A distrofia muscular do Golden Retriever é uma doença degenerativa de caráter hereditário com alterações musculares semelhantes às descritas na distrofia muscular de Duchenne, sendo comprovada a existência de alterações na musculatura lisa do trato gastrointestinal destes animais. Alguns autores sugerem que um dos fatores responsáveis por essas alterações possa estar relacionado com a motililidade intestinal. Este trabalho tem como objetivo estudar os neurônios nitrérgicos e colinérgicos, além da expressão do receptor P2X7, no plexo mioentérico do cólon descendente de cães afetados e não afetados pela distrofia muscular. Foram utilizadas técnicas de imunohistoquímica para marcação das enzimas Óxido Nítrico Sintase (NOS) e Acetilcolina Transferase (ChAT) e da população neuronal total pelo HuC/D e analisada a presença do receptor P2X7. Também foram utilizadas técnicas de microscopia eletrônica de transmissão e histologia básica. Os resultados indicam que neurônios nitrérgicos tendem a ser maiores em cães distróficos e apresentam morfologia Dogiel tipo I; neurônios que expressam o receptor P2X7 colocalizam com neurônios nitrérgicos e colinérgicos. As análises qualitativas demonstram que cães distróficos apresentam maior quantidade de colágeno entre as fibras musculares, entre as camadas musculares circular e longitudinal e, ainda, no interior dos gânglios mioentéricos. Este estudo fornece base para futuras pesquisas e tratamentos da distrofia muscular do Golden Retriever e mais futuramente da Distrofia Muscular de Duchenne, além de poder auxiliar no entendimento das desordens gastrointestinais que são observadas nesta última. / The golden retriever muscular dystrophy is a hereditary degenerative disease characterized by muscle changes similar to those described in Duchenne muscular dystrophy, as well as by alterations in the smooth muscles of the gastrointestinal tract. Some authors suggest that these abnormalities may be associated with intestinal motility. This study evaluated nitrergic and cholinergic neurons in the myenteric plexus of the descending colon of dogs with and without muscular dystrophy, in addition to P2X7 receptor expression. Immunohistochemical techniques were used to label nitric oxide synthase (NOS) and acetylcholine transferase (ChAT), as well as to label total HuC/D-immunoreactive neurons and neurons containing the P2X7 receptor. Transmission electron microscopy and basic histology were used for analysis. Results showed that nitrergic neurons tend to be larger in dystrophic dogs and to be characterized by Dogiel type I morphology, and neurons that express the P2X7 receptor colocalize with nitrergic and cholinergic neurons. Transmission and light microscopy revealed higher collagen density between muscle fibers, between circular and longitudinal muscle layers and within myenteric ganglia of affected dogs. These findings provide support for future research and treatment of the golden retriever muscular dystrophy and hence of the Duchenne muscular dystrophy and contribute to the understanding of the gastrointestinal disorders found in these patients.

Véritable enjeu de santé publique, l’obésité et ses complications seraient la conséquence d’un état inflammatoire chronique de bas-grade qui pourrait résulter de la présence dans le sang de composés bactériens, les lipopolysaccharides (LPS), état appelé endotoxémie métabolique. Le premier objectif de cette thèse était de comprendre pourquoi les LPS, initialement contenus dans le microbiote, sont capables de traverser l’intestin et d’entrer dans le système sanguin. Mon second objectif était d’étudier l’impact de la composition du microbiote dans le contrôle de la satiété par le nerf vague, lien de communication entre l’intestin et le cerveau. Pour cela, un modèle de rats soumis à un régime obésogène a été utilisée.Mes travaux ont montré que la consommation d’un régime obésogène induisait une perte de la fonction de barrière intestinale au niveau de l’iléon caractérisée par une baisse des défenses mucosales et une augmentation de la perméabilité au LPS. L’obésité est également caractérisée par une altération du comportement alimentaire, avec notamment une réduction de la sensibilité aux signaux de satiété. Nous avons montré que ni l’obésité ni le pourcentage de lipides du régime n’étaient responsables de cette perte de sensibilité aux signaux de satiété mais que l’altération du microbiote en serait le contributeur principal. Ainsi, l’endotoxémie métabolique serait le résultat d’une augmentation du passage transepithelial de LPS, qui, une fois dans le sang, pourraient atteindre, entre autres, le nerf vague où ils perturberaient les signaux intestinaux de satiété. / A real public health issue, obesity and its associated metabolic and behavioral disorders are the consequences of a state of low grade chronic inflammation that might originate from the presence in host plasma of gut-derived bacteria components, lipopolysaccharides (LPS). This present state is called metabolic endotoxemia. The first aim of my thesis was to understand why, in diet-induced obesity (DIO), LPS initially contained in the gut lumen, are able to cross the intestine and enter into the circulatory system. My second aim was to investigate the effect of gut microbiota composition and LPS on the satiety regulation by the vagus nerve, the main communication pathway between the gut and the brain. To answer these questions, we have mainly used a DIO rat model.We showed that consumption of WD induced a loss of ileal barrier function characterized by a reduction in mucosal defenses associated to elevated LPS permeability. Obesity is also characterized by an alteration in feeding behavior including a decreased sensitivity to intestinal satiety signals. We showed that neither obesity nor the lipid percentage of the diet triggers loss of sensitivity to satiety signals but that gut microbiota alterations could rather be the main driver. Hence, metabolic endotoxemia could result from an increased transepithelial passage of LPS, which once spread in the blood could reach, among other things, the vagus nerve where they could disrupt intestinal signals of satiety.

L'intestin est la première barrière de l'organisme contre les contaminants alimentaires, dont les mycotoxines. Le déoxynivalenol (DON) est un contaminant majeur des céréales, souvent retrouvé en association avec d'autres trichothécènes B (TCTs B), le 3- et 15-acétyldéoxynivalénol (3-ADON et 15-ADON), le nivalénol (NIV) et la fusarénone X (FX). Au niveau cellulaire, le DON interagit avec l'ARN ribosomique, bloquant ainsi la synthèse protéique et activant la cascade de la voie de signalisation de MAPKinases impliquée dans des mécanismes de la réponse inflammatoire. Au niveau intestinal, cette mycotoxine pourrait donc perturber le renouvellement continu de l'épithélium, et l'homéostasie de la réponse inflammatoire. On suggère ainsi qu'elle pourrait jouer un rôle dans la pathogénie des maladies inflammatoires chroniques de l'intestin. Si les effets du DON sont relativement connus, ceux du NIV et de leurs dérivés acétylés sont moins bien documentés. De même, peu de données existent quant à la toxicité combinée de ces mycotoxines dont la co-occurrence est avérée. Sur des modèles in vitro de cellules épithéliales intestinales humaines et porcines et sur un modèle ex vivo d'explants de jéjunum de porc, nous avons comparé les toxicités individuelles de cinq TCTs B (DON, 3- et 15-ADON, NIV et FX) et analysé leur toxicité combinée en termes de synergie, additivité ou antagonisme vis-à-vis de l'intestin. Les résultats montrent qu'à des concentrations de l'ordre du micromolaire, les TCTs B inhibent la croissance des cellules épithéliales intestinales par ordre croissant de toxicité 3-ADON, DON, 15-ADON, NIV et FX. Aux faibles doses correspondant à des niveaux d'exposition rencontrés chez le consommateur français ou européen, des synergies d'un facteur 3 à 10 ont été observées. Ces travaux ont également permis de caractériser l'activité pro-inflammatoire au niveau intestinal des TCTs B, et l'analyse benchmark de données de transcriptomique a montré que l'exposition de l'intestin à des doses aussi faibles que 0.04µM de FX, 0.1µM de DON ou 0.1µM de NIV s'accompagne d'une activation significative des mécanismes de l'inflammation. Ces doses sont de l'ordre des concentrations attendues dans le chyle sur la base des valeurs toxicologiques de référence actuelles. En conclusion, ces données montrent que le renouvellement de l'épithélium intestinal et l'activité pro-inflammatoire au niveau intestinal pourraient être des marqueurs très sensibles dans le cadre de l'évaluation de la toxicité individuelle et des interactions entre TCTs B. / As for other food-born contaminants, the gastro-intestinal tract represents the first barrier against deoxynivalenol (DON). This mycotoxin frequently co-occurs with other type B trichothecenes (TCTs B) namely 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX). At the cellular level, DON binding to ribosomal RNA results in the inhibition of protein synthesis and triggers the mitogen-activated protein kinases (MAPKs) pathway that have been linked to immune response mechanisms. Thus, intestinal epithelial cell renewing is considered a putative target in DON toxicity. Moreover, based on the ability of DON to disturb the state of homeostasis of the inflammatory response in the intestine mimicking what is found in inflammatory bowel diseases (IBD), it is proposed that this mycotoxin may play a role in such diseases. However, very few is known about the intestinal toxicity of the other co-occuring TCT B, and their combined effects eventually. By means of in vitro human and porcine intestinal epithelial cells models and an ex vivo porcine jejunal explants model, we assessed the individual toxicity of five TCT B (DON, 3- and 15-ADON, NIV and FX) toward the intestine and we analyzed their combined toxicity in terms of additivity, synergy or antagonism. The tested TCT B significantly impaired the intestinal epithelial cell growth in the micromolar range, in increasing order of potency 3-ADON, DON, 15-ADON, NIV and FX. The toxicity of low doses of TCT B was synergistic. For mycotoxin concentrations corresponding to exposure levels reported for French and European consumers, the amplitude of this synergy ranged between 3 and 10. Benchmark dose analyses of the transcriptional data also showed that the exposure of the intestine to mycotoxin concentrations as low as 0.04µM for FX, 0.1µM for DON and 0.1µM for NIV could be associated to a significant activation of the inflammatory response mechanisms. Taken together, these results suggest that epithelial cell renewing and pro-inflammatory effects at the intestinal level may be consider very sensitive biomarkers for the assessment of the individual toxicity and interactions between the co-occurring TCTs B.

L’hypertriglycéridémie post prandiale constitue un facteur de risque des maladies cardiovasculaires et est présente en cas de syndrome métabolique, d’obésité et d’insulino-résistance. L’intestin conditionne la biodisponibilité des lipides et l’hypertriglycéridémie post prandiale. En effet, il contrôle la quantité et la qualité des chylomicrons sécrétés, en adaptant son métabolisme en fonction de la teneur en lipides du régime. Or à l’heure actuelle, le mécanisme de détection des lipides alimentaires par les entérocytes nécessaire à cette adaptation, n’est pas élucidé. Ce travail de thèse a permis de démontrer que la glycoprotéine transmembranaire CD36, est un récepteur aux AGLC qui déclenche l’activation des ERK1/2. Cette activation est responsable de l’induction du taux d’ARNm de 3 protéines clés de l’absorption des lipides (l’Apobec1, la Microsomal Triglyceride Transfer Protein (MTP), la Liver-Fatty Acid Binding Protein (L-FABP)) et de la dégradation post prandiale de CD36. La pertinence physiologique de ce récepteur a été évaluée chez des souris CD36 (-/-) soumises à un régime hyperlipidique riche en AGLC saturés ou insaturés. Nos données démontrent que CD36 intestinal est indispensable à l’absorption de forte quantité de lipides, au contrôle de l’hypertriglycéridémie post prandiale, de l’inflammation intestinale et de l’endotoxémie. Ces effets sont fortement aggravés en cas de régime hyperlipidique riche en AGLC insaturés qui peuvent même être léthal. Le CD36 intestinal pourrait donc être une cible thérapeutique dans le traitement de l’hypertriglycéridémie et de l’endotoxémie post prandiales. / Post prandial hypertriglyceridemia represents a risk factor for cardio-vascular diseases and it is associated with metabolic syndrom, obesity, and insulino-resistance. The intestine influences lipid bioavailibility and post prandial hypertriglyceridemia. It controls the quantity and the quality of secreted chylomicrons by adapting its metabolism according to the lipid content of the diet. Nevertheless, the mechanism of dietary lipid detection by the enterocyte is not understood. Our work demonstrates that the transmembrane glycoprotein CD36 is a Long Chain Fatty Acid (LCFA) receptor which triggers ERK1/2 activation. This activation is responsible for the induction of mRNA rate of 3 key proteins of lipid absorption (Apobec1, Microsomal Triglyceride Transfer Protein (MTP), Liver-Fatty Acid Binding Protein (L-FABP)) and for CD36 degradation. The physiological relevance of this receptor has been assessed in CD36 (-/-) mice fed with a High Fat Diet (HFD) rich in saturated or unsaturated LCFA. Our data demontstrates that CD36 is crucial for the absorption of an important quantity of lipids, to the control of hypertriglyceridemia, of intestinal inflammation and of endotoxemia. These effects are getting worse in the case of HFD rich in unsaturated LCFA, which can be lethal. Intestinal CD36 could represent a therapeutic target in the treatment of post prandial hypertriglyceridemia and endotoxemia.

O presente trabalho teve por objetivo verificar o efeito de diferentes níveis de IGF-I no colostro de vacas que receberam rbST durante o período pré-parto, no desenvolvimento do trato intestinal e na expressão do gene do IGF-I e de seu receptor no fígado e intestino de bezerros. Quarenta e duas vacas da raça Holandesa, gestantes e multíparas foram distribuídas ao acaso em dois grupos de 21 animais, o grupo rbST que recebeu hormônio de crescimento (500 mg rbST) e o grupo controle que recebeu injeção de vitamina E. As aplicações tiveram início aos 35 dias pré-parto, em intervalos de 14 dias até a data de parição. Os bezerros recém-nascidos foram distribuídos aleatoriamente, de acordo com as seguintes idades em que foram abatidos: após o nascimento e sem a ingestão de colostro, dois e sete dias de vida com ingestão de colostro das respectivas mães. Após o abate, amostras do fígado e segmentos do intestino delgado (jejuno e íleo) foram coletadas para quantificação de DNA, RNA, proteína total e o RNAm do IGF-I e do receptor tipo I. O delineamento experimental utilizado foi o inteiramente casualizado, numa estrutura fatorial 2x3, correspondendo aos grupos das mães (rbST ou controle) e as idades dos bezerros (após o nascimento, dois e sete dias de vida). No fígado, as concentrações de RNA e proteína (mg/g tecido) foram maiores no segundo dia de vida e a relação proteína/RNA aumentou no sétimo dia de vida (P<0,05). O jejuno apresentou interações entre os tratamentos nas concentrações de DNA, proteína e relações proteína/RNA e RNA/DNA (P<0,05). Os bezerros que consumiram colostro proveniente de vacas que receberam rbST apresentaram, no jejuno, maiores concentrações de DNA no segundo dia de vida e diminuição a níveis intermediários entre nascimento e dois dias, comparados aos sete dias de vida. Este efeito também foi observado na relação proteína/RNA. No grupo controle, também se verificou aumento de DNA aos dois dias, mas não houve diferenças aos sete dias, e a relação proteína/RNA foi semelhante entre as idades. A concentração de proteína no jejuno do grupo rbST aumentou no segundo dia de vida e diminuiu no sétimo, enquanto que no grupo controle, este aumento foi verificado apenas no sétimo dia de vida. A relação RNA/DNA diminuiu apenas no grupo controle (P<0,05). A expressão do gene do IGF-I foi maior ao nascimento e aos sete dias de idade no fígado dos bezerros do grupo rbST (P<0,05). As concentrações do gene do receptor tipo I diminuíram com a idade dos bezerros (P<0,05). Ao nascimento, o intestino dos bezerros apresentou condição de resposta celular à presença do IGF-I proveniente do colostro. Os resultados sugerem que as células do jejuno de bezerros do grupo rbST apresentaram um diferente estágio de maturação. / The objective of this study was to verify the effect of different levels of IGF-I in colostrum of cows treated with rbST during dry period, in the development of the intestinal tract and IGF-I and receptor gene expression in the liver and intestine of calves. Forty-two Holstein cows, in gestation and multiparous, were randomly assigned in two groups of 21 animals. Group rbST received injection of growth hormone (rbST), and group control received vitamin E injection. Both treatments started 35 days pre-partum, and were administered every 14 days until parturition. Newborn calves were randomly assigned to the following ages of slaughter: just after birth and without colostrum ingestion; two and seven days of life with colostrum ingestion from their respective mothers. After slaughter, samples from liver and small intestine (jejunum and ileum) were collected for quantification of DNA, RNA, total protein and mRNA of IGF-I and receptor type I. A completely randomized design was used with 2X3 factorial arrangements of treatments, which the factors were the mother’s group (control and rbST) and age (just after birth, two and seven days of life). In the liver, RNA and protein concentrations (mg/g tissue) were higher in the second day of age and protein/RNA ratio increased in the seventh day (P<0.05). The jejunum showed interaction among the treatments in the concentrations of DNA and protein, and protein/RNA RNA/DNA ratios (P<0.05). Calves fed colostrum from mothers that received rbST presented, in jejunum, higher DNA concentration in the second day of life, and decrease to intermediate levels between birth and two days, compared to seven days of age. This effect was also observed in protein/RNA ratio. In the control group, it was also verified DNA increase at two days, but there was not difference in the seventh day, and protein/RNA ratio was similar among ages. Protein concentration in jejunum of rbST group increased in the second day and decreased in the seventh, but in the control group, this increase was verified only in the seventh day of age. RNA/DNA ratio decreased in the control group (P<0.05). Expression of IGF-I was higher at birth and seven days old in the liver of the calves from rbST group (P<0.05). The concentrations of receptor type I mRNA decreased with calves’ age (P<0.05). At birth, the small intestine of calves showed a condition of cellular response to the presence of IGF-I in colostrum. These results suggest that cells in jejunum of calves from rbST group presented a different phase of maturation.

O objetivo deste estudo foi avaliar o processo de aquisição de anticorpos em cordeiros recém-nascidos aleitados com colostro bovino e ovino, bem como a taxa de proliferação celular no epitélio intestinal. Este estudo contribui com informações sobre a aquisição de imunidade passiva nesta espécie, com o conhecimento do desenvolvimento e maturação do trato gastrintestinal no período neonatal, e para a avaliação de uma alternativa de manejo de colostro para estes pequenos ruminantes. Foram utilizados 30 cordeiros recém-nascidos. Às 0 e 6 horas de vida, 12 animais receberam 250 mL de colostro bovino (grupo CB) e outros 12 animais receberam 250 mL de colostro ovino (grupo CO). Amostras de sangue foram coletadas às 0, 6, 24, e 72 horas de vida para quantificação de imunoglobulina G (IgG) e proteína total sérica (PT). Seis animais foram sacrificados aleatoriamente, logo após o nascimento, sem ingestão de colostro, constituindo o grupo controle. Os demais grupos foram abatido às 24 e 72 horas. Amostras do intestino delgado foram coletadas para a quantificação da taxa de divisão celular nas criptas intestinais. O delineamento experimental adotado foi inteiramente casualizado, sendo as variáveis séricas analisadas como medidas repetidas no tempo. Para a variável histológica foi considerado um arranjo fatorial 2 X 2 + 1, tendo como efeitos principais o colostro fornecido, as idades de abate e o grupo controle. As concentrações de IgG sérica às 6, 24 e 72 horas foram significativamente superiores para o grupo CB (16,58±6,19; 34,12±5,67 e 28,77±5,45 mg mL-1) comparado com CO (10,76±6,08, 20,77±6,53 e 20,25±7,3 mg mL-1). A eficiência aparente de absorção (EAA) da IgG mostrou-se inferior no grupo CB (15,06±4,97%) em relação aos animais do grupo CO (25,70±13,08%). O grupo CB apresentou às 24 e 72 horas maiores (P<0,05) valores de PT (7,29±0,87 e 6,89±0,30 g 100mL-1) em relação ao grupo CO (5,73±1,35 e 5,69±0,57 g 100mL-1). Ao nascimento, os animais apresentaram 32,52%, 45,47% e 30,60% de células em divisão para as regiões do duodeno, jejuno médio e íleo, respectivamente. Às 24 horas, os animais do grupo CO apresentaram menor (P<0,0001) porcentagem de células em mitose no duodeno (42,12%) e no íleo (35,66%) em relação aos animais CB, 46,44% e 39,74%, respectivamente. Às 72 horas, foi observada uma porcentagem menor (P<0,0001) de células em divisão nas criptas do duodeno dos animais CO (36,28%), comparados com o grupo CB (43,18%). Não foi observada diferença significativa entre os tratamentos na porcentagem de células mitóticas nas criptas do jejuno às 24 e 72 horas, bem como nas criptas do íleo às 72 horas (P>0,05). Independente do tratamento, o jejuno foi o segmento com maior (P<0,0001) porcentagem de células mitóticas em todos os períodos. Os valores superiores na taxa de divisão celular no grupo CB indicam que o colostro bovino, provavelmente pela elevada concentração de fatores bioativos e de anticorpos, influencia positivamente o processo de renovação epitelial e que o mesmo pode ser utilizado como fonte alternativa de IgG para cordeiros recém-nascidos. / The objective of this study was to evaluate the antibody acquisition mechanism in newborn lambs fed bovine or ovine colostrum as well as the cell proliferation rate in the intestine epithelium. This study contributes with information about passive immunity acquisition in this specie, with knowledge about development and maturation of the small ruminant intestine tract, and for the evaluations of colostrums management alternative to these small ruminant. Thirty newborn lambs were used. At 0 and 6 hours of life, 12 animals received 250 mL of bovine colostrum (BC group) and another 12 animals received 250 mL of ovine colostrum (OC group). Blood samples were collected at 0, 6, 24, e 72 hours of life for immunoglobulin G (IgG) and total serum protein (TP) quantification. Six animals were randomly slaughtered just after birth, without colostrum intake, constituting the control group. The other groups were randomly slaughtered at 24 and 72 hours. Samples of the small intestine were collected for quantification of cellular division rate in intestinal crypts. A completely randomized desining was used, with the serum variables analyzed as repeated measures on time. For the histological variable it was considered a 2 X 2 + 1 factorial arrangement, having as the main factors colostrum supply, slaughter date and the control group. The IgG serum concentration at 6, 24 and 72 hours were significantly higher for the BC group (16,58±6,19; 34,12±5,67 and 28,77±5,45 mg mL-1) compared with the OC group (10,76±6,08, 20,77±6,53 and 20,25±7,3 mg mL-1). The apparent efficiency of IgG absorption (AEA) were lower for the BC group (15,06±4,97%) in relation to the animals from the OC group (25,70±13,08%). The BC group showed at 24 and 72 hours higher (P<0,05) TP values (7,29±0,87 and 6,89±0,30 g 100mL-1) in relation to the OC group (5,73±1,35 and 5,69±0,57 g 100mL-1). At birth, the animals showed 32,52%, 45,47% and 30,60% cells in division for duodenum, jejunum and ileum, respectively. At 24 hours, the animals from the OC group showed lower (P<0,0001) percentage of cells in mitosis in the duodenum (42,12%) and ileum (35,66%) in relation to the BC animals, 46,44% and 39,74%, respectively. At 72 hours, it was observed lower percentage (P<0,0001) of cells in division in the duodenum crypts of the OC animals (36,28%) compared with the BC group (43,18%). It was not observed significantly difference between treatment in mitotic cell percentage of jejunum crypts at 24 and 72 hours as well as in ileum crypts at 72 hours. Independent of the treatment the jejunum was the segment with higher mitotic cells percentage in all periods. The highest values in cellular division rate in the BC group, probably due to high concentrations of bioactive factors and antibodies, indicates that bovine colostrum influences positively the epithelium renovation process and that it can be used as an alternative source of IgG for newborn lambs.

L’intestin des mammifères est colonisé par une dense communauté microbienne avec lequel il a établi, au cours d’une longue co-évolution, des relations mutualistes. Ces relations, bien qu’essentiellement basées sur des avantages métaboliques permettent également la maturation complète du système immunitaire, dont le développement est initié in utero par un programme génétique. Chez la souris, seule la SFB a été décrite comme présentant un fort pouvoir immunostimulant permettant la coordination d’un large panel de réponses immunes, notamment IgA et Th17 dont le site d’induction préférentiel serait les plaques de Peyer. La première partie de ma thèse a contribué à la finalisation de travaux consacrés à l’étude des caractéristiques des réponses IgA et Th17 induites par SFB. Nous avons ainsi mis en évidence la capacité de la SFB à stimuler la maturation post-natale de follicules lymphoïdes isolés et de tissus lymphoïdes tertiaires qui se substitueraient aux plaques de Peyer en tant que sites inducteurs des réponses IgA et Th17 stimulées par SFB. Cependant, ce microbiote constitue aussi une source antigénique importante pouvant représenter une menace pour l’intégrité de l’hôte. Notamment, la SFB peut provoquer une inflammation chronique délétère en périphérie de l’intestin, chez des souris prédisposées génétiquement. Cela suggère l’existence de mécanismes capables de réguler la colonisation par la SFB, afin d’éviter tout phénomènes compromettant l’intégrité physiologique de l’hôte. À l’aide de souris immunodéficientes axéniques ou seulement colonisées par SFB, nous avons pu mettre en évidence un rôle de la voie de signalisation des TLR dans le contrôle de la colonisation par SFB. Cependant, ni les peptides anti-microbiens, ni les IgA ne semblent impliqué dans le contrôle de la colonisation par SFB. / The mammalian intestine is heavily colonized by a huge microbial community. During a long coevolution process, the host has evolved mutualistic relationships with its microbiota. Thes relationships, mainly based on metabolstic advantages, also allow the full maturation of the host immune system, which development is initiated in utero by a genetic program. In mice, only SFB has been yet described to display strong immunostimulant properties allowing the coordination of a large panel of immune responses, more specifically IgA and Th17 responses, which preferential induction sites might be the Peyer's patches. The first part of my thesis contributed to complete a work dedicated to the characterization of the IgA et Th17 responses induced by SFB. We have underscored SFB capacity to stimulate the post-natal maturation of isolated lymphoid follicles and also tertiary lymphoid tissues that would substitute to Peyer's patches as inductors sites of both IgA and Th17 responses induced by SFB. However, this microbiote also constitutes a potential antigenic threat for the host integrity. Notably, the SFB could provoke chronic deleterious inflammation in peripheric compartment in genetically predisposed individuals. It suggests the establishment of highly regulated mechanisms regulating SFB colonization, to avoid compromising events for the host homeostasis. In a second part of my thesis, with the help of immunodeficient axenic mice, we have shown a role of TLR signaling pathways in the control of SFB colonization. However, antimicrobial peptides, IgA, IL-17 or IL-22 seem to be involved int eh control of SFB colonization.

Der Morbus Parkinson bzw. das idiopathische Parkinson-Syndrom ist pathophysiologisch durch eine progressive Degeneration der dopaminergen Neurone in der Substantia nigra und im Locus coeruleus charakterisiert. Obwohl die genaue Ursache der Erkrankung bis heute unbekannt ist, wird eine multifaktorielle Genese bei polygenetischer Prädisposition angenommen. Des Weiteren liefert die sogenannte Braak-Hypothese eine mögliche Kausalkette. Diese Hypothese besagt, dass unbekannte Erreger die Fehlfaltung von physiologischem α-Synuclein im peripheren Nervensystem, insbesondere auch im enterischen Nervensystem, initiieren können, welche sich dann retrograd durch axonalen Transport über den Nervus vagus zu dem dorsalen motorischen Kern des Nervus vagus ausbreitet. Von dort werden zusätzliche Hirnregionen, einschließlich der Substantia nigra, beeinträchtigt. Es wird vermutet, dass diese neuronale α-Synuclein-Aggregation im Gehirn ein zentraler Bestandteil der Pathogenese des Morbus Parkinson darstellt. Als histopathologisches Korrelat des Morbus Parkinson können in der Substantia nigra sogenannte Lewy-Körperchen im Zytoplasma der Nervenzellen nachgewiesen werden, die aus intrazellulären Proteinablagerungen und hauptsächlich aus unlöslichem und aggregiertem α-Synuclein bestehen. α-Synuclein-Aggregate im Magen-Darm-Trakt wurden als potenzieller Biomarker für die Früherkennung eines Morbus Parkinson vorgeschlagen. Postuliert wurde hier, dass die α-Synuclein-Pathologie im Darm zeitlich bereits vor der Pathologie in der Substantia nigra nachweisbar ist. Studien, die diese Hypothese weiter untersuchten, führten jedoch zu divergierenden Ergebnissen. Die Zielsetzung der vorgelegten Studie war es, Ablagerungen des α-Synucleins und des phosphorylierten α-Synucleins im menschlichen Gastrointestinaltrakt mithilfe von Immunhistochemie zu untersuchen, um eine Aussage darüber treffen zu können, ob α-Synuclein als potenzieller prädiktiver Biomarker für die Erkrankung geeignet sein könnte. Verwendet wurden hierzu einerseits Antikörper gegen natives α-Synuclein und andererseits Antikörper gegen das an Serin 129 phosphorylierte α-Synuclein, welche die pathologische, aggregierte Form des Proteins besser darstellt. Es sollte festgestellt werden, ob sich die Prävalenz der (phosphorylierten) α-Synuclein-Ablagerungen im Darm von Parkinson-Patienten und Kontrollpatienten unterscheiden. Eine mangelnde Spezifität des Antikörpers gegen das phosphorylierte α-Synuclein wurde dabei durch die Implementierung eines Kontrollexperiments mit einer proteolytischen Vorbehandlung an Gewebeproben histopathologisch bestätigter Parkinson-Patienten ausgeschlossen. In dieser retrospektiven Studie wurde die α-Synuclein-Expression in post-mortem entnommenen Hirn-, Dünn- und Dickdarmproben von 25 neuropathologisch bestätigten Parkinson-Patienten und 20 alters- und geschlechtsspezifisch abgestimmten Kontrollpatienten untersucht. Die Immunoreaktivität wurde durch einen neuen Ansatz quantifiziert, der die detaillierte Bewertung von a-Synuclein-positiven morphologischen Strukturen des enterischen Nervensystems beinhaltet. Alle immungefärbten Schnitte wurden von zwei Untersuchern unabhängig evaluiert. Beide waren bezüglich der Gruppenzuordnung (Gewebeproben von Parkinson-Patienten oder von Kontrollpatienten) verblindet. Zu diesem Zweck wurden von jedem Präparat jeweils 10 zufällig ausgewählte, nicht selektive mikroskopische Bilder/HPF (High-power Fields) in einer 100-fachen Vergrößerung aufgenommen. Insgesamt wurden 1620 HPFs von jedem Untersucher hinsichtlich einer positiven Immunoreaktivität in den drei morphologischen Strukturen „Nervenfasern/Einzelfasern“, „Plexus“ und „Ganglienzellen“ beurteilt. Die Auswertung der Ergebnisse ergab, dass die Immunoreaktivität von α-Synuclein und phosphoryliertem α-Synuclein bei Parkinson-Patienten im Vergleich zu den Kontrollen in fast jeder der untersuchten morphologischen Strukturen signifikant reduziert war. Bis auf die Einzelfasern im Dickdarm bei der immunhistochemischen Färbung mit Antikörper gegen nativem α-Synuclein, sah man durchgehend signifikant weniger Immunoreaktivität in den verschiedenen morphologischen Strukturen im Darmgewebe der Parkinson-Patienten verglichen zu denen der Kontrollpatienten. Der immunhistochemische Nachweis von α-Synuclein und phosphoryliertem α-Synuclein scheint demzufolge ein häufiger und potenziell normaler Befund zu sein. Weder α-Synuclein noch phosphoryliertes α-Synuclein kann daher als molekularer Biomarker der Parkinson-Pathologie betrachtet werden. Die reduzierte intestinale Immunoreaktivität bei Parkinson-Patienten spiegelt eher die Parkinson-bedingte neuronale Degeneration wider. Das Resultat dieser Studie legt nahe, dass der Nachweis von α-Synuclein- und phosphoryliertem α-Synuclein in intestinalen Nervenfasern, dem intestinalen Plexus und intestinalen Ganglienzellen einem Normalbefund entspricht und nicht einer Morbus Parkinson-assoziierten Pathologie. Als bis heute größte Post-mortem-Studie, welche α-Synuclein und phosphoryliertes α-Synuclein in Gewebeproben des Gastrointestinaltraktes von histopathologisch bestätigten Parkinson-Patienten untersuchte, liefert die vorliegende Studie einen wichtigen Beitrag zur Erforschung von α-Synuclein im Gastrointestinaltrakt bei Parkinson-Patienten. Der Nachweis von α-Synuclein in gastrointestinalem Gewebe erscheint für die Diagnostik, insbesondere für die klinische Prädiktion einer Parkinson-Krankheit, ungeeignet.:1. Einführung 1.1. Morbus Parkinson 1.2. Ätiologie und Epidemiologie des idiopathischen Parkinson-Syndroms 1.3. Neuropathologie des idiopathischen Parkinson-Syndroms 1.4. α-Synuclein und phosphoryliertes α-Synuclein 2. Die Bedeutung von (phosphoryliertem) α-Synuclein als molekularer Biomarker bei Morbus Parkinson: aktuelle Studienlage 3. Ableitung der Rationale für die publizierte Studie 3.1. Grundlagen 3.2. Studienziele 3.2.1. Nachweis von α-Synuclein und phosphoryliertem α-Synuclein im Dünndarm und Dickdarm von Parkinson-Patienten und Kontrollpatienten 3.2.2. Vergleich von α-Synuclein und phosphoryliertem α-Synuclein im Dünndarm und Dickdarm von Parkinson-Patienten und Kontrollpatienten 4. Methodik 4.1. Patientenkollektiv 4.2. Gewebeverarbeitung 4.3. Immunhistochemie 4.3.1. α-Synuclein 4.3.2. phosphoryliertes α-Synuclein 4.4. Beurteilung der immunhistochemischen Färbung und Quantifizierung der morphologischen Strukturen 5. Publikationsmanuskript 6. Zusammenfassung der Arbeit 7. Literaturverzeichnis Erklärung über die eigenständige Abfassung der Arbeit Lebenslauf Danksagung

We introduce a novel dynamic model of stem cell and tissue organisation in murine intestinal crypts. Integrating the molecular, cellular and tissue level of description, this model links a broad spectrum of experimental observations encompassing spatially confined cell proliferation, directed cell migration, multiple cell lineage decisions and clonal competition. Using computational simulations we demonstrate that the model is capable of quantitatively describing and predicting the dynamic behaviour of the intestinal tissue during steady state as well as after cell damage and following selective gain or loss of gene function manipulations affecting Wnt- and Notch-signalling. Our simulation results suggest that reversibility and flexibility of cellular decisions are key elements of robust tissue organisation of the intestine. We predict that the tissue should be able to fully recover after complete elimination of cellular subpopulations including subpopulations deemed to be functional stem cells. This challenges current views of tissue stem cell organisation.

Hippo signaling is a tumor suppressive signaling pathway that controls organ size by regulating cellular proliferation, apoptosis, and differentiation during development, regeneration, and homeostasis. The Hippo pathway inhibits transcriptional co-activators and Hippo pathway effectors YAP/TAZ, activation of which is often seen in cancer. Within the adult mammalian intestine, homeostasis of which requires intricate reciprocal interaction between the gut epithelium and adjacent mesenchyme, the Hippo-YAP pathway is crucial for intestinal epithelial homeostasis and regeneration. However, its role in adult mesenchymal homeostasis remains poorly understood. Here, I genetically dissect the role of mesenchymal Hippo-YAP signaling in adult intestinal homeostasis. I find that deletion of core kinases LATS1/2 or YAP activation in mesenchymal progenitor cells, but not terminally differentiated cells, disrupts signaling in the stem cell niche and mesenchymal homeostasis by inducing mesenchymal overgrowth and suppressing smooth muscle actin expression. Furthermore, inhibition of Hippo signaling in Gli1+ mesenchymal progenitors, the main source of Wnt ligands within the stem cell niche, stimulates Wnt ligand production and subsequent epithelial Wnt pathway activation, thereby driving epithelial regeneration following DSS-mediated injury as well as exacerbating APC-mediated tumorigenesis. Altogether, our data reveal a previously underappreciated requirement and the underlying mechanism for stromal Hippo-YAP signaling in adult intestinal homeostasis.