Zugl.: München, Universiẗat, Habil.-Schr., 2008.

Thesis (doctoral)--Universität, Siegen, 2005. / Includes bibliographical references (p. 335-358).

Diss. Univ. Zürich, 2008. / Im Buchh.: Zürich etc. : Schulthess. Literaturverz.

Universiẗat, Diss., 2005--Münster.

Η ανθρώπινη εντερική χλωρίδα είναι ένα «ουσιώδες» όργανο, το οποίο συμβάλλει στη θρέψη, την ανοσία, την ανάπτυξη των εντερικών επιθηλιακών κυττάρων του ξενιστή, αλλά και συμμετέχει σε διάφορα μεταβολικά μονοπάτια αυτού. Επηρεάζεται από ποικίλους παράγοντες, όπως η ηλικία, ο γονότυπος του ξενιστή, το περιβάλλον, το στρες, η δίαιτα, καθώς και η πρόσληψη προβιοτικών, πρεβιοτικών, αντιβιοτικών. Η ανθρώπινη εντερική χλωρίδα αποτελείται κυρίως από δύο φύλα βακτηρίων: τα Bacteroidetes και τα Firmicutes. Τα τελευταία χρόνια πολλοί ερευνητές έχουν επικεντρώσει το ενδιαφέρον τους στη μελέτη της σχέσης που έχει η παχυσαρκία με την εντερική χλωρίδα. Τα αποτελέσματα είναι αντικρουόμενα. Συγκεκριμένα, έχει αποδειχθεί ότι σε παχύσαρκα άτομα υπάρχει χαμηλότερο ποσοστό Bacteroidetes και μεγαλύτερο Firmicutes, σε σχέση με άτομα φυσιολογικού βάρους. Στη συγκεκριμένη μελέτη ερευνήθηκε η επιρροή της παχυσαρκίας, της απώλειας βάρους, της διατροφής και της μεσογειακής δίαιτας στη σύσταση της εντερικής χλωρίδας. Η πειραματική πορεία που ακολουθήθηκε είναι η εξής: απομόνωση DNA, ηλεκτροφόρηση, φωτομέτρηση και Real Time – PCR. Η παχυσαρκία και ο αυξημένος Δείκτης Μάζας Σώματος σχετίστηκε, σε στατιστικά σημαντικό βαθμό, με μειωμένα ποσοστά Bacteroides και Bifidobacterium στα κόπρανα. Ακόμα, η απώλεια βάρους σχετίστηκε με μειωμένη ποσότητα Lactobacillus. Η υιοθέτηση της Μεσογειακής Διατροφής δε φάνηκε να σχετίζεται με διαφορές στην εντερική χλωρίδα σε στατιστικά σημαντικό βαθμό. Ενώ, όσον αφορά τη διατροφική πρόσληψη, βρέθηκε η κατανάλωση φρέσκων φρούτων, μοσχαριού και αναψυκτικών να σχετίζεται αρνητικά με την ποσότητα Clostridium. Η κατανάλωση οσπρίων σχετίστηκε θετικά με την ποσότητα Bacteroides, ενώ την ποσότητα των Bifidobacterium φάνηκε να επηρεάζει αρνητικά η κατανάλωση φρέσκων φρούτων και θετικά η κατανάλωση λαχανικών. / Gut flora is an «essential» part of human life that contributes positive to nutrition, to immunity and to the growth of the epithelial gut cells of the host. Moreover, it sprigthfully participates at host’s different metabolic paths. Gut flora is affected by various factors, such as the age, host’s genotype, environment, stress, diet and intake of probiotics, prebiotics or antibiotics. The gut flora of human consists of two main bacterial genders: The Bacteroidetes and the Firmicutes. Last years, many scientists have focused their interest on researches which deal with the correlation between the obesity and the gut flora. But, there are conflicting results on these researches. In particular, it is proved that the humans who suffer from obesity have smaller percentage of Bacteroidetes and bigger percentage of Firmicutes concerning with humans with normal weight. This research tries to correlate the obesity, the weight loss, the nutrition and the Mediterranean diet with the gut flora. The research has the next experimental development: DNA isolation, electrophoresis, photometry and Real Time – PCR. Obesity and increased Body Mass Index were related, in significant level, with reductions in Bacteroides and Bifidobacterium percentage in feces. Additionally, weight loss was related with decreased Lactobacillus percentage in feces. Adherence to Mediterranean Diet was not related with significant changes in gut microbiota. The consuming of fresh fruits, beef and soft drinks was, in significant level, related negatively with Clostridium levels in feces. The consuming of legumes was related positively with Bacteroides levels and, finally, Bifidobacterium levels in feces were affected negatively by fresh fruits consuming and positively by salads consuming.

The focus of this thesis is a comparative study of approaches in discovery of carbohydrate-active enzymes (CAZymes). CAZymes synthesise, bind to, and degrade all the multitude of carbohydrates found in nature. As such, when aiming for sustainable methods to degrade plant biomass for the generation of biofuels, for which there is a strong drive in society, CAZymes are a natural source of environmentally friendly molecular tools. In nature, microorganisms are the principal degraders of carbohydrates. Not only do they degrade plant matter in forests and aquatic habitats, but also break down the majority of carbohydrates ingested by animals. These symbiotic microorganisms, known as the microbiota, reside in animal digestive tracts in immense quantities, where one of the key nutrient sources is complex carbohydrates. Thus, microorganisms are a plentiful source of CAZymes, and strategies in the discovery of new enzymes from bacterial sources have been the basis for the work presented here, combined with biochemical characterisation of several enzymes. Novel enzymatic activities for the glycoside hydrolase family 31 have been described as a result of the initial projects of the thesis. These later evolved into projects studying bacterial multi-gene systems for the partial or complete degradation of the heterogeneous plant polysaccharide xyloglucan. These systems contain, in addition to various hydrolytic CAZymes, necessary binding-, transport-, and regulatory proteins. The results presented here show, in detail, how very complex carbohydrates can efficiently be degraded by bacterial enzymes of industrial relevance. / <p>QC 20130826</p>

Les microorganismes anaérobies représentent la population majoritaire de notre tube digestif et ont un impact remarquable sur notre santé. Leur culture demeure à ce jour longue, fastidieuse et coûteuse et nombreux sont ceux qui restent incultivables. Or la culture est un outil indispensable pour l'étude du microbiote digestif. Ainsi, le laboratoire dans lequel ma thèse s’est déroulée a créé un nouveau concept de culture « Microbial Culturomics » qui a permis d’isoler 193 nouvelles espèces bactériennes anaérobies. Un travail sur l’utilisation des antioxydants pour permettre la culture aérobie des bactéries anaérobies a également été amorcé : une optimisation des techniques de culture prometteuse autour de laquelle mes travaux ont vu le jour. Notre premier projet a consisté à développer un dispositif de culture innovant permettant la culture des archaea méthanogènes en aérobiose et en absence de source externe de dihydrogène. Notre deuxième projet a consisté à élaborer un flacon d’hémoculture unique dans lequel la croissance de toutes les bactéries, aérobies et anaérobies, pouvaient être détectées. Notre troisième projet quant à lui repose sur la comparaison du mode de culture anaérobie et de celui en aérobie avec les antioxydants à travers l’exemple de trois souches bactériennes strictement anaérobies. L’utilisation des antioxydants pour faciliter la culture des microorganismes anaérobies a donc apporter des résultats très prometteurs qui pourrait être utilisés, après validation par des études multicentriques dans les laboratoires de microbiologie clinique et environnementaux. / Anaerobic microorganisms are characterized by their ability to grow and survive in the absence of oxygen. Indeed free oxygen molecules are not used for their metabolism and can be toxic to varying degrees, sometimes leading to cell death. Although it is known that these microorganisms are the predominant in our digestive microbiota and that they have a great impact on our health, their culture remain long, fastidious, costly, and in most cases impossible. Becteria culture is an indispensable tool for isolating strains, performing studies from living models, and identifying new ones. Thus, the laboratory in which my thesis tooks place created a new concept of culture "Microbial Culturomics" which made it possible to isolate 193 new anaerobic bacterial species. A work based on the use of antioxidants to enable the aerobic culture of anaerobic bacteria was also initiated: a promising optimization of the culture techniques from which my work was born. Our first project consisted in developing an innovative culture device allowing the cultivation of methanogenic archaea in aerobic and without an external source of dihydrogen. In our second project, we performed a single culture bottle in which the growth of all bacteria, aerobic and anaerobic, could be detected. Our third project was based on the comparison of anaerobic and aerobic culture with antioxidants through the example of three strictly anaerobic bacterial strains.Therefore the use of antioxidants enable to facilitate anaerobic bacteria cultivation. These results are very encouraging for clinical and environmental microbiology laboratories.

OBJECTIVE: Chapter 1: Investigate the impact that trimethylamine N-oxide (TMAO), dietary contribution of short chain fatty acids (SCFAs), and role of bile acids has on cardiovascular health and disease. Chapter 2: Evaluate the association between intakes of dietary protein from both animal and plant sources on lipid profile changes. METHODS: Chapter 1: Literature review using PubMed and EMBASE to search for published studies for dietary intake or supplementation impact on TMAO or its precursors and their role in the development or prevention of cardiovascular diseases. Chapter 2: Framingham Offspring Study, prospective cohort study using statistical methods to investigate the changes in lipid profiles with dietary animal and plant protein. PUBLISHED STUDIES/RESULTS: Chapter 1: The increased risk of cardiovascular diseases (CVD) correlates with increasing levels of circulating levels of TMAO. The risk of CVD in animal and human studies have shown to be distinct in groups with and without CVD, leading to either beneficial or adverse effects from the consumption of dietary phosphatidylcholine, choline, betaine, carnitine, or intact TMAO. A Western dietary approach has been linked with the development of dyslipidemia whereas, adherence to a Mediterranean diet reduces the risk of major CVD events. The dietary precursors involved in TMA production by the gut microbiota then respectively to TMAO through hepatic enzyme FMO3 provide both beneficial and detrimental effects. Mechanisms of action for TMAO on CVD risk involves changes associated with cholesterol and sterol metabolism leading to foam cell formation, and enhancement of scavenger receptors, CD36 and scavenger receptor-A, on macrophages affects the rate of cholesterol influx and efflux. Choline derived in a dose-dependent manner from eggs improves cardiometabolic biomarkers with no changes in fasting TMAO. Further, choline from eggs also increases the lipoprotein particle size for both HDL-cholesterol and LDL-cholesterol increasing the rate of reverse cholesterol transport (RCT). Betaine concentrations in humans are associated with health outcomes based on an individual’s overall systemic health at baseline. Supplementation with L-carnitine produced favorable effects in lean subjects compared to obese subjects, improved cardiometabolic status in patients with myocardial infarction, and improved lipid profiles among individuals with prevalent coronary heart disease (CAD). Fish consumption increases concentrations of TMAO due to its high levels of intact TMAO though, protective effects for CVD are obtained from fatty fish providing omega-3-fatty acids impacting positive changes in the lipid profiles. Antibiotic therapy suppresses the gut microbiota and eliminates the production of TMA from the dietary precursors that are required. Chapter 2: Men and women both showed a decreasing trend for LDL-cholesterol as the tertiles increased for animal protein intake. Plant protein intake showed a similar decreasing trend for LDL-cholesterol with increasing protein tertiles; however, men had inconsistency among the trend whereas women had a consistent decreasing trend. HDL-cholesterol content increases in males and females with both increasing tertiles for animal and plant protein, though plant protein presented much stronger effects when compared to animal protein. Log-transformed triglycerides were inversely associated with increasing animal protein intake, men revealing greater effects than females. Plant protein intake showed a stronger effect than animal protein intake in an increasing trend in the log of triglycerides over the 6 exams. Overall, total cholesterol content varied at each examination period, animal protein intake tertiles displayed decreased level of total cholesterol, there was a greater effect in men than women. Higher intake of plant protein had a similar trend to animal protein intake showing a decrease in the total cholesterol concentration. Women had a much greater effect in reducing total cholesterol with plant protein when compared to men. CONCLUSION: Chapter 1: Multiple human and animal trials addressed in the association between diet, dietary precursors, gut microbiota composition, and their derived metabolite TMAO on the presence or absence of CVD display contradictory results and identifies areas needing further study. Chapter 2: Regardless of the source of protein, the lipid profiles improved with the intake of either animal or plant protein as the protein intake was increased over the tertiles in each exam. The overall trend with increasing animal or plant protein intake led to decrease in LDL-cholesterol, log transformed triglycerides, and total cholesterol whereas, the HDL-cholesterol concentrations were increased. Men favored animal protein intake to show greater reductions in LDL-cholesterol and total cholesterol, whereas women favored plant protein. The increase in HDL-cholesterol concentration was stronger with the intake of plant protein in men and women. The changes in log transformed triglycerides were similar in men and women.

L'infection par le virus de l'hépatite B (VHB) est un problème de santé publique majeur avec plus de 257 millions de porteurs chroniques dans le monde ayant un risque important de développer une cirrhose et/ou un hépatocarcinome. L'histoire naturelle de l'infection est très différente selon l'âge auquel l'infection est contractée. Alors que chez l'adulte l'infection est spontanément résolutive dans la majorité des cas, la contamination materno-infantile ou en bas âge aboutit le plus souvent à une infection chronique. Le cccDNA est la forme de persistance du génome viral dans les hépatocytes infectés et la base de transcription de tous les ARN viraux. La protéine virale HBx joue un rôle crucial dans le recrutement des facteurs épigénétiques sur le cccDNA et favorise son activité transcriptionnelle. Les traitements actuels à base d'interféron et d'analogues nucléos(t)idiques ne permettent pas l'éradication du cccDNA et leur interruption est presque toujours suivie d'une réactivation de la réplication du virus. De nouvelles molécules thérapeutiques ciblant le cccDNA sont donc nécessaires pour espérer obtenir une cure fonctionnelle chez les patients chroniquement infectés. Il existe des liens étroits entre l'infection par le VHB et le métabolisme des acides biliaires (AB). Ainsi, notre équipe a précédemment montré que le récepteur nucléaire des acides biliaires, le farnesoid X receptor alpha (FXRalpha) se fixe sur deux éléments de réponse présents dans la région Enhancer II - promoteur de Core du génome viral et module son activité transcriptionnelle. De plus, le VHB et les AB entrent en compétition pour le même récepteur d'entrée hépatocytaire NTCP, modifiant la concentration cellulaire des AB avec des conséquences sur la fonction et l'expression de FXRalpha. Enfin, HBx interagit avec FXRalphaet modifie son activité. Au cours de cette thèse nous avons dans un premier temps identifié une régulation réciproque existante entre la réplication du VHB et FXRalpha. Puis nous avons montré in vitro, dans des cellules HepaRG différenciées et des hépatocytes primaires humains, que FXRalphaest un facteur proviral pour le VHB et que les agonistes de FXRalpha inhibent l'expression de l'ensemble des marqueurs viraux de manière dépendante ou indépendante de la protéine virale HBx. Enfin, dans un modèle in vivo de souris C3H/HeN transduites par un vecteur recombinant AAV2/8-VHB, nous avons obtenu l'effet inhibiteur des agonistes de FXRalphamais uniquement chez les souris adultes et pas chez les souris jeunes. Compte tenu de l'évolution de la flore intestinale avec l'âge et de son importance dans le métabolisme des AB, ces résultats suggèrent que le fort taux d'évolution chronique chez les jeunes enfants pourrait être lié à l'immaturité du métabolisme des AB. La mise en évidence d'un lien entre microbiote, métabolisme des AB et infection par le VHB contribuerait grandement à une meilleure compréhension de l'histoire naturelle de cette infection. De plus, l'identification de FXRalphacomme un facteur de l'hôte favorisant l'infection et l'existence de molécules capables de moduler l'activité de FXRalphasuggèrent que FXRalphapourrait constituer une cible thérapeutique intéressante ciblant le cccDNA et permettant d'améliorer le traitement des patients infectés par le VHB / Hepatitis B virus (HBV) infection is a major global health problem with more than 257 million chronic carriers worldwide that remain at significant risk for developing cirrhosis and/or hepatocellular carcinoma. The natural history of infection is very different depending on the age at which the infection is contracted. Whereas in adults most HBV infections spontaneously resolve, in infants and young children they usually result in chronic infection. cccDNA is the molecular form of viral persistence in infected hepatocytes and serves as a transcription template for all viral RNAs. The viral protein HBx plays a crucial role in the recruitment of epigenetic factors to the cccDNA and promotes its transcriptional activity. Currently, interferon and nucleot(s)ide analogues are the first-line agents in the treatment of chronic hepatitis B without allowing eradication of cccDNA and their interruption are almost always followed by a reactivation of the replication of the virus. New therapeutic molecules targeting cccDNA are therefore needed to hope for a functional cure in chronically infected patients. HBV infection and bile acid (BA) metabolism are tightly linked. Therefore, our team has previously shown that the bile acid nuclear receptor, the farnesoid X receptor alpha (FXRalpha) bind to two response elements present in the Enhancer II - Core promoter region of HBV genome and modulate its transcriptional activity. Moreover, HBV and BA compete for the same entry receptor of hepatocytes NTCP and modify BA cell concentration with consequences on the function and expression of FXRalpha. Finally, HBx interacts with FXRalpha and modify its activity. During my PhD. we have first identified a reciprocal regulation between HBV replication and FXRalpha. Second, we have showed in vitro, in HepaRG differentiated cells and in primary human hepatocytes, that FXRalpha is a proviral factor for HBV and that FXRalpha agonists inhibit the expression of all HBV markers in a dependent or independent manner of the viral protein HBx. Finally, in an in vivo model of C3H/HeN mice transduced with a recombinant AAV2/8-HBV vector, we obtained the inhibitory effect of FXRalpha agonists but only in adult and not in young mice. Considering the evolution of the gut flora with age and its importance in the metabolism of BA, these results suggest that the high rate of chronic progression in young children might be related to the immaturity of BA metabolism. The identification of a link between BA metabolism, gut microbiome composition and evolution of HBV infection will represent a big step toward the understanding of HBV natural history. Moreover, the identification of FXRalpha as a proviral factor for HBV and the capacity of FXRalpha ligands to modulate the transcriptional activity of cccDNA suggest that FXR ligands might represent a new class of molecules with the aim to obtain functional cure for HBV infected patients

Introdução: o estresse psicológico aumenta a permeabilidade intestinal em roedores e humanos, potencialmente levando a inflamação de baixo grau e aos sintomas em distúrbios gastrintestinais funcionais. No entanto, o efeito do estresse agudo combinado ao estresse da vida crônica, que mimetiza potencialmente melhor a situação humana, é desconhecido. Além disso, há poucos dados disponíveis sobre os efeitos do estresse em intestino delgado versus cólon. Métodos: ratos Wistar foram alocados em quatro protocolos de estresse: 1/ controles; 2/ estresse agudo (isolamento e movimentos limitados); 3/ Crowding stress:crônico e 4/ estresse agudo + estresse crônico. Amostras de jejuno e cólon foram colhidas para estudar a permeabilidade em câmaras deUssing, a expressão gênica de moléculas de junção firmes e a densidade de mastócitos. Níveis de corticosterona no plasma foram medidos. Principais resultados:corticosterona plasmática foi avaliada nas três condições de estresse, teve níveis mais altos na condição de estresse combinado. Permeabilidade do jejuno foi aumentada em todas as condições de estresse e correlacionada com os níveis de corticosterona. O aumento da expressão das claudinas 1, 5 e 8, daocludina e da ZO-1 foi detectado no estado de estresse agudo no jejuno. Em contraste, a permeabilidade do cólon foi aumentada no protocolo de estresse combinado, e a expressão de moléculas das junção firmes permaneceu inalterada. O aumento da densidade de mastócitos foi observado no cólon nos ratos submetidos aos estresses crônico e combinado. Conclusão e inferências:os estresses agudo, crônico e combinado influenciam diferentemente a permeabilidade intestinal, a expressão de moléculas de junção firmes e a atividade dos mastócitos, no jejuno e no cólon. Estes resultados fornecem uma visão mais aprofundada dos mecanismos de hiperpermeabilidade intestinal relacionadas ao estresse. / Background: Psychological stress increases intestinal permeability in rodents and humans, potentially leading to low-grade inflammation and symptoms in functional gastrointestinal disorders through disturbances in brain-gut axis. However, the effect of acute stress on the background of Crhonic life stress, potentially better approaching the human situation, is unknown. Moreover, only limited information is available on the effects in small intestine versus colon in animal model. Methods: Wistar rats were allocated to 4 stress protocols: 1/ sham; 2/ acute stress (isolation and limited movement); 3/ Crhonic crowding stress and 4/ acute + Crhonic stress (n = 8 per group). Jejunum and colon were harvested to study permeability in Ussing chambers, gene expression of tight junction molecules and mast cell density. Plasma corticosterone levels were measured. Key Results: Plasma corticosterone was elevated in all three stress conditions, with the highest levels in the combined stress condition. Permeability of the jejunum was increased in all stress conditions and correlated with corticosterone levels. Increased expression of claudin 1, 5 and 8, occludin and ZO-1 was detected in the acute stress condition in the jejunum. In contrast, colonic permeability was increased in the acute on Crhonic stress protocol only and the expression of tight junction molecules was unaltered. Increased mast cell density was observed in the Crhonic and acute on Crhonic stress condition in the colon only. Conclusion and Inferences: Acute, Crhonic and combined stress differentially affect intestinal permeability, expression of tight junction molecules and mast cells in the jejunum and the colon. These findings provide further insight in the mechanisms of stress-related intestinal hyperpermeability and barrier.