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Introduction: Appetite is often impaired in patients with gastrointestinal inflammation. Up to 75% of hospitalised Crohn's disease (CD) patients are malnourished. Recent animal research has suggested that immune mediated upregulation of enteroendocrine cell (EEC) activity plays a mechanistic role in the appetite and feeding disturbance observed during gut inflammation. The role of EEC in producing factors regulating satiety and intestinal growth is well recognised but work on their use as therapeutic targets or agents in inflammatory bowel disease (IBD) is still in its infancy. EEC peptide dynamics are further controlled through dipeptidyl peptidase (DP4) protease metabolism but no data are yet available on its expression in IBD. My aim is to understand the roles of EEC in appetite control and the maintenance of gut mucosal integrity in intestinal inflammation. Methodology: Patients with CD and healthy controls were studied. Symptoms were assessed using visual analogue scores (VAS). Gut hormone responses to a test meal were studied using a multiplex-ELISA technique, and correlated to symptoms. At the tissue level, EEC markers and transcription factors were quantified using immunohistochemistry, quantitative polymerase chain reaction (qPCR) and western blotting techniques. The same techniques were used to study DP4 expression. The effects of glucagon-like peptide-2 (GLP-2) on a gut model of the epithelial barrier were studied by measuring the transepithelial electrical resistance (TEER) across GLP-2 exposed Caco-2 cell monolayers after cytokine exposure. Tight junction protein expression in naïve and GLP-2 exposed cells was quantified by western blotting. Main Results: CD patients with active inflammation displayed a significant reduction in appetite. At the tissue level, GLP-1 and chromogranin A (CgA) were significantly upregulated. At the mRNA level significant increased expression was noted for CgA, glucagon-like peptide-1 (GLP-1), ubiquitination factor 4a and neurogenin 3. At the plasma level, total polypeptide YY (PYY) was increased. A significant correlation was seen between postprandial PYY responses and symptoms of nausea and bloating. Ghrelin, was 3-fold higher in the CD group compared to controls, and showed a reversed postprandial response with a significant correlation with the CD activity index (CDAI). Protein DP4 expression was significantly decreased at the tissue and plasma level in CD. GLP-2 increased tight junction protein expression in Caco-2 cells and maintained stable TEER and tight junction protein expression after cytokine exposure. Conclusions: The data presented are compatible with a potential role of EEC in appetite dysregulation in intestinal inflammation. An enhanced EEC response to food intake may directly affect appetite in such patients through increased gut-brain signalling. These may present tractable therapeutic targets. The decrease in mucosal DP4 expression in CD may make bioactive GLP-2 more available in the affected gut, hence improving gut mucosal integrity in intestinal inflammation. This pilot work has shown that GLP-2 has a role in maintaining gut mucosal integrity in intestinal inflammation through a positive effect on tight junction protein expression.

Les histones désacétylases HDAC1 et HDAC2 catalysent le retrait d’un groupement acétyle de résidus lysine, dans des protéines histones et non-histones. Les HDAC contrôlent la prolifération, la mort et la différenciation cellulaire. Des propriétés anti-inflammatoires et anti-tumorales ont été attribuées à des inhibiteurs contre les HDAC (HDACi), notamment dans les cellules épithéliales intestinales (CEI). Nous supposons que différents niveaux de HDAC1 ou HDAC2 dans les CEI induisent différentes réponses dans le maintien de l’homéostasie intestinale. Nous avons donc généré des souris hétérozygotes avec un seul allèle de Hdac1 ou Hdac2 dans le contexte de la délétion de l’autre. Les résultats indiquent que les souris Hdac1-/-;Hdac2+/- Villine-Cre présentent un phénotype similaire à celui d’un double mutant, à savoir des défauts d'architecture dans le jéjunum et le côlon, de la dysplasie et hyperplasie, une réduction du nombre de cellules à mucus, mais sans modification du nombre de cellules de Paneth et de la perméabilité épithéliale. Un allèle de Hdac2 n'est donc pas suffisant pour maintenir une homéostasie normale en l'absence de Hdac1. Nous avons aussi vérifié l’effet de la délétion de Hdac1 et Hdac2 à l’âge adulte dans le modèle inductible AhCre. Dans ce contexte, la perte de Hdac1 et Hdac2 entraîne une mortalité accrue après 8 jours, avec un arrêt de prolifération et l’induction de dommages à l’ADN. Nous avons alors exploré l’impact moléculaire de la perte des deux Hdac dans les CEI par une approche protéomique et transcriptomique. Nous avons observé des changements notables dans plusieurs voies de signalisation, associées à la prolifération, à des mécanismes de stress, au métabolisme, surtout lipidique. Ces changements sont en partie régulés post-traductionnellement. Bien que très instructifs, les modèles in vivo ne permettent pas de déterminer si les modifications de l’expression des gènes observées sans Hdac1 et/ou Hdac2 sont intrinsèques aux CEI ou si ces changements dépendent de signaux extrinsèques de la muqueuse ou de la lumière intestinale. Nous avons donc établi des cultures d’entéroïdes à partir de la crypte intestinale, ce qui permet la croissance, l'expansion et la différenciation des CEI progénitrices, sans l’influence de l’environnement. Nous avons entrepris des analyses protéomiques de type SILAC, suite à une inhibition pharmacologique des HDAC de type I, le CI994, ou suite à une délétion génétique de Hdac1 ou Hdac2. L’inhibition pharmacologique entraine un arrêt de prolifération associé à une différenciation altérée en faveur des cellules absorbantes, rappelant le modèle murin sans Hdac1 et Hdac2. Les voies liées à la réplication de l’ADN et au cycle cellulaire sont diminuées. Même si la perte de Hdac1 ou Hdac2 n’affecte pas notablement la croissance et la différenciation des entéroïdes, des voies associées au métabolisme et aux réponses à l’environnement sont augmentées. Au contraire, des entéroïdes sans Hdac1 et Hdac2 ne croissent pas en culture et dégénèrent en moins de 3 jours. Ceci suggère que l’environnement mucosal pourrait soutenir les CEI Hdac1-/-;Hdac2-/- de la niche épithéliale in vivo. Nos données suggèrent que des variations intrinsèques ou extrinsèques de l'activité de HDAC1 et HDAC2 modifient la réponse des CEI à l’environnement et entraînent des perturbations de l'homéostasie intestinale.

Calcium bioavailability was investigated using isotopic intestinal absorption, and balance study techniques with bone mineralization and biomechanics as endpoint determinants of calcium utilization. In experiment 1, lactose enhancement of paracellular calcium absorption was confirmed, and it was suggested that a critical luminal concentration of lactose was necessary for its action. Despite the enhanced intestinal absorption of calcium in animals fed the 50% lactose containing diet, bone mineralization was not different from controls; and further, a decrease in bone strength of these animals was found to be secondary to nutrient malabsorption. In this study, there was no evidence to indicate a difference in the bioavailability of calcium from milk (colloidal) or yogurt (ionized) sources. In experiment 2, the absorption of calcium from the ileum was significantly enhanced in[formula omitted] normal Wistar rats fed milk protein diets containing casein as compared to whey and soy protein diets. The increase in absorbed calcium was shown to have little physiological significance in bone mineralization and biomechanics when animals were fed a diet adequate in dietary calcium. In experiment 3, paracellular calcium absorption was similar between genetically spontaneously hypertensive (SHR) and normotensive control Wistar-Kyoto (WKY) rats, suggesting that differences in calcium metabolism between these two strains was not due to differences in ileal calcium transport. The effect of casein and soy protein diets containing high (2.0%), adequate (0.5%) and low (0.05%) levels of calcium, respectively on calcium bioavailability and subsequent utilization was determined in SHR and WKY animals. Ileal calcium absorption was greater in casein fed animals than those fed soy at the adequate and low levels of calcium. Femur calcification was enhanced by casein diets at the high and medium levels of dietary calcium only. Femur biomechanics were not influenced by dietary protein source, but were however, adversely affected by the low dietary calcium level. In experiment 4, the effect of dietary fortification with casein phosphopeptides (CPP) was investigated in casein and soy fed SHR animals. CPP added to casein and soy protein diets appeared to result in a greater ileal absorption of calcium. This increase in calcium bioavailability from the casein diet had little effect however, on bone mineralization and biomechanical strength, due to the excretion of excess absorbed calcium. In experiment 5, severe thermal processing of dietary proteins was shown to reduce in vitro digestibility. Animals fed heat denatured casein and soy diets exhibited reduced intestinal calcium absorption, calcium balance as well as bone mineralization and biomechanics. These effects were influenced by the nutrient malabsorption experienced by these animals. In experiment 6, a low (6%) protein, low phosphorus diet resulted in decreased food intake and animal growth. However, ileal calcium absorption (% dose) was similar between 6% and 20% protein fed animals. A low level of dietary protein influenced calcium balance and utilization for bone mineralization and biomechanical strength. These results indicate that paracellular calcium absorption may not necessarily equate with those obtained from a calcium balance study. Notwithstanding, calcium bioavailability from the ileum was shown to be enhanced in animals fed casein, which was likely due to the production of bioactive peptides (CPP) that are involved in sequestering calcium and retaining it in a soluble form. By reducing protein digestibility with heat denaturation, this effect was lost. Reducing the protein content in the diet however, did not reduce the enhancement of calcium bioavailability observed in casein fed animals. Finally, bone mineralization and biomechanical parameters were shown to be sensitive indicators of calcium utilization from diets that varied in calcium content or bioavailability of this mineral. / Land and Food Systems, Faculty of / Graduate

The purpose of this paper is to report the results of studies on the aerobic, mesophilic intestinal flora of guinea pigs subjected to conditions similar to those encountered by man in spacecraft.

Orientador: Jaime Amaya-Farfan / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos e Agricola / Made available in DSpace on 2018-07-16T16:44:37Z (GMT). No. of bitstreams: 1 Salva_TerezinhadeJesusGarcia_M.pdf: 3265504 bytes, checksum: 25723fce2f12ecd8816ff9a75a65fcb3 (MD5) Previous issue date: 1982 / Resumo: Este trabalho foi desenvolvido com a finalidade de definir o efeito da fibra dietária na absorção, deposição inicial e retenção de chumbo administrado oralmente na forma de litargírio no organismo animal. Foram realizados experimentos biológicos onde se trabalhou com ratos machos a fêmeas da raça Wistar alimentados com uma refeição única por 30 minutos ou ad libitum por 44 dias conforme o experimento. Após o período de alimentação foram determinadas as concentrações de Pb no fígado, rins, sangue e fezes dos animais por espectrofotometria de absorção atômica. Os resultados mostraram que a celulose diminui a absorção, a deposição inicial e a retenção de chumbo no organismo sendo que para se obter uma maior eficiência da fibra nesse sentido ela deve estar presente na dieta em concentrações de 1 a 2%. O experimento com uma única refeição revelou que ocorrem 2 máximos de deposição de chumbo; um deles entre 3 e 4 horas e o outro entre, 5 e 8,5 horas após o início da refeição. Esses dois picos correspondam provavelmente a dois sítios de absorção, um deles no intestino delgado e o outro no intestino grosso. Além de confirmar que é necessária uma concentração de 1 a 2% de celulose na dieta para que se obtenha uma mínima daposição de chumbo e que a maior deposição do metal se dá nos rins e a menor no sangue, o experimento de 44 dias de alimentação mostrou também que a celulose na dieta aumenta 3 excreção fecal do metal. Um experimento o in vitro, onde se misturou várias quantidades de PbO e celulose em meio alcalino e acido, seguido de extensiva lavagem, revelou que a fibra tem capacidade ue reter grande quantidade do metal devido em grande parte a baixa solubilidade do compofíto nesses meio / Abstract: The purpose of this work was to define the effect of dietary fiber in the absorption, initial deposition and retention of lead orally administered as litharge. Two kinds of biological, experiments were designed using both male and female Wintar rats, which were either trained to eat a single meal in 30 minutes or fed ad libitum for days. Diets were prepared with levels of 0, 0.5; 1.0 and 2.0% cellulose. Lead was added to the diets in concentrations of 1.3% for the former and 160 ppm for the latter. The heavy metal contain in the blood, liver, kidney, and feces of the animal was determined by atomic absorption spectrophotometry. The results showed that cellulose decreased in intestinal absorption, initial deposition and retention in the tissues of lead in the animal. The protective effect of the dietary cellulose, however, appeared to have reached a maximum between 1 and 2%. Of the tissues studied, kidney attained the highest and blood the lowest lead concentration, regardless of form of feeding. The short term experiment revealed two maximal deposition versus post-prandial time, one between 1 to 4 hours and another between 5.5 and 8.5 hours. Such maxima were believed to be originated by two distinct absorption sites the small and large intestines, respectively. The long term experiment confirmed that 1-2% cellulose in the diet was needed In order to provide a maximal protective effect and that the highest and lowest concentration of lead occurred in kidney and blood, respectively. More over, this experiment showed that cellulose in the diet inhanced fecal lead excretion. An in vitro experiment in which various amounts of litharge were mixed with cellulose in either line or acid media, followed by extensive washing, suggested that this form of lead could be re retained to a large extent by the fiber due to the low solubility of the monoxide / Mestrado / Mestre em Ciência de Alimentos

Memoria para optar al Título Profesional de Médico Veterinario / El objetivo fue implementar el método del intestino evertido en intestino de cerdos post-faena para estudiar la absorción de hierro desde matrices cargadas con hierro. Se elaboró una matriz control (C, sin hierro), con alginato de sodio (2% p/v) y suero de leche (40% p/v) y mezclas de matriz control con diferentes tipos de hierro hemo (H) al 1% y no hemo (NH) al 1% p/v. Se formaron 5 matrices: I: 100%H, II: 100%NH, III: 25%H:75%NH, IV: 50%H:50%NH y V: 75%H:25%NH. Las matrices se caracterizaron según contenido de hierro. Para realizar el método del intestino evertido se utilizaron segmentos de 10 cm de duodeno de cerdos adultos post-faena. Los trozos de intestinos se incubaron con 3g de cada matriz y se cuantificó el hierro absorbido en los sacos intestinales por espectrofotometría de absorción atómica. Las matrices presentaron un contenido de hierro (mg/g) de: C (0,09 ± 0,01), I (0,37 ± 0,02), II (4,3 ± 0,16), III (3,16 ± 0,45), IV (2,14 ± 0,14) y V (1,08 ± 0,03). La mayor absorción de hierro se produjo desde la matriz V (26,3%), que fue superior a la IV (7,8%), la cual no presentó diferencias con la III (3,7%) y I (2,0%), y éstas a su vez no mostraron diferencias con la II (1,3%) y C (0,8%). Fue posible implementar y validar el modelo del intestino evertido para estudios de absorción de hierro ex vivo en cerdos post-faena, observándose la mejor absorción para la matriz V, que contenía la mezcla con mayor porcentaje de hierro hemo. / The objective was to implement the everted intestine method in intestine of post-slaughter pigs to study the absorption of iron from matrices loaded with iron. A control matrix (C, without iron), with sodium alginate (2% w/v) and whey (40% w/v), and mixtures of control matrix with different types of iron heme (H) at 1 % and not heme (NH) at 1% w/v were made. Five matrices were formed: I: 100% H, II: 100% NH, III: 25% H: 75% NH, IV: 50% H: 50% NH and V: 75% H: 25% NH. The matrices were characterized according to iron content. To perform the everted gut sac method, 10 cm segments of duodenum of post-slaughter adult pigs were used. The pieces of intestines were incubated with 3g of each matrix and the iron absorbed in the intestinal sacs was quantified by atomic absorption spectrophotometry. The matrices had an iron content (mg/g) of: C (0,09 ± 0,01), I (0,37 ± 0,02), II (4,3 ± 0,16), III (3,16 ± 0,45), IV (2,14 ± 0,14) y V (1,08 ± 0,03). The highest iron absorption occurred from matrix V (26.3%), which was higher than IV (7.8%), which in turn did not present differences with III (3.7%) and I (2, 0%), and these showed no differences with II (1.3%) and C (0.8%). It was possible to implement and validate the everted intestine model for ex vivo iron absorption studies in post-slaughter pigs, observing the best absorption for the matrix, which contained the mixture with the highest percentage of heme iron. / Financiamiento: Proyecto Fondecyt 11140249

The current period of human history is considered to be the plastics age due to its versatile characteristics, especially the lightweight, durability and low production cost. Plastics can be manufactured to suit multifarious functions, for example, for personal care products, food/drink storage and medical purposes. Thus, the use of plastics is unavoidable now, finally contributing to the severe pollution worldwide. In 2018 alone, the global plastics production amount has exceeded 359 million tons, around 10% of which ultimately become waste persisting in the environment. When plastic wastes exposed to the sun's radiation, climate change and mechanic abrasion, degradation and fragmentation may occur. Once the size of the fragmentation products is less than 5 mm, they are commonly defined as microplastics (MPs) by the National Oceanographic and Atmospheric Administration. Currently, microplastics have been regarded as the most pervasive environmental pollution problems, not only because of their physical hazards but also due to their interactions with other pollutants in the environment. Pollution can be attributed by the release of additives from MPs, as well as the MPs with adsorbed toxic contaminants. Moreover, MPs additives together with adsorbed chemicals can be easily uptaken by animals, which may cause further propagated effects on the ambient ecosystem. Through the bioaccumulation and biomagnification effect, MPs can even be accumulated in the organisms from different trophic levels and cause serious impacts on aquatic ecology and human health. Despite growing number of evidences that have confirmed the presence and consequential effects of microplastics, researches on microplastic pollution are still lacking. Investigations on occurrence, determination and environmental fate of MPs in aquatic systems are clearly needed. Therefore, the major objective of this study is to elucidate the distribution of MPs in natural environment, to develop novel determination methods to characterize the micro-(nano-)plastics (MNPs), and to study the interactions of MPs with other contaminants in different conditions, as well as their consequential fate in different matrices (e.g. freshwater, cold-blooded intestine, and warm-blooded intestine). The spatial-temporal distribution of the MPs along the Maozhou River was investigated for both the surface water and sediments from 17 sites. Results showed that MPs were widely and unevenly distributed along the river. The MP abundances in dry season ranged from 4.0 ± 1.0 to 25.5 ± 3.5 items·L-1 in water and 35 ± 15 to 560 ± 70 item·kg-1 in sediments, which were relatively higher than those observed in wet season (water: 3.5 ± 1.0 to 10.5 ± 2.5 items·L-1; sediments: 25 ± 5 to 360 ± 90 item·kg-1; p value < 0.05). The dominant types of MPs were identified as: polyethylene (PE, water: 45.0%, sediments: 42.0%), polypropylene (PP, water and sediments: 12.5%), polystyrene (PS, water: 34.5%; sediments 14.5%) and polyvinyl chloride (PVC, water: 2.0%; sediments: 15%). Moreover, metals such as Al, Si, Ca were discovered on the rough surface of the MPs, indicating the interactions between the MPs and the aquatic environment. After obtaining the occurrence of the MPs in the aquatic systems, we proposed an accurate method for MNPs identification and quantification with the employment of the matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). By optimizing the conditions (e.g. the laser energy, matrix, analyte, cationization agent and their ratio), the peaks of PS and polyethylene terephthalate (PET) were successfully identified. A quantitative correlation was built between the normalized signal intensity and ln[polymer concentration], with a correlation coefficient above 0.96 for low-molecular-weight (LM-) polymers and 0.98 for high-molecular-weight (HM-) polymers. Furthermore, two types of environmental MPs samples were prepared, including the particles of an aviation cup as the fresh plastics and the aged MPs extracted from river sediment. By using MALDI-TOF MS, the PS-related micro-(nano-)plastics (in both aviation cup and sediment) consisted of C8H8 and C16H16O oligomers, while the PET-related MNPs (only found in sediment) were identified with compositions of C10H8O4 and C12H12O4. The contents of PS and PET MNPs in sediment were quantified as 8.56 ± 0.04 and 28.71 ± 0.20 mg·kg-1, respectively. Also, the interaction between MPs and bisphenols was investigated. PVC was selected as the representative target because it is comparatively easy to decompose into MPs with the release of additives, especially the bisphenols. The released bisphenols may then be readsorbed by the PVC MPs and cause consequential pollution to the ecosystem. To elaborate on the interactions mechanism, a systematic study was carried out to determine the adsorption mechanisms of five bisphenol analogues (BPA, BPS, BPF, BPB, and BPAF) on PVC MPs. The equilibrium adsorption numbers of the bisphenols on PVC MPs are 0.19 ± 0.02 mg/g (BPA), 0.15 ± 0.01 mg/g (BPS), 0.16 ± 0.01 mg/g (BPF), 0.22 ± 0.01 (BPB), 0.24 ± 0.02 mg/g (BPAF), respectively. Intraparticle diffusion modeling (kinetics) divided the adsorption process into three stages: external mass transport, intraparticle diffusion and dynamic equilibrium. The isotherm results showed a better fit of the adsorption to the Freundlich model. Furthermore, the adsorption mechanisms of the five bisphenol analogues were explored intensively, with respect to hydrophobic interaction, electrostatic force and noncovalent bonds. Besides the adsorption process, the transfer and release behaviors of contaminated MPs are of critically importance in the exploration of their role as culprits and/or vectors for the aforementioned toxicity. Therefore, experiments were performed to examine desorption behaviors and cytotoxicity performance of contaminated MPs in aquatic surroundings and intestinal environment after ingestion by organisms (cold-/warm-blooded). The kinetic study showed that the rate of desorption for bisphenols could be enhanced threefold under simulated warm intestinal conditions. The Freundlich isotherms indicated multiple-layer desorption of the bisphenols on the heterogeneous surfaces of PVC MPs. Hysteresis was detected in the adsorption/desorption of bisphenols in a water environment, but no adsorption/desorption hysteresis was observed in the simulated intestinal conditions of warm-blooded organisms. Due to the enhanced bioaccessibility, the desorption results implied that the environmental risk of contaminated PVC MPs might be significantly increased after ingestion at a high bisphenols dosage. Although with different IC50, the five bisphenols released under the intestinal conditions of warm-blooded organisms can cause higher proliferation reduction in fish and human cell lines than the bisphenols released in water. In summary, this study elucidated the spatial-temporal distribution behaviors of MPs, developed effective determination methods for MNPs revealed the interactions mechanisms of MPs with other contaminants, and explored their consequential fate in different environments. The obtained results are helpful of better understanding on the land-based input of MPs from the intensively affected inland waters, realizing the role of microplastics as both source and carrier for emerging organic pollutants, and providing a novel alternative for MPs determination in future studies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21992号 / 医博第4506号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 坂井 義治, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Objetivo: Evaluar si la infección por Clostridioides difficile se asocia a una mayor frecuencia de complicaciones (muertes, colectomías, obstrucción intestinal, visitas a emergencia, número de exacerbaciones) en pacientes de 18 años a más con enfermedad inflamatoria intestinal (Colitis ulcerativa y/o Enfermedad de Crohn) en comparación a los no infectados. Diseño: Se realizará una Revisión sistemática de la literatura científica disponible que evalúen si la infección por Clostridioides difficile se asocia a una mayor frecuencia de complicaciones (muertes, colectomías, obstrucción intestinal, visitas a emergencia, número de exacerbaciones) en pacientes de 18 años a más con enfermedad inflamatoria intestinal (Colitis ulcerativa y/o Enfermedad de Crohn) en comparación a los no infectados. Para esto se seguirá el método Cochrane y la lista de chequeo PRISMA para la realización de revisiones sistemáticas.