L’étude de la composition de la flore digestive ainsi que son implication avec la santé et les maladies est devenue un enjeu majeur. Nous avons étudié la flore de malades traités par de très nombreux antibiotiques, à la fois par culturomics et par pyroséquençage. Ce travail a montré une diminution importante du nombre de bactéries différentes colonisant le tube digestif après traitement. Cette diminution était d’autant plus importante que le traitement était prolongé. Les bactéries offrant une protection immunitaire contre certains pathogènes, il est à présent proposé des vaccinations contres des microorganismes invasifs après traitement antibiotique au long cours. Par ailleurs, les techniques de séquençage ont montré pour deux des échantillons un haut taux de colonisation par Akkermansia muciniphila, un microorganisme appartenant au phylum des Verrucomicrobia. Ce résultat a par ailleurs été confirmé en utilisant des techniques d’hybridation de fluorescence in situ (FISH). Les tentatives de culture ce microorganisme se sont révélées infructueuses. Au final ce travail nous aura permis de cultiver 7 nouvelles espèces que nous avons décrites en utilisant la taxonogénomique, une approche incluant des données phénotypiques et le séquençage du génome.Nous avons également étudié la flore de patients infectés par le virus du VIH par métagénomique et avons observé une augmentation considérable des bactéries qui supportent l’oxygène, alors que les bactéries intolérantes étaient très diminuées. Ces changements étaient associés aux marqueurs de progression de la maladie, ouvrant la voie à une supplémentation en antioxydants. / The study of the composition of the intestinal flora as well as its involvement with health and disease has become a major issue.We studied the flora of patients treated by broad-spectrum antibiotics by both culture and pyrosequencing. This work showed a significant decrease in the number of different bacteria colonizing the digestive tract after treatment. This decrease was even more important that treatment was extended. Bacteria interacting with immune protection against some pathogens, it is now proposed vaccinations against invasive microorganisms after prolonged antibiotic treatment. Furthermore, the sequencing techniques have shown for two samples a high-level colonization by Akkermansia muciniphila a microorganism belonging to the phylum Verrucomicrobia. Despite the successive failures of culture attempt of this organism, this finding was also confirmed using fluorescence in situ hybridization technique (FISH). Ultimately this work has enabled us to discover 7 new species that have been described using the taxonomogenomics approach which includes phenotypic data and genome sequencing.We also studied the flora of HIV-infected patients by metagenomics, and observed a significant increase in bacteria that withstand oxygen, while intolerant bacteria were deceased. These changes were associated with markers of disease progression, opening the way for antioxidant supplementation.

L’accident de désaturation (ADD) est un accident de plongée lié à la charge en gaz diluants pendant la plongée, et à la formation de bulles dans l’organisme au cours de la décompression. Il est susceptible d’engendrer des séquelles neurologiques. Au cours de plongées utilisant l’hydrogène comme gaz diluant, la diminution de la charge tissulaire en hydrogène par l’inoculation au niveau de l’intestin de bactéries métabolisant ce gaz réduit le risque d’ADD.L’objectif de ce travail était d’évaluer si inversement : 1) la fermentation intestinale lors de la plongée peut favoriser la survenue d’un ADD, par l’intermédiaire de la production d’hydrogène endogène ; 2) la stimulation chronique de la fermentation avant plongée majore le risque d’ADD.Nos résultats sont en faveur d’un effet dual de la fermentation intestinale sur la décompression. Délétère à court terme lors de la plongée, la fermentation intestinale prolongée pourrait être favorable en dehors de la plongée en prévenant la survenue et la sévérité d’un ADD. L’hydrogène, molécule aux propriétés antioxydantes, et le butyrate, un acide gras à chaîne courte, sont en effet deux produits de la fermentation des hydrates de carbone qui ont des vertus neuroprotectrices.La prévention des accidents de désaturation pourrait passer par une exclusion des plongeurs présentant une fermentation importante le jour de la plongée, une élimination des gaz produits au niveau de l’intestin ou une modification de l’alimentation dans les 24 heures précédant une plongée. En revanche, tous les facteurs susceptibles de modifier le microbiote intestinal et d’augmenter la fermentation, en dehors de la plongée, pourraient être testés en prévention de l’ADD. En outre, l’hydrogène et le butyrate pourraient jouer un rôle bénéfique dans le cadre du traitement de l’ADD / Decompression sickness (DCS) is a diving accident related to the dissolution of diluent gas in blood and tissues during a dive, followed by bubble formation in the body during decompression. It can lead to neurological damage. In dives using hydrogen as the diluent gas, the concentration of hydrogen in the tissues can be reduced by the presence in the gut of bacteria capable of metabolising this gas and this reduces the risk of DCS.The aim of this work was conversely to check if: 1) fermentation in the gut at the time of diving could exacerbate DCS as a result of endogenous hydrogen generation; 2) long-term stimulation of fermentation before diving raises the risk of DCS.Our findings point to a two-edged effect of intestinal fermentation on decompression: although deleterious in the short term, i.e. at the time of diving, longer-term intestinal fermentation between dives might have a positive effect by preventing the occurrence of DCS and limiting its severity. Indeed, hydrogen which has antioxidant properties and butyrate, a short-chain fatty acid, are both by-products of the fermentation of carbohydrate and both have neuroprotective activity.DCS prevention could be promoted by excluding divers exhibiting strong fermentation on the day of a dive, by the elimination of gases being produced in gut or by modification of diet in the 24 hours before a dive. On the other hand, any factor that might affect the gut microbiota and stimulate fermentation between dives could be tested to investigate its potential in protecting against DCS. Furthermore, hydrogen and butyrate could play a positive role when it comes to treating DCS

Les relations entre bactéries intestinales, aliments et hôte jouent un rôle crucial dans lemaintien de la santé humaine. La caractérisation fonctionnelle d’Uhgb_MP, une enzyme dela famille 130 des glycoside hydrolases découverte par métagénomique fonctionnelle, arévélé une nouvelle fonction de dégradation par phosphorolyse des polysaccharides de laparoi végétale et des glycanes de l'hôte tapissant l'épithélium intestinal. Les déterminantsmoléculaires de la spécificité d’Uhgb_MP vis-à-vis des mannosides ont été identifiés grâce àla résolution de sa structure cristallographique, sous forme apo et en complexe avec sesligands. Un nouveau procédé de synthèse par phosphorolyse inverse d'oligosaccharidesmannosylés à haute valeur ajoutée, a aussi été développé. Enfin, la caractérisationfonctionnelle de la protéine BACOVA_03624 issue de Bacteroides ovatus ATCC 8483, unebactérie intestinale hautement prévalente, a révélé que la famille GH130 comprend à la foisdes glycoside-hydrolases et des glycoside-phosphorylases capables de dégrader lesmannosides et les galactosides, et de les synthétiser par phosphorolyse inverse et/outransglycosylation. L’ensemble de ces résultats, ainsi que l’identification d’inhibiteurs desenzymes de la famille GH130, ouvrent de nouvelles perspectives pour l'étude et le contrôledes interactions microbiote-hôte / The interplay between gut bacteria, food and host play a key role in human health. Thefunctional characterization of Uhgb_MP, an enzyme belonging to the family 130 of glycosidehydrolases, discovered by functional metagenomics, revealed novel functions of plant cellwall polysaccharide and host glycan degradation by phosphorolysis. The moleculardeterminants of Uhgb_MP specificity towards mannosides were identified by solving itscrystal structure, in apo form and in complex with its ligands. A new process of high addedvalue mannosylated oligosaccharide synthesis by reverse-phosphorolysis was alsodeveloped. Finally, the functional characterization of the BACOVA_03624 protein fromBacteroides ovatus ATCC 8483, a highly prevalent gut bacterium, revealed that GH130 familyboth contains glycoside phosphorylases and glycoside hydrolases, which are able to degrademannosides and galactosides, and to synthesize them by reverse-phosphorolysis and/ortransglycosylation. All these results, together with the identification of GH130 enzymeinhibitors, open new perspectives for studying, and potentially also for controlling,interactions between host and gut microbes

Au cours des dernières années, de nombreuses études ont porté sur les relations entre le microbiote intestinal et l'obésité. Des groupes bactériens ont été incriminés et des hypothèses proposées mais les mécanismes liant microbiote et obésité restent en grande partie inconnus. Le microbiote intestinal est constitué de plusieurs centaines d’espèces et est spécifique de chaque individu. Récemment, il a été révélé l’implication potentielle de ce microbiote dans le développement de l’obésité. Lors d’une étude précédente, notre équipe a ainsi montré que les souris sans germes (dites « axéniques ») sont résistantes à l’obésité et aux désordres métaboliques induits par un régime hypercalorique (Roy et al. 2012). Par ailleurs, nous avons observé que certaines souris conventionnelles soumises à un tel régime développent une obésité et une insulinorésistance importantes (phénotype dit répondeur) tandis que d'autres restent minces et tolérantes au glucose (phénotype dit non répondeur) indépendamment de la quantité de nourriture consommée. Les mécanismes expliquant cette hétérogénéité de phénotype sont actuellement peu connus. Ce projet de thèse vise donc à élucider les mécanismes liant le microbiote intestinal au développement de l'obésité et des désordres associés en partant du principe que l'hétérogénéité (en termes de composition et de fonctions) du microbiote intestinal, spécifique de chaque individu (aussi bien chez l'homme que chez le rongeur), joue un rôle dans ces réponses différentes à un même régime hypercalorique. Pour ce faire, des souris conventionnelles ont reçu un régime contrôle ou un régime hyperlipidique pendant 12 semaines. A l’issu de ce régime, des souris ont été sélectionnées en tant que répondeuses ou non-répondeuses Une approche par métagénomique à partir d’échantillons fécaux prélevés avant et après régime hyperlipidique et issus des souris répondeuses et non-répondeuses ont été analysés. Les profils de microbiotes avant et après régime hyperlipidique ont été comparés afin de déterminer les effets de ce régime sur le microbiote intestinal. De plus, le profil du microbiote des souris répondeuses a été comparé à celui des souris non-répondeuses afin de détecter des espèces bactériennes, des gènes ou des voies métaboliques sous- ou sur-représentés dans les deux phénotypes. Par ailleurs, des échantillons de fèces, d’urine et de plasma prélevés avant et après le régime hyperlipidique ont été analysés par métabolomique et nous avons ainsi analysé les fluctuations métaboliques induites par l’effet du régime. L’ensemble de ces travaux a eu pour but d’identifier s’il existe un microbiote de prédisposition au développement ou à la résistance du développement d’une obésité induite. Au cours de ce projet de thèse nous avons utilisé l’approche de métagénomique à partir d’échantillon fécaux afin d’identifier les profils du microbiote intestinal des souris NR et des souris R avant et après régime hyperlipidique en vue de mesurer l’impact de ce type de régime sur le microbiote, et surtout de détecter les espèces bactériennes, les gènes ou les voies métaboliques potentiellement sous- ou sur-représentés dans les deux phénotypes. D’autre part, l’approche de métabolomique a été utilisée sur des échantillons d’urine, de fèces et de plasma avant et après régime afin de visualiser les fluctuations métaboliques induites par l’effet du régime d’une part et d’identifier les métabolites (biomarqueurs) associés à chacun des phénotypes d’autre part. / In recent years, numerous studies have examined the relationship between the gut microbiota and obesity. Bacterial groups have been incriminated but the mechanisms linking microbiota and obesity remain largely unknown. Intestinal microbiota consists of several hundred species and is specific for each individual. Recently, it was revealed that the potential contribution of microbiota in the development of obesity. In a previous study, our team has shown that mice without germs (called "germ-free") are resistant to obesity and metabolic disorders induced by a high calorie diet (Roy et al. 2012). Furthermore, we observed that certain conventional mice subjected to such a plan develop an important obesity and insulin resistance (responder phenotype) while others remain thin and glucose tolerant (non responder phenotype) regardless of the amount of food consumed. The mechanisms explaining this phenotype heterogeneity are currently poorly known. This PhD project aims to elucidate the mechanisms linking the intestinal microbiota in the development of obesity and disorders associated with the assumption that heterogeneity (in terms of composition and functions) of the intestinal microbiota, specific for each individual ( both in humans than in rodents), plays a role in these different responses to the same high-fat diet. To do this, conventional mice received control diet or a high fat diet for 12 weeks. At the end of this diet, mice were selected as a responder or non-responder A metagenomics approach from fecal samples taken before and after high fat diet and from responder and non-responder mice were analyzed. Microbiota profiles before and after fat diet were compared to determine the effects of this diet on the intestinal microbiota. In addition, the profile of the microbiota of responder mice was compared to that of non-responder mice in order to detect bacterial species, genes or pathways under- or over-represented in both phenotypes. Furthermore, samples of feces, urine and plasma collected before and after fat diet were analyzed by metabolomics and we have analyzed the metabolic changes induced by the effect of diet. All of this work has been aimed to identify if there is a predisposition microbiota to the development or the resistance of induced obesity. In this PhD project we used the metagenomic approach from fecal sample to identify the profiles of the intestinal microbiota in mice NR and R mice before and after fat diet in order to measure the impact of this type of diet on the microbiota, and especially to detect bacterial species, genes or metabolic pathways potentially under- or over-represented in both phenotypes. On the other hand, the approach of metabolomics has been used on samples of urine, feces and plasma before and after diet in order to visualize the metabolic changes induced by the effect of diet on the one hand and to identify metabolites (biomarkers) associated with each other hand phenotypes.

Orientadores: Jaime Amaya-Farfan, Fernanda de Pace / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-27T02:35:33Z (GMT). No. of bitstreams: 1 Monteiro_NaiceEleidianeSantana_M.pdf: 1835989 bytes, checksum: ec62cf398adabcdca614249ae2d4692b (MD5) Previous issue date: 2015 / Resumo: Caracterizada como um dos mais importantes problemas que a saúde pública enfrenta atualmente no Brasil e no mundo, a obesidade está associada a um quadro de inflamação subclínica, que predispõe à resistência à insulina e ao desenvolvimento de diabetes mellitus, além de representar fator etiológico para diversas outras doenças crônicas não transmissíveis. Sabendo da importância que os alimentos com propriedades bioativas podem ter no tratamento da obesidade é que a utilização das proteínas do soro do leite na dieta vem sendo alvo de diversos estudos, devido aos benefícios que podem trazer à saúde humana. Dentre as propriedades da whey protein, citam-se a capacidade de regular a função imune, atuar como agente antimicrobiano, estimular a síntese de proteína muscular, suprimir o apetite e atuar na redução da gordura corporal, além das propriedades de aumentar a expressão gênica de proteínas anti-estresse (HSPs) e de ativar o transportador de glicose GLUT4, ambas descobertas pelo nosso grupo da FEA. O presente estudo teve como objetivo investigar o impacto que as proteínas do soro do leite, seja na sua forma íntegra ou na forma hidrolisada, possam exercer sobre a homeostase e a proteção do organismo de camundongos, contra os danos causados por uma dieta hiperlipídica. Para alcançar este objetivo, 34 camundongos C57BL/6 recém-desmamados foram divididos em quatro grupos de forma aleatória e alimentados com as seguintes dietas: grupo controle normolipídico (AIN93-G), grupo controle hiperlipídico (HFCAS), grupo hiperlipídico com substituição da caseína pela proteína do soro de leite concentrada (HFWPC), e grupo hiperlipídico, com adição da proteína do soro do leite hidrolisada (HFWPH), por 9 semanas. Os camundongos foram acompanhados quanto ao consumo alimentar e ganho de peso por meio de avaliação poderal em dias alternados, parâmetros bioquímicos e de endotoxemia avaliados por técnicas convencionais, biomarcadores inflamatórios analisados por western blot, além da avaliação do perfil histopatológico do fígado e do estudo metagenômico da microbiota intestinal. Os resultados demonstraram que alimentação com a formulação contendo whey protein, em ambas as formas, por 9 semanas, não diminuiu ganho de peso em comparação aos demais tratamentos, mas foi efetiva em reverter a disbiose causada pela dieta gordurosa, reduzir o processo inflamatório para níveis indistinguíveis do controle, em atenuar a infiltração gordurosa no tecido hepático, além de modular a microbiota intestinal. A caseína não mostrou tais propriedades / Abstract: Recognized as one of the most important issues currently facing public health in Brazil and in the world, obesity is associated to a subclinical inflammation framework, which predisposes to insulin resistance and the development of diabetes mellitus, in addition to representing an etiological factor for several other chronic diseases. Owing to the bioactive properties that the milk whey proteins may have on human health, such as the ability to regulate immune function, act as an antimicrobial agent, stimulate protein synthesis, suppress appetite thus helping to reduce body fat, besides the two newly discovered functions from our laboratory regarding the up-regulation of protective HSPs and activation of the glucose transporter 4 (GLUT4), it is thought that these proteins may also have anti-inflammatory action and, therefore, this study was designed to investigate the impact that whey protein, either in its normal form or in the hydrolyzed form, may exert on homeostasis protecting the mouse from the adverse effects of a high-fat diet. To accomplish this, 34 C57BL/6 male mice were randomly divided into four groups that received the following diets for 9 weeks: Normolipidic control (AIN93-G), Hyperlipidic control (HFCAS), Hyperlipidic with whey protein concentrate instead of casein (HFWPC) and a Hyperlipidic group with hydrolyzed whey protein (HFWPH) as the only source of protein. Diet intake and weight gain were monitored and recorded every other day. Biochemical parameters and endotoxemia were evaluated using commercial kits, and inflammatory biomarkers were analyzed by western blotting, besides the evaluation of histopathological liver profile and metagenomic study of the intestinal microbiota. The results showed that feeding the formulation contendowheyprotein, in both its forms, for 9 weeks, not decreased weight gain compared to the other treatments, but was effective in reversing dysbiosis caused by high-fat diet, reduce inflammation to levels indistinguishable control and mitigate the fatty infiltration of the casein hepático, in addition to modulate the gut microbiota. Casein did not show such properties / Mestrado / Nutrição Experimental e de Alimentos / Mestra em Alimentos e Nutrição

Introdução: O trato gastrointestinal humano é composto por uma comunidade microbiana diversificada que atua no controle da saúde. Estudos recentes demonstraram que o equilíbrio da microbiota intestinal é afetado na doença renal crônica (DRC), ocasionando o quadro de disbiose intestinal. Estes estudos sugeriram uma associação da disbiose intestinal com complicações metabólicas como acúmulo de toxinas urêmicas, progressão da DRC, inflamação e risco cardiovascular. Diante disso, medidas com o objetivo de restaurar o equilíbrio da microbiota intestinal são sugeridas, tais como a ingestão oral de probióticos, mas poucos estudos têm abordado o efeito destes suplementos na progressão da DRC e no risco cardiovascular destes pacientes. Objetivo: Avaliar o efeito do consumo de probióticos em fatores associados com progressão da DRC e risco cardiovascular de pacientes com DRC. Material e métodos: Trata-se de um estudo clínico controlado por placebo registrado no Clinical Trials NCT03400228. O estudo incluiu 30 pacientes adultos com DRC nos estágios 3 a 5 não em diálise, com função renal estável e proteinúria igual ou superior a 500 mg. A coleta de dados ocorreu entre novembro de 2015 até dezembro de 2017. O protocolo do estudo constou de período de washout de 4 semanas e randomização dos pacientes para o grupo de intervenção (GI, suplemento com probiótico) ou para o grupo controle (GC, maltodextrina). Foi realizado avaliação basal e após 24 semanas de consumo de probiótico ou placebo. Todos os pacientes receberam a orientação de consumir 2 sachês por dia do probiótico ou do placebo (maltodextrina). Foram avaliadas variáveis demográficas, clínicas, nutricionais, hábito intestinal e exames laboratoriais com amostras sanguíneas e urinárias. Resultados: Dos 30 pacientes incluídos, 20 completaram as 24 semanas do estudo, sendo 10 no grupo intervenção e 10 no grupo placebo. Após o uso de probiótico houve aumento na taxa de filtração glomerular estimada (p<0,001) e diminuição nos níveis séricos de creatinina (p<0,001), ureia (p=0,015), proteína C reativa (p=0,03), hormônio da paratireóide (p=0,03) e potássio (p=0,012), em comparação ao grupo placebo. Os efeitos positivos do probiótico na taxa de filtração glomerular estimada e na diminuição dos níveis séricos de creatinina e ureia permaneceram após análise de regressão multivariada. Não houveram diferenças significativas nos parâmetros urinários entre os grupos. Sintomas de constipação (p<0,001) e consistência fecal (p=0,016) apresentaram melhora no grupo intervenção versus placebo. Conclusão: A suplementação de probióticos melhorou os marcadores de função renal e reduziu inflamação, além de auxiliar na melhora dos sintomas de constipação intestinal em pacientes com DRC. / Introduction: The human gastrointestinal tract is colonized by a diversified microbial community that acts in control of health. Recent studies have shown that intestinal microbiota balance is affected in chronic kidney disease (CKD) leading to intestinal dysbiosis. These studies have suggested association of intestinal dysbiosis with several metabolic disorders such as accumulation of uremic toxins, progression of CKD, inflammation and cardiovascular risk. Therefore, interventional measurement that improve intestinal microbiota balance are suggested such as supplementation of probiotics, however few studies evaluated the effect of these supplements on the progression of CKD and cardiovascular risk in CKD patients. Aim: The purpose of the study was to evaluate the effects of probiotic supplementation on the factors associated with progression of CKD and cardiovascular risk in patients with CKD. Desing and Methods: This was a randomized, double-blind, placebo-controlled study. Thirty patients with CKD stages 3 to 5 not on dialysis, with stable renal function and protein-creatinine ratio > 0.50 were included. Data collection was between November 2015 and December 2017. Study protocol was 4-week washout period, patients randomized to intervention group (IG, probiotic supplement) or control group (CG, maltodextrin), and follow for 24 weeks. Renal function, C-reactive protein (CRP), bone and mineral metabolism, nutritional, and lipid profile markers and intestinal habit were measured at baseline and 24 weeks of study. Results: From 30 patients included in this study, 20 completed the 24 study weeks, 10 in the TG and 10 in PG. After probiotic supplementation, there was increase in estimated glomerular filtration rate (p<0.001) and decrease in serum creatinine 8 (p<0.001), urea (p=0.015), C-reactive protein (p=0.030), parathyroid hormone (p=0.03), and potassium (p=0.012) levels compared to CG. The beneficials effects of probiotics on estimated glomerular filtration rate and serum creatinine, urea, and Creactive protein remained after multivariate linear regression. There were no significant differences in the urinary parameters between the two groups. Symptoms of constipation (p<0.001) and stool consistency (p=0.016) improved in IG compared to CG. Conclusion: Probiotic supplementation improved markers of renal function and reduced inflammation. In addition, it improved the symptoms of intestinal constipation in patients with CKD.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22833号 / 医博第4672号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 濵﨑 洋子, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The presented thesis discusses the issues of chronic inflammatory diseases of the digestive system and suggests the possibility of the alternative natural remedies improving the health conditions or prolonging the remission phase of IBD. The main goal is to design a probiotic supplement enriched by natural anti-inflammatory agents. Phytochemicals, concretely phenolic compounds, flavonoids, and carotenoids suppress harmful inflammatory processes by direct targeting the function of the immune cells or by inhibiting damaging oxidative stress in general. Therefore, there were several plants potentially rich for these biologically active substances selected. Concretely, blueberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), turmeric (Curcuma longa), peppermint (Mentha piperita), chamomile (Matricaria recutita), cinnamon (Cinnamomum zeylanicum), ginger (Zingiber officinale) and flax (Linum usitatissimum). The suitable parts of these plants were extracted by using a total of three different solvents – water, ethanol, and hexane for obtaining lipidic extracts. The concentration of total polyphenols, total flavonoids, total carotenoids, and the ascertainment of specific polyphenols was determined in the prepared extracts. These characteristics were enhanced by the measurement of antioxidant activity of the aqueous and ethanolic extracts. The interaction of individual samples with probiotic cultures (Bifidobacterium breve, Lactobacillus acidophilus) and the ability of extracts to influence the viability of probiotics in the model digestion process has been examined. The most interesting samples (aqueous extracts of blueberries, mint, chamomile, and cinnamon) were selected for further experiments. The possible cytotoxicity towards human intestinal epithelial cells was tested in vitro by the MTT tests utilizing the CaCo-2 cell line. Extracts showing the highest levels of beneficial phytochemicals and antioxidant activity, supporting the growth of probiotic cultures, and showing minimal cytotoxic effects on human intestinal cells were then co-encapsulated with probiotics into alginate particles of a diameter of 1 mm. Water extracts of mint, chamomile, and cinnamon have been chosen. The encapsulation efficiency of successfully entrapped probiotics and phenolic compounds was determined in prepared particles. Furthermore, the particles were studied during the process of model digestion, when the release of the desired substances in the various parts of the digestive system was observed and assessed whether the components would reach the crucial point of action – the colon. For the use of the proposed probiotic mixture as a dietary supplement, a recommended dose of 1 g has been determined. Lastly, possible adjustments such as lyophilization or sheathing by an extra protective polymerous layer, e.g. chitosan, were suggested to prolong the shelf life of the particles and volatile substances stability.

In questa tesi di Dottorato, l’obiettivo era valutare l’impatto di diversi ingredienti alimentari sul metabolismo di alcuni batteri intestinali e viceversa, mediante l’applicazione di tecniche omiche. Utilizzando le tecniche di metabolomica e trascrittomica, è stata studiata la risposta all’acido linoleico del ceppo Bifidobacterium breve DSM 20213. Utilizzando un approccio combinato di metagenomica e metabolomica, è stato possibile studiare le modifiche a carico del microbiota intestinale, del profilo fenolico e degli acidi grassi, in biscotti senza glutine (a base di erba medica) durante digestione e fermentazione in vitro. Inoltre, è stato valutato come alcuni batteri potessero interferire negativamente su una terapia farmacologica a base di Diclofenac, un farmaco usato per alcune patologie intestinali. Per questo tipo di studio è stata utilizzata la spettrometria di massa ad alta risoluzione, che ha consentito di ipotizzare un coinvolgimento dell’enzima batterico β-glucuronidasi. Una sola tecnica omica, seppure di ultima generazione, non permette di valutare tutte le modificazioni del microbiota intestinale data la complessità dei fattori coinvolti. Per questa ragione, integrare più approcci omici potrebbe risultare una buona strategia per analizzare il reale impatto del microbiota sulla salute dell’ospite. Questo permetterebbe di valutare le interazioni microbiota-ospite, i principali metabolismi e le interconnessioni tra gruppi batterici coinvolti nella risposta ad uno stimolo esterno come l’assunzione di particolari ingredienti con l’alimentazione. / The aim of the present PhD thesis was to explore the metabolic response of intestinal bacteria to food components by using ‘omics’ approaches. In particular, the first part of this thesis was focused on the effect of linoleic acid on Bifidobacterium breve DSM 20213 strain. Firstly, an untargeted metabolomics-based approach was used to explore the primary changes in metabolic profile of this strain grown in presence of linoleic acid. Secondly, the gene expression of B. breve DSM 20213 induced by linoleic acid exposure was investigated. Integrated use of metabolomics/transcriptomics was applied to better understand the response mechanisms to linoleic acid stress. In the third part of the thesis, using a combination of metagenomics and metabolomics, the in vitro large intestine fermentation of gluten-free rice cookies containing alfalfa seed was investigated. In the last part of my PhD, the negative effect of β-glucuronidase producing bacteria was evaluated by means of qualitative high-resolution mass spectrometry. Based on my experience there is not a gold standard approach for evaluating a complex environment such as the gastrointestinal tract. For this reason, an integrated use of different techniques should be mandatory to have an accurate framework of gut microbiota composition, its potential metabolic network and the impact on the host physiology and health.

Les cancers du rein métastatiques résistants aux inhibiteurs de tyrosine kinase peuvent faire l’objet de traitements fondés sur le blocage des points de contrôles immunitaires (ICB). Cependant, les ICB induisent des réponses chez une minorité de patients, et des efforts sont en cours pour identifier les mécanismes à l'origine de la résistance. Les ICB compromettent l'intégrité de la barrière intestinale, affectant ainsi la composition du microbiote, favorisant ainsi soit l'accumulation intestinale, soit la translocation de commensaux immunogènes capables de moduler le tonus immunitaire systémique et de reprogrammer le microenvironnement tumoral. Pour déterminer si des profils microbiens distincts du microbiote intestinal pourraient expliquer la résistance aux ICB, ma thèse a montré que dans une cohorte de 249 patients atteints de cancer traités par ICB que la prescription d’antibiotiques (ATB) a significativement diminué la survie sans progression (PFS) et la survie globale (OS) par rapport aux patients sans ATB. Nous avons confirmé ces données en analysant 121 cencers du rein traités par ICB à Gustave Roussy et 239 cancers du poumon traités par ICB au Memorial Sloan Kettering Cancer Center. Nous avons validé que la dysbiose liée à l'ATB diminue l'activité des ICB. Par la suite, nous avons analysé de manière prospective les microbiotes fécaux de 100 patients atteints de tumeurs sensibles à l'anti-PD-1 en utilisant la métagénomique (MG). Nous avons démontré que les bactéries intestinales présentes avant le traitement anti-PD-1 étaient systématiquement différentes entre les patients qui ont répondu (R) au traitement et les patients qui n’ont pas (NR). Entre les R, nous avons observé une surreprésentation d’Akkermansia muciniphila. Au sein d'une large cohorte de patients RCC (n = 85) traités avec un anticorps anti-PD-1, nous avons analysé le microbiote fécal de 69 patients. Des empreintes de MG spécifiques étaient liées aux meilleures réponses et à la survie sans progression. A. muciniphila et Bacteroides spp étaient plus abondants chez les R. Pour valider la pertinence de ces constatations, nous avons mis en évidence deux principaux éléments de preuve. Premièrement, nous avons démontré que chez les patients atteints de NSCLC, la présence de lymphocytes T CD4 + et CD8 + à mémoire spécifiques de l’IFNγ + vis-à-vis de A. muciniphila prédit une PFS plus longue. Deuxièmement, une transplantation de microbiote fécal (FMT) a été réalisée à l'aide de selles de patient pour recoloniser des souris axéniques ou traitées par ATB dans deux modèles murins. Les matières fécales de R entrainaient une réponse immunitaire plus forte contre la tumeur par rapport aux matières fécales de NR. Ensuite, une supplémentation orale d'A. muciniphila post-FMT avec des matières fécales de NR restaurait l'efficacité de l'anti- PD-1. Dans ce contexte, les cellules dendritiques sécrétaient plus d'IL-12, augmentant le recrutement de lymphocytes T CCR9 + CXCR3 + CD4 + à partir des ganglions lymphatiques mésentériques jusque dans les lits tumoraux, ainsi qu'une augmentation du rapport CD4 + /Treg dans le lit tumoral de souris co-traitées avec mAb anti-PD-1 et A. muciniphila. Enfin, nous avons montré qu'une supplémentation orale avec Bacteroides (B. salyersiae mais pas B. xylanisolvens) ou A. muciniphila pourrait restaurer l'efficacité des ICB dans un modèle FMT- défavorable/dysbiotique". La découverte de bactéries immunogènes capables de prédire et d'accroître les avantages cliniques de l'ICB contribuera au développement de nouveaux outils de biomarqueurs et d'un futur concept thérapeutique, grâce auxquels le traitement du cancer peut être amélioré par la modulation du microbiote intestinal. / Metastatic RCC resistant to tyrosine kinase inhibitors are amenable to new therapies based on immune checkpoint blockade (ICB). However, ICB induces responses in a sizeable minority of patients and efforts are ongoing in order to identify mechanisms driving resistance. ICB compromise the integrity of the intestinal barrier, hereby affecting the composition of the intestinal microbiome, thereby promoting either the intestinal accumulation or the translocation of immunogenic commensals capable of modulating the systemic immune tone and reprogramming the tumor microenvironment. To address whether distinct microbial patterns of the intestinal microbiome could account for the resistance to ICB in RCC, during my PhD I showed in a cohort of 249 patients with cancers treated with ICB that antibiotic (ATB) prescription in a therapeutic window of -2 months up to +1 month after starting ICB significantly decreased progression-free survival (PFS) and overall survival (OS) compared to patients without ATB. We confirmed this data analyzing 121 RCC treated with ICB at Gustave Roussy and 239 NSCLC treated with ICB at Memorial Sloan Kettering Cancer Center. We validated that ATB-related dysbiosis decreases activity of ICB. More precisely, patients in RCC ATB group treated with ICB had a higher rate of primary progressive disease. Subsequently, we prospectively analyzed the fecal microbiomes of 100 patients with tumours amenable to anti-PD-1 mAb using quantitative metagenomics (MG) by shotgun sequencing. We demonstrated that gut bacteria present before anti-PD-1 therapy was consistently different between patients who responded (R) to treatment and patients who did not (NR). In R we observed an overrepresentation of un- and classified Firmicutes. The commensal most significantly associated with favorable clinical outcome and PFS was Akkermansia muciniphila. Within a large cohort of RCC patients (n = 85) treated in the NIVOREN study with anti-PD-1 Ab at Gustave Roussy, we analyzed the fecal microbiome of 69 patients. Specific MG-fingerprints were related to best responses and PFS. A. muciniphila and Bacteroides spp were more abundant in R (Derosa et al. ASCO Merit Award 2018). To validate the relevance of these findings, we brought up two major lines of evidence. First, we demonstrated that in NSCLC patients, the presence of specific IFNγ+ memory CD4+ and CD8+ T cells toward A. muciniphila predicted a longer PFS. Secondly, fecal microbiota transplantation (FMT) was performed using patient feces to recolonize germ-free or ATB-treated mice in two tumor models (MCA-205 and RENCA, the renal cell carcinoma model). Feces from R conveyed a stronger immune response against the tumor compared to feces from NR. Next, in MCA-205 model oral supplementation with A. muciniphila post-FMT with NR feces restored the efficacy of PD-1 Abs. In this setting, dendritic cells secreted more IL-12, increasing the recruitment of CCR9+CXCR3+CD4+ T lymphocytes from the mesenteric lymph nodes into tumor beds as well as an increase of CD4+/Treg ratio within the tumor bed of mice co-treated with anti-PD-1 mAb and A. muciniphila. Finally, we showed that oral supplementation with Bacteroides (B. salyersiae but not B. xylanisolvens) or A. muciniphila could restore the efficacy of ICB in "unfavorable/dysbiotic" FMT. The discovery of immunogenic bacteria capable of predicting and increasing clinical benefit of ICB will help for the development of novel biomarker tools and a future therapeutic concept, whereby treatment of cancer can be improved by the modulation of gut microbiome.