We reported previously that prostaglandin E2 (PGE2) upregulates IL-23 in vitro in bone marrow-derived dendritic cells (DC), and in vivo in models of collagen-induced arthritis and inflammatory bowel disease, leading to preferential Th17 development and activity. There is very little information on the molecular mechanisms involved in the PGE2-induced upregulation of Il23a gene expression. In the present study we investigated the signaling pathways and transcription factors involved in the stimulatory effect of PGE2. Although PGE2 does not induce IL-23p19 expression by itself, it synergizes with both extra- and intracellular TLR ligands and with inflammatory cytokines such as TNFα. We established that the effect of PGE2 in conjunction with either LPS or TNFα is mediated through the EP4 receptor and the cAMP-dependent activation of both PKA and EPAC. Using the EP4 agonist PGE1OH in conjunction with TNFα, we found that PKA-induced PCREB and EPAC-induced PC/EBPβ mediate the stimulatory effect of PGE2 on IL-23p19 expression. This is the first report of CREB and C/EBPβ involvement in Il23a promoter activation. Mutation within the putative CREB and C/EBP sites combined with in vivo DNA binding (ChIP) assays identified the distal CREB site (-1125) and the two proximal C/EBP sites (-274 and -232) as essential for PKA-activated CREB and EPAC-activated C/EBPβ induced IL-23p19 expression. / Microbiology and Immunology

Le gène AFP est exprimé sélectivement par le foie fœtal et dans l’hépatome, formant un excellent modèle pour étudier les mécanismes de différenciation qui sont déréglés dans le cancer. L’hépatome, par exemple, est réfractaire à l’action anti-proliférative et AFP-suppressive des hormones glucocorticoïdes. Cette thèse porte sur la régulation transcriptionnelle du locus AFP et plus particulièrement sur l’identification des facteurs de différenciation hépatique l’induisant, avec l’objectif ultime de forcer la différenciation tumorale. À l’aide d’empreintes génomiques in vivo, je démontre pour la première fois l’occupation d’un site C/EBP au promoteur AFP, contrairement au dogme voulant que HNF1 occupe cette position critique. Des études d’immunoprécipitation de chromatine ont établi la présence de C/EBP au promoteur et aux amplificateurs du gène AFP. De plus, j’ai montré par transfection et transgenèse que C/EBP active le gène AFP en tandem avec le récepteur nucléaire FTF et que cette coopérativité dépend du positionnement précis des deux facteurs l’un par rapport à l’autre au promoteur AFP. Une substitution ou une modification de la distance entre les deux sites de fixation des facteurs change radicalement le comportement du gène pendant le développement et dans l’hépatome : ceci semble suggérer une contrainte nucléosomale déterminante dans l’activité onco-fœtale du promoteur AFP. On observe aussi une diminution du recrutement de C/EBP ou des corégulateurs CBP/p300 aux régions régulatrices AFP au cours du développement hépatique et de la croissance d’une lignée d’hépatome. La suite de ces travaux a permis d’identifier HNF4 sur les régions amplificatrices du gène AFP dans un modèle d’hépatome et chez des rats adultes. Ces résultats suggèrent un rôle négatif de HNF4 sur la transcription du gène AFP et une dérégulation possible de ce facteur dans les hépatomes. Nos travaux indiquent aussi que les complexes C/EBP présents aux régions amplificatrices AFP sont directement ciblés par le récepteur des glucocorticoïdes (via son domaine de liaison à l’ADN) dont la présence empêche le recrutement des cofacteurs sur ces régions. L’ensemble des résultats présentés dans cette thèse suggère donc un rôle central des protéines C/EBP dans la régulation développementale et hormonale du locus AFP. Celles-ci, en combinaison avec FTF, constituent ainsi de bons candidats pour forcer la différenciation tumorale. / The AFP gene is expressed specifically in the fetal liver as well as in some hepatoma cells, which makes this gene an excellent model to study differentiation processes that are deregulated in cancer. Hepatoma cells, for example, are often refractory to the AFP-suppressive as well as anti-proliferative effects of glucocorticoid hormones. This thesis explores transcriptional regulation of the AFP locus with a particular attention given to the identification of hepatic differentiation factors involved in the activation of this locus, the ultimate goal being to enforce tumor differentiation. Using in vivo footprinting experiments, I demonstrate for the first time the occupation of a C/EBP site in the AFP promoter, a site which was generally believed to be occupied by HNF1 proteins. Chromatin immunoprecipitation experiments confirmed the binding of C/EBP proteins to the AFP gene promoter as well as its enhancers. Furthermore, we showed by using transfection and transgenesis experiments that C/EBP proteins activate the AFP gene in tandem with the nuclear receptor FTF and that this cooperativity depends on the precise positionning of the two sites relative to one another. A deletion or modification of the distance between these two factors drastically modifies the expression timing of the AFP gene during hepatic development, suggesting a nucleosome position effect crucial for the onco-fetal activity of the gene. We also observe a differential recruitement of C/EBP or the coregulators CBP/p300 on AFP regulatory regions during hepatic development and growth of a hepatoma cell line. We then identified HNF4 on the enhancer regions of the AFP locus in a rat hepatoma model and in adult rat liver. This suggests that HNF4 negatively influences AFP gene transcription and underscores a possible deregulation of this factor in hepatoma cells. Our work also indicates that the C/EBP complexes present at two enhancer regions of the AFP gene are directly targeted by the glucocorticoid receptor (via its DNA-binding domain), leading to a loss of cofactor recruitement and AFP gene repression. On the whole, this thesis underscores a central role for C/EBP proteins in AFP locus developmental and hormonal regulation. Therefore, C/EBP combined with FTF represent good candidates to enforce tumor cell differentiation.

Intestinal epithelial differentiation entails the formation of highly specialized cells with specific absorptive, secretory, digestive and immune functions. Cell-cell and cell-microenvironment interactions appear to be crucial in determining the outcome of the differentiation process. Using the Caco-2 cell line that can undergo spontaneous differentiation when grown past confluency, we observed a loss of VCAM1 (vascular cell adhesion molecule-1) expression while ICAM1 (intercellular cell adhesion molecule-1) expression was seen to be stable in the course of differentiation. Protein kinase C theta (PKC&theta / ) acted downstream of PKC to inactivate Inhibitor of kappa B (IB) and activate NF-&kappa / B in the undifferentiated cells and this axis was inhibited in the differentiated cells. The increase in ICAM1 expression in the differentiated cells was due to a transcriptional upregulation by C/EBP. The protein expressions of both ICAM-1 and VCAM-1, however, were found to decrease in the course of differentiation, with both proteins getting post-translationally degraded in the lysosome. Functionally, a decrease in adhesion to HUVEC cells was observed in the differentiated Caco-2 cells. Thus, the regulation of ICAM-1 and VCAM-1, although both NF-B target genes, appear to be different in the course of epithelial differentiation. microRNAs are known to regulate many cellular pathways. miR-146a, which is known to target NF-&kappa / B, was shown to be highly upregulated in differentiated Caco-2 cells. As a predicted target of miR-146a, mRNA and protein expression of MMP16 was inversely correlated with miR-146a during differentiation of Caco-2 cells. miR-146a could bind to the 3&rsquo / UTR of MMP16 and ectopic expression of miR-146a resulted in a decreased mRNA and protein expression of MMP16 in the undifferentiated Caco-2 and HT-29 cells. Functionally, decreased gelatinase activity determined by gelatin zymography and reduced invasion and migration through Transwells was observed. In the final part of the thesis, the inhibition of NF-&kappa / B via PPAR&gamma / in 15-Lipoxygenase-1 (15LOX1) expressing cells was investigated. The expression of 15LOX1, a member of the inflammatory arachidonate cascade, could lower phosphorylation of I&kappa / B&alpha / and NF-&kappa / B DNA binding activity which was reversed with a 15LOX1 inhibitor. This inhibition was mediated by phospho-PPAR&gamma / , which in turn was phosphorylated by ERK1/2.

No description available.

Ischemic brain injury causes neurodegeneration. In this study, the mechanism of neurodegeneration was investigated by examining the role of sterol regulatory element binding protein-1 (SREBP-1), CCAAT enhancer binding protein&amp / #946 / (C/EBP&amp / #946 / ), glutathione (GSH), malondialdehyde (MDA), glutathione-S-transferase (GST), and superoxide dismutase (SOD). Carotid artery occlusion (CAO) plus hypotension was produced for 10 minutes. Control groups were sham operated. Animals were sacrificed after 24 hours, 1 week, 2 and 4 weeks of reperfusion periods. The expression of C/EBP&amp / #946 / and SREBP-1 in rat brain cortex and cerebellum were examined by western blotting. C/EBP&amp / #946 / expressions significantly increased in both cytosolic (1.19, 1.58 fold) and nuclear (1.73, 1.81 fold) extracts of brain cortex at 24 hours and 1 week CAO groups, respectively. In cerebellum, C/EBP&amp / #946 / expression significantly increased in 1 week, cytosolic (1.63 fold), and nuclear (1.35 fold) extracts. SREBP-1 expression increased significantly in both cytosolic (2.07 fold) and nuclear (1.41 fold) extracts of brain cortex in 1 week. SREBP-1 expression significantly increased in cytosolic (2.15 fold) and nuclear (1.79 fold) extracts of cerebellum in 1 week. There were no significant alterations in SREBP-1 C/EBP&amp / #946 / expressions for 2 and 4 weeks in both cytosolic and nuclear extracts of brain cortex and cerebellum. There were insignificant changes in GSH and GST levels in cortex. However, MDA and SOD levels significantly increased by 43.0 % and 47.3 %, respectively, in 24 hours. Our findings indicate that increase in SREBP-1 and C/EBP&amp / #946 / expressions may be related to oxidative stress during ischemic neurodegenerative processes.

La fonction et la différenciation des cellules de Leydig sont connues pour être régulées par différents stimuli incluant 1' hormone lutéinisante (LB) et autres facteurs paracrines et autocrines comme les cytokines dont 1 ' IL-1. NUR 77 est un facteur de transcription présent au niveau des cellules de Leydig et impliqué dans la régulation de la stéroïdogenèse. Malgré que Nur77 soit connu pour être régulé par les cytokines dans différents types cellulaires, cette régulation n 'est pas encore bien caractérisée au niveau des cellules de Leydig. Afin de mieux comprendre la régulation de Nur77 par les cytokines, j ' ai décidé d'étudier l' effet de deux facteurs de transcription, C/EBP~ et NF -KB, connus pour être impliqués dans les voies de signalisation des cytokines, sur le promoteur Nur77. J'ai trouvé que les facteurs de transcription C/EBP~ et NF -KB coopèrent ensemble dans l'activation du promoteur Nur77. Cette coopération nécessite la présènce d'au moins un des deux éléments nouvellement identifiés dans cette étude : C/EBP~ (à -110 pb en fonction du site d'initiation de la transcription) ou p50 (KB à -18 pb). L'activation du promoteur Nur77 par ces facteurs de transcription appuie mon hypothèse selon laquelle NUR 77 peut être un effecteur dans la voie de signalisation des cytokines comme 1 'IL-l.

Nous avons utilisé une approche ethnobotanique pour identifier des espèces de plantes utilisées par les Cris afin de traiter les symptômes du diabète de type 2. Larix laricina du Roi (L. laricina) a récemment été identifiée comme une des meilleures plantes qui a stimulé le transport de glucose dans les cellules C2C12 et fortement potentialisé la différenciation des 3T3-L1 en indiquant une sensibilité potentiellement accrue à l’insuline. Ensuite, ces études de criblage ont été effectuées sur des extraits éthanolique (EE) en utilisant une série de bioessais in vitro. Cependant, les préparations traditionnelles des plantes sont souvent faites avec l’eau chaude. Le but de cette thèse de doctorat était d’isoler les principes actifs de L. laricina par un fractionnement guidé par l’adipogenèse; d’évaluer et de comparer l’activité et les mécanismes antidiabétiques des EE et des extraits aqueux (HWE) de ces 17 plantes. Pour le fractionnement de L. laricina, on a isolé plusieurs composés connus et identifié un nouveau composé actif cycloartane triterpene, qui a amélioré fortement l’adipogenèse et a été responsable en partie de l’activité adipogénique (potentiellement similaire à l’effet sensibilisateur à l’insuline des glitazone) de l’extrait éthanolique issu de l’écorce de L. laricina. Pour le métabolisme lipidique, nos résultats ont confirmé que 10 parmi les 17 EE ont augmenté la différenciation des adipocytes alors que 2 extraits seulement l’ont inhibée. Les HWE ont montré une faible activité adipogénique ou antiadipogénique. Les EE de R. groenlandicum et K. angustifolia ont le PPAR γ (peroxisome proliferator-activated receptor γ), le SREBP-1 (sterol regulatory element binding protein-1) et le C/EBP (CCAAT-enhancer binding proteins) α, alors que ceux de P. balsamifera et A. incana les ont inhibés. L’effet inhibiteur de P. balsamifera a également été prouvé d’avoir impliqué l’activation de la protéine kinase activée par l’AMP (AMPK). Les EE et HWE de R. groenlandicum ont stimulé les mêmes facteurs de transcription alors que les extraits aqueux d’autres plantes sélectionnées ont perdu ces effets en comparaison avec leurs extraits éthanoliques respectifs. L’analyse phytochimique a également identifié le groupe des espèces actives et inactives, notamment lorsque les espèces ont été séparées par famille de plante. Finalement concernant l’homéostasie de glucose, nos résultats ont confirmé que plusieurs EE ont stimulé le transport de glucose musculaire et inhibé l’activité de la glucose-6-phosphatase (G6Pase) hépatique. Certains des HWE ont partiellement ou complètement perdu ces activités antidiabétiques par rapport aux EE, tandis qu’une seule plante (R.groenlandicum) a juste conservé un potentiel similaire entre les EE et HWE dans les deux essais. Dans les cellules musculaires, les EE de R.groenlandicum, A. incana et S. purpurea ont stimulé le transport de glucose en activant la voie de signalisation de l’AMPK et en augmentant le niveau d’expression des GLUT4. En comparaison avec les EE, les HWE de R.groenlandicum ont montré des activités similaires; les HWE de A. incana ont complètement perdu leur effet sur tous les paramètres étudiés; les HWE de S. purpurea ont activé la voie de l’insuline au lieu de celle de l’AMPK pour augmenter le transport de glucose. Dans les cellules H4IIE, les EE et HWE des 5 plantes ont activé la voie de l’AMPK, et en plus les EE et HWE de 2 plantes ont activé la voie de l’insuline. La quercétine-3-O-galactoside et la quercétine 3-O-α-L-arabinopyranoside ont été identifiées comme des composés ayant un fort potentiel antidiabétique et donc responsables de l'activité biologique des plantes HWE actifs avec le transport du glucose. En conclusion, on a isolé plusieurs composés connus et identifié un nouveau triterpène actif à partir du fractionnement de L. laricina. Nous avons fourni également une preuve directe pour l'évaluation et la comparaison d'une action analogue à l'insuline ou insulino-sensibilisateur des EE et HWE de plantes médicinales Cris au niveau de muscle, de foie et de tissus adipeux. Une partie de leur action peut être liée à la stimulation des voies de signalisation intracellulaire insulino-dépendante et non-insulino-dépendante, ainsi que l’activation de PPARγ. Nos résultats indiquent que les espèces de plantes, les tissus ou les cellules cibles, ainsi que les méthodes d'extraction sont tous des déterminants significatifs de l'activité biologique de plantes médicinales Cris sur le métabolisme glucidique et lipidique. / We have used a collaborative ethnobotanical approach to identify plant species used by the Cree of Eeyou Istchee (CEI) to treat symptoms of type 2 diabetes. Several screening studies were performed on 17 species identified in a survey of the Cree Nation. Firstly, Larix laricina du Roi (L. laricina) was recently identified as one of the top plants, which stimulated glucose uptake in C2C12 muscle cells and strongly potentiated the differentiation of 3T3-L1 pre-adipocytes suggesting enhanced insulin sensitivity. Secondly, these screening studies were performed on ethanol extracts (EE) using an in vitro bioassay platform, however, traditional preparations are often based on hot water. So the purpose of this PhD thesis was to isolate the active principles from L. laricina through adipogenesis-guided fractionation, and to evaluate and compare the antidiabetic activity and mechanisms of EE and hot water extracts (HWE) of these 17 Cree plants. For the fractionation of L. laricina, we isolated several known compounds and identified a new active cycloartane triterpene, which strongly enhanced adipogenesis in 3T3-L1 cells and was responsible partly for the adipogenic (potentially glitazone-like insulin sensitizing) activity of the ethanol extract of the bark of L. laricina. In the adipocyte lipid metabolism course, the results confirmed that 10 of the 17 EE stimulated adipocyte differentiation and adipogenesis, whereas 2 had inhibitory effects. Corresponding HWE exhibited partial or complete loss of such adipogenic or anti-adipogenic activity. R. groenlandicum and K. angustifolia EEs activated Peroxisome proliferator-activated receptor γ (PPAR γ), sterol regulatory element binding protein-1 (SREBP-1) and CCAAT-enhancer binding protein (C/EBP) α, whereas P. balsamifera and A. incana decreased these transcription factors. P. balsamifera’s inhibitory effect was also found to involve AMP-activated protein kinase (AMPK) activation. R. groenlandicum HWE and EE stimulated similar transcription factors, but HWE of other selected plants lost such effects compared to their respective EE. Phytochemical analysis also uncovered clustering of active versus inactive species, notably when species were segregated by plant family. The results showed that several EE stimulated muscle glucose uptake and inhibited hepatic glucose-6-phosphatase (G6Pase) activity. Some of the HWE partially or completely lost these antidiabetic activities in comparison to EE; while one plant (R.groenlandicum) retained similar potential between EE and HWE in both assays. In C2C12 muscle cells, EE of R.groenlandicum, A. incana and S. purpurea stimulated glucose uptake by activating AMPK pathway and increasing GLUT4 expression level. In comparison to EE, HWE of R.groenlandicum exhibited similar activities; HWE of A. incana completely lost its effect on all parameters; interestingly, HWE of S. purpurea activated insulin pathway instead of AMPK pathway to increase glucose uptake. In the H4IIE cells, all selected 5 plants HWE and EE activated AMPK pathway, and in addition, 2 plants EE and HWE also activated insulin pathways. Quercetin-3-O-galactoside and quercetin 3-O-α-L-arabinopyranoside were identified as potential candidates to be responsible for the biological activity of the active HWE plants in the glucose transport assay. In conclusion, we isolated several known compounds and identified a new active triterpene from fractionation of L. laricina. We also provide direct evidence evaluating and comparing of an insulin-like or insulin-sensitizing action of EE and HWE of Cree medicinal plants at the level of muscle, liver and adipose tissue. Part of their actions may be related to stimulation of insulin-dependent and insulin-independent intracellular signaling pathways, as well as to PPARγ activation. The results indicate that plant species, target tissues or cells, as well as extraction methods, are all significant determinants of the biological activity of Cree medicinal plants on glucose and lipid metabolism.

No description available.

No description available.

Breast cancer is the second leading cause of death due to cancer in women. A number of effective therapeutic strategies have been implemented in clinics to cope with the disease yet recurrent disease and toxicity reduce their effectiveness. Hence, there is a need to identify and develop more effective therapies with reduced toxic side effects to improve overall survival rates. This dissertation investigates the mechanisms of action of two natural forms of vitamin E and a cholesterol lowering drug, simvastatin, as a therapeutic strategy in human breast cancer cells. Vitamin E in nature consists of eight distinct forms which are fat soluble small lipids. Until recently, vitamin E was known as a potent antioxidant but emerging work suggests they may be resourceful agents in managing a number of chronic diseases including cancer. Anticancer properties of vitamin E have been identified to be limited to the γ- and δ- forms of both tocopherols and tocotrienols. Gamma-tocopherol ([gamma]T) and gamma-tocotrienol ([gamma]T3) have both already been identified to induce death receptor 5 (DR5) mediated apoptosis in breast cancer cells. Studies here show that similar to [gamma]T3, [gamma]T induced DR5 activation is mediated by c-Jun N-terminal kinase/C/EBP homologous protein (JNK/CHOP) proapoptotic axis which in part contributed to [gamma]T mediated dowregulation of c-FLIP, Bcl-2 and Survivin. Also, both agents activate de novo ceramide synthesis pathway which induces JNK/CHOP/DR5 proapoptotic axis and downregulates antiapoptotic factors FLICE inhibitory protein (c-FLIP), B-cell lymphoma 2 (Bcl-2) and Survivin leading to apoptosis. Simvastatin (SVA) has been identified to display pleiotropic effects including anticancer effects but mechanisms responsible for these actions have yet to be fully understood. In this dissertation, it was observed that simvastatin induced apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 proapoptotic axis and down regulation of antiapoptotic factors c-FLIP and Survivin which are in part dependent on JNK/CHOP/DR5 axis. The anticancer effects mediated by simvastatin can be reversed by exogenously added mevalonate and geranylgeranyl pyrophosphate (GGPP), implicating the blockage of mevalonate as a key event. Furthermore, work has been done to understand the factors responsible for drug resistance and identify therapeutic strategies to counteract the same. It was observed that development of drug resistance was associated with an increase in the percentage of tumor initiating cells (TICs) in both tamoxifen and Adriamycin resistant cells compared to their parental counterparts which was accompanied by an increase in phosphorylated form of Signal transducer and activator of transcription 3 (Stat3) proteins as well as its downstream mediators c-Myc, cyclin D1, Bcl-xL and Survivin. Inhibition of Stat3 demonstrated that Stat3 and its downstream mediators play an important role in regulation of TICs in drug resistant breast cancer. Moreover, SVA, [gamma]T3 and combination of SVA+[gamma]T3 has been observed to target TICs in drug resistant human breast cancer cells and downregulate Stat3 as well as its downstream mediators making it an attractive agent to overcome drug resistance. From the data presented here, the mechanisms responsible for the anticancer actions of [gamma]T, [gamma]T3 and SVA have been better understood, providing the necessary rationale to test these agents by themselves or in combination in pre-clinical models. / text