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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
31

Refined <i>in vitro</i> Models for Prediction of Intestinal Drug Transport : Role of pH and Extracellular Additives in the Caco-2 Cell Model

Neuhoff, Sibylle January 2005 (has links)
<p>Drug transport across the intestinal epithelium is roughly predicted from permeability values obtained from Caco-2 cell monolayers. This thesis examines the important role of <i>pH</i> and extracellular additives for increasing the reliability and predictivity of the <i>in vitro</i> screening system, Caco-2.</p><p>It was shown that the passive transport of ionizable compounds may be biased by a false efflux or uptake component, when applying a physiological <i>pH</i>-gradient across the membrane. <i>pH</i> also affected the amount of compound available at the transporter-binding site. Therefore, <i>pH</i> dependence should be considered in studies of such compounds and of drug-drug interactions involving efflux transporters. It was also shown that proton-dependent apical uptake or basolateral efflux should be studied both with and without a <i>pH</i> gradient over the whole monolayers. </p><p>The two extracellular additives, bovine serum albumin (BSA) and the solubilizing agent, Cremophor<sup>®</sup> EL, also influenced Caco-2 permeabilities. BSA applied to the receiver side increases, and to the donor side decreases drug permeation according to the drug’s protein binding capacity. Thus, the absorptive transport for both passive and active compounds is favoured, giving a physiologically sound improvement of the Caco-2 cell model. Inclusion of BSA increased both the predictivity and quality of permeability studies, particularly of highly lipophilic, BCS class II compounds. Passive and active transport processes could also be distinguished after accounting for unbound concentrations. The overall effect of Cremophor<sup>®</sup> EL on the permeability to a drug was compound-specific and probably dependent on micellar incorporation. Cremophor<sup>®</sup> EL can therefore not be recommended. </p><p>Neither <i>pH</i> nor BSA affect the functionality of transporters such as P-glycoprotein. However, efflux ratios of ionizable or protein bound drugs are altered in the presence of a <i>pH</i>-gradient or BSA, indicating that an experimental system without protein or <i>pH</i> gradient can over- or underestimate active and passive efflux in drug transport.</p>
32

Refined in vitro Models for Prediction of Intestinal Drug Transport : Role of pH and Extracellular Additives in the Caco-2 Cell Model

Neuhoff, Sibylle January 2005 (has links)
Drug transport across the intestinal epithelium is roughly predicted from permeability values obtained from Caco-2 cell monolayers. This thesis examines the important role of pH and extracellular additives for increasing the reliability and predictivity of the in vitro screening system, Caco-2. It was shown that the passive transport of ionizable compounds may be biased by a false efflux or uptake component, when applying a physiological pH-gradient across the membrane. pH also affected the amount of compound available at the transporter-binding site. Therefore, pH dependence should be considered in studies of such compounds and of drug-drug interactions involving efflux transporters. It was also shown that proton-dependent apical uptake or basolateral efflux should be studied both with and without a pH gradient over the whole monolayers. The two extracellular additives, bovine serum albumin (BSA) and the solubilizing agent, Cremophor® EL, also influenced Caco-2 permeabilities. BSA applied to the receiver side increases, and to the donor side decreases drug permeation according to the drug’s protein binding capacity. Thus, the absorptive transport for both passive and active compounds is favoured, giving a physiologically sound improvement of the Caco-2 cell model. Inclusion of BSA increased both the predictivity and quality of permeability studies, particularly of highly lipophilic, BCS class II compounds. Passive and active transport processes could also be distinguished after accounting for unbound concentrations. The overall effect of Cremophor® EL on the permeability to a drug was compound-specific and probably dependent on micellar incorporation. Cremophor® EL can therefore not be recommended. Neither pH nor BSA affect the functionality of transporters such as P-glycoprotein. However, efflux ratios of ionizable or protein bound drugs are altered in the presence of a pH-gradient or BSA, indicating that an experimental system without protein or pH gradient can over- or underestimate active and passive efflux in drug transport.
33

Análise da expressão gênica, formação de biofilmes e adesão/invasão a célula Caco-2 por Listeria monocytogenes em diferentes condições encontradas no trato gastrintestinal, em alimentos e em presença de bacteriocinas / Analysis of gene expression, biofilm formation and adhesion/invasion to Caco-2 cell by Listeria monocytogenes in different conditions found in the gastrointestinal tract, in food and in the presence of bacteriocins

Lizziane Kretli Winkelstroter 10 December 2012 (has links)
Listeria monocytogenes é uma bactéria transmitida principalmente via alimentos, podendo causar infecções graves em pessoas imunodeprimidas e durante a gestação, devido à sua capacidade de sobreviver intracelularmente. A formação de biofilmes por L. monocytogenes é um fator preocupante para as indústrias de alimentos, pois biofilmes comprometem a sanitização de superfícies, aumentando os riscos de contaminação. Além disso, alguns estudos indicam que a capacidade de formação de biofilme pode estar correlacionada com a capacidade de algumas bactérias causarem doença. O controle de L. monocytogenes representa um desafio, especialmente em alimentos refrigerados e prontos para o consumo, pois esta bactéria é de natureza psicrotrófica, ubíqua e adapta-se rapidamente a diferentes condições ambientais, por meio da modulação da expressão de seus genes. O efeito das condições ambientais na expressão de genes de virulência de L. monocytogenes não é totalmente compreendido. Neste trabalho, isolados de L. monocytogenes de diversas origens foram avaliados quanto à sua capacidade de formar biofilmes, de aderir/invadir em células eucarióticas e também de expressar o gene internalina (int A), que está relacionado com o potencial de virulência. A presença de bacteriocinas de bactérias láticas (BAL) e incubação a 5°C foram os principais fatores que influenciaram a formação de biofilmes por L. monocytogenes, em comparação com caldo BHI (controle). Em geral, a adesão/invasão de células Caco-2 foram significativamente menores em pH baixo (4,5), incubação a 5°C e na presença de 0,3% Oxgall (sais biliares). Entretanto, dois isolados de L. monocytogenes (INCQS 353 e Reg 26c) apresentaram aumento nas taxas de invasão quando cultivados na presença de NaCl a 5% (P<0,05). Um isolado de L. monocytogenes (H-2) obtido de hortaliças apresentou a maior capacidade de formação de biofilme e de invadir células Caco-2, sugerindo que há uma possível relação entre a formação de biofilme e de potencial de virulência. No ensaio para avaliação da expresão do gene inlA, todos os isolados foram regulados negativamente pela presença de bacteriocinas, Oxgall 0,3%, pH 4,5 e incubação a 5°C. No entanto, para um isolado de L. monocytogenes (HU 471), a expressão do gene int A foi oito vezes maior na presença de sacarose, indicando que os componentes de alimentos podem aumentar a virulência e o potencial de infecção de L. monocytogenes. / Listeria monocytogenes is a bacterium transmitted mainly by foods and can cause serious infections in immunocompromised individuals and in pregnant woman due to their ability to survive intracellularly. The biofilm formation by L. monocytogenes is a concern for the food industry because microorganism in biofilms may survive after treatment with sanitizers and increases the risk of food contamination. In addition, some studies indicate that the ability of biofilm formation can be correlated with virulence potential. The control of L. monocytogenes is a challenge, especially in chilled and ready to eat foods since it has psychrotrophic nature, it is ubiquitous and can adapt quickly to different environmental conditions by modulating the expression of its genes.The effect of environmental conditions on gene expression and virulence of Listeria monocytogenes is not fully understood. In this report, L. monocytogenes isolates from diverse sources were evaluated for their ability to form biofilms, for adhesion/invasion of eukaryotic cells and also for differential expression of internalin A gene (int A), which is related to virulence potential. The presence of bacteriocins of latic acid bacteria (BAL) and incubation at 5°C were the main factors that influenced biofilm formation by L. monocytogenes, in comparison with BHI broth (control). In general, adhesion and invasion of Caco-2 cells were significantly lower in low pH (4.5), incubation at 5°C and in presence of Oxgall 0.3% (bile salts). On the other hand, two L. monocytogenes isolates (INCQS 353 and Reg 26c) showed higher invasion rates when cultivated in the presence of NaCl 5% (P<0.05). One L. monocytogenes (H-2) isolated from minimally processed leafy vegetables showed the strongest ability to form biofilm and to invade Caco-2 cells, under selected conditions, suggesting there may be a relationship between biofilm formation and virulence potential. For the vast majority of isolates, expression of int A gene were down regulated by the presence of bacteriocins, Oxgall 0.3%, pH4.5 and incubation at 5°C. Nonetheless, for one L. monocytogenes isolate (HU 471) expression of int A gene was eight times higher in presence of sucrose, indicating that food components can increase the infectiveness of L. monocytogenes.
34

Um mecanismo: invasão de células epiteliais por amostras de Escherichia coli enterohemorrágica (EHEC) LEE-negativas. / A mechanism: invasion of epithelial cells by LEE-negative enterohaemorrhagic Escherichia coli (EHEC) strains.

Verônica De Franco Rennó 11 June 2008 (has links)
Escherichia coli enterohemorrágicas (EHEC) que possuem a Ilha de Patogenicidade LEE são importantes patógenos humanos. A habilidade de adesão, invasão e perfil genético têm sido estudados, já que sorotipos que não possuem LEE tem sido isolados de pacientes com doença severa e intracelularmente. Das nove amostras relacionadas com doença, quatro (44.5%) apresentaram stx2+. Todas foram positivas para o gene lpfA e iha, e negativas para toxB. Três (33,3%) apresentaram saa e cinco hly. A maioria apresentou padrão de adesão difusa e invasão negativa em células HEp-2. Em CaCO-2 apresentaram aderência com variados graus de intensidade, e a maioria das amostras testadas apresentou invasão maior que 3,3%. Frente ao inibidor de polimerização de actina citocalasina D, houve significativa redução nos níveis de invasão, sugerindo que estas amostras utilizam um mecanismo da célula hospedeira para internalização, e que provavelmente, fatores de virulência como adesinas, favorecem a adesão das mesmas, compensando a ausência do LEE, e facilitando a instalação da infecção. / Enterohaemorrhagic Escherichia coli (EHEC) that possess the pathogenic island LEE are important human pathogens. The adhesion ability, invasion and genetic profile have been studied, since serotypes that do not possess LEE have been isolated from humans with severe disease and found intracellular. Nine strains related with SHU, four (44,5%) were stx2+. All strains were positive for IpfA and iha genes and negative for toxB. Three (33,3%) showed saa and five hly. The most strains showed a diffusely adhesion pattern and negative invasion in HEp-2 cells. It presented various degrees of adhesion, and the most tested strains showed invasion high than 3,3% in CaCO-2. That was a significant reduction of invasion in the presence of actin polymerization inhibitor Citochalasin D, suggesting that these strains use a host cell mechanism to invade, and probably virulence factors, like adhesins, favors this adhesion and compensate LEE absence, promoting the installation of infection.
35

Padronização de novo método ex vivo para avaliação da permeabilidade intestinal de fármacos utilizando epitélio intestinal de rã-touro (Rana catesbeiana): comparação com células Caco-2 / Standardization of new ex vivo method to assess intestinal permeability of drugs using intestinal epithelium of bullfrog (Rana catesbeiana): comparison with Caco-2 cells

Paula Cristina Torres de Souza 07 August 2014 (has links)
Métodos in vitro utilizando epitélio intestinal animal são importantes ferramentas para avaliar a permeabilidade de fármacos, propriedade que é um importante parâmetro de biodiosponibilidade. Considerando que o maior objetivo na indústria farmacêutica é desenvolver novos fármacos com boa biodisponibilidade oral, o projeto teve como objetivo padronizar o modelo de permeação com membrana de intestino de rã (Rana catesbeiana) em células de Franz, comparando seus resultados com ensaios de células Caco-2. Os fármacos modelo utilizados foram os antivirais zidovudina e aciclovir. A quantidade de fármaco permeado foi determinada por método de eletroforese capilar para o método com intestino de rã touro e por HPLC-UV para os ensaios com células Caco-2. O parâmetro de permeação foi o coeficiente de permeabilidade aparente (Papp) dos fármacos para ambos modelos experimentais. Para estabelecimento do protocolo experimental dos estudos de permeabilidade intestinal de rã, foi proposto um projeto fracionado 24-1 com 4 ensaios adicionais usando o software Minitab, e as variáveis foram: secção intestinal, pH da solução de Ringer e temperatura. A análise do planejamento experimental feita pela estimativa dos parâmetros da regressão obtidos através dos resultados do modelo fatorial possibilitou a determinação dos coeficientes da equação matemática que definiu a influência das variáveis sobre o coeficiente de permeabilidade aparente dos fármacos. Os efeitos das variáveis pH e temperatura interpretados conjuntamente apresentaram interferência leve, porém as variáveis fármaco e secção intestinal interpretados juntos tiveram interferência importante, mostrando maior permeação dos fármacos através da secção inicial do intestino da rã. Os resultados de Papp foram: para o metoprolol, pelo método das células Caco-2 foi de 28 x 10-6 cm/s, valor que está de acordo com demais dados de células Caco-2 na literatura e o valor obtido com células de Franz que mais se adequada a estes resultados e demais disponíveis provenientes de outras técnicas na literatura, foi de 28,1 x 10-6 cm/s, em uma das condições do planejamento estatístico utilizando segmento final da membrana epitelial da rã. No caso do aciclovir, o resultado de Papp de 0,48 x 10-6 cm/s também obtido em uma das condições envolvendo segmento intestinal final da rã foi exatamente igual a o valor 0,48 x 10 - 6 cm/s, encontrado com células Caco-2 no presente estudo e estão de acordo com outros valores disponíveis na literatura por Trapani e colaboradores, 2004 e também com células Caco-2. Para zidovudina o valor de Papp obtido em uma das condições utilizando segmento intestinal inicial da rã, de 13 x 10-6 cm/s, foi a que mais se assemelhou ao obtido pela técnica de células Caco-2, 13,6 x 10-6 cm/s, e também está de acordo com demais dados da literatura. / There are lots of different in vitro technics in the literature using animal intestinal epithelium to estimate permeability of drugs, property that is an important parameter of bioavailabity. Considering that the main objective of Pharmaceutical Companies is the development of new drugs with good oral bioavailabity, the aim of this work was to standardize the permeability of antivirals using in vitro/ex vivo method of intestinal epithelium of Rana catesbeiana in Franz cells and compare these results to those obtained from studies using Caco-2 cells. Zidovudine and Acyclovir were selected as model drugs. The amount of drug permeated will be determined by the method of capillary electrophoresis for assays using Rana Catesbeiana and HPLC-UV for studies with Caco-2 cells. The permeation parameter determined was the apparent permeability coefficient (Papp) of drugs for both experimental models. To establish the experimental protocol to studies of intestinal permeability of frog, it was proposed a fractional 24-1 design with 4 additional tests using Minitab software and the variables were: intestinal section, pH of Ringer solution and temperature. The analysis of the experimental design made by the estimate of the regression parameters obtained from the factorial model results allowed the determination of the coefficients of the mathematical equation that defines the influence of the variables on the apparent permeability coefficient of acyclovir and zidovudine. The effects of pH and temperature interpreted jointly presented a slight interference, but the variables drug and intes tinal section interpreted together had major interference, showing greater permeation of drugs through the initial section of the intestine of the frog . The results of Papp were: for metoprolol, with the method of Caco-2 cells was 28 x 10-6 cm/s, a value which is consistent with other data of Caco-2 cells provided in the literature and the condition obtained with Franz cells that are most suitable for these and other results obtained from other techniques available in the literature, was 28.1 x 10-6 cm/s, provided with the final intestinal segment using frog epithelial membrane. In the case of acyclovir, the result of Papp of 0.48 x 10-6 cm/s obtained in one condition with final frog intestinal segment was exactly equal to the value of 0.48 x 10-6 cm/s, found with Caco-2 cells in the present study and are in agreement with other values available in the literature for Trapani and colegues, 2004 and also with Caco-2 cells. The Papp value for zidovudine obtained with the initial gut segment of frog, 13 x 10-6 cm /s was which more resembled that obtained by the technique of Caco-2 cells, 13.6 x 10-6 cm/s and is also consistent with other literature data.
36

Padronização das condições para cultura de células Caco-2 visando à obtenção de membranas viáveis ao estudo da permeabilidade in vitro da rifampicina / Standardization of culture Caco-2 cells conditions to obtain viable membranes to study the in vitro permeability of rifampicin

José Eduardo Gonçalves 29 April 2010 (has links)
A permeabilidade através do epitélio intestinal tem se tornado um importante aspecto a ser determinado nas avaliações biofarmacotécnicas envolvendo fármacos e medicamentos. A técnica mais empregada para essa determinação in vitro é aquela que utiliza a cultura de células Caco-2. Entretanto, ainda são discutíveis as condições para a realização desses experimentos, uma vez que a padronização das mesmas é fator fundamental para a confiabilidade dos resultados. Nesta tese, foram avaliadas as condições para realização dos estudos de permeabilidade através de membranas de células Caco-2 para a rifampicina, principal fármaco utilizado no tratamento da tuberculose. Para tanto, foram investigados fatores tais como a citotoxicidade da rifampicina em diferentes concentrações, a influência da concentração do fármaco sobre a permeabilidade, do pH de realização dos experimentos e da presença de proteínas do muco intestinal, além da influência de proteínas plasmáticas. Foi também investigado o potencial indutor da rifampicina sobre a expressão da glicoproteína-P (Pgp) e seu impacto na permeabilidade da própria rifampicina. Os estudos foram desenvolvidos utilizando membranas de células Caco-2 provenientes da American Type Culture Collection (ATCC) cultivadas em placas Transwel®, a quantificação da fração permeada foi por cromatografia líquida de alta eficiência com métodos validados. A análise da indução da expressão da Pgp foi realizada por PCR-RT. Demonstrou-se que as concentrações da rifampicina (10,0; 25,0 e 50,0 &#181;g/mL) não ocasionaram danos às células Caco-2 no estudo de citotoxicidade pela técnica que emprega o sal do brometo de 3-(4,5-dimetil-2-tiazoli)-2,5-difenil-2H-tetrazólio (MTT). As concentrações de rifampicina (5,0; 10,0 e 25,0 &#181;g/mL) não resultaram em valores estatisticamente diferentes de permeabilidade aparente (Papp) em células Caco-2 nas condições do estudo. A rifampicina apresentou valor de Papp significativamente maior em pH 6,8 dentre os valores de pH avaliados (5,8 ; 6,8; 7,4). A presença de muco simulado e de soro fetal bovino não resultou em valores de permeabilidade significativamente distintos do resultado obtido sem a sua adição ao experimento. A expressão da Pgp em células Caco-2 é induzida pela adição da rifampicina (10&#181;g/mL), ocasionando diminuição da sua permeabilidade por mecanismo de efluxo. Pelos resultados de permeabilidade obtidos em todas as condições avaliadas, a rifampicina pode ser considerada um fármaco de alta permeabilidade de acordo com o Sistema de Classificação Biofarmacêutica. / The permeability through the intestinal epithelium has become an important aspect to be determined in evaluations involving drugs and pharmaceutical products. The most common technique for this determination in vitro is one that uses the culture of Caco-2 cells. Nevertheless, the conditions for carrying out such experiments are still questionable, since the standardization of them is essential to the reliability of the results. In this thesis, we evaluate the conditions for the studies of permeability of rifampicin through membranes of Caco-2 cells, the main drug used in the treatment of tuberculosis. To this end, we examined factors such as cytotoxicity of rifampicin at different concentrations, the influence of drug concentration on the permeability, as well as the pH of the experiments, the presence of proteins of intestinal mucus, and the influence of plasma proteins. It was also investigated the potential of rifampicin on the expression of P-glycoprotein (Pgp) and its impact on the permeability of rifampicin itself. The studies were developed using membranes of Caco-2 cells from American Type Culture Collection (ATCC) grown on plates Transwel®, and the quantification of the fraction of drug permeated was obtained by high performance liquid chromatography with validated methods. The analysis of induction of expression of Pgp was performed by RT-PCR. It was demonstrated that the concentrations of rifampicin (10,0; 25,0 and 50,0 &#181;g/mL) did not cause damage to Caco-2 cells in the study of the cytotoxicity technique that uses a bromide salt of 3 - (4,5-dimethyl-2 - thiazol) -2,5-diphenyl-2H-tetrazolium (MTT). The concentrations of rifampicin (5,0; 10,0 and 25,0 &#181g/mL) did not result in statistically different values of apparent permeability (Papp) in Caco-2 cells under the conditions of the study. Rifampicin showed a value of Papp significantly higher at pH 6.8 in comparison with other measured pH values (5,8 and 7,4). The presence of mucus simulated and fetal calf serum did not result in permeability values significantly different from the result obtained without its addition to the experiment. The expression of P-gp in Caco-2 cells is induced by the addition of rifampicin (10 &#181;g/ml), decreasing its permeability by efflux mechanism. Taking into account the results of permeability obtained in all conditions, the rifampicin can be considered a high permeability drug according to the biopharmaceutical classification system.
37

Biodisponibilité nutritionnelle de systèmes colloïdaux riches en acides gras polyinsaturés : études in vivo et in vitro / Nutritional bioavailability of polyunsaturated fatty acid-rich colloidal systems : in vivo and in vitro studies

Couedelo, Leslie 14 November 2011 (has links)
Les derniers apports nutritionnels conseillés recommandent une consommation plus importante en acides gras polyinsaturés de la série n-3 que celle actuellement constatée dans l’alimentation française. Dans ce contexte, il convenait d’appréhender les facteurs susceptibles de moduler l’absorption et le devenir de leur chef de file, l’ALA, en faisant appel à deux approches, l’une in vivo (rat), l’autre in vitro (Caco-2). L’étude relative au devenir métabolique de l’ALA, selon sa forme physique et chimique de présentation, a été réalisée avec des lipides « modèles » (TAG structurés) ou naturels (huile de lin) riches en ALA, et selon différents systèmes lipidiques (huile en phase continue ou en émulsion de type huile dans eau et de composition en phospholipides variables). Les résultats obtenus in vivo et in vitro à l’égard de l’huile de lin (émulsionnée ou non) montrent que l’émulsification accélère non seulement le passage intestinal de l’ALA mais améliore également sa concentration lymphatique. L’étude cellulaire a par ailleurs démontré que la présence de lysophospholipides dans les micelles mixtes permet d’améliorer la sécrétion de l’ALA dans les lipoprotéines. D’autre part, le devenir métabolique de l’ALA dépend de sa régiolocalisation sur le triglycéride alimentaire. En effet, les résultats de l’étude faisant appel aux TAG structurés montrent que la position interne n’est que partiellement conservée dans la lymphe, suggérant qu’une hydrolyse des 2-MAG serait opérée par une MG lipase. En conséquence, l’ensemble des résultats obtenus lors de cette étude montre que l’absorption et le transport de l’ALA seraient uniquement modulés selon la forme physique de l’acide gras alors que son devenir et son utilisation métabolique dépendraient de sa régiolocalisation sur le TAG alimentaire. Ces deux facteurs réunis permettraient dés lors de prévenir l’ALA d’une β-oxydation précoce, en vue de favoriser son élongation en dérivés supérieurs dans les tissus cibles. / The last recommended nutrient intakes advise a higher consumption of n-3 polyunsaturated fatty acids series than currently found in French diet. In this context, it was appropriate to apprehend the factors that could modulate the absorption and fate of their leader, ALA, using two approaches, one in vivo (rat), the other one in vitro (Caco-2). The study on the metabolic fate of ALA, according to its physical and chemical submission form, was conducted with "models" lipids (structured TAG) or natural (flaxseed oil) rich in ALA, and according to different lipid systems (oil in continuous phase or in emulsion type oil in water and with variable phospholipid composition). Results obtained in vivo and in vitro for flaxseed oil (emulsion or not) show that emulsification enhances the recovery of ALA at the intestinal level but also improves its lymphatic concentration. The cell study also demonstrated that the presence of lysophospholipids in mixed micelles can improve the secretion of ALA in lipoproteins. On the other hand, the metabolic fate of ALA depends on its location on the glycerol backbone of the dietary triglyceride. The results of the study using structured TAG show that the internal position is partially preserved in lymph, suggesting that a hydrolysis of 2 - MAG by MG lipase could occur. Accordingly, all of the results obtained in this study shows that the absorption and transport of ALA would only be modulated according to the physical form of the fatty acid while its fate and its metabolic use would depend on its location on the dietary TAG. These two factors combined would then allow preventing the early β-oxidation of ALA in order to promote its elongation in higher derivatives in the target tissues.
38

Interactions alimentaires sur la bioaccessibilité et l'activité pro-vitaminique A du beta-carotène : effets de microconstituants phénoliques / Food interactions on the bioaccessibility and the pro-vitaminic A activity of beta-carotene : effects of phenolic micronutrients

Poulaert, Marie 18 December 2012 (has links)
Le β-carotène (Bc) est un caroténoïde connu pour son activité pro vitaminique A. La consommation de fruits et légumes riches en Bc est donc particulièrement encouragée, principalement dans les pays en développement. Cependant, au cours d'un repas, la biodisponibilité du Bc est influencée par la présence des macro et microconstituants des aliments. L'objectif général de ce travail a été d'étudier les interactions alimentaires pouvant survenir au cours des différentes étapes du processus d'absorption du Bc. La bioaccessibilité du Bc, évaluée à l'aide d'un modèle de digestion in vitro, a révélé que la quantité de Bc micellarisé des aliments de base (patate douce orange (PDO) et banane plantain) des pays du Sud se retrouve augmentée dans certaines préparations tratitionnelles. De plus, dans une simple association de deux aliments, nos résultats ont montré que la naringine de jus d'agrumes diminuait la bioaccessibilité du Bc de la PDO car ce flavanone se micellarise et entre en compétition avec le Bc. L'effet de flavanones sur l'absorption intestinale du Bc a ensuite été étudié à l'aide d'un modèle cellulaire de type Caco-2. L'ensemble des glycosides de flavanones testés, mais principalement l'hespéridine (Hes), ont augmenté l'absorption du Bc. L'expérimentation in vivo chez la gerbille n'a quant à elle pas montré d'effet de l'Hes sur la bioefficacité du Bc de la PDO. Par contre, nos données suggèrent que l'Hes, dans le cas d'un régime carencé en caroténoïdes et vitamine A, pourrait stimuler l'activité de la β,β-carotène mono-oxygénase par un mécanisme impliquant le facteur de transcription PPARγ. / Β-carotene (Bc) is a carotenoid mainly known for its provitaminic A activity. Therefore, the consumption of fruits and vegetables rich in Bc are promoted, especially in developing countries. However, during a meal, the Bc bioavailability was modulated by other macro or micronutrients from food. The main objective of this study was to evaluate food interaction occurring during the different steps of Bc absorption. The Bc bioaccessibility, evaluated through an in vitro digestion, showed that the micellarization of Bc staple foods from South countries (orange fleshed sweet potato (OFSP) and plantain) increases in some traditional food preparations. Moreover, we observed that the Bc bioaccessibility from OFSP decreases in the presence of Citrus juice because of the naringin which competes for incorporation into mixed micelles. Effects of flavanones were then assessed on intestinal Bc uptake using Caco-2 cells. Among flavanone glycosides tested, mainly hesperidin (Hes), increased Bc uptake. In vivo experimentation with gerbil did not shown any effect of Hes on the bioefficacity of Bc from OFSP. By contrast, our data suggest that under a low Bc or vitamin A free diet, Hes might enhance BCMO1 activity through its action as agonist of PPARγ.
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Diferença de patogenicidade entre Escherichia coli enteroinvasora e Shigella flexneri em modelo experimental de infecção intestinal / Pathogenicity difference between Escherichia colienteroinvasive and Shigella flexneri in an experimental model of intestinal infection

Moreno, Ana Carolina Ramos 22 August 2008 (has links)
Neste trabalho, esclarecemos tópicos da patogenicidade de EIEC que sustentam a sua menor virulência quando comparada à S. flexneri, e mostramos a importância das células dendríticas (CD) nesse processo. Estudou-se o comportamento de EIEC e S. flexneri quando em contato com células Caco-2, avaliando-se uma cinética de expressão dos genes envolvidos na invasão e disseminação bacteriana. Em geral, todos os genes foram menos expressos em EIEC, fato corroborado pelo fenótipo de disseminação bacteriana, onde EIEC foi menos eficiente do que Shigella. Também foi avaliada a modulação da resposta inflamatória de células dendríticas intestinais murinas pela produção de citocinas, expressão de moléculas co-estimulatórias e apresentação de antígenos, após desafio das células com as bactérias. Os resultados sugerem que EIEC induz a uma resposta protetora ao hospedeiro, enquanto que Shigella estaria \"driblando\" o sistema imune, além de provavelmente super-estimular o sistema imune adaptativo, fato que poderia levar a um agravamento da doença. As ações integradas das células Caco-2, células dendríticas e estímulos bacterianos foram estudadas em co-cultura celular. Observou-se que EIEC e suas proteínas secretadas induzem a migração das CDs ao compartimento apical da co-cultura; nada foi observado quando o desafio se deu com Shigella. Também foram avaliadas as concentrações de citocinas inflamatórias no microambiente infeccioso formado. A citocina TNF-&#945;, bem como CCL20 e MCP-1 foram mais proeminentes após estímulo com EIEC, fato que poderia explicar parcialmente a migração das CDs ao lado apical da co-cultura após estímulo com EIEC e suas proteínas secretadas. Nossas evidências experimentais indicam que a doença desencadeada por EIEC é mais restrita a um determinado local da infecção, ou seja, não é capaz de se disseminar a ponto de estender a lesão tecidual de forma mais drástica, como Shigella. Esse fenômeno pode estar associado à menor expressão de seus dos fatores de virulência e à resposta imune inata induzida no sítio de infecção, o que levaria, fatalmente, à resolução da doença. / In this study, we clarify topics of pathogenicity from EIEC that support its lower level of virulence when it is compared to S. flexneri, and we have shown the importance of dendritic cells (DC) in this process. We studied the conduct of EIEC and S. flexneri when they were in contact with Caco-2 cells and we analyzed the kinetics of the genes expression that was involved in the spread and invasion of the bacteria. In general, all genes were expressed less in EIEC, as demonstrated by the phenotype of the bacterial spread, where EIEC was less efficient than Shigella. We also analyzed the modulation of the inflammatory response by the murine intestinal dendritic cells by the production of cytokine, expression of co-stimulators molecules and antigens presentation, after the interaction of the cells with the bacteria. The results showed that EIEC induces a response that protects the host while Shigella manipulate the host intestinal innate and adaptive immune system and it probably over-stimulates the adaptive immune system which could let the disease worse. The integrated actions of Caco-2 cells, dendritic cells and bacterial stimulus, were studied in a co-culture cell. We observed that EIEC and its secreted proteins induce the migration of the DCs to the apical compartment of the co-culture; nothing was observed related to Shigella. We also evaluated the concentrations of the inflammatory cytokines at the infective micro environment that was formed. The cytokine TNF-&#945;, as CCL20 and MCP-1 were more prominent after been stimulated with EIEC, a fact that could partially explain the migration of DCs to the apical side of the co-culture after the stimulus with EIEC and its secreted proteins. Our experimental evidence shows that the disease triggered by the EIEC is more restricted at a definite infection place, which means that it is not capable of disseminating beyond a certain point to extend the tissue\'s injury and let it worsen, as Shigella do. This phenomenon can be associated with the lower level of expression of its virulence factors and to the immune response induced in the infection site, what could finally lead to the eradication of the disease.
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Estudo dinâmico da expressão gênica global durante a interação STEC-enterócito utilizando séries temporais / Dinamic study of global gene expression along STEC-enterocyte interaction using time series

Iamashita, Priscila 27 November 2017 (has links)
As Escherichia coli produtoras da toxina Shiga (STEC) são importantes patógenos humanos, causando desde diarréias até a síndrome hemolítica urêmica (SHU). Há diversos sorotipos associados a SHU, tais como O157:H7 e O113:H21. No Brasil o sorotipo O113:H21 ainda não aparece associado a SHU, embora seja frequentemente isolado de carcaças e fezes bovinas. Nosso grupo já investigou comparativamente as redes de coexpressão gênica (RCG) de STEC EH41 (associado à SHU) e Ec472/01 (isolado de fezes bovinas). A análise comparativa do perfil transcricional de EH41 e Ec472/01 revelou que somente EH41 expressa um conjunto de genes que inclui o regulador transcricional dicA. A maioria destes genes está situada em um único módulo transcricional e podem estar associados a fatores de virulência. Assim, este trabalho centrou-se numa abordagem de biologia de sistemas, integrando análises genômica e fenotípica da resposta de enterócitos Caco-2 à EH41 e Ec472/01. A análise genômica baseou-se no estudo temporal de RCG para compreender os mecanismos moleculares envolvidos na patogenicidade desses dois isolados. As alterações fenotípicas ocorridas nas células Caco-2 ao longo da exposição a cada um dos isolados de STEC foram visualizadas através de MEV. A análise genômica mostrou que o mecanismo molecular da resposta de Caco-2 durante a interação com EH41 ou Ec472/01 é claramente distinto. Nas redes do grupo Caco-2/EH41 as alterações topológicas incluíram a perda do status scale free e a sua recuperação, com o estabelecimento de uma nova hierarquia de genes na rede. Esses resultados se enquadram no modelo de redes para transição saúde-doença: a nova rede representa a resposta adaptativa da célula ao patógeno, o que não significa um retorno à normalidade. Já no grupo Caco-2/Ec472 as redes, após a perda do status scale free, não recuperam esse status até o final do período estudado, o que sugere um estado de transição mais prolongado para reorganização da hierarquia da rede. Mais ainda, através da caracterização dos módulos transcricionais, foi possível compreender dinamicamente os mecanismos moleculares envolvidos na resposta diferencial de Caco-2 aos dois isolados aqui estudados. STEC EH41 induz rapidamente a resposta inflamatória e apoptótica a partir da primeira hora de interação enterócito-bactéria. Por outro lado, células Caco-2 em contato com Ec472/01 ativam, a partir de uma hora, a fagocitose e, a partir da segunda hora, expressam moduladores da homeostase imune. A análise fenotípica das células Caco-2 mostrou, de forma nítida, uma maior destruição dos microvilos dos enterócitos em contato com EH41 do que com Ec472/01. Integrando os resultados genômicos e fenotípicos pode-se concluir que EH41 induz em Caco-2 - em comparação com Ec472/01 - maiores e mais rápidas alterações na expressão gênica global, além de uma resposta inflamatória e apoptótica excessiva, levando assim a alterações morfológicas mais pronunciadas nas células Caco-2. Em seu conjunto, esses resultados contribuem para uma melhor compreensão dos mecanismos moleculares envolvidos na patogenicidade das STECs associadas à SHU. Assim, as perspectivas de desenvolvimento deste trabalho deverão incluir a investigação de fatores de virulência e vias moleculares envolvidas na indução das respostas imunes que podem conduzir à SHU / Shiga toxin-producing Escherichia coli (STEC) O113:H21 strains are associated with human diarrhea and some of these strains may cause hemolytic uremic syndrome (HUS). In Brazil O113:H21 strains are commonly found in cattle but, so far, were not isolated from HUS patients. Previously, our group conducted comparative gene co-expression network (GCN) analyses of two O113:H21 STEC strains: EH41, isolated from a HUS patient in Australia, and Ec472/01, isolated from bovine feces in Brazil. Differential transcriptome profiles for EH41 and Ec472/01 revealed a gene set exclusively expressed in EH41, which includes the dicA putative virulence factor regulator. GCN analysis showed that this set of genes constitutes an EH41 specific transcriptional module which may be associated to virulence factors. Therefore, in the present work a system biology approach was conducted to investigate the differential Caco-2 response - genomic and phenotypic - to EH41 (Caco-2/EH41) or to Ec472/01 (Caco- 2/Ec472) along enterocyte-bacteria interaction. The genomic analysis was based on temporal GCN data in order to gain a better understanding on the molecular mechanisms underlying the capacity to cause HUS. The phenotypic alterations in Caco-2 during enterocyte-bacteria interaction were assessed by scanning electronic microscopy (SEM). The genomic analysis showed that the molecular mechanism of Caco-2 response to EH41 or to Ec472/01 during enterocyte-bacteria interaction is clearly different. The GCN topological analyses for Caco-2/EH41 group revealed loss of the scale-free status after one hour of interaction, persistence of this condition along the second hour and establishment of a new gene hierarchy thereafter. These events resemble the network mechanism of health-disease transition. The new established network represents an adaptive cell response to the pathogen and not the return to a \"normal\" state. Conversely, the networks for Caco-2/Ec472 group showed a slow and progressive loss of the scale-free status without its restoration at the end of the time interval here studied. Through transcriptional module characterization it was possible to reveal the dynamic of the molecular mechanism involved in the Caco-2 differential responses to the STEC isolates. EH41 induces a rapid inflammatory and apoptotic response just after the first hour of enterocyte-bacteria interaction. Instead, the Caco-2 response to Ec472/01 is characterized by phagocytosis activation at the first hour, followed by the expression of immune response modulators after the second hour. SEM phenotypic analysis of Caco-2 cells along enterocyte-bacteria interaction showed more intense microvilli destruction in cells exposed to EH41, when compared to cells exposed to Ec472/01. The integration of genomic and phenotypic data allowed us to conclude that EH41, comparatively to Ec472/01, induces greater and precocious global gene expression alterations in Caco-2, what is related to excessive inflammatory and apoptotic responses. These responses are associated with the pronounced morphological alterations observed by SEM in Caco-2 cells exposed to EH41. Altogether, these results contribute for a better understanding of the molecular mechanism involved in STEC pathogenicity associated to HUS. Therefore, the future perspectives for the development of the present work should include the investigation of virulence factors and molecular pathways involved in the induction of immune responses leading to HUS

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