INTRODUÇÃO: Isquemia e reperfusão (I/R) intestinal podem ocorrer em cirurgias vasculares e abdominais, trauma, choque, grandes queimados e transplante intestinal. O órgão funciona como barreira contra agressores externos, e uma vez lesionado, sofre aumento da permeabilidade, que permite a passagem de mediadores inflamatórios e bactérias, causando sepse, inflamação sistêmica e disfunção de múltiplos órgãos, que é a maior causa de morte em unidades de terapia intensiva cirúrgica. O diazóxido tem mecanismo de ação semelhante ao pré-condicionamento isquêmico, e demonstrou proteção em I/R de diversos órgãos. De forma semelhante, em cenário de isquemia e reperfusão intestinal, supomos que ele desempenharia função protetora no intestino, através do pré-condicionamento farmacológico, e em órgãos distantes, exercendo o pré-condicionamento remoto. OBJETIVOS: Avaliar os efeitos do diazóxido em intestino, fígado e coração de ratos submetidos a uma hora de isquemia e doze horas de reperfusão intestinal. MÉTODOS: 32 ratos machos Wistar divididos em três grupos, Sham (n=6); Salina (n=13) submetido a uma hora de isquemia e doze horas de reperfusão intestinal, tendo recebido soro fisiológico; Diazóxido (n=13) submetido a I/R e diazóxido. O modelo de I/R incluiu laparotomia mediana e clampeamento da artéria mesentérica superior. No soro, estudamos citocinas, AST, ALT, troponina e IFABP. Em amostras de intestino e fígado, quantificamos a expressão gênica de citocinas e COX-2. No intestino foi estudada ainda a expressão de proteínas ligadas a barreira intestinal (tight junctions): ZO-1, ocludina e JAM-A. Realizamos preparações histológicas em hematoxiina e eosina de intestino, fígado e coração. RESULTADOS: Evidenciamos redução de expressão de IL-6 intestinal, redução de IL-10 hepática, além de menor expressão de COX-2 intestinal e de ZO-1. IL-6 tem níveis associados a dano da barreira intestinal, assim como COX-2. A menor expressão de ZO-1 indica menor esforço de reparação, proporcional ao grau de lesão em modelos não letais de dano tecidual. CONCLUSÃO: O diazóxido exerce efeito protetor sobre o intestino de ratos após uma hora de isquemia e doze horas de reperfusão, porém não foi possível demonstrar efeito protetor sobre fígado ou coração / INTRODUCTION: Intestinal ischemia and reperfusion can occur in great vascular and abdominal surgeries, trauma, shock, great burns and intestinal transplantation. Since the organ works as a barrier against external threats, and once damaged, permeability increases, which permits passage of bacteria and inflammatory mediators, causing sepsis, systemic inflammation and multiple organ dysfunction, the greatest death cause in surgical intensive care units. Diazoxide has similar mechanisms to ischemic preconditioning, and proved benefit during I/R in several tissues. Similarly, in an intestinal ischemia-reperfusion situation, we suppose it can protect intestine with pharmacological preconditioning, and remote organs, with remote preconditioning. OBJECTIVE: To evaluate diazoxide effects over intestine, liver and heart of rats submitted to one-hour ischemia and twelve-hour reperfusion of intestine. METHODS: 32 male Wistar rats divided in three groups, Sham (n=6); Saline (n=13) submitted to one hour of intestinal ischemia and 12 hours of reperfusion and 0.9% saline; Diazoxide (n=13), submitted I/R and diazoxide. I/R model included median laparotomy and superior mesenteric artery clamping. In blood, we studied cytokines, TGO, TGP, troponin and IFABP. In intestine and liver, we quantified genic expression. Of cytokines and COX-2. In intestine, we also studied expression of tight junctions proteins. Also, histologic analysis in hematoxilin-eosin for intestine, liver and heart. RESULTS: We found lower expression of intestinal IL-6, hepatic IL-10, and lower levels of intestinal COX-2 and ZO-1. IL-6 high levels are associated with intestinal barrier lesion, as for COX-2. Lower levels of ZO-1 correlate with minor repair effort, proportional to lesion grade in non-letal models of damage. CONCLUSION: Diazoxide exerts protective effect over intestine of rats submitted to one hour of ischemia and twelve hours of intestinal reperfusion, but no effect was demonstrated over liver and heart

L’obésité est associée à un état inflammatoire chronique de bas grade ainsi qu’à descomplications métaboliques secondaires telles que l’insulino-résistance, l’intolérance au glucose etla stéatose hépatique. La composition bactérienne intestinale semble tenir une place importantedans l’apparition de ces complications. Il a en effet été montré qu’une altération du microbiote parun régime obésogène était associée à une endotoxémie métabolique caractérisée par uneaugmentation des concentrations circulantes de lipopolysaccharides bactériens. Les AGPI enn-3 possèdent des propriétés anti-obésogènes et anti-inflammatoires qui pourraient passer par unemodulation du microbiote intestinal et une prévention de l’endotoxémie métabolique. Nous avonsmontré que des souris transgéniques fat-1, présentant des teneurs tissulaires en AGPI enn-3 élevées, soumises durant 18 semaines à un régime obésogène, étaient résistantes à l’obésité etaux troubles métaboliques associés. Ces animaux présentent un maintien de la fonction barrièreintestinale et une prévention de l’endotoxémie métabolique. De plus, une analyse du microbiotecaecal révèle une augmentation du phylum Akkermansia. Enfin, nous avons montré qu’un transfertde matériel fécal de souris fat-1 à des souris sauvages maintenues sous régime obésogène protègeces dernières du développement d’une obésité et des comorbidités associées. Ainsi, uneaugmentation de la teneur en Akkermansia au niveau intestinal par les AGPI en n-3, peutreprésenter une stratégie intéressante de prévention de l’obésité, ainsi que de l’insulino-résistance,l’intolérance au glucose et la stéatose hépatique qui lui sont associées. / Obesity is associated with chronic low-grade inflammatory state and secondary metabolicdisorders, such as insulin-resistance, glucose intolerance and hepatic steatosis. Gut microbiotaseems to play an important role in these obesity features. Moreover, microbiota dysbiosis has beenshown to be associated with increased systemic lipopolysaccharides levels, called metabolicendotoxemia. N-3 PUFAs are anti-obesity and anti-inflammatory molecules able to modulate gutmicrobiota and prevent metabolic endotoxemia. We showed that fat-1 transgenic mice,endogenously synthetizing n-3 PUFAs, resist to obesity development and comorbidities whensubmitted to diet-induced obesity for 18 weeks. These mice exhibit a maintained intestinal barrierfunction and a prevention of metabolic endotoxemia. Moreover, cecal microbiota analysis revealedan increase of Akkermansia phylum. Microbiota transplantation of fat-1 mice to wild type micewas shown to exert protective effects against diet-induced obesity and associated disorders. Thus,increasing gut microbiota Akkermansia population by appropriate n-3 PUFAs may represent apromising strategy to prevent obesity, insulin-resistance, glucose intolerance and hepatic steatosis.

INTRODUÇÃO: Isquemia e reperfusão (I/R) intestinal podem ocorrer em cirurgias vasculares e abdominais, trauma, choque, grandes queimados e transplante intestinal. O órgão funciona como barreira contra agressores externos, e uma vez lesionado, sofre aumento da permeabilidade, que permite a passagem de mediadores inflamatórios e bactérias, causando sepse, inflamação sistêmica e disfunção de múltiplos órgãos, que é a maior causa de morte em unidades de terapia intensiva cirúrgica. O diazóxido tem mecanismo de ação semelhante ao pré-condicionamento isquêmico, e demonstrou proteção em I/R de diversos órgãos. De forma semelhante, em cenário de isquemia e reperfusão intestinal, supomos que ele desempenharia função protetora no intestino, através do pré-condicionamento farmacológico, e em órgãos distantes, exercendo o pré-condicionamento remoto. OBJETIVOS: Avaliar os efeitos do diazóxido em intestino, fígado e coração de ratos submetidos a uma hora de isquemia e doze horas de reperfusão intestinal. MÉTODOS: 32 ratos machos Wistar divididos em três grupos, Sham (n=6); Salina (n=13) submetido a uma hora de isquemia e doze horas de reperfusão intestinal, tendo recebido soro fisiológico; Diazóxido (n=13) submetido a I/R e diazóxido. O modelo de I/R incluiu laparotomia mediana e clampeamento da artéria mesentérica superior. No soro, estudamos citocinas, AST, ALT, troponina e IFABP. Em amostras de intestino e fígado, quantificamos a expressão gênica de citocinas e COX-2. No intestino foi estudada ainda a expressão de proteínas ligadas a barreira intestinal (tight junctions): ZO-1, ocludina e JAM-A. Realizamos preparações histológicas em hematoxiina e eosina de intestino, fígado e coração. RESULTADOS: Evidenciamos redução de expressão de IL-6 intestinal, redução de IL-10 hepática, além de menor expressão de COX-2 intestinal e de ZO-1. IL-6 tem níveis associados a dano da barreira intestinal, assim como COX-2. A menor expressão de ZO-1 indica menor esforço de reparação, proporcional ao grau de lesão em modelos não letais de dano tecidual. CONCLUSÃO: O diazóxido exerce efeito protetor sobre o intestino de ratos após uma hora de isquemia e doze horas de reperfusão, porém não foi possível demonstrar efeito protetor sobre fígado ou coração / INTRODUCTION: Intestinal ischemia and reperfusion can occur in great vascular and abdominal surgeries, trauma, shock, great burns and intestinal transplantation. Since the organ works as a barrier against external threats, and once damaged, permeability increases, which permits passage of bacteria and inflammatory mediators, causing sepsis, systemic inflammation and multiple organ dysfunction, the greatest death cause in surgical intensive care units. Diazoxide has similar mechanisms to ischemic preconditioning, and proved benefit during I/R in several tissues. Similarly, in an intestinal ischemia-reperfusion situation, we suppose it can protect intestine with pharmacological preconditioning, and remote organs, with remote preconditioning. OBJECTIVE: To evaluate diazoxide effects over intestine, liver and heart of rats submitted to one-hour ischemia and twelve-hour reperfusion of intestine. METHODS: 32 male Wistar rats divided in three groups, Sham (n=6); Saline (n=13) submitted to one hour of intestinal ischemia and 12 hours of reperfusion and 0.9% saline; Diazoxide (n=13), submitted I/R and diazoxide. I/R model included median laparotomy and superior mesenteric artery clamping. In blood, we studied cytokines, TGO, TGP, troponin and IFABP. In intestine and liver, we quantified genic expression. Of cytokines and COX-2. In intestine, we also studied expression of tight junctions proteins. Also, histologic analysis in hematoxilin-eosin for intestine, liver and heart. RESULTS: We found lower expression of intestinal IL-6, hepatic IL-10, and lower levels of intestinal COX-2 and ZO-1. IL-6 high levels are associated with intestinal barrier lesion, as for COX-2. Lower levels of ZO-1 correlate with minor repair effort, proportional to lesion grade in non-letal models of damage. CONCLUSION: Diazoxide exerts protective effect over intestine of rats submitted to one hour of ischemia and twelve hours of intestinal reperfusion, but no effect was demonstrated over liver and heart

L’administration par voie orale des médicaments reste encore de nos jours la voie royale de la prise des médicaments car moins onéreuse et plus adaptée au confort du patient. Mais cette voie reste toujours inaccessible pour certains médicaments comme les médicaments biologiques et les bio similaires voir certains anticancéreux et antirétroviraux.Le but de ce travail est d’améliorer la biodisponibilité par voie orale des médicaments à faible biodisponibilité par la mise au point d’un promoteur d’absorption. Pour y arriver nous avons adopté comme stratégie de développer un promoteur qui agit à la fois sur le passage passif et sur le passage actif des médicaments. Les études in vitro ont été réalisées en chambre de perméation d’Ussing adaptées par la société Biomécatronics SAS (BéthuneFrance). Dans la première partie de ce travail (Brevet), nous avons montré que l’utilisation d’une composition pharmaceutique et/ou diététique comprenant un extrait de plante(Hibiscus sabdariffa) pouvait augmenter la biodisponibilité in vitro des médicaments et des xénobiotiques qui passent par la voie paracellulaire comme le cisplatine (21 fois),l’oxaliplatine (11fois), la fluorescéine isothiocyanate-Dextran 4000 (3 fois), mais également les médicaments connus pour leur transport actif par la voie transcellulaire comme l’Efavirenz (7 fois) et l’Atazanavir (4 fois). Dans la seconde partie de ce travail, nous avons cherché à vérifier si notre promoteur d’absorption des médicaments a un effet sur la couche de mucus intestinale.Cette couche peut être un facteur limitant de passage des médicaments au travers de la barrière intestinale.Dans un premier temps (article 1), nous avons induit l’augmentation de la couche mucus au niveau du colon de rat après un prétraitement pendant une semaine avec le métronidazole. Puis nous avions confirmé (article 2) que l’administration par voie orale de deux antibiotiques le Cotrimoxazole (CTX) et le métronidazole (MTZ) pendant une semaine augmente la couche de mucus au niveau du côlon ; aussi nous avons montré qu’il existe une relation entre l’augmentation de la couche de mucus et la diminution de la conductance qui est l’index de transport passif des ions, des électrolytes et de certaines molécules à faibles poids moléculaires.De plus l’augmentation de la couche de mucus au niveau de l’intestin est responsable de la diminution du passage transépithélial des deux antirétroviraux dont l’utilisation est recommandée en première ligne par l’OMS (le.Ritonavir et l’Atazanavir) surles sujets porteurs du VIH (virus de l’immunodéficience humain). Après les traitements auMTZ et au CTX la sécrétion de l’Atazanavir augmente respectivement dans le côlon proximal de 2 et 4 fois et dans le côlon distal de 3 et 5 fois. On obtient également une sécrétion du Ritonavir de 5 et 10 fois dans le proximal et de 2 et 5 fois plus dans le distal.Le travail se poursuit par l’étude de l’effet de notre promoteur d’absorption des médicaments sur la couche de mucus intestinal.En conclusion, ce travail montre que l’on peut augmenter la biodisponibilité in vitroen utilisant les promoteurs de l’absorption des xénobiotiques qui agissent à la fois au niveau du transport passif et actif. / Oral dosing is still seen as the silver bullet of drug administration, as it is cheaper andbetter adapted to patient comfort. However, oral route is still inaccessible to many drugssuch as biologics and biosimilars respectively certain anticancer drugs and antiretrovirals(ARV).The aim of this present study was to find new drugs enhancers that improve the oralbioavailability of drugs and xenobiotics. All the studies were realized in vitro using Ussingchambers technic. To achieve the set objective we used the strategy to develop drugenhancer which can modulate at the same time transcellular and paracellular pathways.In the first part of this study (patent) we have shown that the use of a pharmaceutical and /or a dietetic formulation containing a plant extract (Hibiscus sabdariffa) could increase thebioavailability in vitro in rats not only of cisplatin (21 fold), oxaliplatin (11 fold) andFluorescein Isothiocyanate-Dextran 4000 (FD4, 3 fold). All that drugs were transportedthrough intestinal barrier using paracellular pathway. In addition the study showed thatthis formulated enhancer can increased the bioavailability of Efavirenz (7 fold) andAtazanavir (4 fold) which are active transported.In order to assess the effect of new drugs enhancer on mucus thickness that limits thetransport of xenobiotic through intestinal barrier, we decide to evaluate his effect on passiveand active transport of drugs.In the second part of this study we have shown that after a week of pre-treatment of ratswith Metronidazole (MTZ, publication 1) and Cotrimoxazole (CTX, publication 2), the twomost commonly used antibiotics in the prophylaxis against opportunistic infections in HIV /AIDS, both increase colonic mucus thickness that affect directly passive intestinalpermeability by reducing conductance an index of passive transport through intestinalepithelium. In addition those antibiotics also entail a change in the transepithelialconductance and ARV fluxes. After MTZ and CTX treatment the secretion of Atazanavir(ATZ) increases respectively in the proximal colon by 2 to 4 fold and in the distal colon by 3to 5 fold respectively. Ritonavir (RTV) is poorly absorbed in control, after a week of pretreatmentwith MTZ and CTX one rather notices a secretion of RTV 5 to 10 fold higher in theproximal and 2 to 5 fold higher in the distal colon. The next study will be conducted toevaluate the effect of new drugs enhancer on mucus thickness layer.In conclusion, oral bioavailability of drugs and xenobiotics can be enhanced bypharmaceutical composition that contains herbal extract which increase passive and activetransport of drugs through intestinal barrier.

No description available.

This doctoral thesis was conducted in the framework of a co-supervised PhD between the Department of Agriculture, Forestry and Food Sciences of the University of Turin and the Unit of Pharmacognosy, Bioanalysis and Drug Discovery of the Faculty of Pharmacy at the Université libre de Bruxelles. The present manuscript was conceived as a thesis of articles and is composed of 9 different scientific publications. The general introduction of the work was issued from a published narrative review, while the result and discussion part is composed by eight chapters based on different scientific articles issued from the PhD project. The cocoa bean shell (CBS) is the external tegument that covers the cocoa bean, and is one of the major by-products in cocoa industry. It is normally discarded or underutilized, which could result in economical and environmental issues. However, CBS represents a notable source of polyphenols and methylxanthines (theobromine and caffeine) which can give it different biofunctionalities such as antioxidant and antidiabetic properties, among others. It also contains high amounts of dietary fiber (about 50% w/w), minerals, vitamins, and proteins. CBS has low-fat content, and interesting cocoa-aroma compounds. All this could make CBS useful as a food ingredient, and source of biofunctional compounds. The first part of the experimental work of this thesis is devoted to the chemical characterization of CBS and the establishment of its polyphenolic and volatile organic compound (aroma) profiles. The utilization of such profiles, determined by both complete characterization methods and screening methods, was also proposed for authentication purposes of CBS depending on its geographical origin and variety. In a second step, the utilization of CBS as a low-cost food ingredient for functional food production was envisaged. CBS-based beverages and biscuits were proposed as model foods. The influence of the CBS addition to the model foods was evaluated from both technological and nutritional points of view. Changes on the physicochemical characteristics of the model foods were assessed as well as their content in compounds of interest and potential biofunctionalities. Moreover, these studies served also to evaluate the effect of the different food matrices on the bioaccessibility and intestinal permeability of the bioactive compounds contained in CBS. In the third and last part of this work, a different utility was given to the study of the cocoa by-product. The antimicrobial potential of CBS was assessed against different bacterial and fungal strains and a metabolomic strategy was applied in order to individualize the putative active compounds against the Streptococcus mutans proliferation. This work was a contribution for the valorization of a high add-value product such as the CBS, and a step towards a zero-waste cocoa industry within the frame of sustainable circular economy. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Le syndrome de l’intestin irritable (SII) et le syndrome de la vessiedouloureuse (SVD) sont tous deux caractérisés par une hypersensibilité viscérale àla distension. Sur le plan épidémiologique, ces deux syndromes sont étroitementassociés puisque les patients SII ont une prévalence du syndrome de la vessiedouloureuse 7 fois plus élevée que la population générale. Cependant, le mécanismeresponsable de la sensibilisation du tube digestif et de l’appareil urinaire n’a jamaisété étudié. Compte tenu de l’innervation commune de ces deux organes, il estprobable que ce mécanisme mette en jeu sur le long terme des phénomènes de laplasticité neuro-gliale aux niveaux communs d’intégration de la sensibilité pelvienne.L’objectif général de ce travail était d’établir et de caractériser un modèleanimal de sensibilisation croisée vessie/colon, aigu et chronique, afin de mieuxcomprendre les mécanismes impliqués dans l’hypersensibilité viscérale croisée. / Irritable bowel syndrome (IBS) and Bladder pain syndrome (BPS) are bothcharacterized by visceral hypersensitivity to distension. Epidemiology showed thatthese two syndromes are closely associated since IBS patients have a prevalence ofbladder pain syndrome that is 7 times higher than the general population. However,the mechanism responsible for sensitization of the gastrointestinal tract and theurinary tract has never been studied. Given the common innervation of these twoorgans, it is likely that this mechanism involves long-term phenomena of neuro-glialplasticity at the common levels of integration of pelvic sensitivity.The overall objective of this work was to establish and characterize an animalmodel of bladder / colon cross-sensitization, acute and chronic, to better understandthe mechanisms involved in cross-visceral hypersensitivity.