Abstract—This paper will detail the process and methods to the problem with having an Offline Certificate Authority that can still be accessed remotely. Specifically, to update CRL on the server when the need arises without bringing the entire CA online. This has been managed via local access in the past but to ease the use a need for remote management has arisen. This paper will explain how this problem was solved with the use of a Data diode to prevent the CA to be fully online. A Data Diode will only allow traffic in one direction keeping any data from leaking from the CA while still making it available for specific uploads, in this case CRL files that handle the revocation of certificates issued by the CA. This will allow for more up to date lists when the server is brought online for the issuance of certificates once per year. This paper will try to detail the steps that need to be taken in order to set up an edge server that allows the transmission of files with the use of UDP.  Keywords—CRL Content Revocation List, CA Certificate Authority, Data Diode, Offline Server, UDP user Datagram Protocol.

La recombinaison homologue est essentielle à la réparation des lésions de l’ADN ainsi qu’à la ségrégation correcte des chromosomes en méiose. Une étape importante de la recombinaison méiotique est la formation des crossovers (CO). Au cours de ma thèse, j’ai mis en évidence un nouveau mécanisme de régulation de la recombinaison méiotique. J'ai montré que les cycles d'activation et de désactivation des cullin-RING ligases (CRL) sont absolument nécessaires à la recombinaison méiotique. Les CRL sont activées par neddylation et désactivées par la deneddylation. De plus, elles peuvent aussi être inhibées par la séquestration via la protéine CAND1. Mon travail a démontré que ces trois niveaux de régulation des CRL jouent des rôles cruciaux dans la recombinaison homologue méiotique chez A. thaliana. J’ai montré qu'AXR1, un composant clé de la machinerie de neddylation, est nécessaire à la localisation correcte des CO méiotiques et à la recombinaison homologue somatique. J’ai aussi prouvé que le processus de deneddylation médié par CSN5A est nécessaire à la formation des CO. J'ai obtenu des données montrant que cette régulation de la localisation des CO agit à travers la régulation d’un complexe CRL4. Enfin, j’ai pu montrer que l'inhibiteur des CRL, CAND1, est requis pour la formation de plus de 90 % des CO. En utilisant des outils génétiques et cytologiques, j'ai montré que CAND1 agit probablement sur la régulation du biais inter-homologue. L’ensemble de ces données, met l’accent sur un nouveau mécanisme de la régulation de la recombinaison homologue, connectant pour la première fois la méiose et l’ubiquitination via les cullin-RING Ligases. / Homologous recombination is essential to all living organisms in order to repair DNA damages. In addition, a large majority of organisms use homologous recombination in meiosis to ensure proper chromosome segregation. A main step of meiotic recombination is crossover (CO) formation. During my PhD, I was able to highlight a new pathway controlling meiotic recombination. I showed that cycles of activation and deactivation of cullin-RING ligases (CRLs) are absolutely required for correct meiosis. CRLs are activated by neddylation, and deactivated by deneddylation. In addition, they can also be inhibited by sequestration by the CAND1 protein. My work demonstrated that these three levels of CRL regulation play crucial roles in meiotic homologous recombination in A. thaliana. First, I showed that AXR1, a key component of the neddylation machinery, is required for the correct localisation of meiotic COs and for somatic homologous recombination. Second, I showed that the deneddylation process mediated by CSN5A is also necessary for normal CO formation. I obtained evidence that this regulation of CO position is likely to be mediated by a CRL4 complex. Last, I could show that the CRL inhibitor, CAND1, is required for the formation of up to 90% of the COs. Using genetic and cytological tools, I showed that CAND1 probably acts on the regulation of the inter-homolog bias. Considering all these data, my work draws the attention to a new mechanism regulating meiotic homologous recombination, connecting for the first time meiosis to CRL-mediated ubiquitylation.

Ce travail décrit un criblage systématique du système ubiquitine-protéasome (UPS) basé sur une organisation en cascade. Nous avons évalué l’effet de l’inhibition par ARN interférent de composants individuels d’UPS sur la viabilité de cellules cancéreuses de la prostate, avec un accent particulier sur les cellules TMPRSS:ERG-positive (VCaP), comme un modèle de phénotype prévalent du cancer. Sept gènes ont été identifiés comme étant particulièrement importants pour le fonctionnement des cellules cancéreuses de la prostate. Parmi eux, le gène-candidat le plus prometteur était UBE2U. Cette thèse met en évidence l’implication d’UBE2U dans la carcinogénèse de la prostate et décrit les premières caractérisations d’UBE2U comme une cible thérapeutique potentielle.La prévalence des composants de la voie CRL/NEDD8 parmi les hits (4 sur 7) suggère que la neddylation est importante dans la biologie des cellules cancéreuses de la prostate. Deux de ces gènes, CUL2 et RBX1, n’ont des effets spécifiques que dans des cellules TMPRSS2:ERG-positives, et, donc, sont potentiellement ERG-dépendantes. Nous avons également révélé un rôle crucial du facteur d’échange de CRL (CAND1), en particulier lorsque la neddylation est compromise. L’inhibition de CAND1 induit l’apoptose dans des cellules VCaP, qui est renforcé par l’inhibiteur spécifique de la neddylation MLN4924. CAND1 est donc une nouvelle cible thérapeutique potentielle. Par ailleurs, nous avons démontré que l’inhibition de la voie CRL/NEDD8 dans les cellules cancéreuses de prostate a des conséquences qui dépendent fortement du contexte cellulaire. L’inhibiteur MLN4924 induit l’apoptose dans toutes les lignées cellulaires testées, bien que les cellules TMPRSS2:ERG-positives se soient révélées significativement plus résistantes. Nous avons démontré que la résistance accrue des cellules VCaP reflète la plasticité des cellules cancéreuses régulée par un réseau d’interactions ERG:NF-kB:c-Myc:Wnt/β-cat:AR. L’inhibition partielle de la neddylation enclenche une reprogrammation transcriptionnelle de cellules VCaP, amenant à la quiescence des cellules et à l’inhibition de l’apoptose dépendant de la prolifération. Cet effet est le résultat de la réactivation du programme AR. Nous avons conclus que la voie CRL/NEDD8 régule le réseau transcriptionnel qui contrôle la plasticité des cellules cancéreuses. Ces résultats peuvent aider à trouver des traitements plus efficaces de cancers TMPRSS2:ERG-positifs.Finalement, nous avons observé que l’inhibition de la neddylation modifie les propriétés de la membrane et la morphologie des cellules VCaP. Cet effet est accompagné par des changements du taux et de la localisation de plusieurs protéines associées à la membrane, y compris l’occludine, la N-cadherine, la paxilline and FAK. Nous en avons conclus que la voie CRL/NEDD8 pourrait être impliquée dans le tri/trafic des protéines membranaires. Cette partie du projet nécessite de plus amples études, étant donné que la compréhension des mécanismes sous-jacents est importante et peut mettre à jour un nouveau rôle de la voie CRL/NEDD8 dans la régulation des fonctions cellulaires.Conclusion générales :1. Nous avons caractérisé l’implication de tous les composants E1-E2 d’UPS dans la régulation de la viabilité des cellules cancéreuses de prostate (avec cinq différentes lignées cellulaires).2. Nos travaux ont mise en évidence de nouvelles cibles thérapeutiques potentielles pour le traitement du cancer, telles qu’UBE2U et CAND1.3. Nous avons démontré le rôle de la voie CRL/NEDD8 dans la régulation de la plasticité et de la morphologie des cellules cancéreuses. / In this work we describe a systematic approach for screening of ubiquitin-proteasome system (UPS) based on cascade organization. We have evaluated the effect of RNAi knockdown of individual UPS components on viability of PCa cells with major focus on TMPRSS:ERG-positive cell line, VCaP, as a model of prevalent phenotype of prostate cancer. Seven genes have been identified to be particularly important for the functioning of PCa cells. Among them, UBE2U was the strongest hit. This thesis provides the first evidence for UBE2U involvement in prostate carcinogenesis and describes initial characterization of UBE2U as a potential drug-target.The prevalence of the components of CRL/NEDD8 pathway in the hits (four out of seven) suggested the importance of neddylation for PCa biology. Two of these hits, CUL2 and RBX1, being specific to TMPRSS2:ERG-positive cells, are potentially ERG-dependent. We have also revealed the crucial role of CRL-exchange factor CAND1, in particular, when the neddylation is compromised. Knockdown of CAND1 induces apoptosis in VCaP cells that is further potentiated by neddylation-specific inhibitor MLN4924. CAND1 is, therefore, a novel potential drug target. Furthermore, we have demonstrated that the inhibition of CRL/NEDD8 pathway in prostate cancer cells has a complex outcome that strongly depends on cellular context. MLN4924 inhibitor induced apoptosis in all tested cell lines, though TMPRSS2:ERG positive cells were significantly more resistant. We have demonstrated that the increased resistance of VCaP cells reflects the plasticity of cancer cells ensured by sophisticated interaction network ERG:NF-kB:c-Myc:Wnt/β-cat:AR. We found that partial inhibition of neddylation triggered transcriptional reprogramming of VCaP cells leading to cell quiescence and inhibition of proliferation-dependent apoptosis. This was a result of re-activation of AR program and induction of differentiation-like state. We conclude that CRL/NEDD8 pathway regulates cancer transcriptional network that underlies cancer cells plasticity. This knowledge would help to find better treatments for TMPRSS2:ERG-positive cancers.Finally, we observed that neddylation inhibition changed membrane properties and morphology of VCaP cells. This was accompanied by dose-dependent changes in the level and the localization of several membrane-associated proteins, including occludin, N-cadherin, paxillin and FAK. We thus conclude that CRL/NEDD8 pathway might be involved in sorting/trafficking of membrane proteins. This part of the work requires further investigation, as understanding of the underlying mechanisms is of general importance and may uncover a new role of CRL/NEDD8 pathway in regulation of cellular functions.General conclusions:1. We have obtained a comprehensive dataset on the involvement of all human E1-E2 UPS components in the regulation of viability of PCa cells, represented by five different cell lines.2. Our work has revealed new potential drug targets for PCa treatment: UBE2U and CAND1.3. We have demonstrated the role of CRL/NEDD8 pathway in the regulation of cancer cell plasticity and morphology.

Orientador: Elizeu Antonio Rossi / Banca: Alice Yoshiko Tanaka / Banca: Iracilda Zeppone Carlos / Resumo: Tendo em vista verificar algumas características essenciais aos microrganismos para serem considerados como probióticos, o objetivo deste trabalho foi avaliar *in vitro* de características probióticas do *Enterococcus faecium *CRL183 e do* Lactobacillus helveticus *ssp* jugurti* 416. Primeiramente foi testada a capacidade destes microrganismos em resistir aos pH 1.5, 2.0, 3,0 e 4,0 em meio de cultivo específico para cada espécie. Em seguida foi realizado o teste de sobrevivência ao trânsito gastrintestinal, onde, foram simuladas as condições do estômago e do intestino delgado, determinando-se a viabilidade dessas bactérias frente à pepsina em pH 2.0 e a pancreatina em pH 8.0. A resistência aos sais biliares foi avaliada inoculando o *Enterococcus faecium *CRL183 e do* Lactobacillus helveticus * ssp* jugurti* 416 em meio de cultivo suplementado com 0,1; 0,2; 0,3 e 0,5% de Oxgall. Já para teste de produção da hidrolase de sais biliares, foi vericada a ocorrência da mudança de cor das colônias ou precipitação dos sais biliares taurodeoxicolico e glicodeoxicolico (TDCA e GDCA). No estudo de auto-agregação e coagregação essas cepas foram colocadas separadamente (auto-agregação) e associados (coagregação) e verificada a densidade óptica (*DO*560). Foi testada a produção de substâncias antagônicas pelos dois microrganismos estudado frente a *Escherichia coli* 0157: H7, *Listeria monocytogenes* V2 e *Salmonella Enteritidis *por meio do teste *spot-on-the lawn*.* *Os resultados demonstraram que os microrganismos resistiram a todos os pH. Essas bactérias mostraram também ser tolerantes ao teste de sobrevivência ao trânsito gastrintestinal e também no teste de sobrevivência a sais biliares, porém neste teste, o *Enterococcus faecium *CRL 183 obteve um tempo de retardo menor em relação ao *Lactobacillus helveticus ssp. jugurti* 416. Já os resultados obtidos...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The purpose of this work was to assess in vitro the probiotic characteristics of Enterococcus faecium CRL183 and Lactobacillus helveticus ssp jugurti 416, having as an aim to verify some characteristics which are essential to the microorganisms, in order to be considered as probiotic. Firstly, the capacity of these microorganisms to resist to pH 1.5, 2.0, 3.0and 4.0 was tested in a specific culture medium for each species. Then the survival test to the gastrointestinal passage was done, and the conditions of the stomach and small intestine were simulated, so determining the viability of these bacteria comparing with pepsin at pH 2.0 and pancreatin at pH 8.0. The resistance to bile salts was assessed by inoculating Enterococcus faecium CRL183 and Lactobacillus helveticus ssp jugurti 416 in a culture medium supplemented with 0,1; 0,2; 0,3 and 0,5 % of Oxgall. However, for the production test of the hydrolase of bile salts, it was verified the occurrence of change in color of the colonies, or the precipitation of the bile salts taurodeoxycholic and glycodeoxycholic (TDCA and GDCA). In the auto-aggregation and co-aggregation studies, these strains were placed separately (auto-aggregation) and associated (co-aggregation), and then the optical density was verified (DO560), for completion and adhesion intestinal epithelium was tested Enterococcus faecium CRL183 and Lactobacillus helveticus ssp jugurti 416 with the Escherishia coli 0157: H7. The production of antagonist substances by both studied microorganisms was tested in comparison with Escherishia coli 0157: H7, Listeria monocytogenes V2 and Salmonella enteriotidis, by using the spot-on-the-lawn test. The results demonstrated that the microorganisms resisted to all pH. These bacteria also showed to be tolerant to the survival test to the gastrointestinal passage and also to the survival test to bile salts, though in this test,...(Complete abstract click electronic access below) / Mestre

La faculté d’adaptation aux conditions environnementales des bactéries provient de lacomplexité de leur réseau de régulation génique, impliquant de nombreux régulateursspécifiques et la machinerie d’expression génique. Nous avons montré que le gène crl, codantun régulateur global d’Escherichia coli, est exprimé de façon transitoire lors de laphase exponentielle. Notre étude a permis d’identifier deux régulateurs responsables dece profil parmi une centaine testés. Ainsi, le complexe CRP-AMPc, réprime de façon indirectel’expression de crl, tandis que la nucléoprotéine Fis se fixe sur le promoteur de crlet active sa transcription directement. Le profil d’expression de crl étant similaire à celuide nombreux régulateurs globaux, nous nous sommes intéressés au rôle de la machinerieglobale d’expression des gènes et à son impact sur la croissance. Dans ce but, nous avonsconstruit un système nous permettant de contrôler la croissance d’E. coli en modulantl’expression des sous-unités _ et _’ de l’ARN polymérase et donc le niveau de transcriptiondans la cellule. Lorsque l’ARN polymérase est en faible concentration, le taux decroissance devient quasiment nul et les cellules filamentent. Ce contrôle de la croissanceest dose dépendant et a été mis en évidence autant à l’échelle de la population qu’à cellede la cellule unique. Nous avons enfin étudié par RNA-seq l’impact du niveau d’ARNpolymérase sur la transcription de l’ensemble du génome de cette souche. Cette étudemontre que toutes les classes fonctionnelles de gènes sont affectées par notre système, àl’exception des gènes qui codent les protéines ribosomales. / Bacteria can adapt to many different environmental conditions. This capacity of adaptationis conferred to the organism by a complex regulatory network, composed of specificregulators and the global gene expression machinery. We have studied the expression dynamicsof Crl, a global regulator of Escherichia coli, and observed a peak of transcriptionduring the exponential phase of growth. In order to identify potential regulators of crlexpression, we have measured the expression profile of crl in about one hundred differentmutant strains. This screen has revealed that CRP-cAMP represses indirectly the transcriptionof crl and the nucleoprotein Fis activates transcription of the crl promoter bybinding to the crl promoter region. We noted that the expression of most global regulatorsof E. coli have an expression profile similar to the one of Crl. We have thereforestudied the relationship between global gene expression machinery and cellular growth.We constructed a bacterium where the transcription of the two large subunits of RNApolymerase, _ et _’, is under external control. A small concentration of RNA polymeraseleads to a small growth rate of this engineered bacterium and the cells start to filament,whereas a high concentration of RNA polymerase produces phenotypically wild-type cells.We have characterized the control of growth rate by our system at the population level andin single cells. An analysis of the global transcription pattern of this strain by RNA-seqshows that the transcription of genes in all functional classes, with the possible exceptionof genes coding for ribosomal proteins, are almost equally affected by the modificationsof the intracellular concentration of RNA polymerase.

Un problème fondamental pendant l'évaluation in vitro de la toxicité de composés organiques volatils (COVs) est le manque de connaissance de l'évolution de la concentration des COVs à laquelle les systèmes vivants sont exposés au cours des études expérimentales. Ce travail présente un nouveau dispositif expérimental conçu pour étudier l'exposition des systèmes vivants aux COVs. Le dispositif est formé de deux compartiments séparés par une membrane hydrophobe poreuse et permet des durées relativement longues de manipulations sans restreindre la respiration cellulaire. Une modélisation théorique qui couple la conservation de masse et du moment entre les différentes phases et la respiration des cellules hybridomes (ATCC CRL-1606) au sein du dispositif a été développée. Le modèle permet de prédire l'évolution de la concentration des COVs, de l'oxygène et du dioxyde de carbone dans le dispositif. Les résultats simulés pour le transfert des COVs ont revélé une bonne concordance avec les résultats expérimentaux et ont montré que le type de membrane et son diamètre, le coefficient de partage des COVs et la hauteur de la phase liquide ont une influence significative sur l'évolution de la concentration de ceux-ci dans la phase liquide. Néanmoins la disponibilité de l'oxygène au niveau des cellules dépend principalement de la densité cellulaire initiale, de la vitesse spécifique de consommation de ce gaz et de la hauteur du liquide alors que les paramètres liés à la membrane ont une influence sur le contrôle du pH. / A major problem during in vitro evaluation of the toxicity of volatile organic compounds (VOC) is the lack of knowledge of the evolution of the concentration of such compounds during the course of experimental studies with living systems. This work presents the design of a novel experimental device for the study of cell culture exposure to VOCs. The device is made of two compartments separated by a porous hydrophobic membrane and allows relatively long durations of handling without restricting cellular breathing. A theoretical modeling which couples mass and moment conservation between the different phases inside the device with the breathing kinetics of hybridoma cells (ATCC CRL-1606) was developed. The model allows predicting the evolution of the concentration of the VOCs, the oxygen and the carbon dioxide inside the device. The simulations of the mass transfer of the VOCs simulated presented a good agreement with experiments and showed that the type of membrane and its diameter, the VOCs partition coefficient and the height of the liquid phase have a significant influence on the evolution of their concentration in the liquid phase. Nevertheless, the availability of oxygen for the cells depends mainly on the initial cellular density, the specific kinetics of consumption of this gas and on the height of the liquid phase, whereas the parameters related to membrane have an influence on the control of the pH.

In this dissertation, the ion mobility technique is used to determine the structures of small acetylene cluster ions, (C2H2)1-3+, mass-selected from the largest ever reported ionized acetylene clusters. The technique is also used to characterize the reaction of acetylene clusters with water in an effort to elucidate thermochemistry and kinetics of some interesting ion-molecule reactions suspected to occur in interstellar clouds and other interplanetary bodies.A combination of ion mobility measurements, collision induced dissociation (CID), and theoretical calculations are used to provide the most conclusive evidence for the frequently hypothesized trimerization of ionized acetylene to form the benzene ion. The results also provide evidence for the isomerization of the acetylene dimer ion, (C2H2)2+, to form the cyclobutadiene and vinylacetylene ions.Investigation of the reactions of acetylene radical ions (C2H2∙+) with water reveals competing kinetics for two primary, C2H4O∙+ and C2H3O+, and a secondary, H+(H2O)n, product with an overall reaction rate coefficient of 2.0 × 10-11 cm3s-1. By comparing experimentally observed reactions to theoretically (G3MP2) predicted thermochemistry, the C2H4O∙+ ion is suggested to be the ethenolium ion (vinyl alcohol ion, CH2CHOH∙+) and the C2H3O+ ion is suggested as either the 1-hydroxy-ethenylium (CH2COH+) or cyclic 2H-oxirenium (c-CH2CHO+) ion. Investigation of the temperature dependence of the equilibrium constant for the association reaction (C2H2)3 + + (H2O)n-1 ↔ (C2H2)3∙+(H2O)n using the van't Hoff plot revealed binding energies and reaction entropies identical to those recently published for the benzene+/water system thus providing even more evidence for the formation of benzene ions from ionized acetylene clusters.We also provide a density functional (UB3LYP/Wachters+ f) investigation of Fe+, Co+, and Ni+(C2H2)n clusters (where n = 1-3) to supplement mass spectrometric analysis of the acetylene-solvated cations. For the Co+(C2H2)n clusters, the mass spectrum revealed an intriguing behavior of oscillating magic numbers which we suspect to be the consequence of a Co+-mediated polymerization reaction to form covalent Co+CnHn complexes. The UB3LYP/Wachters+f predicted barrier and exothermicity for the initial step of the proposed trimerization reaction are 25.4 and 101.4 kcal/mol respectively. Our results suggest the efficiency of this reaction is facilitated by cooperative interactions and favorable orientations of acetylenes in the cluster.

Made available in DSpace on 2014-06-11T19:31:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-08Bitstream added on 2014-06-13T19:01:16Z : No. of bitstreams: 1 bedani_r_dr_arafcf.pdf: 613120 bytes, checksum: d031cc68ff60a01cf7493fa3bed1a364 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / O objetivo desse trabalho foi investigar a influência do consumo do “iogurte” de soja fermentado com Enterococcus faecium CRL 183 sobre o perfil e atividade metabólica da microbiota intestinal de animais e humanos, de maneira a evidenciar prováveis mecanismos de ação indireta na diminuição do risco de ocorrência de câncer de cólon. Os ratos foram divididos em 6 grupos (n=10): I – animais que receberam ração à base de caseína; II – animais que receberam ração à base de carne + 3 mL/kg peso corpóreo/dia do produto não fermentado; III – animais que receberam ração à base de carne + 3 mL/kg peso corpóreo/dia de E. faecium ; IV – animais que receberam apenas ração à base de carne; V – animais que receberam ração à base de carne + 3 mL/kg peso corpóreo/dia de “iogurte” de soja esterilizado; VI – animais que receberam ração à base de carne + 3 mL/kg peso corpóreo/dia do “iogurte” de soja. Os animais consumiram os diferentes produtos (“iogurte” de soja, “iogurte” de soja esterilizado, suspensão de E. faecium e produto não fermentado) durante 4 semanas do período experimental. Foram estudadas a capacidade de aderência de Enterococcus faecium CRL 183 ao cólon e de sobrevivência nas fezes de ratos, além das determinações de pH, concentração de amônia e composição da microbiota fecal. A sobrevivência desse microrganismo no trato gastrointestinal foi verificada por técnicas de isolamento e identificação a partir do cólon e das fezes dos animais que receberam este microrganismo na forma de produto fermentado (“iogurte” de soja) e cultivo puro. Para a confirmação da espécie E. faecium foi utilizada a técnica de PCR. A composição da microbiota fecal foi verificada realizando a enumeração dos seguintes grupos de bactérias: aeróbios e anaeróbios totais, Enterococcus sp, Lactobacillus spp, Bifidobacterium spp... / The aim of this study was to investigate the effect of the consumption of soy yoghurt, fermented with Enterococcus faecium CRL 183, on composition and metabolic activity of intestinal microbiota of animals and humans, and to verify potential mechanisms involved in the anti-carcinogenic action. The rats were placed in 6 groups, distinguished by their diets: for 90 days, group I was given a standard casein-based rodent feed and groups II-VI, the beef-based feed. From the 30th day, groups II, III, V and VI also received the following products: II) unfermented soy product; III) pure suspension of E. faecium CRL 183; V) sterilized soy yoghurt; VI) soy yoghurt. The animals received the different products during 4 weeks. The capacity of E. faecium to adhere on colon and to survive gastrointestinal passage was determined by monitoring rat faecal samples and colon by microbiological and molecular analysis. Species confirmation of E. faecium was performed by PCR amplification. The composition of the faecal microbiota was determined by counting culturable bacteria in the following groups: total aerobes and anaerobes, Enterococcus spp. Enterobacteria, Clostridium spp., Bacteroides spp., Lactobacillus spp. and Bifidobacterium spp. Enzymatic activities of b-galactosidase, b-glucosidase and b-glucuronidase were verified in rat faecal samples. Nitroreductase and azoreductase activities were determined in the caecal contents of mice. The culture of E. faecium CRL 183 was tested for antimicrobial substance production by spot-on-the-lawn assay. Forty healthy adult volunteers who were between 40 and 50 years old participated of this study. The subjects consumed 100 mL/day of soy yoghurt (group F) or unfermented product (group P) during 4 weeks. In feces of volunteers enzymatic activities of bgalactosidase, b-glucosidase and b-glucuronidase were verified. The administration of soy yoghurt and pure suspension... (Complete abstract click electronic access below)

Bluberries (Vaccinium sp.) are rich in polyphenols that are responsible for lowering the risk of developing several chronic degenerative diseases. However, the effect of food matrix interactions on the bioaccessibility and bioavailability of polyphenols is not well understood. In this research free and complexed polyphenols found in blueberry extracts were characterized and their antioxidant activity as well as antiproliferative activities against colon cancer cells (HT-29) and normal colon cells (CRL-1790) were evaluated. The blueberry food matrix and different carbohydrate-rich synthetic matrices were characterized and their biological activities assessed alone and in complexed state with polyphenols. The degradation of polyphenols during their transit through the gastrointestinal tract (GIT) was evaluated using an in vitro digestion model. Biological activities of blueberry polyphenols and their parent metabolites produced during colonic fermentation were estimated by in vitro antioxidant assays and cell proliferation analysis using HT-29 and CRL-1790 cell lines. HPLC analysis revealed the presence of 7 phenolic compounds and 13 anthocyanins in all samples. Although the concentration of the polyphenols varied among the samples, free and complexed polyphenols showed significant antioxidant and antiproliferative activities. Polyphenol complexes were analyzed using transmission electron microscopy (TEM) revealing the presence of electron dense complexes ranging from 100 – 200 nm. Pectinase treatment disrupted the structure of the complexes, suggesting the pectin nature of the polyphenol complexes. The antioxidant- and antiproliferative activities of the blueberry food matrix alone was below 10% compared to almost 90% and 70% of free and complexed polyphenols, respectively. Polyphenols and anthocyanins were highly stable during simulated gastric digestion step with approximately 93% and 99% of recovery, respectively. The intestinal digestion process decreased the polyphenol- and anthocyanin- contents by 49% and 15 % respectively. During colonic digestion, the complex polyphenol mixtures were degraded to a limited number of phenolic compounds. Only acetylated anthocyanins were detected in low amounts after the colonic digestion process. After simulated colonic digestion, the isolated catabolites showed lowered antioxidant activity and cell growth inhibition potential. Understanding the interactions that occur among polyphenols and different food matrices may help to produce more stable foods with better bioavailability. / The National Council of Science and Technology of Mexico (CONACYT)

Orientador: Elizeu Antonio Rossi / Banca: Iracilda Zeppone Carlos / Banca: Daniela Cardoso Umbelino Cavallini / Banca: Susana Marta Isay Saad / Banca: Elaine Cristina Pereira de Martinis / Resumo: O objetivo desse trabalho foi investigar a influência do consumo do "iogurte" de soja fermentado com Enterococcus faecium CRL 183 sobre o perfil e atividade metabólica da microbiota intestinal de animais e humanos, de maneira a evidenciar prováveis mecanismos de ação indireta na diminuição do risco de ocorrência de câncer de cólon. Os ratos foram divididos em 6 grupos (n=10): I - animais que receberam ração à base de caseína; II - animais que receberam ração à base de carne + 3 mL/kg peso corpóreo/dia do produto não fermentado; III - animais que receberam ração à base de carne + 3 mL/kg peso corpóreo/dia de E. faecium ; IV - animais que receberam apenas ração à base de carne; V - animais que receberam ração à base de carne + 3 mL/kg peso corpóreo/dia de "iogurte" de soja esterilizado; VI - animais que receberam ração à base de carne + 3 mL/kg peso corpóreo/dia do "iogurte" de soja. Os animais consumiram os diferentes produtos ("iogurte" de soja, "iogurte" de soja esterilizado, suspensão de E. faecium e produto não fermentado) durante 4 semanas do período experimental. Foram estudadas a capacidade de aderência de Enterococcus faecium CRL 183 ao cólon e de sobrevivência nas fezes de ratos, além das determinações de pH, concentração de amônia e composição da microbiota fecal. A sobrevivência desse microrganismo no trato gastrointestinal foi verificada por técnicas de isolamento e identificação a partir do cólon e das fezes dos animais que receberam este microrganismo na forma de produto fermentado ("iogurte" de soja) e cultivo puro. Para a confirmação da espécie E. faecium foi utilizada a técnica de PCR. A composição da microbiota fecal foi verificada realizando a enumeração dos seguintes grupos de bactérias: aeróbios e anaeróbios totais, Enterococcus sp, Lactobacillus spp, Bifidobacterium spp... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to investigate the effect of the consumption of soy yoghurt, fermented with Enterococcus faecium CRL 183, on composition and metabolic activity of intestinal microbiota of animals and humans, and to verify potential mechanisms involved in the anti-carcinogenic action. The rats were placed in 6 groups, distinguished by their diets: for 90 days, group I was given a standard casein-based rodent feed and groups II-VI, the beef-based feed. From the 30th day, groups II, III, V and VI also received the following products: II) unfermented soy product; III) pure suspension of E. faecium CRL 183; V) sterilized soy yoghurt; VI) soy yoghurt. The animals received the different products during 4 weeks. The capacity of E. faecium to adhere on colon and to survive gastrointestinal passage was determined by monitoring rat faecal samples and colon by microbiological and molecular analysis. Species confirmation of E. faecium was performed by PCR amplification. The composition of the faecal microbiota was determined by counting culturable bacteria in the following groups: total aerobes and anaerobes, Enterococcus spp. Enterobacteria, Clostridium spp., Bacteroides spp., Lactobacillus spp. and Bifidobacterium spp. Enzymatic activities of b-galactosidase, b-glucosidase and b-glucuronidase were verified in rat faecal samples. Nitroreductase and azoreductase activities were determined in the caecal contents of mice. The culture of E. faecium CRL 183 was tested for antimicrobial substance production by spot-on-the-lawn assay. Forty healthy adult volunteers who were between 40 and 50 years old participated of this study. The subjects consumed 100 mL/day of soy yoghurt (group F) or unfermented product (group P) during 4 weeks. In feces of volunteers enzymatic activities of bgalactosidase, b-glucosidase and b-glucuronidase were verified. The administration of soy yoghurt and pure suspension... (Complete abstract click electronic access below) / Doutor