L’Insuffisance Rénale Chronique (IRC) résulte de la destruction progressive et irréversible des reins. Elle est associée à une rétention de toxines urémiques à l’origine des nombreuses complications de la maladie rénale chronique (cardiovasculaires, osseuses ou métaboliques). Nos travaux se sont focalisés sur l’impact de la dysfonction rénale et de l’urémie sur la fonction barrière de l’intestin et la motilité intestinale. Deux modèles d’IRC ont été implémentés : un modèle animal, chez la souris, par néphrectomie chimique (régime alimentaire enrichi en adénine) et un modèle cellulaire d’urémie en incubant des cellules coliques Caco-2 avec 10% de plasma de patients hémodialysés (HD). Le transit, la motilité, la perméabilité intestinale et la régulation des protéines des jonctions serrées ont été explorés. Les animaux urémiques présentent un transit gastro-intestinal ralenti et une perméabilité intestinale augmentée associés à une dérégulation de l’expression et de l’abondance des protéines des jonctions serrées dans le côlon (surexpression de la Claudine 1). La perméabilité de la monocouche cellulaire de Caco-2 incubées avec du plasma HD est significativement augmentée et est associée à une augmentation de l’expression et de l’abondance de la Claudine-1. En IRC, la motilité digestive et la fonction barrière de l’intestin sont significativement altérées. Ces dysfonctions pourraient contribuer à la production intestinale et l’absorption des toxines urémiques accélérant ainsi la progression du syndrome urémique et installant un véritable « cercle vicieux ». / Chronic Kidney Disease (CKD) result from a progressive kidney dysfunction. CKD is associated with an increase in the concentration of uremic toxins inducing CKD-associated metabolic alterations. Our work focused on the impact of renal dysfunction on gut permeability and gut motility. In vivo, CKD was induced in mice by chemical nephrectomy (adenine-enriched diet); In vitro, Caco-2 cells were incubated for 24h with 10% (v/v) of uremic plasma to mimic the uremic environment. Gastrointestinal transit time, gut motility, intestinal permeability and expression of tight junction proteins were explored. In vivo, kidney failure was associated with an impaired gastrointestinal transit and an increased intestinal permeability associated with a dysregulation of tight junction proteins (mainly claudine-1 overexpression). The Caco-2 monolayer permeability was significantly increased in cell monolayers incubated with uremic plasma. Claudine-1 expression and abundance was increased. In CKD, gut motility and gut permeability (e.g. « leaky » gut) are significantly impaired. Generally speaking, these gut dysfunctions could promote the production and the absorption of uremic toxins contributing to the uremic syndrom.

La Radiothérapie occupe la deuxième place dans la liste de traitement du cancer le plus important après chirurgie. Le progrès technique récent, comme la radiothérapie avec modulation d'intensité (IMRT) ou la radiothérapie guidée par l'image (IGRT), en combinaison avec de nouveaux médicaments à action spécifique tels que les anticorps monoclonaux, sont une garantie d'augmentation de l'index thérapeutique. Cependant, la radiothérapie peut provoquer un’ altération du processus normal de réparation et d'induire le développement d'un cadre de fibrose dans un sous-ensemble de patients sensibles et dans les survivants à long terme au cancer. La principale caractéristique de la fibrose radio-induit est l'accumulation excessive et anormale de collagène composé principalement des éléments fibrillaire et immatures de la matrice extracellulaire (ECM).Les organes qui peuvent être touchés par ce phénomène sont le foie, la peau, les intestins, les reins et les poumons. D'un point de vue clinique, la fibrose peut être considérée comme une condition irréversible, sans solution. Nous et d'autres ont récemment montré que, outre l'activation de la TGF-β/Smad canonique, d'autres voies sont activées dans les tissus fibreux tels que la cascade de signalisation intracellulaire Rho/ROCK. Fait intéressant, la façon dont Rho/ROCK semble spécifiquement activé dans la fibrose intestinale radio-induite, fournis une justification pour un stratégie anti-fibrotique ciblé. L’ inhibition pharmacologique de Rho avec les statines, en fait, est en mesure de prévenir et même inverser les phénomènes de fibrose post-actinique intestinale.Avec ces prémisses, dans nos études, nous avons montré le rôle des statines (Simvastatine et Pravastatine) et d'un inhibiteur spécifique de ROCK (Y-27632) dans un modèle murin de fibrose pulmonaire obtenue avec une approche pharmacologique (Bléomycine - BLM) . Par la suite, nous avons développé un modèle de fibrose pulmonaire induite par l'irradiation complet du thorax et évalué la réponse à l'administration de la Pravastatine. La confirmation de la participation de la voie Rho/ROCK/CTGF dans la fibrose pulmonaire a été montré par immunohistochimie: le traitement à la Pravastatine normalise l'expression de trois marqueurs: RhoB, TGFβ-RII et CTGF.Après, dans deux modèles de fibrose radio-induite (intestinal et pulmonaire), nous avons analysé, grâce à l’immunohistochimie, les mécanismes sous-jacents l'action antifibrotique de la Pravastatine via l’axe MMP2-TIMP2. Très intéressant, quand la pravastatine a été administré à titre préventif ou curatif, nous avons trouvé un impact différent sur la fibrolyse.Enfin, in vitro, nous avons étudié par zymographie l'expression des gélatinases (MMP2 et MMP9) dans des cultures primaires des fibroblastes pulmonaire murins exposées à différentes doses de rayonnement et de Pravastatine. Le métalloprotéases semble être à son tour impliquée dans les mécanismes pro-fibrolytiques induits par les statines.Dans notre modèle animal de fibrose pulmonaire, la Pravastatine est capable d'inverser le processus fibrotique et les métalloprotéases semblent être impliqués à leur tour, in vivo et in vitro, dans les mécanismes pro-fibrolyse induits par le médicament.La multiplicité des acteurs impliqués dans la physiopathologie de lésions fibrotiques explique pourquoi la mise en place d'une stratégie thérapeutique efficace est si complexe. La recherche dans les processus mécaniques de dommages aux tissus normaux ont ouvert la voie à de nouvelles approches thérapeutiques. Ces nouvelles cibles comprennent la réduction de l’inflammation, de l'activation vasculaire et de la thrombose, ainsi que la découverte de nouvelles cibles moléculaires. Il existe une variété de modèles précliniques et des stratégies efficaces, mais de nombreux efforts doivent être déployés pour atteindre l'objectif difficile de protéger les tissus normaux des effets secondaires de la radiothérapie. / Radiotherapy is the second most important treatment modality after surgery in the treatment of cancer. Recent technical advancements, such as intensity-modulated radiation therapy (IMRT) or image-guided radiation therapy (IGRT), combined with new targeted drugs have significant promise for therapeutic outcome. However radiation treatment could result in disabling normal tissue injury and in the development of progressive fibrosis in a subset of sensitive patients and in long-term cancer survivors. The main feature of tissue fibrosis is excessive accumulation of abnormal and cross-linked collagen mainly composed of fibrillar and immature extracellular matrix (ECM) components.The organs that can be affected by this phenomenon are liver, skin, intestine, kidneys and lungs. From a clinical point of view, fibrosis can be seen as an irreversible condition, without solution. We and others recently showed that beside the activation of the canonical TGF-β/Smad pathway, other intracellular signaling cascades including the Rho/ROCK pathway are switched on in fibrotic tissues. Interestingly, the Rho/ROCK pathway seems differentially activated in radiation-induced intestinal fibrosis, thereby providing a rationale for a specific, targeted anti-fibrotic strategy. Pharmacological inhibition of Rho using statins indeed prevent and even reverse intestinal radiation fibrosis.In our studies, we showed the role of Statin (Pravastatin e Simvastatin) and a specific inhibitor ROCK inhibitors (Y-27632) in a mice model of pulmonary induced-fibrosis obtained by a pharmacological approach (Bleomycin – BLM). Indeed, we developed a model of lung fibrosis by complete irradiation of chest and tested Pravastatin action. Confirmation of the involvement of Rho/ROCK/CTGF pathway in lung fibrosis are shown by immunohistochemistry: Pravastatin-treament normalized the expression of three markers: RhoB, TGF-RII and CTGF.Then, in models of radiation induced gut and lung fibrosis, we analysed, from a immunohistological point of view, the underlying mechanisms of the antifibrotic action of Pravastatin via MMP2-TIMP2 axis. Interestingly we found a different impact on fibrolysis when Pravastatin was administered preventively or curatively.Finally, in vitro, we investigate by zymography the expression of Gelatinases (MMP2 and MMP9) in primary lung fibroblasts cultures exposure at the different radiation and Pravastatin doses. Metalloproteases would appear to be in turn involved in pro-fibrolytic mechanisms induced by statin.The multiplicity of actors involved in the pathogenesis of fibrotic lesions explains why the definition of an effective therapeutic strategy is so complex.Researches in mechanistic processes of normal tissue damage paved the way for new therapeutic approaches. These new targets include reduction of vascular activation, inflammation and thrombosis and new molecular targets definition. Effective strategies are multiple on preclinical models, but numerous efforts have to be made to achieve the complicated goal of protection of normal tissues from the side effects of radiation therapy. / La radioterapia è la seconda modalità di trattamento più importante dopo chirurgia neltrattamento delle neoplasie. I recenti progressi tecnici, come la terapia ad intensità modulata(IMRT) o l’image-guided radioterapia (IGRT), in combinazione con nuovi farmaci ad azionemirata come gli anticorpi monoclonali, costituiscono ulteriore garanzia di incrementodell’indice terapeutico. Tuttavia il trattamento radiante può causare un’alterazione delnormale processo di riparazione e indurre lo sviluppo di un quadro di fibrosi in unsottogruppo di pazienti sensibili e nei lungo-sopravviventi al cancro. La caratteristicacardinale della fibrosi radioindotta è l’eccessivo ed anomalo accumulo di collagene compostoprincipalmente di componenti fibrillari e immature della matrice extracellulare (ECM).Gli organi che possono essere interessati da questo fenomeno sono fegato, pelle,intestino, reni e polmoni. Da un punto di vista clinico, la fibrosi può essere vista come unacondizione irreversibile, senza soluzione. Noi ed altri recentemente abbiamo mostrato cheaccanto alla attivazione della via canonica TGF-β/Smad, altre vie vengono attivate nei tessutifibrotici come la cascata di segnalazione intracellulare della via Rho/ROCK. Interessantenotare che la via Rho/ROCK sembra specificatamente attivata nella radiazione indotta fibrosiintestinale, fornendo così una spiegazione razionale per una specifica, mirata strategia antifibrotica.L'inibizione farmacologica di Rho con le statine infatti è in grado di prevenire eaddirittura invertire i fenomeni di fibrosi intestinale post-attinica.Grazie a queste premesse, nei nostri studi, abbiamo mostrato il ruolo delle statine(Pravastatina e Simvastatina) e di uno specifico inibitore di ROCK (Y-27632) in un modellomurino di fibrosi polmonare indotta ottenuto con un approccio farmacologico (bleomicina -BLM). In seguito, abbiamo sviluppato un modello di fibrosi polmonare indottadall’irradiazione completa del torace e valutata la risposta alla somministrazione dellaPravastatina. In questo modello ed in un modello di fibrosi intestinale indotto da radiazioni,abbiamo analizzato, da un punto di vista immunoistologico, i meccanismi sottostanti l'azione9antifibrotica della pravastatina e il ruolo delle metalloproteasi (MMP2 e TIMP2). Infine, invitro, abbiamo indagato, mediante zimografia, l'espressione delle gelatinasi (MMP2 e MMP9)in culture primarie di fibroblasti polmonari murini esposti a differenti dosi di radiazione epravastatina.Nel nostro modello animale di fibrosi polmonare, la Pravastatina è in grado di renderereversibile il processo fibrotico e le metalloproteasi parrebbero essere a loro volta coinvolte,in vivo and in vitro, nei meccanismi pro-fibrolitici indotti dal farmaco.La molteplicità di attori coinvolti nella patogenesi delle lesioni fibrotiche spiegaperché la definizione di una strategia terapeutica efficace è così complessa. Ricerche neiprocessi meccanicistici di danno ai tessuti normali hanno aperto la strada a nuovi approcciterapeutici. Questi nuovi obiettivi comprendono la riduzione dell’ attivazione vascolare,dell'infiammazione e della trombosi, oltre alla definizione di nuovi target molecolari. Esistonomolteplici ed efficaci strategie su modelli preclinici, ma numerosi sforzi devono essere fattiper raggiungere il complicato obiettivo di proteggere i tessuti normali dagli effetti collateralidella radioterapia.

Le microbiote digestif humain joue un rôle essentiel et bénéfique pour son hôte mais il est également impliqué dans un nombre croissant de pathologies. Les connaissances sur la composition de cet écosystème ont récemment été révolutionnées grâce à l’utilisation de techniques moléculaires. Cependant, ces techniques comportent des limites importantes. C’est ainsi que le concept de « culturomique » a été introduit ; il consiste en la multiplication de milieux et conditions de culture et l’identification rapide de colonies bactériennes par spectrométrie de masse (MALDITOF) ou par amplification et séquençage du gène de l’ARN ribosomal 16S. Dans la première partie de ce travail, nous avons mis en évidence une association entre la présence de Clostridium butyricum dans les selles et la survenue d’entérocolite ulcéro-nécrosante que ce soit par méthodes de pyroséquençage et culture ou par PCR quantitative en temps réel spécifique de C. butyricum; identifié après séquençage du génome complet de toutes nos souches de C. butyricum, la présence du gène de la β-hémolysine (toxine). Dans la deuxième partie de ce travail, nous avons montré par cuturomique que les bactéries à Gram-négatif (BGN) étaient fréquemment disséminées au sein du microbiote cutané transitoire des patients hospitalisés en réanimation ; le réservoir serait essentiellement digestif. En conclusion, le microbiote digestif constitue un réservoir sousestimé de bactéries pathogènes. La microbiologie moderne incluant les nouvelles méthodes de culture permet d’étendre de manière considérable les connaissances sur la composition de cet écosystème et son implication en pathologie humaine. / He human gut microbiota plays an important and beneficial role in its host but it is also involved in a growing number of diseases. Knowledge of the composition of this ecosystem have recently been revolutionized by the use of molecular techniques. However, these techniques have significant limitations. Thus, the concept of "culturomics" has been introduced; it consists of the multiplication of culture conditions and the rapid identification of bacterial colonies by mass spectrometry (MALDI-TOF) or by PCR 16S RNA gene sequencing. In the first part of this work, we have demonstrated an association between the presence of Clostridium butyricum in the stool and the occurrence of necrotizing enterocolitis whether by pyrosequencing methods and Culture or by quantitative PCR specific real time C. butyricum; identified after sequencing the complete genome of all our strains of C. butyricum, the presence of the gene of β-hemolysin (toxin). In the second part of this work, we showed by cuturomics that Gram-negative bacteria (BGN) were frequently spread out over the transitional skin microbiota of patients hospitalized in intensive care; the reservoir would essentially digestive. In conclusion, the gut microbiota is an underestimated reservoir of pathogenic bacteria. Modern microbiology including new culture-based methods is currently extending exponentially our knowledge on gut microbiota giving rise to new insights into the pathogenesis or the transmission of infectious diseases.

A necessidade de economias sustentáveis tem aumentado o interesse no desenvolvimento de plataformas microbianas para novos processos, tanto para a produção de biocombustíveis quanto para a síntese de compostos que demandam alta capacidade energética e processamento químico em sua produção. O isolamento de microrganismos, capazes de degradação materiais lignocelulósicos, resistentes a diferentes inibidores e com rendimento elevado na biossíntese de moléculas específicas faz-se necessário para atender tais propósitos. Neste trabalho, microrganismos cultiváveis isolados do trato digestivo de larvas dos insetos pragas de cana-de-açúcar, besouro da raiz, Migdolus fryanus, bicudo da cana, Sphenophorus levis, broca-gigante da cana, Telchin licus licus e de broca-da-cana, Diatraea saccharalis, foram caracterizados quanto a atividade enzimática em fontes de carbono e identificados por técnicas moleculares. Larvas no terceiro ínstar de M. fryanus e S. levis e quinto ínstar de T. licus licus e D. saccharalis foram coletadas em plantios de cana-de-açúcar no interior de São Paulo. Um total de 341 microrganismos cultiváveis foram avaliados quanto a capacidade de degradação de substratos lignocelulósicos, como única fonte de carbono em meio sólido usando o índice de atividade enzimática (I.E.). Os isolados foram identificados por sequênciamento das regiões do 16S rRNA para bactérias, ITS rDNA para fungos e 26S rDNA para leveduras. Bactérias com atividade enzimática foram relacionadas aos gêneros do filo Firmicutes Bacillus, Enterobacter Serratia e Citrobacter. Os isolados relacionados à B. amyloliquefaciens destacaram-se na degradação da celulose. Entre fungos filamentosos, leveduras e leveduriformes, a atividade enzimática foi destacada para a degradação da hemicelulose. Fungos filamentosos e leveduras pertencem ao filo Ascomycota e os leveduriformes ao filo Chlorophyta. Fungos que apresentaram hidrólise de xilano foram relacionados aos gêneros Pyrenophora, Aspergillus e Penicillium. As leveduras relacionam-se a nove gêneros, majoitariamente aos gêneros Meryerozyma e Candida. Leveduras com capacidade hidrolítica destacada foram relacionadas à Meyerozyma guilliermondii, Cryptococcus laurentti, Candida pseudointermedia, C. parapsilosis, C. solani e Aureobasidium pullulans. Entre as leveduras, 40 isolados apresentam sequências que diferem em mais de 1% das sequências referência, podendo-se inferir a respeito de possíveis novas espécies. No ensaio cinético, realizado para a triagem de leveduras com capacidade de bioconversão da D-xilose em xilitol e etanol, o maior rendimento em xilitol foi demonstrado pelo microrganismo relacionado à Prototheca zopfi var. hydrocarbonea. A capacidade desse organismo de produzir xilitol não havia sido descrita. Os resultados obtidos nesse trabalho, revelaram que o trato digestivo das pragas de cana-de-açúcar como uma fonte de microrganismos que apresentam capacidade de degradação enzimática para celulose e xilano cujo potencial para utilização na biotecnologia industrial ainda precisa ser revelado / The pursuit for sustainable economies has increased the interest in development of microbial-based platforms for new processes, both for biofuel production and for the synthesis of compounds, which demands high energetic capacity and chemical processing in its production. The isolation of microorganisms capable of degradation of lignocellosic materials, resistant to different inhibitors and highly efficient in the biosynthesis of specific molecules is necessary to achieve such purposes. In this research, cultivable microorganisms isolated from the larval gut of sugarcane pests: sugarcane borer root Migdolus fryanus, sugarcane weevil Sphenophorous levis, giant sugarcane borer Telchin licus licus and sugarcane borer Diathrea saccharalis, were characterized according to their extracellular enzymatic activity in carbon sources and molecularly identified. Third-instar larvae of Migdolus fryanus and S. levis and fifth-instar larvae of T. licus licus and D. saccharalis were collected in sugarcane fields in São Paulo, Brazil. A total number of 341 strains were evaluated for their capacity of degradation of lignocelulitic substrates, as single carbon sources in solid medium. The enzymatic activities of the strains were estimated by Enzymatic Activity Index (EAI); further, the strains were molecularly identified by sequencing of the 16S rRNA region for bacterias; ITS rDNA region for filamentous fungi and 26S rDNA region for yeasts. Bacteria strains which presented enzymatic activity were related to Firmiculites genera Bacillus, Enterobacter, Serratia and Citrobacter. Strains related to B. amyloliquefaciens have demonstrated higher levels of cellulosic degradation. Among filamentous fungi, yeasts and yeast-like organisms higher activity was showed to degradation of hemicelluloses. Filamentous fungi and yeasts belong to phylum Ascomycota, and the yeast-like organisms to phylum Chlorophyta. Six filamentous fungi which presented higher hidrolisys of xylan were related to Pyrenophora, Aspergillus and Penicillium genera. Yeasts were related mostly to the Meryerozyma and Candida genera. Yeasts with higher hydrolysis cababilities were reated to Meyerozyma guilliermondii, Cryptococcus laurentti, Candida pseudointermedia, C. parapsilosis, C. solani e Aureobasidium pullulans. Among the yeasts, 40 strains showed sequences that differ by more than 1% from reference sequences, which allows infering about possible new species. In the kinetic assay carried out for screening for yeasts capable of bioconverting D-xylose into xylitol and ethanol, higher yields of xylitol were obtained for the yeast-like organism related to Prototheca zopfi var. hydrocarbonea. This microorganism\'s ability to produce xylitol had not been reported yet. The results obtained in this work have demonstrated the gut of sugarcane pests as source of microorganisms capable of enzymatic degradation of cellulose and xylan, whose potential for use in the industrial biotechnology has yet to be revealed.

Diabetes tipo 1 (DM1) e uma doenca autoimune que se inicia devido a defeitos na tolerancia imunologica a auto-antigenos, resultando na destruicao autoimune das celulas pancreaticas em individuos geneticamente suscetiveis. Os receptores NOD-like (NLRs) sao receptores intracelulares responsaveis pelo reconhecimento de padroes moleculares associados a patogenos (PAMPs) e padroes moleculares associados ao dano (DAMPs). Estudos recentes tem demonstrado que os receptores NOD1 e NOD2 desempenham um importante papel na ativacao da imunidade inata contra patogenos e na regulacao da imunidade adaptativa, uma vez que sua ativacao leva a producao de citocinas relacionadas a diferenciacao de linfocitos T auxiliares produtores de IL-17 (Th17). Porem, a importancia desses receptores no DM1 ainda e incerto. Nesse sentido, investigamos o papel dos receptores NOD1 e NOD2 na patogenese do DM1, com enfoque na diferenciacao de linfocitos Treg/Th17/Th1 e na plasticidade desses subtipos celulares. Nossos resultados mostram que camundongos deficientes de NOD2, mas nao NOD1 ou RIP2, sao resistentes ao DM1, como comprovado por menor incidencia, hiperglicemia, diminuicao do infiltrado inflamatorio e normalizacao dos niveis de insulina quando comparado aos controles. Foi observado tambem que animais NOD2-/- tiveram uma reducao da populacao de linfocitos Th17, Tc17, Th1 e T citotoxicos nos linfonodos pancreaticos, o que correlaciona com a inibicao da producao de IL-23p19 e IFN- no pancreas. Em paralelo, foi evidenciado o aumento do numero de celulas T reguladoras, macrofagos do perfil M2 nos linfonodos pancreaticos e elevada producao de IL-10 no pancreas de animais NOD2-/-. Alem disso, foi observado que animais NOD2-/- apresentaram uma menor populacao de linfocitos T duplo-positivos (Foxp3+RORt+ e IL-17+IFN+). Posteriormente, foi detectado menor producao de IL- 1, IL-6, IL-23p19 e IL-12p40 por celulas dendriticas de animais deficientes de NOD2. De forma interessante, foi observada a translocacao de bacterias para os linfonodos pancreaticos de animais diabeticos. Adicionalmente, animais tratados com antibioticos tornaram-se resistentes ao DM1, o que nos fornece indicios da contribuicao da microbiota intestinal na inducao da doenca. Por fim, comprovamos alta expressao genica de NOD2 nos linfonodos pancreaticos e no pancreas na fase inicial (pre-diabetica) em outro modelo de DM1, utilizando camundongos NOD (nonobese diabetic mice). Portanto, nossos dados indicam que a ativacao do receptor NOD2 por componentes bacterianos da microbiota intestinal induz a producao de citocinas pro-inflamatorias com subsequente diferenciacao/conversao de linfocitos do perfil Th17/Th1 e progressao do DM1. Dessa forma, estes dados apontam o bloqueio do receptor NOD2 como uma potencial terapia imunomoduladora para o DM1 em humanos. / Type 1 diabetes is an autoimmune disease that precipitates due to defects in the self tolerance to auto- antigens, resulting in the autoimmune destruction of the pancreatic cells in genetically susceptible individuals. NOD-like (NLRs) receptors are intracellular receptors responsible for the recognition of pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Recent studies have shown a role of NOD1 and NOD2 receptors in the innate immune response against pathogens and in the adaptive immune response, since its activation leads to the generation of cytokines related to the differentiation of IL-17-producing T helper cells (Th17). However, the role of these receptors in T1D remains elusive. Therefore, we investigated the role of NOD1 and NOD2 receptors in the pathogenesis of T1D, focusing on the differentiation of Treg/Th1/Th17 lymphocytes and in the plasticity of these subtypes. Our data demonstrate that NOD2-/- mice, but not NOD1-/- or RIP2-/-, are resistant to T1D, as shown by the lower incidence, hyperglycemia, less insulitis and normal insulin production when compared to wild type mice. It was also observed that NOD2-/- mice have a reduction in the Th17, Tc17, Th1 and cytotoxic T lymphocyte population within the pancreatic lymph nodes (PLNs), which correlates with the inhibition of IL-23p19 and IFN production in the pancreas. In parallel, there was an increase in Treg cells, M2 macrophages in the PLNs and IL-10 production in the pancreatic tissue of NOD2-/- mice. Also, NOD2-/- mice presented a downregulation of Foxp3+RORt+ and IL-17+IFN+ double-positive T cells. Later, it was shown that IL-1, IL-6, IL-23p19 and IL-12p40 production was downregulated in mice deficient to the NOD2 receptor. Interestingly, we observed a bacterial translocation to the pancreatic lymph nodes in diabetic mice, what could be triggering NOD2 activation, thus contributing to T1D development. As expected, mice pre-treated with antibiotics failed to become diabetic, suggesting a possible role of the gut microbiota in the development of the disease. Lastly, we observed a higher relative expression of NOD2 in the PLNs and pancreas of pre-diabetic mice, using another mouse model of the disease, the nonobese diabetic (NOD) mouse. Collectively, our data suggest that components from the gut microbiota are capable of translocating to the PLNs, thus triggering the activation of NOD2, which in turn induces the production of proinflammatory cytokines related to the differentiation of Th1/Th17 cells, thus contributing to T1D development in a mouse model of the disease. Therefore, the blockade of NOD2 appears as an interesting therapeutical target in the treatment of type 1 diabetes in humans.

The alpaca is a small South American camelid (SAC) that is an important production animal in Peru, especially among the highly impoverished communities of the high Andes, and raised for its fiber and meat. Alpacas are highly reliant on the microbes within their digestive tracts to digest the plant material they consume; volatile fatty acids (VFAs) are released as a byproduct of this microbial fermentation and used as a major source of energy by the alpaca. To explore optimal parameters for alpaca microbiome analysis, performed 16S rRNA gene surveys on alpaca C1 and fecal samples that had been extracted using one of three different DNA extraction methods (PowerFecal® DNA Isolation Kit (MO BIO); ZR Fecal DNA MiniPrep™ (Zymo); and a non-commercial extraction method called salting out) and amplified using one of two different polymerase enzyme mixes (AccuPrime™ Pfx SuperMix and 5 PRIME HotMasterMix). We found that choice of polymerase enzyme had a profound effect on the recovered microbiome, with the majority of 5 PRIME-amplified fecal samples failing to amplify. Extraction method had an effect on the recovered microbiome of fecal samples (but not C1 samples), with samples extracted using the MO BIO kit and the salting out method recovering different communities. The Zymo extraction kit returned microbial communities comparable to each of the other extraction methods. These results suggested that the AccuPrime enzyme and either the MO BIO or Zymo kits were optimal for alpaca gut microbiome analysis. We also performed two 16S rRNA gene surveys, the first from alpacas fed either a grass hay (GH) or alfalfa hay (AH) diet, and the second a C1 survey of alpacas fed two-week periods of mixed grass hay plus one of four supplements. We discovered body site and diet effects on the microbiota of alpacas fed either the GH or AH diet, with samples grouping by general body site (C1, small intestine, and distal intestine) and diet. However, we found no significant effect on the C1 microbiome of alpacas administered grain supplements. To study how energy extraction related to the microbiome, we correlated OTUs from GH/AH-fed alpaca with C1 VFA abundances. We discovered no significant correlations, and a 16S survey of low body condition (LBC) and good body condition (GBC) alpacas showed no difference in C1 microbial communities. We concluded that the microbiota of the alpaca digestive tract follow trends seen in microbiome studies of ruminants, but found no evidence of a relationship between body condition, energy extraction, and the C1 microbiome in alpacas.

Chez l’homme, la dégradation des fibres alimentaires est une des fonctions principales du microbiote colique. Elles ont de nombreux effets bénéfiques en santé humaine et pourtant les mécanismes microbiens mis en jeu dans leur dégradation restent encore largement méconnus. L’objectif de cette thèse était d’approfondir les connaissances sur la dégradation des polysaccharides pariétaux (hémicelluloses et pectines) par une espèce bactérienne prédominante du côlon de l’homme, Bacteroides xylanisolvens. L’analyse du transcriptome de B. xylanisolvens XB1AT a révélé l’existence de six et deux loci génomiques respectivement dédiés à la dégradation des pectines et des xylanes. Ces loci ou PULs (« Polysaccharide Utilization Loci ») sont connus chez Bacteroides pour coder pour des systèmes enzymatiques spécifiques d’un polysaccharide en particulier. L’analyse des CAZymes (Carbohydrate-Active Enzymes) codées par les PULs « pectinolytiques » a permis de proposer une cible polysaccharidique (homogalacturonane, rhamnogalaturonane de type I et II, arabinane) à cinq des six PULs identifiés. Les deux PULs « xylanolytiques » cibleraient les xylanes de faible complexité. La mutation du gène susC-like dans le PUL 49 et du gène HTCS (Hybrid Two-Component System) dans le PUL 43 a démontré l’importance respective de ces deux loci dans la fonction pectinolytique et xylanolytique de la bactérie. Le mutant HTCS a aussi permis de montrer pour la première fois que deux PUL peuvent être liés au niveau transcriptionnel. En présence de xylane, les données de protéomique ont souligné la surproduction par la bactérie d’une endo-xylanase possédant deux CBMs (Carbohydrate-Binding Modules). Cette enzyme modulaire pourrait être considérée comme un marqueur fonctionnel de la xylanolyse dans l’écosystème microbien intestinal. En conclusion, B. xylanisolvens déploie une machinerie enzymatique qui reflète la complexité des polysaccharides pariétaux de plantes. La plasticité métabolique de B. xylanisolvens vis-à-vis des fibres alimentaires contribue certainement à sa survie et son maintien dans le côlon humain. Des études d’écologie fonctionnelle ciblant la communauté fibrolytique intestinale sont encore nécessaires afin de mieux décrypter l’impact des fibres alimentaires et en particuliers des polysaccharides pariétaux sur le métabolisme microbien intestinal et par conséquent sur la santé humaine. / Dietary fiber degradation is a key function of the human gut microbiota. They have many beneficial effects on human health and yet microbial mechanisms involved in their degradation remain largely unknown. The aim of this thesis was to increase our knowledge on the degradation of plant cell wall polysaccharides (hemicelluloses and pectins) by a prominent human gut bacterial species, Bacteroides xylanisolvens. The transcriptome analysis of B. xylanisolvens XB1AT revealed the existence of six and two genomic loci dedicated to the degradation of pectins and xylan, respectively. These loci or PUL ("Polysaccharide Utilization Loci") are known to encode enzyme systems in Bacteroides that are specific to a particular polysaccharide. Analysis of the CAZymes (Carbohydrate-Active Enzymes) encoded by the "Pectinolytic" PULs allowed us to propose a polysaccharide target (homogalacturonan, type I and type II rhamnogalaturonane, arabinan) to five of the six identified PULs. The two identified "xylanolytic" PULs would target low complexity xylan. Mutation of the susC-like gene of PUL 49 and of the HTCS gene (Hybrid Two-Component System) of PUL 43 showed the importance of these two loci in pectinolytic and xylanolytic functions of the bacterium, respectively. The HTCS mutant also revealed for the first time that two PULs can be linked at the transcriptional level. With xylan, proteomic data highlighted the overproduction by the bacterium of an endo-xylanase with two CBMs (Carbohydrate-Binding Modules). This modular enzyme could be considered as a functional marker of xylan breakdown in the intestinal microbial ecosystem. In conclusion, B. xylanisolvens harbors an enzymatic machinery that reflects the complexity of plant cell wall polysaccharides. The metabolic plasticity of B. xylanisolvens towards dietary fibers certainly contributes to its fitness in the human gut. Functional and ecological studies targeting the intestinal fibrolytic community are still necessary to better understand the impact of dietary fibers and in particular plant cell wall polysaccharides on the intestinal microbial metabolism and consequently on human health.

Tarmfloran spelar en viktig roll för människorshälsa genom att bl.a. reglera värdens immunitet, producera näringsämnen och stärkatarmbarriären. Dysbios i tarmfloran har associerats med flera sjukdomar,inklusive diabetes typ 2. Metformin är förstahandsvalet av läkemedel mot diabetestyp 2 och har en pleotropisk effekt. Det finns stöd för att tarmarna är ettbetydande målorgan för metformin, dels för att intravenös administrering av metforminger sämre effekt än oral. Syftet med denna litteraturstudie var därför attundersöka om metformin har effekter på tarmflorans sammansättning hos människormed diabetes typ 2. Resultatet är baserat på fem olika studier hämtade frånPubmed. Alla studierna ger stöd åt att metformin förändrar tarmfloranssammansättning och indikerar även att tarmfloran är involverad i metforminsbehandlingseffekter och biverkningar. Hur omfattande förändringarna var ochvilka taxa som påverkades varierade, troligen p.g.a. olika studiedesigner. Resultatentyder på att metformin ökar den relativa förekomsten av Escherichia, Akkermansiamuciniphila och SCFA-producerande taxa, inklusive Blautia. Indikationer fanns också på att metformin minskar den relativaförekomsten av Intestinibacter.Slutsatsen är att metformin förändrar tarmflorans sammansättning, men att flerstudier krävs för att bekräfta både de taxonomiska och funktionella förändringarna.Det finns också ett behov av mer kunskap om hur resultat påverkas avsekvenserings- och statistikmetoder för att lättare kunna göra jämförelser mellan studier. / The gut microbiota has important impact on hosthealth by regulating host immunity, providing nutrients and strengthening ofthe gut integrity. A gut microbiota dysbiosis has been associated with severaldiseases, including type 2 diabetes. Metformin is the drug of first choiceagainst type 2 diabetes and has pleiotrophic effects. It has been suggestedthat the intestines is a important target of metformin because intravenousadministration exerts smaller therapeutic effects than oral. The aim of thislitterature study was to investigate if metformin alters the gut microbiotacomposition in humans. The result is based on five studies collected from Pubmed.All studies support that metformin treatment is associated with an altered gutmicrobiota and may be involved in terapeutic and side effects of metformin. Theamplitude of the metformin-induced alterations and affected taxa differed amongthe studies, probably due to large differences in study designs. Main findingssuggest that metformin increase the relative abundance of Escherichia, Akkermansiamuciniphila and SCFA-producing taxa as Blautia.Metformin also might decrease the relative abundance of Intestinibacter. In conclusion, metformin alters the gutmicrobiota, but more studies are needed to determine the taxonomic andfunctional alterations during metformin treatment. There is also a need of moreknowledge of how results are affected by sequencing and statistical methods.

Atopic allergy is the most common chronic disease among children in the developed world. This high prevalence could be associated with low microbial exposure. The early gut microbiota appears to be important for immune maturation. Immunomodulatory components in human milk might differ between mothers and could therefore explain the contradictory results seen regarding breastfeeding and allergy development. The aim of this thesis was to investigate whether early colonization with certain gut microbiota species influences childhood immune responses and allergy development up to age five. Also, as human milk oligosaccharides (HMOs) might stimulate the growth of certain gut microbiota species, the consumption of neutral colostrum HMOs was investigated for their role in allergy development up to 18 months. The concentrations of neutral colostrum HMOs varied considerably between women; however this variation could not be explained by their allergic status. Neither was the consumption of neutral colostrum HMOs related to allergy development in their children up to 18 months. Infants who harboured lactobacilli group I and Bifidobacterium adolescentis one week after birth developed allergic disease less frequently during their first five years than infants who did not harbour these bacteria at the same time. Also, colonization with several Bifidobacterium species was associated with higher levels of house dust endotoxin and larger family size. The early Bifidobacterium flora influenced levels of salivary secretory IgA at six and 12 months but not during later childhood. Moreover, the intensity of early Bacteroides fragilis colonization was inversely associated with spontaneous Toll-like receptor 4 mRNA expression in peripheral blood cells collected 12 months after birth. In conclusion, these results indicate that the early infant gut microbiota influences systemic and mucosal immune maturation during infancy, and that it might be altered in infants developing allergic disease.

Introduction: Gut microbial composition has been associated with immune-mediated diseases. Breastfeeding yields a microbiota rich in bifidobacteria and promotes colonization by lactobacilli. Bifidobacteria and lactobacilli are considered health-promoting and are used as probiotics, i.e. live microbial food supplements which when ingested in adequate amounts confer a beneficial effect on the host. During weaning the developing gut immune system is exposed to an increasing variety of antigens from both foods and gut microbiota. Aims: We aimed to determine if daily feeding of 1x108 colony-forming units (CFU) of the probiotic Lactobacillus paracasei ssp. paracasei strain F19 (LF19) to healthy term infants from 4 to 13 months of age could maintain some of the beneficial effects conferred by breastfeeding on gut microbial composition, with possible effects on gut microbial function, T cell function, Th1/Th2 immune balance and eczema incidence. Study design: Infants were randomized to daily intake of cereals with (n=89) or without LF19 (n=90) from 4-13 months of age. Clinical outcome measures were monitored by diaries and a questionnaire. Stool and blood samples were obtained at 4, 6½, 9, 13 and 5½, 6½, 12 and 13 months of age, respectively. Stool samples were analyzed for lactobacilli counts by conventional culture methods and the presence of LF19 was verified by randomly amplified polymerase chain reaction (RAPD-PCR). Fecal short-chain fatty acid (SCFA) pattern, a proxy for gut microbial function, was determined by gas-liquid chromatography. After polyclonal or specific activation of T cells, the cytokine mRNA expression levels [interleukin 2 (IL2), IFN-, IL4 and IL10] were determined on isolated mRNA by quantitative real time reverse transcriptase-PCR. Serum concentrations of total and specific IgE antibodies, Haemophilus influenzae type b, diphtheria and tetanus toxoid specific IgG antibodies were analyzed by enzyme immunoassay. Results: Feeding LF19 maintained high fecal lactobacilli counts during weaning. Persistent colonization with LF19 induced differences in the fecal SCFA pattern. The cumulative incidence of eczema was lower in the probiotic group, in conjunction with a higher IFN-γ/IL4 mRNA ratio in polyclonally activated T cells. Even though there was an effect by LF19 on Th1/Th2 immune balance, there was no effect on IgE sensitization. Infants in both groups increased their capacity to express both Th1 and Th2 cytokines during the second half of infancy but the expression was still lower than that of adults. Infants in the probiotic group had lower IL2 levels after polyclonal T cell activation at 13 months of age compared with infants in the placebo group. Infants fed LF19 did not have fewer infections, but had fewer days with antibiotic prescription compared with infants fed placebo. In addition, compared to placebo, persistent colonization by LF19 enhanced specific vaccine responses to protein antigens during the course of vaccination. Conclusions: We conclude that feeding LF19 was safe, based on no observed adverse effects in our study. Infants in both groups demonstrated maturation of adaptive immune responses during weaning. Adding probiotics in complementary foods during weaning reduced the risk of eczema by 50%, with a concomitant shift towards an enhanced Th1/Th2 ratio. The reduction of eczema might be explained by probiotic effects on both T cell-mediated immune responses and reinforced gut microbial function.