Inflammatory bowel disease (IBD) is a growing disorder with unknown etiology. However, increasing evidence strongly highlights the role of gut microbiota with possible involvement of microbial plasmidome in the inflammatory process. Although the composition of the gut microbiota has been extensively studied, important functional groups such as butyrate producers remain poorly characterized, particularly in pediatric IBD. Furthermore, evaluation of the gut plasmidome in healthy and IBD children is missing. In this study, we used molecular techniques involving quantitative PCR (qPCR) and next-generation sequencing of functional and 16S rRNA genes to analyze the level and composition of butyrate-producing microbes in mucosal washes collected from the right colon of healthy children and Crohn's disease (CD) patients during diagnostic colonoscopy. Also, we isolated and characterized the gut plasmidome from the right colon mucosal washes collected from pediatric non-IBD control, ulcerative colitis (UC), and CD subjects. Although no difference was observed in the total amount of butyrate producers that utilize the butyrate kinase (BUK) pathway for butyrate synthesis, butyrate producers that use the butyryl CoA:acetate CoA-transferase (BCoAT) pathway were decreased in CD patients with inflamed colon as compared to controls. This functional gene approach shows that pediatric CD is characterized by generalized decreased abundance of Eubacterium rectale and increased abundance of Faecalibacterium prausnitzii in patients with inflamed colon. Also, phylogenetic analysis highlighted 15 Operational Taxonomic Units (OTUs) as potential novel butyrate producers, five of which were decreased in CD patients. Using 16S rRNA sequencing approach validated the functional gene results and showed decreased abundance of Coprococcus in CD patients with inflamed colon. Furthermore, non-IBD plasmidome has higher level of genes involved in butyrate synthesis and regulation of different cellular processes and stress response. On the other hand, IBD plasmidome is enriched with antibiotic resistance genes and phage elements, and pediatric CD plasmidome in particular has higher abundance of the adenosine-5'-phosphosulfate reductase gene. Altogether, our study represents the first comprehensive description of gut butyrate producers and plasmidome of pediatric subjects that emphasize a characteristic dysbiosis of butyrate producers in pediatric CD and a potential link between the gut plasmidome and IBD pathogenesis.

Colorectal cancer (CRC) has a 5-year survival rate of 68% yet it still has a mortality rate of 50,000 per year. While CRC has a host of causes, one that stands out is TGFβ deficient signaling, which is disrupted in a majority of high-microsatellite-instability or inflammation-associated CRCs. Since TGFβ is a multifunctional cytokine, it has been elusive to determine whether its effect on cancer development is operating through inflammation, differentiation or developmental pathways. Additionally, it is now becoming apparent that a great number of CRC cases can be associated with and possibly caused by gut bacteria dysbiosis. Here, I present a metagenomic and metatranscriptomic study of the interactions between TGFβ deficient signaling, inflammatory signaling, and the microbiome in a CRC mouse model. TGFβ deficient mice have reduced amounts of Firmicutes as well as mRNA counts of a key butyrate enzyme. Lack of butyrate, as shown by previous literature, could be inhibiting apoptosis and promoting growth. Also, TGFβ deficient mice have increased mRNA counts of polyamine producing genes, which could act synergistically with butyrate reduction. I find that H. hepaticus inoculation, as a source of inflammatory signaling, affects another species, M. schaedleri, to produce pro- inflammatory lipopolysaccharides. Additionally, H. hepaticus itself has increased oxidative phosphorylation; reactive oxygen species from this process could be adding to cancer-promoting DNA damage. Taken together, TGFβ deficient signaling and H. hepaticus inoculation, disrupt enough pathways to cross the threshold of carcinogenicity in 40% of the mice in our study. The results of this study emphasize the importance of microbiome function and represent possible new avenues of treatment.

Orientador: Gustavo do Valle Polycarpo / Resumo: A restrição do uso de antibióticos e quimioterápicos na alimentação animal, alavancada pela União Europeia em 2006, provocou um aumento nas pesquisas em busca de aditivos alternativos sobre o desempenho de frangos de corte. Objetivou-se desenvolver uma meta-análise de ácido butírico como melhorador de desempenho em alternativa aos antibióticos sobre o desempenho de frangos de corte. A busca digital por estudos incluiu artigos científicos publicados na base de dados Scopus nos anos de 2014 a 2019 por meio de três principais palavras-chave broiler, butyric acid e performance, e seus sinônimos. Foram selecionados artigos descrevendo experimentos in vivo com frangos de corte, suplementados com ácido butírico com respostas de desempenho. Os dados foram sistematizados em planilhas eletrônicas e classificados de acordo com o tipo de desafio sanitário (sem ou com inoculação de microrganismos patogênicos). Ao todo foram utilizados 36 experimentos para avaliar o efeito do ácido butírico. Com base em 36 estudos foram criados dois ensaios, um primeiro considerando o tratamento controle contra o tratamento suplementado com ácido butírico e um segundo ensaio considerando-se apenas 10 experimentos, os quais também continham um tratamento controle positivo contendo antibiótico para avaliar o efeito do ácido butírico como alternativa aos promotores de crescimento (ensaio 2). A análise estatística foi realizada por meio do software SAS University Edition, versão 9.4. Foi utilizado um modelo mi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The restriction of the use of antibiotics and chemotherapy in animal feed, leveraged by the European Union in 2006, caused an increase in research in search of alternative additives on the performance of broilers. The objective was to develop a meta-analysis of butyric acid as a performance enhancer as an alternative to antibiotics on broiler performance. The digital search for studies included scientific articles published in the Scopus database in the years 2014 to 2019 through three main keywords broiler, butyric acid and performance, and their synonyms. Articles were described describing in vivo experiments with broilers, supplemented with butyric acid with performance responses. The data were systematized in electronic spreadsheets and classified according to the type of health challenge (without or with inoculation of pathogenic microorganisms). Altogether 36 experiments were used to evaluate the effect of butyric acid. Based on 36 studies, two trials were created, a first considering the control treatment against treatment supplemented with butyric acid and a second trial considering only 10 experiments, which also contained a positive control treatment containing antibiotics to evaluate the effect of the acid butyric as an alternative to growth promoters (trial 2). Statistical analysis was performed using the software SAS University Edition, version 9.4. A mixed model was used through the MIXED procedure, in which the effect of each experiment was considered as a clas... (Complete abstract click electronic access below) / Mestre

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23799号 / 医博第4845号 / 新制||医||1058(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 妹尾 浩, 教授 中川 一路, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The objective of this thesis was to further develop the polymer composite system consisting of cellulose acetate butyrate (CAB) and high modulus, continuous, regenerated cellulose fiber (lyocell). Of particular concern were both the interfacial adhesion between the fiber and matrix and the consolidation process in the manufacture of these composite materials. Interfacial adhesion was found to be substantial due to the relative lack of the fiber pull-out phenomenon observed after tensile failure in the unmodified fiber composites. This result was then supported in the second study in which similar unmodified fiber composites experienced very little fiber pull out with evidence of a large amount of cohesive failure of the matrix accompanied by matrix particles adhering to the fiber surfaces. Void volume formation was mitigated to a small extent by the use of optimal consolidation conditions. Composites formed at moderate temperature (200 °C), low consolidation pressure (11.8 p.s.i.) and high consolidation time (13 min.) were found to have the lowest void volume formation of ca. 2.8 %. These composites were generally found to have the highest interfacial shear strength, ca. 16 MPa. A tensile modulus of 22 GPa and an ultimate strength of 246 MPa was obtained for this composite having a fiber volume content of ca. 62 %. / Master of Science

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

L’émergence et le développement des maladies métaboliques est sous le contrôle de multiples facteurs génétiques et environnementaux. Le diabète et la résistance à l’insuline sont des maladies métaboliques caractérisées par des défauts dans la sécrétion de l’insuline ou son utilisation périphérique, ou les deux. L’insuline est l’hormone clé de l’utilisation du glucose, et régule également transcriptionnellement et épigénétiquement l’expression des gènes.En travaillant sur le muscle, l’implication de l’épigénétique dans la régulation de l’expression des gènes de la voie de l’insuline a été mis en évidence. L’hexokinase 2 (HK2) est régulée par l’insuline et participe au métabolisme glucidique. Le rôle de l’épigénétique y est démontré avec l’augmentation de l’acétylation des histones autour du site d’initiation de la transcription (SIT) de HK2 et l’accumulation d’une isoforme permissive des histones, H2A.Z. Ces deux phénomènes sont le signe d’une transcription permissive.Nous avons ensuite étudié le rôle de l’acétylation des histones dans les régulations amenées par l’insuline dans les myotubes L6. Nous avons utilisé le butyrate, un inhibiteur des histones deacetylase (HDACi), dans un contexte d’insulino-résistance induite par une lipotoxicité. Le butyrate a en partie restauré la sensibilité à l’insuline visible au niveau des phosphorylations de la PKB (protein kinase B) et de la MAPK (Mitogen-activated protein kinase), inhibées par le traitement au palmitate. Le butyrate a augmenté l’expression de l’ARNm et de la protéine d’IRS1. La surexpression génique d’IRS1 est épigénétique-dépendante car liée à une augmentation de l’acétylation des histones au SIT d’IRS1.L’ensemble de ces résultats démontre l’existence d’un lien entre les modifications épigénétique et l’action de l’insuline. Cela suggère qu’une intervention pharmacologique sur la machinerie épigénétique pourrait être un moyen d’améliorer le métabolisme, et l’insulino-résistance / Diabetes and insulin resistance are metabolic diseases characterized by altered glucose homeostasis due to defects in insulin secretion, insulin action in peripheral organs, or both. Insulin is the key hormone for glucose utilization and regulates gene expression via transcriptional and epigenetic regulations.We determined the epigenetic implications in the regulation of expression of insulin signaling pathway genes. Hexokinase 2 (HK2) is known to be upregulated by insulin and directs glucose into the glycolytic pathway. In L6 myotubes, we demonstrated that insulin-induced HK2 gene expression rely on epigenetic changes on the HK2 gene, including an increase in histone acetylation around the transcriptional start site (TSS) of the gene and an increase in the incorporation of the histone H2A.Z isoform – a histone variant of transcriptionally active chromatin. Both are epigenetic modifications compatible with increased gene expression.To elucidate the role of histone acetylation in the regulation of insulin signaling and insulin-dependent transcriptional responses in L6 myotubes, we investigated the effects of butyrate, an histone deacetylase inhibitor (HDACi), in a model of insulin resistance induced by lipotoxicity. Butyrate partly alleviated palmitate-induced insulin resistance by ameliorating insulin-induced PKB (protein kinase B) and MAPK (Mitogen-activated protein kinase) phosphorylations, downregulated with exposure to palmitate. Butyrate induced an upregulation of IRS1 gene and protein expression. The transcriptional upregulation of IRS1 was proven to be epigenetically regulated, with butyrate promoting increased histone acetylation around the TSS of the IRS1 gene.These results support the idea of the existence of a link between epigenetic modifications and insulin action. Pharmacological targeting of the epigenetic machinery might be a new approach to improve metabolism, especially in the insulin resistant condition.Key words: Muscle, insulin resistance, epigenetic, chromatin, histone acetylation, histone deacetylase inhibitor (HDACi), butyrate, palmitate

Les potentialités Fibrobacter succinogenes, l'espèce majeure du rumen, ont été utilisées pour la dégradation de végétaux et dans le cadre du projet MELiSSa créé par l'ESA. Dans un premier temps des cultures sur glucose nous ont permis de maîtriser notre procédé et de valider les techniques utilisées. Les cultures sur déchets végétaux (chou, soja et paille) par Fibrobacter succinogenes ont permis de mettre en évidence des cinétiques de dégradation différentes selon le substrat utilisé. Ensuite, les cultures sur un substrat issu d'une première dégradation par biométhanogenèse (projet MELiSSA), ont permis une amélioration du système et ont révélé des rendements de dégradation importants. La recherche d'un contaminant par biologie moléculaire a été menée pour expliquer la production de butyrate dans nos cultures. Enfin le programme "Anaerobic Waste Compartment Modelling and Simulation" nous a montré une très grande similitude avec les résultats expérimentaux

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

A influência do butirato de sódio (NaBu), do cloreto de manganês (MnCl2), combinados ou isolados, e da cicloheximida (CHX) na síntese de tireotrofina humana recombinante (r-hTSH) derivada de células CHO foi investigada pela primeira vez. Com exceção da CHX, que gerou uma redução de 1,5 vezes na produtividade volumétrica, todos os reagentes geraram um aumento (1,4 vezes para o MnCl2 e 3 vezes para o NaBu e NaBu+MnCl2) na produtividade volumétrica. Também foi observada uma diminuição no número de células viáveis com a utilização de todos os reagentes. A adição destes reagentes ao meio de cultura de células CHO produtoras de hTSH gerou também alterações na glicosilação do r-hTSH. As alterações geradas pela presença de CHX foram todas negativas, resultando em uma diminuição de 5,5% no conteúdo de ácido siálico, 10% na ocupação dos sítios de glicosilação, além de uma diminuição no conteúdo de todos os monossacarídeos neutros. Os demais reagentes, por outro lado, resultaram em alterações positivas. A presença de NaBu no meio de cultura acarretou um aumento de 12% no conteúdo de ácido siálico e de 3% na ocupação dos sítios de glicosilação. Com relação às estruturas de carboidratos presentes no hTSH, foi observado um aumento de 14% nas estruturas bi-antenárias, aumento no conteúdo de manose e fucose e uma diminuição no conteúdo de galactose e N-acetilglucosamina. A presença de MnCl2 no meio de cultura resultou em um aumento de 1,7% no conteúdo de ácido siálico e de 1,3% na ocupação dos sítios de glicosilação. Houve ainda um aumento no conteúdo de manose e N-acetilglucosamina e uma diminuição no conteúdo de fucose e galactose. Um aumento de 7% na freqüência de estruturas bi-antenárias foi também observado quando MnCl2 foi utilizado. O uso simultâneo de NaBu e MnCl2 proporcionou um aumento de 14% no conteúdo de ácido siálico e de 3% na ocupação dos sítios de glicosilação, bem como um aumento no conteúdo de todos os monossacarídeos neutros. Não houve, entretanto, alterações na freqüência das estruturas de carboidratos. Apesar de todas as alterações causadas pelos diferentes reagentes, não foram observadas diferenças na atividade biológica ou no comportamento farmacocinético das diferentes preparações de hTSH estudadas. Este estudo é extremamente relevante, tanto do ponto de vista do desenvolvimento de novos biofármacos, quanto do ponto de vista do controle de qualidade das glicoproteínas hormonais já utilizadas na clínica médica. / The influence of sodium butyrate (NaBu), manganese chloride (MnCl2), combined or isolated, and cycloheximide (CHX) on the synthesis of human thyrotropin CHO-derived (r-hTSH) was investigated for the first time. Exception made for CHX, which caused 1.5-fold reduction on volumetric productivity, all other reagents caused an increase in the volumetric productivity of 1.4-fold with MnCl2 and 3-fold with NaBu and NaBu+MnCl2. The number of viable cells decreased in presence of all these reagents. They also caused alterations in the glycosylation of r-hTSH. When CHX was utilized, a decrease in the content of sialic acid (5.5%), in the site occupancy (10%) and in the content of neutral monosaccharides was observed. In contrast, the addition of NaBu to culture medium caused an increase of about 12% in the sialic acid content and of 3% in the site occupancy. Concerning to carbohydrate structures, an increase of 14% of bi-antennary structures was achieved. In addition, an increased content of mannose and fucose and a decrease in the content of galactose and N-acetyl glucosamine was also observed. When MnCl2 was utilized, an increase of 1.7% in the sialic acid content and of 1.3% in site occupancy occurred. An increased content of mannose and N-acetyl glucosamine was also observed, while the content of fucose and galactose decreased. Concerning to carbohydrate structures, an increase of 7% on bi-antennary structures was achieved. When NaBu and MnCl2 were utilized simultaneously, we observed an increase of about 14% in the sialic acid content, of 3% in the site occupancy, as well as an increase in the content of all neutral monosaccharides. The frequency of carbohydrates structures, however, was not altered. Despite all the changes caused by these different reagents, in vivo biological activity and pharmacokinetic behavior were found not significant different in all the hTSH preparations studied in the present work.