Les fibrates sont utilisés cliniquement pour traiter l'hypertriglycéridémie. Leurs actions sont médiées par le peroxisome proliferator activated receptor [alpha] (PPAR [alpha]), un récepteur nucléaire hautement exprimé dans le foie, qui régule l'expression des gènes impliqués dans la lipolyse, la [bêta]-oxydation et la synthèse des cétones.Les cétones, produites à partir de la dégradation des acides gras, sont des substrats énergétiques alternatifs pour le cerveau lorsque les concentrations de glucose diminuent. Lors du vieillissement, dans des cas de déclin cognitif ou de maladie d'Alzheimer, la captation du glucose par le cerveau diminue ce qui perturbe son homéostasie énergétique.Les cétones, en concentration plasmatique suffisante, pourraient pallier efficacement au manque énergétique cérébral. Le potentiel des fibrates à stimuler la production des cétones n'a jamais été étudié chez l'homme. Objectif. Évaluer la capacité des fibrates, des agents activateurs du PPAR[alpha], à stimuler la production des cétones chez les humains. Méthodes : Trois études ont été réalisées. La première étude (a) comparait les concentrations plasmatiques de cétones d'un groupe traité avec les fibrates avec un groupe témoin, non traité aux fibrates.Les deux autres études cliniques comparaient les concentrations de cétones à jeun et lors d'une journée métabolique de 6 heures avant et après (b) un traitement de 12 semaines avec le bezafibrate sur 10 personnes légèrement hypertriglycéridémiques et (c) l'arrêt d'un traitement au fénofibrate pour une période de 6 semaines, sur 10 personnes déjà sous médication au fénofibrate. Résultats : Dans les trois études, les concentrations plasmatiques de cétones à jeun n'ont pas été augmentées significativement par les fibrates. Dans les deux études cliniques, comme attendu, les fibrates ont diminué significativement les triglycérides. Par contre, les fibrates n'ont pas augmenté les concentrations de cétones à jeun ni aux différents temps de la journée. Cependant, après 12 semaines de traitement avec le bezafibrate, la réponse post prandiale des cétones, obtenue par l'aire sous la courbe des données normalisées par rapport aux concentrations à jeun, a été augmentée de 85%. Conclusion : La cétogenèse n'est pas stimulée par la prise d'un fibrate quand le contexte métabolique n'est pas favorable. Un besoin énergétique est nécessaire pour initier la cétogenèse mais la présence d'un fibrate aurait un certain potentiel pour favoriser cette voie métabolique. Perspectives : Le recrutement se poursuit pour mieux définir l'effet des fibrates sur les cétones et d'autres stratégies devront être mises de l'avant, comme la combinaison d'un fibrate avec un apport en substrats facilement oxydables, pour induire un état de cétose chronique afin de favoriser la captation des cétones par le cerveau.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: pp. 152-183.

Peroxissome proliferator-activated receptor-alfa (PPAR-?) é um fator de transcrição nuclear envolvido na regulação do metabolismo de lipídeos e da inflamação. PPAR? pode estar relacionado com a modulação da cicatrização de feridas cutâneas, que é um processo multifatorial, dependente de mecanismos de sinalização celular e de inflamação. Deste modo, o objetivo deste trabalho foi analisar o papel do receptor PPAR? na cicatrização de feridas cutâneas induzidas experimentalmente e a sua relação com o metabolismo sistêmico após tratamento com agonista do PPAR?. Para tanto, foram realizadas feridas na pele da região dorsal de camundongos 129/SvEv, que foram tratados diariamente com o agonista de PPAR?, Gemfibrozil, por via oral ou tópica. Os animais foram acompanhados durante 240h pós-cirúrgico (p.c.) para a análise do reparo cutâneo e alterações metabólicas que poderiam ser induzidas pela ativação de PPAR?. Os camundongos tratados apresentaram melhor cicatrização após ativação de PPAR? com 100 ou 50 mg/kg/dia de agonista por via oral ou tópica, respectivamente. O tratamento oral induziu cicatrização mais rápida somente após 24h, 48h e 72h p.c., enquanto que os animais tratados com Gemfibrozil tópico apresentaram cicatrização mais precoce em todos os tempos avaliados. A indução de feridas alterou o metabolismo sistêmico dos camundongos que demonstraram significativa perda de peso e redução de triglicérides, independentemente do tratamento. Porém, a ativação de PPAR? não alterou a glicemia ou a função hepática. Na análise histopatológica das feridas foi verificado infiltrado inflamatório, composto principalmente por neutrófilos e outras células polimorfonucleares. Entretanto, o tratamento com Gemfibrozil tópico levou a um menor infiltrado inflamatório e diferenciada deposição de colágeno após 10 dias p.c. Além disso, houve diminuição do acúmulo de neutrófilos, macrófagos e eosinófilos quando comparados aos animais que receberam apenas o veículo. O tratamento tópico promoveu menor acúmulo de linfócitos TCD4+, TCD8+ e T??, e ainda diferenciado influxo de células dendríticas para a lesão. No entanto, não houve diferença em relação a células T reguladoras nos linfonodos drenantes, mas os animais tratados apresentaram diminuição de Foxp3 nas células CD4+CD25-. Em conclusão, PPAR? atua no reparo cutâneo, e sua ativação local acelera a cicatrização por meio da modulação da inflamação na pele. Finalmente, os resultados sugerem que PPAR? pode ser alvo importante para novas terapias que visam melhorar a cicatrização de feridas, especialmente quando ativado no local da lesão. / Peroxissome proliferator-activated receptor alpha (PPAR?) is a nuclear transcription factor involved in the regulation of lipid metabolism and inflammation. PPAR? may be associated to the modulation of wound healing, which is a multifactorial process dependent on mechanisms of cell signaling and inflammation. Then this work aimed to analyze the role of PPAR? receptor in experimental cutaneous wound healing and its relationship to the systemic metabolism of mice treated with a PPAR? agonist. For this, skin wounds were performed in the dorsal region of 129/SvEv mice, treated daily with the PPAR? agonist, Gemfibrozil, by oral or topical route. Mice were followed for 240h post-surgery (p.s.) for skin repair and metabolic changes that could be induced by PPAR? activation. There was improved wound healing in mice treated with 100 or 50 mg/Kg of PPAR? agonist by oral or topical route respectively. The oral treatment induced a better repair in the early 24h, 48h and 72h p.s. while mice treated by topical application of Gemfibrozil presented faster healing in all times evaluated. Wound\'s induction affected the systemic metabolism of mice leading to significant weight loss. PPAR? agonist did not alter glucose, triglycerides or liver function, although all injured animals had a significant decrease on triglycerides levels in the early times p.s., independent on the treatment. In histopathological examination of the wounds it was observed inflammatory infiltrate, composed mainly of neutrophils and other polymorphonuclear cells. However, topical treatment with PPAR? agonist led to lower inflammatory infiltrate and differentiated collagen deposition 10 days p.s. Furthermore, there was decrease of neutrophil, macrophages and eosinophils influx when compared to untreated mice. Topical treatment led to decrease in the TCD4+, TCD8+ e T?? lymphocytes accumulation in the lesions, and differentiated dendritic cell influx to the wounds. However there was no difference regarding CD4+CD25+ T cells in lymph nodes, but treated mice showed decrease Foxp3 expression. In conclusion, the triglycerides serum level was altered in the course of wound healing and may be associated to skin lesion, while PPAR? agonist acts in wound repair by accelerating healing and modulating neutrophil influx to the skin. Finally, our results suggested that PPAR? may be an important target for novel therapies aimed at improved wound healing, especially when administered topically.

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

Peroxissome proliferator-activated receptor-alfa (PPAR-?) é um fator de transcrição nuclear envolvido na regulação do metabolismo de lipídeos e da inflamação. PPAR? pode estar relacionado com a modulação da cicatrização de feridas cutâneas, que é um processo multifatorial, dependente de mecanismos de sinalização celular e de inflamação. Deste modo, o objetivo deste trabalho foi analisar o papel do receptor PPAR? na cicatrização de feridas cutâneas induzidas experimentalmente e a sua relação com o metabolismo sistêmico após tratamento com agonista do PPAR?. Para tanto, foram realizadas feridas na pele da região dorsal de camundongos 129/SvEv, que foram tratados diariamente com o agonista de PPAR?, Gemfibrozil, por via oral ou tópica. Os animais foram acompanhados durante 240h pós-cirúrgico (p.c.) para a análise do reparo cutâneo e alterações metabólicas que poderiam ser induzidas pela ativação de PPAR?. Os camundongos tratados apresentaram melhor cicatrização após ativação de PPAR? com 100 ou 50 mg/kg/dia de agonista por via oral ou tópica, respectivamente. O tratamento oral induziu cicatrização mais rápida somente após 24h, 48h e 72h p.c., enquanto que os animais tratados com Gemfibrozil tópico apresentaram cicatrização mais precoce em todos os tempos avaliados. A indução de feridas alterou o metabolismo sistêmico dos camundongos que demonstraram significativa perda de peso e redução de triglicérides, independentemente do tratamento. Porém, a ativação de PPAR? não alterou a glicemia ou a função hepática. Na análise histopatológica das feridas foi verificado infiltrado inflamatório, composto principalmente por neutrófilos e outras células polimorfonucleares. Entretanto, o tratamento com Gemfibrozil tópico levou a um menor infiltrado inflamatório e diferenciada deposição de colágeno após 10 dias p.c. Além disso, houve diminuição do acúmulo de neutrófilos, macrófagos e eosinófilos quando comparados aos animais que receberam apenas o veículo. O tratamento tópico promoveu menor acúmulo de linfócitos TCD4+, TCD8+ e T??, e ainda diferenciado influxo de células dendríticas para a lesão. No entanto, não houve diferença em relação a células T reguladoras nos linfonodos drenantes, mas os animais tratados apresentaram diminuição de Foxp3 nas células CD4+CD25-. Em conclusão, PPAR? atua no reparo cutâneo, e sua ativação local acelera a cicatrização por meio da modulação da inflamação na pele. Finalmente, os resultados sugerem que PPAR? pode ser alvo importante para novas terapias que visam melhorar a cicatrização de feridas, especialmente quando ativado no local da lesão. / Peroxissome proliferator-activated receptor alpha (PPAR?) is a nuclear transcription factor involved in the regulation of lipid metabolism and inflammation. PPAR? may be associated to the modulation of wound healing, which is a multifactorial process dependent on mechanisms of cell signaling and inflammation. Then this work aimed to analyze the role of PPAR? receptor in experimental cutaneous wound healing and its relationship to the systemic metabolism of mice treated with a PPAR? agonist. For this, skin wounds were performed in the dorsal region of 129/SvEv mice, treated daily with the PPAR? agonist, Gemfibrozil, by oral or topical route. Mice were followed for 240h post-surgery (p.s.) for skin repair and metabolic changes that could be induced by PPAR? activation. There was improved wound healing in mice treated with 100 or 50 mg/Kg of PPAR? agonist by oral or topical route respectively. The oral treatment induced a better repair in the early 24h, 48h and 72h p.s. while mice treated by topical application of Gemfibrozil presented faster healing in all times evaluated. Wound\'s induction affected the systemic metabolism of mice leading to significant weight loss. PPAR? agonist did not alter glucose, triglycerides or liver function, although all injured animals had a significant decrease on triglycerides levels in the early times p.s., independent on the treatment. In histopathological examination of the wounds it was observed inflammatory infiltrate, composed mainly of neutrophils and other polymorphonuclear cells. However, topical treatment with PPAR? agonist led to lower inflammatory infiltrate and differentiated collagen deposition 10 days p.s. Furthermore, there was decrease of neutrophil, macrophages and eosinophils influx when compared to untreated mice. Topical treatment led to decrease in the TCD4+, TCD8+ e T?? lymphocytes accumulation in the lesions, and differentiated dendritic cell influx to the wounds. However there was no difference regarding CD4+CD25+ T cells in lymph nodes, but treated mice showed decrease Foxp3 expression. In conclusion, the triglycerides serum level was altered in the course of wound healing and may be associated to skin lesion, while PPAR? agonist acts in wound repair by accelerating healing and modulating neutrophil influx to the skin. Finally, our results suggested that PPAR? may be an important target for novel therapies aimed at improved wound healing, especially when administered topically.

No description available.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: Myocardial oxidative fuel supply is increased in obese conditions. How this metabolic environment and altered cardiometabolic phenotype associated with prediabetic obesity impacts on cardiac function and tolerance to ischaemia/reperfusion injury remains uncertain. While obese individuals are likely to be treated with PPARα agonists, controversy exists as to how activation of the PPARα receptor influences cardiovascular function and post-ischaemic recovery. Aims: To determine in a model of hyperphagia-induced obesity 1) whether protracted obesity is associated with left ventricular (LV) mechanical dysfunction; 2) the responsiveness of these hearts to insulin stimulation; 3) whether insulin can afford cardioprotection against ischaemia/reperfusion damage; and 4) how obesity and chronic PPARα agonist (K-111) treatment influences myocardial function, substrate metabolism, mitochondrial function and post-ischaemic outcomes. Methods: Male Wistar rats were fed standard rat chow or a high caloric diet. 1) In vivo LV mechanical function was assessed echocardiographically in 32 week fed animals. Ex vivo LV function was measured in the presence of glucose, insulin and/or fatty acid (FA); 2) Ex vivo myocardial insulin sensitivity was assessed by measuring insulin stimulated glycolytic flux in 16 week fed rats. Insulin was also administered prior to and during regional ischaemia to determine its effect on post-ischaemic function and infarct size; 3) K-111 was added to the drinking water during the last 10 weeks of feeding (feeding period of 18 weeks); a) Ventricular mitochondrial function was determined polarographically in the presence of either glutamate or palmitoyl-L-carnitine as substrates; b) Myocardial carbohydrate and lipid metabolism, and in a separate series of perfusions, myocardial infarct size were determined in the presence of physiological or high insulin (30 or 50μIU/ml) and FA (0.7 or 1.5mM) concentrations. Results: 1) Obese animals maintained normal in vivo LV mechanical function. Glucose perfused hearts from obese animals had depressed aortic outputs compared to the control group (32.58±1.2 vs. 46.17±0.91 ml/min; p<0.001) which was abolished by the presence of FA; 2) Hearts from obese animals had reduced insulin stimulated glycolytic flux rates (1.54±0.42 vs. 2.16±0.57 μmol/g ww/min, p<0.01). Although insulin reduced infarct size in the obese group (20.94±1.60 vs. 41.67±2.09 %, p<0.001), its cardioprotective effect was attenuated in the presence of FA; 3) By simulating the in vivo metabolic environment of control and obese animals in ex vivo perfusions, elevated insulin and FA levels associated with obesity increased infarct sizes in the obese group compared to the control group (47.44±3.13 vs. 37.17±2.63 %, p<0.05); 4) While chronic K-111 treatment reversed systemic metabolic abnormalities associated with obesity, neither obesity nor the drug influenced myocardial and mitochondrial function or postischaemic outcomes. K-111 was able to reduce palmitate oxidation in the obese group. Conclusion: Elevated levels of circulating FFA may be important in maintaining normal LV mechanical function in the obese condition. While obesity had no impact on myocardial mitochondrial function and post-ischaemic outcomes during comparable perfusion conditions, the specific metabolic environment associated with obesity may augment post-ischaemic injury. K-111 is effective in reducing obesity related metabolic abnormalities, but has no effects on myocardial function, mitochondrial function or ischaemic tolerance. / AFRIKAANSE OPSOMMING: Agtergrond: Miokardiale oksidatiewe substraat voorsiening is verhoog in vetsug. Hoe hierdie metaboliese omgewing en veranderde miokardiale metaboliese fenotipe in prediabetiese vetsug miokardiale funksie en iskemie/herperfusie skade beïnvloed, is onseker. Alhoewel vetsugtige individue met PPARα agoniste behandel kan word, is die resultate verkry van hierdie reseptor aktivering op miokardiale funksie en iskemiese skade teenstrydig. Doelwitte: Om te bepaal of 1) verlengde vetsug linker ventrikulêre (LV) funksie beïnvloed; 2) hierdie harte sensitief vir insulien stimulasie is; 3) insulien die hart teen iskemie/herperfusie beskadiging beskerm; en of 4) vetsug en chroniese K-111 behandeling miokardiale funksie, substraat metabolisme, mitochondriale funksie en post-iskemiese herstel in vetsugtige, insulienweerstandige rotte beïnvloed. Metodes: Manlike Wistar rotte is met gewone rotkos, of ʼn hoé kalorie dieet gevoer. 1) In vivo LV funksie in 32 week gevoerde rotte is met behulp van eggokardiografie bepaal. Ex vivo LV funksie is met of sonder insulien en/of vetsure in die perfusaat bepaal; 2) Die ex vivo insuliensensitiwiteit is in 16 weke gevoerde rotte bepaal deur miokardiale glikolise te meet. Insulien is ook voor en tydens streeksiskemie toegedien, ten einde sy effek op miokardiale beskerming te bepaal; 3) K-111 is in die drink water van rotte toegedien vir die laaste 10 weke van hul dieet (voedingsperiode van 18 weke); a) Ventrikulêre mitochondriale funksie is polarografies bepaal in die aanwesigheid van glutamaat of palmitiel-L-karnitien; b) Miokardiale koolhidraat- en lipied metabolisme, en in ʼn aparte groep rotte, infarktgrootte, is bepaal in die teenwoordigheid van fisiologiese of hoë insulien- (30 of 50μIU/ml) en vetsuurvlakke (0.7 of 1.5mM). Resultate: 1) Vetsugtige rotte het normale in vivo LV funksie gehandhaaf. Glukose geperfuseerde harte van vet rotte se LV funksie was laer as die van kontroles (Aorta omset: 32.58±1.2 vs. 46.17±0.91 ml/min; p<0.001), maar dit het verbeter in teenwoordigheid van vetsure; 2) Harte van vetsugtige rotte het verlaagde insuliengestimuleerde glikolise getoon (1.54±0.42 vs. 2.16±0.57 μmol/g ww/min, p<0.01). Alhoewel insulien infarktgrootte in die vetsugtige groep verlaag het (20.94±1.60 vs. 41.67±2.09 %, p<0.001), is sy beskermende effekte in die teenwoordigheid van vetsure verlaag; 3) deur die in vivo metaboliese omgewing van kontrole en vetsugtige rotte in die perfusaat van die harte ex vivo te simuleer, is dit aangetoon dat die verhoogde vlakke van insulien en vetsure, geassosieer met vetsugtigheid, infarktgroottes in die vetsugtige groep teenoor die kontrole groep verhoog het (47.44±3.13 vs 37.17±2.63 %, p<0.05); 4) Hoewel chroniese gebruik van K-111 die metaboliese abnormaliteite gepaardgaande met vetsug normaliseer het, het beide vetsug en die middel geen invloed op miokardiale of mitochondriale funksie of vatbaarheid vir iskemiese beskadiging gehad nie. K-111 het miokardiale palmitaatoksidasie in die vetsugtige behandelde groep verlaag. Gevolgtrekking: Verhoogde bloed vetsuurvlakke in vetsug mag n belangrike rol in die handhawing van sistoliese funksie speel. Dit blyk dat die spesifieke in vivo omgewing geassosieer met vetsug wel tot verhoogte vatbaarheid vir iskemie/herperfusie skade mag lei. K-111 is effektief om die sistemiese metaboliese abnormaliteite gepaard met vetsugtigheid te verbeter, maar het geen effek op miokardiale funksie, mitochondriale funksie of vatbaarheid vir iskemie gehad nie.

La dermatite atopique (DA) est une maladie inflammatoire chronique de la peau caractérisée par un épaississement épidermique et un infiltrat dermique de lymphocytes T mémoire activés, macrophages, mastocytes et éosinophiles avec des phases aiguë et chronique associées respectivement à des profils cytokiniques de type Th2 et Th1. Chez la majorité des patients atteints de DA, une augmentation de production d'IgE et IgG totales et spécifiques d'allergènes et d'antigènes microbiens est observée. Les récepteurs de forte et de faible affinité pour l'IgE, FcepsilonRI et FcepsilonRII/CD23 et le récepteur de faible affinité pour l'IgG, FcgammaRIII/CD16, jouent un rôle essentiel dans les maladies allergiques. Dans la peau humaine, ces FcR sont exprimés par des cellules présentatrices d'antigène et des cellules effectrices résidentes ou recrutées au derme durant l'inflammation. Au cours de ce travail, nous avons étudié le rôle de ces FcR dans un modèle murin de DA, qui reproduit la pathologie humaine, en comparant des animaux déficients pour ces FcR aux animaux correspondants de type sauvage (WT). Les symptômes de la DA sont complètement absents chez les souris déficientes en FcRgamma et partiellement inhibés dans les souris déficientes en FcepsilonRI ou CD16. Cette inhibition est corrélée avec une augmentation de l'expression cutanée de l'IL-10 et Foxp3. Alors que FcepsilonRI régule les réponses Th1 et Th2, le recrutement des mastocytes vers les ganglions drainants et la production d'IgE, CD16 régule uniquement la réponse Th2, la prolifération lymphocytaire et la production d'IgG1. FcepsilonRI et CD16 régulent spécifiquement la production de leurs ligands en contrôlant respectivement l'expression ganglionnaire d'IL-4 et d'IL-21. D'une façon importante, l'absence de CD23 aboutit à une inhibition drastique de la pathologie cutanée et, comme celle de FcepsilonRI, à une diminution des réponses cutanées Th1 et Th2 ainsi que de la production sérique de l'IgE mais pas celle de l'IgG1. Par ailleurs, nous avons étudié le rôle régulateur du récepteur nucléaire PPAR-alpha dans ce modèle de DA. En effet, il est exprimé par plusieurs types cellulaires au sein du système immunitaire et possède des propriétés anti-inflammatoires dans d'autres pathologies. Suite à la sensibilisation cutanée, nous avons remarqué que les souris déficientes en PPAR-alpha montrent une exacerbation des réponses cutanée et pulmonaire, et de la production de l'IgE et IgG2a par rapport aux souris WT. Ce phénomène est corrélé avec une exacerbation des réponses moléculaires cutanées Th2 et surtout Th1 ainsi qu'à une augmentation d'expression de NF-kappaB. D'une façon intéressante, l'expression de PPAR-alpha a été diminuée dans les lésions cutanées de patients atteints de DA suggérant donc que cette diminution d'expression puisse contribuer à la pathologie. Enfin, l'application d'un agoniste spécifique de PPAR-alpha diminue significativement la pathologie expérimentale. Nos travaux ont donc précisé la contribution des IgE/FcepsilonR et IgG/FcgammaR à la physiopathologie de la DA. Ces derniers, comme PPAR-alpha, constituent des cibles thérapeutiques potentielles pour cette affection.

La barrière hémato-encéphalique (BHE) est une interface localisée au niveau des cellules endothéliales des capillaires cérébraux. Elle présente des caractéristiques physiques et métaboliques spécifiques restreignant les échanges entre le sang et le cerveau dans le but de maintenir l’homéostasie du système nerveux central. Dans des conditions pathologiques comme l’ischémie cérébrale, la perte de son intégrité provoque l’apparition d’un oedème vasogénique qui aggrave considérablement le pronostic vital des patients. Malheureusement, les mécanismes impliqués dans l’hyperperméabilité vasculaire demeurent inconnus, ce qui limite l’utilisation de la seule thérapie disponible à 5% des patients. Depuis qu’aucun agent pharmacologique n’a réussit à être neuroprotecteur, notre compréhension des rapports entre le sang et le cerveau est remise en cause. La complexité des interactions entre la BHE et les cellules nerveuses a mené au concept d’une unité fonctionnelle dite neurovasculaire. Ainsi de nouvelles stratégies de protection émergent à partir d’observations au niveau vasculaire. Ainsi la première partie de nos travaux a consisté à étudier l’effet vasculoprotecteur potentiel du fénofibrate, un hypolipémiant agoniste du récepteur nucléaire PPAR-a (Peroxisome Proliferator- Activated Receptor-alpha), dont le bénéfice est observé en clinique depuis quelques années et plus récemment dans une étude expérimentale menée chez la souris. Les mécanismes de cette protection aujourd’hui inconnus, pourraient impliquer la BHE réputée très peu perméable à ce fibrate. Un renforcement de la BHE limiterait la formation de l’oedème cérébral. Pour cela nous avons adapté un modèle in vitro syngénique murin de BHE aux études de perméabilité en condition d’OGD (oxygen and glucose deprivation) mimant les conséquences immédiates de l’occlusion, toute première étape de l’accident vasculaire cérébral (AVC) ischémique. Le modèle consiste en une co-culture de cellules endothéliales primaires de capillaires cérébraux et de cellules gliales primaires. Nos travaux démontrent qu’un traitement préventif au fénofibrate protège l’endothélium en limitant l’hyperperméabilité induite par l’OGD. Cette action protectrice cible exclusivement l’endothélium et dépend de l’activation de PPAR-a démontré par l’absence d’effet protecteur sur les cellules endothéliales dont le gène codant pour PPAR-a a été invalidé. La seconde partie de l’étude s’est intéressée aux dommages vasculaires de la reperfusion, étape plus tardive de l’ischémie cérébrale connue pour aggraver l’oedème vasogénique et mener à des hémorragies fatales. A l’aide de notre modèle in vitro, nous avons étudié l’effet de la réoxygénation sur la perméabilité vasculaire dans le but de se rapprocher des conditions ischémiques in vivo. Après une incubation en condition d’OGD, la co-culture est replacée dans un milieu réoxygéné pendant une période allant de 2h à 24h. La mesure de la perméabilité vasculaire a démontré un profil multiphasique de l’ouverture de la BHE dépendant de la présence des cellules gliales. L’analyse en microscopie électronique des cellules endothéliales a suggéré une modulation fine de la fonctionnalité des jonctions serrées endothéliales. De plus, l’étude en IRM de diffusion chez la souris in vivo a révélé des mouvements d’eau qui suggèrent une perturbation de l’homéostasie hydrique du parenchyme cérébral au voisinage de l’occlusion dans les étapes précoces mais aussi dans les étapes tardives. En conclusion, l’ensemble des travaux met en avant la possibilité d’une préservation pharmacologique de l’intégrité de la BHE au début de l’ischémie cérébrale. Celle-ci montre l’intérêt des approches in vitro utilisant un modèle cellulaire pertinent et caractérisé. La validation de la cible cellulaire et moléculaire du fénofibrate à l’aide de notre modèle ouvre une première voie d’exploration des mécanismes impliqués dans ce phénomène de protection microvasculaire précoce. Cependant, la dysfonction retardée de la BHE est également un élément à prendre en compte pour se rapprocher de la physiopathologie de l’ischémie in vivo et espérer à terme une amélioration de l’approche thérapeutique de cette pathologie. / The Blood brain barrier (BBB) is an interface localised at brain capillary endothelial cells. The BBB possesses both physical and metabolic restrictive properties aiming at the maintenance of the central nervous system homeostasis. But under pathological conditions like ischemic stroke, the loss of BBB integrity induces a cerebral vasogenic edema which considerably worsens the vital prognosis of patients. The mechanisms underlying this vascular hyperpermeability are currently unknown thus limiting the use of the only medical intervention available at only 5% of stroke patients. Since no pharmacological molecule succeeded in being neuroprotective, our understanding of the relationships between blood and brain is questioned. The complex interactions between the BBB and nervous cells have lead to the concept of a functional unit, termed the neurovascular unit. Thus, new strategies are recently emerging from observation of vascular events. Thus, the first aim of our study was to test the potential vasculoprotective action of fenofibrate, a hypolipemic drug known as an activator of the nuclear receptor PPAR-a a (Peroxisome Proliferator-Activated Receptor-alpha), as benefit against stroke was observed in clinics since a few years, and recently reported in an experimental study. Yet unknown mechanisms, the protective effect may be exerted on the BBB since reported as impermeable to this compound. An early tightening of the BBB would limit the extent of brain edema. Hence, we have adapted a mouse syngenic BBB in vitro cell model to permeability studies under the stress condition found at the early stage of ischemic stroke defined in vitro as oxygen and glucose deprivation (OGD). This stress simulates the early consequences of occlusion. This model consists of a co-culture of primary brain capillary endothelial cells together with primary glial cells. We have demonstrated that a preventive treatment with fenofibrate has a protective effect on the BBB by limiting the hyperpermeability induced by the OGD condition. This effect targets endothelial cells exclusively and depends on PPAR-a activation, as revealed by the absence of protective action of fenofibrate on PPAR-a deficient endothelial cells. The second part of the study has focused on vascular reperfusion injury, a later stage of ischemia known to worsen vasogenic oedema and to lead to fatal haemorrhage. Using our in vitro BBB model, we have studied the effect of reoxygenation on vascular permeability in order to closely simulate in vivo ischemic condition. Following incubation under OGD condition, the co-cultures were placed into an oxygenated culture medium from 2h to 24h. The BBB permeability demonstrated a multiphasic opening of the BBB which depended on glial cells presence. Electronic microscopy analysis of BBB endothelial cells suggested a fine modulation of tight junction functionality. Moreover, the MRI diffusion analysis in mice has revealed particular water movements suggesting an early disturbance in water homeostasis of brain parenchyma in the vicinity of occlusion. In conclusion, this work put forward the idea of a pharmacological BBB protection at the early stage of ischemic stroke. This demonstrates the relevance of in vitro approaches using a pertinent and well characterised cell model. The validation of cellular and molecular targets of fenofibrate opens a way of first exploration of mechanisms involved in this early microvascular protection phenomenon. But the late BBB dysfunction also needs to be taken into account for a complete fitting with in vivo stroke pathophysiology and an improvement of the therapeutic approaches to this pathology.

Yau Wing Yiu, Winifred. / On t.p. "alpha"s appear as the Greek letter. / Thesis submitted in: October 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 91-98). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese version) --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Abbreviations --- p.xii / List of Figures --- p.xiv / List of Tables --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Peroxisome proliferator-activated receptors (PPARs) --- p.1 / Chapter 1.1.1 --- What are PPARs? --- p.1 / Chapter 1.1.2 --- PPAR ligands - peroxisome proliferators --- p.1 / Chapter 1.1.3 --- PPAR isoforms --- p.2 / Chapter 1.2 --- Biological roles of PPARα --- p.3 / Chapter 1.2.1 --- Lipid metabolism --- p.3 / Chapter 1.2.2 --- Glucose metabolism --- p.4 / Chapter 1.2.3 --- Inflammation --- p.5 / Chapter 1.2.4 --- Oxidative stress --- p.5 / Chapter 1.2.5 --- Cell proliferation and apoptosis --- p.6 / Chapter 1.3 --- PPARα in health and diseases --- p.6 / Chapter 1.3.1 --- Wound-healing --- p.6 / Chapter 1.3.2 --- Anti-atherogenesis --- p.7 / Chapter 1.3.3 --- Neuroprotection --- p.7 / Chapter 1.3.4 --- Carcineogenesis --- p.7 / Chapter 1.4 --- PPARα-regulated and starvation inducible gene (PPSIG) --- p.8 / Chapter 1.4.1 --- PPSIG is a PPARα target gene --- p.8 / Chapter 1.4.2 --- Computer-assisted predictions on PPSIG --- p.9 / Chapter 1.4.3 --- Current characterization of PPSIG --- p.10 / Chapter 1.5 --- Objectives of the present study --- p.11 / Chapter Chapter 2 --- Materials and Methods --- p.12 / Chapter 2.1 --- Materials --- p.12 / Chapter 2.2 --- Animals and treatment --- p.13 / Chapter 2.3 --- Cloning of PPSIG into pThioHis and pTYB expression vectors --- p.13 / Chapter 2.3.1 --- PCR amplification of PPSIG cDNA insert --- p.13 / Chapter 2.3.1.1 --- PPSIG cDNA insert for pThioHis vector --- p.13 / Chapter 2.3.1.2 --- PPSIG cDNA insert for pTYB vector --- p.15 / Chapter 2.3.2 --- Restriction enzyme digestion of PPSIG cDNA insert and pThioHis vector --- p.18 / Chapter 2.3.3 --- Restriction enzyme digestion of PPSIG cDNA insert and pTYB vector --- p.20 / Chapter 2.3.4 --- Ligation and transformation --- p.20 / Chapter 2.3.5 --- Screening for recombinants by phenol/chloroform method --- p.21 / Chapter 2.3.6 --- Confirmation of recombinant plasmid by restriction enzyme digestion --- p.22 / Chapter 2.3.6.1 --- Digestion of pThioHis-PPSIG plasmid with Xba I and Sac II --- p.22 / Chapter 2.3.6.2 --- Digestion of pTYB-PPSIG plasmid with EcoR V --- p.22 / Chapter 2.3.7 --- Transformation into expression E. coli strains --- p.23 / Chapter 2.4 --- Over expression of PPSIG proteins in E. coli --- p.23 / Chapter 2.5 --- Semi-purification of PPSIG fusion proteins by preparative SDS-PAGE --- p.24 / Chapter 2.6 --- Rabbit immunization --- p.25 / Chapter 2.7 --- Northern blotting analysis --- p.26 / Chapter 2.7.1 --- Probe preparation --- p.26 / Chapter 2.7.2 --- "Formaldehyde-agarose gel electrophoresis, blotting of RNA and hybridization" --- p.26 / Chapter 2.8 --- Subcellular fractionation --- p.29 / Chapter 2.9 --- Western blotting of liver microsomes --- p.31 / Chapter 2.10 --- Immunoprecipitation --- p.32 / Chapter 2.11 --- Mass spectrometry --- p.33 / Chapter 2.11.1 --- Trypsin digestion and peptide extraction --- p.33 / Chapter 2.11.2 --- Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry --- p.34 / Chapter Chapter 3 --- Results --- p.36 / Chapter 3.1 --- Cloning of PPSIG into pThioHis and pTYB vectors --- p.36 / Chapter 3.1.1 --- Cloning of PPSIG into pThioHis vector --- p.36 / Chapter 3.1.2 --- Cloning of PPSIG into pTYB vector --- p.36 / Chapter 3.2 --- Protein expression of Thio-PPSIG and Intein-PPSIG --- p.41 / Chapter 3.3 --- Identification of recombinant Thio-PPSIG and Intein-PPSIG by mass spectrometry --- p.49 / Chapter 3.4 --- Preparation and characterization of Thio-PPSIG and Intein-PPSIG antisera --- p.61 / Chapter 3.5 --- Identification of native PPSIG and its induction pattern --- p.65 / Chapter 3.5.1 --- PPSIG was highly inducible upon 72-h starvation in a PPARα dependent manner --- p.65 / Chapter 3.5.2 --- "PPSIG showed slight induction upon 2-wk Wy-14,643 treatment" --- p.71 / Chapter 3.6 --- Confirmation of the specificity of PPSIG antiserum --- p.74 / Chapter Chapter 4 --- Discussion --- p.81 / References --- p.91 / Appendix A Deduced amino acid sequences of PPSIG fusion proteins --- p.99 / Chapter A1 --- Deduced amino acid sequence of Thio-PPSIG from pThioHis-PPSIG plasmid --- p.99 / Chapter A2 --- Deduced amino acid sequence of Intein-PPSIG from pTYB-PPSIG plasmid --- p.101 / Appendix B Mass spectra of trypsin digested native PPSIG --- p.104 / Chapter B1 --- Mass spectrum of trypsin digested native PPSIG immunoprecipitated from liver microsomes from PPARα wild-type mice fed with normal diet (starvation experiment) --- p.104 / Chapter B2 --- Mass spectrum of trypsin digested native PPSIG immunoprecipitated from liver microsomes from PPARα wild-type mice starved for 72 hours (starvation experiment) --- p.105 / Chapter B3 --- "Mass spectrum of trypsin digested native PPSIG immunoprecipitated from liver microsomes from PPARα wild-type mice fed with control diet (Wy-14,643 feeding experiment)" --- p.106 / Chapter B4 --- "Mass spectrum of trypsin digested native PPSIG immunoprecipitated from liver microsomes from PPARα wild-type mice fed with 0.1% (w/w) Wy-14,643 for 2 weeks (Wy-14,643 feeding experiment)" --- p.107