published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

The gastrointestinal tract houses one of the most dense and diverse communities of bacteria on the planet. The mutualistic relationship between the host and commensal microbe permits the microbe an ideal environment to grow and provides the host with increased caloric intake, maturation of the adaptive immune system, and resistance against invading pathogens. To maintain a system in which both parties benefit, the epithelium has evolved numerous strategies to ensure epithelial cells respond to microbes appropriately and that potentially hazardous commensals remain distanced from the soma proper. Breakdown of these propitiating mechanisms elicits unchecked inflammation and can lead to pathology and reduction of host fitness. We show that oral and intestinal epithelial cells respond to the circulating hormone adiponectin in the presence of bacterial constituents, and that adiponectin has the potential to downregulate NF-κB signaling. We also show many commensal bacteria have no effect on TNF or IL-8 proinflammatory gene expression in intestinal cells. Commensals within the family Enterobacteriaceae can increase TNF and IL-8 expression, but also expression of the NF-κB regulator A20 and MAPK phosphatase MKP-1. Importantly, Enterobacteriaceae also increased expression of the IgA transporter pIgR. In the mouse model, we show pIgR expression along the intestinal epithelium is necessary for SIgA accumulation in the outer mucus layer where commensal bacteria reside. Loss of the mucus layer, but not pIgR is sufficient to allow direct bacterial-epithelial cell contact and induce spontaneous inflammation along the colon. Secretory IgA is supplied maternally through breast milk early in life to compensate for the neonate’s inability to produce sufficient endogenous amounts. By utilizing a breeding scheme in which mouse dams were unable to provide their offspring with SIgA, we show the necessity of maternally-supplied SIgA to control bacterial invasion to mesenteric lymph nodes before weaning. In addition, 8-10 week old adult offspring not receiving SIgA as neonates showed both a unique intestinal microbiota and different patterns of intestinal epithelial cell gene expression with and without chemically-induced acute colitis. In summary, we reveal new mechanisms the mammalian host utilizes in order to maintain peace between the commensal microbe and host immune system.

The purpose of this study was to examine the pharmacokinetics of d-xylose in man as a function of age with particular emphasis on its absorption characteristics. This study required the development of a specific and sensitive method for the quantitation of xylose from plasma and urine. Following a clean-up procedure, plasma or urine samples are concentrated and undergo two sequential derivatization steps and then are quantitated by capillary column gas liquid chromatography (GLC). D-Xylose is frequently quantitated by a tedious colorimetric assay involving the use of thiourea, a proven animal carcinogen. We have evaluated a more expedient colorimetric assay employing less toxic reagents. Based upon these comparisons the "phloroglucinol" method has been recommended as a replacement for the currently used clinical method for quantitating d-xylose. The human studies revealed age related changes in some but not all d-xylose disposition parameters. Systemic, renal, and nonrenal clearances all declined with advancing age. The terminal elimination half life increased with age. Age had very little influence on the various volumes of distribution. In general, parameters relating to oral absorption showed no age-related dependence. In contrast to what is generally believed, the bioavailability of d-xylose did not decline with age. Lastly, this dissertation addresses the problem of how infusion data may best be fit. Concentration-time data may be fit by a nonlinear regression algorithm in two ways; (1) concentration-time data may be collected and fit both during infusion and after infusion is terminated, (2) concentration-time data may be collected only after the infusion is terminated and be fit as a bolus. Concentration-time data were computer simulated with random error and we found that fitting the entire curve gave the most accurate estimates of disposition parameters.

The ability of essential oils (EO) to manipulate the intestinal microbiota may potentiate their application in food as nutraceutical and as prophylactic agents for colonic disease. Little is known about the influence of EO on gut bacteria, the mechanism of their antibacterial action and genotoxicity to the host. Here, the antibacterial activities of EO in pure and in a mixed faecal culture were investigated. These antibacterial activities were further studied to compare the selective nature of EO and their effects on membrane integrity. The growth of gut pathogens and commensals was inhibited in a dose-dependent manner in pure culture, with most of the pathogens, Escherichia coli O157:H7, Clostridium difficile, C. perfringens and Salmonella typhimurium are sensitive to nerolidol, thymol, eugenol and geraniol at a half maximal inhibitory concentration (IC50) of 50-500 ppm. These concentrations of EO and mainly nerolidol were also inhibitory to some gut commensals, in particular affecting Faecalibacterium prausnitzii adversely in pure culture. In contrast, in the mixed culture system beneficial groups of bacteria, including F. prausnitzii, as determined by qPCR of 16S rRNA genes were not affected. Thymol and geraniol at 500 ppm suppressed the growth of total bacteria, resulting in minimal fermentation. A lower dose of 100 ppm of EO compounds was effective in suppressing the pathogen, C. difficile with no concern for commensal bacteria or their fermentation products, acetate, propionate and butyrate. This study also discovered that the proteome of commensal, Faecalibacterium prausnitzii and pathogenic gut bacteria, Escherichia coli, in response to EO compounds are affected differently. Thymol and eugenol down-regulated virulence factors in E. coli. The tested EO compounds were not genotoxic in the comet assay at non-toxic doses. Differential effects of EO compounds on gut pathogens and commensals and their non-toxicity but geno-protective properties could be applicable in improving gut health in man.

NTRODUCTION: Colonic bacteria produce short-chain fatty acids (SCFAs) by fermentation of dietary carbohydrates and fiber. The production of SCFAs is greatest in proximal colon where propulsion is likely to be highly dependent on chemical/nutrient stimuli. Unabsorbed SCFAs entering the distal colon are likely to modify peristalsis initiated by fecal pellet-induced distension. AIM: To determine the effect of individual SCFAs on propulsive contractions in guinea pig proximal colon and on pellet propulsion in distal colon. METHODS: Proximal colon was excised, cannulated and placed in Krebs buffer in an organ bath. After equilibration, the colon was distended with 1ml of Krebs buffer alone or containing sodium salts of acetate, butyrate, or propionate at 10-100mM. Motility was video recorded, spatiotemporal maps generated, and the number of full-length propulsive contractions during a 5 min period was determined. The distal colon was removed from guinea pig and placed in an organ bath containing Krebs buffer. Following equilibration, video-tracking software was used to measure the velocity of propulsion of a clay pellet placed in the orad end of a segment. Krebs buffer alone or containing individual SCFAs at 30mM was perfused caudad to the pellet at 0.1 ml/min. RESULTS: The basal rate of propulsive contractions in Krebs buffer was 2.9 ± 0.7 per 5 min. Butyrate and propionate had concentration-dependent and opposing effects on propulsive contractions; acetate had no effect. Butyrate significantly increased propulsive contractions (maximal increase of 207% (p ≤ 0.05) at 30 mM) whereas propionate abolished propulsive contractions (91-100% inhibition at 10-30mM). Control Krebs buffer containing 10- 100mM NaCl had no effect. The mean rate of pellet propulsion during perfusion of Krebs buffer alone was 1.5 ± 0.2 mm/sec. Addition of sodium butyrate (30 mM) to the perfusate increased the velocity of pellet propulsion by 40 ± 4% (p<0.05) whereas addition of 30mM sodium propionate decreased velocity of pellet propulsion by 75 ± 8%. Acetate had no significant effect in either proximal or distal colon. Mixtures of SCFAs showed a general decrease in overall motility. CONCLUSION: SCFAs have differential effects on propulsive contractions in the proximal and distal colon of the guinea pig, with butyrate being excitatory and propionate being inhibitory. These studies suggest that the movement of feces in the colon depends not only on distension but the chemical (i.e. dietary) composition of the feces.

Caveolae are integral part of the smooth muscle membrane and caveolins, the defining proteins of caveolae, act as scaffolding proteins for several G protein-coupled receptor signaling molecules and regulate cellular signaling through direct and indirect interactions with signaling proteins. Caveolin-1 is the predominant isoform in the smooth muscle and drives the formation of caveolae. However, little is known about the role of caveolin-1 in the regulation of excitation-contraction and excitation-transcription coupling in gastrointestinal smooth muscle. In the present study we have characterized muscarinic m2 and m3 receptor signaling in gastric smooth muscle and tested the hypothesis that caveolin-1 positively regulates muscarinic receptor signaling and contractile protein expression in smooth muscle. The role of caveolae/caveolin-1 in the regulation of muscarinic signaling was examined using complementary approaches: a) methyl b-cyclodextrin (MbCD) to deplete cholesterol in dispersed muscle cells, b) caveolin-1 siRNA to suppress caveolin-1 expression in cultured muscle cells, and c) caveolin-1 knockout (KO) mice. RT-PCR, western blot and radioligand binding studies demonstrated the selective expression of m2 and m3 receptor in gastric smooth muscle cells. Carbachol (CCh), acting via m3 receptors caused stimulation of phosphoinositide (PI) hydrolysis, Rho kinase and ZIP kinase activity, and induced phosphorylation of MYPT1 (at Thr696) and MLC20 (at Ser19), and muscle contraction, and acting via m2 receptors caused inhibition of forskolin stimulated cAMP formation. Stimulation of PI hydrolysis, Rho kinase and ZIP kinase activities, phosphorylation of MYPT1 and MLC20 phosphorylation and muscle contraction in response to CCh was attenuated in dispersed cells treated with MbCD or in cultured cells transfected with caveolin-1 siRNA. Similar inhibition of all responses was obtained in gastric muscle cells from caveolin-1 KO mice compared to gastric muscle cells to WT mice. Although, caveolin-1 had no effect on m2 receptor signaling, agonist-induced internalization of m2, but not m3 receptors was blocked in dispersed cells treated with MbCD or in cultured cells transfected with caveolin-1 siRNA. These results suggest that caveolin-1 selectively and positively regulates Gq/13-coupled m3 receptor signaling, Gi-coupled m2 receptor internalization. The expression of contractile proteins, g-actin and caldesmone and the transcription factors SRF and myocardin that regulate the expression of contractile proteins are down regulated, whereas EGF-stimulated EGF receptor phosphorylation and ERK1/2 activity are up-regulated in cells transfected with caveolin-1 siRNA. These results suggest using pharmacological, molecular and genetic approaches provide conclusive evidence that caveolae and caveolin-1 play an important role in orchestrating G protein coupled receptor signaling to have dual pro- excitation-contraction and excitation-transcription coupling, and anti-proliferative role in gastric smooth muscle.

Memoria para optar al Titulo Profesional de Médico Veterinario / Los informes endoscópicos de 36 caninos y 12 felinos, fueron descritos en relación a las características individuales, los resultados del examen endoscópico y el diagnóstico histopatológico de las biopsias obtenidas en dichas endoscopías. No existió registro de razas felinas a diferencia de los caninos, que reportaron 25% de mestizos (9) y 75% de individuos (27) pertenecientes a 17 razas puras. En relación al sexo de los pacientes, los felinos registraron igual proporción de machos y hembras, mientras que los caninos reportaron 39% de hembras (14) y 61% de machos (22). En cuanto a la edad, los felinos promediaron 7,3 años, mientras que los caninos registraron una edad media de 3,9 años. Los signos clínicos más prevalentes tanto en felinos como en caninos,,fueron vómito y diarrea con un 39,5% y 29,1% del total de casos respectivamente. Las principales lesiones registradas en la única esofagoscopía fueron edema e hiperemia, similar a lo reportado en 40 gastroscopías, es decir, edema en 45% de los casos, hiperemia en 20% y erosiones/úlceras en 17,5% de ellos, como las lesiones más prevalentes. En un total de 4 duodenoscopías no se reportaron lesiones de la mucosa, a diferencia de las colonoscopías que registraron edema en un 60%, erosiones/úlceras en un 20% e hiperemia en un 15% de los casos, de un total de 20 procedimientos. Los diagnósticos endoscópicos de cada segmento, comparados con los diagnósticos histopatológicos de las biopsias, reportaron niveles aceptables de especificidad y valor predictivo positivo, a diferencia de lo sucedido con los niveles de sensibilidad y valor predictivo negativo, lo que se traduciría en bajos niveles de validez y seguridad de la prueba endoscópica por sí sola. Por otra parte, los resultados de la prueba de McNemar y el índice kappa indicarían la existencia de diferencias significativas entre los cambios y un bajo acuerdo entre las observaciones endoscópicas e histopatológicas. Aunque la endoscopia es una excelente técnica para visualizar el estado macroscópico de la mucosa gastrointestinal, la evaluación histológica de biopsias endoscópicas siempre es necesaria para confirmar el diagnóstico

Orientador: Célia Regina Nogueira / Resumo: Estudos epidemiológicos demonstram um acelerado aumento do número de indivíduos com obesidade em quase todo o mundo. A fisiopatologia da obesidade ainda não está bem elucidada, mas sabe-se que o controle do peso corporal está relacionado a diversos hormônios, sinalizadores celulares e neurônios do Sistema Nervoso Central que regulam o apetite e o balanço energético. Nesse contexto, o tempo de esvaziamento gástrico (TEG) poderia influenciar na fisiopatologia da doença, visto estar relacionado à secreção de hormônios reguladores da fome e da saciedade. Considera-se, a cintilografia como diagnóstico padrão-ouro de anormalidades no TEG. Esse procedimento ocorre com a ingestão de fonte radioativa não selada. Diante disso, avaliamos o TEG sólido em pacientes obesos por meio da cintilografia de esvaziamento gástrico sólido e sua relação com o índice de massa corporal (IMC) dos pacientes. Fizeram parte do estudo 45 mulheres com idade entre 18 e 50 anos, IMC > 35 kg/m2, acompanhadas no ambulatório de gastrocirurgia da Faculdade de Medicina de Botucatu. Foram obtidos dados antropométricos e exames laboratoriais com amostras de sangue, sendo dosados transaminase glutâmico-oxalacética (TGO), transaminase glutâmico pirúvica (TGP), glicemia de jejum, hemoglobina glicada, hormônio tireoestimulante (TSH) e hormônio tiroxina livre (T4 livre). Os resultados mostraram que a maioria dessa população era constituída por mulheres brancas (80%), não tabagistas (91,1%), não etilistas (95,6%), com ida... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Epidemiological studies have shown an accelerated increase in the number of obese individuals in almost the entire world. The pathophysiology of obesity has not yet been fully elucidated, but it is known that body weight control is related to several hormones, cellular signals and neurons of the Central Nervous System that regulate appetite and energy balance. In this context, gastric emptying time (GET) could influence the pathophysiology of the disease, since it is related to the secretion of hormones regulating hunger and satiety. Scintigraphy is considered the gold standard diagnosis of TEG abnormalities. This procedure occurs with ingestion of unsealed radioactive source. Therefore, we evaluated solid GET in obese patients through solid gastric emptying scintigraphy and its relation to the patient's body mass index (BMI). The study included 43 women aged between 18 and 50 years, BMI> 35 kg / m2, accompanied at the gastro-surgery outpatient clinic of the Botucatu Medical School. Anthropometric data and laboratory tests were obtained with blood samples, being measured glutamic-oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), fasting glycemia, glycated hemoglobin, thyroid stimulating hormone (TSH) and free thyroxine (free T4). The results showed that the majority of this population consisted of white women (80%), non-smokers (91.1%), non-alcoholics (95.6%), mean age 38.27 years, mean weight 125, 92 kg, mean height of 1.63 meters, mean BMI of 47.56 kg / m... (Complete abstract click electronic access below) / Mestre

Orientador: Ciniro Costa / Resumo: O trabalho objetivou avaliar o desempenho, grau de infecção natural por helmintos gastrintestinais, variáveis hematológicas e análise econômica de borregas com duas estratégias de recria: um em semiconfinamento e outro em confinamento e posteriormente em semiconfinamento com duas taxas de lotação, em área de aveia preta proveniente de sistema integrado de produção agropecuária (SIPA). Foram utilizados 13 borregas da raça Corriedale por tratamento, sendo: T1) Semiconfinamento com menor taxa de lotação por área (686,4kg de PV/ha) durante 70 dias; T2) Semiconfinamento com maior taxa de lotação por área (1113,6kg de PV/ha) durante 70 dias; T3) Confinamento por 28 dias, seguido por semiconfinamento com menor taxa de lotação por área (686,4kg de PV/ha) durante 42 dias; T4) Confinamento por 28 dias, seguido por semiconfinamento com maior taxa de lotação por área (1113,6kg de PV/ha) durante 42 dias. Para as análises foram utilizados os comandos PROC MIXED e PROC GLIMMIX do SAS e as médias ajustadas pelo método dos quadrados mínimos foram usadas nas análises de comparações múltiplas (P ≤ 0,05). Os tratamentos 3 e 4 apresentaram melhor desempenho, maior consumo de matéria seca e menor contagem de ovos por grama de fezes, em relação aos tratamentos 1 e 2. Ao final do experimento, os animais apresentaram percentual médio de Haemonchus (73,1%), seguida por Trichostrongylus (11,4%) e Cooperia (3,9%). As espécies de helmintos gastrintestinais presentes foram: Haemonchus contortus, Trichostr... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor