A microbiota intestinal é um ecossistema complexo que desempenha um importante papel na gênese da obesidade. A ocorrência e participação dos Mollicutes na microbiota intestinal é praticamente desconhecida. Deste modo, o objetivo do presente estudo foi analisar a participação dos Mollicutes e dos Filos Firmicutes e Bacteroidetes na microbiota intestinal de mulheres obesas e eutróficas. A casuística foi de 20 mulheres obesas e 20 mulheres em eutrofia. Foram obtidas amostras de fezes, sangue e aplicado questionário semiestruturado sobre fatores relacionados com obesidade, microbiota intestinal e ambiente, além de Bioimpedância e questionário de frequência alimentar. Constatou-se uma associação positiva estatisticamente significante entre a presença de Mollicutes e mulheres obesas. Foi observada maior proporção de Firmicutes/Bacteroidetes na microbiota intestinal das mulheres obesas. Os resultados obtidos permitiram obter evidências importantes da participação dos micro-organismos da classe Mollicutes. As alterações da microbiota intestinal também contribuíram na definição de subconjuntos de indivíduos com diferentes perfis de risco metabólico e a da heterogeneidade associada a fenótipos humanos relacionados com a adiposidade. / The gut microbiota is a complex ecosystem that plays an important role in the pathogenesis of obesity. The occurrence and participation of Mollicutes in the gut microbiota is pratically unknown. The aim of this study was to analyze the participation of Mollicutes and Firmicutes and Bacteroidetes phylos in the gut microbiota of obese and normal weight women. For the study, it was collected samples of 20 women with obesity and 20 women of normal weight. It was collected stool samples, blood, semi-structured questionnaire on factors associated with obesity, gut microbiota and the environment, and anthropometric measurements using bioelectrical impedance and food frequency questionnaire. It was detected a statistically significant positive association between the presence of Mollicutes and obese women, and there was a higher proportion of Firmicutes/Bacteroidetes in the gut microbiota of obese women. The results provide important evidence about the participation of Mollicutes class in the gut microbiota of the population studied and interactions in intestinal microbiota can define subsets of individuals with different metabolic risk profiles and thus contribute to investigation of the heterogeneity associated phenotypes related to adiposity.

Studying the microbiome in natural populations could improve our understanding of the biological factors that influence microbiome variation. If host genetic variation is important in microbiota assembly, then understanding genetic divergence among natural populations could be informative. Despite advances in sequencing technology, we have not yet taken full advantage of this technology in natural populations. Here we integrate genome-wide population genomic and microbiome analyses in wild threespine stickleback (Gasterosteus aculeatus) fish distributed throughout western Oregon, USA. We found that gut microbiome varied in diversity and composition more among than within wild host populations. Furthermore, this among population variation was better explained by host population genetic divergence than by environment and geography. We also identified a subset of gut microbial taxa that were most strongly sorted both across environments and across genetically divergent populations. We believe this study contributes generalizable methods and findings in host systems. This thesis includes supplemental materials. / 2021-04-30

Orientador: Urbano dos Santos Ruiz / Resumo: O objetivo desta pesquisa foi avaliar o aditivo alternativo, composto por ácido benzoico e óleos essenciais de eugenol, timol e piperina, associado ou não a antibiótico melhorador de desempenho na alimentação de leitões recém-desmamados. Foram utilizados 108 leitões, de linhagem genética comercial, em três fases: I - dos 21 aos 35 dias; II - dos 36 aos 50 dias; e III - dos 51 aos 63 dias de idade. As dietas foram isonutritivas, diferindo quanto à adição dos aditivos, da seguinte maneira: dieta sem qualquer aditivo melhorador de desempenho, dieta com adição de 40 ppm do antibiótico colistina, dieta com inclusão de 0,3% do aditivo alternativo, dieta com adições de 0,3 % do aditivo alternativo e de 40 ppm de colistina. Foram avaliados: o desempenho zootécnico, digestibilidades de nutrientes das dietas; incidência de diarreia; tempo de trânsito da digesta, morfologia intestinal, pesos relativos de órgãos do sistema digestório; composição da microbiota do conteúdo do ceco; e índices econômicos. Os animais foram distribuídos em blocos casualizados, de acordo com seus pesos ao início do experimento, com quatro tratamentos e nove repetições, sendo a unidade experimental a baia, composta por três animais na fase I e dois nas fases II e III. Como os animais não foram redistribuídos nos blocos ao final de cada fase, as análises estatísticas foram efetuadas de forma cumulativa, ou seja, do início do experimento ao final das fases I, II e III, em um esquema fatorial 2×2. Os dados foram su... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this research was evaluate alternative feed additive, composed by benzoic acid and the essential oils of eugenol, thymus and piperine, associated or not with performance enhancer antibiotic, in newly weaned piglets feeding. One hundred and eight piglets, from a commercial lineage, were used in three phases: I – from 21 to 35 days; II from 36 to 50 days; and III – from 51 to 63 days. The diets were composed mainly by corn, soybean meal, spray dried blood plasma and dairy products, presenting the same levels of metabolizable energy, digestible amino acids, calcium and digestible phosphorous, differing over feed additive addition, as follows: diet without performance enhancer feed additive: diet with 40 ppm of colistin; diet with 0.3% alternative feed additive; diet with 40 ppm colistin and 0.3% alternative feed additive. The parameters evaluated were: growth performance; nutrient diets digestibility; diarrhea incidence; digesta transit time; intestinal morphology, weights of digestive organs (absolute and relative to body weight); microbial cecum content compositon; economical indices. The animals were distributed in blocks, according with their initial body weight, assigned to four treatments, with nine repetitions, and the experimental unit was the pen, with three animals in phase I e two, in phases II and III. As the animals were redistributed in blocks at the end of each phase, the statistical analysis were performed in a cumulative way, that is, from the b... (Complete abstract click electronic access below) / Mestre

Enteroendocrine hormones released from the intestine following food intake have several roles in the control of metabolism, some of which are exploited therapeutically for the treatment of type 2 diabetes. Within this thesis, focus has been on the receptors of the gut hormones glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2). In recent years there has been a surge of interest in the enteroendocrine hormones particularly due to the success of GLP-1 mimetics in the treatment of type 2 diabetes. GLP-1 is an incretin hormone, which enhances glucose induced insulin secretion by binding GLP-1 receptors (GLP1R) on pancreatic β-cells. Despite the therapeutic success, several extra-pancreatic clinical effects of GLP-1 remain unexplained. Here, a GLP1R monoclonal antagonistic antibody that can block GLP1R signalling in vivo has been developed and characterised, providing a new tool for the study of GLP1R physiology. GIP is the second incretin hormone, initially referred to as the 'ugly duckling' incretin hormone due to it's ineffectiveness in inducing insulin secretion in type 2 diabetic patients. Aside from the incretin actions, GIP is thought to be involved in the regulation of high-fat diet (HFD) induced obesity. A new transgenic mouse model expressing a fluorescent reporter under the control of the Gipr promoter has been used here to identify GIPR expressing cells. This model showed GIPR expression in the pancreas, adipose tissue, duodenum and nodose ganglia. Surprisingly GIPR expressing cells were found centrally, in areas of the hypothalamus involved in the regulation of food intake and energy expenditure. We consequently sought to investigate the function of GIPR expressing hypothalamic cells. GLP-2, unlike GLP-1 and GIP, is not an incretin hormone. Rather, GLP-2 has been implicated in the regulation of epithelial cell proliferation and apoptosis within the intestine. Therapeutically, an analogue of GLP-2 is used for the treatment of short bowel syndrome. A common missense mutation in the GLP-2 receptor (GLP2R), D470N, has been found to be associated with type 2 diabetes, and here we sought to understand the mechanism underlying this association. The D470N mutant has decreased β-arrestin recruitment, though the significance of this finding will need further research. Overall; the new monoclonal antagonistic GLP1R antibody will help to further understand GLP1R physiology, the new transgenic GIPR mouse model has contributed to the understanding of GIPR localisation, and cell based assays have identified functional implications of a polymorphism in the GLP2R associated with an increased risk of diabetes. It is hoped that further understanding of the physiology of these gut hormone receptors will be critical in the development of new therapeutics for diabetes and obesity.

A microbiota intestinal é um ecossistema complexo que desempenha um importante papel na gênese da obesidade. A ocorrência e participação dos Mollicutes na microbiota intestinal é praticamente desconhecida. Deste modo, o objetivo do presente estudo foi analisar a participação dos Mollicutes e dos Filos Firmicutes e Bacteroidetes na microbiota intestinal de mulheres obesas e eutróficas. A casuística foi de 20 mulheres obesas e 20 mulheres em eutrofia. Foram obtidas amostras de fezes, sangue e aplicado questionário semiestruturado sobre fatores relacionados com obesidade, microbiota intestinal e ambiente, além de Bioimpedância e questionário de frequência alimentar. Constatou-se uma associação positiva estatisticamente significante entre a presença de Mollicutes e mulheres obesas. Foi observada maior proporção de Firmicutes/Bacteroidetes na microbiota intestinal das mulheres obesas. Os resultados obtidos permitiram obter evidências importantes da participação dos micro-organismos da classe Mollicutes. As alterações da microbiota intestinal também contribuíram na definição de subconjuntos de indivíduos com diferentes perfis de risco metabólico e a da heterogeneidade associada a fenótipos humanos relacionados com a adiposidade. / The gut microbiota is a complex ecosystem that plays an important role in the pathogenesis of obesity. The occurrence and participation of Mollicutes in the gut microbiota is pratically unknown. The aim of this study was to analyze the participation of Mollicutes and Firmicutes and Bacteroidetes phylos in the gut microbiota of obese and normal weight women. For the study, it was collected samples of 20 women with obesity and 20 women of normal weight. It was collected stool samples, blood, semi-structured questionnaire on factors associated with obesity, gut microbiota and the environment, and anthropometric measurements using bioelectrical impedance and food frequency questionnaire. It was detected a statistically significant positive association between the presence of Mollicutes and obese women, and there was a higher proportion of Firmicutes/Bacteroidetes in the gut microbiota of obese women. The results provide important evidence about the participation of Mollicutes class in the gut microbiota of the population studied and interactions in intestinal microbiota can define subsets of individuals with different metabolic risk profiles and thus contribute to investigation of the heterogeneity associated phenotypes related to adiposity.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-01-06T11:37:49Z No. of bitstreams: 1 2016 - Gabriel Alves Landulfo.pdf: 2085230 bytes, checksum: a7f7c364b23211ad81d1168cb1f6db85 (MD5) / Made available in DSpace on 2017-01-06T11:37:49Z (GMT). No. of bitstreams: 1 2016 - Gabriel Alves Landulfo.pdf: 2085230 bytes, checksum: a7f7c364b23211ad81d1168cb1f6db85 (MD5) Previous issue date: 2016-02-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also aggressive towards humans. It inhabits some countries in the Neotropical region. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to ascertain the main molecules expressed in the gut of argasid ticks after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon. Sixty females were fed and dissected to extract the gut tissue. The transcriptome was obtained through pyrosequencing and the de novo assembly method on mRNA of the gut tissue. We identified 2,235 contigs, of which 1,729 matched database sequences, while 506 did not present any hits. The transcripts were annotated and grouped according to their biological function. Catalytic, binding and transporter activity were the most representative functions, accounting for 780, 709 and 106 contigs, respectively. The transcripts were classified into 31 categories, using both bioinformatics and data curation practices. The most representative categories were, respectively, unknown, catalytic activity and transporter channels. One hundred and three (103) digestives transcritps associated to digestion of proteins (67), carbohydrates (19) and lipid (17) were identified in the transcriptome analysis. Peptidases associated with hemoglobin digestion, such as serine, cysteine, aspartic protease and metalloenzymes, were identified in the gut of the engorged females. Genes associated with transport (hemelipoglycoprotein) and storage (ferritin) of nutrients resulting from hemoglobin digestion, such as heme, were also found in the digestive tract. The presence of a cathepsin O-like cysteine peptidase was recorded in ticks, for the first time. Two thousand and two hundred thirteen (2213) transcripts were deposited to the Transcriptome Shotgun Assembly (TSA) portal of the NCBI.The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. Genes for cathepsin L in O. mimon appear to have diverged from other more common recent ancestors. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on different datasets, such as mitochondrial genome and nuclear rRNA sequences. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity / Ornithodoros mimon ? um carrapato argas?deo parasita de quir?pteras, aves e marsupiais, al?m de ser bastante agressivo aos humanos. O conhecimento dos transcritos presentes no intestino dos carrapatos auxilia no entendimento do papel de mol?culas vitais no processo digest?o e na rela??o parasito-hospedeiro, al?m de fornecer tamb?m informa??es sobre a evolu??o dos artr?podes hemat?fagos. Desta maneira, o presente estudo teve como objetivo conhecer e identificar as principais mol?culas expressas no intestino de uma esp?cie de carrapato argas?deo ap?s o repasto sangu?neo, atrav?s de uma an?lise transcrit?mica do intestino de f?meas ingurgitadas de O. mimon. Sessenta f?meas foram alimentadas e dissecadas para coleta o tecido intestinal. O RNAm da amostra do intestino foi extra?do, purificado e quantificado. Esse serviu de molde para s?ntese do cDNA, que foi utilizado no pirosequenciamento. O transcritoma foi obtido atrav?s do m?todo de montagem de novo do cDNA do tecido intestinal. Identificou-se 2235 sequ?ncias consensos (contigs) ou transcritos, dos quais 1729 apresentaram similaridade (hit) com sequ?ncias dos bancos de dados, enquanto que 506 n?o tiveram nenhuma similaridade. Os transcritos foram anotados e agrupados conforme as fun??es biol?gicas atribu?das as eles no processo de anota??o g?nica. Atividade catal?tica, ades?o e transporte foram as fun??es mais representativas com 780, 709 e 106 transcritos, respectivamente. Em uma an?lise n?o automatizada, os transcritos foram subcategorizados em 31 categorias. As categorias mais representativas foram desconhecido, atividade catal?tica e transportadores-canais. Identificamos 103 transcritos digestivos associados ? digest??o de prote?nas (67), carboidratos (19) e lip?dios (17). Proteinases das classes serino, ciste?ne, asp?rtica e metalo representaram as enzimas atuantes na digest?o intracelular do constituinte prote?co do repasto sangu?neo. Genes associados com o transporte (hemelipoglicoprote?na) e estocagem (ferritina) dos nutrientes resultantes da digest?o foram encontrados bem expressos no trato digestivo. Registrou-se pela primeira vez a presen?a de uma ciste?na peptidase do tipo catepsina O em carrapatos. Foram depositados no banco de dados g?nico p?blico 2213 transcritos de O. mimon. A an?lise filogen?tica das peptidases revelou que a maioria das proteinases de O. mimon ? pr?xima aos genes codificadores de proteinases de carrapatos. Transcritos de catepsinas L de O. mimon parecem ter divergido de ancestrais recentes diferentes. A infer?ncia filogen?tica baseada em conjunto de dados transcritos hom?logos tem uma resolu??o topol?gica similar a de outros conjuntos de dados, como genoma mitocondrial e sequ?ncias nuclear de RNA riboss?mico (rRNA). Os achados obtidos no presente estudo podem contribuir para compreens?o dos importantes processos dos carrapatos argas?deos, como digest?o, nutri??o e imunidade, al?m de fornecer informa??es sobre a filogenia dos carrapatos.

The gut microbiome is a community of closely interacting microbes living in the gastrointestinal tract. Its structure has direct relevance to health. Disturbances to the microbiome, such as due to antibiotic use, have been implicated in various diseases. The goal of this study was to determine how the gut microbiome reacts to and recovers from disturbance caused by antibiotics. Because diet also influences the microbiome, this study included the interaction between diet and antibiotics. Half of the mice in each diet treatment were given antibiotics to disturb their microbiomes. After cessation of antibiotics, mice were paired in combinations within diets to determine whether the microbiomes of control mice influenced the disturbed microbiomes of formerly antibiotic mice. Chapter 1. Diet significantly altered the structure of the gut microbiome but its effect was significantly smaller than the effect of antibiotics. There was a significant interaction between diet and antibiotics; the antibiotic effect was larger in the cornstarch diet than in the glucose diet. Dysbiotic microbiomes resulting from antibiotics were characterized by an increase in Bacteroidetes and Proteobacteria, and a decrease in Firmicutes. Antibiotic administration also resulted in an initial increase OTU diversity, mainly because it reduced the abundance of dominant OTUs, resulting in greater evenness. Chapter 2. Seven weeks after the cessation of antibiotics (experiment termination), the effect of the antibiotics on the microbiome was still evident. The structure of the dysbiotic microbiome had not returned to that of control mice. Antibiotics significantly increased the relative abundance of some taxa and significant decreased the relative abundance of others. It was unexpected that the taxonomic hierarchy within the microbiome did not recover after 7 weeks following cessation of antibiotics. It would appear, therefore, that antibiotics established a new, semi-stable hierarchy. Chapter 3. When paired together, the assumption was that dysbiotic microbiomes of antibiotic mice would be positively influenced by microbiomes of control mice, based on the assumption that the control mouse would act as a probiotic for the antibiotic mouse, either via coprophagy or consumption of food contaminated by feces. Contrary to that hypothesis, the microbiomes of control mice became more similar to that of antibiotic mice. One can offer at least two hypotheses to explain this result, but neither was tested. First, compared to the control microbiome, the dysbiotic microbiome may have been more stable and thus more resistant to change due to invasion by OTUs from the control microbiome. Other research has shown that dysbiotic microbiomes have a high degree of stability. If this were true, the use of probiotics is questionable. Second, one or more of the antibiotics could still have been active at the initial phase of pairing, and coprophagy caused the microbiome of the control mice to rapidly become dysbiotic. If this is true, the experiment should have been conducted with a waiting period between the cessation of antibiotic administration and pairing.

Environmental contamination is a public health concern. In particular persistent organic pollutants like Polychlorinated Biphenyls (PCBs) have been associated with multiple chronic inflammatory diseases, including non-alcoholic fatty liver disease (NAFLD). NAFLD prevalence has steadily increased and is expected to continue to rise with an estimated 25% of the world’s population and 80-100 million people affected in the United States alone. Importantly, the liver is the primary site for endobiotic and xenobiotic metabolism, hence its proper function is critical for the body’s response to innate and extrinsic molecules. One way to combat the deleterious effects of PCB toxicity and fatty liver disease is by increasing consumption of beverages and foods that contain beneficial bioactive nutrients, like dietary polyphenols. However, the biological properties of these dietary compounds are subject to their bioavailability which is directly dependent on the activity of the liver. The first aim of this dissertation was to test the hypothesis that in the presence of a compromised liver, PCB-126 toxicity is altered. Indeed, hepatic and systemic PCB-126 toxicity was exacerbated in this severe liver injury mouse model with an observed increase in hepatic inflammation, systemic inflammation, and early markers of endothelial cell dysfunction. Interestingly, we also observed an increase in the novel gut-liver axis derived cardiovascular disease (CVD) marker trimethylamine-N-oxide (TMAO). Taken altogether, aim 1 proved that a compromised liver can alter PCB toxicity, with implications of the gut microbiota in disease pathology. In aim 2 we investigated whether GTE can protect against MCD-induced hepatic toxicity and development of NAFLD. Results indicated that MCD mice exhibited severe liver injury and gut dysbiosis and unexpectedly, GTE had no protective effects. Interestingly MCD mice displayed differential epigallocatechin-3-gallate (EGCG) metabolism at the hepatic and gut microbiota level, which may alter polyphenol bioavailability and therapeutic potential. Overall, the results provide insight into how a dysfunctional liver and gut dysbiosis can alter polyphenol metabolism, possibly reducing its therapeutic efficiency. In aim 3 we sought to determine potential protective effects of a prebiotic in this mouse model. MCD-fed mice were exposed to PCB-126 with or without inulin supplementation. Although findings from this study are preliminary, our evidence indicates that inulin restores body weight and body composition in this MCD+PCB mouse model and alters the expression of Cyp1a1 in PCB exposed mice, suggesting that inulin’s protective effects may be a result of its ability to interact with the AhR pathway. However further analysis will need to be done to examine the effects of inulin on hepatic, systemic, and gut microbiota endpoints. Overall the data contained in this dissertation suggests that in the presence of a compromised liver both pollutant toxicity and nutrient metabolism are altered, with implications of the gut-microbiota in disease risk. These findings suggest that individuals with end stage liver injury may be more susceptible to pollutant-induced toxicity and nutritional intervention may be unsuccessful at mitigating disease risk.

A series of experiments were conducted to explore factors affecting transfer of value-added ingredients from the diet to table eggs, with the goal of contributing to improvements in the enrichment process. Flaxseed-based ω-3 PUFA enrichment did not reduce lutein enrichment. The combine enrichment of lutein and ω-3 PUFA had decresed lipid oxidation potential. Also, when fed a ω-3 PUFA diet, birds scored as energetic Efficient had longer and wider villi, resulting in greater absorptive surface area/villi than Non-efficient hens. However, histomorphological differences did not affect transfer of ω-3 PUFA from diet to egg. Finally, birds fed graded levels of ω-3 PUFA to characterize change in lipid profile of egg and blood plasma in time reached a plateau in total ω-3 PUFA/egg in 5.9 to 6.6d, with High birds reaching the target of 300 mg/egg in 5d. Egg enrichment can be modulated by changes to the hen diet. / Animal Science

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.