One of the main obstacles to maintaining patients with short bowel syndrome on parenteral nutrition, or successfully transplanting these patients with a small bowel graft, is the many severe infections that occur. Evidence is accumulating that translocating bacteria from the patient’s bowel causes a significant part of these infections. In this thesis bacterial translocation is studied in a Thiry-Vella loop of defunctionalised small bowel in the rat. Bacterial translocation to the mesenteric lymph nodes (MLNs) occurs in almost 100% of the rats after three days. No systemic spread of bacteria is observed unless there is additional immunosupression with depletion of Kupffer cells in the liver. However, blocking the function of α/β T cells does not increase the translocation. Removal of MLNs does not either aggravate bacterial translocation in the Thiry-Vella loop model. Conversely, after small bowel transplantation translocating bacteria spread systemically if the MLNs are removed. The Thiry-Vella loop should also be a suitable model for the testing of potentially translocation-inhibiting substances. Reinforcement of the intestinal barrier with glutamine or phosphatidylcholine proved insufficient in decreasing bacterial translocation. Even selective bowel decontamination with tobramycin failed to abolish bacterial translocation. Thus, it seems that the driving force for translocation in this model is strong regardless of the relatively small trauma of intestinal defunctionalisation. Flow cytometric studies of the immune cells in the spleen MLNs showed a decrease in MHC class II positive T cells in the MLNs of the Thiry-Vella loop. Concurrently the number of macrophages increased with time as observed by immunohistochemistry. The fraction of MHC class II negative macrophages increased in the spleens of rats treated with glutamine. In conclusion, the Thiry-Vella loop model offers possibilities of immunological as well as mechanistic studies on bacterial translocation from small intestine.

As sementes de Chenopodium quinoa (quinoa) são conhecidas pelo seu elevado teor de proteína bem como de saponinas. Quimicamente as saponinas de quinoa são triterpenos sendo ácido fitolacagênico, hederagenina, ácido oleanólico e ácido serjânico, as agliconas mais comumente encontradas. Para as saponinas de quinoa existem relatos contraditórios de atividade imunoadjuvante. Complexos imunoestimulantes têm sido bastante estudados nos últimos anos por atuarem como carreadores de antígenos. Esses complexos são constituídos, de saponinas, colesterol, fosfolipídios e um antígeno (ISCOM); na ausência de um antígeno são denominados de matrizes ISCOM. Para as saponinas de quinoa a possibilidade de formação de matrizes ISCOM não está completamente elucidada. Esse trabalho teve como objetivo a caracterização química das principais saponinas presentes nas sementes de C. quinoa bem como a avaliação das atividades antifúngica e imunoadjuvante. Agregados micelares formados por auto-associação das saponinas, bem como os complexos formados quando da formulação com colesterol e fosfatidilcolina também foram avaliados. O método de purificação das saponinas de quinoa utilizando resina poliaromática permitiu a obtenção de duas frações saponosídicas principais denominadas FQ70 e FQ90. Nessas frações foram caracterizadas dez saponinas triterpênicas bidesmosídicas pela técnica de UPLC/Q-TOF-MS. Um método por CLAE foi desenvolvido e validado para a determinação do conteúdo de saponinas nas frações de quinoa. A atividade antifúngica das frações de quinoa foi avaliada pelo método da microdiluição em placa para a determinação da concentração inibitória mínima (CIM). As frações foram inativas frente a todas as leveduras avaliadas. No entanto, todos os fungos dermatófitos testados foram suscetíveis às frações de quinoa. Os agregados formados por auto-associação das saponinas em solução aquosa bem como as nanoestruturas formadas após a complexação das saponinas de quinoa com colesterol (CHOL) e fosfatidilcolina (PC) foram estudados em diferentes proporções. As técnicas de espalhamento de luz dinâmico (DLS) e microscopia eletrônica de transmissão (MET) demonstraram estruturas esféricas e micelas filiformes. Em condições experimentais similares àquelas relatadas para a formação de matrizes ISCOM de saponinas de Quillaja saponaria, foram observadas estruturas tubulares e micelas anelares. A composição de saponinas das frações de quinoa parece determinar o tipo de nanoestrutura observada por MET. A toxicidade das frações de quinoa foi avaliada pela determinação da atividade hemolítica, toxicidade frente à Artemia salina e toxicidade aguda em camundongos. FQ70 foi praticamente atóxica frente à A. salina, no entanto, FQ90 apresentou toxicidade. Ambas as frações de quinoa foram menos hemolíticas quando comparadas com Quil A (extrato purificado Q. saponaria). Para avaliar a atividade imunoadjuvante camundongos foram imunizados somente com ovoalbumina (OVA) ou com OVA e os adjuvantes Quil A (adjuvante controle), FQ70 ou FQ90. Hipersensibilidade do tipo tardia (DTH) foi avaliada 28 dias após o priming. A proliferação de esplenócitos com os mitógenos Concanavalina A (Con A)-, lipopolissacarídeo e OVA, foi avaliada 28 dias pós priming. Ambas as frações de quinoa promoveram um estímulo da resposta imune humoral e celular, porém de forma diferenciada. / Chenopodium quinoa (quinoa) seeds are a rich protein source and well-known for their high saponin content. Chemically, quinoa saponins are triterpene glycosides being phytolaccagenic, hederagenin, oleanolic and serjanic acids the most common aglycones found in seeds. Its immunoadjuvant properties have been examined and the results obtained were conflicting. Mixed micelles composed of saponin, cholesterol and phospholipids, either containing antigen (ISCOM) or not (ISCOM matrix), have been under intensive development in recent years due to their ability to act as antigen presenting-carriers with remarkable immunostimulating properties. The formation of ISCOM or other clearly defined micellar structures with quinoa saponins remained uncorroborated. The objectives of this study were the chemical structure characterization of main saponins present in C. quinoa seeds and the evaluation of antifungal and immunoadjuvant properties related to them. Also, micellar aggregates formed by self-association in aqueous solutions by quinoa saponins as well as nanostructures formed after their complexation with cholesterol (CHOL) and phosphatidylcholine (PC) were evaluated. The separation method of quinoa saponins using a polyaromatic resin allowed the preparation of two purified and enriched fractions, FQ70 and FQ90. Ten triterpenic saponins were chemically characterized by UPLC/Q-TOF-MS in quinoa saponin fractions. A LC-method was developed and validated aiming the saponin content assay in quinoa saponin fractions. The antifungal activity of quinoa fractions was evaluated by broth microdilution method for the determination of the minimal inhibitory concentration (MIC). Both fractions were inactive against all yeasts tested. However all dermatophyte fungi were susceptible to quinoa saponin fractions. The aggregates formed by self-association in aqueous solutions by two quinoa saponin fractions, as well as several distinctive nanostructures formed after their complexation with cholesterol and phosphatidylcholine at different ratios were studied. Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) showed novel nanosized spherical vesicles formed by self-association and worm-like micelles in quinoa saponin fractions. When experimental conditions, similar to those reported for the preparation of Quillaja saponaria ISCOM matrices, tubular and ring-like micelles arose from quinoa saponin fractions. The saponin composition of quinoa fractions seems determines the nanosized structures viewed by TEM. The toxicity of quinoa fractions were assayed by haemolytic, toxicity to brine shrimps, and acute toxicity in mice tests. FQ70 was almost atoxic however, for FQ90 presented toxicity against shrimps. The quinoa saponin fractions were less haemolytic than Quil A (purified extract from Q. saponaria). To evaluate immunoadjuvant activity, mice were immunized subcutaneously with ovoalbumin (OVA) alone or adjuvanted with Quil A (adjuvant control), FQ70 or FQ90. Delayed-Type Hypersensitivity (DTH) were assayed 28 days post-priming and Concanavalin A (Con A)-, Lipopolysaccharide-, and OVA-stimulated splenocyte proliferation were also measured 28 days post-priming. The results suggested that the two quinoa saponin fractions enhanced significantly the production of humoral and cellular immune responses to OVA in mice.

As sementes de Chenopodium quinoa (quinoa) são conhecidas pelo seu elevado teor de proteína bem como de saponinas. Quimicamente as saponinas de quinoa são triterpenos sendo ácido fitolacagênico, hederagenina, ácido oleanólico e ácido serjânico, as agliconas mais comumente encontradas. Para as saponinas de quinoa existem relatos contraditórios de atividade imunoadjuvante. Complexos imunoestimulantes têm sido bastante estudados nos últimos anos por atuarem como carreadores de antígenos. Esses complexos são constituídos, de saponinas, colesterol, fosfolipídios e um antígeno (ISCOM); na ausência de um antígeno são denominados de matrizes ISCOM. Para as saponinas de quinoa a possibilidade de formação de matrizes ISCOM não está completamente elucidada. Esse trabalho teve como objetivo a caracterização química das principais saponinas presentes nas sementes de C. quinoa bem como a avaliação das atividades antifúngica e imunoadjuvante. Agregados micelares formados por auto-associação das saponinas, bem como os complexos formados quando da formulação com colesterol e fosfatidilcolina também foram avaliados. O método de purificação das saponinas de quinoa utilizando resina poliaromática permitiu a obtenção de duas frações saponosídicas principais denominadas FQ70 e FQ90. Nessas frações foram caracterizadas dez saponinas triterpênicas bidesmosídicas pela técnica de UPLC/Q-TOF-MS. Um método por CLAE foi desenvolvido e validado para a determinação do conteúdo de saponinas nas frações de quinoa. A atividade antifúngica das frações de quinoa foi avaliada pelo método da microdiluição em placa para a determinação da concentração inibitória mínima (CIM). As frações foram inativas frente a todas as leveduras avaliadas. No entanto, todos os fungos dermatófitos testados foram suscetíveis às frações de quinoa. Os agregados formados por auto-associação das saponinas em solução aquosa bem como as nanoestruturas formadas após a complexação das saponinas de quinoa com colesterol (CHOL) e fosfatidilcolina (PC) foram estudados em diferentes proporções. As técnicas de espalhamento de luz dinâmico (DLS) e microscopia eletrônica de transmissão (MET) demonstraram estruturas esféricas e micelas filiformes. Em condições experimentais similares àquelas relatadas para a formação de matrizes ISCOM de saponinas de Quillaja saponaria, foram observadas estruturas tubulares e micelas anelares. A composição de saponinas das frações de quinoa parece determinar o tipo de nanoestrutura observada por MET. A toxicidade das frações de quinoa foi avaliada pela determinação da atividade hemolítica, toxicidade frente à Artemia salina e toxicidade aguda em camundongos. FQ70 foi praticamente atóxica frente à A. salina, no entanto, FQ90 apresentou toxicidade. Ambas as frações de quinoa foram menos hemolíticas quando comparadas com Quil A (extrato purificado Q. saponaria). Para avaliar a atividade imunoadjuvante camundongos foram imunizados somente com ovoalbumina (OVA) ou com OVA e os adjuvantes Quil A (adjuvante controle), FQ70 ou FQ90. Hipersensibilidade do tipo tardia (DTH) foi avaliada 28 dias após o priming. A proliferação de esplenócitos com os mitógenos Concanavalina A (Con A)-, lipopolissacarídeo e OVA, foi avaliada 28 dias pós priming. Ambas as frações de quinoa promoveram um estímulo da resposta imune humoral e celular, porém de forma diferenciada. / Chenopodium quinoa (quinoa) seeds are a rich protein source and well-known for their high saponin content. Chemically, quinoa saponins are triterpene glycosides being phytolaccagenic, hederagenin, oleanolic and serjanic acids the most common aglycones found in seeds. Its immunoadjuvant properties have been examined and the results obtained were conflicting. Mixed micelles composed of saponin, cholesterol and phospholipids, either containing antigen (ISCOM) or not (ISCOM matrix), have been under intensive development in recent years due to their ability to act as antigen presenting-carriers with remarkable immunostimulating properties. The formation of ISCOM or other clearly defined micellar structures with quinoa saponins remained uncorroborated. The objectives of this study were the chemical structure characterization of main saponins present in C. quinoa seeds and the evaluation of antifungal and immunoadjuvant properties related to them. Also, micellar aggregates formed by self-association in aqueous solutions by quinoa saponins as well as nanostructures formed after their complexation with cholesterol (CHOL) and phosphatidylcholine (PC) were evaluated. The separation method of quinoa saponins using a polyaromatic resin allowed the preparation of two purified and enriched fractions, FQ70 and FQ90. Ten triterpenic saponins were chemically characterized by UPLC/Q-TOF-MS in quinoa saponin fractions. A LC-method was developed and validated aiming the saponin content assay in quinoa saponin fractions. The antifungal activity of quinoa fractions was evaluated by broth microdilution method for the determination of the minimal inhibitory concentration (MIC). Both fractions were inactive against all yeasts tested. However all dermatophyte fungi were susceptible to quinoa saponin fractions. The aggregates formed by self-association in aqueous solutions by two quinoa saponin fractions, as well as several distinctive nanostructures formed after their complexation with cholesterol and phosphatidylcholine at different ratios were studied. Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) showed novel nanosized spherical vesicles formed by self-association and worm-like micelles in quinoa saponin fractions. When experimental conditions, similar to those reported for the preparation of Quillaja saponaria ISCOM matrices, tubular and ring-like micelles arose from quinoa saponin fractions. The saponin composition of quinoa fractions seems determines the nanosized structures viewed by TEM. The toxicity of quinoa fractions were assayed by haemolytic, toxicity to brine shrimps, and acute toxicity in mice tests. FQ70 was almost atoxic however, for FQ90 presented toxicity against shrimps. The quinoa saponin fractions were less haemolytic than Quil A (purified extract from Q. saponaria). To evaluate immunoadjuvant activity, mice were immunized subcutaneously with ovoalbumin (OVA) alone or adjuvanted with Quil A (adjuvant control), FQ70 or FQ90. Delayed-Type Hypersensitivity (DTH) were assayed 28 days post-priming and Concanavalin A (Con A)-, Lipopolysaccharide-, and OVA-stimulated splenocyte proliferation were also measured 28 days post-priming. The results suggested that the two quinoa saponin fractions enhanced significantly the production of humoral and cellular immune responses to OVA in mice.

As sementes de Chenopodium quinoa (quinoa) são conhecidas pelo seu elevado teor de proteína bem como de saponinas. Quimicamente as saponinas de quinoa são triterpenos sendo ácido fitolacagênico, hederagenina, ácido oleanólico e ácido serjânico, as agliconas mais comumente encontradas. Para as saponinas de quinoa existem relatos contraditórios de atividade imunoadjuvante. Complexos imunoestimulantes têm sido bastante estudados nos últimos anos por atuarem como carreadores de antígenos. Esses complexos são constituídos, de saponinas, colesterol, fosfolipídios e um antígeno (ISCOM); na ausência de um antígeno são denominados de matrizes ISCOM. Para as saponinas de quinoa a possibilidade de formação de matrizes ISCOM não está completamente elucidada. Esse trabalho teve como objetivo a caracterização química das principais saponinas presentes nas sementes de C. quinoa bem como a avaliação das atividades antifúngica e imunoadjuvante. Agregados micelares formados por auto-associação das saponinas, bem como os complexos formados quando da formulação com colesterol e fosfatidilcolina também foram avaliados. O método de purificação das saponinas de quinoa utilizando resina poliaromática permitiu a obtenção de duas frações saponosídicas principais denominadas FQ70 e FQ90. Nessas frações foram caracterizadas dez saponinas triterpênicas bidesmosídicas pela técnica de UPLC/Q-TOF-MS. Um método por CLAE foi desenvolvido e validado para a determinação do conteúdo de saponinas nas frações de quinoa. A atividade antifúngica das frações de quinoa foi avaliada pelo método da microdiluição em placa para a determinação da concentração inibitória mínima (CIM). As frações foram inativas frente a todas as leveduras avaliadas. No entanto, todos os fungos dermatófitos testados foram suscetíveis às frações de quinoa. Os agregados formados por auto-associação das saponinas em solução aquosa bem como as nanoestruturas formadas após a complexação das saponinas de quinoa com colesterol (CHOL) e fosfatidilcolina (PC) foram estudados em diferentes proporções. As técnicas de espalhamento de luz dinâmico (DLS) e microscopia eletrônica de transmissão (MET) demonstraram estruturas esféricas e micelas filiformes. Em condições experimentais similares àquelas relatadas para a formação de matrizes ISCOM de saponinas de Quillaja saponaria, foram observadas estruturas tubulares e micelas anelares. A composição de saponinas das frações de quinoa parece determinar o tipo de nanoestrutura observada por MET. A toxicidade das frações de quinoa foi avaliada pela determinação da atividade hemolítica, toxicidade frente à Artemia salina e toxicidade aguda em camundongos. FQ70 foi praticamente atóxica frente à A. salina, no entanto, FQ90 apresentou toxicidade. Ambas as frações de quinoa foram menos hemolíticas quando comparadas com Quil A (extrato purificado Q. saponaria). Para avaliar a atividade imunoadjuvante camundongos foram imunizados somente com ovoalbumina (OVA) ou com OVA e os adjuvantes Quil A (adjuvante controle), FQ70 ou FQ90. Hipersensibilidade do tipo tardia (DTH) foi avaliada 28 dias após o priming. A proliferação de esplenócitos com os mitógenos Concanavalina A (Con A)-, lipopolissacarídeo e OVA, foi avaliada 28 dias pós priming. Ambas as frações de quinoa promoveram um estímulo da resposta imune humoral e celular, porém de forma diferenciada. / Chenopodium quinoa (quinoa) seeds are a rich protein source and well-known for their high saponin content. Chemically, quinoa saponins are triterpene glycosides being phytolaccagenic, hederagenin, oleanolic and serjanic acids the most common aglycones found in seeds. Its immunoadjuvant properties have been examined and the results obtained were conflicting. Mixed micelles composed of saponin, cholesterol and phospholipids, either containing antigen (ISCOM) or not (ISCOM matrix), have been under intensive development in recent years due to their ability to act as antigen presenting-carriers with remarkable immunostimulating properties. The formation of ISCOM or other clearly defined micellar structures with quinoa saponins remained uncorroborated. The objectives of this study were the chemical structure characterization of main saponins present in C. quinoa seeds and the evaluation of antifungal and immunoadjuvant properties related to them. Also, micellar aggregates formed by self-association in aqueous solutions by quinoa saponins as well as nanostructures formed after their complexation with cholesterol (CHOL) and phosphatidylcholine (PC) were evaluated. The separation method of quinoa saponins using a polyaromatic resin allowed the preparation of two purified and enriched fractions, FQ70 and FQ90. Ten triterpenic saponins were chemically characterized by UPLC/Q-TOF-MS in quinoa saponin fractions. A LC-method was developed and validated aiming the saponin content assay in quinoa saponin fractions. The antifungal activity of quinoa fractions was evaluated by broth microdilution method for the determination of the minimal inhibitory concentration (MIC). Both fractions were inactive against all yeasts tested. However all dermatophyte fungi were susceptible to quinoa saponin fractions. The aggregates formed by self-association in aqueous solutions by two quinoa saponin fractions, as well as several distinctive nanostructures formed after their complexation with cholesterol and phosphatidylcholine at different ratios were studied. Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) showed novel nanosized spherical vesicles formed by self-association and worm-like micelles in quinoa saponin fractions. When experimental conditions, similar to those reported for the preparation of Quillaja saponaria ISCOM matrices, tubular and ring-like micelles arose from quinoa saponin fractions. The saponin composition of quinoa fractions seems determines the nanosized structures viewed by TEM. The toxicity of quinoa fractions were assayed by haemolytic, toxicity to brine shrimps, and acute toxicity in mice tests. FQ70 was almost atoxic however, for FQ90 presented toxicity against shrimps. The quinoa saponin fractions were less haemolytic than Quil A (purified extract from Q. saponaria). To evaluate immunoadjuvant activity, mice were immunized subcutaneously with ovoalbumin (OVA) alone or adjuvanted with Quil A (adjuvant control), FQ70 or FQ90. Delayed-Type Hypersensitivity (DTH) were assayed 28 days post-priming and Concanavalin A (Con A)-, Lipopolysaccharide-, and OVA-stimulated splenocyte proliferation were also measured 28 days post-priming. The results suggested that the two quinoa saponin fractions enhanced significantly the production of humoral and cellular immune responses to OVA in mice.

A astaxantina (AST) é um carotenóide derivado do β-caroteno produzido por algas e cianobactérias, mas que também pode ser encontrada em animais marinhos. Em animais, é reportada como interceptadora de radicais de oxigênio mais eficiente que o β-caroteno. O objetivo central dessa dissertação foi avaliar a capacidade antioxidante da AST em lipossomos enriquecidos com citocromo c (cit c) desafiados com 3-morfolinosidnonimina (SIN-1), um doador de óxido nítrico, em diferentes microambientes (pH e composição das vesículas). Diferenças na interação destas vesículas com o cit c periférico, com reflexos na atividade antioxidante da AST também foram avaliadas. O SIN-1 gera, por termólise, quantidades equimolares de radical superóxido e óxido nítrico, quando há oxigênio no meio. Vesículas unilamelares de fosfatidilcolina (PC), PC contendo 5% ou 10% de fosfatidilglicerol (PG), com ou sem AST, foram incubadas com SIN-1 e/ou cit c. Medidas do índice de lipoperoxidação pelo teste das substâncias reativas ao ácido tiobarbitúrico (TBARS) revelaram que SIN-1 não causa aumento de TBARS, enquanto o cit c foi capaz de aumentar significativamente este índice. Este fato pode ser explicado pela atividade peroxidásica do cit c. Apenas em vesículas de PCPG10%, ao realizar a incubação do cit c concomitantemente com SIN-1, o índice de TBARS foi maior ao observado em vesículas incubadas apenas com cit c. É conhecido que a interação entre cit c e membranas aniônicas pode alterar a conformação da proteína, aumentando sua atividade peroxidásica. A presença da AST fez com que os índices de lipoperoxidação chegassem a valores próximos aos do controle. A alteração no pH do meio revelou que a AST possui ação antioxidante mais pronunciada em pHs 7,4 e 8,0, em comparação com pHs levemente ácidos. A presença de PG evidenciou ainda mais esta tendência e em pH 6,2, a AST apresentou inclusive pequena atividade próoxidante. Estes resultados podem ser discutidos à luz de alterações da permeabilidade da membrana e da reatividade de espécies reativas induzidas por mudanças da fluidez e de pH. O efeito dos produtos gerados por SIN-1 sobre o cit c foi estudado em condições de normóxia e hipóxia. Resultados de EPR e de fluorescência demonstram que a presença do radical superóxido previne lesões oxidativas causada por peróxido orgânico (t-butOOH) tanto no cit c quanto nas membranas, pois é capaz de reduzir o ferro hemínico do cit c. Através de CD e espectrofotometria UV-Vis e EPR, foi observado que a incubação com SIN-1 promove alterações estruturais no cit c causando ruptura na sexta coordenação do ferro hemínico, levando à geração de uma espécie de cit c com rombicidade menor em comparação ao cit c nativo e que apresenta maior atividade peroxidásica. Este trabalho contribui com informações para entendimento do mecanismo antioxidante da AST em diferentes microambientes, além de demonstrar o efeito paradoxal do superóxido que é capaz de proteger o cit c, através da redução do ferro hemínico, mas também pode expor a proteína à oxidação promovida por peroxinitrito. / Astaxanthin (AST) is a β-carotene derived carotenoid, produced by algae and cyanobacteria, but can also be found in marine animals. In phytoplankton it has the function to absorb light radiation for photosinthesys occurence. In animals AST acts as a scavenger of oxygen free radicals, even more efficiently than β-carotene itself. The main objective of this work is to evaluate the antioxidant capacity of AST over cytochrome c (cyt c) incorporated in liposomes and challenged with 3-morpholinosidnonimine (SIN-1), a nitric oxide donor, under different experimental conditions, namely vesicles composition and pH. Distinct interactions between cyt c and vesicles affecting the AST antioxidant activity were also evaluated. SIN-1 spontaneously generates equal amount of nitric oxide and superoxide anion when oxygen is present. Unilamellar vesicles made from phosphatidylcholine (PC) or PC with 5% or 10% of phosphatidylglycerol (PG), with or without AST, were incubated with SIN-1 and/or cyt c. The extent of lipid peroxidation was evaluated by the classical method of thiobarbituric reactive substances (TBARS). Control experiments with SIN-1 alone showed no increase in TBARS content, whereas cyt c significantly increased TBARS. Concomitant addition of cyt c, SIN-1 to PCPG10% vesicles led to lipid peroxidation indices even higher than those found when cyt c was incubated with PCPG10% vesicles. A peroxidase activity of cyt c resulting from the interaction between this protein and anionic membranes can explain this result. In this system, the presence of AST inhibited formation of TBARS, whose levels were near the control values. Astaxanthin was found to exhibit a more effective antioxidant capacity under basic pH (7.4 and 8.0), in comparison with pH 6.2 and 6.8. In the presence of PG, this trend became more evident. Interestingly, at pH 6.2, AST showed a slight pro-oxidant activity. These results can be explained by differences in membrane permeability and reactivity of reactive species, caused by pH and membrane fluidity alterations. The effects of products of SIN-1 decomposition on cyt c structure and its peroxidase activity were investigated under hypoxia and normoxia. EPR and fluorescence experiments revealed that superoxide anion radical, due to its ability to reduce heme iron, prevents oxidative damage of cyt c and membrane lipids by peroxide-derived free radicals. By means of CD and UV-Visible spectroscopy, we have found that concomitant incubation of SIN-1 and cyt c promoted structural alterations in the protein which changes the irons sixth axial coordination, leading to generation of a less rhombic cyt c, which is reportedly a better peroxidase than native cyt c. This work contributes with information aiming to better understand the antioxidant mechanism of AST under different membrane microenvironments and unveil a paradoxal effect of superoxide ion, which can protect cyt c from oxidative lesions by transferring electron to ferricyt c, but can also expose cyt c to oxidation by peroxynitrite.

No description available.

Oxidativ modifizierte Phospholipide haben PAF-ähnliche Struktur und zeigen in vitro PAF-ähnliche Wirkung. Dabei handelt es sich hauptsächlich um fragmentierte Phosphatidylcholine, die eine lange Fettsäure- oder Alkylkette in sn-1 Stellung und einen kurzen Acylrest (C4-C9) mit oxidiertem C-Atom in sn-2 Stellung haben und als PAF-ähnliche Lipide bezeichnet werden. In der vorliegenden Arbeit wurden durch eine neue HPLC-Fluoreszenzmethode ein Vertreter dieser in vivo existierender PAF-ähnlichen Lipide im Plasma quantifiziert. Dieses fragmentierte Phosphatidylcholin (FPC) besitzt einen Palmitoylrest in sn-1 Position, sowie eine C3 oder C4 Kette in sn-2 Position und erfüllt damit die strukturelle Voraussetzungen für biologische Aktivität. Weiterhin konnte gezeigt werden, dass FPC in biologisch aktiven Fraktionen enthalten war, deren biologische Aktivität jedoch nicht von PAF bewirkt wurde, sondern von PAF-ähnlichen Lipiden. Dies unterstützt die Vermutung, dass FPC biologische Aktivität zeigt. Im Plasma wird FPC von Lipoproteinen transportiert. Oxidativer Stress in biologischen Systemen, ausgelöst durch Tabakrauch, Organischämie/ Reperfusion, Vitamin E-Mangel oder Inkubation von Zellen mit H2O2, führte zu einer Erhöhung der FPC-Konzentration. Damit wurde gezeigt, dass die PAF-ähnlichen Lipide in vivo durch Radikal-induzierte Lipidperoxidation gebildet werden können. Trotz eines Konzentrationsanstiegs von FPC nach oxidativem Stress, kam es bei einer systemischen Inflammation, die von Radikal-bildenen Prozessen begleitet ist, jedoch nicht zu einer Anreicherung von FPC-ähnlichen Lipidperoxidationsprodukten. Ursache für die fehlende Anreicherung von oxidativ modifizierten Lipiden bei einer systemischen Inflammation könnte ihr rascher Abbau durch Phospholipasen, oder der Abbau von Vorstufen, bzw. die Substratrminderung, sein. / Oxidative modified phospholipids have PAF like structure and show in vitro PAF like activity. They consist of fragmented phosphatidylcholine with a long fatty acid or alkyl chain in sn-1 position and a short acylrest (C4-C9) with a oxidized C-atom in sn-2 position. According to their structure they are called PAF like lipids. In this paper one representative of these in vivo existing PAF like lipids are quantified in plasma by means of a new fluorescence HPLC method. These fragmented phosphatidylcholines consits of a palmitoylrest in sn-1 position and a C3 or C4 chain in sn-2 position and therefore they fullfill the strutural conditions to have biological activity. We further showed, that the biological active fractions contain FPC. The biological activity is not caused by PAF but by PAF like lipids. This fact supports the hypothesis that FPC are biological active. FPC is carried by lipoproteins in plasma. Oxidative stress caused by cigarette smoke, ischemia/ reperfusion, Vitamin E deficiency or incubation of cells with H2O2 leads to an increase of the concentration of FPC. Thereby it could be shown that PAF like lipids can be formed in vivo by means of radical induced lipidperoxidation. Many radical producing processes are involved in systemic inflammation. Despite of an increase of FPC's concentration after oxidative stress, there was no accumulation of FPC like lipidperoxidation products during systemic inflammation. One reason for the missing accumulation of oxidative modified lipids during systemic inflammation can be the fast degradation by phospholipases or the reduction of substrat.

Der obligat intrazelluläre Parasit Toxoplasma gondii ist der Erreger der Toxoplasmose, und dient zugleich als wichtiger Modellorganismus für weitere Human- und Tierpathogene, wie z.B. Plasmodium oder Eimeria. Die Vermehrung von T. gondii erfordert eine effiziente Biosynthese von Phospholipiden für die Herstellung neuer Membranen, was durch die de novo Synthese durch den Parasiten, und/oder den Import von Lipiden aus der umgebenden Wirtszelle gewährleistet werden kann. Während der Parasit zahlreiche Möglichkeiten für Synthese oder Import von PtdEtn und PtdSer verwendet, scheint die Biosynthese des abundantesten Membranlipids PtdCho auschließlich über den CDP-Cholin Weg zu erfolgen. Dieser erstreckt sich in T. gondii über 3 zelluläre Kompartimente, mit einer cytosolischen Cholin-Kinase (TgCK), einer im Zellkern lokalisierenden Cholin-Cytidylyltransferase (TgCCT) und einer Cholin-Phosphotransferase (TgCPT) im ER. Anders als die substrat-spezifische Ethanolamin-Kinase (TgEK), kann TgCK neben Cholin außerdem Ethanolamin phosphorylieren. TgCK zeigt eine geringe Affinität zu Cholin (Km ~0.77 mM), während eine verkürzte TgCK (TgCKS), welcher eine als Signalpeptid vorhergesagte N-terminale Sequenz (20 Aminosäuren) fehlt, eine etwa 3-fach höhere Aktivität aufweist (Km ~0.26 mM). Während jedoch die Wildtyp-TgCK cytosolische Cluster in Toxoplasma bildet, zeigt die verkürzte TgCK eine gleichmäßigere cytosolische Lokalisierung. Wir schlussfolgern daraus, dass der hydrophobe N-Terminus nicht notwendig ist für eine funktionale TgCK, sondern eine strukturelle Funktion bei der Protein-Lokalisierung hat. Eine konitionelle Mutante, in welcher der TgCK Promoter gegen den Tetracyclin-regulierbaren Promoter pTetO7Sag4 ausgetauscht wurde (Deltatgcki), zeigt erstaunlicherweise normales Wachstum und PtdCho Biosynthese. Die TgCK Aktivität und die daraus resultierende PtdCho Synthese sind nur zu ~30% regulierbar. Unsere Ergebnisse deuten auf die Verwendung eines alternativen Startcodons bzw. Promoters hin, welcher zur Expression einer verkürzten (~53-kDa) aber vermutlich aktiven Cholin Kinase führt, wodurch der Verlust der TgCK (~70-kDa) kompensiert wird. Der konditionelle Knockout von TgCCT, dem regulatorischen Enzym des CDP-Cholin Wegs, hatte einen 50%igen Wachstumsdefekt zur Folge. Diese Studie zeigt eine erstaunliche Flexibilität des Parasiten bezüglich seiner Membranzusammensetzung, und bestätigt zugleich die Annahme, dass PtdCho nicht von der Wirtszelle importiert werden kann. Diese Anpassungsfähigkeit stellt einen möglichen Faktor dar, der es T. gondii erlaubt sich in einem breiten Spektrum von Wirten zu vermehren. / Toxoplasma gondii is an obligate intracellular apicomplexan parasite that causes life-threatening disease in neonates and in immunocompromised people. Successful replication of Toxoplasma requires substantial membrane biogenesis, which must be satisfied irrespective of the host-cell milieu. Like in other eukaryotes, the two most abundant phospholipids in the T. gondii membrane are phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). Bioinformatics and precursor labeling analyses confirm their synthesis via the CDP-choline and CDP-ethanolamine pathway, respectively. This work shows that the 3-step CDP-choline pathway, involving the activities of TgCK, TgCCT and TgCPT, localizes to the cytosol, nucleus and ER membrane, respectively. The initial reaction is catalyzed by a dual-specificity choline kinase (TgCK, ~70-kDa), capable of phosphorylating choline as well as ethanolamine. The purified full-length TgCK displayed a low affinity for choline (Km ~0.77 mM). TgCK harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. The displacement of the TgCK promoter in a conditional mutant of T. gondii (deltatgcki) attenuated the enzyme expression by ~80%. Unexpectedly, the ?tgcki mutant was not impaired in intracellular growth, and exhibited a normal PtdCho biogenesis. To recompense for the loss of full-length TgCK, the mutant appears to make use of an alternative promoter and/or start codon, resulting in the expression of a shorter but active TgCK isoform identified by the anti-TgCK antiserum, which correlated with its persistent choline kinase activity. Accordingly, the ?tgcki showed an expected incorporation of choline into PtdCho, and susceptibility to dimethylethanolamine (a choline analog). Interestingly, the conditional mutant displayed a regular growth in off state despite a 25% decline in PtdCho content, which suggests a compositional flexibility in T. gondii membranes and insignificant salvage of host-derived PtdCho. The two-step conditional mutagenesis of TgCCT, which caused a reduced growth rate to about 50%, further substantiated this finding. The enzymatic activity of TgCCT and its role in PtdCho synthesis remain to be proven, however. Taken together, the results demonstrate that the CDP-route is likely essential in T. gondii. The competitive inhibition of choline kinase to block the parasite replication appears a potential therapeutic application.The work also reveals a remarkably adaptable membrane biogenesis in T. gondii, which may underly the evolution of Toxoplasma as a promiscuous pathogen.

La couche de nacre de la coquille de l'huître perlière Pinctada radiata des côtes tunisiennes est considérée comme un biomatériau ostéogénique prometteur. L’objectif de ce travail intitulé « Ostéogénie, intégration et qualité de la nacre d’un bivalve des côtes tunisiennes : Pinctada radiata (Leach, 1814) » consiste dans un premier temps à valoriser l’espèce P. radiata par sa qualité nutritionnelle par un suivi saisonnier de la composition de sa chair en lipides totaux et en phospholipides particulièrement les PC, PE, PS et PI. Les analyses effectuées ont montré que les lipides de P.radiata sont caractérisés par une richesse en acides gras polyinsaturés (AGPI) de la série n-3 qui dépasse 3 fois celle des AGPI de la série n-6. Ces AGPI de la série n-3 en particulier l’EPA (C20:5n-3) et le DHA (C22:6n-3), sont connus comme étant les AG les plus importants dans l’alimentation humaine puisqu’ils préviennent des maladies cardiovasculaires et des pathologies ostéo-articulaires. Par ailleurs, P. radiata de la région de Maharès présente la meilleure qualité de nacre en Tunisie. Les analyses biochimiques ont montré que cette région, constitue la meilleure localisation de cette espèce qui est loin des zones portuaires et des différentes origines de stress (pêche, exploitation, zone touristique etc…). En plus de cet aspect, la zone de Maharès renferme des pintadines présentant une bonne qualité en termes d’épaisseur de nacre. Nos résultats montrent que la composition, saisonnière, en acide gras des phospholipides et en particulier des glycérophospholipides (PE, PI, PS et PC) de la nacre est riche en acides gras saturés C14 :0, C16 :0 et C18 :0 particulièrement en hiver et dans un moindre degré au printemps. La nacre, substance ostéogénique, a été également caractérisée par un taux élevé de plusieurs AGPI de la série n-3 et n-6, particulièrement (18:3n-3, 18:4n-3, 20:5n-3, 22:5n-3, 22:6n-3 et le 20:4n-6). Pour démontrer les potentialités ostéogéniques des extraits de la nacre, nous avons utilisé un modèle "in vitro" utilisant 4 extraits lipidiques : l’extrait lipidique de la nacre de P.radiata (Ln), l’extrait lipidique de la chair de P.radiata (Lc), l’ESM (Ethanol soluble Matrix) de la nacre de P.radiata (Br) et l’ESM de la nacre de P.margaritifera (Bm). Nous avons comparé, in vitro, le pouvoir ostéogénique des extraits ESM des deux espèces P. radiata et P. margaritifera sur deux types de cellules les préchondrocytes ATDC5 et les préostéoblastes murins MC3T3. Les différents extraits (Ln, Lc, Br et Bm) induisent l’engagement des cellules MC3T3 vers le lignage ostéoblastique par l’activation des promoteurs des gènes spécifiques du tissu osseux, tels que: le collagène de type 1, l’ostéocalcine (OC), l’ostéopontine(OP) et le Runx2. Ces extraits induisent aussi l’engagement des cellules ATDC5 vers la différenciation endochondrale par l’activation des promoteurs des gènes spécifiques du tissu osseux, tels que: le collagène de type 1 alpha-1 (Col1a1), l’Aggrécane et le collagène de type X alpha-1 (ColXA1). De plus, nous remarquons que la fraction organique ou ESMr(Br) en comparaison avec celle de P.margaritifera (Bm) présente également les propriétés stimulantes de la nacre et la stimulation est même beaucoup plus importante. Ces résultats mettent en évidence, dans les modèles expérimentaux mis en oeuvre, l’intérêt des lipides. Ces derniers semblent jouer un rôle important dans cette stimulation. De plus, nous pouvons penser à la possibilité de l’association des molécules de nacre ou de biominéralisation avec les acides gras de la nacre et de la chair dans les défauts osseux à travers les sites actifs de l’os ou du cartilage humain présentant les différentes pathologies ostéarticulaires / The nacre layer of the shell of the pearl oyster Pinctada radiata of tunisian coast is considered a promising osteogenic biomaterial. The objective of this work entitled "Osteogenie, integration and quality of nacre of a tunisian coast bivalve: Pinctada radiata (Leach, 1814)" is a first step to enhance the species P.radiata its nutritional quality by seasonal monitoring of the composition of the flesh of total lipids and phospholipids in particular PC, PE, PS and PI. The analyzes showed that lipids of P.radiata are characterized by rich in polyunsaturated fatty acids (PUFAs) of the n-3 more than 3 times that of PUFAs n-6 series. These PUFAs of the n-3 series particularly EPA (C20: 5n-3) and DHA (C22: 6n-3) are known to be the most important AG in the food as prevent of the cardiovascular disease, and joint/ bone pathologies. Moreover, P. radiata of Mahares region has the best quality of nacre in Tunisia. Biochemical analyzes showed that this region is the best location of this species that is far from the port areas and different sources of stress (fishing, exploitation, tourist area etc ...). In addition to this aspect, the area contains pintadines having good quality in terms of thickness of nacre. Our results show that the seasonal composition of fatty acid of phospholipids in particular glycerophospholipids (PE, PI, PS and PC) nacre is rich in saturated fatty acids C14: 0, C16: 0 and C18: 0 especially in winter and spring in a lesser degree. Nacre, osteogenic substance, was also characterized by a high rate of PUFA of the n-3 and n-6 rate, especially (18: 3n-3, 18: 4n-3, 20: 5n-3, 22 5n-3, 22: 6n-3 and 20: 4n-6). To demonstrate the osteogenic potential of extracts of nacre, we have established an "in vitro" model using 4 lipid extracts: the lipid extract of nacre P.radiata (Ln); the lipid extract of the flesh of P.radiata (Lc), ESM (Ethanol soluble Matrix) of the mother-of P.radiata (Br) and ESM nacre of P. margaritifera (Bm). We compared “in vitro” osteogenic power ESM extracts of both species P. radiata and P. margaritifera on two types of cells the préchondrocytes ATDC5 and the murine preosteoblasts MC3T3. The different extracts (Ln, Lc, Br and Bm) induce engagement MC3T3 osteoblast lineage cells to the activation of the promoters of specific genes of bone tissue, such as collagen type 1, osteocalcin (OC), osteopontin (OP) and Runx2. These extracts also induce the commitment of ATDC5 cells to endochondral differentiation by activating specific genes promoters of bone tissue, such as collagen type 1 alpha 1 (COL1A1), the aggrecan and collagen type alpha 1-X (ColXA1). Moreover, we note that the organic fraction or ESMR (Br) compared with that of P. margaritifera (Bm) also has stimulant properties of nacre and the stimulation is even more important. These results demonstrate, in experimental models used, the interest of lipids. They seem to play an important role in this stimulation. Moreover, we can think about the possibility of the association of molecules or nacre biomineralization with the fatty acids of the nacre and flesh in bone defects through the active sites of bone or cartilage presenting the human osteoarticular different pathologies

This diploma thesis deals with the preparation and characterization of stealth liposomes and their combination with trimethylchitosan (TMC). This complex could find application in the field of inhalation administration. Stealth liposomes were prepared from neutral phophatidylcholine, negatively charged fosfatidic acid and polyethyleneglycol bounded to phosphatidylethanolamine. We have managed to prepare stealth liposomes with suitable properties that should guarantee passive targeting without evocation an immune response, despite the content of the negative component. We also found a suitable method of preparation for stealth liposome–TMC complex, where the change of size and zeta potential confirmed the non–covalent bound between two components despite the content of the polyethyleneglycol.