-, carboxi- e dipeptidases, enquanto que a digestão de carboidratos, com exceção da trealose, ocorre somente na superfície das microvilosidades. / An analytical approach involving differencial centrifugation of Rhynchosciara Americana midgut caeca homogenates prepared in mild and in vigorous conditions support the assumption there are one main class of aryl-α- e β-glucosidases, aryl-α - e β-galactosidases and cellobiases, which are plasma membrane bound, and two classes of maltases, aminopeptidades, carboxypeptidases and dipeptidases. Maltases, occur in plasma membrane and in lysosomes (as a soluble enzyme) and the others are found in plasma membrane and cytosol. A succeful purification of caeca cells brush-borders by tecniques used for mammal enterocytes confirmed the assumptions based on differential centrifugation data. Treatment of purified microvilli with a hyperosmotic solution of Tris, followed by density gradient centrifugation, resulted in the separation of four membrane fractions and one fraction containing intact microvilli and, probably, isolated microfilaments. Electropherograms of the material recovered from the gradient showed differences among the membrane fractions, and they permitted to propose that actin is a major component of microvilli microfilaments. Enzymatic assays in the different fractions from the gradient confirmed the occurrence of cellobiase and carboxypeptidase plasma membrana bound. The results permite to propose that the majority of the terminal digestion of proteins and carbohydrates, with the exception of trehalose, occurs on the surface of the insect midgut caeca cells microvilli.

Made available in DSpace on 2014-06-11T19:33:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-24Bitstream added on 2014-06-13T19:04:37Z : No. of bitstreams: 1 torres_kaa_dr_jabo.pdf: 619941 bytes, checksum: e0160fe883801063a539b7087e557875 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Experimento 1: Foram avaliados os efeitos da substituição do milho pelo sorgo na ração para frangos de corte sobre o desempenho, o rendimento de carcaça e partes, a morfometria (densidade de vilos, altura dos vilos, profundidade das criptas, número de células caliciformes e índice mitótico nas criptas) e a integridade da mucosa do duodeno, jejuno e íleo, e a atividade das enzimas de membrana (aminopeptidase, fosfatase alcalina e maltase) da mucosa do jejuno. Foram utilizados 792 pintainhos de um dia machos, Cobb-500, distribuídos ao acaso em quatro tratamentos (0, 50, 75 ou 100% de substituição do milho pelo sorgo do 1º ao 42º dia) e seis repetições. No 7º, 21º e 42º dia o peso dos frangos e as sobras das rações foram aferidas, e foram obtidas amostras do intestino delgado de um frango por repetição. Experimento 2: Foi avaliado o efeito da substituição total do milho pelo sorgo sobre as Enterobactérias, Lactobacillus spp, Clostridium perfringens (Log10 UFC/g), a viscosidade e o pH do conteúdo ileal. Cem pintainhos foram distribuídos em dois grupos e alimentados com rações à base de milho ou sorgo do 1º ao 42º dia de idade. Concluiu-se desses experimentos que a substituição do milho da ração por sorgo, na fase inicial (1 a 21 dias), não altera o desempenho e a função da mucosa do intestino delgado dos frangos. A substituição parcial do milho pelo sorgo (50 ou 75%) durante a fase total de criação (1 a 42 dias) melhora a conversão alimentar dos frangos. A substituição total (100%), até os 42 dias, prejudica a estrutura dos vilos do duodeno e jejuno, mas, diminui a colonização por Clostridium perfringens no íleo e a viscosidade do conteúdo ileal. A substituição de 75 ou 100% até os 42 dias diminui a atividade de aminopeptidase e maltase. O rendimento de carcaça e partes, a atividade da fosfatase alcalina intestinal e as populações... / Experiment 1: The effects of substitution of corn by sorghum in diets for broiler chickens on performance, carcass and parts (breast and thigh+drumstick), morphology (density of villi, villous height, depth crypts, number of goblet cells and mitotic index in the crypts) and integrity (score of epithelial loss) of the duodenum, jejunum and ileum mucosa, and the activity of membrane enzymes (aminopeptidase, intestinal alkaline phosphatase, and maltase) of the jejunum. We used 792 day-old chicks male Cobb-500, were randomly assigned to four treatments (0, 50, 75 or 100% replacement of corn by sorghum 1 to 42 days) and six replications. At 7, 21 and 42 days the weight of the chickens and the remains of the diets were measured, and samples were collected from the small intestine of a chicken for replication. Experiment 2: The effect of total substitution of corn by sorghum on the counts of Enterobacteriaceae, Lactobacillus spp and Clostridium perfringens, viscosity and pH of ileal contents. One hundred chicks were divided into two groups and fed diets based on corn or sorghum, 1 to 42 days of age. From the results of these experiments it was concluded that the substitution of corn by sorghum in the initial phase (1 to 21 days) does not affect the performance and function of small intestinal mucosa of chickens. The partial substitution of corn by sorghum (50 or 75%) during the total period (1 to 42 days) seems to improve feed conversion of chickens. Total substitution (100%), up to 42 days, affect the structure of the villi of the duodenum and jejunum, but reduced colonization by Clostridium perfringens in the ileum and the viscosity of ileal contents. The replacement of 75 or 100% up to 42 days reduces the activity of aminopeptidase and maltase. The carcass and parts, the activity of intestinal alkaline phosphatase and the count of Enterobacteriaceae and Lactobacillus spp, are not affected by treatments

Resumo: Experimento 1: Foram avaliados os efeitos da substituição do milho pelo sorgo na ração para frangos de corte sobre o desempenho, o rendimento de carcaça e partes, a morfometria (densidade de vilos, altura dos vilos, profundidade das criptas, número de células caliciformes e índice mitótico nas criptas) e a integridade da mucosa do duodeno, jejuno e íleo, e a atividade das enzimas de membrana (aminopeptidase, fosfatase alcalina e maltase) da mucosa do jejuno. Foram utilizados 792 pintainhos de um dia machos, Cobb-500, distribuídos ao acaso em quatro tratamentos (0, 50, 75 ou 100% de substituição do milho pelo sorgo do 1º ao 42º dia) e seis repetições. No 7º, 21º e 42º dia o peso dos frangos e as sobras das rações foram aferidas, e foram obtidas amostras do intestino delgado de um frango por repetição. Experimento 2: Foi avaliado o efeito da substituição total do milho pelo sorgo sobre as Enterobactérias, Lactobacillus spp, Clostridium perfringens (Log10 UFC/g), a viscosidade e o pH do conteúdo ileal. Cem pintainhos foram distribuídos em dois grupos e alimentados com rações à base de milho ou sorgo do 1º ao 42º dia de idade. Concluiu-se desses experimentos que a substituição do milho da ração por sorgo, na fase inicial (1 a 21 dias), não altera o desempenho e a função da mucosa do intestino delgado dos frangos. A substituição parcial do milho pelo sorgo (50 ou 75%) durante a fase total de criação (1 a 42 dias) melhora a conversão alimentar dos frangos. A substituição total (100%), até os 42 dias, prejudica a estrutura dos vilos do duodeno e jejuno, mas, diminui a colonização por Clostridium perfringens no íleo e a viscosidade do conteúdo ileal. A substituição de 75 ou 100% até os 42 dias diminui a atividade de aminopeptidase e maltase. O rendimento de carcaça e partes, a atividade da fosfatase alcalina intestinal e as populações ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Experiment 1: The effects of substitution of corn by sorghum in diets for broiler chickens on performance, carcass and parts (breast and thigh+drumstick), morphology (density of villi, villous height, depth crypts, number of goblet cells and mitotic index in the crypts) and integrity (score of epithelial loss) of the duodenum, jejunum and ileum mucosa, and the activity of membrane enzymes (aminopeptidase, intestinal alkaline phosphatase, and maltase) of the jejunum. We used 792 day-old chicks male Cobb-500, were randomly assigned to four treatments (0, 50, 75 or 100% replacement of corn by sorghum 1 to 42 days) and six replications. At 7, 21 and 42 days the weight of the chickens and the remains of the diets were measured, and samples were collected from the small intestine of a chicken for replication. Experiment 2: The effect of total substitution of corn by sorghum on the counts of Enterobacteriaceae, Lactobacillus spp and Clostridium perfringens, viscosity and pH of ileal contents. One hundred chicks were divided into two groups and fed diets based on corn or sorghum, 1 to 42 days of age. From the results of these experiments it was concluded that the substitution of corn by sorghum in the initial phase (1 to 21 days) does not affect the performance and function of small intestinal mucosa of chickens. The partial substitution of corn by sorghum (50 or 75%) during the total period (1 to 42 days) seems to improve feed conversion of chickens. Total substitution (100%), up to 42 days, affect the structure of the villi of the duodenum and jejunum, but reduced colonization by Clostridium perfringens in the ileum and the viscosity of ileal contents. The replacement of 75 or 100% up to 42 days reduces the activity of aminopeptidase and maltase. The carcass and parts, the activity of intestinal alkaline phosphatase and the count of Enterobacteriaceae and Lactobacillus spp, are not affected by treatments / Orientador: Marcos Macari / Coorientador: Renato Luis Furlan / Banca: Alex Maiorka / Banca: Alice Eiko Murakami / Banca: Isabel Cristina Boleli / Banca: João Martins Pizauro Junior / Doutor

Um enfoque analítico, envolvendo centrifugação diferencial de homogeneizados de cecos gástricos de Rhynchosciara americana preparados em condições brandas e vigorosas, suporta a asserção de que existe uma classe principal de aril-α- e β -glicosidases, aril-α- e β-galactosidases e celobiases que é ligada à membrana plasmática, e duas classes de maltases, aminopeptidases, carboxipeptidases e dipeptidases. Maltases ocorrem na membrana plasmática e no interior de lisossomos na forma solúvel, enquanto que as peptidases (amino-carboxi- e dipeptidase) são encontradas na membrana plasmática e citossol. A ocorrência das enzimas citadas acima na membrana plasmática das células cecais, foi confirmada por resultados obtidos em preparações de microvilosidades realizadas com técnica desenvolvida para enterocito de mamíferos. O tratamento de microvilosidades com solução hiperosmótica de Tris, seguido de centrifugação em gradiente de densidade, permitiu a separação de quatro frações de membrana e de uma fração que contém microvilosidades intactas e, provavelmente, microfilamentos isolados. Eletroferogramas do material recolhido do gradiente mostrou diferenças entre as frações de membrana e sugeriu a ocorrência de actina como componente majoritário dos microfilamentos das microvilosidades. Ensaios enzimáticos nas diferentes frações do gradiente confirmaram a ocorrência de celobiase e carboxipeptidase ligadas à membrana plasmática. Os resultados de distribuição intracelular de hidrolases nos cecos de R. americana permitiram propor que a digestão terminal de proteínas ocorre na membrana plasmática e no citossol, por ação de amino-, carboxi- e dipeptidases, enquanto que a digestão de carboidratos, com exceção da trealose, ocorre somente na superfície das microvilosidades. / An analytical approach involving differencial centrifugation of Rhynchosciara Americana midgut caeca homogenates prepared in mild and in vigorous conditions support the assumption there are one main class of aryl-α- e β-glucosidases, aryl-α - e β-galactosidases and cellobiases, which are plasma membrane bound, and two classes of maltases, aminopeptidades, carboxypeptidases and dipeptidases. Maltases, occur in plasma membrane and in lysosomes (as a soluble enzyme) and the others are found in plasma membrane and cytosol. A succeful purification of caeca cells brush-borders by tecniques used for mammal enterocytes confirmed the assumptions based on differential centrifugation data. Treatment of purified microvilli with a hyperosmotic solution of Tris, followed by density gradient centrifugation, resulted in the separation of four membrane fractions and one fraction containing intact microvilli and, probably, isolated microfilaments. Electropherograms of the material recovered from the gradient showed differences among the membrane fractions, and they permitted to propose that actin is a major component of microvilli microfilaments. Enzymatic assays in the different fractions from the gradient confirmed the occurrence of cellobiase and carboxypeptidase plasma membrana bound. The results permite to propose that the majority of the terminal digestion of proteins and carbohydrates, with the exception of trehalose, occurs on the surface of the insect midgut caeca cells microvilli.

Tout d’abord considérés comme simples composants d’une barrière imperméable, il a été démontré que les lipides membranaires auraient en fait un rôle biologique bien plus important, pouvant modifier l’environnement des enzymes membranaires et moduler l’activité de ces dernières. Dans la thèse présentée ici, ces relations ont été étudiées dans les mitochondries de l’huître creuse Crassostrea gigas. Les bivalves subissent d’importants changements environnementaux et l’adaptation à ces changements peut passer par un remodelage des membranes, ce qui fait de ces animaux des modèles intéressants pour les études des relations entre la structure des membranes et les activités d’enzymes associées. Des huîtres ont été nourries en écloserie avec deux régimes d’algues monospécifiques, T-Iso et Chaetoceros gracilis, et un mélange équilibré de ces deux algues. Malgré d’importantes modifications de composition en acides gras induites par les différents régimes alimentaires, une grande stabilité des processus membranaires mitochondriaux a été observée. D’un autre côté, la comparaison entre des huîtres élevées en écloserie et des huîtres élevées dans leur milieu naturel a révélé d’importantes modifications de capacités mitochondriales, qui pourraient être liées à une modulation des classes de phospholipides et de leur insaturation. Ces différences ne peuvent pas s’expliquer par une influence des cycles tidaux dans la mesure où, malgré un changement de production d’ATP, l’activité des mitochondries a été montrée comme étant similaire chez les huîtres collectées en émersion et en immersion. L’homéostasie mitochondriale observée dans cette étude pourrait être un moyen pour les huîtres de faire face aux variations biotiques (disponibilité en nourriture) et abiotiques (disponibilité en oxygène) de l’environnement naturel de C. gigas, et de maintenir leurs fonctions physiologiques malgré ces variations. / First considered as simple components of an impermeable barrier, it has been shown that membrane lipids would have a more important biological role. These lipids could modify the environment of membrane enzymes and modulate their activity. In this thesis, these relationships have been studied in mitochondria of the oyster Crassostrea gigas. Bivalves undergo major environmental changes and adaptation to these changes may require a membrane remodelling, which makes these animals interesting models to study the relationship between membrane structure and membrane processes. In this study oysters were fed in hatchery with two monospecific algal diets, T-Iso and Chaetoceros gracilis, and an equilibrated mix of both algae. Despite significant changes in fatty acid composition induced by these diets, mitochondrial capacities remained stable. On the other hand, the comparison between hatchery-reared oysters and oysters reared in their natural environment revealed significant changes in mitochondrial capacity, which could be related to modulation of phospholipid class composition and unsaturation. These differences can not be explained by the influence of tidal cycles. Indeed, despite a change in ATP production, mitochondrial activity was shown to be similar in oysters collected during emersion and immersion.Mitochondrial homeostasis observed in this study could be a way for oysters to cope with biotic (food availability) and abiotic (oxygen availability) variations in the natural environment of C. gigas, and to maintain their physiological functions despite these variations.

La production microbienne de molécules d'intérêt pourrait être améliorée par des stratégies d'ingénierie du vivant. L'ingénierie enzymatique joue un rôle central dàns la conception d'organismes hôtes efficaces car l'efficacité de la voie dépend en premier lieu de l'efficacité des enzymes. Aujourd'hui, il est utile de savoir quelles conceptions d'enzymes synthétiques sont efficaces et quels paramètres doivent être testés pour les caractériser. La colocalisation spatiale d'enzymes à l'intérieur de la voie métabolique pourrait améliorer la production de la molécule d'intérêt finale en permettant une biotransformation rapide des intermédiaires de la voie de biosynthèse. Des protéines multidomaines regroupant plusieurs activités enzymatiques sont décrites dans la littérature. Ces travaux ont permis la création de fusions synthétiques d'enzymes caroténogéniques pour la production de bêta-carotène chez Saccharomyces cerevisiae. Différents types de fusions et de configurations enzymatiques ont été testés. L'étude a permis ia création d'une fusion enzymatique tripartite efficace produisant deux fois moins d'intermédiaires et deux fois plus de bêta-carotène. Les mesures précises de la concentration de chaque caroténoïde, associées à la quantification des enzymes, ont permis de caractériser l'efficacité de chaque enzyme synthétique. D'autres stratégies de colocalisation spatiale d'enzymes ont également été testées en utilisant des domaines d'interaction tels que la cohesinedockérine ou la protéine oligomériques CcmK2. Certaines enzymes caroténogéniques préservent leur fonctionnalité au sein de ces configurations. Des systèmes enzymatiques construites modifient le flux métabolique des caroténoïdes et produisent des caroténoïdes différents de ceux des enzymes naturelles. Un contrôle plus affiné des activités enzymatiques pourrait permettre un contrôle précis de la nature du caroténoïde final produit / Microbial production of molecules of interest can be improved by severa! engineering strategies. Enzymatic engineering has a central role in the conception of efficient host because pathway's efficiency depends in first place on the efficiency of the enzymes. Knowing which synthetic enzymes conceptions are efficient and knowing to characterize the best candidates are essential. Enzyme colocalisation inside metabolic pathway might improve the production of final molecule of interest by allowing rapid biotransformation of intermediates of the pathway. Multidomain proteins regrouping severa! enzymatic activities are described in the literature. This work has focused in part on the creation of synthetic fusion of sorne carotenogenic enzymes for the production of beta carotene in Saccharomyces cerevisiae. Different types of enzymatic fusions and configurations have been tested and. characterized. The study allowed the creation of an efficient tripartite enzyme. fusion which produces two times Jess intermediates and two times more beta carotene. Precise measurement of each caro teno id' s concentration coupled with quantification of enzymes allows the characterization of the efficiency of each synthetic enzyme. Other strategies for enzyme spatial co localisation have also been tested using domains of interaction like cohesin-dockerin or the oligomeric protein CcmK2. Sorne carotenogenic enzymes are still functional using those configurations. Sorne of the enzymatic systems modify the metabolic flow ofcarotenoids and produce carotenoids different from the natural systems. Sorne strategies have changed the metabolic flux of carotenoids inside the pathway. Interestingly, a fine control of activity of enzyme might allow a fine control of the nature of the final carotenoid