The main objectives of this study were to investigate the applicability of chitosan as an effective wood preservative against subterranean termites, conduct metagenomic analysis of the bacterial hindgut community of Reticulitermes flavipes exposed to chitosan-treated wood, and perform chitosanase activity assay of metagenomics suggested bacterial species potentially responsible for chitosan breakdown. Chitosan showed termiticidal effects on subterranean termites at varying retention levels. Termite mortality increased when exposed to samples treated with higher chitosan concentration solutions. Approximately 40 - 100% of chitosan retained in treated-wood was leached depending on the initial retention. Post-leaching results indicate chitosan is not suitable for protection against both subterranean termites in outdoor conditions, but should be effective in non-leaching/indoor applications. For metagenomic analysis of the bacterial hindgut community of Reticulitermes flavipes, two methods were used for sequence data interpretation. The Illumina BaseSpace program identified twenty-six bacteria phyla with significant differences in abundance between the chitosan-treated and control groups. The second method, mothur, identified fifteen bacterial phyla also with significant differences in abundance between both treatment groups. Similar bacterial taxa were uniquely assigned to samples from termites fed on chitosan-treated wood using both methods. These results suggest a treatment driven effect on the hindgut bacteria diversity. While majority of the bacterial taxa were common to both methods, inconsistencies detected using the BaseSpace program suggests that the Greengenes database in its present state is not reliable for 16S rRNA gene sequence analysis. As for chitosanase activity of bacterial species with significance abundance from chitosan-treated wood exposed termites, three bacteria species, Lactococcus raffinolactis, Lactococcus lactis, and Dysgonomonas gadei, were examined. After culturing on chitosan media plates and broth, no conclusive activity could be detected from all three species. Further studies need to be conducted to understand the mechanism of chitosan toxicity to termites and insects in general and to prevent chitosan leaching from treated wood. A comparative metatransciptomic study needs to be implemented to supplement the metagenomic study performed herein, so as to elucidate the exact bacteria species involved in chitosan breakdown and the enzymes produced. Also, other bacterial species suggested by the metagenomic data to possess chitosanase activity should be investigated.

In all living organisms, biological activities such as proper functioning and co-ordination of different organs will depend on different cells and molecular interactions. In some organisms the loss of functional organs or damage of organs can be lethal, whereas in others a special process called regeneration can retrieve lost organs. The molecular details of regeneration are still not completely understood in many organisms. Echinoderms are close to vertebrates in the evolutionary tree and are well known for their amazing regeneration capacity. So we chose to investigate the molecular processes of regeneration mechanism with an interest towards our favorite groups of molecules, glycosaminoglycans (GAGs). GAGs are linear polysaccharides, expressed on all cell surfaces and extracellular space and are also known to be involved in many cellular activities. We aimed to characterize the GAGs present in Echinodermata species Amphiura filiformis and investigated their role during arm regeneration. In Paper I we characterized the structure and function of GAGs from A. filiformis and identified that A. filiformis contains CS/DS type of GAGs, but no HS. The sulfation degree of these CS/DS is close to the one of heparin, i.e. they are highly sulfated. These chains are able to bind FGF-2 growth factor and induce FGF-2 mediated cell signaling. In Paper II we further characterized these GAGs for their localization and for their role in arm regeneration in A. filiformis. Immuno- and histochemical stainings on arm sections revealed that CS/DS GAGs are localized around the podia, surrounding the water vascular system, and around the muscle tissues. Inhibition of sulfated GAG biosynthesis by chlorate treatment affected the regeneration efficiency of the arms, which may be an indication of the importance of CS/DS structures in A. filiformis arm regeneration. We also characterized some bacterial sulfatases in Paper III and a lyase in Paper IV from human and canine gut symbiotic bacteria. Here we sought to find the substrate specificity and optimal conditions for these enzymes’ activities. Our findings suggest that these polysaccharide lyase and sulfatases can be used as potential tools to characterize different GAG structures and their application could further add knowledge on diseases mechanisms related to host pathogen interactions.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22494号 / 農博第2398号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5274(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 栗原 達夫, 教授 加納 健司 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Enteric viruses are infectious human pathogens that initiate infection in the gastrointestinal tract. They follow a fecal-oral route of transmission and are spread by contamination of food, water, or contact between individuals. Furthermore, enteric viruses also cause significant morbidity, mortality, and economic burdens yearly. Coxsackievirus (CV) is commonly isolated among enteric viruses and is an etiological agent of hand, foot, and mouth disease, hemorrhagic conjunctivitis, and myocarditis. The virus predominantly infects infants and young children and accounts for 11% of the fatality rate in neonates. Despite CV’s impact on human health, there are no treatments or vaccines for CV infections. Using a mouse model to study a key CV, Coxsackievirus B3 (CVB3), our laboratory has found two critical factors that impact CVB3 replication and pathogenesis. First, we have demonstrated that intestinal bacteria enhance intestinal CVB3 replication. We found that certain specific bacteria (Salmonella enterica) and its cell wall components, like lipopolysaccharides (LPS), enhanced CVB3 stability and infectivity in vitro. Additionally, we found that particular constituents of LPS are required for stability to occur. These data suggest that specific bacteria may be integral in maintaining CVB3 infectivity in the intestine. Besides virus-microbiome interaction, CVB3 is also impacted by sex hormones. Using castrated mice models, we observed a sex bias to CVB3 infection, with male mice succumbing to CVB3-induced disease at an increased rate compared to female mice. Our data suggest that testosterone, a predominant male sex hormone, enhanced CVB3 intestinal replication and viral dissemination to organs in male and female mice, but lethality only in male mice. Moreover, testosterone also affected the immune response by reducing the activation of the CD8+ T cells. CD8+ T cells are required to clear the viral infection and are integral in vaccine development. In contrast, we found an enhanced CD8+ T cell response in female mice to CVB3 infection, suggesting a sex-dependent T cell response that may underlie the sex bias in disease. Overall, these data represent an essential advancement in the CV field and will help develop future therapeutics and aid in vaccine design to limit CV infections.

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

Exercise is known to induce changes in the gut, typically referred to as the ‘forgotten organ’, and changes in gut microbiota can also occur with exercise possibly imparting systemic benefits. The question remains whether or not microbiota from an exercised animal can independently affect skeletal muscle morphology. Our first objective was to examine whether an endurance exercise program could modify the microbiota in donor mice. Second, we aimed to elucidate if such an endurance-trained microbiota could be transferred to germ-free mice via fecal inoculation. Finally, we sought to determine how the morphology and functional characteristics of skeletal muscle were influenced as a result of fecal inoculation. We hypothesized that germ-free mice recipients inoculated with the microbiota from endurance trained donors would undergo morphological changes in muscle fibre type composition and physiological changes in skeletal muscle function associated with a more oxidative phenotype. Eight-week-old male C57BL/6NCrl donor mice (n = 20) were randomized into two groups: one group completed an endurance exercise training protocol on a treadmill machine 3x/week for 11 weeks (n = 10) while one group remained cage-bound (n = 10). Ten-week-old male (n = 7) and female (n = 9) germ-free mice were colonized with the cecal microbiota of the donor mice in that, equal numbers of germ-free mice were inoculated with exercised-microbiota as sedentary-microbiota. Glucose metabolism and performance measures were evaluated in the donors as well as the recipients post-inoculation. Muscle tissue was extracted for immunohistochemistry and mitochondrial assays. During the intra-peritoneal glucose tolerance test (IPGTT), significant differences in blood glucose were found at 30min between exercise-inoculated and sedentary-inoculated (23.4 ± 2.2; 29.0 ± 1.9 mmol/L, p<0.05).and change in blood glucose relative to baseline (12.04 ± 2.4; 18.3 ± 1.9 mmol/L, p<0.01). There were significant sex-based differences in the blood glucose response in inoculated animals such that there were significant differences in blood glucose between the exercise-inoculated females and sedentary-inoculated females at 15mins (28.4 ± 2.4; 30.6 ± 1.1 mmol/L, p<0.05) and 30mins (24.7 ± 3.6; 29.9 ± 2.4 mmol/L, p<0.01), however no differences between exercise-inoculated males and sedentary-inoculated males. In addition, there were significant differences in the change in blood glucose relative to baseline between the exercise-inoculated females and sedentary-inoculated females at 15mins (12.3 ± 1.9; 20.6 ± 0.8 mmol/L, p<0.01) and 30mins (10.2 ± 2.6; 19.9 ± 2.1 mmol/L, p<0.001). This novel characterization of a link between gut microbiota and skeletal muscle suggests a transmissible capacity of microbiota to impart properties of ‘healthy’ muscle into compromised populations. / Thesis / Master of Science (MSc) / The gut microbiome or microbiota describes the composition of the human gut – remarkably, over 100 trillion bacterial cells live in symbiosis with the cells of the human body. Research from the past decade has elucidated the salient nature of the human gut microbiome on immunity, metabolic homeostasis, and overall health and disease. Transformative research in the field has demonstrated the ability to transfer these bacterial colonies from one individual to another and elicit change, such as altering body mass and adiposity, respective to their donor. The interaction between gut microbiota and other organ systems i.e. brain, liver, adipose tissue has been the focus of several recent investigations, suggesting that lifestyle changes such as diet and exercise can influence communication between the gut and various other organs and contribute to changes in function. Skeletal muscle is the largest muscle in the human body accounting for 40% of total mass and although the main role of skeletal muscle is locomotion and postural stabilization, it is integral for the regulation of blood glucose as well as a reservoir for other macronutrients. Acute and chronic physical exercise cause a myriad of adaptive responses throughout the human body including in skeletal muscle and the gut. Therefore, the existence and influence of a gut-muscle link or ‘axis’ on human health cannot be ignored. What is unclear exactly, is if exercise-induced adaptations in the gut of an individual can be transferred to elicit change in the gut of a recipient and further induce adaptations at the level of the skeletal muscle.

No description available.

La présence de sucres complexes constitue une source nutritive importante pour le microbiote qui assure leur dégradation via des CAZymes. Dans le cadre de cette thèse, nous avons construit in silico un modèle de type minimicrobiome contenant 177 génomes représentatifs des communautés bactériennes dans un microbiote intestinal conventionnel. L’analyse du contenu de ce minimicrobiome nous a permis d’estimer leur abondance et leur diversité. De plus, la comparaison du contenu CAZymes par groupe bactérien de type « phylum » a révélé une variabilité inter-phylum, notamment une diversité de familles CAZymes et une abondance en gènes bien plus élevées chez les Bacteroidetes. Dans un deuxième temps, nous avons développé une puce à ADN sur laquelle nous avons greffé des sondes non redondantes ciblant plus de 6500 gènes codant des CAZymes. Nous avons ensuite testé la "puce CAZyme" par hybridation d’ADN bactérien extrait d’échantillons de selles. Nos résultats suggèrent que cette méthode serait plus sensible dans la détection de CAZymes provenant de bactéries rares par rapport à la métagénomique. Ainsi, il est intéressant de noter qu’en utilisant la puce CAZyme, nous avons pu détecter un gène codant pour une famille GH6, alors que les études métagénomiques n’ont jamais réussi à détecter ce gène dans le microbiome intestinal humain et animal. Enfin, l’examen de huit échantillons de selles a permis l’identification d’un noyau CAZome contenant 46 familles de GHs et PLs, ce qui suggérerait que le microbiote intestinal est caractérisé par une stabilité fonctionnelle en dépit de variations taxonomiques importantes entre les individus testés et indépendamment de leur état de santé. / The bacterial communities that inhabit our gut ensure their growth and survival by extracting their carbon source from the food that transits through the intestines. The complex carbohydrates included in the human diet are almost exclusively degraded by the gut microbiota using CAZymes. We built a minimicrobiome model using 177 genomes associated to gut microbiota. The CAZyme content analysis revealed their huge diversity and abundance in our minimicrobiome model. At the phylum level, the Bacteroidetes genomes showed the greatest CAZyme diversity and abundance. Interestingly, as most of CAZymes found in Bacteroidetes genomes contain a signal peptide allowing their secretion in the intestinal lumen and/or in periplasmic space, members of this phylum are suggested to be the primary degraders of complex carbohydrates. Further, we developed a microarray containing probes to target more than 6,500 CAZyme genes. We then validated the CAZyme microarray by the hybridization of bacterial DNA extracted from the stool samples of individuals. Our results suggest that a microarray-based study can detect genes from low-abundance bacteria better than metagenomic-based studies. A striking example was the detection of gene encoding a GH6-family in all subjects examined, whereas metagenomic studies have consistently failed to detect this gene in both human and animal gut microbiomes. In addition, an examination of eight stool samples allowed the identification of a corresponding core CAZome containing 46 CAZymes families that suggests a functional stability of the gut microbiota despite large taxonomical variations between individuals and independently of health state.

In the human gut, bacterial fermentation of dietary fibers and proteins produces metabolites, primarily as short-chain fatty acids (SCFA), that are highly beneficial for host health. However, unlike dietary fiber, bacterial fermentation of protein additionally generates potentially toxic substances such as ammonia, hydrogen sulfide, amines, and indoles. It is believed that most gut bacteria favor utilization of dietary fiber over that of protein for energy. Therefore, when fermentable dietary fiber is readily available to colonic bacteria, protein fermentation, and its subsequent potentially toxic metabolites, remains relatively low. Dietary intake primarily determines the quantity of dietary fiber and protein substrate available to the gut microbiota and the resulting profile of metabolites produced. Increased protein consumption is associated with deleterious health outcomes such as higher risk of colorectal cancer and type II diabetes. Conversely, diets following US dietary recommendations are high in fiber, which promote a healthy microbiome and are protective against disease. Diets following the recommendation are also moderate in protein intake so that, ultimately, far more fiber than protein is available for colonic bacterial fermentation. On the contrary, dietary fiber intake is chronically low in a standard Western diet, while protein consumption is above dietary recommendations, which results in nearly equal amounts of dietary fiber and protein available for gut microbial fermentation. Furthermore, the popularity of high-protein diets for athletes, as well as that of high-protein low-carbohydrate diets for weight loss, may flip fiber and protein substrate proportions upside down, resulting in more protein than fiber available in the gut for fermentation. The objective of this study was to elucidate how substrate ratios in protein-fiber mixtures affect protein fermentation and metabolites, as well as examine the degree to which fiber source may influence these outcomes. Each dietary fiber source [fructooligosaccharides (FOS), apple pectin (Pectin), a wheat bran and raw potato starch mixture (WB+PS), and an even mixture of the three aforementioned fibers (Even Mix)] and protein were combined in three ratios and provided as substrate for in vitro fecal fermentation to understand how low, medium, and high fiber inclusion levels influence fermentation outcomes. They were compared to 100% protein and fiber (each different fiber) controls. Branched-chain fatty acids (BCFAs), metabolites produced exclusively from protein fermentation, were used as a measure of protein fermentation; the data were normalized based on the initial quantity of protein within the substrate. In protein-fiber substrate mixtures, only FOS and Even Mix inhibited BCFAs (mM/g protein basis) and only when they made up at least half of the substrate. Unexpectedly, the rate of protein fermentation was increased when the protein-fiber substrate contained 25% WB+PS fiber, possibly due to the starch component of the fiber. There was evidence that when pH drops during fermentation, as was the case for protein-FOS mixtures, it played a significant role in suppressing protein fermentation. Ammonia production was not largely affected by increasing the proportion of dietary fiber. A significant reduction did not occur until FOS made up at least 50% of the protein-fiber substrate; for Pectin, WB+PS, and Even Mix fibers, 75% inclusion was required for a significant decrease in ammonia. Interestingly, protein was butyrogenic. Protein as the sole substrate produced more butyrate than either Pectin or Even Mix as the sole substrates, and in fact, addition of Pectin to protein significantly reduced butyrate concentrations. However, the possible benefits of butyrate produced via protein fermentation needs to be tempered by the production of potentially toxic compounds and the association between protein fermentation and colorectal cancer. Overall, the thesis findings showed protein fermentation to be relatively stable and not easily influenced by increasing the availability of dietary fiber, and no clear evidence of microbial preference for carbohydrates over protein was found.

No description available.