Agarose and porphyran are related galactans that are only found within red marine algae. As such, marine microorganisms have adapted to using these polysaccharides as carbon sources through the acquisition of unique Carbohydrate Active enZymes (CAZymes). A recent metagenome study of the microbiomes from a Japanese human population identified putative CAZymes in several bacterial species, including Bacteroides plebeius that have significant amino acid sequence similarity with those from marine bacteria. Analysis of one potential CAZyme from B. plebeius (BpGH50) is described here. While displaying up to 30% sequence identity with β-agarases, BpGH50 has no detectable agarase activity. Its crystal structure reveals that the topology of the active site is much different than previously characterized agarases, while containing the same core catalytic machinery. It is unclear whether the enzyme has endo- or exo- activity; the large binding ‘groove’ is typical of an endo-acting enzyme, while a loop at one end of the groove may provide a terminal pocket for the substrate, which is suggestive of exo-activity. Furthermore, the enzyme contains a basic pocket that may dock a sulphated substrate, like porphyran. While no quantifiable porphyran activity was observed, properties of the putative active site suggest that this unusual enzyme may be specific on an unusual substrate, such as a porphyran-agarose hybrid. / Graduate

The wide use of nanoscale materials in food and health care products raises the concern of their possible uptake across the gastrointestinal tract, but very limited data are available on their uptake kinetics, and the potential hazards for humans. In this study, the uptake mechanism of titanium dioxide (TiO2) across the isolated perfused fish intestine and human intestinal Caco-2 cells were evaluated. The in vitro preparation of the whole gut sac and the isolated perfused intestine of rainbow trout were performed using both bulk and nano TiO2 in a concentration of 1 mg l-1 for up to 4 h. The results showed that the Ti from both bulk and TiO2 NPs were mainly accumulated in the mid and hind intestine, with 80% or more of the accumulation in the mucosa rather than the underlying muscularis. Perfused intestines showed a saturable, time-dependent accumulation of the Ti from TiO2 and the uptake of Ti from exposure to NPs was faster than that of the bulk form. The uptake of Ti from exposure to TiO2 NPs increases 10 fold when the CO2 in the gas mixture was lowered to 0.5%. Subsequently, further investigation on the mechanisms of uptake of TiO2 was applied using different kinds of inhibitors. Adding 10 mmol l-1 cyanide did not stop Ti uptake from TiO2 exposures, and 100 µmol l-1 vanadate (ATPase inhibitor) caused a 2.8 fold reduction in the net uptake rate of Ti for the TiO2 NP exposure. Luminal additions of 120 IU ml-1 nystatin (endocytosis inhibitor) blocked the uptake of Ti from both bulk and TiO2 NPs treatments. The results indicate that Ti accumulation from TiO2 exposures was sensitive to both nystatin and vanadate; the former suggesting that there is an endocytosis involvement in the uptake of TiO2 across the intestinal epithelium. Human intestinal Caco-2 cell showed a steady, saturable and time-dependent accumulation of Ti over 24 h exposures to 1 mg l-1 TiO2 (for all forms of TiO2). A scanning electron microscope study indicated the appearance of the particles underneath the cells, increasing the evidence of the Ti uptake from different forms of TiO2 by Caco-2 cells. Both nystatin and vanadate increase the accumulation of TiO2 which suggests interference of these drugs with endocytic pathways. All the data in the thesis demonstrates Ti uptake across the intestinal epithelium from TiO2 exposures involving CO2-dependent and nystatin-sensitive mechanisms. The results in this thesis have contributed to some understanding on the behaviour, uptake and effects of the TiO2 NPs across the intestine; and highlight the possible dietary hazard of the NPs to human health.

The human gut microbiome is a complex microbial ecosystem residing in the lumen of our gastrointestinal tract. The type and amounts of microbes present in this ecosystem varies based on numerous factors, including host genetics, diet, and environmental factors. The human gut microbiome plays an important role in normal host physiological functions, including providing energy to colonocytes in the form of short-chain fatty acids. However, gut microbial metabolites have also been associated with numerous disease states. Current tools for analyzing the gut microbiome, such as high-throughput sequencing techniques, are limited in their predictive ability. Additionally, “-omic” approaches of studying the complex array of molecules, such as transcriptomics (RNA), proteomics (proteins), and metabolomics (previously identified physiologically active molecules), give important insight as to the levels of these molecules but do not provide adequate explanations for their production in a complex environment. With a better physiological understanding of why specific metabolites are produced by the gut microbiome, more directed therapies could be developed to target their production. Therefore, it is immensely important to study the specific bacteria that reside within the gut microbiome to gain a better understanding of how their metabolic actions might impact the host. Within this framework, this study aimed to better understand the production of secondary bile acid metabolites by bacterial in the gut microbiome. High levels of secondary bile acids are associated with numerous pathophysiological disorders including colon cancer, liver cancer, and cholesterol gallstone disease. In the current study, three bile acid metabolizing strains of bacteria that are known members of the gut microbiome were studied. A novel strain of Eggerthella lenta was identified and characterized, along with the type strain, for its ability to modulate bile acid and steroid metabolism based on the atmospheric gas composition. Additionally, it was shown that the oxidation of hydroxyl groups on primary bile acids by E. lenta C592 inhibited subsequent 7α-dehydroxylation by Clostridium scindens. The gene involved in the production of a Δ4,6-reductase enzyme, responsible for catalyzing two of the final reductive steps in the 7α-dehydroxylation pathway, was putatively identified and characterized in Clostridium scindens ATCC 35704. Lastly, the transcriptomic profile of Clostridium scindens VPI 12708 in the presence of numerous bile acids and steroid molecules was studied. These studies contribute significantly to the understanding of why specific bile acid metabolites are made by members of the gut microbiome and suggest ways of modulating their production.

Variations in drug metabolism may alter drug efficacy and cause toxicity; better understanding of the mechanisms and risks shall help to practice precision medicine. At the 21st International Symposium on Microsomes and Drug Oxidations held in Davis, California, USA, in October 2-6, 2016, a number of speakers reported some new findings and ongoing studies on the regulation mechanisms behind variable drug metabolism and toxicity, and discussed potential implications to personalized medications. A considerably insightful overview was provided on genetic and epigenetic regulation of gene expression involved in drug absorption, distribution, metabolism, and excretion (ADME) and drug response. Altered drug metabolism and disposition as well as molecular mechanisms among diseased and special populations were presented. In addition, the roles of gut microbiota in drug metabolism and toxicology as well as long non-coding RNAs in liver functions and diseases were discussed. These findings may offer new insights into improved understanding of ADME regulatory mechanisms and advance drug metabolism research. (C) 2017 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

Immune-mediated inflammatory diseases (IMID) represent a group of ostensibly unrelated, chronic and highly disabling diseases that preferentially affect different organ systems. IMID are assumed to manifest as a result of the accumulation of genetic, environmental and immunological factors. A fundamental commonality between IMID is the idiopathic nature of disease, and moreover, substantial similarities are apparent in disease etiopathogenesis. The complex assemblage of microbes and their genes that exists within and on the human body, collectively known as the microbiome has emerged as a critical factor in human health and, altered microbial populations within the gastrointestinal tract lumen and mucosa have been linked to several IMID. Accordingly, we conducted several studies investigating the association of the gut microbiome with IMID. Our main study investigated differences in the microbial profile and functional potential of multiple IMID utilizing 16S rDNA amplicon sequencing and analysis of stool. We also investigated the mucosal-associated microbiome in IBD to characterize the microbial populations and their functions residing in distinct gastrointestinal compartments from inflamed and noninflamed mucosa. We also explored a potential environmental factor; specifically assessing whether microbes present in drinking water in low or high incidence areas of IBD might contribute to disease etiology. The findings of these studies are manifold. First, we show important differences of the stool microbial profile in IMID. In doing so, we were able to identify distinct states of gut dysbiosis and have revealed numerous microbes that are consistently or uniquely disproportionate between IMID. Second, we have shown the microbial profile associated with inflamed and noninflamed mucosa and have reported that a localized dysbiosis is not observed in the presence of inflammation. Third, we have revealed that distinct gastrointestinal compartments are comprised of similar microbial communities. Lastly, we have reported the drinking water microbiome to differ between low and high incidence areas of IBD, thus suggesting a potential role in IBD etiology. Understanding the role of the gut microbiome in human disease will enable the development and application of more appropriate therapeutic strategies that specifically target microbes within the gut. / May 2017

Primates who disperse from their natal group may shape their adult stable gut microbiome through physical contact and shared environments with their new group members. However, it is possible that individuals retain the dominant microbiome composition that they developed as an infant in their natal group even after joining their new group, due to a combination of genetic inheritance and exposure to their natal group environment. Microbial exposure during early life, before an immune system has been developed, can exert strong selection on a developing individual, in effect creating a selection bottleneck. Therefore, the environmental signals transmitted from mother to infant are critical in developing an infant’s immunocompetence. Determining what adaptations take place in an individual’s gut microbiota during their life could help determine the maternal importance of gut microbe transmissions which may be essential to the evolutionary success of a species. We studied Eulemur rubriventer (red-bellied lemurs) who live in family groups. We tested whether individuals now living in different social groups as adults overlap in microbe composition, and if areas of overlap are distinct compared with unrelated individuals. We also tested whether the gut microbiomes of co-residents (dispersed adult group-mates) would be more similar than that of individuals living in different groups. Using census and genetic data, we determined the social group membership and relatedness of 15 individuals in Ranomafana National Park, Madagascar. Quantitative real-time PCR and Microbial 16S ribosomal RNA gene sequencing indicated that E. rubriventer kinship accounted for just 2.4% of variability in gut microbiome diversity. Our findings indicate that host adult social group explained 25% of the variation in composition of E. rubriventer microbiomes. Additional research incorporating an increased sample size to include additional kin dyads is necessary to fully understand the influence of genetic kinship and early life colonization on the GI microbiome. If initial microbial colonizing species are retained in adults, this demonstrates that early life colonization can persist through adulthood and perhaps preserve important microbial species across larger evolutionary time scales.

Le Loup ou Bar Dicentrarchus labrax est une espèce euryhaline dont les femelles pondent généralement en eau de mer. Pendant son cycle de vie, des migrations vers les estuaires et les lagunes, peuvent exposer très tôt les jeunes stades à des variations de salinité. Les mécanismes de l'osmorégulation sont bien connus chez les prélarves, les larves et les adultes de D. labrax en eau de mer (EM, 38) et en eau de mer diluée (EMD, 5), cependant les possibilités d'osmorégulation et leurs mécanismes sont inconnus chez les embryons. Le but de cette étude a été d'évaluer la mise en place de la fonction osmorégulatrice chez les embryons de D. labrax.Tout d'abord le développement embryonnaire des différents sites osmorégulateurs a été décrit, en se focalisant sur le tube digestif, en incluant le pharynx et les premières fentes branchiales. La formation de ces structures débute au stade 12 somites (S) et a été décrite jusqu'à l'ouverture de la bouche, 5 jours après l'éclosion.En second lieu, le lieu et la cinétique d'apparition des premières cellules osmorégulatrices, les « ionocytes » ont été recherchés. Ces cellules ont été identifiées au stade 12S sur la membrane de la vésicule vitelline et au niveau des premières fentes branchiales et du tube digestif primitif au stade 14S. La fonctionnalité de ces cellules a été étudiée grâce à des immunomarquages des principales protéines transmembranaires impliqués dans l'osmorégulation [l'ATPase Na+/K+ (NKA), le cotransporteur Na+/K+/2Cl- (NKCC) et le canal à chlore (CFTR)], et avec une étude ultrastructurale. Des ionocytes potentiellement fonctionnels sont présents à partir du stade 25S au niveau de la membrane de la vésicule vitelline et du tube digestif primitif, mais les ionocytes des premières fentes branchiales ne sont pas totalement fonctionnels à l'éclosion. L'existence d'un phénomène de boisson passive qui permettrait la régulation hydrique chez D. labrax est envisagé.Finalement, l'osmorégulation embryonnaire existant en EM et en EMD a été étudiée. Des mesures nanoosmométriques des fluides embryonnaires indiquent une capacité à hyper- et hypo-osmoréguler. Cependant, en EMD, des analyses en qRT-PCR et des immunomarquages de NKA, NKCC et CFTR révèlent que les mécanismes de l'hyper-osmorégulation peuvent limiter les pertes ioniques mais ne sont pas suffisamment efficaces pour permettre une acclimatation totale à l'EMD à ce stade très précoce. / The European sea bass Dicentrarchus labrax is a euryhaline species which usually spawns in seawater. Due to its life cycle that includes migrations to lagoon and estuaries, young stages can be exposed early to salinity variations. Osmoregulatory patterns are well known in prelarvae, larvae and adults D. labrax in seawater (SW, 38) and in dilute seawater (DSW, 5), but the possibility and mechanisms of embryonic osmoregulation are still unknown. The goal of this study was to investigate the occurence of the omoregulatory function in the embryos of D. Labrax.First, the embryonic development of the different osmoregulatory sites was described, focusing on the digestive system including the pharynx and the first gill slits. The formation of these structures is initialized at stage 12 somites (S) and was described throughout the opening of the mouth five days after hatching.Secondarily, the time and the location of the occurrence of the first osmoregulatory cells, the ionocytes were followed. These cells were identified at stage 12S on the yolk sac membrane and at stage 14S in the first gill slits and in the posterior primitive gut. The functionality of these cells was studied, using immunostaining of the main ionic transporters involved in osmoregulation [the Na+/K+ ATPase (NKA), the Na+/K+/2Cl- cotransporter (NKCC) and the chloride channel (CFTR)], and through ultrastructural investigations. Potentially functional ionocytes are present from stage 25S in the yolk sac membrane and in the gut, but gill slits ionocytes are not fully functional at hatching. Passive drinking is suspected to regulate water balance in D. labrax.Finally, the embryonic osmoregulation in SW and DSW was investigated. Nanoosmometry measurements of the embryonic fluids demonstrated some capabilities of hyper- and hypo-osmoregulation. However, in DSW, qRT-PCR and imunostaining of NKA, NKCC and CFTR, reveal that hyper-osmoregulatory mechanisms can only limit ion loss but are not efficient enough to allow a full acclimation at this early life stage.

Il tratto gastro intestinale infantile al momento del parto è considerato virtualmente sterile e viene rapidamente colonizzato da microrganismi di origine materna e/o ambientale nei primi giorni di vita. Studi accoppiati di microbiologia classica e molecolare hanno dimostrato come la cavità amniotica sia popolata da microrganismi alcuni dei quali appartenenti a taxa non ancora coltivati e caratterizzati. Lo scopo del presente lavoro di tesi è stato quello di valutare la composizione del microbiota infantile durante i primi due anni di vita, in particolare, di popolazioni parzialmente “trascurate”. La tesi è suddivisibile in tre tematiche principali: presenza di popolazioni idrogenotrofiche, la distribuzione della famiglia delle Lachnospiraceae in soggetti sani prima del secondo anno di vita, e la possibile correlazione tra gli archaea metanogeni e la dieta in modello animale. Le tecniche impiegate nel presente lavoro di tesi sono state la PCR-DGGE e la PCR quantitativa (qPCR) e il sequenziamento Illumina. Le principali conclusioni derivabili dai tre studi sono correlate alla necessità di sviluppare nuove coppie di primers che meglio possano descrivere la complessa ecologia delle comunità microbiche intestinali. L’ambizioso obiettivo potrà considerarsi raggiunto quando si potranno identificare e stimare in modo preciso e corretto anche le popolazioni batteriche poco abbondanti nel microbiota intestinale infantile. / At birth, the gastrointestinal tract is virtually sterile, but is rapidly colonized during the first days of life until a relatively stable state is reached. Several studies using both bacterial culture techniques and bio-molecular methods revealed that the amniotic cavity harbors microorganisms and, among them, uncultivated and uncharacterized taxa. The aim of the present PhD thesis was to evaluate the composition of some neglected populations inhabiting the infant gut microbiota until the second year of life. The PhD thesis is composed of three main chapters related to the presence of hydrogenotrophic populations, the occurrence of the Lachnospiraceae family in healthy subjects before the second year and the possible linkage between methanogens and diet in a piglet’s model. PCR-DGGE, the quantitative PCR (qPCR) and the Illumina deep sequencing have been the prevalent molecular techniques used in this work. Main conclusions obtained from these studies were mainly linked to the need of new primer sets that better describe the ecological complexity of the gut microbial community. This ambitious objective will be reached when it is possible to properly identify and quantify less represented bacterial populations within the infant gut ecosystem.

Partial migration, whereby populations consist of residents and migrants, is common among migrating organisms. Partial migration of aquatic organisms, however, remains largely under-studied even though many aquatic animals exhibit horizontal and vertical migrations during their lifetime. Macroinvertebrates of the genus Mysis exhibit diel vertical migrations (DVM). Some species have recently been observed to exhibit partial diel migrations where some individuals reside on the bottom throughout the night while others migrate into the water column. To test the hypothesis that individuals are fixed as residents or migrants, we compared demographic information and C and N isotope compositions of M. diluviana caught at night in pelagic and benthic regions of Lake Champlain. Our results suggest there are two distinct ecotypes of M. diluviana separated by migration behavior. The migrating ecotype was smaller than the resident ecotype and enriched in δ15N while the resident ecotype had a higher C:N ratio. Because we did not allow for gut evacuation prior to our analyses, we conducted a follow-up experiment to test the effect of gut content on isotope composition of M. diluviana. The experiments confirmed that differences between benthic- and pelagic-caught M. diluviana were not a result of gut contents at the time of capture. Fixed partial migration behavior in M. diluviana in Lake Champlain indicates that DVM of M. diluviana may be more complex than previously thought. Additionally, partially migrating Mysis spp. may represent a model study organism to test hypotheses about the causes and consequences of partial DVM behavior in aquatic invertebrates.

Animal hosts provide associated microorganisms with suitable ecological niches in their intestines. Microbes help their hosts to digest food, protect against pathogens, and influence the host’s metabolisms. Compositional variation of gut microbial communities is common among hosts, and may affect the health status of hosts. Diet and genetic factors are well known to influence the assembly of gut microbial communities. This thesis focuses on disentangling the contributions of factors including host genetics (sex), diet, environment, and other ecological processes to the assembly of gut microbial communities in freshwater fish. The association between gut microbial communities and fish condition is also evaluated in this thesis. Applying metacommunity theory, we found environmental factors including fish habitat, fish species, their diet, dispersal factors including microbes from fish diet, and ecological drift contributed to the assembly of fish gut microbial communities. The proportion of their contribution varied between fish species, where ecological drift explained more in perch than in roach. Under natural conditions fish populations face the risk of predation, which can induce competition and impose predation stress within prey individuals. This can therefore lead to changes in their diet qualities and quantities. In this thesis, it was shown that fish diet in terms of qualities and quantities significantly influenced the overall gut microbial composition, and this influence was dependent on fish sex, a host genetic factor. Predation stress was also suggested to significantly decrease the species richness. Furthermore, when fish were experiencing a diet shift, we showed that different bacterial phyla from novel food had different colonization success in the intestine, and this colonization success was positively influenced by predation stress. Fish condition was suggested in this thesis to be affected by gut microbial composition, especially by the contributions of the bacterial phyla Tenericutes and Actinobacteria.