[ES] La presente Tesis doctoral plantea distintas estrategias para el diseño y desarrollo de oleogeles estables, con un perfil lipídico de alta calidad nutricional y la posterior incorporación de los oleogeles en la formulación de distintos alimentos. Se pretende diseñar alimentos mediante el reemplazo de grasas sólidas, ricas en ácidos grasos saturados y trans por oleogeles, que, por un lado, mantengan las propiedades texturales y organolépticas, y, por otro lado, presenten un perfil lipídico mejorado. En una primera etapa, se desarrollaron oleogeles elaborados con 1% de hidroxipropilmetilcelulosa y 0,6% de goma xantana empleando dos condiciones de secado diferentes: secado convencional en estufa a 80 °C durante 10 h 30 min y secado a vacío a 60 °C durante 24 h. Los aceites estructurados fueron aceite de oliva, lino, girasol y girasol alto oleico. La microestructura permitió apreciar oleogeles bien estructurados cuando se utilizaron los aceites de oliva, girasol y girasol alto oleico. La estabilidad física y las propiedades reológicas corroboraron la formación de oleogeles de alta estabilidad física, a lo largo de 35 días de almacenamiento, y con un comportamiento de gel sólido. Se observó que tanto el grado de insaturación del aceite como las condiciones de secado afectaron a la estabilidad física y química del oleogel. De esta manera, se obtuvieron oleogeles poco estructurados y no homogéneos al utilizar aceite con un alto grado de insaturación, como el aceite de lino, por secado convencional, mientras que no fue factible desarrollar oleogeles de lino con secado a vacío. Además, el secado en estufa convencional a 80 °C durante 10 h 30 min generó oleogeles de girasol y de girasol alto oleico con mayor estabilidad estructural y física que el secado a 60 °C durante 24 h. Los oleogeles de oliva y de girasol alto oleico producidos por secado convencional y los oleogeles de oliva y de girasol producidos por secado a vacío presentaron valores de estabilidad oxidativa primaria y secundaria dentro de los límites de aceptabilidad establecidos por el Codex Alimentarius. En una segunda etapa se desarrollaron cremas de cacao untables y panes dulces. En las cremas se estudió la microestructura, textura, reología y atributos sensoriales. La reformulación de las cremas con un reemplazo total (100%) y parcial (50%) de grasa de coco por oleogeles de oliva o girasol obtenidos por secado a vacío permitió mantener sus propiedades estructurales. Concretamente, la sustitución parcial de grasa de coco por oleogel de girasol permitió obtener cremas con atributos sensoriales como "apariencia cremosa", "textura cremosa" y "sabor a cacao", por lo que podría ser una alternativa viable para reformular cremas untables con un perfil nutricional más saludable. Los oleogeles de oliva o girasol alto oleico obtenidos por secado convencional y previamente caracterizados, se emplearon en la formulación de panes dulces elaborados al horno o al vapor. La reformulación con oleogeles permitió obtener panes con unas características estructurales y apariencia similar a la de los panes elaborados con margarina. La realización de una prueba triangular mostró diferencias en la apariencia de la miga y el sabor, mientras que no se observaron diferencias para el atributo textura entre los panes elaborados con oleogeles y con margarina. No se encontraron diferencias en la cantidad de ácidos grasos liberados tras la digestión in vitro entre los panes dulces elaborados con oleogeles y con margarina. Sin embargo, la velocidad inicial de la digestibilidad lipídica fue diferente dependiendo del tipo de procesado, horneado o vaporización, empleado en la elaboración de los panes. El desarrollo de oleogeles a base de hidrocoloides además de ofrecer a la industria alimentaria una alternativa como sustituto de grasas plásticas, podría investigarse como estrategia para modular la digestión de lípidos y brindar beneficios para la salud. Todas las estrategias abordadas en el transcurso de esta Tesis permitieron comprender y ahondar en los conocimientos que conducen a cómo reformular un alimento para mejorar su perfil lipídico sin comprometer sus características sensoriales y fisicoquímicas. / [CA] La present Tesi doctoral planteja diferents estratègies per al disseny i desenvolupament d'oleogels estables, amb un perfil lipídic d'alta qualitat nutricional, i la posterior incorporación dels oleogels en diferents aliments. Es pretén dissenyar aliments mitjançant el reemplaçament de greixos sòlids, rics en àcids grassos saturats i trans per oleogels, que d'una banda, mantinguen les propietats texturals i organolèptiques, i d'altra banda, presenten un perfil lipídic millorat. En una primera etapa, es van desenvolupar oleogels elaborats amb un 1% de hidroxipropilmetilcel·lulosa i un 0,6% de goma xantana emprant dues condicions d'assecat diferents: assecat convencional en estufa a 80 °C durant 10 h 30 min i assecat a buit a 60 °C durant 24 h. Els olis estructurats van ser d'oliva, de lli, de gira-sol i de gira-sol alt oleic. La microestructura va permetre apreciar oleogels ben estructurats quan es van utilitzar els olis d'oliva, gira-sol i gira-sol alt oleic. L'estabilitat física i les propietats reològiques van corroborar la formació d'oleogels d'alta estabilitat física, al llarg de 35 dies d'emmagatzematge, i amb un comportament de gel sòlid. Tant el grau d'insaturació de l'oli com les condicions d'assecat van afectar l'estabilitat física i química del oleogel. D'aquesta manera, es van obtindre oleogels poc estructurats i no homogenis en utilitzar oli amb un alt grau d'insaturació, com l'oli de lli, per assecat convencional, mentre que no va ser factible desenvolupar oleogels de lli amb assecat a buit. A més, l'assecat en estufa convencional a 80 °C durant 10 h 30 min va generar oleogels de gira-sol i de gira-sol alt oleic amb major estabilitat estructural i física que l'assecat a 60 °C durant 24 h. Els oleogels d'oliva i gira-sol alt oleic, produïts per assecat convencional i els oleogels d'oliva i gira-sol produïts per assecat a buit van presentar valors d'estabilitat oxidativa primària i secundària dins dels límits d'acceptabilitat establits pel Codex Alimentarius. En una segona etapa, es van desenvolupar cremes de cacau untables i pans dolços. En aquest treball, es va estudiar la microestructura, textura, reologia i atributs sensorials de les cremes untables. La reformulació de cremes amb un reemplaçament total (100%) i parcial (50%) de greix de coco per oleogels d'oliva o gira-sol obtinguts per assecat a buit, va permetre mantindre les propietats estructurals de les cremes untables. Concretament, la substitució parcial de greix de coco per oleogel de gira-sol va ser descrita amb atributs sensorials com a "aparença cremosa", "textura cremosa" i "sabor de cacau", la qual cosa podria ser una alternativa viable per a reformular cremes per a untar amb un perfil nutricional més saludable. Els oleogels d'oliva o gira-sol alt oleic obtinguts per assecat convencional i prèviament caracteritzats, es van empra en la formulació de pans dolços cuinats al forn o al vapor. Es van estudiar les propietats estructurals, i la digestibilitat lipídica in vitro del producte final. La reformulació amb oleogels va permetre obtindre pans amb unes característiques estructurals i aparença similar a la dels pans elaborats amb margarina. La realització d'una prova triangular va mostrar diferències en l'aparença de la molla i el sabor, mentre que no es van observar diferències per a l'atribut textura, entre els pans elaborats amb oleogels i amb margarina. No es van trobar diferències en la quantitat d'àcids grassos alliberats després de la digestió in vitro entre els pans dolços elaborats amb oleogels i amb margarina. No obstant això, la velocitat inicial de la digestibilitat lipídica va ser diferent depenent de la mena de processament, enfornat o vaporatge, emprat en l'elaboració dels pans. El desenvolupament d’oleogels a base de hidrocol∙loides, a més d ́oferir a la indústria alimentària una alternativa com a substitut de greixos plàstics, podria investigar-se com a estratègia per modular la digestió de lípids i brindar beneficis per la salut. Totes les estratègies abordades en el transcurs d'aquesta Tesi van permetre comprendre i aprofundir en els coneixements que condueixen a com reformular un aliment per a millorar el seu perfil lipídic sense comprometre les seues característiques sensorials i fisicoquímiques. / [EN] The research of this doctoral thesis proposes different strategies for the design and development of stable oleogels, with high nutritional lipid profile and the subsequent incorporation of the oleogels in the formulation of different . It aims to design foods by replacing solid fats, rich in saturated and trans fatty acids with oleogels, which, on the one hand, maintain the textural and organoleptic properties, and on the other hand, present an improved lipid profile. In a first stage, oleogels made with 1% hydroxypropylmethylcellulose and 0.6% xanthan gum were developed using two different drying conditions: conventional drying in an oven at 80 ° C for 10 h 30 'and vacuum drying at 60 ° C for 24 h. The structured oils were olive, flaxseed, sunflower and high oleic sunflower oil. The microstructure showed the oleogels structured, when olive, sunflower or high oleic sunflower oils were used. Physical stability and rheological properties corroborated the formation of oleogels with high physical stability, over 35 days of storage, and with a solid gel behaviour. Both the degree of unsaturation of the oil and the drying conditions affected the physical and chemical stability of the oleogel. In this way, unstructured non-homogeneous oleogels were obtained by using oil with a high degree of unsaturation, such as flaxseed oil, by conventional drying, while it was not feasible to develop flaxseed oleogels with vacuum drying. Furthermore, drying in a conventional oven at 80 ° C for 10 h 30 min generated sunflower and high oleic sunflower oleogels with more structural and physical stability than drying at 60 ° C for 24 h. Olive and high oleic sunflower oleogels, produced by conventional drying, and olive and sunflower oleogels produced by vacuum drying, presented primary and secondary oxidative values within the acceptability limits established by the Codex Alimentarius. In a second stage, spreadable cocoa creams and sweet breads were developed. The microstructure, texture, rheology and sensory attributes of spreadable creams were studied. The reformulation of cocoa creams with a total (100%) and partial (50%) replacement of coconut fat by olive or sunflower oleogels obtained by vacuum drying allowed to maintain the structural properties of spreadable creams. Specifically, the partial substitution of coconut fat for sunflower oleogel gave place to creams described with sensory attributes such as "creamy appearance", "creamy texture" and "cocoa flavor"; therefore, it could be a viable alternative to reformulate spreads with a healthier nutritional profile. The olive or sunflower oleogels obtained by conventional drying and previously characterized, were used in the formulation of sweet breads made in the oven or steamed. Replacement of margarine for oleogels produced breads with similar structural characteristics and appearance that those made with margarine. A triangular discriminatory test showed differences in the appearance of the crumb and the flavor, while no differences were observed for the texture attribute, between the breads made with oleogels or with margarine. No differences were found in the amount of fatty acids released after in vitro digestion between sweet breads made with oleogels and margarine. However, the initial rate of lipid digestibility was different depending on the type of processing, baking or steaming, used in the preparation of the breads. In addition to offering the food industry an alternative as a substitute for plastic fats, the debe lopment of hydrocolloid-based oleogels could be investigated as a strategy to modulate lipid digestion and provide health benefits. / Bascuas Véntola, SM. (2021). Estructuración de aceites mediante el uso de hidrocoloides para sustituir grasas plásticas en los alimentos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/174797

L’objectif de cette étude était de démontrer que le léchage excessif de surface (LES) chez le chien représente un signe clinique d’un trouble digestif sous-jacent plutôt qu’un trouble obsessionnel compulsif. Vingt chiens présentés pour LES (groupe L) ont été divisés en 2 sous-groupes de 10 chiens chacun : L0, sans, et LD, avec des signes cliniques digestifs concomitants. Dix chiens en santé ont été assignés à un groupe contrôle (groupe C). Une évaluation comportementale complète, un examen physique et neurologique ont été réalisés avant un bilan diagnostic gastro-intestinal (GI) complet (hématologie, biochimie, analyse urinaire, mesure des acides biliaires pré et post-prandiaux et de l’immunoréactivité spécifique de la lipase pancréatique canine, flottaison fécale au sulfate de zinc, culture de selles, échographie abdominale et endoscopie GI haute avec prise de biopsies). En fonction des résultats, un interniste recommandait un traitement approprié. Les chiens étaient suivis pendant 90 jours durant lesquels le comportement de léchage était enregistré. Des troubles GI ont été identifiés chez 14/20 chiens du groupe L. Ces troubles GI sous-jacents incluaient une infiltration éosinophilique du tractus GI, une infiltration lymphoplasmocytaire du tractus GI, un retard de vidange gastrique, un syndrome du côlon irritable, une pancréatite chronique, un corps étranger gastrique et une giardiose. Une amélioration >50% en fréquence ou en durée par rapport au comportement de léchage initial a été observée chez une majorité de chiens (56%). La moitié des chiens ont complètement cessé le LES. En dehors du LES, il n’y avait pas de différence significative de comportement (p.ex. anxiété), entre les chiens L et les chiens C. Les troubles GI doivent être considérés dans le diagnostic différentiel du LES chez le chien. / The objective of this study was to characterize excessive licking of surfaces (ELS) in dogs and demonstrate that it can be a sign of underlying gastrointestinal (GI) pathology rather than an obsessive-compulsive disorder. Twenty dogs presented with ELS (L group) were divided in 2 subgroups of 10 dogs each: L0 without and LD with concomitant digestive signs. Ten healthy dogs were assigned to a control group (C group). Behavioral, physical and neurological examinations were performed prior to a complete work-up of the GI system (CBC, serum chemistry panel, urinalysis, assessment of total serum bile acids and canine specific pancreatic lipase immunoreactivity, fecal flotation by zinc sulfate, fecal culture, abdominal ultrasonography and upper GI endoscopy with biopsies). Based on results, appropriate treatment was recommended. Dogs were monitored subsequently for 90 days during which the licking behavior was recorded. Gastrointestinal disorders were identified in14/20 L dogs. Underlying GI disorders included eosinophilic infiltration of the GI tract, lymphoplasmacytic infiltration of the GI tract, delayed gastric emptying, irritable bowel syndrome, chronic pancreatitis, gastric foreign body and giardiasis. Significant improvement (>50%) in frequency or duration of the basal ELS behavior was observed in the majority of dogs (56%). Resolution of ELS occurred in half of the L dogs. Except for ELS, there was no significant difference in the behavior (e.g. anxiety) of L dogs and C dogs. GI disorders should be considered in the differential diagnosis of ELS in dogs

Introduction: Les efforts globaux pour contrôler la tuberculose sont présentement restreints par la prévalence croissante du VIH/SIDA. Quoique les éclosions de la tuberculose multi résistante (TB-MDR) soient fréquemment rapportées parmi les populations atteintes du SIDA, le lien entre VIH/SIDA et le développement de résistance n’est pas clair. Objectifs: Cette recherche visait à : (1) développer une base de connaissances concernant les facteurs associés à des éclosions de la TB-MDR parmi les patients atteints du VIH/SIDA; (2) utiliser ce cadre de connaissances pour accroître des mesures préliminaires pour mieux contrôler la tuberculose pulmonaire chez les patients atteints du VIH/SIDA; et (3) afin d’améliorer l’application des ces mesures, affiner les techniques bactériologiques existantes pour Mycobacterium tuberculosis. Méthodologie: Quatre études ont été réalisées : (1) Une étude longitudinale pour identifier les facteurs associés avec une éclosion de la TB-MDR parmi les patients atteints du SIDA qui ont reçu le traitement directement supervisé de courte durée (DOTS) pour la tuberculose pulmonaire au Lima et au Pérou entre 1999 et 2005; (2) Une étude transversale pour décrire différentes étapes de l’histoire naturelle de la tuberculose, la prévalence et les facteurs associés avec la mycobactérie qu’on retrouve dans les selles des patients atteints du SIDA; (3) Un projet pilote pour développer des stratégies de dépistage pour la tuberculose pulmonaire parmi les patients hospitalisés atteints du SIDA, en utilisant l’essaie Microscopic Observation Drug Susceptibility (MODS); et (4) Une étude laboratoire pour identifier les meilleures concentrations critiques pour détecter les souches MDR de M. tuberculosis en utilisant l’essaie MODS. Résultats : Étude 1 démontre qu’une épidémie de TB-MDR parmi les patients atteints du SIDA qui ont reçu DOTS pour la tuberculose pulmonaire ait été causée par la superinfection du clone de M. tuberculosis plutôt que le développement de la résistance secondaire. Bien que ce clone ait été plus commun parmi la cohorte de patients atteints du SIDA, il n’avait aucune différence de risque pour superinfection entre les patients avec ou sans SIDA. Ces résultats suggèrent qu’un autre facteur, possiblement associé à la diarrhée, peu contribuer à la prévalence élevée de ce clone chez les patients atteints du SIDA. Étude 2 suggère que chez la plupart des patients atteints du SIDA il a été retrouvé une mycobactérie dans leurs selles alors qu’ils étaient en phase terminale au niveau de la tuberculose pulmonaire. Or, les patients atteints du SIDA ayant été hospitalisés pendant les deux dernières années pour une autre condition médicale sont moins à risque de se retrouver avec une mycobactérie dans leurs selles. Étude 3 confirme que la tuberculose pulmonaire a été commune à tous les patients hospitalisés atteints du SIDA, mais diagnostiquée incorrectement en utilisant les critères cliniques présentement recommandés pour la tuberculose. Or, l’essaie MODS a détecté pour la plupart de ces cas. De plus, MODS a été également efficace quand la méthode a été dirigée aux patients soupçonnés d’avoir la tuberculose, à cause de leurs symptômes. Étude 4 démontre les difficultés de détecter les souches de M. tuberculosis avec une faible résistance contre ethambutol et streptomycine en utilisant l’essai MODS avec les concentrations de drogue présentement recommandées pour un milieu de culture. Cependant, l’utilité diagnostique de MODS peut être améliorée ; modifier les concentrations critiques et utiliser deux plaques et non une, pour des tests réguliers. Conclusion: Nos études soulèvent la nécessité d’améliorer le diagnostic et le traitement de la tuberculose parmi les patients atteints du SIDA, en particulier ceux qui vivent dans des régions avec moins de ressources. Par ailleurs, nos résultats font ressortir les effets indirects que les soins de santé ont sur les patients infectés par le VIH et qu’ils peuvent avoir sur le développement de la tuberculose. / Background: Global efforts to control tuberculosis are currently being hampered by a continuing rise in the prevalence of HIV/AIDS. Although outbreaks of multidrug resistant tuberculosis (MDR-TB) are commonly reported among AIDS populations, the link between HIV/AIDS and the development of drug-resistance remains unclear. Objectives: This thesis aimed to: (1) build a knowledge foundation regarding underlying factors associated with outbreaks of MDR-TB among HIV/AIDS patients; (2) use this knowledge framework to develop preliminary health measures for controlling pulmonary tuberculosis among HIV/AIDS patients; and (3) in an effort to better implement these health measures, refine existing culture-based diagnostics for Mycobacterium tuberculosis. Methods: Four studies were conducted: (1) a longitudinal study to identify the underlying factors associated with an epidemic of MDR-TB among AIDS patients receiving Directly- Observed Therapy Short-course (DOTS) for pulmonary tuberculosis in Lima, Peru between 1999 and 2005; (2) a cross-sectional study to characterize the prevalence and factors associated with gastrointestinal shedding with mycobacteria among AIDS patients at different stages in the natural history of tuberculosis; (3) a pilot study to develop screening strategies for pulmonary tuberculosis among hospitalized HIV/AIDS patients using the Microscopic Observation Drug Susceptibility (MODS) assay; and (4) a laboratory-based study to define the optimal critical concentrations needed for detecting drug resistance in M. tuberculosis using MODS. Results: Study 1 revealed that an epidemic of MDR-TB among AIDS patients receiving DOTS for pulmonary tuberculosis was due to super-infection with a specific clone of M. tuberculosis rather than the development of secondary drug-resistance. Although this epidemic clone was more common among patients in the AIDS cohort, risk of superinfection did not differ between AIDS and non-AIDS patients after adjusting for baseline risk of exposure, suggesting that another factor possibly associated with diarrhea may be contributing to the strain’s high prevalence among AIDS patients. Study 2 showed that the majority of AIDS patients in the later stages of pulmonary tuberculosis exhibited gastrointestinal shedding with mycobacteria. Stool shedding was rare in the absence of pulmonary tuberculosis. AIDS patients were also less likely to shed mycobacteria if they had been hospitalized during the previous two years for another medical condition. Study 3 confirmed that pulmonary tuberculosis was common among hospitalized AIDS patients but frequently misdiagnosed using currently recommended diagnostic algorithms. The MODS assay detected most cases and was equally effective when targeted to patients clinically suspicious for tuberculosis. Study 4 demonstrated that low grade drug resistance in M. tuberculosis to ethambutol and streptomycin was difficult to detect with MODS using currently recommended drug-concentration standards in broth. Its diagnostic utility could be improved by modifying drug-concentration standards, and including two versus one critical concentration well for standardized testing. Conclusion: Our studies underscore the need to improve the diagnosis and treatment of tuberculosis among AIDS patients living in resource-constrained settings, all in an effort to prevent morbidity, mortality and the transmission of drug-resistant strains. They also highlight the indirect effect that general health care among HIV-infected patients can have on the development of tuberculosis.

Les tumeurs stromales gastrointestinales (GIST) sont les néoplasies mésenchymateuses les plus complexes du système gastrointestinal. Le traitement curatif standard de cette pathologie est la chirurgie avec l'obtention de marges microscopiques négatives. Les résultats impressionnants obtenus sur la prolongation de la survie avec l'administration d'imatinib (IM) chez les patients atteints de maladie métastatique et non-réséquable ont suggéré aux cliniciens que ce même médicament pourrait aussi collaborer à l'obtention de marges négatives plus aisément lors de cancer avancé. Jusqu'à présent, aucune étude prospective n'a caractérisée l'effet d'une thérapie néoadjuvante prolongée à l'IM sur la qualité de la résection chirurgicale subséquente. L'objectif de ce projet de maîtrise était d'évaluer l'efficacité de l'imatinib utilisé avant la chirurgie (néoadjuvant) jusqu'à l'obtention d'une réponse maximale, en vue d'augmenter le taux de résection microscopique complète (R0) dans le traitement chirurgical des GIST à haut risque de résection microscopique incomplète (R1) ou impossible (R2). Pour ce faire, une étude prospective multicentrique de phase II a été réalisée. Le traitement néoadjuvant à l'IM a été instauré chez des patients porteurs d'une GIST localement avancée ou métastatique. Au total, quatorze patients ont reçu une dose de 400-600 mg/d d'IM pour une durée de 6-12 mois avant la chirurgie. Quatorze patients ont été inclus dans l'étude. Onze ont eu une chirurgie à visée curative, un patient a démontré une maladie non-réséquable suite à une laparotomie exploratrice et deux patients ont refusé la chirurgie. Après un suivi moyen de 48 mois, tous les patients opérés étaient vivants et sept sans évidence de récidive. L'utilisation prolongée (12 mois) d'IM dans un contexte néoadjuvant est faisable, sécuritaire, efficace et comporte peu de toxicité. De plus, cette approche est associée à des hauts taux de résection complète (R0), tout en permettant une chirurgie moins extensive. Des études de phase III actuellement en cours sont nécessaires afin de confirmer nos résultats. / Gastrointestinal stromal tumor (GIST) is the most common mesenchymal malignancy of the GI tract. The current standard of care for GIST is surgical complete resection with negative margins. The agent response rate as well as survival advantages obtained with imatinib mesylate in patients with metastatic and/or non-resectable GIST has lead clinicians to evaluate this therapy as neoadjuvant treatment in patients with locally advanced or metastatic but potentially resectable GIST. This study was designed to evaluate the efficacy of neoadjuvant use of imatinib mesylate until maximal clinical response in potentially resectable GIST patients (locally advanced or metastatic), in order to provide preliminary data regarding the efficacy of this approach in the surgical treatment of GIST at high-risk of incomplete microscopic (R1) or macroscopic (R2) margins. A prospective multicenter phase II trial was designed. Fourteen consecutive patients diagnosed with advanced GIST received imatinib at dose of 400 mg/d to 600 mg/d, given from 6 to 12 months prior to surgery. Amoung the 14 patients included, 11 underwent surgery and had a complete microscopic resection (R0). After a median follow-up of 48 months, all operated patients were alive and 7 without evidence of recurrence. The prolonged use (12 months) of neoadjuvant imatinib is feasible, safe, eficient ans associated with low toxicity. Furthermore, it is associated with a high rate of microscopic resection (R0) and a less extensive surgical approach. Phase III study with higher cohorts are necessary to confirm our primary results.

With the advent of industrialization and food processing techniques the sizes of the cereal bran have been drastically reduced. In my thesis, I have tested the effect, if any, of wheat bran and maize bran particle size, in vitro, on the gut microbiota community structure by 16S rRNA sequencing and their function, by Short chain fatty acids (acetate, propionate, butyrate) production. In turn, we also linked the microbiota and SCFA differences to different chemical composition amongst variously sized fractions of wheat and maize bran.

INTRODUÇÃO: A microbiota intestinal tem sido alvo de diversos estudos moleculares, principalmente através da introdução de plataformas de sequenciamento de nova geração, devido à sua importância e amplo relacionamento com o hospedeiro humano. Entretanto, o armazenamento de amostras fecais antes da extração do DNA é crítico ao caracterizar a composição da microbiota intestinal. Com base nesses dados, o presente estudo buscou compreender os efeitos de diferentes métodos de armazenamento de amostras fecais para caracterizar a microbiota intestinal através do sequenciamento da nova geração, bem como estabelecer um método alternativo de conservação do material genético bacteriano nessas amostras, utilizando guanidina. MÉTODO: Foram coletadas amostras de fezes de 10 voluntários saudáveis. Cada amostra foi dividida em cinco alíquotas, uma alíquota extraída imediatamente após a coleta (fresca) e duas alíquotas submetidas ao congelamento, à temperaturas de -20°C e -80°C e extraídas após 48 horas. As outras duas alíquotas restantes foram armazenadas em guanidina à temperatura ambiente e a 4°C e extraídas após 48 horas. Para observar a presença de alterações na microbiota intestinal, durante um período de armazenamento maior das amostras de fezes, três amostras foram armazenadas em guanidina à temperatura ambiente e a 4ºC e extraídas após o período de 60 dias. A região hipervariável v4 do gene 16S rRNA bacteriano foi amplificada por PCR. Os amplicons gerados foram sequenciados utilizando a plataforma Ion PGM Torrent e os dados analisados utilizando o software QIIME. A determinação da significância estatística foi realizada utilizando-se o teste não-paramétrico de Kruskal-Wallis. RESULTADOS: Não foram encontradas diferenças significativas em nenhum dos níveis taxonômicos (filo, classe, família, ordem e gênero) entre amostras frescas analisadas e os métodos de armazenamento testados. As análises de coordenadas principais (PCoA) mostraram que as amostras se agruparam de acordo com os indivíduos analisados, tendo as amostras referentes a cada indivíduo agrupado-se com maior proximidade do que com outras amostras do mesmo grupo de armazenamento. CONCLUSÃO: Nossos dados sugerem que o congelamento e o uso de guanidina para armazenamento de amostras de fezes, para a caracterização da microbiota intestinal, podem efetivamente preservar o material genético bacteriano nessas amostras ao longo de um período de 48 horas para amostras submetidas ao congelamento e durante 60 dias para amostras armazenadas em guanidina / INTRODUCTION: The gut microbiota has been the target of several molecular studies, mainly through the introduction of next generation sequencing platforms, due to its importance and wide relationship with the human host. However, the storage of fecal samples prior to DNA extraction is critical when characterizing the composition of the intestinal microbiota. Based on these facts, the present study aimed to understand the effects of different methods of storage of fecal samples to characterize the intestinal microbiota by next generation sequence, as well as establishing an alternative conservation method of the bacterial genetic material in these samples using guanidine. METHODS: Stool samples from 10 healthy volunteers were collected. Each collected sample was divided into five aliquots, one aliquot extracted immediately after collection (fresh) and two aliquots subjected to freezing at -20°C and -80°C temperatures and extracted after 48 hours. The others two remaining aliquots were stored in guanidine at room temperature and at 4°C and extracted after 48 hours. In order to observe the presence of alterations in the intestinal microbiota, during a longer storage period of the stool samples, three samples were stored in guanidine at room temperature and at 4°C and extracted after 60 day period. The v4 hypervariable region of bacterial and archeal 16S rRNA gene were amplified by PCR. The generated amplicons were sequenced using Ion PGM Torrent platform and the data analyzed using the software QIIME. Determination of statistical significance was performed using non-parametric Kruskal-Wallis test. RESULTS: No significant differences were found in any of the taxonomic levels (phylum, class, family, order and genus) between analyzed fresh samples and the others different storage methods. The principal coordinates analysis (PCoA) unweighted showed that the samples clustered based on the host each sample originated from, rather than by storage group. CONCLUSION: Our data suggest that both freezing and the use of guanidine to store stool samples for gut microbiota characterization can effectively preserve the bacterial genetic material in these samples over a 48 hours period for samples subjected to freezing and for up to 60 days for samples stored in guanidine

O tratamento antimicrobiano pode destruir o equilíbrio da microbiota gastrintestinal, podendo induzir sintomas clínicos, principalmente a diarréia. A influência de Lactobacillus casei Shirota sobre a microbiota intestinal foi avaliada em um estudo prospectivo, randomizado, duplo-cego e controlado. Sessenta e três crianças hospitalizadas com idade de 2 a 14 anos, sob tratamento com antibióticos &#946;-lactâmicos, foram randomizadas para receber o leite fermentado por L. casei Shirota, 108-9 UFC/mL, ou o placebo, durante o tratamento antimicrobiano. As amostras de fezes foram colhidas antes da administração do leite fermentado, durante o tratamento antibiótico e uma semana após o término do tratamento com o antimicrobiano e a ingestão do leite fermentado. O número de L. casei Shirota aumentou significativamente (p<0,001) durante o período de ingestão do leite fermentado. Foi observado na microbiota do grupo que recebeu o placebo um aumento na contagem de Pseudomonas aeruginosa (p<0,05) e Clostridium sp (p<0,05), principalmente no último período da terapia antimicrobiana. A alteração da microbiota intestinal em decorrência do tratamento antibiótico foi constatada pela diminuição de acetato (p<0,05), butirato (p<0,05) e formato (p<0,05). Embora nenhuma criança deste estudo tenha apresentado diarréia, na avaliação geral, a microbiota daquelas que receberam o leite fermentado mostrou uma recuperação precoce da microbiota intestinal. Foi observado que a variação da contagem bacteriana realizada não foi significativa para as crianças do grupo que recebeu o leite fermentado, enquanto que no grupo placebo a contagem bacteriana ficou alterada, mostrando desequilíbrio da microbiota. Cerca de 50% das crianças ainda apresentaram L. casei Shirota nas fezes após uma semana da ingestão do leite fermentado. Este estudo mostrou que a ingestão do leite fermentado contendo L. casei Shirota promoveu um reequilíbrio mais rápido da microbiota intestinal quando comparada com a do grupo que ingeriu o placebo. / Antimicrobial treatment can destroy the balance of gastrointestinal microflora, which may induce clinical symptoms, mainly diarrhoea. The influence of Lactobacillus casei Shirota on the intestinal microflora was assessed in a prospective, randomised, double-blind controlled study. Sixty-three hospitalised children, with ages between 2 and 14 years, under treatment with &#946;-lactam antibiotics were randomised to receive milk fermented by L. casei Shirota, 108-9 CFU/mL, or placebo during the antimicrobial treatment. Stool samples were collected before the administration of fermented milk, during the antibiotic treatment, and one week after the end of treatment with the antimicrobial agent and the ingestion of fermented milk. The number of L. casei Shirota increased significantly (p<0.05) during the period in which fermented milk was ingested. An increase in the Pseudomonas aeruginosa (p<0.05) and Clostridium sp (p<0.05) count was observed in the microflora of the group that received placebo, mainly in the last period of antimicrobial therapy. The alteration of intestinal microflora as a result of antibiotic treatment was found by the reduction of acetate (p<0.05), butyrate (p<0.05) and formate (p<0.05). The variation in bacterial count proved not to be significant for the children under antimicrobial treatment who received fermented milk, while the placebo group showed imbalance of microflora with the result of the altered bacterial count. About 50% of the children still presented L. casei Shirota in their stools after interrupting the ingestion of fermented milk for one week. This study showed that ingestion of fermented milk containing L. casei Shirota promoted a much faster re-balance of the intestinal microflora when compared to the group that ingested a placebo.

A cada dia cresce o interesse por alimentos ricos em carboidratos não disponíveis em virtude da relação inversa entre seu consumo e o risco de doenças crônicas não transmissíveis (DCNT). No presente trabalho, foi avaliado o potencial fisiológico da farinha de banana verde (FBV) como ingrediente funcional. Em ratos adultos, foi realizado ensaio de média duração (28 dias) para avaliação do efeito trófico da FBV sobre o intestino grosso e de parâmetros relacionados à tolerância à glicose. Em humanos, foram realizados ensaios clínicos de curta e média duração para avaliação dos efeitos sobre resposta glicêmica; liberação de hormônios gastrintestinais relacionados à saciedade; status antioxidante; fome e saciedade; e funcionamento intestinal. A FBV foi produzida com banana verde, Musa acuminata, de acordo com patente depositada pelo grupo (Patente (RPI - 1941), 2008). A FBV é uma fonte concentrada de carboidratos não disponíveis, com 56% de AR e 8% de FAT na base integral. A adição de FBV nas rações provocou efeito trófico no ceco dos animais, evidenciado por aumento no índice metafásico, número de células da cripta e profundidade das criptas. Além disso, a ração com FBV proporcionou melhora nos parâmetros relacionados à tolerância à glicose. Em voluntários saudáveis, a ingestão de uma única refeição adicionada de 8 g de FBV proporcionou aumento na saciedade e boa correlação entre os parâmetros fome/saciedade e níveis plasmáticos de grelina e insulina, melhorou o funcionamento intestinal, além de resultar em alta fermentabilidade in vitro em relação à lactulose. Após ingestão diária da FBV por 14 dias, os resultados da ingestão de RC0 (refeição controle antes do tratamento) e de RC14 (RC0 após 14 dias de tratamento) mostraram que ocorre melhora na tolerância à glicose, evidenciada pela menor liberação de insulina durante o GTT. O efeito positivo sobre funcionamento intestinal, sobre saciedade e sobre liberação de hormônios gastrintestinais no plasma permaneceu após ingestão prolongada da FBV. A adição da FBV na refeição resultou em aumento da capacidade antioxidante in vitro. A FBV apresenta inúmeros atributos positivos para elaboração de produtos que ampliem as opções para uma alimentação saudável, bem como propiciem saúde intestinal, visando a diminuição do risco de DCNT. / The study of unavailable carbohydrates has been of great concern due to their inverse relation with the risk for non-transmissible chronic diseases (NTCD). In the present study, the functional potential of unripe banana flour (UBF) was evaluated. In rats, a medium-term assay was carried in order to evaluate parameters related to glucose tolerance and the trophic effect of UBF on the large bowel. In healthy volunteers, short and medium-term clinical assays were carried to evaluate the effects of UBF on glycemic response; release of gastrointestinal hormones related to satiety (ghrelin, leptin and insulin); antioxidant status; hunger and satiety; and intestinal health. UBF was produced with unripe banana, Musa acuminata, subgroup Cavendish, maturation stage I, in industrial scale and according to a patent deposited by the group (Patent (RPI - 1941), 2008). UBF is a concentrated source of unavailable carbohydrates, with 56% RS and 8% DF (wet weight). Adding UBF in rat rations resulted in a trophic effect in the animals\' cecum, which was evidenced by increase in the metaphasic index, number of crypt cells and crypt depth. Moreover, the ration with UBF resulted in better glucose tolerance parameters. In healthy volunteers, adding UBF (8 g) to an only meal provided significant satiety and good correlation between the parameters hunger/ satiety and plasmatic levels of ghrelin and insulin, improved bowel habit, as well as resulted in high in vitro fermentability in relation to lactulose. After daily intake of UBF for 14 days, the results of the intake of RC0 (control meal before treatment) and RC14 (RC0 after 14 days treatment) showed that there is a positive post-prandial variation in the plasmatic concentrations of gastrointestinal hormones, as well as improvement in glucose tolerance, evidenced by lower insulin release during GTT. The positive effect on bowel habit, satiety and release of gastrointestinal hormones in plasma was kept after prolonged intake of UBF. Adding UBF to the meal provided significant increase in the in vitro antioxidant capacity. UBF presents several positive attributes for the elaboration of products that may increase the options for healthy eating habits, as well as provide intestinal health, always aiming to decrease the risk for NTCD.

蠕動是一種能夠幫助食物通過胃腸道以及促進胃腸道產生能動性的類似波浪的收縮運動。它由一種叫做Cajal (ICC)間質細胞的起搏器細胞產生的慢波所控制。ICCs亦幫助由腸神經系統(ENS)到平滑肌的信息傳導。嚙齒動物和人類實驗表明，老化所導致的ICC細胞數量下降和腸神經退化與排便睏難和便秘有關。通過研究ICC和ENS在正常老化情況下和加速膽碱能神經元喪失的阿爾茲海默症(AD)老鼠模型(Tg2576)中的變化，我們對治療神經退化性疾病也許會有新的認識。本課題的目的在于，研究老化情況下正常老鼠模型及澱粉樣前體蛋白質(APP)過量表達下的AD老鼠模型的胃腸道在形態及功能上的變化。 / 六個月大的Tg2576和同齡野生型對照的全樣載片免疫組化實驗顯示， 十二指腸 (P < 0.05)和迴腸 (P < 0.01)中的腸神經細胞顯著降低，迴腸 (P < 0.001)中的GFAP陽性的腸神經膠質細胞也顯著消失。S100陽性的腸神經膠質細胞在胃竇（胃部中的起搏區域）(P < 0.05), 迴腸 (P < 0.05)和結腸 (P < 0.05)中顯著喪失。這些結果表明，在早期的AD階段，ENS已經出現變質。ICC細胞數量在六個月大的Tg2576和同齡野生型對照的所有腸胃部分並沒有顯著性差異 (P > 0.05)。同時，早期AD階段的基本蠕動節奏也並沒有發生改變。除此之外，結腸和十二指腸的GFAP/S100陽性的腸神經膠質細胞比例並沒有顯著增加，表明在早期AD階段，可能出現了炎症。 / 利用石蠟切片進行β澱粉樣蛋白免疫組化，天狼猩紅溶液化驗和硫代黃素T溶液化驗可以測試不溶的澱粉樣斑塊是否存在。結果指出在六個月大的Tg2576所有腸胃部分都觀察到澱粉樣斑塊聚集而在不同的腸胃部分聚集的程度都有所分別。除了結腸外，六個月大的野生型對照所有腸胃部分都觀察不到澱粉樣斑塊聚集。澱粉樣斑塊形成的增長可能和早期AD階段出現的腸神經細胞和腸神經膠質細胞喪失互相關聯。 / 應用電泳轉移酶標免疫印斑技術，測試六個月大的Tg2576和同齡野生型對照的迴腸和結腸中，膽碱乙酰轉移酶 (ChAT，出自興奮神經元)， 神經元型一氧化氮合酶(nNOS，出自抑制神經元)， 膠質細胞源性神經營養因子 (GDNF， 出自腸神經膠質細胞)和可溶解的β澱粉樣蛋白寡聚體的表達是否改變。和野生型對照相比，Tg2576的nNOS的表達在迴腸 (P < 0.05) 而不是結腸 (P > 0.05) 中顯著增加。而ChAT，GDNF和各β澱粉樣蛋白寡聚體 (十二聚物，九聚物和六聚物)在六個月大的Tg2576和同齡野生型對照之間並沒有顯著改變 (P > 0.05)。綜上結果表明，在早期AD階段，腸胃道中的抑制信號有所增加，但是β澱粉樣蛋白寡聚體可能不是引致腸胃道中的腸神經細胞和腸神經膠質細胞喪失的原因。 / 在腸胃道的組織學和生化實驗之後，我們利用了微電極陣列 (MEA) 系統來量度出自胃竇和迴腸的慢波信號。量度出來的主導頻率(DF)和功率分佈可以成為測量在老化的ICR老鼠和早期AD階段下腸胃道的功能有沒有變化的參數。在硝苯地平存在下，尼古丁顯著地刺激三個月大 (P < 0.05) 和 六個月大 (P < 0.05) 的ICR老鼠中胃竇和迴腸的慢波活動但未能引起十二個月大 (P > 0.05) 的ICR老鼠中的慢波活動，說明神經退化可能在十二個月的年齡開始。附加了河豚毒素的情況下，尼古丁不能再刺激三個年齡組中胃竇和迴腸的慢波活動 (P > 0.05)，由此證明了尼古丁是對腸神經細胞起作用再去激發ICC的活動。六個月大的Tg2576和同齡野生型對照之間的胃竇和迴腸的基准讀數沒有顯著分別 (P > 0.05)。然而，尼古丁顯著地增加野生型對照中胃竇和迴腸的DF和胃電過速範圍 (P < 0.05) 但是不能刺激Tg2576中胃竇和迴腸的電流活動 (P > 0.05)，示意在早期AD階段腸胃道中已經出現了腸神經細胞和/或腸神經膠質細胞喪失。 / 綜上所言，研究結果提出AD老鼠模型有形態學，生物化學和功能上的轉變。本課題提供了在研究神經退化疾病上的基礎，也支持ENS是中樞神經系統早期病變前的關口這個假設。 / Peristalsis is the wave-like contraction that moves food along the gastrointestinal (GI) tract and generates GI motility. Peristalsis is modulated by slow waves that originate from pacemaker cells called interstitial cell of Cajal (ICC). ICCs also modulate and transduce inputs from the enteric nervous system (ENS) to the smooth muscle. Recent studies in rodents and humans demonstrated that a decrease in ICC number and enteric neurodegeneration during ageing is associated with difficult bowel movements and constipation. By studying ICC and the ENS during normal aging and in a mouse model (Tg2576) of Alzheimer’s disease (AD) where cholinergic loss may be exaggerated, we may gain new perspectives on the treatment of degenerative diseases. The aim of the present study therefore, was to investigate the morphological and functional changes of the GI tract of mice during ageing and in an AD mouse model over-expressing amyloid precursor protein (APP) using an isolated tissue approach. / Whole mount immunohistochemistry of 6-month-old Tg2576 mice and their age-matched wild type (WT) controls revealed that there were significant losses of enteric neurons in the duodenum (P < 0.05) and ileum (P < 0.001), and of GFAP-positive enteric glial cells in the ileum (P < 0.001). There was also a loss of S100-positive glial cells in the antrum (pacemaker region in the stomach) (P < 0.05), ileum (P < 0.05) and colon (P < 0.05). These results indicated the alteration of the ENS during the early stages of AD. There were no differences in ICC arears of all GI regions between 6-month-old Tg2576 mice and their age-matched WT controls (P > 0.05), and there was no alteration of basal peristaltic rhythm during the early stages of AD. The non-significant increase of GFAP to S100 enteric glial cell ratio in the duodenum and colon might indicate an ongoing inflammatory process in these two GI regions during the early stages of AD. / The presence of insoluble amyloid plaques was studied using Aβ immunohistochemistry, Sirius red assay and Thioflavin-T assay on paraffin wax sections. The aggregation of amyloid plaques was observed in all the GI regions of 6-month-old Tg2576 mice and the levels of amyloid plaque varied in different regions. No amyloid plaques were found in the GI tract of 6-month-old WT animals excepting the colon. The increase in formation of amyloid plaques might be correlated to the losses of enteric neurons and enteric glial cells during the early stages of AD. / Western blot analysis was performed on frozen sections of tissues from the ileum and colon to investigate whether there were changes in choline acetyltransferase (ChAT, from excitatory neurons), neuronal nitric oxide synthase (nNOS, from inhibitory neurons), glial cell line-derived neurotrophic factor (GDNF, from enteric glia) and soluble Aβ oligomers between 6-month-old Tg2576 mice and WT controls. nNOS expression significantly increased in the ileum (P < 0.05) but not in the colon (P > 0.05) of Tg2576 mice compared with WT controls. There were no differences in the expressions of ChAT, GDNF and Aβ oligomers (docecamer, nonamer and hexamer) in the ileum and colon between Tg2576 mice and WT controls (P > 0.05). These results imply that there is an increase in the inhibitory signal in the GI tract during the early stages of AD but soluble Aβ oligomers might not be the cause of neuronal and glial losses in the GI tract. / Following histological and biochemical studies of different GI regions, slow wave signals from the antrum and ileum were measured using a microelectrode array (MEA) system. The dominant frequencies (DFs) and power distributions were measured and these served as parameters for measuring functional changes in the GI tract during ageing in ICR mice and the early stages of AD. In the presence of nifedipine, nicotine significantly stimulated the slow wave activities in the antrum and ileum of 3-month-old (P < 0.05) and 6-month-old (P < 0.05) ICR mice but failed to trigger the slow wave activities in 12-month-old (P > 0.05) ICR mice, suggesting the neurodegeneration might begin with the age between 6 and 12 months. With the addition of tetrodotoxin, nicotine failed to stimulate the slow wave activities in the antrum and ileum of three age groups (P > 0.05) and it showed that nicotine only acted on enteric neurons to trigger the ICC activities. There were no differences in the antral and ileal baseline recordings between 6-month-old Tg2576 mice and their age-matched WT controls (P > 0.05). However, nicotine significantly increased DFs and tachygastria ranges of the antrum and ileum in WT controls (P < 0.05) but failed to increase electrical activitiy of the antrum and ileum in Tg2576 mice (P > 0.05), thus suggesting a loss of neuronal and/or glial cells in the GI tract during the early stages of AD. / In conclusions, these findings suggest the mouse model for AD has morphological, biochemical and functional changes in the GI tract. The present studies provide a foundation for the investigation of degenerative diseases and support the hypothesis that the ENS may be the gateway for the early pathological changes in the central nervous system. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Hui, Chin Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 180-200). / Abstracts also in Chinese. / PUBLICATIONS RELATED TO THE WORK IN THIS THESIS --- p.i / ABSTRACT --- p.ii / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / LIST OF ABBREVIATIONS --- p.vii / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Interstitial cells of Cajal (ICCs) as electrical pacemaker cells in GI tract --- p.1 / Chapter 1.2.1 --- ICC subtypes in GI tract --- p.2 / Chapter 1.3 --- Hypotheses of slow wave generation --- p.4 / Chapter 1.3.1 --- Mechanisms of the NSCC pacemaking hypothesis --- p.5 / Chapter 1.3.2 --- Mechanisms of the alternative hypothesis --- p.6 / Chapter 1.4 --- Involvement of ion channels in slow wave generation of ICC --- p.6 / Chapter 1.4.1 --- Calcium channels --- p.6 / Chapter 1.4.2 --- Sodium channels --- p.7 / Chapter 1.4.3 --- Potassium channels --- p.7 / Chapter 1.4.4 --- Chloride channels --- p.8 / Chapter 1.4.5 --- Non-selective cation channels --- p.8 / Chapter 1.5 --- Distribution of several types of receptors in ICC --- p.11 / Chapter 1.5.1 --- Purinergic receptors --- p.11 / Chapter 1.5.2 --- Muscarinic receptors --- p.11 / Chapter 1.5.3 --- Tachykinin receptors --- p.12 / Chapter 1.5.4 --- Vasoactive intestinal peptide receptors --- p.12 / Chapter 1.5.5 --- Serotonin receptors --- p.13 / Chapter 1.6 --- Introductions and functions of enteric nervous system --- p.15 / Chapter 1.6.1 --- Interaction amongst the central, peripheral and enteric nervous system: brain-gut axis --- p.15 / Chapter 1.6.2 --- Enteric neuronal subtypes in the GI tract --- p.15 / Chapter 1.6.2.1 --- Motor neurons --- p.16 / Chapter 1.6.2.2 --- Interneurons --- p.16 / Chapter 1.6.2.3 --- Intrinsic primary afferent neurons --- p.18 / Chapter 1.6.3 --- Enteric glial cells --- p.18 / Chapter 1.6.3.1 --- Enteric glial subtypes in the GI tract --- p.18 / Chapter 1.6.3.2 --- Communication between enteric neurons and glial cells --- p.19 / Chapter 1.6.3.3 --- Possible functions of enteric glial cells in the GI tract --- p.19 / Chapter 1.6.3.3.1 --- Secretion of neurotrophic factors --- p.20 / Chapter 1.6.3.3.2 --- Secretion of reduced glutathione --- p.20 / Chapter 1.6.3.3.3 --- Secretion of transforming growth factor-beta 1 --- p.21 / Chapter 1.7 --- Interactions amongst ICC, enteric neurons and enteric glial cells --- p.21 / Chapter 1.8 --- Gastrointestinal disorders --- p.22 / Chapter 1.8.1 --- Mechanisms for cell depletion --- p.22 / Chapter 1.8.1.1 --- Autoimmune attack --- p.22 / Chapter 1.8.1.2 --- Hyperglycaemia and diabetes mellitus --- p.24 / Chapter 1.8.1.3 --- Oxidative stress --- p.25 / Chapter 1.8.1.4 --- Ageing --- p.26 / Chapter 1.9 --- Alzheimer’s disease --- p.28 / Chapter 1.9.1 --- Genetics and pathogenesis of Alzheimer’s disease --- p.28 / Chapter 1.9.1.1 --- Aggregation of amyloid beta protein --- p.29 / Chapter 1.9.1.2 --- Genetic factors of AD --- p.29 / Chapter 1.9.1.3 --- Tau hyperphosphorylation and neurofibrillary tangles --- p.31 / Chapter 1.9.2 --- Current treatment for Alzheimer’s disease --- p.33 / Chapter 1.9.2.1 --- Symptomatic treatment --- p.33 / Chapter 1.9.2.2 --- Disease-modifying treatment --- p.34 / Chapter 1.9.2.3 --- Other potential drugs for AD treatment --- p.35 / Chapter 1.9.3 --- Possible animal models for AD investigation --- p.36 / Chapter 1.9.4 --- Possible correlations between Alzheimer’s disease and the enteric nervous system --- p.36 / Chapter 1.10 --- Aim of study --- p.37 / Chapter CHAPTER 2 --- Investigation into the morphologies of enteric nervous system and interstitial cell of Cajal in Tg2576 mice --- p.38 / Chapter 2.1 --- Introduction --- p.38 / Chapter 2.1.1 --- Molecular markers for ICC, ENC, and EGC --- p.38 / Chapter 2.1.2 --- Aims and objectives --- p.39 / Chapter 2.2 --- Materials and methods --- p.41 / Chapter 2.2.1 --- Animals --- p.41 / Chapter 2.2.2 --- Tissue preparation --- p.41 / Chapter 2.2.3 --- Immunohistochemistry --- p.42 / Chapter 2.2.4 --- Image acquisition and analysis --- p.43 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- General observations --- p.44 / Chapter 2.3.2 --- Area and pattern of ICCs and the ENS in the stomach --- p.46 / Chapter 2.3.3 --- Area and pattern of ICCs and the ENS in the duodenum --- p.52 / Chapter 2.3.4 --- Area and pattern of ICCs and the ENS in the jejunum --- p.56 / Chapter 2.3.5 --- Area and pattern of ICCs and the ENS in the ileum --- p.60 / Chapter 2.3.6 --- Area and pattern of ICCs and the ENS in the colon --- p.66 / Chapter 2.4 --- Discussion --- p.70 / Chapter 2.4.1 --- Major findings --- p.70 / Chapter 2.4.2 --- Possible alterations of the ENS during AD --- p.70 / Chapter 2.4.3 --- Morphological changes of the ENS in relation to genotype --- p.71 / Chapter 2.4.4 --- Morphological changes of ICCs in relation to genotype --- p.72 / Chapter 2.4.5 --- Morphological changes of the ENS and ICCs in relation to GI regions --- p.72 / Chapter 2.4.6 --- Inflammatory conditions in different GI regions --- p.73 / Chapter 2.5 --- Conclusion --- p.74 / Chapter CHAPTER 3 --- Formation of amyloid plaques in the brain and the GI tract of Tg2576 mice --- p.75 / Chapter 3.1 --- Introduction --- p.75 / Chapter 3.1.1 --- The absence of amyloid plaques in rodents --- p.75 / Chapter 3.1.2 --- Overexpression of human APP in transgenic mice --- p.76 / Chapter 3.1.3 --- Distribution of human APP and Aβ deposition in human and transgenic mice --- p.77 / Chapter 3.1.4 --- Transgene and promoter in Tg2576 mouse --- p.77 / Chapter 3.1.5 --- Methods for Aβ plaque detection --- p.78 / Chapter 3.1.6 --- Aim and objectives --- p.78 / Chapter 3.2 --- Materials and methods --- p.80 / Chapter 3.2.1 --- Animals --- p.80 / Chapter 3.2.2 --- Tissue processing --- p.80 / Chapter 3.2.3 --- Preparation of paraffin wax blocks and slide sections --- p.81 / Chapter 3.2.4 --- Aβ immunohistochemistry --- p.82 / Chapter 3.2.5 --- Sirius red assay --- p.83 / Chapter 3.2.6 --- Thioflavin-T assay --- p.84 / Chapter 3.2.7 --- Image acquisition --- p.84 / Chapter 3.3 --- Results --- p.85 / Chapter 3.3.1 --- Aβ immunohistochemistry --- p.85 / Chapter 3.3.1.1 --- The absence of positive immunoreactivity in the brain --- p.85 / Chapter 3.3.1.2 --- The presence of positive immunoreactivity in the GI tract of Tg2576 mice --- p.85 / Chapter 3.3.2 --- Sirius red assay --- p.92 / Chapter 3.3.2.1 --- The presence of positive immunoreactivity in the brain of Tg2576 mice --- p.92 / Chapter 3.3.2.2 --- Characteristics of Sirius red staining in the GI tract --- p.92 / Chapter 3.3.2.3 --- The presence of positive immunoreactivity in the GI tract of Tg2576 mice --- p.92 / Chapter 3.3.3 --- Thioflavin-T assay --- p.98 / Chapter 3.3.3.1 --- The presence of positive immunoreactivity in the brain of Tg2576 mice --- p.98 / Chapter 3.3.3.2 --- The presence of positive immunoreactivity in the GI tract of Tg2576 mice --- p.98 / Chapter 3.4 --- Discussion --- p.104 / Chapter 3.4.1 --- The presence of a small amount of amyloid plaques in the brain of young Tg2576 mice --- p.104 / Chapter 3.4.2 --- The presence of amyloid plaques in the GI tract --- p.104 / Chapter 3.4.3 --- Plaque formation in relation to genotype --- p.105 / Chapter 3.4.4 --- Possible effects of amyloid plaques in the brain and GI tract --- p.106 / Chapter 3.5 --- Conclusion --- p.108 / Chapter CHAPTER 4 --- Expression of Aβ oligomers, ChAT, nNOS and GDNF in the GI tract of Tg2576 mice --- p.109 / Chapter 4.1 --- Introduction --- p.109 / Chapter 4.1.1 --- Common and peripheral types of ChAT --- p.109 / Chapter 4.1.2 --- Three subtypes of NOS --- p.111 / Chapter 4.1.3 --- Functions of glial cell line-derived neurotrophic factor in the ENS --- p.112 / Chapter 4.1.4 --- Neurotoxicity of soluble Aβ peptides --- p.113 / Chapter 4.1.5 --- Aims and objectives --- p.113 / Chapter 4.2 --- Materials and methods --- p.115 / Chapter 4.2.1 --- Animals --- p.115 / Chapter 4.2.2 --- Preparation of materials --- p.115 / Chapter 4.2.3 --- Sample preparation --- p.117 / Chapter 4.2.4 --- Separating and stacking gels preparation --- p.118 / Chapter 4.2.5 --- Western blot --- p.119 / Chapter 4.2.6 --- Image acquisition and analysis --- p.120 / Chapter 4.3 --- Results --- p.122 / Chapter 4.3.1 --- Increase in nNOS expression in the ileum of Tg2576 mice --- p.122 / Chapter 4.3.2 --- No changes in the expressions of Aβ oligomers, ChAT, nNOS and GDNF in the colon of Tg2576 mice --- p.122 / Chapter 4.4 --- Discussion --- p.127 / Chapter 4.4.1 --- The absence of “cholinergic hypothesis of AD in the GI tract of Tg2576 mice --- p.127 / Chapter 4.4.2 --- Increased expression of nNOS in the ileum of Tg2576 mice --- p.128 / Chapter 4.4.3 --- Neuronal and glial losses may be related to the reduced GDNF expression --- p.129 / Chapter 4.4.4 --- No relationship between the Aβ oligomers and neuronal damages in the GI tract --- p.129 / Chapter 4.5 --- Conclusion --- p.129 / Chapter CHAPTER 5 --- Microelectrode array (MEA) study on slow wave activity in the GI tract --- p.131 / Chapter 5.1 --- Introduction --- p.131 / Chapter 5.1.1 --- Components in peristalsis-controlling unit --- p.131 / Chapter 5.1.2 --- Techniques in evaluating slow wave activity --- p.131 / Chapter 5.1.2.1 --- Patch clamp --- p.132 / Chapter 5.1.2.2 --- Calcium imaging --- p.132 / Chapter 5.1.3 --- Application of microelectrode array in evaluating slow wave activity --- p.134 / Chapter 5.1.4 --- Aims and objectives --- p.136 / Chapter 5.2 --- Methods and materials --- p.137 / Chapter 5.2.1 --- Animals --- p.137 / Chapter 5.2.2 --- Tissue preparation --- p.137 / Chapter 5.2.3 --- Electrical recordings --- p.138 / Chapter 5.2.4 --- Analysis and Statistics --- p.139 / Chapter 5.3 --- Results --- p.142 / Chapter 5.3.1 --- Experiments on ICR mice --- p.142 / Chapter 5.3.1.1 --- Nicotine stimulates the slow wave activity in the antrum in the presence of NIF but not in the presence of NIF and 500 nM TTX --- p.142 / Chapter 5.3.1.2 --- Nicotine stimulates the slow wave activity in the ileum in the presence of NIF but only partially stimulates activity in the presence of NIF and 500 nM TTX --- p.152 / Chapter 5.3.1.3 --- The use of 1 μM TTX completely blocked the nicotine stimulation in the ileum --- p.160 / Chapter 5.3.1.4 --- The dominant frequency of baseline increased in the ileum of 12-month-old ICR but not in the antrum in the presence of NIF --- p.162 / Chapter 5.3.2 --- Experiments on Tg2576 mice and their wild type controls --- p.164 / Chapter 5.3.2.1 --- No differences in both antral and ileal baseline DFs between 6- month-old non-transgenic and Tg2576 mice --- p.164 / Chapter 5.3.2.2 --- Nicotine stimulates slow wave activity in the antrum of 6-month-old wild type controls but not of Tg2576 mice --- p.164 / Chapter 5.3.2.3 --- Nicotine stimulates slow wave activity in the ileum of 6-month-old wild type controls but not of Tg2576 mice --- p.167 / Chapter 5.4 --- Discussion --- p.171 / Chapter 5.4.1 --- Pharmacological effects of nicotine in the GI tract --- p.171 / Chapter 5.4.2 --- Excitatory effects of nicotine in the slow wave activities of the stomach and ileum --- p.172 / Chapter 5.4.3 --- Changes of ICC functions and neuronal activities during ageing --- p.174 / Chapter 5.4.4 --- Enteric neurodegeneration leads to alteration in the ENS function in Tg2576 mice --- p.175 / Chapter 5.4.5 --- Conclusion --- p.176 / Chapter CHAPTER 6 --- Concluding discussion --- p.177 / REFERENCES --- p.180

Bifidobacterium spp. são microrganismos probióticos que podem ser incorporados em produtos alimentícios. Entretanto, para que seus efeitos benéficos à saúde humana ocorram, é necessário que o número de células viáveis na hora do consumo seja, no mínimo, 106UFC/g. As bifidobactérias são sensíveis à elevada acidez e, por isso, torna-se necessária a busca por métodos que possam proteger a integridade da célula, sendo um deles a microencapsulação. Em uma primeira etapa do trabalho, Bifidobacterium lactis foi encapsulado em micropartículas de alginato e alginato modificado (alginatoquitosana, alginato-quitosana-sureteric e alginato-quitosana-acryl-eze) e sua sobrevivência e liberação das micropartículas em fluidos simulados do trato gastrintestinal foram mensuradas utilizando-se soluções tampão com pH 1,5, 5,6 e 7,5, na presença e na ausência de pepsina (3g/L), pancreatina (1g/L) e bile (10g/L). A liberação de células das micropartículas teve uma relação direta com o pH do tampão. A microencapsulação aumentou a taxa de sobrevivência de B. lactis, em comparação com células não encapsuladas, em soluções tampão com pH 1,5 sem a presença de enzimas. Em suco gástrico simulado com enzimas digestivas, por outro lado, foi observado que a pepsina proporcionou um efeito protetor sobre as células de B. lactis, e nesse caso, as taxas de sobrevivência do microrganismo estavam diretamente relacionadas com o grau de injúria das células. Em uma segunda etapa do trabalho, leites fermentados com Streptococcus salivarius ssp. thermophilus e Lactobacillus delbrueckii ssp. bulgaricus foram enriquecidos com culturas de Bifidobacterium lactis submetidas a quatro tratamentos diferentes: desidratação em temperatura ambiente, liofilização/congelamento, encapsulação em alginatoquitosana e encapsulação em alginato-quitosana-acryl-eze. A população sobrevivente de B. lactis foi determinada semanalmente no leite fermentado e também após tratamento simulando condições do trato gastrintestinal. Os resultados indicaram que na ausência de pepsina, as populações de B. lactis foram reduzidas drasticamente após o contato com tampão pH 1,5, não sendo possível a detecção de células viáveis livres ou encapsuladas após 120 minutos de teste. A presença de pepsina influenciou positivamente a recuperação de células viáveis de B. lactis em todas as condições testadas, mas as culturas na forma desidratada apresentaram melhores resultados que as culturas microencapsuladas ou liofilizadas. No caso do leite fermentado contendo as células desidratadas, a população de B. lactis, após o tratamento em suco gástrico com enzimas, foi superior à detectada no produto antes desse tratamento. Conclui-se que a microencapsulação não foi eficiente para proteger B. lactis em leite fermentado contra injúrias causadas pelo trato gastrintestinal simulado. / Bifidobacterium spp. are microorganisms that can be added to foods. However, the benefits for the human health occur when the numbers of viable cells in the moment of the consumption is at least 106CFU/g. Bifidobacteria are acid sensitive, and methods to protect cell integrity, such as microencapsulation, are needed. In the first part of the present study, Bifidobacterium lactis was encapsulated in microparticles of alginate and modified alginate (alginate-chitosan, alginate-chitosan-sureteric and alginate-chitosan-acryl-eze) and the survival and release from microparticles in simulated gastrointestinal conditions were measured, using buffers (pH 1.5, 5.6 and 7.5), in the absence and presence of pepsin (3g/L), pancreatin (1g/L) and bile. The release from microparticles presented a direct relationship with pH. When the pH was 1.5 and no enzyme was present, encapsulation improved the survival of B. lactis, when compared to free cells. However, pepsin had a protective effect on B. lactis, and the survival rate was directly related to the cells injury degree. In the second part of the study, fermented milk samples containing Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. Bulgaricus were supplemented with B. lactis submitted to four different treatments: dehydration at room temperature, freeze drying, encapsulation in alginate-chitosan and encapsulation in alginate-chitosaacryl-eze. The number of viable B. lactis cells in the fermented milk was determined weekly and also after treatment with simulated gastrointestinal conditions. Results indicated that in the absence of pepsin, the number of viable cells decreased significantly after contact with buffers (pH 1.5), and no viable cell was detected after 120 minutes. Pepsin improved the recovery of viable cells in the assayed gastric conditions, being the dehydrated cultures more resistant than other cultures. In fermented milk containing the dehydrated cells, the number of viable cells increased after treatment with simulated gastrointestinal fluids. Microencapsulation was not an effective procedure to protect B. lactis in fermented milk against injury caused by the simulated gastrointestinal tract.