The Tec kinase ITK is activated by TCR stimulation and also required for TCR downstream signaling. Previous studies have reported differential roles of ITK and another Tec family kinase RLK in CD4+ TH differentiation and effector function. However, these findings are confounded by the complex T cell developmental defects in Itk-/- mice. Furthermore, the function of ITK in tissue-resident T cells in the intestine and anti-viral immune response to a persistent infection has not been studied previously. In addition to T cells, recent studies have indicated an expression of ITK in ILC2, but not in other ILC subsets. Yet, the role of ITK in ILC2 has not been characterized. Here, I have examined the role of ITK and RLK in CD4+ TH subsets using a small molecule inhibitor PRN694. I found that PRN694 impaired TH1 differentiation in vitro, and PRN694 administration prevented TH1-mediated colitis progression in vivo. In an MHV68 infection model, Itk-/- mice failed to control viral replication in the intestine, while gut-homing of CD8+ T cells was greatly impaired. Finally, I found that ILC2 number was markedly reduced in the intestine of Itk-/- mice. Gut-specific defect of Itk-/- ILC2 is associated with a low availability of IL-2 in the intestine of Itk-/- mice. Collectively, these data suggest that ITK is important in T cell migration to the intestine and ILC2 homeostasis in the intestine, thereby contributing to the protective response to a latent virus and intestinal tissue homeostasis.

Alcohol is a commonly consumed beverage, a drug of abuse and an important molecule affecting nearly every organ-system in the body. This project seeks to investigate the interplay between alcohol’s effects on critical organ-systems making up gut-liver-brain axis. Alcohol initially interacts with the gastrointestinal tract. Our research describes the alterations seen in intestinal microbiota following alcohol consumption in an acute-on-chronic model of alcoholic hepatitis and indicates that reducing intestinal bacteria using antibiotics protects from alcohol-induced intestinal cytokine expression, alcoholic liver disease and from inflammation in the brain. Alcohol-induced liver injury can occur due to direct hepatocyte metabolic dysregulation and from leakage of bacterial products from the intestine that initiates an immune response. Here, we will highlight the importance of this immune response, focusing on the role of infiltrating immune cells in human patients with alcoholic hepatitis and alcoholic cirrhosis. Using a small molecule inhibitor of CCR2/CCR5 chemokine receptor signaling in mice, we can protect the liver from damage and alcohol-induced inflammation. In the brain, we observe that chronic alcohol leads to the infiltration of macrophages in a region-specific manner. CCR2/CCR5 inhibition reduced macrophage infiltration, alcohol-induced inflammation and microglial changes. We also report that chronic alcohol shifts excitatory/inhibitory synapses in the hippocampus, possibly through complement-mediated remodeling. Finally, we show that anti-inflammasome inhibitors altered behavior by reducing alcohol consumption in female mice. Together, these data advance our understanding of the gut-liver-brain axis in alcoholism and suggest novel avenues of therapeutic intervention to inhibit organ pathology associated with alcohol consumption and reduce drinking.

The intestinal epithelium represents one of the first lines of defense against pathogenic bacteria. Immune regulation at this critical barrier is necessary to maintain organismal fitness, and mis-regulation here has been linked to numerous debilitating diseases. Functional relationships between the nervous system and immune system have been found to be critical in the proper coordination of immune defenses at barrier surfaces, however the precise mechanisms underlying theses interactions remains unclear. Through conducting a forward genetic screen utilizing the model organism Caenorhabditis elegans, we uncovered a surprising requirement for the olfactory neuron gene olrn-1 in the regulation of intestinal epithelial immunity. During nematode development, olrn-1 is required to program the expression of odorant receptors in the AWC olfactory neuron pair. Here, we show that olrn-1 also functions in AWC neurons in the cell non-autonomous suppression of the canonical p38 MAPK PMK-1 immune pathway in the intestine. Low activity of OLRN-1, which activates the p38 MAPK signaling cassette in AWC neurons during larval development, also de-represses the p38 MAPK PMK-1 pathway in the intestine to promote immune effector transcription, increased clearance of an intestinal pathogen and resistance to bacterial infection. However, derepression of the p38 MAPK PMK-1 pathway also results in severe developmental and reproductive defects, demonstrating the critical function of OLRN-1 to both prime C. elegans intestinal epithelial cells for the induction of anti-pathogen responses, and to limit the deleterious effects of immune hyper-activation. These data reveal an unexpected connection between olfactory receptor development and innate immunity, as well as demonstrate how neuronal regulation of immune responses within the intestinal epithelium is critical for both reproductive and developmental fitness.

No description available.

Es wird vermutet, dass das Zusammenspiel von NF-κB-Signalen und Autophagie die Entzündung in verschiedenen zellulären Kontexten und als Reaktion auf unterschiedliche Stimuli reguliert. Der molekulare Mechanismus, durch den diese beiden Signalwege bei der Regulierung der Entzündungsreaktion zusammenwirken, ist jedoch noch nicht bekannt. Mithilfe biochemischer Analysen und bildgebender Verfahren haben wir zum ersten Mal die Interaktion zwischen dem autophagischen Marker LC3 und der NF-κB/p65-Untereinheit als Reaktion auf verschiedene Stressbedingungen charakterisiert. Wir konnten zeigen, dass die Anhäufung von LC3 im Zellkern nach der NF-κB-Aktivierung mit p65 interagiert, was durch die Ubiquitinierung des p65-Proteins gefördert und durch den Cargo-Rezeptor p62 erkannt wird. Zusammengenommen weisen diese Daten auf eine neue Rolle von p62 beim Transport von im Kern ubiquitiniertem p65 zu Autophagosomen hin, wo es abgebaut wird, um die entzündungsbedingte NF-κB-Hyperaktivierung zu kontrollieren. Diese Erkenntnisse sind wichtig für die Entwicklung neuer therapeutischer Strategien gegen Krankheiten, die mit einer gestörten Autophagie und einer konstitutiven NF-κB-Aktivität einhergehen. Die NF-κB-Signalübertragung spielt nicht nur eine entscheidende Rolle bei Entzündungen und der Tumorbildung, sondern ist auch für Entwicklungsprozesse wichtig. Durch die Etablierung von 3D-Organoid-Kulturen aus dem Dünndarm und unter Verwendung verschiedener Mauslinien weisen wir im zweiten Teil der Arbeit nach, dass NF-κB eine wichtige Funktion bei der Zelldifferenzierung und der Erhaltung von Stammzellen in vivo und ex-vivo spielt. Wir konnten zeigen, dass die Proliferation und das Absterben von Darmepithelzellen (IEC) bei Mäusen mit ubiquitärer Unterdrückung der NF-κB-Aktivität unverändert sind, während die Zahl der Becherzellen auf Kosten der Paneth-Zellen zunimmt. Zusammenfassend lässt sich sagen, dass unsere Ergebnisse eine neue IEC-immanente Rolle von NF-κB bei Entscheidungen über das Zellschicksal und die Differenzierung aufzeigen, die über die Regulierung der Wnt-Signale und der Sox9-Expression stromabwärts von NF-κB erfolgt. Die hier beschriebenen Erkenntnisse verbessern unser Verständnis der NF-κB-Funktionen in der Stammzellbiologie, die, wenn sie dereguliert sind, auch Auswirkungen auf die Entzündung des Darms und die Tumorentstehung haben. / The interplay between NF-κB signaling and autophagy has been suggested to regulate inflammation in different cellular contexts and in response to different stimuli. However, the molecular mechanism by which these two pathways interact to regulate the inflammatory response remains elusive. By using biochemical analysis and imaging techniques, we characterized for the first time the interaction of autophagic marker LC3 and NF-κB/p65 subunit in response to different stress conditions. We demonstrated that the accumulation of LC3 within the nucleus interacts with p65 following NF-κB activation, which is promoted by ubiquitination of p65 protein and recognized by the cargo receptor p62. Together, these data identify a novel role for p62 in trafficking nuclear-ubiquitinated p65 to autophagosomes for degradation to control inflammation-driven NF-κB hyperactivation. These findings are important for developing novel therapeutic strategies against diseases involving defective autophagy and constitutive NF-κB activity. In addition to its critical role in inflammation and tumor formation, NF-κB signaling is essential in developmental processes. Establishing 3D organoid culture from the small intestine and using different mouse lines, we prove in the second part of the thesis that NF-kB plays an important function in cell differentiation and stem cell maintenance in vivo and in ex-vivo. We demonstrated that while intestinal epithelial cell (IEC) proliferation and death are unaltered in mice with ubiquitous suppression of NF-κB activity, goblet cell numbers increase at the expense of Paneth cells. In summary, our results revealed a novel IEC-intrinsic role of NF-κB in cell fate decisions and differentiation which occur via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The findings described here improve our understanding of NF-κB functions in stem cell biology which, when deregulated, also have an impact on intestinal inflammation and tumorigenesis.

Organoids retain the morphological and molecular patterns of their tissue of origin, are self-organizing, relatively simple to handle and accessible to genetic engineering. Thus, they represent an optimal tool for studying the mechanisms of tissue maintenance and aging. Long-term expansion under standard growth conditions, however, is accompanied by changes in the growth pattern and kinetics. As a potential explanation of these alterations, epigenetic drifts in organoid culture have been suggested. Here, we studied histone tri-methylation at lysine 4 (H3K4me3) and 27 (H3K27me3) and transcriptome profiles of intestinal organoids derived from mismatch repair (MMR)-deficient and control mice and cultured for 3 and 20 weeks and compared them with data on their tissue of origin. We found that, besides the expected changes in short-term culture, the organoids showed profound changes in their epigenomes also during the long-term culture. The most prominent were epigenetic gene activation by H3K4me3 recruitment to previously unmodified genes and by H3K27me3 loss from originally bivalent genes. We showed that a long-term culture is linked to broad transcriptional changes that indicate an ongoing maturation and metabolic adaptation process. This process was disturbed in MMR-deficient mice, resulting in endoplasmic reticulum (ER) stress and Wnt activation. Our results can be explained in terms of a mathematical model assuming that epigenetic changes during a long-term culture involve DNA demethylation that ceases if the metabolic adaptation is disturbed.

<p>Parasite infections around the world are a huge economic burden and decrease the quality of life for many people. Probiotic bacteria are being investigated as a possible treatment for many enteric issues due to their beneficial effects by altering the immune system. Goblet cells are the main source of mucins in the gut, and play an important role in host defense. Alterations in goblet cells and mucin have been implicated in a number of gastrointestinal (GI) diseases and infections. The aim of this study is to develop a probiotic based strategy to modulate goblet cell function in relation to host defense in enteric infection. Utilizing a murine model of parasite infection, <em>Trichuris muris</em>,<em> </em>we examined the effect of daily administration with probiotic <em>Lactobacillus reuteri</em> in different strains of mice and investigation of goblet cell alterations, immune and inflammatory responses in gut, and host defense mechanisms.</p> <p>Treatment with<strong> </strong>live <em>L. reuteri</em> significantly enhanced worm expulsion in resistant C57BL/6 mice and this was associated with significant increase in goblet cells numbers and an increase in IL-10. This lead to investigation of the probiotic effects in IL-10 knock out (KO) and Muc2 KO mice during the infection. There was no difference of worm burden or goblet cell amounts in infected IL-10 KO mice infected treated with probiotic or medium. In infected Muc2 KO mice treated with <em>L. reuteri</em>, there was an earlier increase of goblet cells, and a corresponding decrease in worm numbers. Finally, assessment of this probiotic in susceptible ARK mice revealed no alterations in worm burden, but the treatment prevented the increase in IFN-γ and IL-1β and significantly increased goblet cell numbers.</p> <p>These data demonstrate that altering the flora with probiotic <em>L. reuteri</em> treatment can modulate intestinal goblet cell biology and immune responses in gut, and promote worm expulsion, possibly through an IL-10 mediated mechanism. The increases in goblet cell numbers may also play a role in the early expulsion of the parasite. In addition to enhancing our understanding on the beneficial effect of probiotics in host defense in enteric infection, this research provides new information on gut function in the context of goblet cells and mucins.</p> / Master of Science (MSc)

En lien avec l’augmentation constante de l’obésité, de plus en plus de personnes sont atteintes de résistance à l’insuline ou de diabète de type 2. Ce projet doctoral s’est surtout intéressé à l’une des conséquences majeures des pathologies cardiométaboliques, soit la dyslipidémie diabétique. À cet égard, les gens présentant une résistance à l’insuline ou un diabète de type 2 sont plus à risque de développer des perturbations lipidiques caractérisées essentiellement par des taux élevés de triglycérides et de LDL-cholestérol ainsi que de concentrations restreintes en HDL-cholestérol dans la circulation. Les risques de maladies cardiovasculaires sont ainsi plus élevés chez ces patients. Classiquement, trois organes sont connus pour développer l’insulino-résistance : le muscle, le tissu adipeux et le foie. Néanmoins, certaines évidences scientifiques commencent également à pointer du doigt l’intestin, un organe critique dans la régulation du métabolisme des lipides postprandiaux, et qui pourrait, conséquemment, avoir un impact important dans l’apparition de la dyslipidémie diabétique. De façon très intéressante, des peptides produits par l’intestin, notamment le GLP-1 (glucagon-like peptide-1), ont déjà démontré leur potentiel thérapeutique quant à l’amélioration du statut diabétique et leur rôle dans le métabolisme intestinal lipoprotéinique. Une autre évidence est apportée par la chirurgie bariatrique qui a un effet positif, durable et radical sur la perte pondérale, le contrôle métabolique et la réduction des comorbidités du diabète de type 2, suite à la dérivation bilio-intestinale. Les objectifs centraux du présent programme scientifique consistent donc à déterminer le rôle de l’intestin dans (i) l’homéostasie lipidique/lipoprotéinique en réponse à des concentrations élevées de glucose (à l’instar du diabète) et à des peptides gastro-intestinaux tels que le PYY (peptide YY); (ii) la coordination du métabolisme en disposant de l’AMPK (AMP-activated protein kinase) comme senseur incontournable permettant l’ajustement précis des besoins et disponibilités énergétiques cellulaires; et (iii) l’ajustement de sa capacité d’absorption des graisses alimentaires en fonction du gain ou de la perte de sa sensibilité à l’insuline démontrée dans les spécimens intestinaux humains prélevés durant la chirurgie bariatrique. Dans le but de confirmer le rôle de l’intestin dans la dyslipidémie diabétique, nous avons tout d’abord utilisé le modèle cellulaire intestinal Caco-2/15. Ces cellules ont permis de démontrer qu’en présence de hautes concentrations de glucose en basolatéral, telle qu’en condition diabétique, l’intestin absorbe davantage de cholestérol provenant de la lumière intestinale par l’intermédiaire du transporteur NPC1L1 (Niemann Pick C1-like 1). L’utilisation de l’ezetimibe, un inhibiteur du NPC1L1, a permis de contrecarrer cette augmentation de l’expression de NPC1L1 tout comme l’élévation de l’absorption du cholestérol, prouvant ainsi que le NPC1L1 est bel et bien responsable de cet effet. D’autre part, des travaux antérieurs avaient identifié certains indices quant à un rôle potentiel du peptide intestinal PYY au niveau du métabolisme des lipides intestinaux. Toutefois, aucune étude n’avait encore traité cet aspect systématiquement. Pour établir définitivement l’aptitude du PYY à moduler le transport et le métabolisme lipidique dans l’intestin, nous avons utilisé les cellules Caco-2/15. Notre étude a permis de constater que le PYY incubé du côté apical est capable de réduire significativement l’absorption du cholestérol et le transporteur NPC1L1. Puisque le rôle de l'AMPK dans l'intestin demeure inexploré, il est important non seulement de définir sa structure moléculaire, sa régulation et sa fonction dans le métabolisme des lipides, mais aussi d'examiner l'impact de l’insulino-résistance et du diabète de type 2 (DT2) sur son statut et son mode d’action gastro-intestinal. En employant les cellules Caco-2/15, nous avons été capables de montrer (i) la présence de toutes les sous-unités AMPK (α1/α2/β1/β2/γ1/γ2/γ3) avec une différence marquée dans leur abondance et une prédominance de l’AMPKα1 et la prévalence de l’hétérotrimère α1/β2/γ1; (ii) l’activation de l’AMPK par la metformine et l’AICAR, résultant ainsi en une phosphorylation accrue de l’enzyme acétylCoA carboxylase (ACC) et sans influence sur l'HMG-CoA réductase; (iii) la modulation négative de l’AMPK par le composé C et des concentrations de glucose élevées avec des répercussions sur la phosphorylation de l’ACC. D’autre part, l’administration de metformine au Psammomys obesus, un modèle animal de diabète et de syndrome métabolique, a conduit à (i) une régulation positive de l’AMPK intestinale (phosphorylation de l’AMPKα-Thr172); (ii) la réduction de l'activité ACC; (iii) l’augmentation de l’expression génique et protéique de CPT1, supportant une stimulation de la β-oxydation; (iv) une tendance à la hausse de la sensibilité à l'insuline représentée par l’induction de la phosphorylation d'Akt et l’inactivation de la phosphorylation de p38; et (v) l’abaissement de la formation des chylomicrons ce qui conduit à la diminution de la dyslipidémie diabétique. Ces données suggèrent que l'AMPK remplit des fonctions clés dans les processus métaboliques de l'intestin grêle. La preuve flagrante de l’implication de l’intestin dans les événements cardiométaboliques a été obtenue par l’examen des spécimens intestinaux obtenus de sujets obèses, suite à une chirurgie bariatrique. L’exploration intestinale nous a permis de constater chez ceux avec un indice HOMA élevé (marqueur d’insulinorésistance) (i) des défauts de signalisation de l'insuline comme en témoigne la phosphorylation réduite d'Akt et la phosphorylation élevée de p38 MAPK; (ii) la présence du stress oxydatif et de marqueurs de l'inflammation; (iii) la stimulation de la lipogenèse et de la production des lipoprotéines riches en triglycérides avec l’implication des protéines clés FABP, MTP et apo B-48. En conclusion, l'intestin grêle peut être classé comme un tissu insulino-sensible et répondant à plusieurs stimuli nutritionnels et hormonaux. Son dérèglement peut être déclenché par le stress oxydatif et l'inflammation, ce qui conduit à l'amplification de la lipogenèse et la synthèse des lipoprotéines, contribuant ainsi à la dyslipidémie athérogène chez les patients atteints du syndrome métabolique et de diabète de type 2. / In relation with the constant increase in obesity, more and more people suffer from insulin resistance and type 2 diabetes (DT2). This doctoral research program especially emphasizes lipid disorders, one of the major consequences of cardiometabolic diseases. In this respect, people with insulin resistance or DT2 are at higher risk of developing lipid disturbances characterized mainly by high levels of triglycerides and LDL-cholesterol concentrations and HDL cholesterol in the blood circulation. The risks of cardiovascular disease are higher in these patients. Classically, three organs are known to develop insulin resistance: muscle, adipose tissue and liver. Nevertheless, important studies begin to point out the small intestine as a major organ in the regulation of postprandial lipids, which may have a significant impact on the development of diabetic dyslipidemia. In addition, the intestine produces peptides, including GLP-1 (glucagon-like peptide-1), that have already demonstrated their therapeutic potential with regard to diabetic status and intestinal lipoprotein metabolism. Further evidence is also is provided by the advent of bariatric surgery that has a positive effect on radical and sustainable weight loss, metabolic control and reduction of comorbidities of DT2, following biliopancreatic diversion. The central objectives of this scientific program are therefore to determine the role of the intestine in (i) lipid/ lipoprotein homeostasis in response to high concentrations of glucose (mimicking diabetes) and to gastrointestinal peptides such as PYY; (ii) the coordination of metabolism by involving AMPK (AMP-activated protein kinase) as an essential sensor for fine tuning of cellular energy needs; and (iii) adjusting absorption capacity of dietary fat in the gain or loss of insulin sensitivity demonstrated in intestinal specimens collected during bariatric surgery. In order to confirm the role of the intestine in diabetic dyslipidemia, we first used the intestinal Caco-2/15 cell model. The use of this epithelial cell line has shown a marked stimulation of cholesterol uptake via the transporter NPC1L1 (Niemann-Pick C1-like 1) in the presence of high glucose concentrations (as is the case in diabetic conditions) in basolateral compartment (compared to apical). The use of ezetimibe, an inhibitor of NPC1L1, helped to counteract this elevation of cholesterol absorption, thus proving that NPC1L1 is indeed behind this effect. If previous reports have identified some clues as to the potential role of intestinal PYY (peptide YY) in lipid metabolism disorders, no study has yet addressed this issue systematically. To definitively establish the ability of PYY to modulate lipid transport and metabolism in the intestine, we have used Caco-2/15 cells. Our recent investigation has shown that PYY (administered in the apical compartment) is able to significantly reduce cholesterol absorption via NPC1L1 transporter. Since the role of AMPK in the intestine remains unexplored, it is important to define not only its molecular structure, regulation and function in lipid metabolism, but also its impact on insulin resistance and T2D on its status and mode of action in the gastrointestinal tract. Using Caco-2/15 cells, we have been able to show (i) the presence of all AMPK subunits (α1/α2/β1/β2/γ1/γ2/γ3) with a marked difference in their abundance, but with a predominance of AMPKα1 and the prevalence of α1/β2/γ1 heterotrimer; (ii) the activation of AMPK by metformin and AICAR, resulting in increased phosphorylation of the downstream target acetylCoA carboxylases (ACC) without no influence on HMG-CoA reductase; (iii) the negative modulation of AMPK by compound C and glucose concentrations with high impact on ACC phosphorylation. On the other hand, administration of metformin to Psammomys obesus with insulin resistance and T2D led to (a) an upregulation of intestinal AMPK signaling pathway essentially typified by ascending AMPKα-Thr172 phosphorylation; (b) a reduction in ACC activity; (c) an elevation in the gene and protein expression of CPT1, supporting a stimulation of β-oxidation; (d) a trend of increase in insulin sensitivity portrayed by augmentation of Akt and GSK3β phosphorylation; (e) an inactivation of the stress-responsive p38-MAPK and /ERK1/2 exemplified by their phosphorylation lessening; and (f) a decrease in diabetic dyslipidemia following lowering of intracellular events that govern lipoprotein assembly. Therefore these data suggest that AMPK fulfills key functions in metabolic processes in the small intestine. The clear evidence for the involvement of the gut in cardiometabolic events has been obtained through the scrutiny of intestinal specimens obtained from obese subjects after bariatric surgery. Intestine of insulin-resistant subjects shows defects in insulin signaling as demonstrated by reduced Akt phosphorylation but increased p38 MAPK phosphorylation. These defects were accompanied with increased oxidative stress and inflammation markers in intestine of insulin-resistant subjects. Enhanced de novo lipogenesis rate and apo B-48 biogenesis along with increased triglyceride-rich lipoprotein production was also observed in the intestine of insulin-resistant subjects. Concomitantly, fatty acid binding proteins (FABP) and microsomal transfer protein (MTP) expression was increased in the intestine of insulin-resistant subjects. In conclusion, the small intestine may be classified as an insulin-sensitive tissue. Its deregulation, possibly triggered by oxidative stress and inflammation, may lead to amplification of lipogenesis and lipoprotein synthesis and may therefore represent a key mechanism for atherogenic dyslipidemia in patients with metabolic syndrome and T2D.

La stéatose hépatique et la détérioration du profil lipidique plasmatique sont des pathologies métaboliques favorisées par la carence œstrogénique post-ménopausique. Cependant les mécanismes à la base de ces pathologies n’ont été que très peu étudiés. Le but de cette thèse a été d’investiguer les mécanismes moléculaires possibles à l’origine de l’hypercholestérolémie et de l’accumulation des lipides (triglycérides : TG et cholestérol) dans le foie en utilisant un modèle animal de la ménopause, la rate Sprague Dawley ovariectomisée (Ovx). Nous avons également examiné si le changement des habitudes de vie comme la pratique de l’exercice physique pouvait prévenir ou corriger les modifications induites par l’Ovx. Enfin, rosuvastatine (statine) a été utilisée comme thérapie pharmacologique de l’hypercholestérolémie dans le but de comprendre son effet au niveau moléculaire chez la rate Ovx. L’objectif de la première étude était de déterminer comment l’Ovx peut affecter les niveaux de TG et de cholestérol dans le foie des rates nourries avec une diète riche en lipides (HF : 42% gras). Les rates ont été soumises à la diète HF ou normale pendant 6 semaines avant d’être Ovx ou Sham (ovariectomie simulée), puis maintenues aux mêmes conditions diététiques pour 6 autres semaines. L’Ovx a provoqué une accumulation de TG dans le foie, mais pas la diète HF seule. Cependant, lorsque l’Ovx était combinée à la diète HF, l’accumulation des TG était beaucoup plus importante comparé à ce qui était observé chez les rates Ovx soumises à la diète normale. L’expression génique (ARNm) de CPT1 (Carnitine palmitoyltransferase 1), PGC1α (Peroxisome proliferator-activated receptor gamma, coactivator 1) et PPARα (Peroxysome proliferetor activated receptor alpha) intervenant dans l’oxydation des acides gras dans le foie était augmentée par la diète HF (p ˂ 0.001; p ˂ 0.01; p ˂ 0.05 respectivement) ; mais atténuée (p ˂ 0.05; p ˂ 0.05; p ˂ 0.07 respectivement) lorsque les rates ont été Ovx, favorisant ainsi l’accumulation des TG dans le foie. La combinaison de la diète HF à l’Ovx a également provoqué une hypercholestérolémie et une accumulation de cholestérol dans le foie malgré la diminution de l’expression de la HMGCoA-r (3-hydroxy-3-methylglutaryl-CoA reductase), enzyme clé de la synthèse du cholestérol. Ceci était associé à l’inhibition de l’expression génique de CYP7a1 (Cytochrome P450, family 7, subfamily a, polypeptide 1), suggérant une diminution de la synthèse des acides biliaires. Ayant constaté dans la première étude que l’Ovx élevait les niveaux de cholestérol hépatique et plasmatique, nous nous sommes fixés comme objectif dans la deuxième étude d’évaluer les effets de l’Ovx sur l’expression génique des transporteurs et enzymes responsables du métabolisme du cholestérol et des acides biliaires dans le foie et l’intestin, et de vérifier si l’exercice sur tapis roulant pouvait prévenir ou corriger les changements causés par l’Ovx. L’hypercholestérolémie constatée chez les rates Ovx comparativement aux Sham était accompagnée de la diminution de l’expression génique des récepteurs des LDL (R-LDL), des résidus de lipoprotéines (LRP1), de SREBP-2 (Sterol regulatory element binding protein 2) et de PCSK9 (Proprotein convertase subtilisin/kexin type 9) dans le foie, suggérant une défaillance dans la clairance des lipoprotéines plasmatiques. L’Ovx a aussi inhibé l’expression génique de la MTP (Microsomal triglyceride transfer protein) et stimulé celle de SR-B1 (Scavenger receptor class B, member 1); mais aucun changement n’a été observé avec CYP7a1. Ces changements moléculaires pourraient par conséquent favoriser l’accumulation de cholestérol dans le foie. L’exercice physique n’a pas corrigé les modifications causées par l’Ovx sur l’expression génique de ces molécules au niveau hépatique à l’exception de SREBP-2. Par contre, au niveau intestinal (iléum), l’exercice sur tapis roulant a inhibé l’expression génique des marqueurs moléculaires intervenant dans l’absorption des acides biliaires (OSTα/β, FXR, RXRα, Fgf15) et du cholestérol (LXRα, NCP1L1) au niveau de l’iléum chez les rates Sham entraînées. Ces adaptations pourraient prévenir le développement de l’hypercholestérolémie protégeant en partie contre la survenue de l’athérosclérose. Au vue des effets délétères (hypercholestérolémie et diminution de l’expression du R-LDL, PCSK9, LRP1, SREBP-2 et HMGCOA-r dans le foie) causés par l’Ovx sur le métabolisme du cholestérol constatés dans l’étude 2, la 3ième étude a été conçue pour évaluer l’efficacité de rosuvastatine (Ros) sur l’expression génique de ces marqueurs moléculaires chez les rates Ovx sédentaires ou soumises à l’entraînement volontaire. Ros a été administrée aux rates Ovx pendant 21 jours par voie sous-cutanée à la dose de 5mg/kg/j à partir de la 9ième semaine après l’Ovx. Ros n’a pas diminué la concentration plasmatique de LDL-C et de TC chez les rates Ovx. Par contre, Ros a stimulé (P ˂ 0.05) l’expression génique de PCSK9, SREBP-2, LRP1, HMGCoA-r et ACAT2 (Acyl-CoA cholesterol acyltransferase) mais pas significativement (P = 0.3) celle du R-LDL dans le foie des rates Ovx sédentaires et entraînées. Ros n’a pas réduit la concentration plasmatique de LDL-C probablement à cause de l’induction plus importante de PCSK9 par rapport au R-LDL. Cependant, la stimulation de LRP1 par Ros protège partiellement contre la survenue des maladies cardiovasculaires. En conclusion, les études de cette thèse indiquent que la baisse du niveau des œstrogènes entraîne des changements radicaux du métabolisme hépatique des TG et du cholestérol provoqués par des altérations de l’expression des gènes clés des voies métaboliques associées. / Hepatic steatosis and plasma lipid profile deterioration are metabolic diseases favored by post-menopausal estrogen deficiency. However, mechanisms underlying these diseases have not been systematically adressed. The aim of this thesis was to investigate molecular mechanisms causing hypercholesterolemia and lipids (triglycerides: TG and cholesterol) accumulation in the liver using animal model of menopause, the ovariectomized (Ovx) Sprague Dawley rat. We also examined whether lifestyle modifications such as physical activity can prevent or correct changes induced by Ovx. Finally, rosuvastatin (statine) was used as a pharmacological therapy of hypercholesterolemia in order to understand its effect at the molecular level in Ovx rats. The first study was designed to determine how the Ovx may affect levels of TG and cholesterol in the liver of rats fed a high-fat diet (HF: 42% fat). Rats were submitted to a HF or a normal diet for 6 weeks prior to Ovx or being sham operated, and then kept on the same diets for another 6 weeks. The Ovx increased liver TG content, but not the HF diet alone. However, the combination of Ovx and HF diet resulted in a greater liver TG accumulation than that observed in Ovx submitted to normal diet. The mRNA levels of CPT-1, PGC1 and PPARα involved in liver lipid oxidation significantly increased in rats fed the HF diet (p ˂ 0.001; p ˂ 0.01; p ˂ 0.05 respectively); but this increase was substantially less if HF fed rats were Ovx (p ˂ 0.05; p ˂ 0.05; p ˂ 0.07 respectively), thus favouring TG accumulation in the liver. The combination of HF diet and Ovx also induced hypercholesterolemia and an increase in liver total cholesterol content, in spite of the reduction of liver HMGCoA-r gene expression, the key enzyme for cholesterol synthesis. This was also associated with a decrease of liver CYP7a1 gene expression, suggesting a reduction in bile acids synthesis. Having found in the first study that the Ovx increases liver and plasma cholesterol levels, we aimed in the second study at determining the effects of Ovx on gene expression of hepatic and intestinal transporters and enzymes involved in cholesterol and bile acids metabolism; and to verify whether treadmill exercise could prevent or correct changes induced by Ovx. The Ovx resulted in hypercholesterolemia associated with a reduction in gene expression of hepatic low-density lipoprotein receptor (LDL-R), lipoprotein remnants receptor (LRP1), SREBP-2 and PCSK9, suggesting a failure in the clearance of plasma lipoproteins particles. The Ovx also inhibited the expression of MTP and stimulated that of SR-B1 in the liver, but no change was observed with CYP7a1. These molecular changes might, therefore, favor cholesterol accumulation in the liver. Exercise training did not correct the deleterious effects caused by Ovx on gene expression of these molecular markers in the liver with the exception of SREBP-2. However, in the intestine (ileum) treadmill exercise reduced gene expression of molecular markers involved in the absorption of bile acids (OSTα/β, FXR, RXRα, Fgf15) and cholesterol (LXRα, NCP1L1) in Sham trained rats compared to sedentary rats. This could prevent the development of cholestasis and hypercholesterolemia protecting partially against the onset of atherosclerosis. In view of the deleterious effects (hypercholesterolemia and decreased in gene expression of LDL-R, PCSK9, LRP1, SREBP-2 and HMGCoA-r in the liver) caused by Ovx on cholesterol metabolism observed in the second study, the 3rd study was designed to test the effect of rosuvastatin (Ros) on gene expression of these molecular markers in Ovx sedentary rats or in Ovx rats submitted to voluntary training. Ros was injected to Ovx rats subcutaneously at dose of 5mg/kg/day during 21 days from the ninth week after ovariectomy. Ros failed to decrease plasma LDL-C and TC in Ovx rats. In contrast, Ros increased (P ˂ 0.05) PCSK9, SREBP-2, LRP1, HMGCoA-r and ACAT2 but not significantly (P ˂ 0.3) LDL-R mRNA in the Ovx sedentary and trained rat liver. Ros failed to decrease plasma LDL-C in Ovx rats probably because of a stronger induction of PCSK9 than LDL-R gene expression. However by increasing LRP1 expression, Ros could decrease circulating lipoprotein remnants and, therefore, protects partially against the onset of cardiovascular diseases. In conclusion, the studies of this thesis indicate that the decrease of ovarian estrogen levels causes radical changes in hepatic TG and cholesterol metabolism caused by alterations in the expression of key genes associated with metabolic pathways.

Colorectal cancer (CRC) arises in part from the dysregulation of cellular proliferation, associated with the canonical Wnt pathway, and differentiation, effected by the Notch signalling network. In this thesis, we develop a mathematical model of ordinary differential equations (ODEs) for the coupled interaction of the Notch and Wnt pathways in cells of the human intestinal epithelium. Our central aim is to understand the role of such crosstalk in the genesis and treatment of CRC. An embedding of this model in cells of a simulated colonic tissue enables computational exploration of the cell fate response to spatially inhomogeneous growth cues in the healthy intestinal epithelium. We also examine an alternative, rule-based model from the literature, which employs a simple binary approach to pathway activity, in which the Notch and Wnt pathways are constitutively on or off. Comparison of the two models demonstrates the substantial advantages of the equation-based paradigm, through its delivery of stable and robust cell fate patterning, and its versatility for exploring the multiscale consequences of a variety of subcellular phenomena. Extension of the ODE-based model to include mutant cells facilitates the study of Notch-mediated therapeutic approaches to CRC. We find a marked synergy between the application of &gamma;-secretase inhibitors and Hath1 stabilisers in the treatment of early-stage intestinal polyps. This combined treatment is an efficient means of inducing mitotic arrest in the cell population of the intestinal epithelium through enforced conversion to a secretory phenotype and is highlighted as a viable route for further theoretical, experimental and clinical study.