Fibroblast Growth Factor 19 (FGF19 chez l’humain ; FGF15 chez la souris) est un régulateur central du métabolisme hépatique. Cette molécule a un impact important au niveau de la différentiation neurologique et de l’oreille interne au stade foetal. À l’âge adulte, le patron d’expression est restreint au système gastro-intestinal. Contrairement aux autres membres de la superfamille des FGFs, FGF19/15 agit de manière endocrine car il n’est pas retenu par la matrice extracellulaire et peut rejoindre la circulation sanguine. L’expression de FGF19/15 est induite par les acides biliaires au niveau de l’intestin grêle, plus précisément l’iléon. Les acides biliaires lient le récepteur nucléaire Farnesoid-XReceptor (FXR) qui peut ensuite s’hétérodimériser avec Retinoid-X-Receptor (RXR) pour se lier au promoteur de FGF19/15, ce qui enclenche son expression. Une fois dans le sang, l’hormone rejoint le foie et son action est médiée par le complexe de récepteur Fibroblast Growth Factor Receptor 4 (FGFR4) et β-Klotho (BKL). Une fois les récepteurs activés, FGF19/15 module la glycémie en inhibant la néoglucogenèse hépatique et en activant la synthèse du glycogène, le flux protéique en activant eIF4B et la lipémie en inhibant les enzymes clefs de la lipogénèse. FGF19/15 joue aussi un rôle majeur au niveau du métabolisme biliaire. Ce dernier permet de réduire la production d’acide biliaire en inhibant la Cholesterol 7-α oxygenase (CYP7A1). Les travaux présentés dans ce mémoire portent dans un premier temps sur la caractérisation des conséquences amenées par l’infection sur l’axe endocrine FGF15/FGFR4. Un premier manuscrit traite de l’expression des différents gênes clefs du système et de leur perte lors d’une infection à Salmonella typhimurium, l’agent pathogène causant la fièvre typhoïde chez la souris, et des conséquences sur l’homéostasie biliaire. Il est possible de remarquer une perte de l’expression de FGF15 au niveau de l’intestin et de FGFR4 et β-Klotho au niveau du foie, en plus de plusieurs transporteurs responsables d’amener différents composants clefs de la bile à la vésicule biliaire ou à la circulation sanguine. Le deuxième volet du travail consistait à déterminer le mécanisme derrière la perte du complexe de récepteurs FGFR4/β-Klotho. Les résultats préliminaires démontrent que la perte de β-Klotho semble être médiée seulement par le processus inflammatoire normal et la perte de FGFR4 semble être Salmonella dépendante, par le biais de la voie c- Jun N-terminal kinases (JNK) et le facteur de transcription Hepatic Nuclear Factor 1 alpha (HNF1α)

Secretion of appropriate amounts of insulin from pancreatic β-cells is crucial for glucose homeostasis. The β-cells release insulin in response to glucose and other nutrients, hormones and neurotransmitters, which trigger intracellular signaling cascades, that result in exocytotic fusion of insulin-containing vesicles with the plasma membrane. Increases of the intracellular concentration of calcium ions ([Ca2+]i) trigger exocytosis, whereas the messenger cyclic adenosine monophosphate (cAMP) amplifies various steps of the secretion process. The protein Epac2 mediates some effects of cAMP, but little is known about its regulation in β-cells. In this study, the spatio-temporal dynamics of Epac2 was investigated in insulin-secreting MIN6-cells and primary β-cells using various cell signaling biosensors and live-cell fluorescence microscopy approaches. Increases in the cAMP concentration triggered translocation of Epac2 from the cytoplasm to the plasma membrane. Oscillations of cAMP induced by glucose and the insulin-releasing hormone GLP-1 were associated with cyclic translocation of Epac2. Analyses of Epac2 mutants showed that the high-affinity cyclic nucleotide-binding domain and Ras-association domains were crucial for the translocation, whereas neither the DEP domain, nor the low-affinity cAMP-binding domain were required for membrane binding. However, the latter domain targeted Epac2 to insulin granules at the plasma membrane, which promoted their priming for exocytosis. Depolarization-induced elevations of [Ca2+]i also stimulated Epac2 translocation, but the effects were complex and in the presence of high cAMP concentrations, [Ca2+]i increases often reduced membrane binding. The stimulatory effect of Ca2+ was mediated by increased Ras activity, while the inhibitory effect reflected reduced concentrations of the membrane phospholipid PtdIns(4,5)P2. Anti-diabetic drugs of the sulfonylurea class, suggested to directly activate Epac2, induced translocation indirectly by depolarizing β-cells to increase [Ca2+]i. Epac2 is an activator of Rap GTPases, and its translocation increased Rap activity at the plasma membrane. It is concluded that the subcellular localization of Epac2 is controlled by a complex interplay between cAMP, Ca2+ and PtdIns(4,5)P2 and that the protein controls insulin release by binding to the exocytosis machinery. These results provide new insights into the regulation of β-cell function and may facilitate the development of new anti-diabetic drugs that amplify insulin secretion.

The v-rel oncogene is the most efficiently transforming member of the Rel/NF-κB family of transcription factors. Identification of genes or signal transduction pathways that contribute to v-Rel transformation provide insight into the mechanisms of tumorigenesis by Rel/NF-κB proteins. In these studies, the contribution of TGF-β/Smad signaling to v-Rel transformation was assessed. TGF-β/Smad signaling regulates several cellular processes, including growth, differentiation, and apoptosis and has been implicated in a number of different cancers. Using microarray technology and Northern blot analysis, key components of the TGF-β/Smad pathway (tgf-β2 and tgf-β3 ligands, TGF-β type II receptor, and receptor-activated smad3) were identified with upregulated mRNA expression in v-Rel-transformed fibroblasts and lymphoid cells relative to control cells. A corresponding change in their protein levels was also observed. Further analysis revealed elevated levels of the phosphorylated, active form of Smad3, which correlated with its increased DNA-binding activity in v-Rel transformed cells. In contrast, the overexpression of c-Rel resulted in little to no alteration in the RNA and protein expression of members of the TGF-β/Smad pathway. Further studies demonstrated that elevated TGF-β/Smad signaling is required for the transforming ability of v-Rel. Blocking TGF-β signaling with a kinase inhibitor of TGF-β type I receptor inhibited the activation of Smad3 and dramatically reduced the ability of v-Rel transformed cells to form colonies in soft agar. Overexpression of a constitutively active form of Smad3 in the inhibitor-treated cells restored their ability to form colonies in soft agar close to the levels seen in untreated cells. Additional experiments with dominant negative Smad3 also revealed its ability to hinder the oncogenic potential of v-Rel. In complementary experiments, a stimulatory effect on v-Rel transformation was observed with cells treated with recombinant TGF-β2 ligand or overexpressed with wild-type Smad3. Taken together, these studies demonstrate that TGF-β signaling is crucial for the transformation potential of v-Rel and is primarily mediated by Smad3 activity. / text

The liver is an essential organ for cholesterol homeostasis. If this process becomes dysregulated, cardiovascular disease (CVD) develops. Zinc-fingers and homeoboxes 2 (Zhx2) as an important hepatic gene regulator and contributes to CVD. BALB/cJ mice, with mutant Zhx2 allele, have fewer atherosclerotic plaques compared to other strains on a high fat diet. In my dissertation, I focused on the liver phenotype in BALB/cJ mice on a high-fat diet and found increased liver damage compared to wild-type Zhx2 mice. These data indicates that reduced Zhx2 in the liver leads to CVD resistance, but increases liver damage. Therefore, Zhx2 has an important role in lipid metabolism and liver function. Hepatic alpha-fetoprotein (AFP) is expressed abundantly in the fetal liver and repressed after birth regulated through three enhancers (E1, E2, and E3). E3 activity is restricted to a single layer of hepatocytes surrounding central veins (pericentral region) along with glutamine synthetase (GS). In my dissertation, I explore pericentral gene regulation in the adult liver. A GS enhancer (GSe) also exhibits pericentral activity which, along with E3, is regulated by the β-catenin signaling pathway. Orphan receptors, Rev-erbα, Rev-erbβ, and RORα, contribute to E3 activity elucidating a potential mechanism for zonation.

In this research, a series of synthetically engineered copolymers were synthesized containing polysaccharides (e.g., β-cyclodextrin and chitosan) to address the removal of trihalomethanes (THMs) from water environments. There are two main parts in this research thesis: i) the preparation and characterization of polysaccharide-based copolymers; ii) sorption studies of the copolymers with chloroform and total THMs (TTHMs) in aqueous solution. In the first part of this thesis, grafted polyester, polyester and grafted polyamide copolymers were prepared by cross-linking β-cyclodextrin (β-CD) and chitosan (CS) with various cross-linkers, including poly (acrylic acid) (PAA), terephthaloyl (TCl), and sebacoyl chloride (SCl), respectively. The synthesized copolymer materials were characterized by Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS), Scanning Electron Microscopy (SEM), Thermogravimetric analysis (TGA), Differential scanning calorimetry (DSC), elemental (C and H) analyses, and NMR spectroscopy. Nitrogen porosimetry was used to analyze the surface area and pore structure characteristics of the copolymers and starting materials in solid state. The sorption properties of the copolymers in aqueous solution were studied using different dye probes (e.g., p-nitrophenol and methylene blue) by UV–Vis spectrophotometry. The copolymers showed markedly varied interactions with dye probes in accordance with their composition, surface area, and pore structure characteristics. Diverse materials were afforded by variation of the synthetic conditions. The sorption isotherms were evaluated with various isotherm models (e.g., Langmuir, BET, Freundlich and Sips). The Sips isotherm showed the best overall agreement with the experimental results and the sorption parameters provided estimates of the sorbent surface area and the sorption capacity for various copolymers in aqueous solution. The copolymer sorbents display tunable physicochemical properties according to the synthetic conditions. In the second part of this thesis, the direct aqueous injection (DAI) method with gas chromatography (GC) with electron capture or electrolytic conductivity detectors (ECD) enabled quantitative detection of chloroform and TTHMs in water. A preliminary adsorption study and kinetic study of chloroform provided the information to establish the experimental protocol for the sorption study. The sorption parameters were evaluated using the Sips model. The sorption capacity (Qm) values of chloroform for these synthetically engineered copolymers at similar conditions ranged from 0.00335-1.70 mmol/g. The relative ordering of the Qm values was observed: β-CD/PAA 1:5 > SCl-5 > SCl-10 ~ CP-1 > β-CD/PAA 1:10 > CP-5 > AC > β-CD/PAA 1:5 at high mixing speed. An extension of the sorption study for copolymers toward the multi-component THMs in water was carried out. The copolymers showed distinct adsorption capacities to THMs: chloroform (0.0485-0.287 mmol/g); DBCM (0.0712-0.277 mmol/g); BDCM (0.0684-0.387mmol/g); and bromoform (0.0522-1.07 mmol/g). The copolymers exhibited relatively high selectivity toward individual components of THMs due to their variable molecular size and polarizability. The copolymers showed favorable adsorption (e.g., β-CD/PAA 1:5, CP-1) and each type of polysaccharide (e.g., β-CD and CS) copolymers displays great potential for the removal of halomethane-based contaminants.

Les plaques amyloïdes sont l’une des principales caractéristiques pathologiques de la maladie d’Alzheimer (MA). Elles sont composées de peptides amyloïdes-β (Aβ) 1-40 et 1-42 fibrillaires et ont été décrites comme responsable de la réponse inflammatoire et de la neurodégénérescence. L’Herpès simplex virus 1 (HSV-1) a été impliqué comme facteur de risque de la MA et retrouvé à l’intérieur des plaques amyloïdes. Des données récentes suggèrent que les peptides Aβ possèdent une activité antimicrobienne et antivirale in vitro. La première partie des travaux décrits dans cette thèse concerne l’étude du rôle antiviral des peptides Aβ contre les virus HSV-1. Pour cela, des cellules fibroblastiques, épithéliales et neuronales ont été exposées aux peptides Aβ40 et Aβ42 et infectées avec le virus HSV-1. L’analyse quantitative par PCR a montré que les peptides Aβ40 et Aβ42 inhibent la réplication des virus HSV-1, qui possèdent une enveloppe mais n’inhibent pas l’infection par les adénovirus-5 qui n’en possèdent pas. Nos résultats ont aussi montré que les peptides interagissent directement avec les virus HSV-1 dans un modèle acellulaire et inhibent leur entrée dans les cellules. Dans une deuxième partie de l’étude, nous avons utilisé un modèle de cocultures de lignées cellulaires de neuroblastomes (H4) et microgliales (U118-MG) en tant que modèle minimal in vitro afin de déterminer si les cellules H4 produisent des peptides Aβ et si cette production induit une protection antivirale contre le virus HSV-1. Les résultats ont montré que les cellules H4 sécrètent Aβ42 en réponse à une infection par les virus HSV-1 et que les cellules U118-MG internalisent le peptide Aβ42. De plus, le peptide Aβ42 exogène induit une forte production de cytokines pro-inflammatoires mais l’infection des cellules par les virus HSV-1 n’augmente pas significativement cette production. Par ailleurs, la protection antivirale du peptide Aβ42 a été confirmée lors d’expériences de transferts impliquant des milieux de cultures conditionnés provenant de cellules H4 infectées par le virus HSV-1. Ces milieux confèrent une protection Aβ-dépendante, contre les infections par les virus HSV-1 dans des essais de nouvelles cultures de cellules H4. Dans le but de mettre en évidence le mécanisme responsable de l’effet antiviral des peptides Aβ, nous avons proposé que l’homologie de séquence entre les peptides Aβ et la région proximale transmembranaire de la glycoprotéine de fusion B (gB) du virus HSV-1 pourrait être responsable de l’interférence des peptides Aβ avec la réplication des virus HSV-1 en s’insérant dans l’enveloppe externe des virus. Cette explication a été appuyée par des expériences de FRET. Les résultats ont montré que l’interaction peptide Aβ42 et gB-GFP implique une distance de 10 nm ou moins, suggérant que le peptide Aβ42 peut s’insérer dans l’enveloppe virale. L’ensemble de nos données suggère qu’une production de peptides Aβ par les neurones pourrait les protéger lors d’une infection par les virus HSV-1 latents et, contre d’autres pathogènes. Par contre, une surproduction pourrait contribuer à la formation des plaques amyloïdes, expliquant ainsi pourquoi les infections ont un rôle dans la pathogénicité de la forme sporadique de la MA.

The current study focuses on improving the wear resistance of femoral head component and enhancing the osseo-integration properties of femoral stem component of a hip implant made of a new generation low modulus alloy, Ti-35Nb-7Zr-5Ta or TNZT. Different techniques that were adopted to improve the wear resistance of low-modulus TNZT alloy included; (a) fabrication of graded TNZT-xB (x= 0, 1, 2 wt%) samples using LENS, (b) oxidation, and (c) LASER nitriding of TNZT. TNZT-1B and TNZT-O samples have shown improved wear resistance when tested against UHMWPE ball in SBF medium. A new class of bio-ceramic coatings based on calcium phosphate (CaP), was applied on the TNZT sample surface and was further laser processed with the objective of enhancing their osseo-integration properties. With optimized LASER parameters, TNZT-CaP samples have shown improved corrosion resistance, surface wettability and cellular response when compared to the base TNZT sample.

Résumé : La prééclampsie (PE) est un désordre de la grossesse caractérisée par une dysfonction endothéliale faisant en sorte que l’endothélium devient moins sensible aux signaux de vasodilatation. La réponse provoquée par la liaison de la sérotonine au sous-type de récepteur S[indice inférieur 2] entraîne la libération de molécules aux propriétés vasoconstrictrices, qui, par une boucle de rétroaction positive, entraîne la libération de davantage de sérotonine par les plaquettes. Cette boucle amplifie la réponse et contribue ainsi à l’hypertension présente chez les femmes ayant une PE. Précédemment, il a été démontré par notre laboratoire que le Bisphénol A (BPA) s’accumulait davantage dans le placenta des femmes avec PE en comparaison aux femmes normotensives. Cette accumulation pourrait découler d’une perturbation de sa métabolisation qui impliquerait notamment la β-glucuronidase (GUSB). Des études chez les animaux ont quant à elles démontré que le BPA pouvait inhiber l’activité de la monoamine oxydase (MAO) à forte dose. Nous avons étudié l’effet du BPA à faible concentration (10 ng/ml) sur la MAO-A des cellules placentaires et démontré que le BPA inhibait la MAO-A de façon significative sans affecter son expression protéique. Afin d’expliquer l’accumulation particulière du BPA chez les femmes PE, nous avons comparé l’activité spécifique et l’expression protéique de la β-glucuronidase (GUSB) placentaire en utilisant un devis cas-témoins. Une tendance non significative suggère que la GUSB pourrait partiellement contribuer à l’accumulation du BPA chez les femmes PE. Nous avons étudié la relation entre la concentration sérique maternelle de BPA et la concentration à laquelle le fœtus est exposé par régression linéaire et corrélation de Spearman. Un tel modèle ne pourrait être utilisé pour déterminer de façon quantitative l’exposition fœtale. En revanche, en vue de la forte corrélation entre ces deux variables, une haute concentration sérique maternelle de BPA devrait se refléter par une haute exposition fœtale. Cette corrélation implique aussi que le métabolisme placentaire ne joue pas un rôle significatif dans la protection du fœtus. Le BPA pourrait ainsi contribuer à l’hypertension chez les femmes PE présentant une dysfonction endothéliale en inhibant la MAO-A et ainsi, favorisant la hausse de sérotonine circulante. Cette étude suggère les bases d’un mécanisme par lequel le BPA s’accumulerait davantage chez les femmes PE et affecterait ainsi la MAO-A placentaire et potentiellement, la MAO-A fœtale vu ses propriétés physico-chimiques. / Abstract : Preeclampsia (PE) is an hypertensive disorder of pregnancy characterized by a generalized endothelial dysfunction where the response to vasodilatation signals is compromised. The binding of serotonin to its S[subscript 2] receptor subtype 2 releases vasoconstrictor molecules which, by a positive retroaction loop, stimulates the release of more serotonin from platelets. This positive retroaction loop stimulates the vasoconstriction of blood vessels and contributes to the hypertension in women with PE. Previously, we showed that Bisphenol A (BPA) accumulates more in the placenta of women with PE than in normotensive women. This accumulation may be the result of an impaired metabolization due to the action of the β-Glucuronidase (GUSB). Animal studies showed that BPA at high dose could lower the activity of the monoamine oxidase A (MAO-A), an enzyme implicated in the metabolism of serotonin. We studied the impact of BPA at low dose (10 ng/ml) in trophoblastic primary cells and showed that even at low dose, BPA can lower its activity without affecting the protein expression. To determine if GUSB could be the cause of the BPA accumulation in women with PE, we studied its activity and protein expression in placental biopsies from women with and without PE. A nonsignificant tendency showed that the GUSB activity and protein expression were higher in women with PE. To study the impact of placental metabolism in the fetal exposure, we studied the relation between maternal and fetal concentrations of BPA with linear regression analysis and Spearman’s correlation. We showed that maternal BPA could not precisely predict the fetal exposure and that the placental metabolism is probably limited in light of the strong correlation between both variables. This strong correlation also implied that high maternal exposure would result in high fetal exposure. This study shows that the accumulation of BPA in preeclamptic women could contribute to maternal hypertension by interacting with serotonin levels. This accumulation could partially be attributed to a higher GUSB, but other factors are probably implicated. The strong correlation between maternal and fetal exposure implies that the placental metabolism of BPA is limited and does not protect the fetus significantly. This study suggests the basis of a mechanism explaining the abnormal accumulation of BPA in the placenta in women with PE and its impact on the placental MAO-A and potentially, the foetal MAO-A because of its physico-chemical properties.

No description available.

A β-galactosidase (E.C 3.2.1.23) é uma das enzimas mais empregadas na indústria de alimentos sendo utilizada na hidrólise da lactose. Neste trabalho foram utilizadas duas metodologias para imobilização desta enzima. Na primeira delas foi empregado como suporte um material híbrido à base de sílica que possui um grupo orgânico catiônico covalentemente ligado. A adsorção da enzima a este material apresentou eficiência que variou de 74 a 53% com o aumento da quantidade de enzima aplicada ao suporte. A baixa estabilidade térmica da enzima imobilizada obtida e as prováveis fracas interações envolvidas na sua adsorção a este suporte podem explicar o decréscimo de atividade observada durante as sucessivas bateladas de hidrólise da lactose. Na primeira batelada o grau de hidrólise foi de 90,9% e no final da última batelada (4ª), a enzima foi capaz de converter apenas 13% do substrato. A segunda metodologia utilizada foi imobilização covalente da enzima em um filme de celulose/líquido iônico modificado com uma poliamina e ativado com glutaraldeído. A presença da poliamina foi confirmada por análises de infravermelho. Após a imobilização, a enzima reteve 60% de sua atividade inicial. Bons resultados de hidrólise da lactose em batelada foram obtidos tanto a 7ºC como a 35ºC e foi possível reutilizar a enzima imobilizada por 16 ciclos consecutivos, a 7ºC, sem mudanças significativas na atividade enzimática. O valor de Km para a enzima imobilizada no material híbrido à base de sílica foi de 9,17 mM e para a enzima imobilizada nos filmes de celulose foi de 11,22 mM, ambos apresentaram um acréscimo quando comparados ao Km enzima livre (1,25 mM), devido à dificuldade de acesso do substrato ao sítio ativo da enzima. Não houve mudança no pH e temperatura ótimos da enzima imobilizada em relação à enzima livre em nenhum dos métodos testados. / β-galactosidase (E.C 3.2.1.23) is the most widely used enzymes in the food industry and its employed in the lactose hydrolysis process. In this study, two methodologies were used to test their immobilization. In the first, the enzyme was immobilized by adsorption in one silica based hybrid material that contains a cationic organic group covalently linked. The efficiency of immobilization showed a decrease of 74 to 53% by increasing the protein load applied to the support. The low thermo stability of the immobilized enzyme and the probable weak interactions involved in their adsorption, could explain the decrease in enzyme activity observed in the successive batch hydrolysis of lactose. In the first run, the degree of lactose hydrolysis was 90.9% and, at the end of the last run (4th), the enzyme was able to convert only 13% of the substrate. The second methodology used was the covalent immobilization of the enzyme on a cellulose/ionic liquid film, modified with a polyamine and activated using glutaraldehyde. The presence of a polyamine was confirmed by infrared analysis. After immobilization, the enzyme retained 60% of its initial activity. Highly efficient lactose conversion was achieved in a batch process at 7ºC and 35ºC and was possible to reuse the immobilized enzyme in 16 repeated cycles, at 7ºC, without any drastic decrease in enzyme activity. Km value for the immobilized enzyme in silica based hybrid material was 9.17 mM and for the enzyme immobilized in the film of cellulose/ionic liquid was 11.22 mM, both showing an increase compared with the Km value for free enzyme (1.25 mM), due to the difficulty of access of the substrate to the active sites of the enzyme. The immobilized enzyme did not show any changes in the optimal pH and temperature when compared to the free enzyme in both methods tested.