Orientador: Aulus Cavalieri Carciofi / Banca: Marita Vedovelli Cardozo / Banca: Márcia de Oliveira Sampaio Gomes / Banca: Thaila Cristina Putarov / Banca: Lilian Rose Marques de Sá / Resumo: O presente estudo avaliou e comparou a composição da microbiota associada à mucosa gastrointestinal de cães adultos e idosos, alimentados com rações contendo proteína de origem animal ou vegetal e fibras de diferentes fermentabilidades. O estudo também comparou a composição da microbiota associada a mucosa do duodeno, jejuno e cólon. O ensaio seguiu esquema fatorial 3 x 2, com três rações e duas idades. Foram utilizados 18 cães adultos (2,6 ± 0,9 anos) e 18 cães idosos (10,2 ± 1,0 anos). Foram produzidas três dietas: dieta NFF com fibra insolúvel não fermentável a base de cana-de-açúcar e farinha de vísceras de frango; dieta FF com polpa de beterraba, fibra fermentável e parcialmente solúvel, e farinha de vísceras; dieta SM com 30% de farelo de soja em substituição a farinha de vísceras de frango. Os animais foram submetidos a 30 dias de adaptação à dieta e nos dias 31 e 32 foram realizadas as endoscopias e colonoscopias para coleta de fragmentos do duodeno, jejuno e colon, para posterior análise da microbiota associada à mucosa gastrointestinal através do sequenciamento Illumina. Os dados foram avaliados por análise de variância e médias comparadas pelo teste de Tukey. Dados sem destibuição normal foram submetidos a transformação logarítmica na base 10, log (x + 1) ou raiz quadrada antes da análise estatística, e quando necessário submetidos ao teste não paramétrico de Kruskal-Wallis. A avaliação das dietas e do efeito da idade sobre a mucosa do cólon demonstraram que cães i... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present study evaluated and compared the microbiota composition associated with gastrointestinal tract mucosa of adult and elderly dogs, fed with animal or vegetable protein and fiber of different fermentabilities. The study also compared the composition of the microbiota associated with the mucosa of the duodenum, jejunum and colon The assay followed a 3 x 2 factorial scheme, with three rations and two ages, generating six experimental treatments. Three blocks of 12 dogs each were used, six adult dogs (2.6 ± 0.9 years) and six elderly (10.2 ± 1.0 years). The experimental diets were: diet with non-fermentable insoluble fiber based on sugarcane (NFF) and chicken offal meal; Diet with 30% of soybean meal (SM), replacing the flour of chicken viscera; Diet with fermentable and partially soluble fiber based on beet pulp (FF). Each block was structured as follows: day one to day 30 adaptation to the diet; day 31 and 32 endoscopies and colonoscopies with collection of fragments of the duodenum, jejunum and colon, for further analysis of microbiota associated to the gastrointestinal mucosa by Illumina sequencing.The data was evaluated by analysis of variance by the SAS MIXED procedure. Averages were compared by the Tukey test (P <0.05). Data without normal staining were submitted to log 10 base transformation, log (x + 1) or square root before statistical analysis, and when necessary submitted to non-parametric Kruskal-Wallis test. The evaluation of diets and age effect on colon ... (Complete abstract click electronic access below) / Doutor

For centuries Aloe vera has been exploited for several verified and unverified medicinal uses such as wound healing, treatment of gastrointestinal ulcers and for its many biological effects including anti-microbial, laxative, anti-inflammatory and immunostimulatory activities. Studies have generally focused on its effects in vitro and in adults. When nursing mothers use Aloe vera extracts, their suckling infants are at risk of indirect exposure to Aloe vera via breast feeding or directly as dietary/health supplements. The gastrointestinal tract (GIT) of the neonate is sensitive to dietary manipulations during the suckling period with long lasting effects that can be irreversible. Thus babies may be at risk if administered Aloe vera extracts directly as dietary supplements or indirectly via breast milk. The main objectives of this study were to evaluate the effects of orally administered aqueous and alcohol extracts of Aloe vera on growth performance, the morphometry and morphology of the gastrointestinal tract and accessory organs, and liver function of suckling rats. Suckling Sprague-Dawley rats (77), males (n=38) and females (n=39) of 6 days old were randomly assigned to one of five treatment groups and given once daily by oral gavage a suspension of lyophilized crude alcohol or aqueous extracts of Aloe vera suspended in distilled water. Group I (control) was gavaged with distilled water (vehicle). Group II received a low dose of the aqueous extract (AqL) at 50mg. kg-1; Group III received a high dose of the aqueous extract (AqH) at 500mg. kg-1; Group IV received a low dose of the alcohol extract (AlcL) at 50mg. kg-1 whilst Group V received a high dose of the alcohol extract (AlcH) at 500mg. kg-1. The extracts and distilled water were 2 administered at a volume of 10ml.kg-1. The pups remained with their dams for the duration of the study and after 8 days on the treatments, the pups were humanely killed to harvest their tissues for measurements and physiological analysis. All data were expressed as mean ± SD and analyzed by one way ANOVA, the values were considered statistically significant when p < 0.05 and then a Bonferroni Post hoc test was applied. The suckling rats fed respectively with high doses of AlcH and AqH had a significantly higher body mass gain than the other groups (p < 0.05, one way ANOVA). Linear growth as measured by tibial length was significantly increased in the AqH group compared to the other groups. There was no significant difference in the mass and relative density of the tibia bones of the rats from the different treatment groups. The differences in growth could not be attributed to circulating concentrations of the somatotrophic hormone, Insulin-like growth factor-1 (IGF-1) which was not significantly different between the groups. The treatments did not result in any significant differences in lengths, and mass of the small and large intestine, however the caecum was significantly enlarged (hypertrophy of muscularis, submucosa and mucosa) in the rats that received the Aloe vera extracts. Although, there was no significant difference in the mass of the rats’ livers, the lipid and glycogen content were significantly higher (p < 0.001) for the AqH group compared to the other groups. Histologically, the hepatocytes showed enlarged nuclei, granular cytoplasm and dilated sinusoids for AqH and AlcH as compared to the control group. An indirect assessment of liver function by measurement of blood concentrations of alkaline phosphatase (ALP) and alanine amino transaminase (ALT) did not reveal a significant difference between the groups. The non fasting concentration of metabolic substrates (glucose and triglycerides) was also not significantly different between the groups. The pups given high doses of the extracts had a significantly greater (p < 0.05) thymus mass (hyperplastic) than the other groups. The short term administration of Aloe vera extracts has shown a growth promoting effect, enhanced hepatic storage of metabolic substrates and hypertrophy of the caecum and thymus of neonatal rats. These effects need to be explored further to enhance animal production and health.

A análise de sinais mioelétricos provenientes do tubo gastro-intestinal de animais de laboratório (ratos), conseguidos por meio de eletrodos cronicamente implantados, é peça-chave no entendimento das desordens associadas ao sistema digestivo. Esta análise enfrenta consideráveis dificuldades quando realizadas por métodos clássicos, em especial os baseados na transformada de Fourier. A interação de várias componentes mioelétricas torna muito complicado e trabalhoso o acompanhamento destes sinais ao longo do tubo digestivo e a obtenção de parâmetros típicos como a velocidade de propagação entre eletrodos. Estuda-se aqui uma alternativa mais nova e promissora: a transformada Wavelet. Utilizando esta ferramenta matemática, torna-se possível obter uma melhor resolução tempo-freqüencial dos sinais estudados, permitindo encontrar padrões referentes à propagação do sinal mesmo em leituras ruidosas e compostas de várias freqüências. Foram analisados 82 leituras de 9 animais normais do Laboratório de Investigação Médica da Faculdade de Medicina da USP, sendo possível determinar dois parâmetros: a velocidade de propagação média entre eletrodos (cerca de 1.2 cm/s) e as componentes principais da freqüência basal (0.63 e 0.65 Hz). / The analysis of myoelectric signals from the gastro-intestinal tube of laboratory animals (mice), recorded by chronically implanted electrodes, is a key stone in understanding the disorders associated to the digestive system. This analysis meets considerable difficulties when done by classical methods, specially those based in the Fourier transform. The many myoelectric components interactions makes the following of these signals along the digestive tract and the retrieval of typical parameters (such as the propagation velocity between electrodes) a very complicated and laborious task. Here is studied a newer and more promising alternative: the Wavelet transform. Using this mathematical tool, it becomes possible to obtain a better time-frequency resolution of the studied signals, allowing to find patterns related to the signal propagation even in noisy and multifrequencial readings. 82 readings from 9 normal animals belonging to the Medical Investigation Laboratory of the Medicine Faculty of University of São Paulo were analyzed, becoming feasible to determine two parameters: the mean propagation velocity between electrodes (about 1.2 cm/s) and the main components of the basal frequency (0.63 e 0.65 Hz).

O megacólon chagásico pode acometer até 10% dos pacientes na fase crônica da doença e o principal sintoma é a constipação intestinal que pode variar de dias a meses. Vários aspectos da fisiopatologia do megacólon não são bem compreendidos. O objetivo deste trabalho foi avaliar o tempo de trânsito colônico com marcadores radiopacos em portadores de megacólon chagásico com constipação intestinal crônica acima de oito dias. Foram estudados 64 pacientes (36 mulheres e 28 homens), idade variando de 32 a 76 anos, com história clínica de constipação intestinal crônica, diagnóstico radiológico por enema opaco de megacólon e com provas sorológicas positivas para doença de Chagas. Foi considerado como megacólon quando o diâmetro do cólon era maior ou igual 6,5 cm. Para avaliar o tempo de trânsito colônico, cada paciente ingeriu uma cápsula contendo 24 marcadores em forma de anéis radiopacos (Sitzmarks, Consil Pharmaceutics, Fort Worth, EEUU), e realizadas radiografias simples de abdome (ortostase) após três e cinco dias. Um grupo de 20 pacientes, sem constipação e com sorologia negativa para doença de Chagas, foi avaliado pela mesma metodologia e considerado como controle. A interpretação da localização e contagem dos marcadores foi realizada dividindo-se o intestino grosso nos segmentos: cólon direito, cólon esquerdo e retossigmóide. Empregou-se o teste de Kolmogorov-Smirnov para comparação do número de marcadores colônicos entre si e no 3o e 5o dias. O número de marcadores em cada grupo, em períodos distintos, foi avaliado com o teste de Wilcoxon. O coeficiente de correlação de Pearson foi utilizado para se avaliar a influência dos fatores externos (sexo, idade, tempo do início dos sintomas em anos, dias de constipação e presença de megarreto). O tempo de início dos sintomas variou de um a 33 anos e a duração da constipação, em dias, variou de oito a 90 dias. O megarreto ocorreu em 68,8% dos pacientes. Não se observou diferença, com significado estatístico, na quantidade total de marcadores retidos no 3o e 5o dias do exame entre os pacientes de ambos os sexos com e sem megarreto. A quantidade de marcadores, em cada segmento, colônico mostrou diferença estatística. Estes marcadores apresentaram uma distribuição em forma de um gradiente, crescendo de proximal para distal tanto no 3o quanto no 5o dia. Ocorreu uma progressão dos marcadores em direção ao ânus com um acúmulo no retossigmóide com diferença estatística do 3o para o 5o dia. Conclusões: houve retenção quase total dos marcadores até o 5o dia com predomínio no cólon esquerdo e no retossigmóide, sendo maior no retossigmóide; não houve diferença estatisticamente significante na retenção total dos marcadores entre o 3o e o 5o dia de exame; os fatores: idade, sexo, início dos sintomas, dias de constipação e megarreto não interferiram na quantidade e localização dos marcadores; os segmentos colônicos direito e esquerdo apresentaram trânsito lento, tendo ocorrido estase dos marcadores no retossigmóide / Chagasic megacolon may be present in up to 10% of infected people during chronic phase of the disease. Long lasting constipation of a few days up to months is the main complaint. Phisiopathology of megacolon is not completely understood as well as the predominant dilatation of sigmoid colon. This study addressed the colonic transit time (CTT) in patients with acquired megacolon with chronic constipation grater then 8 days. Study group consisted of 64 patients (36 women and 28 men); age ranged from 32 to 76 years. All patients had clinical history of constipation, barium enema showing megacolon (colon diameter ³ 6,5 cm) and positive serological tests for Chagas disease. CTT was evaluated by oral administration of a capsule with 24 ring-shaped radiopaque markers (Sitzmarks, Consil Pharmaceutics, Fort Worth, USA) and plain abdominal X-Rays were taken in the 3rd and 5th days post capsule ingestion. Twenty patients with no colorectal complaint and negative serology for Chagas disease were submitted to the same CTT exam as a control group. Results of number and position of radiopaque markers were evaluated according to large bowel segments: right colon, left colon and rectosigmoid colon. Kolmogorov-Smirnov test was used to compare the number of markers by segments in the 3rd and 5th days and Wilcoxon test to compare groups by the number of markers. Pearson correlating test was applied to analyze variables as sex, age, symptoms onset (years), period of constipation (days) and megarectum association. Onset of symptoms ranged from 1 to 33 years while constipation varied from 8 to 90 days. There was not difference between the number of retained colonic markers in the 3rd and 5th days post administration. The number of markers in each colonic segment had significant difference statistically in the 3rd and 5th days with greater numbers from proximal to distal colon. Such distribution assumed an aspect of a gradient and some radiopaque markers moved towards rectosigmoid colon from 3rd to 5th days with difference statistically significant. Conclusions: majority of radiopaque markers was retained up to 5th day in the left and rectosigmoid colon with greater accumulation in the rectosigmoid; sex, age, symptoms onset (years), period of constipation (days) and megarectum association had not influenced the number and position of retained markers; right an left colons showed slow transit while rectosigmoid showed fecal stasis

Dentre os microrganismos que a compõem a microbiota intestinal, sobressai Escherichia coli, que possui como principal nicho ecológico o intestino grosso de humanos. Sua importância destaca-se em fazer parte dos microrganismos comensais pioneiros na colonização da mucosa intestinal e também o seu papel patogênico causando doenças intra e extra intestinais. O objetivo deste estudo foi avaliar a resposta imune contra antígenos totais de Escherichia coli. Os resultados desse estudo podem servir como subsídio para o entendimento da resposta imune sistêmica a um microrganismo presente na mucosa. Os antígenos totais de E. coli foram obtidos após lise com solução de guanidina a 8M. Realizou-se a diálise e a dosagem de proteínas. Foi realizado a caracterização eletroforética em gel de poliacrilamida e perfil antigênico por Western Blotting. Avaliou-se a presença de anticorpos IgG total e IgA sérica específicos em 30 soros de humanos e também a resposta de células mononucleares de sangue periférico humano (PBMC) através da metabolização de MTT. Os resultados obtidos demonstraram que a suspenção de antígenos obtida era composta por várias proteínas e o teste de Western Blotting revelou que estas foram reconhecidas por anticorpos presentes nos soros de humanos. Foi possível detectar a presença de anticorpos IgG total e IgA sérica contra os antígenos de E. coli por ELISA. No ensaio de viabilidade e proliferação celular pelo MTT, observou-se que houve proliferação celular em diferentes concentrações do antígeno e a viabilidade não foi inferior a 70%. Os resultados sugerem que os antígenos oriundos de E. coli podem induzir respostas imunes locais e sistêmicas. / Among the microorganisms that make it up the intestinal microbiota, stands out Escherichia coli, which has as main ecological niche, the large human intestine. Its importance stands out in being part of the pioneers commensal microorganisms on the colonization of the intestinal mucosa also his pathogenic role causing extra intra and intestinal diseases. The objective of this study was to evaluate the immune response against total antigens of Escherichia coli. The results of this study could aid to understanding systemic immune response to a commensal microorganism that lives in the mucosa. Total E. coli antigens were obtained after lysis with 8M guanidine solution. After dialysis, protein assay was carried out. It was performed electrophoretic characterization of the antigens using polyacrylamide gel electrophoresis and the antigenic profile by Western blotting. We evaluated the presence of total IgG and IgA specific antibodies in 30 human sera. It also assessed the human response of peripheral blood mononuclear cell (PBMC) by MTT metabolization. The results obtained demonstrated that the antigens were composed of various proteins and Western Blotting showed that antigen proteins were recognized by antibodies present in human serum. It was possible to detect the presence of total IgG and IgA antibodies against E. coli antigens by ELISA. In viability assay evaluated by the MTT metabolization by PBMC, it found that cell proliferation occurred at different antigen concentrations, and viability was not less than 70 %. The results suggest that the antigens from E. coli can induce local and systemic responses. / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

Tenascin X (TNX) is a matricellular protein involved in regulating cellular functions by interacting with other extracellular matrix (ECM) proteins within the cell matrix and has anti-adhesive properties evidenced in tumours and wound healing. TNX is the only member of the tenascin family that is lost in Joint Hypermobility Syndrome (JHS) and exerts a crucial architectural function. Of importance, TNX deficient and JHS patients have gastrointestinal (GI) dysfunction. Despite this association no study has described the role of TNX in the GI tract. Thus, the aim of this thesis was to characterise the expression of TNX in the stomach and colon in mouse and human tissue. Second, we aimed to elucidate the functional role of TNX using TNX knockout (TNX KO) mice. Expression studies revealed TNX in vagal afferent endings in the mouse, and myenteric cell bodies in human stomach. In colon, TNX strongly associated with cholinergic submucous and myenteric neurons in both species, however, was not found in CGRP positive fibres. Cell bodies in nodose ganglia, dorsal root ganglia, ventral and dorsal horn were also TNX positive. Functional studies in stomach, using single fibre electrophysiology showed TNX KO mice had increased vagal afferent mechanoreceptor sensitivity. Octanoic acid breath test revealed rapid gastric emptying in TNX KO. Colonic manometry showed the amplitude and frequency of colonic contractions were reduced in TNX KO mice, particularly in the distal colon. Ussing chamber studies measuring changes in ion flux (indirect measure of secretion) showed no major difference between TNX KO and wild type (WT) mice. The specific localisation of TNX with neuronal structures in the gut is shown here for the first time suggesting that TNX is more than just an architectural protein. Indeed, its role in specific GI functions supports this observation and provides a mechanism for GI symptoms in JHS.

This study investigated how gastrointestinal and skin bacterial infections were affected by differential expression of connexin (Cx) 26 and a disintegrin and metalloprotease (ADAM) 17 in vitro. Cx26 is a component of gap junctions, which facilitate the transfer of small molecules between two cells. Recessive mutations in Cx26 cause non syndromic hearing loss (NSHL), and in certain populations, specific mutations account for the majority of Cx26 related NSHL. Their common occurrence suggests that they may provide a heterozygous, protective advantage to carriers. In this study adherence by the attaching and effacing pathogen Enteropathogenic Escherichia coli (EPEC) was significantly reduced in cells expressing mutant Cx26 compared to wild type Cx26. Furthermore, EPEC adherence and invasion of an alternative enteric pathogen, Shigella flexneri were reduced following treatment with Cx26 short-interfering-RNA in intestinal cells. These findings suggest that the loss of functional Cx26 expression improves protection against enteric bacteria. ADAM17 releases substrates including tumour necrosis factor alpha and ligands of the epidermal growth factor receptor and therefore is involved in the induction of immune responses and maintenance of the epidermal barrier. This study demonstrated that ADAM17 provides protection during Staphylococcus aureus infection of keratinocytes. Subsequently the protective effects of ADAM17 mediated protection were explored. Secretion of the proinflammatory cytokines Interleukins 6 and 8 correlated with ADAM17 activity. Additionally gene expression profiling was performed which identified the IL-17 signalling pathway, which is known to be important during S. aureus infection, as a potential downstream target of ADAM17. In summary, based on these findings, Cx26 and ADAM17 may represent potential therapeutic targets for gastrointestinal and skin bacterial pathogens.

糖尿病是一种呈现流行趋势的代谢紊乱综合症，现如今，全球大约有3.46亿糖尿病患者， 这庞大的数字给各国的公共健康安全支出带来了严重的财政负担。 其中，二型糖尿病（T2DM）占90%。其特点是周围组织的胰岛素抵抗以及后期损伤的胰岛β细胞的功能。在饮食后，小肠会分泌两种肠促胰岛素，葡萄糖依赖性促胰岛素多肽（GIP）和胰高血糖素样肽-1（GLP-1）。两种多肽的主要功能是促进餐后胰岛细胞中胰岛素的分泌，另外他们还可以通过其自身的G蛋白偶联受体，GIPR和GLP-1R发挥其他作用，如葡萄糖依赖性的刺激胰岛素的生成，刺激胰岛β细胞的增殖，抑制细胞的凋亡等。这些功能也使肠促胰岛素成为糖尿病治疗的一种手段，比如Exendin-4和DPP4抑制剂。 然而，除了在胰岛中的作用，肠促胰岛还可能和脂质代谢相关，其中GIP和脂质代谢的报导研究的更加深入。在肥胖的状态下，血液中GIP含量高于正常水平；GIPR基因敲除老鼠和GIPR的抑制剂喂养的小鼠可以抵抗高脂饮食诱导的肥胖和2型糖尿病；GIP还可以直接调节脂肪细胞的脂肪生成和脂解。这些数据表明GIP在肥胖和糖尿病的发生过程中可能存在促进作用,这使得GIP治疗药物的开发需要谨慎的对待。 / 为了进一步研究GIP在脂肪细胞中发挥的生物学效应，在本研究中，我们利用腺病毒介导技术通过在脂肪细胞中过表达GIPR来增加GIP的活性，然后检查GIP在脂肪细胞中所起的作用。实验结果表明,GIP可以通过cAMP-PKA信号通路迅速并且长期的刺激脂肪细胞的炎症反应，增强IKKβ-NFκB信号通路和增加炎症基因的表达。更深入的机制研究表明，JNK 信号通路也参与GIP诱导的炎症反应，抑制JNK通路可以大部分恢复GIP增加的炎症因子的表达和IKKβ的磷酸化水平。由于长期的炎症反应，脂肪细胞的胰岛素信号通路受到GIP的损伤，在GIPR过表达的脂肪细胞中，胰岛素刺激的AKT磷酸化水平和葡萄糖吸收能力都被GIP降低，葡萄糖转运蛋白4（Glut-4）的表达水平也同时减少。因此，本研究结果表明GIP可能在肥胖的发展过程中，通过诱导脂肪细胞的炎症反应来损伤胰岛素敏感性而最终导致2型糖尿病的发生。 / Diabetes mellitus is a type of metabolic syndrome that has prevailed all over the world with the development of economic and over-nutrient lifestyle. It is estimated to 346 million diabetes patients in the worldwide most recently. The huge population put a major burden on the cost of public health care to all the countries. Among the types of diabetes, type 2 diabetes (T2DM) makes up 90% of recorded cases. The characteristics of T2DM are insulin resistance of peripheral tissues and impaired pancreatic cell function and mass. Two major incretins GIP (glucose-dependent insulinotropic peptide) and GLP-1 (glucagon-like peptide 1) are secreted from gut in response to food ingestion. The prominent role of GIP and GLP-1 is to stimulate glucose-dependent insulin release in pancreatic β cell. In addition, they both exert multiple biological effects via their relative G-protein coupled receptors, GIPR and GLP-1R, including glucose-stimulated insulin production, cell proliferation and anti-apoptosis in pancreatic β cells. The beneficent effects of incretins potentiate them as targets for the treatment of diabetes. GLP-1 analog, exendin-4 and DDP4 (dipeptidyl peptidase-4) inhibitors (to prevent GIP and GLP-1 from degradation) have been already used in clinical research. However, in addition to their effects on pancreatic β cell, both peptides are also related to lipid metabolism. The role of GIP has been studied more extensively. In obese state, the circulating level of GIP is elevated. GIPR knockout (KO) mice are resistant to high fat diet (HFD) induced obesity, a similar phenotype is found in GIPR antagonist administrated HFD-mice. Moreover, GIP also directly promotes lipogenesis and lipolysis in adipocytes. The rising evidence suggests a potential role of GIP in adipocyte biology and lipid metabolism, which diminishes the enthusiasm of GIP as a candidate therapeutic reagent for T2DM. / In order to further understand the biological effects of GIP in adipocytes, here, we over-expressed GIPR in 3T3-L1 CAR adipocytes via adenovirus-mediated gene transfer technology to enhance the activity of GIP. The results demonstrate that GIP impairs the physiological functions of adipocytes as a consequence of increasing the production of inflammatory cytokines, chemokines, and phosphorylation of IkB kinase (IKK) β through activation of the cyclic AMP-protein kinase A (cAMP-PKA) pathway. Activation of Jun N-terminal Kinase (JNK) pathway is also observed in GIP-induced inflammatory responses in adipocytes. An inhibitor of JNK blocks GIP-stimulated secretion of inflammatory cytokines and chemokines, as well as phosphorylation of IKKβ. The chronic inflammatory response eventually impairs insulin signaling in adipocytes, as demonstrated by reduction of protein kinase B (PKB/AKT) phosphorylation. The subsequently physiological analysis also indicates that GIP inhibits insulin-stimulated glucose uptake, and gene expression analysis reveals a decrease of glucose transporter 4 (Glut-4) in the meanwhile. The results suggest that GIP may be one of stimuli attributable to obesity induced insulin resistance via induction of adipocyte inflammation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Nie, Yaohui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 95-111). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / INTRODUCTION --- p.1 / Chapter Part 1 --- Obesity and Type 2 diabetes --- p.1 / Chapter 1.1 --- Introduction to diabetes --- p.1 / Chapter 1.1.2 --- Physiology of adipocyte --- p.4 / Chapter 1.1.3 --- Mechanism of obesity induced diabetes --- p.10 / Chapter Part 2 --- Incretins and T2DM --- p.12 / Chapter 2.1 --- History of incretins --- p.12 / Chapter 2.2 --- Physiological actions of incretins --- p.14 / Chapter 2.3 --- Molecular mechanism of incretin actions in pancreas --- p.16 / Chapter 2.4 --- Incretins and T2DM --- p.19 / Chapter Part 3 --- Incretins and lipid metabolism --- p.23 / Objective --- p.26 / Methods and materials --- p.28 / Chapter 1 --- Cell culture --- p.28 / Chapter 1.1 --- 3T3-L1 culture and differentiation --- p.28 / Chapter 1.2 --- 3T3-L1 CAR culture and differentiation --- p.29 / Chapter 2 --- Cloning and recombinant adenovirus construction --- p.30 / Chapter 2.1 --- Plasmid construct --- p.30 / Chapter 2.2 --- Construct of recombinant adenoviruses --- p.30 / Chapter 2.3 --- Generation and infection of the adenoviruses --- p.31 / Chapter 3 --- Physiological and morphological assays --- p.32 / Chapter 3.1 --- Lipolysis assay --- p.32 / Chapter 3.2 --- TUNEL assay --- p.32 / Chapter 3.3 --- Glucose uptake --- p.33 / Chapter 3.4 --- Glut-4 localization --- p.33 / Chapter 4 --- Gene expression analysis --- p.35 / Chapter 4.1 --- Quantitative real-time PCR --- p.35 / Chapter 4.2 --- Immunoblot analysis --- p.35 / Chapter 4.3 --- ELISA assay --- p.36 / Chapter 5 --- Isolation of primary adipocytes --- p.37 / Results --- p.38 / Chapter Part 1 --- Role of GIP in 3T3-L1 cells --- p.38 / Chapter 1.1 --- Differentiation of 3T3-L1 adipocytes --- p.38 / Chapter 1.2 --- GIP slightly stimulates phosphorylation of p-CREB and lipolysis in 3T3-L1 cells. --- p.40 / Chapter 1.3 --- Analysis of gene expression in GIP-treated adipocytes --- p.42 / Chapter 1.4 --- Discussion --- p.44 / Chapter Part 2 --- Role of GIP in GIPR over-expressing 3T3-L1 CAR adipocytes --- p.46 / Chapter 2.1 --- Differentiation of 3T3-L1 CAR adipocytes --- p.46 / Chapter 2.2 --- Functional tests in GIPR over-expressing 3T3-L1 CAR adipocytes. --- p.48 / Chapter 2.3 --- Effect of GIP on cell viability --- p.50 / Chapter 2.4 --- Analysis of gene expression in GIP-treated adipocytes --- p.52 / Chapter 2.5 --- GIP activates inflammatory responses in GIPR over-expressing adipocytes --- p.54 / Chapter 2.6 --- Inhibition of IKKb pathway restores GIP-induced inflammatory responses --- p.56 / Chapter 2.7 --- Effects of GIP on adipocytes are partially dependent on the cAMP-PKA pathway --- p.58 / Chapter 2.8 --- Activation of cAMP-PKA pathway induces adipocyte inflammation. --- p.60 / Chapter 2.9 --- cAMP-Epac pathway is not involved in GIP-induced inflammation --- p.62 / Chapter 2.10 --- GIP stimulates cell stress activated kinases --- p.64 / Chapter 2.11 --- JNK partially mediates GIP-induced adipocyte inflammation --- p.65 / Chapter 2.12 --- Inhibition of JNK pathway partially restores GIP-induced inflammatory responses --- p.67 / Chapter 2.13 --- GIP impairs insulin signaling in GIPR over-expressing 3T3-L1 CAR adipocytes via inducing inflammatory response --- p.69 / Chapter 2.14 --- GIP enhances basal glucose uptake but impairs insulin stimulated glucose uptake in 3T3-L1 CAR GIPR over-expressing adipocytes --- p.71 / Chapter 2.15 --- Discussion --- p.73 / Chapter Part 3 --- Role of GIP in primary adipocytes --- p.78 / Chapter 3.1 --- GIPR expression level in primary adipocytes --- p.78 / Chapter 3.2 --- Analysis of gene expression in primary adipocytes after GIP treatment --- p.80 / Chapter 3.3 --- Discussion --- p.81 / SUMMARY --- p.82 / Chapter Future investigation --- p.83 / Chapter Appendix 1: --- Abbreviations --- p.86 / Chapter Appendix 2: --- Protocols --- p.90 / Preparation of competent cells --- p.90 / Outlines of recombinant adenovirus preparation --- p.91 / Virus titering (TCID50) --- p.92 / Primers for real-time PCR --- p.93 / Chapter Publications and Scientfic activities --- p.94 / Thesis related publication: --- p.94 / Other pubiliations: --- p.94 / Scientific activities: --- p.94 / References --- p.95

O guaraná (Paullinia cupana) é uma planta nativa da América do Sul, e suas sementes tem sido utilizadas por tribos amazônicas desde antes da colonização. O extrato de guaraná é consumido popularmente nos dias de hoje, entretanto pouco se sabe sobre a relação desse extrato com a microbiota intestinal. A microbiota possui um papel central na absorção de nutrientes da dieta e na regulação do metabolismo energético, sendo modificada na obesidade. A microbiota intestinal também é afetada pelo consumo de compostos vegetais, sendo muitas vezes regulada positivamente por dietas ricas em polifenóis. Todavia, o consumo de extratos vegetais pode gerar um desfecho tóxico, assim como alterações negativas na microbiota. O efeito de extratos ricos em compostos secundários deve ser estudado individualmente. Nesse trabalho avaliamos as alterações no ecossistema intestinal causadas pelo extrato comercial de sementes do Guaraná em animais saudáveis e no contexto da obesidade.Induzimos a obesidade em animais através de uma dieta que mimetiza a dieta ocidental, caracterizada por uma maior quantidade de gordura, sal e açúcar refinado, e uma menor quantidade de fibras. Os animais também foram submetidos ao tratamento com um extrato comercial de Guaraná, cafeína ou salina. A disbiose intestinal foi medida no conteúdo cecal através da amplificação e sequenciamento da porção ribossomal 16S. Também foi medida a atividade enzimática no fígado, rins e intestino delgado, assim como a quantidade de citocinas proinflamatórias no soro. A dieta obesogênica gerou um aumento no ganho de peso e no acúmulo de gordura dos animais comparada com a dieta Chow. Tanto o Guaraná quanto a cafeína não foram capazes de reverter o ganho de peso e o acúmulo de gordura. Também observamos a diminuição da atividade da enzima CAT no rim nos animais que receberam guaraná, independente da dieta. A dieta obesogênica induziu alterações na microbiota intestinal semelhantes às da obesidade, diminuindo a proporção de Bacteroidetes sobre Firmicutes, aumentando o gênero Mucispirilum e diminuindo os gêneros Lactobacillus e Bifidobacterium. Os tratamentos com guaraná e cafeína na dieta Chow diminuíram a proporção B/F, assim como o gênero Bifidobacterium. Observamos um aumento no filo Proteobacteria nos animais do grupo controle obeso. Esse aumento foi revertido pelo tratamento com guaraná. Analisando os gráficos de clusterização e landscape, observamos uma maior distância entre a microbiota dos animais que receberam dietas diferentes do que a dos animais que receberam tratamentos diferentes. Os tratamentos com guaraná ou cafeína modificaram o ecossistema intestinal, mas sem a capacidade de diminuir o acúmulo de gordura ou o ganho de peso. A maior alteração na microbiota foi devido à dieta obesogênica e não aos tratamentos, mostrando que os efeitos anti-obesogênicos do guaraná observados em outros estudos provavelmente não são via microbiota intestinal. / Guarana (Paullinia cupana) is a plant native of South America, and its seedshave been used by Amazonian tribes since before colonization. Guarana extract is consumed popularly today, however little is known about the interactions of this extract with the intestinal microbiota. The gut microbiota plays a central role in the absorption of nutrients from the diet and in the regulation of energy metabolism, being altered in obesity. The intestinal microbiota is also affected by the consumption of plant metabolites and is often regulated positively by diets rich in polyphenols. However, the consumption of plant extracts can generate a toxic outcome, as well as negative changes in the microbiota. Therefore, the effect of plant extracts should be studied individually. In this work, we evaluated the changes in the intestinal ecosystem caused by the administration of a commercial extract of Guaraná seeds in healthy animals and in the context of obesity. We induced obesity in animals through a diet that mimics the western diet, characterized by a higher amount of fat, salt and refined sugar, and a smaller amount of fiber. The animals were also treated with a commercial extract of Guarana, caffeine or saline. Intestinal dysbiosis was evaluated by cecal content, amplification and sequencing of the 16S ribosomal portion. The antioxidant enzymatic activity of the liver, kidney and small intestine was measured as well as the concentration of pro-inflammatory cytokines in the serum. The obesogenic diet increased the weight gain and fat accumulation of the animals compared to the Chow diet. Both Guarana and caffeine were unable to reverse weight gain and fat accumulation. We observed a lower activity of the Glutathione Peroxidase enzyme in the kidney and small intestine in the animals that received obesogenic diet compared to the Chow diet, regardless of the treatment. We also observed the decrease of Catalase enzyme activity in the kidney of animals that received Guarana, regardless of diet. The obesogenic diet induced changes in the intestinal microbiota similar to other works in literature, reducing the proportion of Bacteroidetes/Firmicutes (B/F), increasing the genus Mucispirilum and decreasing the genera Lactobacillus and Bifidobacterium. Guarana and caffeine treatments in the Chow diet decreased the B/F ratio, thus the Bifidobacterium genus. We observed an increase in the phylum Proteobacteria, in the animals of the Obese Control group. This increase in the phylum Proteobacteria, was reversed by the treatment with Guaraná. Analyzing the phylogenetic proximity charts and landscape, we observed a greater distance between the animals that received different diets than animals that received different treatments. Treatments with guarana or caffeine modified the intestinal ecosystem, but without the ability to decrease fat accumulation or weight gain. The major change in the microbiota was due to the obesogenic diet and not to the treatments, showing that the antiobese effects of guarana observed in other studies probably are not via intestinal microbiota.

by Joseph Jao Yiu Sung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (p. 201-220). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.