Obesity and type-2 diabetes are major global health crises. The synthetic retinoid compound 4-hydroxy(phenyl)retinamide (Fenretinide, FEN), has been shown to inhibit adiposity and reverse insulin resistance in pre-clinical studies. Fenretinide acts via several different mechanisms, including induction of retinoid signalling and increased hepatic lipid oxidation to exert its metabolic effects. However, the signalling mechanisms behind these effects have yet to be fully elucidated. A number of approaches were taken in this thesis to investigate the signalling mechanisms of Fenretinide. To characterise the relationship between Fenretinide and leptin signalling, Fenretinide treatment was administered in two different leptin-deficient mouse models. Fenretinide effects on hepatic signalling mechanisms were further characterised by performing global transcriptomics analysis in liver from mice receiving HFD ± Fenretinide. In this analysis, the important metabolic hormone fibroblast growth factor (FGF) 21 was identified as a novel retinoid-dependent target of Fenretinide signalling, which was further characterised in multiple mouse models. Retinoic-acid receptor-specific ChIP-sequencing was performed in order to identify other liver genes that are regulated by Fenretinide via retinoid-dependent signalling mechanisms. This work has shown that the beneficial effects of Fenretinide on adiposity occur via a mechanism independent of that through which Fenretinide mediates effects on glucose homeostasis. Fenretinide effects on insulin sensitivity and glucose homeostasis are most likely mediated via the inhibition of ceramide synthesis in the liver and other metabolically active tissues. This work also shows that Fenretinide can normalise the effects of chronic HFD-feeding by targeting the expression of a set of PPARα-target genes in the liver via a retinoid-dependent signalling mechanism. Overall, the work described in this thesis both uncovers more detail about the signalling mechanisms of Fenretinide and identifies novel target genes that may be exploited for the development of new therapeutics to treat obesity and type 2 diabetes.

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Indiana University-Purdue University Indianapolis (IUPUI) / Retinoids play an important role in tissue patterning during development as well as in epithelial formation and health. In the mammalian central nervous system, the meninges are a source of retinoids for brain tissue. Retinoid production has been described in juvenile Axolotl ependymal cells. Retinoid effects may possess a significant role in the regeneration-permissive interaction of the meninges and ependyma of the Axolotl spinal cord after penetrating injury. During spinal cord regeneration in urodele amphibians, the pattern of retinoid production changes as the meninges interact with the injury-reactive ependymal cells reconstructing the injured spinal cord. In order to determine which components of the retinoid metabolism and intracellular signaling pathway act in Urodele spinal cord regeneration, we employed antibody/horseradish peroxidase staining of both intact and regenerating Axolotl spinal cord tissues obtained from adult animals as well as cell culture techniques to determine expression of three retinoid pathway components: Cellular Retinoic Acid Binding Protein II (CRABP 2), Cellular Retinol Binding Protein I (CRBP 1), and Retinaldehyde Dehydrogenase II (RALDH 2). Current results demonstrate the following in the intact cord: 1) CRBP 1 is expressed in the pia and dura mater meningeal layers, in gray matter neurons (including their axonal processes), and the ependymal cell radial processes that produce the glia limitans, 2) CRABP 2 is expressed in the arachnoid and/or dura mater meningeal layers surrounding the spinal cord, and 3) RALDH 2 is expressed in the meninges as well as cytoplasm of grey matter neurons and some ependymal/sub-ependymal cells. In the regenerating cord, CRBP 1 is expressed in ependymal cells that are undergoing epithelial-to-mesenchymal transition (EMT), as is CRABP 2. RALDH 2 staining is very strong in the reactive meninges; in addition, expression is also upregulated in the cytoplasmic and perinuclear regions of reactive grey matter neurons, including motor neurons and in the apical region of ependymal. Preliminary studies culturing reactive meninges and ependymal cells together suggested that the meninges could drive re-epithelialization of the reactive ependymal cells. Experiments to characterize this interaction show an unusual proliferation pattern: Proliferating Cell Nuclear Antigen (PCNA) labeling is present in intact and regenerating cord ependymal cells. However, in culture, the presence of meninges results in no proliferation proximal to the explant, but extensive proliferation in leading cell outgrowth; also, the cultured meninges is positive for RALDH2. In summary, the intact adult cord shows meningeal production of RA, which is upregulated following injury; in addition, during this time, RA production is upregulated in the adult ependymal cells as well. In culture, the reactive meninges appears to modulate the behavior of reactive ependymal cells.

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

Chez les vertébrés, la perception de la lumière est possible grâce à la transformation de l'énergie des photons en un signal électrochimique dans les photorécepteurs. La photo-isomérisation du chromophore RAL-11-cis en RAL-tout-trans déclenche une cascade d'événements de transduction du signal et conduit à la perte de la sensibilité à la lumière des photorécepteurs, un processus de recyclage du chromophore visuel entre alors en jeu afin de rétablir cette sensibilité, c'est le cycle visuel de rétinoïdes. Le cycle visuel s'effectue dans les photorécepteurs et l'épithélium pigmentaire rétinien et fait intervenir plusieurs protéines. L'étape limitante de ce cycle est catalysée par la protéine spécifique de l'EPR RPE65. Mieux comprendre la régulation de cette étape permettra d'ouvrir des pistes thérapeutiques pour différentes pathologies liées l'activité du cycle visuel. Notre laboratoire a montré que FATP1, une protéine du métabolisme lipidique, inhibe l'activité de RPE65 in vitro. Dans ce travail, nous avons évalué l'effet de l'absence de FATP1 sur l'activité du cycle visuel et la fonction visuelle dans un modèle murin. La souris ko FATP1 est caractérisée par une baisse des amplitudes des ondes a et b de l'ERG, un retard de régénérescence de l'ERG et d'accumulation de rétinylesters après éblouissement. La formation du RAL-11-cis reste comparable à celle des souris sauvages. Les souris FATP-/- montrent une plus grande susceptibilité aux effets du vieillissement : des anomalies de structure ont été observées au niveau des photorécepteurs, de la membrane de Brüch et de la choroïde, sans accumulation excessive de lipides ou de lipofuscine ni de dégénérescence rétinienne. La perturbation de la fonction visuelle observé en l'absence de FATP1 chez la souris pourrait avoir un effet plus accru chez l'homme qui vie plus longtemps et dont la rétine reçoit plus de lumière. En conclusion, FATP1 pourrait être une composante importante pour le vieillissement de la rétine chez l'homme. / In vertebrates, perception of light is made possible through the conversion of photon energy into an electrochemical signal in photoreceptors. The photo-isomerization of the chromophore 11-cis-RAL into all-trans-RAL triggers a cascade of signal transduction and leads to loss of light sensitivity of photoreceptors, thus, a recycling process of chromophore called the rétinal visual cycle is involved to restore this sensitivity. The visual cycle takes place in the photoreceptors and retinal pigment epithelium and involves several proteins. The limiting step of this cycle is catalyzed by RPE-specific protein RPE65. Better understanding the regulation of this step might open up new ways for treatment of various pathologies related to the visual cycle activity. Our laboratory has shown that FATP1, a protein involved in lipid metabolism, inhibits the activity of RPE65 in vitro. In this study, we evaluated the effect of the lack of FATP1 on the activity of the visual cycle and visual function in a mouse model. FATP1 ko mice are characterized by lower a and b amplitudes of the elctroretinogram, delayed ERG and retinylesters accumulation recovery after bleache. 11-cis-RAL synthesis rates are similar to those of wt mice. FATP1 ko mice show a greater susceptibility to the aging effects: structural abnormalities were observed in the photoreceptors, Bruch's membrane and choroid without excessive accumulation of lipid or lipofuscin or retinal degeneration. The disturbance of visual function which accopagne the lack of FATP1 in mice could have a more increased effect in human who live longer and wich the retina receives more light. In conclusion, FATP1 might be an important component for the aging of the retina in humans.

O carcinoma epidermóide de boca, neoplasia maligna de boca mais comum, pode originar-se de lesões potencialmente malignas. O ácido retinóico, que atua no crescimento e diferenciação celular, tem sido comumente estudado como um possível quimioterápico na prevenção dessa progressão. Embora o mecanismo pelo qual o ácido retinóico previne essa progressão, e promove a parada do crescimento celular, não esteja estabelecido, sabe-se que os genes homeobox são importantes alvos do ácido retinóico durante o desenvolvimento embrionário e diferenciação tecidual. Este estudo visa determinar se a modulação da expressão desses genes está envolvida na inibição do crescimento pelo ácido retinóico em carcinoma epidermóide de boca. Para isso, foi realizado PCR array para avaliar a expressão de 84 genes homeobox na linhagem celular de carcinoma epidermóide de boca sensível ao ácido retinóico SSC-25, comparando com a linhagem resistente, SSC-9, após o tratamento com ácido retinóico por sete dias. Os resultados mostraram nove genes com perda de expressão e quatro com alta expressão. A validação por qPCR de 7 desses genes confirmou os resultados. Desses, três genes(ALX1, DLX3, TLX1) foram selecionados para terem a expressão avaliada em amostras tratadas por 3, 5 e 7 dias. O gene ALX1 apresentou baixa expressão apenas no dia 7. O gene DLX3 apresentou baixa expressão no terceiro dia com maior decréscimo no sétimo. Já o gene TLX1, mostrou baixa significativa no quinto dia, com valores semelhantes no sétimo. Os dados mostram genes homebox são modulados pelo ácido retinóico em linhagens de carcinoma epidermóide de boca. No entanto, esses genes não parecem ser alvo direto da inibição do crescimento promovida pelo ácido retinóico. / Oral squamous cell carcinoma, the most frequent oral cancer, may arise from potentially malignant oral lesions. Retinoic acid, which plays a role in cell growth and differentiation, has been frequently studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which retinoic acid prevents progression and suppresses cell growth has not been completely elucidated, it is known that homeobox genes represent important targets of retinoic acid during embryogenesis and differentiation. The present study aims to determine if modulation of the expression of these genes is involved in inhibition of OSCC cell growth by retinoic acid. In order to achieve this goal a PCR array was performed to evaluate the expression of 84 homeobox genes in retinoic acid sensitive SCC-25 cells compared to retinoic acid resistant SCC-9 cells following treatment with retinoic acid for 7 days. Results showed that 9 homeobox genes are downregulated and 4 are upregulated by retinoic acid. The validation confirmed these results. Three genes (ALX1, DLX3, TLX1) were selected for having their expression evaluated on samples treated with retinoic acid for 3, 5 and 7 days. Three different patterns of gene expression were observed. Gene ALX1 showed down-regulation only on day 7. Homeobox gene DLX3 showed reduced expression on day 3 and decreased expression on day 7. TLX1 showed a substantial down-regulation on day 5 with similar values on. The data presented show that a number of homeobox genes are modulated by retinoic acid in oral squamous cell carcinoma cell lines. However, these genes do not appear to be direct targets of growth suppression trigged by retinoic acid.

Vitamin A and its derivatives, known as retinoids, are involved in a number of functions in the developing and adult brain (Lane et al., 2005). Roaccutane (13-cis-retinoic acid, 13-cis-RA) is a synthetic retinoid used for the treatment of severe cystic acne, although its use has been controversially associated with adverse psychiatric events including depression. In this thesis, the presence of retinoid receptors in the rat hippocampus was verified and a similar profile of expression was observed in the rat raphe nuclei for the first time. The expression of retinoid receptors in brain regions that are implicitly associated with depression pathology provides proof of concept for retinoids to influence depressive behaviour. The ability of 13-cis-RA treatment to induce a pro-depressive profile in animal models of depression-related behaviour was tested. In the resident-intruder paradigm, adult rats treated for 7 or 14 days with 13-cis-RA (1mg/kg, i.p.) showed reduced aggressive behaviour, with a concomitant increase in flight submit and flight escape behaviours, compared with vehicle-treated controls. These findings are indicative of increased depression-related behaviour. However, chronic treatment did not alter depression-related behaviour in the forced swim test and sucrose consumption anhedonia paradigms The molecular mechanisms mediating 13-cis-RA-induced depression were investigated by examining monoaminergic gene expression, protein levels and neurotransmitter levels in rat brain tissue and plasma and an in vitro model. The majority of serotonergic components (SERT, 5-HT1AR, 5-HT1BR and MAOA) were not altered by chronic 13-cis-RA treatment, with the possible exception of TPH2 gene/protein expression and increased 5-HT levels in platelets. In fact, the expression of D2 dopamine receptor was significantly elevated in the RN46A-B14 cell line (10μM 13-cis-RA, 48 h) and was similarly elevated at the protein level in the juvenile rat hippocampus (1mg/kg/day, i.p., 6 weeks), suggesting dopaminergic pathways may be of importance. There was also a trend in the data to suggest that 13-cis-RA-treated juvenile rats may be more susceptible the molecular alterations than corresponding adult rats. xii