L’hypertriglycéridémie postprandiale représente un facteur de risque émergent des maladies cardiovasculaires et est retrouvé en cas de syndrome métabolique, d’obésité et d’insulino-résistance. L’intestin grêle conditionne la triglycéridémie postprandiale puisque la taille et de la quantité des chylomicrons sécrétés modulent l’activité de la Lipoprotéine Lipase (LPL). La synthèse des chylomicrons est un mécanisme complexe dont l’étape de lipidation de l’Apolipoprotéine B48 (ApoB48) par la Microsomal Triglyceride Transfert Protein (MTP) et celle de leur transfert du réticulum vers le Golgi dans laquelle intervient la Liver Fatty Acid binding Protein (L-FABP) sont limitantes. Des expériences menées in vivo chez des animaux sauvages et transgéniques et ex vivo sur des segments intestinaux, nous ont permis de démontrer qu’il existe une adaptation postprandiale du métabolisme intestinal des lipides. Cette adaptation postprandiale est déclenchée par la glycoprotéine CD36 qui en présence d’acides gras à longue chaîne (AGLC) régule la voie ERK1/2 et conduit à l’induction de l’ApoB48, de la MTP et de la L-FABP. La dégradation rapide du CD36 par la voie ubiquitine-protéasome en présence d’AGLC, qui conduit à la désactivation de la voie ERK1/2, est typique d’un récepteur. Puisque d’une part les souris invalidées pour le Peroxisome Proliferator Activated receptor (PPAR) présentent une altération de l’adaptation et une hypertriglycéridémie postprandiale et que d’autre part les lipides alimentaires induisent le PPAR via CD36, CD36 et PPAR pourraient faire partie d’un mécanisme commun de régulation. En conclusion, CD36 et PPAR contribuent au sensing entérocytaire des AGLC d’origine alimentaire, responsable de l'adaptation postprandiale du métabolisme des lipides qui favorise la formation de gros chylomicrons efficacement épurés de la circulation sanguine. / Postprandial hypertriglyceridemia is an emerging risk factor for cardiovascular diseases and is associated with metabolic syndrome, obesity and insulin resistance. The small intestine participates in the postprandial triglyceridemia since both the size and number of secreted chylomicrons modulate lipoprotein lipase activity (LPL). Chylomicron synthesis is a complex mechanism in which the lipidation of Apolipoprotein B48 (ApoB48) by the Microsomal Triglyceride Transfer Protein (MTP) and the transfer between reticulum and Golgi in which the Liver Fatty Acid Binding Protein (L -FABP) is involved are limiting steps. An intestinal fat-mediated adaptation in postprandial period has been demonstrated by in vivo (transgenic and wild type mice) and ex vivo (intestinal segments) approches. This postprandial adaptation is triggered by the glycoprotein CD36 in the presence of Long chain Fatty Acids (LCFA) that regulates the ERK1/2 pathway and leads to the induction of ApoB48, MTP and L-FABP. The rapid degradation of CD36 by the ubiquitin-proteasome pathway in the presence of LCFA, which leads to ERK1/2 deactivation, has a feature of a receptor. Since firstly, Peroxisome Proliferator Activated Receptor (PPAR) knockout mice display an alteration of postprandial adaptation associated with a hypertriglyceridemia and secondly, dietary fat-mediated PPAR up-regulation is CD36 dependent, CD36 and PPAR might participate to a common regulation mechanism. In conclusion, CD36 and PPAR contribute to the enterocyte LCFA sensing responsible for postprandial adaptation that promotes the formation of large chylomicrons efficiently cleared into the blood.

A atividade dos receptores ativados por proliferadores de peroxissoma (PPAR) e receptor X hepático (LXR) são regulados por ácidos graxos. Entretanto, o papel do LNO2, um produto endógeno da nitração do ácido linoléico por espécies reativas derivadas de óxido nítrico (•NO), na via de sinalização que regula a ativação destes receptores ainda não está elucidada. Assim, considerando a propriedade do LNO2 como doador de •NO, nós investigamos a participação da via de sinalização p21Ras/Raf/ERK na ativação de PPAR e LXR por LNO2. Os resultados obtidos demonstraram que LNO2, na concentração de 0.01µM, foi um potente ativador de PPAR quando comparado ao ligante natural ácido linoléico, o qual apresentou ativação equivalente do PPAR na concentração de 10µM. O LNO2, contudo não teve efeito na ativação de LXR. LNO2 foi um potente ativador de p21Ras quando comparado ao ácido linoléico. A ativação de Ras ocorreu após 5 minutos de incubação com LNO2 em células parentais. Entretanto, em células transfectadas com p21RasC118S, o LNO2 não foi capaz de ativar Ras. A ativação de Ras e PPAR foi dependente da liberação de •NO a partir de LNO2, o que foi evidenciado na presença de C-PTIO, um seqüestrador de •NO. LNO2 ativou ERK, mas não demonstrou efeito relevante na ativação de p38 MAP kinase. A utilização de um inibidor específico de MEK, PD09895, inibiu a ativação de ERK induzida por LNO2, sugerindo que existe uma conexão entre ERK e a ativação de PPAR. Nós concluímos que a ativação do receptor nuclear PPAR por LNO2 é dependente de •NO e da via de sinalização p21Ras/Raf/ERK, a qual é capaz de ativar os subtipos α, &$946; e γ do PPAR, modulando, desse modo, a expressão de genes responsivos a este fator de transcrição. / Fatty acids bind to and regulate the activity of peroxissome proliferator-activated (PPAR) and liver X receptors (LXR). However, the role of LNO2, an endogenous product of the nitration of linoleic acid by nitric oxide (•NO)-derived reactive species, on signalling pathways regulating these nuclear receptors is poorly understood. Thus, considering the properties of LNO2 as •NO donor, we investigated the role of p21RasMAP kinases signaling pathway in the activation of PPARs and LXR by LNO2. LNO2 at physiologically relevant concentrations (0.01 µM) activates PPAR. By contrast, linoleic acid, a natural ligand for PPAR, only activated the receptor at much higher concentrations (10µM). However, it did not affect LXR activation. LNO2 is a more potent activator of p21 Ras than linoleic acid at the same conditions. Ras activation occurred within the first 5 minutes after LNO2 addition to parental cells. However, in p21RasC118s transfected cells, were unable to detect activation of Ras. Ras and PPAR activation depends on •NO released from LNO2 as evidenced by the inhibitory effect of C-PTIO, a •NO scavenger. LNO2 activated ERK but displayed no effects relevant on p38 MAP kinase. In addition, the use of specific inhibitors to MEK, PD09895, blocked PPAR activation and ERK phosphorylation by LNO2, suggesting a connection between ERK and the activation of PPAR. We conclude that LNO2 induced THP-1 cells activating Ras by S-nitrosation and recruiting the MAP kinase ERK, a downstream element of this signalling cascade and activated PPAR (α, β and γ).

La prévalence de l'obésité et du diabète de type II est en constante augmentation dans les pays industrialisés. La manifestation hépatique de ces pathologies est la NAFLD (" Non-Alcoholic Fatty Liver Disease "). Celle-ci représente aujourd'hui un réel problème de santé publique et résulte d'atteintes métaboliques et hépatiques. La NAFLD démarre par l'accumulation excessive de lipides dans les hépatocytes nommée " stéatose hépatique ". Ces lipides s'accumulent sous différentes formes comme les triglycérides, les esters de cholestérol, les diglycérides et les céramides. La lipotoxicité induite par l'accumulation de ces espèces lipidiques est à l'origine d'un dysfonctionnement au niveau cellulaire et d'une insulino-résistance. Dans ce contexte, les objectifs de ce travail de thèse ont été d'étudier in vivo le rôle des céramides dans l'apparition et les complications de la NAFLD. Pour cela, nous avons utilisé différentes approches : pharmacologiques, génétiques et nutritionnelles. Par une approche pharmacologique, nous avons montré que la fumonisine B1, un contaminant alimentaire ciblant la synthèse des céramides, est à l'origine d'une toxicité hépatique dépendante d'un facteur de transcription essentiel du métabolisme lipidique, LXR (" Liver X Receptor "). Puis, nous avons combiné différentes approches nutritionnelles et génétiques permettant d'induire ou de protéger de la stéatose hépatique. Pour cela, nous avons utilisé des souris invalidées de façon totale ou hépatocyte-spécifique pour PPARa (" Peroxisome Proliferator-Activated Receptor alpha "), un facteur de transcription essentiel au catabolisme des lipides. Cela nous a permis de confirmer le rôle essentiel de PPARa hépatocytaire dans la réponse au jeûne et l'implication systémique de PPARa dans la régulation du métabolisme des céramides au cours de l'obésité induite par un régime HFD (" High Fat Diet "). Enfin, nous avons utilisé des souris invalidées au niveau hépatocytaire pour la sous-unité catalytique de la PI3 Kinase alpha, p110a. Cela nous a permis de confirmer le rôle majeur de la voie de signalisation à l'insuline dépendante de p110a dans l'apparition de l'insulino-résistance dissociée de la stéatose hépatique induite par un régime HFD. De façon intéressante, grâce à ce modèle, nous avons démontré que la lipolyse adipocytaire (et non l'inhibition de la voie insulinémique) représente le signal dominant de l'activité hépatique de PPARa durant le jeûne. L'ensemble de ces travaux mettent en avant le rôle des céramides dans la lipotoxicité associée à la stéatose hépatique. / Prevalence of obesity and type II diabetes is constantly increasing in industrialized countries. NAFLD (" Non-Alcoholic Fatty Liver Disease ") is the hepatic manifestation of these pathologies. NAFLD represents a significant public health problem and is defined as a nexus of metabolic and hepatic diseases. NAFLD begins with fatty accumulation in the liver named "hepatic steatosis". Lipids can accumulate in different forms like triglycerides, cholesterol esters, diglycerides and ceramides. Lipotoxicity induced by the accumulation of these lipid species leads to cellular dysfunction and insulin resistance. In this context, we studied the role of ceramides in apparition and evolution of NAFLD in vivo. For this purpose, we used pharmacological, genetic and nutritional approaches. By pharmacological approach, we showed that fumonisin b1, a mycotoxin targeting ceramide synthesis, leads to hepatic toxicity, which is dependent from LXR ("Liver X Receptor"), a major transcriptional regulator of lipid metabolism. Then, we combined genetic and nutritional approaches in order to induce or protect from hepatic steatosis. For this, we used mice with hepatic or total deletion for PPARa (" Peroxisome Proliferator-Activated Receptor alpha "), a transcriptional factor essential in fatty acid catabolism. First, this model allow us to confirm the role of hepatocyte PPARa in response to fasting and second, to demonstrate the systemic involvement of PPARa in regulating ceramide metabolism during obesity induced by an HFD ("High Fat Diet"). Last, we used p110a liver-specific knockout mice, the catalytic subunit of PI3Kinase alpha. With this model, we confirmed the critical role of p110a-dependent insulin signaling in insulin resistance dissociated from hepatic steatosis induced by a HFD. Interestingly, we demonstrated with this model that free fatty acid released from adipocyte lipolysis (rather than inhibition by p110a-dependent insulin signaling) determines PPARa activity in the liver. Finally, this work highlights the key role of ceramides in lipotoxicity associated with hepatic steatosis.

Many studies have shown that peroxisome-proliferator-activated receptor [gamma] (PPAR[gamma]) plays an important role in adipose tissue formation by activating genes implicated in adipogenesis. PPAR[gamma] heterodimerizes with retinoid X receptor [alpha] (RXR[alpha]), in the presence of ligand, on PPAR response elements (PPREs) in the promoter of target genes involved in adipocyte differentiation. General receptor for phosphoinositides 1 (GRP1) is a corepressor of thyroid hormone receptors (TRs), a nuclear receptor like PPAR[gamma]. GRP1 decreases TRs' transcriptional activity by lowering dimerisation on DNA. Since PPARs and TRs have important structural similarities and that GRP1 interacts with PPARs in vitro, we hypothesized that GRP1 could be a coregulator of PPARs and, thus be implicated in adipogenesis. To better understand GRP1's effect on PPAR[gamma]2, transcriptional activity assays have been done and show that increasing concentrations of GRP1 decrease the transcriptional activity of PPAR[gamma]2. We also studied GRP1 expression by Western blots of total protein extracts from 3T3-L1 cells at different times during differentiation: GRP1 is present in 3T3-L1 preadipocytes and its expression decreases during adipogenesis. According to those results, GRP1 may be a PPAR[gamma] corepressor. After those observations, GRP1 effects on adipogenesis were studied by modulating its expression with lentiviral particles. Interestingly, GRP1 knock-down before inducing 3T3-L1 differentiation, almost abrogates adipogenesis and adipocytes markers, PPAR[gamma] and aP2, while its overexpression increases lipid storage without affecting PPAR[gamma] expression. On the opposite, GRP1 modulation after differentiation induction shows that expression knock-down slightly promotes adipogenesis by increasing PPAR[gamma], aP2 and lipid accumulation and that overexpression weakly decreases lipid storage. Our results suggest that GRP1 implication during adipogenesis occurs at two distinct and precise moments. It seems to be a key factor in the early stages of adipocyte differentiation and to be implicated as a PPAR[gamma] transcriptional activity modulator as a corepressor. Future experiments will help detail modulation of protein expression and underlying mechanisms to better understand the role of GRP1 in adipogenesis and, eventually, comorbidities linked to obesity like cardiovascular diseases and type 2 diabetes mellitus.

Les thiazolidinediones (TZDs) sont des médicaments antidiabétiques (agonistes des récepteurs nucléaires de type PPAR[gamma]) utilisés au cours des dix dernières années pour le traitement du diabète de type II. Malheureusement leur utilisation peut provoquer, chez certains patients, une rétention accrue de fluides et une formation d?oedèmes rénaux. Des études récentes suggèrent l'implication d'un canal sodique épithélial (ENaC), exprimé au niveau du tubule collecteur rénal, dans ces effets secondaires. En effet, la stimulation des PPAR[gamma] par les TZDs activent les canaux sodiques épithéliaux probablement via l'expression et l'activation de SGK1 (Serum and Glucocorticoid-regulated Kinase 1). Sachant que les transports des ions sodiques et potassiques sont étroitement liés au niveau rénal, notre objectif est de déterminer si les TZDs seraient impliqués dans la régulation des canaux potassiques (ROMK1). Nous montrons qu'en traitant les ovocytes de xénopes exprimant ROMK et PPAR[gamma], avec un TZDs comme la rosiglitazone (RGZ), le courant potassique mesuré par voltage-clamp (TEVC) est augmenté de deux fois. Cette augmentation est bloquée par l'utilisation d'un antagoniste de PPAR[gamma], le GW9662. Nous démontrons aussi l'implication de SGK1 dans la régulation de l'activité des canaux ROMK1 d'une part en mutant son site de phosphorylation sur ROMK1 (sérine 44) et d'autre part en utilisant son inhibiteur, GSK. Finalement les expériences d'immunofluorescences ont montré un recrutement de ROMK1 à la membrane des ovocytes traités à la RGZ. L'ensemble des données présentées dans ce travail suggère que la RGZ augmente le courant potassique en augmentant l'expression de SGK1.

BACKGROUND: A systems toxicology investigation comparing and integrating transcriptomic and proteomic results was conducted to develop holistic effects characterizations for the wildlife bird model, Northern bobwhite (Colinus virginianus) dosed with the explosives degradation product 2-amino-4,6-dinitrotoluene (2A-DNT). A subchronic 60d toxicology bioassay was leveraged where both sexes were dosed via daily gavage with 0, 3, 14, or 30 mg/kg-d 2A-DNT. Effects on global transcript expression were investigated in liver and kidney tissue using custom microarrays for C. virginianus in both sexes at all doses, while effects on proteome expression were investigated in liver for both sexes and kidney in males, at 30 mg/kg-d. RESULTS: As expected, transcript expression was not directly indicative of protein expression in response to 2A-DNT. However, a high degree of correspondence was observed among gene and protein expression when investigating higher-order functional responses including statistically enriched gene networks and canonical pathways, especially when connected to toxicological outcomes of 2A-DNT exposure. Analysis of networks statistically enriched for both transcripts and proteins demonstrated common responses including inhibition of programmed cell death and arrest of cell cycle in liver tissues at 2A-DNT doses that caused liver necrosis and death in females. Additionally, both transcript and protein expression in liver tissue was indicative of induced phase I and II xenobiotic metabolism potentially as a mechanism to detoxify and excrete 2A-DNT. Nuclear signaling assays, transcript expression and protein expression each implicated peroxisome proliferator-activated receptor (PPAR) nuclear signaling as a primary molecular target in the 2A-DNT exposure with significant downstream enrichment of PPAR-regulated pathways including lipid metabolic pathways and gluconeogenesis suggesting impaired bioenergetic potential. CONCLUSION: Although the differential expression of transcripts and proteins was largely unique, the consensus of functional pathways and gene networks enriched among transcriptomic and proteomic datasets provided the identification of many critical metabolic functions underlying 2A-DNT toxicity as well as impaired PPAR signaling, a key molecular initiating event known to be affected in di- and trinitrotoluene exposures.

Atherosclerosis is a chronic inflammatory disease thought to be initiated by endothelial cell dysfunction. Research described in this dissertation is focused on the role of zinc deficiency in endothelial cell activation with an emphasis on the function of the transcription factors nuclear factor-κB (NF-κB), peroxisome proliferator activated receptor (PPAR), and the aryl hydrocarbon receptor (AhR), which all play critical roles in the early pathology of atherosclerosis. Cultured porcine aortic vascular endothelial cells were deprived of zinc by the zinc chelator TPEN and/or treated with the NF-κB inhibitor CAPE or the PPARγ agonist rosiglitazone, followed by measurements of PPARα expression, cellular oxidative stress, NF-κB and PPAR DNA binding, COX-2 and E-selectin expression, and monocyte adhesion. Cellular labile zinc deficiency increased oxidative stress and NF-κB DNA binding activity, and induced COX-2 and Eselectin gene expression, as well as monocyte adhesion in endothelial cells. CAPE significantly reduced the zinc deficiency-induced COX-2 expression, suggesting regulation through NF-κB signaling. PPAR can inhibit NF-κB signaling. Zinc deficiency down-regulated PPARα expression and PPAR DNA binding activity in endothelial cells. Zinc deficiency compromised PPARγ transactivation activity in PPARγ and PPRE co-transfected rat aortic vascular smooth muscle cells. Furthermore, rosiglitazone was unable to inhibit the adhesion of monocytes to endothelial cells during zinc deficiency. Most of these effects of zinc deficiency could be reversed by zinc supplementation. An in vivo study utilizing the atherogenic LDL-R-/- mouse model generally supported the importance of PPAR dysregulation during zinc deficiency. LDLR-/- mice were maintained for four weeks on either zinc deficient or zinc adequate diets. Half of the mice within each zinc group were gavaged daily with rosiglitazone during the last stage of the study. Selected inflammation and lipid parameters were measured. The anti-inflammatory properties of rosiglitazone were compromised during zinc deficiency. Specifically, rosiglitazone induced inflammatory genes (MCP-1) in abdominal aorta only during zinc deficiency, and adequate zinc was required for rosiglitazone to down-regulate pro-inflammatory markers such as iNOS in abdominal aorta of the mice. Rosiglitazone significantly up-regulated liver IκBα protein expression only in zinc adequate mice. Plasma data also suggest an overall pro-inflammatory environment during zinc deficiency and support the concept that zinc is required for proper anti-inflammatory or protective functions of PPAR. Zinc deficiency also altered PPAR-regulated lipid metabolism in LDL-R-/- mice. Specifically, zinc deficiency increased plasma total cholesterol, and non- HDL (VLDL, IDL and LDL)-cholesterol. Plasma total fatty acids tended to be increased during zinc deficiency, and rosiglitazone treatment resulted in similar changes in fatty acid profile in zinc deficient mice. FAT/CD36 expression in abdominal aorta was upregulated by rosiglitazone only in zinc-deficient mice. In contrast, rosiglitazone treatment markedly increased LPL expression only in zinc-adequate mice. These data suggest that in this atherogenic mouse model treated with rosiglitazone, lipid metabolism can be compromised during zinc deficiency. AhR is another transcription factor involved in the development and homeostasis of the cardiovascular system. Cultured porcine aortic endothelial cells were exposed to the AhR ligands PCB77 or beta-naphthoflavone (β-NF) alone or in combination with the zinc chelator TPEN, followed by measurements of the AhR responsive cytochrome P450 enzymes CYP1A1 and 1B1. Zinc deficiency significantly reduced PCB77- induced CYP1A1 activity and mRNA expression, as well as PCB77 or β-NF-induced CYP1A1 protein expression, which could be restored by zinc supplementation. These data suggest that adequate zinc is required for the activation of the AhR-CYP1A1 pathway. Impairment of the AhR pathway presents an additional mechanism by which zinc deficiency negatively affects transcription factor function and homeostasis of the vascular system. Taken together, zinc nutrition can markedly modulate the pathogenesis of inflammatory diseases such as atherosclerosis.

A system of ordinary differential equations is developed to model the effect of fatty acids on chronic intestinal inflammation that is typical of a Crohn’s patient. Several murine studies have shown an anti-inflammatory response when specific polyunsaturated fatty acids are included regularly in the diet. It is believed that the fatty acids serve as a specific ligand that activates the Peroxisome Proliferator Activated Receptor (PPAR) which is located on multiple cell types that are active in the inflammatory response. The binding of the PPAR results in a suppression of the inflammatory pathway. Results of the model indicate a muted inflammatory response when fatty acids are added regularly to the diet in mild to moderate cases of Crohn’s. Results of mathematical analysis show a stable fixed point with decreased inflammatory markers and pathogen levels when fatty acids are added regularly to the diet.

Peroxisome proliferator-activated receptor alpha (PPARa) and delta (PPARd) belong to the nuclear receptor superfamily. PPARa is a target of lipid-lowering drugs and PPARd promotes fatty acid utilization and is a promising anti-diabetic drug target. However, evidence is growing that PPARd-agonism can stimulate fat accumulation in liver, which may aggravate the toxic situation in diabetics. The aim of the study was to characterise the hepatic transcriptional and lipid response of humanized mouse models to PPARd-agonists. In our studies of mice conditionally-expressing human PPARd (hPPARd), or the dominant-negative derivative of hPPARd (hPPARd?AF2) or wild-type animals, we demonstrated that GW501516, a potent PPARd activator, promoted up-regulation of the genes involved in lipid turnover, stimulated significant weight loss and promoted hepatic steatosis in these mouse models. There was time-dependent accumulation of hepatic triglycerides observed in wild-type and in conditionally-expressing hPPARd mice fed a diet containing PPARd synthetic ligand. This was not seen in animals conditionally-expressing hPPARd?AF2, neither in PPARa-KO or PPARd-KO animals. Concurrently, activation of PPARd in humanised animals caused significant depletion, as compared with controls, of adipose tissue deposits when fed normal or high fat diet. This effect was completely absent in PPARa-KO or PPARd-KO mice, fed diet containing GW501516. Genome-wide transcriptional profiling of GW501516 effects in the livers of these different mouse strains was performed. In PPARa-KO mice fed PPARd-agonist, some direct PPARd target genes were still up-regulated, demonstrating that they are not sufficient for the observed phenotype. In addition the blood HDL-raising effects of GW501516 were preserved in the PPARa-KO mice. This suggests a novel finding that both PPARd and PPARa receptors are essential for GW501516-driven weight loss and hepatic steatosis, with PPARa working downstream of PPARd.

Chan, Pui Ting. / Thesis submitted in: May 2007. / On t.p. "alpha" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 110-118). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.xi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Peroxisome proliferater-activated receptors (PPARs) --- p.1 / Chapter 1.1.1 --- What are PPARs? --- p.1 / Chapter 1.1.2 --- PPAR isoforms --- p.1 / Chapter 1.1.3 --- PPARα ligands --- p.2 / Chapter 1.2 --- Biological role of PPARα --- p.3 / Chapter 1.2.1 --- Lipid metabolism --- p.3 / Chapter 1.2.2 --- Glucose metabolism --- p.5 / Chapter 1.2.3 --- Oxidative stress and carcinogenesis --- p.6 / Chapter 1.3 --- Discovery of PPARα-regulated and starvation-induced gene (PPSIG) --- p.7 / Chapter 1.4 --- Objectives of the present study --- p.9 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.10 / Chapter 2.1 --- Cloning of PPSIG cDNA into a pCMV-Tag epitope tagging mammalian expression vector --- p.10 / Chapter 2.1.1 --- Materials --- p.10 / Chapter 2.1.2 --- Methods --- p.10 / Chapter 2.2 --- Transient transfection of PPSIG cDNA into CHO-K1 and AML-12 cells --- p.16 / Chapter 2.2.1 --- Cell culture and transfection --- p.16 / Chapter 2.2.1.1 --- Materials --- p.16 / Chapter 2.2.1.2 --- Methods --- p.19 / Chapter 2.2.2 --- Western blot analysis --- p.20 / Chapter 2.2.2.1 --- Materials --- p.20 / Chapter 2.2.2.2 --- Methods --- p.20 / Chapter 2.3 --- Stable transfection of PPSIG cDNA into CHO-K1 and AML-12 cells --- p.22 / Chapter 2.3.1 --- Linearization of the pCMVT4B-PPSIG construct --- p.22 / Chapter 2.3.1.1 --- Materials --- p.22 / Chapter 2.3.1.2 --- Methods --- p.22 / Chapter 2.3.2 --- Cell culture and stable transfection --- p.23 / Chapter 2.3.2.1 --- Materials --- p.23 / Chapter 2.3.2.2 --- Methods --- p.23 / Chapter 2.3.3 --- Selection of the G418-resistant clones --- p.26 / Chapter 2.3.3.1 --- Materials --- p.26 / Chapter 2.3.3.2 --- Methods --- p.29 / Chapter 2.3.4 --- Picking and expanding the G418-resistant clones --- p.30 / Chapter 2.3.4.1 --- Materials --- p.30 / Chapter 2.3.4.2 --- Methods --- p.30 / Chapter 2.3.5 --- Screening and confirmation of the stable transfectants --- p.31 / Chapter 2.3.5.1 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.31 / Chapter 2.3.5.1.1 --- Materials --- p.31 / Chapter 2.3.5.1.2 --- Methods --- p.31 / Chapter 2.3.5.2 --- Northern blot analysis --- p.35 / Chapter 2.3.5.2.1 --- Materials --- p.35 / Chapter 2.3.5.2.2 --- Methods --- p.35 / Chapter 2.3.5.3 --- Western blot analysis --- p.37 / Chapter 2.3.5.3.1 --- Materials --- p.37 / Chapter 2.3.5.3.2 --- Methods --- p.37 / Chapter 2.3.5.4 --- Immunoprecipitation --- p.37 / Chapter 2.3.5.4.1 --- Materials --- p.37 / Chapter 2.3.5.4.2 --- Methods --- p.38 / Chapter 2.3.5.5 --- Matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry analysis --- p.39 / Chapter 2.3.5.5.1 --- Materials --- p.39 / Chapter 2.3.5.5.2 --- Methods --- p.39 / Chapter 2.4 --- "Analysis of the all-trans-13,14-dihydroretinol saturase (RetSat) activity by high-performance liquid chromatography (HPLC) analysis" --- p.41 / Chapter 2.4.1 --- Materials --- p.41 / Chapter 2.4.2 --- Methods --- p.42 / Chapter 2.4.2.1 --- Preparation of all-trans-retinol --- p.42 / Chapter 2.4.2.2 --- Treatment of PPSIG-transfected cells with all-trans-retinol --- p.42 / Chapter 2.4.2.3 --- Retinoid analysis --- p.43 / Chapter 2.5 --- Analysis of fatty acid compositions by gas chromatography-mass spectrometry (GC-MS) --- p.43 / Chapter 2.5.1 --- Materials --- p.43 / Chapter 2.5.2 --- Methods --- p.44 / Chapter 2.5.2.1 --- Preparation of fatty acid-BSA complex --- p.44 / Chapter 2.5.2.2 --- Treatment of PPSIG-transfected cells with fatty acid-BSA complex --- p.44 / Chapter 2.5.2.3 --- Extraction of fatty acids --- p.45 / Chapter 2.5.2.4 --- Methylation of the fatty acids --- p.45 / Chapter 2.5.2.5 --- GC-MS analysis --- p.46 / Chapter 2.5.2.6 --- Statistical analysis --- p.47 / Chapter CHAPTER 3 --- RESULTS --- p.48 / Chapter 3.1 --- The PPSIG cDNA was subcloned into a pCMV-Tag epitope tagging mammalian expression vector --- p.48 / Chapter 3.2 --- The pCMVT4B-PPSIG expression construct was transiently transfected into CHO-K1 and AML-12 cells --- p.54 / Chapter 3.3 --- Stable transfection of the pCMVT4B-PPSIG expression construct into CHO-K1 and AML-12 cells --- p.54 / Chapter 3.3.1 --- PPSIG-transfected CHO-K1 and AML-12 cells were obtained after G418 selection --- p.54 / Chapter 3.3.2 --- PPSIG-transfected CHO-K1 and AML-12 cells had high PPSIG mRNA expression --- p.58 / Chapter 3.3.3 --- PPSIG-FLAG fusion protein was over-expressed in the PPSIG- transfected CHO-K1 and AML-12 cells --- p.61 / Chapter 3.3.4 --- The stable transfectants were immunoprecipitated and identified as PPSIG protein by the mass spectrometry analysis --- p.64 / Chapter 3.4 --- PPSIG protein posseses saturase activity towards all-trans-retinol --- p.66 / Chapter 3.5 --- PPSIG protein is not a fatty acid transporter --- p.78 / Chapter CHAPTER 4 --- DISCUSSION --- p.101 / FUTURE STUDIES --- p.107 / REFERENCES --- p.110 / Appendix A: Prediction of the molecular weight of pCMVT4B- PPSIG protein --- p.119 / Appendix B: Theoretical tryptic peptides of PPSIG --- p.120 / Appendix C: Protein-peptide mass reports --- p.122 / Chapter C1. --- Peptide mass summary of trypsin-digested PPSIG immunoprecipitated protein from clone L2H4B18 --- p.122 / Chapter C2. --- Peptide mass summary of trypsin-digested PPSIG immunoprecipitated protein from clone AL2L7 --- p.123 / Appendix D: HPLC spectrum of the RetSat activity towards all- trans retinol --- p.124 / Chapter D1. --- RetSat activity towards all-trans retinol according to the Moise's group study ((Moise et al. 2004) --- p.124