<p>One of the aims of this thesis was to investigate the involvement of efflux proteins, such as the P-glycoprotein (Pgp), in the drug transport in different regions of the rat and the human intestine. The intestinal extrusion of intracellularly formed CYP3A4 metabolites, including whether this extrusion might be mediated by Pgp, was also studied. The model drugs used were local anaesthetics (LA), which have been evaluated for inflammatory bowel disease, such as ropivacaine, lidocaine and bupivacaine. The intestinal permeability to LAs was found to be high throughout all intestinal regions of the rat and human intestine. Results from the Ussing chamber model indicated only minor efflux involvement as the drug permeability was higher in the serosa to mucosa transport direction than in the opposite direction. However, the involvement of efflux in the absorption of LAs could not be verified using in situ single-pass perfusion of rat jejunum. The extrusion of the ropivacaine metabolite, 2´,6´-pipecoloxylidide (PPX), was polarized to the mucosal reservoir of the Ussing chamber for both rat and human intestinal samples, and was probably not caused by any Pgp involvement. The expression levels of CYP3A4 and efflux transporters were consistent with the enzymes’ activity in human intestine. PPX formation was mediated by CYP3A4 in human intestine, and cyp2c and cyp2d in rat intestine. Species differences were observed, as PPX was formed in rat colon, but not human colon. In conclusion, the permeability of ropivacaine, lidocaine and bupivacaine was not subjected to efflux transport of significance for their intestinal uptake. The transport of ropivacaine metabolites to the mucosal compartment was probably not mediated by Pgp. The Ussing chamber model showed consistent results with those from intestinal microsomes as far as intestinal metabolism is concerned, making it a suitable model for investigations of the interplay of efflux and metabolism. </p>

Colon cancer is one of the most commonly diagnosed cancers in the US, yet small intestine cancer is a rare event. While there are many similarities between these two tissues, inherent differences such as redox status, may contribute to the variation in cancer occurrence. We examined the difference in reactive oxygen species (ROS) generation, antioxidant enzyme activity and oxidative DNA damage in the small and large intestine of rats under normal conditions and following exposure to exogenous oxidative stress. Basal ROS and antioxidant enzyme activities were greater in the colon than the small intestine, and the balance of ROS to antioxidant enzymes in the colon was more pro-oxidant than in the small intestine. During oxidative stress, ROS and oxidative DNA damage were greater in the colon than the small intestine. Thus the colon responds to oxidative stress less effectively than the small intestine, possibly contributing to increased cancer incidence at this site. We next wanted to understand how diets containing a combination of fish or corn oil and pectin or cellulose may alter the redox environment of the colon. ROS, oxidative DNA damage, antioxidant enzyme activity and apoptosis were measured in colonocytes of rats fed one of four diets containing either corn oil or fish oil and cellulose or pectin. Measurements were madein rats untreated with carcinogen and rats exposed to a chemical carcinogen and radiation. In rats not treated with a carcinogen, fish oil enhanced ROS, and fish oil/pectin suppressed antioxidant enzymes as compared to corn oil/cellulose. Oxidative DNA damage was inversely related to ROS in the fish oil/pectin diet and apoptosis was enhanced relative to other diets. In carcinogen treated and irradiated rats, a similar protective effect was seen with fish oil/pectin as evidenced by a reduction in oxidative DNA damage and enhancement of apoptosis. This suggests that a diet containing fish oil/pectin may protect against colon carcinogenesis by modulation of the redox environment to promote apoptosis and minimize oxidative DNA damage.

One of the aims of this thesis was to investigate the involvement of efflux proteins, such as the P-glycoprotein (Pgp), in the drug transport in different regions of the rat and the human intestine. The intestinal extrusion of intracellularly formed CYP3A4 metabolites, including whether this extrusion might be mediated by Pgp, was also studied. The model drugs used were local anaesthetics (LA), which have been evaluated for inflammatory bowel disease, such as ropivacaine, lidocaine and bupivacaine. The intestinal permeability to LAs was found to be high throughout all intestinal regions of the rat and human intestine. Results from the Ussing chamber model indicated only minor efflux involvement as the drug permeability was higher in the serosa to mucosa transport direction than in the opposite direction. However, the involvement of efflux in the absorption of LAs could not be verified using in situ single-pass perfusion of rat jejunum. The extrusion of the ropivacaine metabolite, 2´,6´-pipecoloxylidide (PPX), was polarized to the mucosal reservoir of the Ussing chamber for both rat and human intestinal samples, and was probably not caused by any Pgp involvement. The expression levels of CYP3A4 and efflux transporters were consistent with the enzymes’ activity in human intestine. PPX formation was mediated by CYP3A4 in human intestine, and cyp2c and cyp2d in rat intestine. Species differences were observed, as PPX was formed in rat colon, but not human colon. In conclusion, the permeability of ropivacaine, lidocaine and bupivacaine was not subjected to efflux transport of significance for their intestinal uptake. The transport of ropivacaine metabolites to the mucosal compartment was probably not mediated by Pgp. The Ussing chamber model showed consistent results with those from intestinal microsomes as far as intestinal metabolism is concerned, making it a suitable model for investigations of the interplay of efflux and metabolism.

Colon cancer is one of the most commonly diagnosed cancers in the US, yet small intestine cancer is a rare event. While there are many similarities between these two tissues, inherent differences such as redox status, may contribute to the variation in cancer occurrence. We examined the difference in reactive oxygen species (ROS) generation, antioxidant enzyme activity and oxidative DNA damage in the small and large intestine of rats under normal conditions and following exposure to exogenous oxidative stress. Basal ROS and antioxidant enzyme activities were greater in the colon than the small intestine, and the balance of ROS to antioxidant enzymes in the colon was more pro-oxidant than in the small intestine. During oxidative stress, ROS and oxidative DNA damage were greater in the colon than the small intestine. Thus the colon responds to oxidative stress less effectively than the small intestine, possibly contributing to increased cancer incidence at this site. We next wanted to understand how diets containing a combination of fish or corn oil and pectin or cellulose may alter the redox environment of the colon. ROS, oxidative DNA damage, antioxidant enzyme activity and apoptosis were measured in colonocytes of rats fed one of four diets containing either corn oil or fish oil and cellulose or pectin. Measurements were madein rats untreated with carcinogen and rats exposed to a chemical carcinogen and radiation. In rats not treated with a carcinogen, fish oil enhanced ROS, and fish oil/pectin suppressed antioxidant enzymes as compared to corn oil/cellulose. Oxidative DNA damage was inversely related to ROS in the fish oil/pectin diet and apoptosis was enhanced relative to other diets. In carcinogen treated and irradiated rats, a similar protective effect was seen with fish oil/pectin as evidenced by a reduction in oxidative DNA damage and enhancement of apoptosis. This suggests that a diet containing fish oil/pectin may protect against colon carcinogenesis by modulation of the redox environment to promote apoptosis and minimize oxidative DNA damage.

L’expansion des maladies métaboliques dans les sociétés modernes exige plus d’activités de recherche afin d’augmenter notre compréhension des mécanismes et l’identification de nouvelles cibles d’interventions cliniques. L’obésité, la résistance à l’insuline (RI) et la dyslipidémie, en particulier sont tous des facteurs de risque associés à la pathogenèse du diabète de type 2 (DT2) et des maladies cardiovasculaires. Ainsi, la dyslipidémie postprandiale, notamment la surproduction des lipoprotéines hépatiques et intestinales, contribue d’une façon significative à l’hypertriglycéridémie. Quoique plusieurs études cliniques et fondamentales chez l’homme et les modèles animaux aient mis en évidence les rôles importants joués par le foie et l’intestin dans la dyslipidémie, les mécanismes moléculaires en cause ne sont pas bien élucidés. L’une des voies principales régulant le métabolisme lipidique est la voie de la protéine kinase AMPK. L’épuisement de l’ATP intracellulaire entraîne une activation de l’AMPK qui va œuvrer pour rétablir l’équilibre énergétique en stimulant des voies génératrices d’ATP et en inhibant des voies anaboliques consommatrices d’ATP. Les effets positifs de l’activation de l’AMPK comprennent l’augmentation de la sensibilité à l’insuline dans les tissus périphériques, la réduction de l’hyperglycémie et la réduction de la lipogenèse, d’où son importance dans les interventions cliniques pour la correction des dérangements métaboliques. Il est à souligner que le rôle de l’AMPK dans le foie et l’intestin semble plus complexe et mal compris. Ainsi, la voie de signalisation de l’AMPK n’est pas bien élucidée dans les situations pathologiques telles que le DT2, la RI et l’obésité. Dans le présent projet, notre objectif consiste à caractériser le rôle de cette voie de signalisation dans la lipogenèse hépatique et dans le métabolisme des lipides dans l’intestin chez le Psammomys obesus, un modèle animal d’obésité, de RI et de DT2. À cette fin, 3 groupes d’animaux sont étudiés (i.e. contrôle, RI et DT2). En caractérisant la voie de signalisation de l’AMPK/ACC dans le foie, nous avons constaté une augmentation de l’expression génique des enzymes clés de la lipogenèse (ACC, FAS, SCD-1 et mGPAT) et des facteurs de transcription (ChREBP, SREBP-1) qui modulent leur niveau d’expression. Nos analyses détaillées ont révélé, par la suite, une nette augmentation de l’expression de l’isoforme cytosolique de l’ACC, ACC1 (impliqué dans la lipogenèse de novo) concomitante avec une invariabilité de l’expression de l’isoforme mitochondrial ACC2 (impliqué dans la régulation négative de la β-oxydation). En dépit d’un état adaptatif caractérisé par une expression protéique et une phosphorylation (activation) élevées de l’AMPKα, l’activité de la kinase qui phosphoryle et inhibe l’ACC reste très élevée chez les animaux RI et DT2. Au niveau de l’intestin grêle des animaux RI et DT2, nous avons démontré que l’augmentation de la lipogenèse intestinale est principalement associée avec une diminution de la voie de signalisation de l’AMPK (i.e. expression protéique et phosphorylation/activation réduites des deux isoformes AMPKα1 et AMPKα2). La principale conséquence de la diminution de l’activité AMPK est la réduction de la phosphorylation de l’ACC. Étant donné que le niveau d’expression totale d’ACC reste inchangé, nos résultats suggèrent donc une augmentation de l’activité des deux isoformes ACC1 et ACC2. En parallèle, nous avons observé une réduction de l’expression protéique et génique de la CPT1 [enzyme clé de la β-oxydation des acides gras (AG)]. L’ensemble de ces résultats suggère une inhibition de l’oxydation des AG concomitante avec une stimulation de la lipogenèse de novo. Enfin, nous avons démontré que l’intestin grêle est un organe sensible à l’action de l’insuline et que le développement de la résistance à l’insuline pourrait altérer les deux voies de signalisation (i.e. Akt/GSK3 et p38MAPK) essentielles dans plusieurs processus métaboliques. En conclusion, nos résultats indiquent que l’augmentation de la lipogenèse qui contribue pour une grande partie à la dyslipidémie dans la résistance à l’insuline et le diabète serait due, en partie, à des défauts de signalisation par l’AMPK. Nos observations illustrent donc le rôle crucial du système AMPK au niveau hépatique et intestinal, ce qui valide l’approche thérapeutique consistant à activer l’AMPK pour traiter les maladies métaboliques. / Understanding the cellular mechanisms involved in the development of insulin resistance, and later on the occurrence of type 2 diabetes and its metabolic complications, is a perquisite step toward the identification of new therapeutic targets to fight against the development of these metabolic diseases. In the present studies, we used the gerbil Psammomys obesus, a well-established animal model of obesity, insulin resistance (IR) and type 2 diabetes (T2D), to characterize the hepatic and intestinal signaling abnormalities associated with lipid metabolism disorders during the pathogenesis of IR and T2D. Thus, we are able to demonstrate that the development of these metabolic diseases in Psammomys obesus animals, is accompanied by increased hepatic and intestinal lipogenesis with very high efficiency to form triglycerides rich-lipoproteins. In the liver, we observed an increase in mRNA levels of key lipogenic enzymes (ACC, FAS, SCD-1 and mGPAT) and transcription factors (SREBP-1, ChREBP), which modulate the expression level of lipogenic enzymes. Thereafter, our detailed analysis of the AMPK/ACC signaling pathway revealed a rise in the gene expression of the cytosolic ACC1 isoform of ACC(involved in de novo lipogenesis) concomitant with a constant expression of the mitochondrial ACC2 (negative regulator of β-oxidation). In spite of an adaptive state characterized by higher protein expression and phosphorylation (activation) of AMPKα, the kinase that phosphorylates and inhibits ACC, the activity of the later remains very high in IR and T2D animals. In the small intestine of IR and T2D animals, we demonstrated that the increase in intestinal lipogenesis is mainly associated with a decrease of AMPK signaling pathway (i.e. reduced expression and protein phosphorylation/activation of the two AMPKα1 and AMPKα2 isoforms). The main consequence of the decline in AMPK activity is the reduction of ACC phosphorylation. Given that, the expression levels of ACC remain unchanged; our results thus suggest an increased activity of both ACC isoforms, ACC1 and ACC2. Next, we observed a reduction in protein and gene expression of CPT1 [key enzyme in fatty acid (FA) β-oxidation]. Taken together, these results suggest an inhibition of FA β-oxidation concomitant with a stimulation of de novo lipogenesis. Finally, we demonstrated that the small intestine is an insulin sensitive organ and that the development of IR affects two signaling pathways (i.e. Akt/GSK3 and p38MAPK) essentials for several metabolic processes. In conclusion, our results indicate that increased lipogenesis, in IR and T2D, which exacerbate the dyslipidemia associated with these diseases, might be, at least partially, a result of AMPK signaling defects. In addition, our observations illustrate the crucial role of AMPK/ACC in the liver and intestine and validate AMPK as a potential target to treat the metabolic diseases.

Le stress oxydant joue un rôle majeur dans le développement et l’évolution des maladies inflammatoires de l’intestin. Le corps humain est doté d’une panoplie d’enzymes antioxydantes ayant pour fonction de protéger l’intégrité cellulaire. De nouvelles enzymes au fort potentiel antioxydant, les paraoxonases (PON) 1, 2 et 3, ont récemment été identifiées tout au long du tube digestif, mais leurs rôles y restent inconnus. Les cellules intestinales Caco-2/15, qui ont la capacité de se différencier et d’acquérir les caractéristiques physiologiques de l'intestin grêle, ont été utilisées dans le présent travail pour étudier la régulation des PON. Les cellules ont été traitées avec différents effecteurs physiologiques (cytokines, LPS, stress oxydant) et pharmacologiques (fibrates, thiazolidinédiones) et l’expression des leurs gènes et protéines a été évaluée. Les résultats ont mis en lumière la modulation distincte de l’expression des PON par le stress oxydant et l’inflammation. Ceci suggère que chaque PON peut jouer un rôle différent au niveau intestinal et être impliquée dans le maintien de l’homéostasie. La régulation de l’expression des PON a également été largement explorée dans un article de revue. Pour définir le rôle de PON2, celle-ci étant potentiellement la plus importante pour l’homéostasie intestinale, les cellules Caco-2/15 ont été infectées à l’aide de lentivirus contenant des ARN d’interférence, ce qui a fortement réduit l’expression de PON2. En l’absence de PON2, les cellules Caco-2/15 étaient plus susceptibles face à un stress oxydant, la réponse inflammatoire était exacerbée et la perméabilité cellulaire paraissait altérée. Toutes ces composantes sont majeures dans le développement des maladies inflammatoires de l’intestin chez l’humain. De plus, des cellules Caco-2/15 de la PON2, ce qui a renforcé la force de la défense antioxydante cellulaire. Les résultats suggèrent que les PON jouent un rôle dans le maintien de l’homéostasie intestinale et pourraient être impliquées dans l’étiologie et la pathogenèse des maladies inflammatoires de l’intestin. / Oxidative stress is a major part of the pathogenesis of inflammatory bowel disease (IBD). The endogenous antioxidative defence is formed of multiple enzymes that have to protect cellular integrity. Paraoxonases (PON1, 2 and 3) are antioxidant enzymes that have recently been identified throughout the digestive tract, but their roles remain unclear in the intestine. Intestinal Caco-2/15 cells, which have the capacity to differentiate and exhibit the functionality of the small intestine, were used to study PON’s regulation. Cells were treated with various physiological effectors (cytokines, lipopolysaccharides, oxidative stress) and pharmacological molecules (fibrates, thiazolidinediones) and gene and protein expression were determined. Results obtained showed that PONs are distinctly modulated especially by inflammation and oxidative stress and suggests that they could play different roles in the maintenance of intestinal homeostasis. PONs regulation has also been the main topic of a review article. Our results and the literature pointed to PON2 as the most important PON for intestinal health. To better define PON2 functions in the intestine, PON2 expression was knocked-down using lentiviral infection and plasmids containing anti-PON2 shRNA. In the relative absence of PON2, Caco-2/15 cells were more susceptible towards induction of oxidative stress, the inflammatory response was exacerbated and cell permeability seemed altered. All of these components are major players involved in the development of human IBD. Moreover, Caco-2/15 cells were treated with purified PON2, which increased their antioxidative defence. All of these results suggest that PONs are implicated in the antioxidative and anti-inflammatory response in intestinal epithelial cells and makes them potentially important players for the aetiology and pathogenesis of IBD.

Le facteur de transcription homéotique Cdx2 est un suppresseur de tumeurs et son expression est réduite de manière hétérogène dans les tumeurs coliques. Or, le cancer colorectal demeure un problème de santé publique par sa fréquence et sa gravité. Au travers de 3 sous-projets, cette thèse visait à comprendre le mode d’action de Cdx2. Premièrement, la cadhérine Mucdhl a été identifiée et caractérisée en tant que nouvelle cible de Cdx2 : Mucdhl inhibe la croissance des cellules cancéreuses coliques et s’oppose à l’activité de la -caténine. Deuxièmement, un effet inattendu de Cdx2 sur le cytosquelette et la rigidité cellulaire a été montré. Ceci pourrait expliquer comment Cdx2 intervient dans l’organisation structurale des entérocytes ou la migration. En parallèle, une lignée cellulaire rapportrice de l’expression de Cdx2 a été créé qui, après validation, sera un outil précieux pour l’étude des mécanismes moléculaires qui conditionnent l’hétérogénéité tumorale. Par la mise en évidence de nouvelles fonctions et cibles de Cdx2, cette thèse permet de mieux appréhender son rôle physiologique et son action de suppresseur de tumeurs dans l’intestin. / Colorectal cancer is a major public health problem because of its frequency and propensity to metastasize. Cdx2 plays the role of a tumor suppressor and its expression is frequently but heterogeneously reduced in colon tumors. Through three sub-projects, this thesis aimed to better understand the mode of action of Cdx2. First, the Cadherin Mucdhl was identified and characterized as a new direct target gene of Cdx2. Mucdhl was also shown to inhibit the growth of colon cancer cells and to oppose -catenin activity. Second, an unexpected effect of Cdx2 on the cell cytoskeleton and rigidity of a cancer cell monolayer was uncovered. This gives new clues about how Cdx2 contributes to the structural organization and differentiation of enterocytes but also nhibits cell migration. In parallel, a reporter cell line for Cdx2 expression was created. After its validation, this cell line will be a precious tool to study the molecular mechanisms underlying tumor heterogeneity in vivo. Altogether, this thesis unraveled new functions and target genes of Cdx2 that permit to better apprehend its physiological function and its action as a tumor suppressor in the intestine.

Made available in DSpace on 2014-06-11T19:28:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-04Bitstream added on 2014-06-13T18:57:30Z : No. of bitstreams: 1 alarcon_mff_me_jabo.pdf: 1406127 bytes, checksum: 842dbbaa540dbb4b860fec0374842606 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Foram avaliados parâmetros fisiológicos e imunológicos de matrizes de corte vacinadas ou não contra o vírus da bronquite infecciosa das galinhas (VBIG), submetidas a diferentes períodos de jejum após a eclosão, seguido de alimentação até a terceira semana de vida. No Capítulo 2, encontram-se os resultados do desempenho zootécnico e o desenvolvimento de órgãos gastrintestinais. No Capítulo 3, estão descritos os resultados de parâmetros hematológicos e bioquímicos. O Capítulo 4 apresenta as cinéticas de decaimento dos anticorpos maternos e os perfis cinéticos da reposta imune humoral nos compartimentos local e sistêmico. A vacina contra a BIG influenciou parâmetros de desempenho, de morfometria intestinal, o hematócrito, parâmetros bioquímicos, percentuais de heterófilos e linfócitos e induziu a resposta imune humoral na secreção lacrimal. O jejum pós-eclosão prolongado seguido de alimentação influenciou negativamente o desempenho, o desenvolvimento das vísceras gastrintestinais, as variáveis bioquímicas, imunológicas e a maioria das variáveis hematológicas. Os dados indicam que períodos de jejum pós-eclosão superiores a 48 h devem ser evitados, pois ao afetar negativamente parâmetros hematológicos, intestinais e imunológicos, podem comprometer o crescimento das matrizes e inferir negativamente sobre sua resposta imune. No entanto, o jejum moderado pode favorecer a resposta imune vacinal / Immunological and physiological parameters were evaluated in broiler breeder vaccinated or not against infectious bronchitis virus (IBV), submitted to different periods of fasting post-hatching, followed by feed until the third week of life. In Chapter 2, are the results of zootechnical performance and development of gastrointestinal organs. The Chapter 3 describes the results of hematological and biochemical parameters of blood. Chapter 4 presents the kinetics of decay of maternal antibodies and the kinetic profiles of humoral immune response in local and systemic compartments. The IBV vaccine influenced parameters of performance, intestinal morphology, hematocrit, biochemical parameters, percentage of heterophils and lymphocytes, and induced humoral immune response in tear secretion. Prolonged fasting post-hatching, followed by feeding, negatively affected the performance, the development of gastrointestinal organs, biochemical variables, immunological and the most of hematological variables. The data indicate that periods of fasting post-hatching over 48 h should be avoided as they adversely affect the hematological, gastrointestinal and immunologic, may compromise the growth of broiler breeder and infer a negative effect on their immune response. However, moderate fasting can promote the immune response vaccine

Made available in DSpace on 2014-06-11T19:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-24Bitstream added on 2014-06-13T20:10:21Z : No. of bitstreams: 1 barbosa_ra_me_rcla.pdf: 1782892 bytes, checksum: c33b7a80d34353572789d680df7e20ec (MD5) / O diabetes tipo I, causado pela destruição das células beta do pâncreas, promove alterações metabólicas que podem ser acompanhadas de prejuízos em órgãos e tecidos específicos. O intestino delgado é um órgão que apresenta uma série de alterações, principalmente de caráter morfológico e funcional. A atividade física regular por sua vez, pode contribuir para o controle desta situação, sendo um fator importante, já que grande parte da população mundial sofre com esta doença. Este trabalho teve como objetivo analisar os aspectos morfológicos e histoquímicos do intestino delgado e a ultra-estrutura de células caliciformes de ratos diabéticos, a fim de se observar possíveis alterações causadas pela doença. Além disso, este trabalho buscou avaliar de que maneira o exercício físico pode interferir nestas modificações e contribuir para o controle do estado diabético. Neste estudo, ratos Wistar foram distribuídos em 4 grupos: controle sedentário (CS), controle treinado (CT), diabético sedentário (DS) e diabético treinado (DT). O diabetes foi induzido por aloxana monoidratada Sigma (35 mg/kg de peso corporal). Os grupos treinados realizaram um protocolo de exercícios físicos, que consistiu em natação com cargas acopladas ao corpo. Durante o período experimental, foram realizadas avaliações de peso corpóreo e ingestão hídrica e alimentar. Após o período experimental, o sangue dos animais foi coletado para análise de glicemia. Os intestinos delgados dos animais foram retirados, pesados e o comprimento foi medido. Amostras de duodeno, jejuno e íleo foram processadas e analisadas para a microscopia de luz e para a microscopia eletrônica de transmissão. O diabetes causou um aumento geral do intestino delgado, também devido à alteração de características histomorfométricas, tais como aumento de vilosidades e da espessura da camada mucosa... / Type I diabetes, caused by pancreatic beta cells destruction, promotes metabolic changes that may be accompanied by losses in specific organs and tissues. The small intestine is an organ that shows several changes, mainly of morphological and functional aspects. Regular physical activity can contribute to control this situation being an important factor, since most of the world population suffers from this disease. This study aimed to analyze the morphological and histochemical aspects of the small intestine and goblet cells ultrastructure in diabetic rats, in order to observe possible changes caused by the disease. In addition, this study aimed to evaluate how exercise can interfere on these changes and contribute to control the diabetic state. In this study, Wistar rats were divided into 4 groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD) and trained diabetic (TD). Diabetes was induced by alloxan monohydrate Sigma (35 mg/kg body weight). Trained groups performed an exercise protocol, which consisted of swimming with loads attached to the body. During the experimental period, assessments were made on body weight, water and food intake. After the trial period, the animals' blood was collected for glucose analysis. The small intestines of the animals were removed, weighed and the length was measured. Samples of duodenum, jejunum and ileum were processed and analyzed for light microscopy and for transmission electron microscopy. Diabetes caused a general increase in the small intestine, also due to changes on histomorphometric features, such as increased villi and increased thickness of the mucosa. In addition, the disease promoted histochemical changes as demonstrated by reduced amounts of collagen fibers, reticular fibers and general proteins, and increased amounts of neutral polysaccharides and general nucleic acids. About goblet cells, diabetes increased... (Complete abstract click electronic access below)

Made available in DSpace on 2015-03-26T12:10:48Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1584074 bytes, checksum: 9f5933c0ddc882411ad84b3276bfaa43 (MD5) Previous issue date: 2014-03-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The larvae of the yellow fever mosquito Aedes aegypti live in the water, feeding on microorganisms and organic matter decomposition. After metamorphosis, adults emerge and feed on sap. However, for the maturation of eggs, the A. aegypti females need a blood meaL This variation of the diets is possible due to the midgut remodeling undergoes during metamorphosis, allowing adaptation to different diets depending on life stages. After ingestion, the blood is stored and digested into midgut, which is the fírst organ víruses, such as Dengue, yellow fever and other infect. The midgut epithelium of A. aegypti Compríses three Cell typesz digestive (responsible for digestion and absorption of nutrients), regenerative (stem, undifferentiated Cells) and enteroendocrine (neuropeptides secretory) Cells. During metamorphosis, the digestive Cells are replaced by new adult digestive Cells by differentiation of regenerative ones. In the present study morphological and biochemical aspects of the metamorphosis of the midgut were investigated, íncluding the differentíation and the quantificatíon of dividing regenerative and enteroendocrine cells at different developmental stages [fourth ínstar larvae - L4, female whíte pupae - WP and pupae 24 and 48 h (P24 and P48, respective1y) after ecdysis, and 50 newly-emerged (NE) adult females] by means líght microscopy and transmission electron microscopy. In addition, the proteins synthesized by the midgut in these stages and in sugar- and b1ood-fed adult females (SA and BA, respective1y) were also studied. The lysis of digestive Cells, as well as the proliferation of regenerative Cells, begin in the anterior region of L4 midgut Simultaneously, the proliferation of regenerative Cells begins in the anterior region of the larval midgut, moving to the posterior region in P48 and NE. The effects of the neocotinoid insectícíde imídacloprid Was also tested in the mosquito midgut Third instar larvae (L3) were treated with two subletal Concentratíons (3 and 15 ppm) of insecticide, and the midgut was examined in L4, P24, P48 and NE. Subletal Concentrations of imidacloprid inhíbít the proliferation and differentíation of regenerative Cells, often leading to cell damaged through their nuclear DNA fragmentation. This inhibition has led to the reduction of the digestive and endocrine Cell numbers, and the mídgut of NE Consists of mostly vacuolatedlincompletely formed digestive Cells. Therefore, the imidacloprid exposition during juvenile stages, impair the normal midgut development in NEs, and they may not be able to get/digest a blood meaL The mídgut of NE has higher expression of proteins related to energy and protein metabolisms, cell signaling and transport. The mídgut of SA and BA express more proteins associated to the protein biosynthesis process. In the mídgut of pupae, several proteins essential for the organ remodeling are expressed, but the peak expression of these proteins occurs at the end of remodeling process in NE. Not surprising1y, in SA and BA there is a high expression of proteins associated to the digestion of sugar and blood, respective1y. In the present Work, several morphological and biochemical aspects of the mídgut of A. aegypti are investigated, íncluding the repertoire of differentially expressed proteins during mídgut development and in the midguts of individuals under different feeding conditions. Morpho-physiological processes that occur during the development and functioning of this organ are Crucial to the survival of this vector mosquito and are discussed. / Ao longo do seu desenvolvimento pós-embrionário, 0 mosquito Aedes aegypti passa parte do seu Cíclo de vida no ambíente aquático, sendo que suas larvas se alimentam de microrganismos e matéria orgâníca em decomposição. Após a metamorfose, os adultos alados emergem, e passam a se alimentar de seiva. Para maturação dos ovos, as fêmeas de A. aegypti necessitam do repasto sanguíneo. Essa plasticidade quanto ao tipo de alimentação só é possível graças às modificações que 0 íntestíno médio sofre ao longo da metamorfose, permitindo a adaptação do ínseto a diferentes dietas dependendo da fase do desenvolvimento. Após a ingestão, 0 sangue é armazenado e digerido no intestino médio, que é 0 primeiro órgão do ínseto que diversos vírus, Como por exemp10, vírus Dengue e da febre amarela, são Capazes de infectar 0 hospedeiro. No epitélio do intestino médio de A. aegypti há três tipos Celularesz Células digestivas (responsáveis pela digestão e absorção de nutrientes), regenerativas (Células indiferencíadas) e enteroendócrinas (secretoras de neuropeptídeos). Durante a metamorfose, as Células digestivas de A. aegypti são substituídas por novas Células digestivas adultas através da diferenciação das Células regenerativas. No presente trabalho aspectos morfológicos e bioquímicos referentes à metamorfose do íntestíno médio foram investígados, incluindo a diferenciação e a divisão das Células regenerativas, e 0 número de Células enteroendócrinas em diferentes fases do desenvolvimento de A. aegypti (1arva 4° instar - L4, pupa branca - PB, pupas 24h -P24 e 48h -P48 após a ecdise e adultos recém-emergidos - RE). Adicíonalmente, a expressão de proteínas sintetizadas pelo órgão nessas fases e em fêmeas adultas submetidas às dietas à base de açúcar (AA) e de sangue (AS) foi estudada. A morte das Células digestivas e a proliferação das Células regenerativas ocorrem de forma ordenada, em regiões específicas do órgã0, ínícíando na região anterior das L4 e passando para a região posterior nas P48 e RE. Os efeitos subletaís do inseticida neocotinoide imídacloprid também foram testados no processo de remodelargem do íntestino médio de A. aegypti. Para ísso, larvas 3° instar (L3) foram tratadas Com duas Concentrações (3 e 15 ppm) do inseticida e 0 intestino médio foi analisado nas fases seguintes. Mesmo em Concentrações subletaís, 0 imídacloprid alterou 0 processo de remodelação do intestino médio, inibindo a proliferação e diferenciação das Células regenerativas, e Causando danos ao DNA nuclear delas, Como atestado pela reação de TUNEL. Essa íníbíção acarretou a diminuição das populações das Células digestivas e enteroendócrinas, sendo que 0 intestino médio dos RE é Constituído, na maior parte, por Células digestivas vacuolizadas e mal formadas. O ímidacloprid possui potencíal no Controle de A. aegypti, pois, mesmo que 0 índivíduo Chegue à fase adulta, do ponto de vista morfológico, seu intestino aparentemente não está apto para 0 processo de digestã0. O intestino médio dos RE possui maior expressão de proteínas ligadas à produção de energia, metabolismo de proteínas, sinalização e transporte Celulares. Os intestinos médios de AA e AS expressam maís proteínas ligadas ao processo de bíossíntese de proteínas. Nas pupas se inicia a síntese das múltiplas proteínas essenciais para a formação e Constítuição do novo epitélio do órgã0, mas 0 pico da expressão dessas proteínas ocorre no final do processo de formação do órgão no adulto recém- emergido. Já nos AA e AS há alta expressão de proteínas ímportantes para a digestão de açúcar e sangue, respectivamente. O presente trabalho é amp10, tratando de aspectos morfológicos e bioquímicos do intestino médio de A. aegypti, incluindo parte do repertórío de proteínas diferencialmente expressas no órgão durante 0 desenvolvimento pós-embríonário e em díferentes Condições alimentares. Processos morfofisiológicos que ocorrem ao longo do desenvolvimento e funcionamento desse órgão são Cruciais para a sobrevivência dessa espécíe vetora e são discutidos aqui neste trabalho.