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.

Submitted by ANA LUIZA ROCHA FARIA DUQUE null (analuiza.rduque@gmail.com) on 2016-04-24T19:06:34Z No. of bitstreams: 1 Dissertação Ana Luiza Duque.pdf: 1544679 bytes, checksum: decc5d7d0f47e1235b24c75d5577fd3b (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-04-26T20:13:00Z (GMT) No. of bitstreams: 1 duque_alrf_me_arafcf.pdf: 1544679 bytes, checksum: decc5d7d0f47e1235b24c75d5577fd3b (MD5) / Made available in DSpace on 2016-04-26T20:13:00Z (GMT). No. of bitstreams: 1 duque_alrf_me_arafcf.pdf: 1544679 bytes, checksum: decc5d7d0f47e1235b24c75d5577fd3b (MD5) Previous issue date: 2016-03-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A microbiota intestinal apresenta impacto direto na saúde do hospedeiro sendo fortemente influenciada pela dieta. O consumo de suco de laranja vem sendo associado à redução do risco de desenvolvimento de doenças crônicas, principalmente devido à presença de compostos bioativos. Os compostos bioativos presentes no suco de laranja, especialmente os polifenóis, também podem estar relacionados com a composição e o metabolismo da microbiota intestinal. O objetivo desse trabalho foi avaliar a influência do suco de laranja fresco e pasteurizado sobre a microbiota intestinal usando o Simulador do Ecossistema Microbiano Humano (SEMH®). O SEMH® foi utilizado para investigar a fermentação do suco de laranja ao longo do cólon e para avaliar as alterações na composição e no metabolismo microbiano. A atividade antioxidante dos sucos e das amostras dos compartimentos do SEMH® também foi avaliada. Foi observado no tratamento com suco de laranja fresco aumento (p≤0,05) das populações de Lactobacillus spp., Enterococcus spp., Bifidobacterium spp. e Clostridium spp. e diminuição (p≤0,05) de enterobactérias, enquanto no tratamento com suco de laranja pasteurizado houve aumento (p≤0,05) da população de Lactobacillus spp. e diminuição (p≤0,05) de enterobactérias. A análise de PCR-DGGE mostrou redução dos valores de riqueza da população de bactérias totais para ambos os sucos. Em relação ao metabolismo microbiano, foi observado aumento (p≤0,05) da produção de ácidos graxos de cadeia curta (AGCC) e diminuição (p≤0,05) do conteúdo de íons amônio no tratamento com os sucos de laranja fresco e pasteurizado. A atividade antioxidante das amostras dos compartimentos do SEMH® no tratamento com os sucos de laranja foi elevada, com ligeira redução em comparação àquela do suco fresco e do suco pasteurizado. A Análise de Componentes Principais (ACP) permitiu diferenciar o tratamento com os sucos dos períodos controle e washout, mostrando que os sucos de laranja fresco e pasteurizado apresentaram impacto sobre a microbiota intestinal. Os sucos mostraram efeito prebiótico e seletivo sobre a microbiota intestinal com aumento de AGCC e bactérias comensais e diminuição de íons amônio, embora com redução dos valores de riqueza da população de bactérias totais. / The gut microbiota has a direct impact on host's health being strongly influenced by diet. Orange juice consumption has been associated with a reduced risk of chronic diseases, largely because of the presence of bioactive compounds. The bioactive compounds present in orange juice, particularly polyphenols, may also be associated with the composition and metabolism of gut microbiota. The aim of this work was to evaluate the influence of fresh orange juice and pasteurized orange juice on gut microbiota using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). SHIME® was used to investigate orange juice fermentation throughout the colon and to assess changes in microbial composition and microbial metabolism. Antioxidant activity of the SHIME® vessels and juice was also evaluated. An increase (p≤0.05) in Lactobacillus spp., Enterococcus spp., Bifidobacterium spp. and Clostridium spp. population was observed in fresh orange juice treatment, as well as a reduction (p≤0.05) in enterobacteria. Regarding pasteurized orange juice treatment, an increase (p≤0.05) in Lactobacillus spp. population and a decrease (p≤0.05) in enterobacteria was observed. The PCR-DGGE analysis showed a reduction in total bacteria population richness values on both juices. According to microbial metabolism, an increasing (p≤0.05) of short-chain fatty acids (SCFA) production and decreasing (p≤0.05) of ammonium was observed for two juices treatments evaluated. The antioxidant activity of the samples from the SHIME® vessels in the orange juice treatments was high, with a slight reduction compared to that of fresh juice and pasteurized juice. Both fresh and pasteurized orange juice influenced on gut microbiota according to Principal Component Analysis (PCA), which enabled to differentiate the orange juice treatments from control and washout periods. Both juices showed a prebiotic and selective effect on gut microbiota which is in agreement with increases in both SCFAs and commensal bacteria, as well as with decreases in ammonium levels, though total bacteria richness values were reduced.

The study of the gut microbiota of fish began in the 1930’s and since that time a considerable amount of information has been collated on its composition and diversity. These studies have revealed that the microbial communities of the fish gastrointestinal tract are generally difficult to culture on bacteriological media and mainly consist of bacteria, archaea, viruses, yeasts and protists. The bacteria appear to be the most abundant of these microbial groups and their activity may have major implications for host health, development, immunity and nutrition. Therefore, much of the most recent published research has focused on developing improved methods of identifying the extent of the bacterial diversity within the fish gut and unravelling the potential influence of these microorganisms on the health of farmed fish species. However, whilst such studies have improved our knowledge of the dominant bacterial groups present in the rainbow trout gastrointestinal tract, the limited resolution capacity of many of the methods used has meant that our understanding of their baseline composition in healthy fish remains poorly understood. In this study, the bacterial communities that inhabit the intestine, now commonly referred to as the ‘microbiome’, of farmed Rainbow trout (Oncorhynchus mykiss) were characterized using a culture independent high-throughput molecular sequencing method. The microbiome of the intestinal lumen and mucosa was investigated to ascertain the true extent of the bacterial diversity present in this fish species prior to further experiments. It was found that the diversity of the intestinal microbiome was greater than previous studies had reported with a total of 90 and 159 bacterial genera being identified in both the lumen and mucosal regions respectively. The dominant bacterial phyla identified in both of the regions investigated were Proteobacteria, Firmicutes, Fusobacteria, Bacteroidetes and Actinobacteria. Furthermore, the data collected suggested that the intestinal microbiome may be similar in structure between individual fish, and illustrate the utility of next generation molecular methods in the investigation of the fish gut microbiome. A study was conducted to examine the effect of diet on the composition of the intestinal microbiome of rainbow trout. Two diets, one control and one treatment, were prepared which were identical apart from that the treatment diet contained a microalgal component at 5% of the total formulation. These diets were fed to rainbow trout for a total of 15 weeks. At the end of the trial period a total of 12 fish, three from each of four tanks, were sacrificed from each of the control and treatment groups and their intestinal tissue was sampled in order to compare the composition of the microbiome of both groups. The results revealed that both groups of fish shared similar microbiome compositions, with the Tenericutes being by far the most dominant phylum observed. The structure of the intestinal microbiome was not significantly different between both populations of trout tested. An increased level of bacterial diversity was noted in the treatment fish, however, this was not found to be statistically significant. A limited number of bacterial taxa were discriminatory between diets and were significantly elevated in the treatment group. These taxa were predominantly lactic acid bacteria of the genera Streptococcus, Leuconstoc, Lactobacillus, Lactococcus and Weissella. The results of this study suggested that the minor difference in the diets fed resulted in a correspondingly minor alteration in the intestinal microbiome of the tested rainbow trout. This may indicate that diet composition can modify the composition of the intestinal microbiome of these fish. A further study was conducted to investigate the structure of the intestinal microbiome from groups of fish reared in both freshwater cages and aquarium systems, in order to assess whether or not fish raised in different environments share similar microbiomes. This study also employed a novel computational tool, PICRUSt, to analyse the predicted functional capacity of the microbial communities of individual fish sampled from both environments. The data collected suggested that the structure of the intestinal microbiome was similar regardless of where the fish were raised, with the Tenericutes, Firmicutes, Proteobacteria, Spirochaetae and Bacteroidetes representing the dominant bacterial phyla recorded in the rainbow trout intestine. This suggests that the host may regulate the formation of the intestinal microbiome. A significant difference was however noted in community membership between the fish populations tested, which may point to an environmental influence on the intestinal microbiome. These data suggest that both deterministic host factors and stochastic environmental influences play important roles in shaping the composition of the bacterial communities in the intestine of these fish. The PICRUSt analysis revealed that gene pathways relating to metabolism, transport and cellular processes were enhanced in all of the fish studied, which may signal an involvement of these communities in the digestive processes of rainbow trout. In conclusion, this study used high-throughput sequencing methods in order to improve our understanding of the intestinal microbiome of farmed rainbow trout, and the effect of dietary and environmental factors on its composition. This research has generated scientific information relating to baseline bacterial community compositions in healthy fish, which may be used in future experiments including screening these baselines against the effects of novel aquafeed formulations, environmental perturbations or pathogenic challenges.

Förekomsten av övervikt och fetma har ökat kraftigt de senaste åren över hela världen och i fetmans fotspår ökar även fetmarelaterade sjukdomar. Utvecklingen anses bero på en kombination av faktorer som större tillgång på energirik kost, miljömässiga, livsstilsrelaterade, genetiska och patologiska faktorer. Ny forskning har gett en ökad kuskap om tarmflorans betydelse för hälsa och studier på både obesa människor och djur visar att deras sammansättning i tarmfloran skiljer sig jämfört med normalviktiga individer. Detta har lett till ett ökat intresse hos forskare att titta närmare på kostens relation tilltarmfloran och dess sammansättning för att få klarhet i dess koppling till fetma och kunna använda denna kunskap för att förebygga och utveckla behandlingsmetoder mot fetma. Syftet med denna litteraturstudie var att analysera vetenskapliga artiklar och titta närmare på relationen mellan tarmfloran, kost och fetma. Resultaten visade att tarmflorans sammansättning är olika hos normalviktiga och obesa individer och att sammansättningen påverkar fermentationen av ej nedbrytningsbara kolhydrater i kolon. Studierna visade också att det finns en koppling mellan tarmfloran och inflammation som i sin tur också är en faktor relaterad till fetma. Ytterligare studier krävs för att besvara frågan ifall sammansättningen av tarmfloran är en orsakande faktor till utveckling av fetma, eller ett resultat av sjukdomen, samt vilka bakterier och grupper i den som genom bland annat kost går att påverka på ett positivt sätt mot fetma. / Obesity has increased dramatically during the past decades over the whole world, and has resulted in an increase of obesity-related diseases. The potential contributing factors to obesity are a combination of increase in the availability of energy-rich foods, environmental, lifestyle-related, genetic and pathological factors. New research has led to more knowledge about the gut microbiota and its role in health and studies show a difference in the microbial communities of lean vs. obese humans and animals. These findings have created an interest in research to understand gut microbiota composition and its relation to obesity so that the knowledge can be used in the prevention and treatment of obesity. The aim of this project was to analyse scentific articles and investigate the relation between the gut microbiota, diet and obesity. The studies showed differences in gut microbiota composition between lean vs. obese individuals, and that the composition affects the microbiotas ability to ferment non-digestible carbonhydrates in the colon. The studies also showed that the gut microbiota is related to inflammation, and inflammation is another factor that´s also related to obesity. There is a need for further studies to answer the question if the composition of the gut microbiota is the cause or the consequence of obesity, and which bacteria that for example through dietary modulation, can have a positive effect on obesity.

Background: Age is an important risk factor for rheumatoid arthritis (RA), which often develops in middle age. However, how age-associated changes in immunity impact RA is poorly understood. Gut microbiota are known to be involved in the pathogenesis of RA, but the effects of microbiota in older subjects remain mostly unknown. Methods: We used segmented filamentous bacteria (SFB), a gut commensal species with immunomodulatory effects, and K/BxN mice, a T cell receptor (TCR) transgenic model, to study the effect of age and microbiota on autoimmune arthritis. Comparing young and middle-aged K/BxN T cells of the same TCR specificity allows us to study T cells with an age focus eliminating a key variable: TCR repertoire alteration with age. In addition to joints, we also studied pathological changes in the lung, an important extra-articular RA manifestation. We used flow cytometry to evaluate T follicular helper (Tfh) and T helper 17 (Th17) cells, as they both contribute to autoantibody production, a key disease index in both RA and K/BxN arthritis. Results: Middle-aged K/BxN mice had aggravated arthritis and pathological changes in the lung compared to young mice. Middle-aged mice displayed a strong accumulation of Tfh but not Th17 cells, and had defective Th17 differentiation and low expression of interleukin-23, a critical cytokine for Th17 maintenance. Although a soaring Tfh cell population accompanied by robust germinal center B cell responses were found in middle-aged mice, there was decreased cycling of Tfh cells, and SFB only induced the non-Tfh cells to upregulate Bcl-6, the Tfh master transcription factor, in the young but not the middle-aged group. Finally, the accumulated Tfh cells in middle-aged mice had an effector phenotype (CD62LloCD44hi). Conclusion: Age-dependent Tfh cell accumulation may play a crucial role in the increased autoimmune disease phenotype in middle-age. SFB, a potent stimulus for inducing Tfh differentiation, fails to promote Tfh differentiation in middle-aged K/BxN mice, suggesting that most of the middle-aged Tfh cells with an effector phenotype are Tfh effector memory cells induced at an earlier age. Our results also indicate that exposure to immunomodulatory commensals may allow the young host to develop an overactive immune system reminiscent of that found in the middle-aged host.

Doctor of Philosophy / Division of Biology / Brett K. Sandercock / Gastrointestinal microbiota play a vital role in maintaining organismal health, through facilitating nutrient uptake, detoxification and interactions with the immune system. Shorebirds vary widely in life-history characteristics, such as habitat, migration and breeding system, but the dynamics of their gut microbial communities are unknown. In my dissertation, I investigated composition and dynamics of gut microbiota in migratory shorebirds from embryos to 10 day old chicks, and determined environment and host-related factors affecting gut microbial communities of adults. First, I tested whether precocial chicks from three species of arctic-breeding shorebirds acquire gut microbiota before or after hatching using next-generation sequencing. In addition, I documented the dynamics of gut microbial establishment. I showed that gut microbiota were absent in shorebird embryos before hatching, but that stable gut communities established within the first three days after hatching. In addition, gut microbiota of young shorebird chicks were more similar to the environmental microbiome than later in life, suggesting that the environment is a likely source for microbial recruitment. After reaching adulthood, shorebirds migrate long distances, potentially exposing them to a wide range of microorganisms. Host phylogeny and environmental factors have both been identified as drivers of gut microbiota composition in birds in previous studies. The second part of my project aimed to compare the relative importance of host and environmental factors that underlie variation in gut microbiota composition in eight species of migratory shorebirds sampled across the North American Arctic. I found that sampling site was the main driver of variation in gut microbiota of Arctic-breeding shorebirds, and that site-related variation in gut microbiota of shorebirds was a result of differences in core bacterial taxa that occurred in more than half of the analyzed samples. A relatively large influence of local environment on gut microbiota composition of chicks and adults lead to the question: how does site affect pathogen prevalence in shorebirds? Migratory behavior has been hypothesized to have evolved as a response to variation in climatic conditions and food availability, to avoid predation, and to reduce risk of exposure to pathogens. The migratory escape hypothesis predicts avoidance of high disease prevalence areas through migration, and has been proposed as one of the main reasons that many bird species migrate to the Arctic for breeding. To test the migratory escape hypothesis in shorebirds, I screened for prevalence of seven known avian pathogens in shorebirds at different stages of migration. I did not detect the majority of pathogens we tested for, with the exception of Campylobacter jejuni and C. coli. Prevalence of C. jejuni in shorebirds was linked to sampling sites but not shorebird species. My dissertation is the first comprehensive study to broadly characterize the gut microbiota in shorebirds. Overall, local environment emerged as an important factor in shaping microbiota composition in Arctic-breeding shorebirds throughout my dissertation research. The role of local environment in shaping gut microbiota invites future investigations of the interactions among shorebirds and the microorganisms present in their environment, as well as the functions gut microbiota perform within their shorebird hosts.

En faisant le parallèle avec les selles modernes, les coprolithes peuvent être appropriés à l'étude des habitudes alimentaires, de la flore intestinale et des maladies, des animaux et des hommes ayant vécu il y a des siècles. Dans le travail de thèse ici présenté, un coprolithe datant des 14-15ième siècles, provenant de Namur en Belgique, a été étudié. Dans un premier temps l'ensemble de la communauté microbienne associée au coprolithe été characteriser. Les résultats ont montré qu'une partie du microbiote est similaire à l'environnement et l’autre la flore intestinale, des parasites intestinaux et des pathogènes systémiques ont été aussi trouvés. Un second projet a visé à la purification de particules virales à partir du coprolithe et leur analyse par microscopie électronique et métagénomique virale. Des particules virales sphériques, ainsi que des bactériophages, ont ainsi été observés. Les virus associés au coprolithe correspondent à des virus d'eucaryotes, de procaryotes et d'archaea. La communauté virale était dominée par des bactériophages détectés dans le sol et les selles. Parmi les fonctions métaboliques détectées, une correspond d'ailleurs à des résistances aux antibiotiques. Dans un troisième projet, des cultures et des identifications moléculaires ont été réalisées sur des kystes d'amibes observés dans le coprolithe. Les amibes isolées appartiennent au genre Acanthamoeba et pourraient avoir été conservées sous forme de kystes pendant des siècles dans le coprolithe. Les co-cultures d'amibes ont mené à l'isolement d'une nouvelle bactérie bi-flagellée résistante aux amibes, proche des Rickettsiales. / By drawing parallels to modern stools, coprolites can be suitable specimen to study diet habits, gut microbiota and diseases of animals and humans that have lived centuries ago. During this thesis work, a 14-15th century coprolite specimen from Namur, Belgium was analyzed. At the initiation of this thesis work, it was aimed to characterize the entire microbial communities associated to the coprolite and to identify ancient pathogens. Results indicated that parts of the microbiota are similar to those coming from environment and the gut microbiota inhabitants. Further intestinal parasites and systemic pathogens – still relevant nowadays – were also found. In a second work, viral particles were purified from the Namur coprolite and analyzed by electron microscopic and viral metagenomic. Viral particles associated to spherical virions and bacteriophages were observable. Viruses infecting eukaryotes, bacteria and archaea were associated to the specimen. The viral community was dominated by bacteriophages commonly found in soil and in modern stools and antibiotic resistance was one of the metabolic functions detected. In a third project, culture and molecular identification were performed on amoebal cysts observed within the coprolite. The amoebas isolated belong to the genus Acanthamoeba and might have been conserved in form of cysts inside the Namur coprolite for centuries. Amoeba-co culturing leaded to the isolation and identification of a new bi-flagellar amoeba-resistant bacterium closely related to Rickettsiales.

La consommation d'aliments riches en ellagitannins (ETs) pourrait être associée principalement à la prévention des maladies cardiovasculaires et la régulation des cancers hormono-dépendants. Néanmoins, les ETs ne sont pas biodisponibles en tant que tel et, après avoir été partiellement transformés en acide ellagique (EA) dans le tractus gastro-intestinal (GI) supérieur, ils sont métabolisés dans le côlon par la flore intestinale en urolithines, un groupe de molécules plus biodisponibles et bioactives qui peuvent persister jusqu'à 4 jours à des concentrations relativement élevées dans le plasma et l'urine. La variabilité de l'excrétion des urolithines dans l'urine est importante et à partir d'un échantillon de population de 26 volontaires sains, trois groupes principaux d'individus ont pu être distingués : "faible ou non-excréteur d'urolithin », « Excréteur prédominant d'UA et dérivés» et « Excréteur prédominant d'UB et dérivés»". Ces groupes ont également été observés en considérant la cinétique totale d'excrétion sur une période de 4 jours après ingestion du jus et à des périodes différentes tout au long d'une année. Bien que les variabilités inter-et intra-individuelles soient relativement élevées, les individus conservent leur statut au cours des différentes périodes d'intervention même en modifiant les quantités d'ETs ingérées. L'analyse par UPLC-PDA/ESI-Q-TOF/MS2 a permis d'attribuer hypothétiquement une identité à 15 autres métabolites d'ETs dans l'urine, mais le profilage métabolomique n'a pas permis de discriminer d'autres composés exceptés les dérivés d'UA ou d'UB. La fermentation in-vitro des ETs et EA, par les matières fécales a montré une voie métabolique spécifique qui débouche sur la production d'UA. Néanmoins, les métabolites excrétés in vivo sont beaucoup plus complexes ce qui met en évidence de fortes interactions entre le système excréteur de l'hôte et la composition du microbiote intestinal. La recirculation hépatique suivie par une re-conversion des métabolites de phase II dans le côlon permettrait d'expliquer l'excrétion d'UB chez certains volontaires. L'écologie spécifique de la flore intestinale évaluée par la méthode des empreintes PCR-DGGE a permis d'identifier quelques microorganismes associés à une plus grande capacité de bioconversion des ETs en urolithins / Consumption of dietary ellagitannins (ETs) could be associated mainly with prevention of cardiovascular diseases and regulation of hormone-dependent cancers. Nonetheless, ETs are not bioavailable as such; therefore, after being partially converted into ellagic acid (EA) in the upper gastrointestinal (GI) tract, they undergo sequential bioconversion in the colon by gut microbiota into urolithins, a more bioavailable and bioactive group of molecules that persist up to 4 days at relatively high concentrations in urine. Variability of urolithin excretion in urine is high and three main groups, “no or low urolithin excreters,” “predominantly UA derivatives excreters” and “predominantly UB derivatives excreters,” were observed on a cohort of 26 healthy volunteers. These categories were also unambiguously observed following the total excretion of main ETs' metabolites over a 4 day period after ingesting one shot of juice, and at different periods of time along one year. Although relatively high inter- and intra-individual variabilities were observed, individuals preserved their status during various intervention periods with different amounts of ETs ingested. UPLC-PDA and ESI-Q-TOF/MS1 and MS2 allowed the tentative assignment of an identity to 15 other ETs metabolites in urine, but this profiling did not allow the discrimination of any other compounds aside from UA or UB derivatives. In-vitro fermentation of ETs and EA with fecal stools showed a specific metabolic pathway ending in the production of UA. Nonetheless, metabolites excreted in-vivo are much more complex, highlighting strong interactions between host excretory system and composition of gut microbiota. Hepatic recirculation and additional bioconversion of Phase II metabolites in the colon may explain predominant excretion of UB in some volunteers. Microbiota ecology assessed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE) fingerprint method allowed the association of some microorganism species to higher capacity of bioconversion of dietary ETs into urolithins.Key words: Ellagitannins, blackberry, urolithin, colonic metabolites, ETs degradation patterns, gut microbiota, gastrointestinal tract,

Orientadores: Jaime Amaya-Farfan, Fernanda de Pace / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-27T12:05:55Z (GMT). No. of bitstreams: 1 Roquetto_AlineRissetti_M.pdf: 2148135 bytes, checksum: 6a59bf58e9dbc6285574497bb9558141 (MD5) Previous issue date: 2015 / Resumo: A obesidade é um dos maiores problemas de saúde pública no mundo, sendo associada a diversas doenças metabólicas como inflamação, resistência insulínica, dislipidemia, esteatose hepática, entre outras. Recentemente, tem sido demonstrado que alterações nas proporções dos filos que compõem a microbiota intestinal repercutem negativamente sobre o metabolismo e processos fisiológicos do hospedeiro. A dieta moderna é apontada como um dos fatores capazes de modular as bactérias intestinais e desencadear respostas inflamatórias. Diante deste cenário e tendo conhecimento de que a própolis, resina produzida por abelhas que possui ação anti-inflamatória e antimicrobiana, a presente pesquisa teve como objetivo avaliar o efeito da suplementação da própolis em camundongos tratados com dieta hiperlipídica sobre a microbiota intestinal e biomarcadores inflamatórios. Quarenta camundongos da linhagem C57BL/6 foram divididos em 4 grupos (n=10) aleatoriamente: grupo controle ¿ dieta baseada na AIN-93G; grupo hiperlipídico (HF) ¿ dieta com 37% de gordura; e grupos HFP2 e HFP5 tratados com dieta hiperlipídica, seguida de suplementação com própolis 0,2% nas duas e cinco semanas que antecederam ao sacrifício respectivamente. Foram coletadas amostras de sangue e músculo para determinações bioquímicas e indicadores de inflamação, o conteúdo cecal foi extraído para sequenciamento do DNA da microbiota intestinal. Os resultados não mostraram diferenças no ganho de peso entre os grupos experimentais, mas o tratamento com própolis por 5 semanas foi efetivo em reverter a disbiose causada pela dieta HF, com relação aos filos Firmicutes, e Proteobacteria. Os níveis de lipopolissacarídeos (LPS) no soro, bem como a expressão de toll-like receptor-4 (TLR4) e de citocinas pró-inflamatórias no músculo foram reduzidos pelo tratamento prolongado com própolis. Além disso, esta intervenção melhorou os níveis séricos de glicose e triacilgliceróis. Estes resultados sugerem a possibilidade de que a própolis exerça ação benéfica modificando o microbioma que limita a permeabilização da parede intestinal, regulando a translocação de componentes bacterianos para a corrente sanguínea e, consequentemente, conduzindo a uma menor expressão de citocinas inflamatórias / Abstract: Obesity is a major world-wide public health problem and is associated with metabolic disorders as generalized inflammation, insulin resistance, dyslipidemia, hepatic steatosis, among others. Recently, it has been demonstrated that changes in the proportions of phyla that make up the gut microbiota have a profound effect on the metabolism and physiology of the host. The modern diet has been identified as one of the factors that modulate the intestinal bacteria and trigger inflammatory responses. Considering this state of affairs and knowing that propolis, a resin present in bee honey, has anti-inflammatory and anti-microbial action, the present study was designed to evaluate the effect of propolis supplementation on the intestinal microbiome and inflammatory biomarkers of mice pre-conditioned with a high-fat diet. Forty mice of the C57BL/6 strain were randomly divided into four groups (n = 10): control group ¿ diet based on the AIN 93-G; high-fat group ¿ diet with 37% fat; and two other groups treated with high-fat, HFP2 and HFP5, that were supplemented with 0.2% propolis during two and five weeks preceding sacrifice, respectively. Blood and muscle samples were collected for biochemical analyses and inflammation markers, the cecal contents were extracted for DNA sequencing of the intestinal microbiota¿s genome. The results showed no differences in weight gain among the experimental groups, but treatment with propolis for 5 weeks effectively reverted the dysbiosis caused by the HF diet with respect to the Firmicutes and Proteobacteria phyla. The levels of serum lipopolysaccharide (LPS), and Toll-like receptor-4 (TLR4) expression, and proinflammatory cytokines in muscle were reduced by the longer propolis treatment. In addition, this intervention improved serum glucose and serum triacylglycerol levels. The present results suggest that ingested propolis exerts its beneficial action, first modifying the intestinal microbiota, which limits intestinal wall permeability and controls the translocation of bacterial components into the bloodstream and thus averting inflammatory cytokine overexpression / Mestrado / Nutrição Experimental e de Alimentos / Mestra em Alimentos e Nutrição