The human gut is host to a vast consortium of microorganisms, collectively referred to as the microbiota or microflora, which play important roles in health and disease. Current applications focus only on a single type of bacteria, which are not the most dominant numerically, and without detailed knowledge of the specific functions of these bacteria. A good indicator of the function of a bacterial species involves detailed analysis of its enzymes. Bacteroides thetaiotaomicron is one of the predominant bacterial species with a great representation of the carbohydrate processing enzymes, glycoside hydrolases in its proteome. This thesis reports the production and purification of one such enzyme, BT3299, suitable for kinetic and structural studies. The enzyme displayed a broad substrate specificity with a slight preference for 1-->3 and 1-->6 glycosidic linkages and longer chain saccharides. Future work will focus on structural analysis as an aid to the understanding of the enzyme function.

The human gut is host to a vast consortium of microorganisms, collectively referred to as the microbiota or microflora, which play important roles in health and disease. Current applications focus only on a single type of bacteria, which are not the most dominant numerically, and without detailed knowledge of the specific functions of these bacteria. A good indicator of the function of a bacterial species involves detailed analysis of its enzymes. Bacteroides thetaiotaomicron is one of the predominant bacterial species with a great representation of the carbohydrate processing enzymes, glycoside hydrolases in its proteome. This thesis reports the production and purification of one such enzyme, BT3299, suitable for kinetic and structural studies. The enzyme displayed a broad substrate specificity with a slight preference for 1-->3 and 1-->6 glycosidic linkages and longer chain saccharides. Future work will focus on structural analysis as an aid to the understanding of the enzyme function.

There has been a rapid increase in the number of overweight and obese individuals worldwide in the past 50 years. It has been assumed that an increased caloric intake and a more sedentary lifestyle are the main causes of this rise. However, recent evidence has shown that the microbes that live in the human gastrointestinal tract may play a role in the regulation of weight and obesity development. These microbes, termed the gut microbiota, are commensal and symbiotic microbes that are densely populated throughout an individual's gastrointestinal tract. This paper presents the relevant research and possible mechanisms of how these microbes, mainly bacteria, are thought to play a role in weight regulation and obesity.

The weaning transition period in pigs is linked to increased vulnerability to enteric disorders, which is partly attributed to destabilisation of the gut microbiota. Post-weaning colibacillosis is an economically important disease of the small intestine, which is most commonly caused by enterotoxigenic Escherichia coli (ETEC) strains. This disease has been variably linked to a diarrhoeal phenotype and decreased growth rate under clinical or sub-clinical conditions, and has been associated with shifts in particular bacterial populations using culturing methods. The emergence of next-generation sequencing technologies such as 16S rRNA gene metabarcoding now allows higher resolution study of complex microbial communities, without being reliant on the ability to culture fastidious micro-organisms. As part of this project, a 16S rRNA gene metabarcoding method was developed and validated to allow qualitative and quantitative measurement of gut microbiota shifts. A series of experimental ETEC challenge trials were carried out to monitor temporal faecal microbiota dynamics (Chapter 2), to further understand ETEC adhesion and shedding dynamics (Chapter 3) and to study potential changes in both ileal and faecal microbiota populations in response to dietary protein manipulation (Chapter 4). The effects of experimental treatments on pig health and performance were also measured as part of each experiment. Temporal shifts in ileal and faecal microbiota structure and stability were observed over the post-weaning period, as well as shifts in relative abundances of particular bacterial phylotypes (P < 0.05) (Chapter’s 2 and 4). ETEC challenge had no effects on faecal microbiota composition, pig health and performance when comparing to samples obtained from sham-challenged pigs (P > 0.05). However, when taking ETEC shedding level into account, variations in both microbiota structure and stability were observed at specific time points (P < 0.05) (Chapter 2). After a single-dose ETEC challenge, ETEC adhesion in the ileum and faecal shedding were evident up to 4 and 6 days post-challenge, respectively (Chapter 3). Changes in ileal microbiota structure and stability were observed in response to ETEC challenge (P < 0.05), with no changes exerted at faecal level (P > 0.05). Additionally, different dietary protein levels were linked to changes in ileal microbiota structure, stability and phylotype relative abundances (P < 0.05). Interestingly, significant differences in ileal microbiota structure were evident in samples obtained from ETEC-challenged pigs fed the low and high protein diets, with the pigs fed the high protein diet having significantly less stable ileal communities at population level (P < 0.05) (Chapter 4). The treatments had no effect on host performance (P > 0.05), but faecal consistency scores were higher in pigs fed the high protein diet (P < 0.05). In conclusion, both ETEC challenge and manipulation of dietary protein level had profound effects on ileal microbiota composition and faecal microbial communities were variable according to ETEC shedding status. These findings have implications for the development of alternative management strategies for enteric diseases in weaner pigs.

Orientador: Mario Jose Abdalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T21:44:54Z (GMT). No. of bitstreams: 1 Zambon_RenataAlvaresBagarolli_D.pdf: 7972972 bytes, checksum: 0c4feccde8df219c21aef1c3e8740fc0 (MD5) Previous issue date: 2013 / Resumo: A obesidade é caracterizada por processo inflamatório crônico e resistência à insulina (RI), os quais são responsáveis por grande parte de suas doenças associadas. Sabe-se que diversas moléculas do sistema imune inato estão associadas à RI e obesidade, destacando-se o receptor toll-like-receptor 4 (TLR4). Sua via de sinalização está ativada na obesidade, devido à presença aumentada na circulação de seu principal ligante, lipopolissacarídeo (LPS). Acredita-se que esta endotoxemia metabólica seja causada por alterações na microbiota e na permeabilidade intestinais, o que torna o intestino e as bactérias que o habitam, grandes alvos para o tratamento da obesidade. O objetivo deste presente trabalho foi avaliar os efeitos dos probióticos (PB) na sensibilidade à insulina e na sinalização de TLR4 em tecidos insulino-sensíveis, além de verificar suas possíveis ações na microbiota intestinal. Dessa forma, camundongos Swiss foram divididos em 4 grupos: controles (C), controles tratados com PB (C+PB), obesos (DIO) e obesos tratados com PB (DIO+PB). O tratamento teve a duração de 5 semanas. O uso de PB em animais obesos proporcionou grandes melhoras nos parâmetros fisiológicos e moleculares de RI, além de atenuar a ativação da via de sinalização do TLR4, provavelmente pela redução dos níveis circulantes de LPS. O grupo DIO+PB ainda mostrou mudanças positivas na distribuição dos filos de bactérias intestinais e menor permeabilidade intestinal, quando comparado com o grupo DIO. Analisando o hipotálamo, observou-se que o uso de PB nesse modelo de obesidade regulou de forma favorável os mecanismos centrais de controle da fome, bem como alguns parâmetros de inflamação. Assim, conclui-se que a regulação da microbiota intestinal promovida pela administração de probióticos pode trazer benefícios no controle da obesidade, por reduzir ou atenuar mecanismos moleculares de resistência à insulina / Abstract: Obesity is the main risk factor to the development of insulin resistance and type 2 diabetes. The common basis among these events is an inflammatory process characterized by the activation of toll-like receptor 4 (TLR4) by its main ligand lipopolysaccharide, LPS. Its concentration is higher in obese people and it is believed that changes in composition of the gut microbiota and epithelial functions may play a role in the inflammation associated with obesity. The aim of the study was to evaluate the effects of probiotic on the insulin sensitivity, TLR4 signaling, intestinal permeability and microbiota composition in diet-induced obese mice. Male adult Swiss mice composed randomly 2 groups: chow diet (CTL) and high-fat diet by 5 consecutive weeks (DIO). During these 5 weeks, some mice of the DIO and CTL groups received daily a pool of probiotics. The DIO animals that received probiotic presented an expressive improvement in their glucose tolerance test, fasting glucose and in parallel a significant increase in the phosphorylation levels of insulin induced IR, IRS1 and Akt in muscle, liver and adipose tissue. There was a relevant reduction in the TLR4-Myd88 interaction, IKK? and JNK phosphorylation and iNOS expression in DIO mice treated with probiotic. This treatment also improved the expression of ileal tight-junctions proteins (ZO-1, Occludin), decreased LPS portal levels and the concentrations of bacteria of the phylum Firmicutes (associated with obesity) in feces. Analyzing the hypothalamus, it was observed that the use of probiotics in this model of obesity favorably regulated central mechanisms of food intake, as well as some inflammation parameters. In conclusion, our results show that probiotics, through their effects on intestinal permeability and microbiota composition, can improve insulin sensitivity and signaling of DIO mice, reducing their inflammation and suggesting potential beneficial effects in the treatment of insulin resistance and type 2 diabetes / Doutorado / Medicina Experimental / Doutora em Ciências

In recent years, the association between inflammatory bowel diseases (IBDs) and gut microbiota has been extensively studied in adults using post-treatment cohorts of patients. However, microbial composition and functional interplay between host genetics and microorganisms in newly diagnosed early IBD onset remain poorly defined. Using colonoscopic mucosal washes to collect mucosal-luminal microbiota from different intestinal locations, we studied the gut microbiome in a large number of children with either Crohn’s disease (CD) or ulcerative colitis (UC). Although no significant difference in the diversity was evident between the gut microbiota of IBD-affected and control children, the microbiome of IBD subjects is characterized by an increased abundance of potent hydrogen sulfide (H2S) producers and decreased abundance of beneficial butyrate producers. Microbiota and proteomic profiling revealed that the abundance of Atopobium parvulum, a potent H2S producer, was associated with increased CD severity and a concurrent reduction in the expression of the host H2S detoxification pathway. Gnotobiotic and conventionalized colitis-susceptible interleukin-10-deficient (Il-10-/-) mice showed that A. parvulum induces severe colitis, a phenotype requiring the presence of the gut microbiota. In addition, administration of bismuth, an H2S scavenger, prevented A. parvulum-induced colitis in Il-10-/- mice. Our findings have identified A. parvulum as a major mediator of inflammation severity. We also reveal an imbalance between the H2S production and detoxification in the gastrointestinal tract of pediatric IBD patients. Altogether, our findings provide new avenues for diagnostics as well as therapies to treat IBD.

abstract: First year college students have been identified as a vulnerable population for weight gain and the onset of overweight and obesity. Research regarding the gut microbiome has identified differences in the microbial composition of overweight and obese individuals compared to normal weight individuals. Dietary components like dietary fibers, act as prebiotics, or fermentable substrate, that the gut microbiota use for metabolic functions including the production of short-chain fatty acids. The objective of this longitudinal, observational study was to assess changes in the gut microbiota over time in relation to changes in fiber consumption in healthy college students at a large a southwestern university (n=137). Anthropometric and fecal samples were collected at the beginning and end of the fall and spring semesters between August 2015 and May 2016. Both alpha, within sample, diversity and beta, between sample, diversity of participant gut microbes were assessed longitudinally using non-parametric pairwise (pre-post) comparisons and linear mixed effect (LME) models which also adjusted for covariates and accounted for time as a random effect. Alpha and beta diversity were also explored using LME first difference metrics and LME first distance metrics, respectively, to understand rates of change over time in microbial richness/phylogeny and community structure. Pre-post comparisons of Shannon Diversity and Faith’s PD were not significantly different within participant groups of fiber change (Shannon diversity, p=0.96 and Faith’s PD, p=0.66). Beta diversity pairwise comparisons also did not differ by fiber consumption groups (Unweighted UniFrac p=0.182 and Bray Curtis p=0.657). Similarly, none of the LME models suggested significant associations between dietary fiber consumption and metrics of alpha and beta diversity. Overall, data from this study indicates that small changes in fiber consumption among a free-living population did not have an impact on gut microbial richness, phylogeny or community structure. This may have been due to the low intake (~15 g/d) of fiber. Further study is needed to fully elucidate the role that fiber plays in the diversity and composition of the gut microbiota, especially when delivered from a variety of food sources rather than fiber supplements. / Dissertation/Thesis / Masters Thesis Nutrition 2020

Lactate, a metabolic end product of glycolysis in mammals, has emerged as an important energy substrate for the brain. In addition to its energetic role, lactate was shown to modulate the excitability of neurons, to have a neuroprotective role and to participate in long-term memory formation. One previous investigation from our group reported that lactate modulates 4 synaptic plasticity-associated genes and potentiates the activity of the N-Methyl-D-aspartic acid (NMDA) receptor, a major receptor type involved in glutamatergic neurotransmission. The current thesis aimed at first to extend these findings by examining genome-wide transcriptional responses to this metabolite in cortical neurons. Using ribonucleic acid(RNA) sequencing to evaluate expression changes in protein-coding genes, we found that lactate modulates robustly after 1h, 20 genes involved in the mitogen-activated protein kinase (MAPK) signaling pathway and in synaptic plasticity in a NMDA receptor activitydependent manner and that nicotinamide adenine dinucleotide, reduced (NADH), but not pyruvate, reproduces the modulatory effects of lactate on 70% of all differentially expressed genes. In a time course experiment, genes modulated after lactate treatment for 6h and 24h were also identified; these are involved in 9 signaling pathways including circadian rhythm, drug addiction, and retrograde endocannabinoid signaling. Bioinformatics analyses indicated CREB1 and CREM as candidate master regulators of gene expression and the modulatory effect of lactate was prevented by inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, indicating a role for this kinase in mediating lactate signaling. An examination of changes in dendritic spines’ morphology and density - a morphologicalcorrelate of synaptic plasticity – has shown that lactate modulated spine density changes induced by potassium chloride (KCl) and carbachol. An additional investigation described in this thesis indicated that different gut microbiota manipulations (germ-free, prebiotics, high-fat diet) regulated mRNA expression of genes involved in the Astrocyte-Neuron Lactate Shuttle (ANLS) - a metabolic cooperation mechanism between astrocytes and glutamatergic neurons. Overall, the results of this thesis help to establish a role for lactate as a signaling molecule in the brain, highlight mechanisms implicated in its signaling, and open new avenues for investigation of links between the gut microbiota and brain energy metabolism.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Objective: The objective of this study was to characterize the composition of the gut microbiota in type 2 Diabetes Mellitus (T2DM) patients with adequate and inadequate metabolic control, and its relationship with fiber consumption. Results: A total of 26 patients with type 2 diabetes mellitus (T2DM) were enrolled, of which 7 (26.9%) cases had adequate metabolic control (HbA1c < 7%) and 19 (73.1%) inadequate metabolic control (HbA1c ≥ 7%). It was observed that among patients with controlled T2DM, 2 (28.6%) cases presented good intake of fiber and 5 (71.4%) cases a regular intake. In contrast, in patients with uncontrolled T2DM, 13 (68.4%) patients reported a regular intake and 6 (31.6%) a poor intake. In relation to the identification of the gut microbiota, both groups presented a similar characterization. There were differences in the population of bacteria identified in both groups, however, the results were not statistically significant. The most frequently identified bacteria in controlled and uncontrolled T2DM patients were Prevotella (71.4% vs 52.6%), followed by Firmicutes (71.4% vs 42.1%), Proteobacteria (71.4% vs 36.8%) and Bacteroidetes (57.1% vs 37.8%). On the other hand, Fusobacterium, Actinobacteria were not identified in either of the two groups of study. / Revisión por pares

Thesis (Master of Environmental Health)--Cape Peninsula University of Technology, 2020 / Background: The development of metabolic disease is accompanied by changes in gut microbiota phenotype, including a decrease of beneficial bacteria and increase of pernicious bacteria of the gastrointestinal tract. A Western (high-fat and high-sugar) diet, sedentary lifestyle and altered gut microbiota diversity have been associated with an increased risk of developing metabolic diseases such as type 2 diabetes and its associated risk factor, obesity. Many researchers have studied the link between the gut microbiota and diet. Hence our in vitro study is aimed at investigating the potential prebiotic effect of an aspalathin-rich unfermented rooibos extract, Afriplex GRT™ and aspalathin on the faecal bacterial diversity of vervet monkeys fed Western diet. Methodology: A total of six vervet monkeys (Chlorocebus pygerythrus) were selected from monkeys fed either a maize based normal diet (standard diet group; n=3) or a high fat diet (Western diet group; n=3) for more than 5-years. Faecal samples were collected from the animals in both groups at the Primate Unit and Delft Animal Centre (PUDAC) between 7 – 9 AM. Faecal samples from the two groups were divided into culture-independent baseline samples (before culture) and culture-dependent samples (after anaerobic culture). The culture-dependent samples were cultured under anaerobic conditions at 37°C for 10 hours, with or without Afriplex GRT™ extract or aspalathin. Bacterial genomic DNA (gDNA) was extracted from all samples using the NucleoSpin® DNA Stool extraction kit. Purified gDNA was sent for metagenomic sequencing for 16S rRNA gene analysis of microbial diversity using an Ion Torrent Next-generation Sequencing platform. Results: Results indicated that the Western diet affects the abundance of several bacterial species. Afriplex GRT™ and aspalathin significantly enhanced the relative abundance of health promoting butyrate-producing bacteria such as Faecalibacterium prausnitzii in both standard and Western diet groups (p= 0.02 and p=0.04, respectively). A similar trend was observed in other beneficial bacteria such as Eubacterium spp., Sutterella spp., and Dorea longicatena. Conclusion: Based on the data observed, it can be suggested that Afriplex GRT™ has a beneficial prebiotic effect on gut microbiota diversity and gut health.