Colorectal cancer is the 4th most widespread cause of cancer mortality. One of the pathways that are involved in the development of colorectal cancer is the arachidonic acid metabolizing lipoxygenase (LOX) pathway. Inflammatory molecules formed from this pathway exert profound effects that may exacerbate the development and progression of colon and other cancers. 15 lipoxygenase-1 (15-LOX-1) is a member of LOX protein family that metabolizes primarily linoleic acid to 13-(S)-HODE. Several lines of evidence support an antiangiogenic role for 15-LOX-1, especially through 13-(S)-HODE. The expression of 15-LOX-1 is lost in colon cancer cells. Our aim in this thesis was to study whether 15-LOX-1 expression has an anticarcinogenic role, particularly on the metastatic and angiogenic potential of colon cancer cells. For this purpose, 15-LOX-1 was introduced into HCT-116 colon cancer cell lines. Having confirmed 15-LOX-1 expression and activity it was observed that expression of 15-LOX-1 significantly decreased cell proliferation, cell motility, anchorage-independent growth, migration and invasion across Matrigel, the expression of the metastasis-related MTA-1 protein, neoangiogenesis and induced apoptosis. Mechanistically, most of these effects were arbitrated by the 15-LOX-1 mediated inhibition of the inflammatory transcription factor NF-&kappa / B via the orphan nuclear receptor PPAR&gamma / . In conclusion, we propose that 15-LOX-1 has anti-tumorigenic properties and can be exploited for therapeutic benefits.

Nuclear hormone receptors (NHRs) are ligand-activated transcriptions factors which transduce the effects of various hormones as well as nutritional and other environmental signals. They thus function to maintain physiological homeostasis by integrating the tissue expression of specific target genes to regulate a wealth of biological processes including reproduction, development, metabolism and environmental adaptation. Mounting evidence indicates NHRs are the target of endocrine disrupting compounds (EDCs), exogenous chemicals, often of anthropogenic origin, which disrupt NHRs and thus the processes under their control. EDCs can interfere with NHR signalling by activating receptors (agonists), by inhibiting the actions of the receptor (antagonists), or by disrupting endogenous hormone synthesis, secretion, transport or metabolism. Much of the focus to date has been on the risk of EDCs to reproductive functions, via estrogen and androgen NHRs in humans, and also in aquatic organisms. However environmental pollutants also have the potential to interact with other NHRs, particularly in aquatic environments, and cause dysregulation of other critical physiological processes, including energy homeostasis, immune functions and the stress response. To address this possibility a reporter gene assay was developed, allowing the high-throughput screening of pollutants for their interactions with piscine NHRs with critical roles in energy homeostasis, stress reponse and immune functions, namely the peroxisome proliferator-activated receptors (PPARs) and corticosteroid receptors (CRs) from European plaice (Pleuronectes platessa) and European flounder (Platichthys flesus), respectively. Complementary DNA (cDNA) sequences encoding the ligand-binding domains of PPARs and CRs, critical for receptor-ligand interactions and receptor activation, were ligated to the DNA-binding domain (DBD) of the yeast Gal4 transcription activator protein to create experimental expression plasmid constructs. Co-transfection of these expression plasmids into the fathead minnow (FHM) cell line with an upstream-activating sequence (UAS)-firefly luciferase reporter gene plasmid increased luciferase expression in the presence of known PPAR and CR ligands. Several aquatic pollutants including pharmaceuticals, industrial by-products and biocides were tested for their potential to disrupt PPAR and CR functions by interacting with these receptors in an agonistic or antagonistic manner. Several fibrates, a group of pharmaceutical compounds used to treat dyslipidemia in humans by targeting the PPARs, were able to activate plaice Gal4-PPARα and Gal4-PPARβ in the reporter gene assay, indicative of an interaction with PPAR receptors in non-target species. Fibrates which did not activate Gal4-PPARα were able to inhibit the activation of Gal4-PPARα by the PPARα-specific agonist, Wy14643, suggesting differential effects of fibrates on human and flounder PPARs. In addition some metabolites of widespread phthalate ester pollutants were also agonists of the Gal4-PPARα and Gal4-PPARβ constructs. The Gal4-PPARγ construct was unresponsive to almost all the compounds tested, including the mammalian PPARγ agonist, rosiglitazone. The exception to this was the phthalate metabolite monobenzylphthalate, which induced a small increase in firefly luciferase in Gal4-PPARγ transfected cells. All of the above effects required concentrations of at least 10 µM, which are unlikely to be encountered in the aquatic environment. In contrast bis(tributyltin) oxide (TBTO), a notorious environmental pollutant, inhibited Gal4-PPARα and Gal4-CR constructs at concentrations as low as 1 nM and 100 nM, respectively. These concentrations are lower than those reported in aquatic environments, or in fish tissues, making TBTO a candidate endocrine disruptor in fish by inhibiting PPARα and CR signalling. A European flounder cDNA microarray was used to investigate the trasnscriptional responses of flounder hepatocytes to TBTO (10 nM) exposure. Exposure to TBTO and Wy14643, both alone and in combination, indicated a TBTO-driven downregulation of several potential PPARα-target genes with functions in the immune system, the proteasome, and lipid metabolism, although, based on mammalian comparisons, some potential PPARα-target genes were also upregulated, indicating differences in mammalian and fish PPAR-target genes or reflecting the complexity of organisms at a higher organisational level than cell-based assay systems. However, the microarray-based approach was useful in formulating further hypotheses about the effects of TBTO on PPARα signalling. Overall, these results indicate that exogenous chemicals entering the aquatic environment can interfere with NHRs with functions in energy homeostasis, immune functions and stress, in non-target organisms. The cell-based reporter gene assay is a useful tool for identifying potential endocrine disruptors which target PPARs and CRs and would be a useful method in a first tier testing approach, limiting the use of live animal models and enabling investigation into specific receptors which are targets of endocrine disrupting compounds. Although more work is required to confirm the physiological consequences of TBTO inhibition of PPARα, the results presented here indicate that organisms inhabiting TBTO-polluted environments may experience suppression of the immune system, an increase in non-functional or misfolded proteins through suppression of genes involved in the ubiquitin/proteasome system and a disruption in lipid homeostasis.

Dissertation (Ph.D.)--University of South Florida, 2005. / Includes vita. Includes bibliographical references (leaves 179-187). Also available online.

Made available in DSpace on 2014-12-17T14:13:46Z (GMT). No. of bitstreams: 1 MariaFPB_TESE.pdf: 831507 bytes, checksum: 16c85e1c11c77cb917991582252e33d1 (MD5) Previous issue date: 2013-03-20 / A S?ndrome de Berardinelli-Seip (SBS) ? um dist?rbio raro do metabolismo dos lip?dios, caracterizada pela aus?ncia quase total de tecido adiposo subcut?neo, hipertrigliceridemia, hipoleptinemia e diabetes insulino resistente ou lipoatr?fico. Sua etiologia envolve implica??es hipotal?micas, altera??es nos receptores de insulina e muta??es nos genes AGPAT2, Gng3lg, CAV1 e PTRF. O tecido adiposo secreta diversas subst?ncias, tais como: leptina, resistina, adiponectina, ester?ides, TNF , IL-6, PAI-1, angiotensinog?nio, IGF-1. Muitas delas est?o associadas ao diabetes mellitus tipo 2, obesidade e hipertens?o. Os PPARs s?o fatores transcricionais pertencentes ? superfam?lia de receptores nucleares ligantes ativados. Sabe-se que o PPAR , ? importante para o metabolismo lip?dico e glic?dico e que o ligante natural do PPAR ? derivado do ?cido graxo. Nesse sentido, foram avaliados 24 pacientes portadores da SBS, provenientes do Estado do Rio Grande do Norte, com a mediana das idades de 18,5 anos (0,55 a 47 a), sendo 9 (37,5 %) do g?nero masculino e 15 (62,5 %) do g?nero feminino. Quanto ao grupo ?tnico, foram classificados em caucas?ides (brancos) 21 (87,5 %) e negr?ides 3 (12,5 %) pacientes. Foram feitas avalia??es cl?nico-endocrinol?gica, bioqu?mica, hormonal, molecular e o estudo dos polimorfismos Adiponectina ADIPOQ, PPARγ2 Pro12Ala, LPL-PvuII, APOC3-SstI e LDLR-AvaII em portadores da SBS. Nesta popula??o n?s n?o encontramos nenhuma associa??o de par?metros lip?dicos e glic?dicos com os polimorfismos LPL-PvuII, APOC3-SstI e LDLR-AvaII. Por?m, observamos associa??o entre Adiponectina ADIPOQ e PPARγ2 Pro12Ala e n?veis lip?dicos mais elevados, sugerindo um papel biol?gico para estes fatores, indicando estudos mais aprofundados

Notre étude a montré l’implication de la déficience de PPARα dans la modulation de latranscription des gènes de l’insuline et de l’inflammation des adipocytes chez les sourisadultes C57BL/6J (WT) et PPARα-null. A jeun, les souris PPARα-null sont hypoglycémiquespar rapport aux animaux témoins WT. La concentration en insuline et l’expression de sesARNm pancréatiques, par rapport aux animaux témoins, sont diminuées chez les sourisPPARα-null, suggérant que la suppression du gène de PPARα contribuait à la faibletranscription de ces gènes. De plus, la suppression du gène de PPARα aboutit à la diminutiondes facteurs de transcription des gènes de l’insuline comme Pdx-1, Nkx6.1 et MafA. En outre,la capacité pancréatique fonctionnelle est aussi détériorée par la suppression du gène dePPARα puisque le pancréas des souris PPARα-null exprime de faibles taux de Glut2 et deglucokinase. Les souris PPARα-null expriment des taux élevés d’adiponectine et de leptinecomparées aux souris témoins. Dans les tissus adipeux, les souris PPARα-null présentent uneaugmentation de l’expression de CD14 et CD68 généralement exprimés par les macrophages.La suppression du gène de PPARα diminue, au niveau des adipocytes, l’expression de MCP-1, TNFα, IL-1β, IL-6 et RANTES, alors que l’expression de TLR-2 et de TLR-4 (récepteurspro-inflammatoires) était élevée dans les tissus adipeux. Ces résultats suggèrent qu’encondition normale, la déficience en PPARα, chez les souris est impliquée dans la modulationde la transcription des gènes de l’insuline et le statut inflammatoire du tissu adipeux.En outre, l'invalidation du gène de PPARα dans les cellules T a abouti àl'augmentation de T-bet et la diminution de GATA-3 tant aux niveaux de la protéine que del’ARNm. Comme prévu, l’acide Docosahexaénoïque (DHA) a exercé non seulement un effetinhibiteur sur la prolifération des cellules T, mais aussi a diminué la sécrétion d’IFN-γ etstimulé la sécrétion de l’IL-4 dans les deux types cellulaires. Le DHA a aussi diminué T-bet etaugmenté GATA-3 tant au niveau de la transcription qu’au niveau de la protéine. Quoique lescellules T des souris PPARα-null ont exprimé un plus fort niveau de phosphorylation de p38MAP kinase que les cellules T de WT, le DHA a diminué la phosphorylation des MAPkinases (p38 et ERK1/2) dans tous les deux les types cellulaires. Les inhibiteurspharmacologiques des MAP kinases ont aussi diminué T-bet et augmenté GATA-3 dans lescellules T. Ces résultats démontrent que le DHA, via son action sur les MAP kinases, modulel'expression des facteurs de transcription. Ces résultats expliquent aussi le mécanisme d'actionde cet acide gras sur la différenciation des cellules T dans la maladie et la santé / We assessed, in this study, the effects of PPARα deficiency on the expression of mRNAencoding for insulin gene transcription factors in pancreatic β-cells along with thoseimplicated in inflammation in adipose tissues. On fasting, the adult PPARα-null mice werehypoglycemic. Serum insulin concentrations and its pancreatic mRNA transcripts weredownregulated in PPARα-null mice, suggesting that PPARα gene deletion contributes to lowinsulin gene transcription. The PPARα gene deletion downregulates the mRNA expression ofinsulin gene transcription factors, i.e., Pdx-1, Nkx6.1 and MafA. Besides, the pancreaticfunction was diminished by PPARα deficiency as PPARα-null mice expressed low pancreaticGlut2 and glucokinase mRNA. PPARα-null mice also expressed high adiponectin and leptinmRNA levels compared to wild type animals. Adipose tissues of PPARα-null mice exhibitedupregulation of CD14 and CD68 mRNA, generally expressed by macrophages. PPAR-a genedeletion downregulates the adipocyte mRNA of certain pro inflammatory agents, like MCP-1,TNF-a, IL-1b, IL-6, and RANTES, though pro-inflammatory TLR-2 and TLR-4 mRNAswere upregulated in the adipose tissues. Our results suggest that PPAR-a deficiency, in mice,is implicated in the modulation of insulin gene transcription and inflammatory status inadipose tissues.The another part of the study was conducted on CD4+ T-cells, isolated from wild type(WT) and PPARα-null mice, in order to assess the mechanismof action of docosahexaenoicacid (DHA), an n-3 fatty acid, in the modulation of two transcription factors, i.e., T-bet andGATA-3, implicated in T-cell differentiation towards, respectively, TH1 and TH2 phenotype.The T-cells from PPARα-null mice secreted higher IFN-γ and lower IL-4 concentrations thanWT T-cells. Furthermore, the deletion of PPAR-α gene in T-cells resulted in the upregulationof T-bet and downregulation of GATA-3 both at mRNA and protein levels. DHA exerted notonly an inhibitory effect on T-cell proliferation, but also diminished IFN-γ and stimulated IL-4 secretions in both cell types. DHA also downregulated T-bet and upregulated GATA-3 bothat transcription and protein levels. Though the T-cells from PPARα-null mice expressedhigher p38 phosphorylation than WT T-cells, DHA diminished the MAP kinasephosphorylation (p38 and ERK1/2) in both the cell types. The pharmacological inhibitors ofMAP kinases also downregulated T-bet and upregulated GATA-3 in T-cells. Altogether, theseresults demonstrate that DHA, via its action on MAP kinases, modulates the expression oftranscription factors. These results also explain the mechanism of action of this fatty acid onT-cell differentiation in disease and health

Inflammatory bowel disease (IBD), which includes the subtypes Crohn's disease (CD) and ulcerative colitis (UC), is a common disease particularly in the Western world. IBD is characterised by inflammation of the small intestine and/or colon. The two subtypes affect different gut locations but both show an increased intestinal permeability or the 'leaky gut syndrome'. This led to the hypothesis that tight junction (TJ) proteins expressed in the epithelium may affect the intestinal permeability as a cause or effect of IBD.Initially, variants in the CARD15, IL23R and ATG16L1 genes, previously associated with an increased risk of IBD, were genotyped in a cohort of 500 IBD (295 CD and 205 UC) patients and 877 matched controls. These variants were significantly associated in our cohort. A random effects meta-analysis was undertaken on all previously reported CD associations with the variant rs2241880 from ATG16L1 (n=25, p=0.0017, OR: 1.36 95% CI 1.12-1.66) and with rs11209026 from IL23R (n=26, p=0.0006, OR: 0.37 95% CI 0.21-0.67), showing pooled odds ratios consistent with those reported in our cohort. Individuals carrying >1 CARD15 mutant variant were found to have a 2.5 fold increased risk of CD (p=0.0001). Candidate TJ proteins were chosen on the basis of previous reported associations and through the investigation of the claudin proteins which are abundant at TJs. Twenty one candidate genes were selected and 79 variants successfully genotyped in up to 1063 IBD (502 CD and 478 UC) and 870 control patients. Significant associations were detected with variants in the CLDN1, CLDN5 and CDH1 genes with CD; CLDN5, CLDN8 and CDH1 variants were associated to IBD; and the rs7791132 variant (between CLDN4 and ELN) and a CDH1 variant were associated to UC. The CLDN1 rs6809685 variant trended towards association in a Toronto ascertained IBD replication cohort (genotypic p=0.04, allelic p=0.06) suggesting this may be a novel IBD susceptibility variant. Small intestinal biopsies from CD patients with known rs6809685 genotypes showed a dose dependent reduced immunohistochemical staining of claudin 1 with carriage of the mutant G allele. Claudin 1 helps seal TJs and reduced levels may increase risk of CD.Peroxisome proliferator activator receptors (PPARs) can directly affect TJ proteins and could therefore affect intestinal permeability. Twelve PPARγ variants were genotyped in up to 1050 IBD (502 CD and 467 UC) and 725 control patients. Significant genotypic associations were found with the rs2067819 variant in CD (p=0.05) and IBD (p=0.02), and also the rs13099634 variant in UC (P=0.02). There was a strong gender difference particularly for rs2067819 and rs4135247, where allelic associations were highly significant and increased risk of IBD in men (p=0.01 and p=0.007 respectively). However no significant associations were found in the female cohort. Troglitazone a PPARα agonist increased Caco2 cell transepithelial electrical resistance (TEER), a marker of TJ integrity, and increased expression of claudins -3 and -4. In contrast, the PPARα antagonist GW6471 reduced the TEER without causing cell death and PPARγ ligands did not affect TEER measurements. In summary, using a robust cohort of cases and controls the data indicates that variants in genes encoding TJ proteins may affect susceptibility to IBD and that PPARs can regulate these proteins altering intestinal permeability.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2007-2008. / Les agonistes des récepteurs activés par les proliférateurs des peroxysomes-y (PPAR-y) sont utilisés en clinique pour le traitement de la résistance à l'insuline et du diabète de type 2. En plus de favoriser l'amélioration de la sensibilité à l'insuline, ces agonistes induisent de profondes modifications du métabolisme des lipides. Entre autres, ces composés augmentent l'accumulation des graisses dans le tissu adipeux blanc souscutané et diminuent leur accumulation dans le tissu adipeux blanc viscéral. Aussi, ces agonistes réduisent les triglycérides (TG) et les acides gras libres (AGL) circulants. Les mécanismes impliqués dans ces effets ne sont pas parfaitement élucidés. Les travaux décrits dans cette thèse montrent chez le rat que l'agonisme de PPAR-y augmente l'accumulation des graisses dans le tissu adipeux sous-cutané en augmentant la différenciation adipocytaire et l'expression/l'activité des déterminants métaboliques impliqués dans la captation et la rétention des lipides. Dans le tissu adipeux viscéral, ces déterminants n'ont pas été fortement affectés par le traitement. La dissipation de l'énergie et l'oxydation des lipides fut préférentiellement induite dans le tissu adipeux viscéral, contribuant ainsi à réduire l'accrétion des lipides dans ce tissu. Ces travaux montrent que l'augmentation de l'activité de la lipase lipoprotéique (LPL) dans le tissu adipeux sous-cutané et le tissu adipeux brun contribue fortement à l'augmentation de l'accumulation des graisses dans ces tissus et à la réduction de la triglycéridémie. Ces études soulignent le rôle majeur du tissu adipeux brun dans l'amélioration de la lipémie chez les rongeurs traités avec des agonistes PPAR-y. Bien que la LPL du tissu adipeux soit importante dans l'effet hypotriglycéridémiant des agonistes PPAR-y, les études décrites dans cet ouvrage montrent que sa présence n'est pas essentielle à cet effet. En absence de LPL, la réduction des AGL causée par l'augmentation de la rétention des lipides dans les tissus adipeux a réduit l'accumulation des lipides dans le foie, le potentiel de sécrétion des TG et la triglycéridémie. L'amélioration de l'effet antilipolytique de l'insuline et l'augmentation de l'expression des gènes impliqués dans l'estérification et le recyclage des acides gras dans le tissu adipeux ont contribué à réduire les AGL. Cette diminution s'est produite malgré le fait que l'agonisme de PPAR-y a augmenté le potentiel lipolytique du tissu adipeux en stimulant l'expression des lipases impliquées dans ce sentier métabolique. / Peroxisome proliferator-activated receptor-y (PPAR-y) agonists are used clinically for the treatment of insulin resistance and type 2 diabetes. In addition to improving insulin sensitivity, these agonists induce profound changes in lipid metabolism. Indeed, PPAR-y agonists increase lipid accumulation in subcutaneous fat and reduce lipid deposition in visceral fat. Concomitantly with these changes, PPAR-y agonism reduces circulating triglycerides (TG) and non-esterified free fatty acids (NEFA). The precise mechanisms involved in these effects are not known. The work presented in this thesis shows in rats that PPAR-y agonism increases lipid accumulation in subcutaneous fat by increasing adipocyte differentiation and the expression/activity of determinants of lipid uptake and retention. In visceral fat, these determinants were not strongly affected by the treatment. In this tissue, energy dissipation and lipid oxidation were preferentially increased, contributing to reduce fat accretion in visceral depots. We show here that PPAR-y agonists increased lipoprotein lipase (LPL) activity especially in subcutaneous and in brown adipose tissues and that this effect was strongly associated with the stimulation of fat accretion in these depots and with the reduction in circulating TG. This work underlies the major role of brown adipose tissue in the hypolipidemic effect of PPAR-y agonism in rodents. Although adipose tissue LPL is important for the reduction in circulating TG in PPAR-y agonist-treated animals, some observations indicate that LPL is not essential in this effect. In mice lacking LPL in fat and muscles, the stimulation of NEFA uptake and retention in adipose tissue reduced lipid accumulation in the liver, TG secretion potential and triglyceridemia. The improvement of insulin's anti-lipolytic action and the stimulation of the expression of genes involved in fatty acid esterification and recycling contributed to reduce circulating NEFA. This effect occurred even if PPAR-y agonism strongly increased the expression of the lipases involved in TG breakdown in adipose tissue.

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