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Os receptores nucleares compreendem uma superfamília de proteínas intracelulares reguladas relacionados estruturalmente, capazes de reconhecer sequências específicas de DNA e regulam a transcrição de genes alvos respondendo a sinais metabólicos, hormônios e outras moléculas regulatórias integrando muitas vias de sinalização. Os receptores ativadores da proliferação de peroxissomos (PPARs) são receptores nucleares que regem a transcrição de vários genes envolvidos principalmente no metabolismo de ácidos graxos e energia. A ativação do PPARY possui um amplo aspecto de funções biológicas, regulando o metabolismo, reduzindo a inflamação, influenciando o equilíbrio das células imunes, inibindo a apoptose e o estresse oxidativo e melhorando a função endotelial. Estes efeitos parecem ser benéficos não apenas em diabetes e aterosclerose, mas também em várias outras condições. Os agonistas do PPARY são utilizados como sensibilizadores de insulina para o tratamento da diabetes II, sendo um alvo molecular dos fármacos tiazolidinadionas. Diversos efeitos colaterais severos associados ao uso dos fármacos desta classe e à importância do PPARY no metabolismo de glicose e na sensibilização da insulina, o presente trabalho justifica-se como um esforço para avançar na compreensão da interação entre ligantes sintéticos com o receptor PPARY e a proposição de moléculas mais seguras e mais eficazes para a manutenção de níveis euglicêmicos. Foi realizada a expressão, a purificação, seguida de estudos cristalográficos em cinco ligantes selecionados a partir de etapas de docking realizados anteriormente pelo nosso grupo de Biotecnologia Molecular do Instituto de Física de São Carlos. Os ensaios de cristalização do PPARY complexado a ligantes sintéticos resultaram em duas estruturas cristalográficas que apresentaram uma conformação em que os ligantes não interagiram diretamente na hélice 12 como descritos para agonistas totais do PPARY, adotando características de agonistas parciais. Esses ligantes apresentaram interações hidrofóbicas que estabilizam as fitas-β. Este conjunto de informações estruturais apresentados neste trabalho para o PPARY proporcionou um entendimento das interações que esse receptor é capaz de fazer na presença de um ligante, além de que poderão ser úteis no desenvolvimento de novos moduladores seletivos do PPARY semelhante ao que já se encontram no mercado, porém com efeitos colaterais reduzidos. / Nuclear receptors comprise a superfamily of structurally-related regulated intracellular proteins capable of recognizing specific DNA sequences and regulating the transcription of target genes responding to metabolic signals, hormones and other regulatory molecules integrating many signaling pathways. Peroxisome proliferator-activating receptors (PPARs) are nuclear receptors that govern the transcription of several genes involved primarily in fatty acid and energy metabolism. Activation of PPARY has a broad aspect of biological functions, regulating metabolism, reducing inflammation, influencing immune cell balance, inhibiting apoptosis and oxidative stress, and improving endothelial function. These effects appear to be beneficial not only in diabetes and atherosclerosis, but also in several other conditions. PPARY agonists are used as insulin sensitizers for the treatment of diabetes II, being a molecular target of the thiazolidinediones drugs. A number of severe side effects associated with the use of drugs of this class and the importance of PPARY in glucose metabolism and insulin sensitization, the present work is justified as an effort to advance the understanding of the interaction between synthetic ligands with the PPARY receptor and proposing safer and more effective molecules for the maintenance of euglycemic levels. The expression, purification, followed by crystallographic studies in five ligands selected from docking steps previously performed by our Molecular Biotechnology group of the Physics Institute of São Carlos. The crystallization assays of PPARY complexed to synthetic ligands resulted in two crystallographic structures that exhibited a conformation in which the ligands did not interact directly in helix 12 as described for total PPARY agonists, adopting characteristics of partial agonists. These ligands showed hydrophobic interactions that stabilize the β-ribbons. This set of structural information presented in this work for the PPARY was of great value for the understanding of the interactions that this receptor is able to make in the presence of a ligand, besides that they could be useful in the development of new selective modulators of the PPARY similar to that are already on the market, but with reduced side effects.

Aside from an indirect effect of PPARgamma activation to reduce insulin resistance and to facilitate adiponectin release, PPARgamma agonist could also exert direct effects on blood vessels. I provided a first line of experimental evidence demonstrating that PPARgamma agonist rosiglitazone up-regulates the endothelin B receptor (ETBR) expression in mouse aortas and attenuates endothelin-1-induced vasoconstriction through an endothelial ET BR-dependent NO-related mechanism. ETBR up-regulation inhibits endothelin-1-induced endothelin A receptor (ETAR)-mediated constriction in aortas and mesenteric resistance arteries, while selective ETBR agonist produces endothelium-dependent relaxations in mesenteric resistance arteries. Chronic treatment with rosiglitazone in vivo or acute exposure to rosiglitazone in vitro up-regulate the ETsR expression without affecting ETAR expression. These results support a significant role of ETBR in contributing to the increased nitric oxide generation upon stimulation with PPARgamma agonist. This study provides additional explanation for how PPARgamma activation improves endothelial function. / Firstly, I demonstrated that adipocyte-derived adiponectin serves as a key link in PPARgamma-mediated amelioration of endothelial dysfunction in diabetes. Results from ex vivo fat explant culture with isolated arteries showed that PPARgamma expression and adiponectin synthesis in adipose tissues correlate with the degree of improvement of endothelium-dependent relaxation in aortas from diabetic db/db mice. PPARgamma agonist rosiglitazone elevates the adiponectin release and restores the impaired endothelium-dependent relaxation ex vivo and in vivo, in arteries from both genetic and diet-induced diabetic mice. The effect of PPARgamma activation on endothelial function that is mediated through the adiponectin- AMP-activated protein kinase (AMPK) cascade is confirmed with the use of selective pharmacological inhibitors and adiponectin -/- or PPARgamma+/- mice. In addition, the benefit of PPARgamma activation in vivo can be transferred by transplanting subcutaneous adipose tissue from rosiglitazone-treated diabetic mouse to control diabetic mouse. I also revealed a direct effect of adiponectin to rescue endothelium-dependent relaxation in diabetic mouse aortas, which involves both AMPK and cyclic AMP-dependent protein kinase signaling pathways to enhance nitric oxide formation accompanied with inhibition of oxidative stress. These novel findings clearly demonstrate that adipocyte-derived adiponectin is prerequisite for PPARgamma-mediated improvement of endothelial function in diabetes, and thus highlight the prospective of subcutaneous adipose tissue as a potentially important intervention target for newly developed PPARgamma agonists in the alleviation of diabetic vasculopathy. / To summarize, the present investigation has provided a few lines of novel mechanistic evidence in support for the positive roles of PPARgamma and PPARdelta activation as potentially therapeutic targets to combat against diabetic vasculopathy. / Type 2 diabetes mellitus and obesity represent a global health problem worldwide. Most diabetics die of cardiovascular and renal causes, thus increasing the urgency in developing effective strategies for improving cardiovascular outcomes, particularly in obesity-related diabetes. Recent evidence highlights the therapeutic potential of peroxisome proliferators activated receptor (PPAR) agonists in improving insulin sensitivity in diabetes. / While agonists of PPARalpha and PPARgamma are clinically used, PPARdelta is the remaining subtype that is yet to be a target for current therapeutic drugs. Little is available in literature about the role of PPARdelta in the regulation of cardiovascular function. The third part of my thesis focused on elucidating cellular mechanisms underlying the beneficial effect of PPARdelta activation in the modulation of endothelial function in diabetes. PPARdelta agonists restore the impaired endothelium-dependent relaxation in high glucose-treated aortas and in aortas from diabetic db/db mice through activation of a cascade involving PPARdelta, phosphatidylinositol 3-kinase, and Akt. PPARdelta activation increases Akt and endothelial nitric oxide synthase and nitric oxide production in endothelial cells. The crucial role of Akt is confirmed by selective pharmacological inhibitors and transient transfection of dominant negative Akt plasmid in these cells. Treatment with PPARdelta agonist GW501516 in vivo augments endothelial function in diabetic db/db and diet-induced obese mice. The specificity of GW501516 for PPARdelta is proven with the loss of its effect against high glucose-induced impairment of endothelium-dependent relaxation in aortas from PPARdelta knockout mice. In addition, oral administration of GW501516 in vivo fails to improve endothelial function in diet-induced obese PPARdelta deficient mice. / Tian, Xiaoyu. / Adviser: Huang Yu. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 132-165). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Ng Lui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 243-257). / Abstracts in English and Chinese. / Abstract --- p.i_iii / Abstract (Chinese version) --- p.iv-v / Acknowledgements --- p.vi / Table of Contents --- p.vii-xvii / List of Abbreviations --- p.xviii-xx / List of Figures --- p.xxi-xxvi / List of Tables --- p.xxvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Peroxisome Proliferators (PPs) --- p.1 / Chapter 1.2 --- Peroxisome proliferator-activated receptors (PPARs) --- p.3 / Chapter 1.2.1 --- What are PPARs? --- p.3 / Chapter 1.2.2 --- PPAR isoforms --- p.3 / Chapter 1.2.2.1 --- PPARp/δ --- p.3 / Chapter 1.2.2.2 --- PPARγ --- p.4 / Chapter 1.2.2.3 --- PPARα --- p.5 / Chapter 1.2.3 --- PPARα target genes --- p.5 / Chapter 1.2.3.1 --- Transcriptional regulation --- p.5 / Chapter 1.2.3.2 --- PPRE --- p.6 / Chapter 1.2.4 --- Physiological roles --- p.9 / Chapter 1.2.4.1 --- Lipid metabolism --- p.9 / Chapter 1.2.4.1.1 --- Cellular fatty acid uptake and fatty acid activation --- p.9 / Chapter 1.2.4.1.2 --- Intracellular fatty acid transport --- p.11 / Chapter 1.2.4.1.3 --- Mitochondrial fatty acid uptake --- p.12 / Chapter 1.2.4.1.4 --- Mitochondrial fatty-acid P-oxidation / Chapter 1.2.4.1.5 --- Peroxisomal fatty acid uptake --- p.13 / Chapter 1.2.4.1.6 --- Peroxisomal fatty acid oxidation --- p.13 / Chapter 1.2.4.1.7 --- Micorsomal co-hydroxylation of fatty acids --- p.14 / Chapter 1.2.4.1.8 --- Ketogenesis --- p.15 / Chapter 1.2.4.1.9 --- Bile acid metabolism --- p.15 / Chapter 1.2.4.1.10 --- Lipoprotein metabolism --- p.17 / Chapter 1.2.4.1.11 --- Hepatic lipogenesis --- p.18 / Chapter 1.2.4.2 --- Glucose metabolism --- p.19 / Chapter 1.2.4.2.1 --- Glycogenolysis --- p.19 / Chapter 1.2.4.2.2 --- Glycolysis --- p.20 / Chapter 1.2.4.2.3 --- Gluconeogenesis --- p.20 / Chapter 1.2.4.3 --- Urea cycle --- p.21 / Chapter 1.2.4.4 --- Biotransformation --- p.22 / Chapter 1.2.4.5 --- Inflammation --- p.23 / Chapter 1.2.4.6 --- Acute phase response --- p.23 / Chapter 1.2.5 --- Toxicological roles --- p.24 / Chapter 1.2.5.1 --- PPs induce hepatocarcinoma formation through PPARα --- p.24 / Chapter 1.2.5.2 --- Mechanism of PPARa-mediated PP-induced hepatocarcinoma --- p.25 / Chapter 1.2.5.2.1 --- Oxidative stress --- p.25 / Chapter 1.2.5.2.2 --- Hepatocellular proliferation and inhibition of apoptosis --- p.26 / Chapter 1.3 --- Discovery of novel PPARα target genes --- p.27 / Chapter 1.3.1 --- Peroxisome proliferator and starvation-induced gene (PPSIG) --- p.28 / Chapter 1.3.1.1 --- PPSIG is a putative PPARa target gene --- p.28 / Chapter 1.3.1.2 --- Examination of PPSIG FDD fragment cDNA sequence --- p.28 / Chapter 1.4 --- Objectives --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Cloning of the full-length mouse PPSIG cDNA --- p.38 / Chapter 2.1.1 --- Rapid amplification of cDNA ends (RACE) --- p.38 / Chapter 2.1.1.1 --- Total RNA extraction --- p.38 / Chapter 2.1.1.1.1 --- Materials --- p.38 / Chapter 2.1.1.1.2 --- Methods --- p.38 / Chapter 2.1.1.2 --- Primers design --- p.39 / Chapter 2.1.1.3 --- 5' and 3' cDNA ends amplification --- p.42 / Chapter 2.1.1.3.1 --- Materials --- p.42 / Chapter 2.1.1.3.2 --- Methods --- p.42 / Chapter 2.1.2 --- Subcloning of 5' and 3'RACED products --- p.45 / Chapter 2.1.2.1 --- Ligation and transformation --- p.45 / Chapter 2.1.2.1.1 --- Materials --- p.45 / Chapter 2.1.2.1.2 --- Methods --- p.46 / Chapter 2.1.2.2 --- Screening of the recombinants --- p.48 / Chapter 2.1.2.2.1 --- PhenoI:chloroform test --- p.48 / Chapter 2.1.2.2.1.1 --- Materials --- p.48 / Chapter 2.1.2.2.1.2 --- Methods --- p.48 / Chapter 2.1.2.2.2 --- Restriction enzyme digestion --- p.48 / Chapter 2.1.2.2.2.1 --- Materials --- p.48 / Chapter 2.1.2.2.2.2 --- Methods --- p.49 / Chapter 2.1.3 --- DNA sequencing of the 5'and 3'RACED subclones --- p.49 / Chapter 2.1.4 --- Northern blot analysis using PPSIG 5' and 3' RACED cDNA as probes --- p.52 / Chapter 2.1.4.1 --- RNA sample preparation --- p.52 / Chapter 2.1.4.1.1 --- Materials --- p.52 / Chapter 2.1.4.1.2 --- Methods --- p.52 / Chapter 2.1.4.2 --- Formaldehyde-agarose gel electrophoresis and blotting of RNA --- p.52 / Chapter 2.1.4.2.1 --- Materials --- p.52 / Chapter 2.1.4.2.2 --- Methods --- p.53 / Chapter 2.1.4.3 --- Probe preparation --- p.55 / Chapter 2.1.4.3.1 --- DIG labeling of RNA probe from 5'RACED PPSIG cDN A subclone 5'#32 --- p.55 / Chapter 2.1.4.3.1.1 --- Materials --- p.55 / Chapter 2.1.4.3.1.2 --- Methods --- p.55 / Chapter 2.1.4.3.2 --- PCR DIG labeling of 3´ة RACED PPSIG cDNA subclone 3' #12 --- p.56 / Chapter 2.1.4.3.2.1 --- Materials --- p.56 / Chapter 2.1.4.3.2.2 --- Methods --- p.57 / Chapter 2.1.4.4 --- Hybridization --- p.57 / Chapter 2.1.4.4.1 --- Materials --- p.57 / Chapter 2.1.4.4.2 --- Methods --- p.57 / Chapter 2.1.4.5 --- Post-hybridization washing and colour development --- p.59 / Chapter 2.1.4.5.1 --- Materials --- p.59 / Chapter 2.1.4.5.2 --- Methods --- p.59 / Chapter 2.2 --- Cloning of the PPSIG genomic DNA --- p.61 / Chapter 2.2.1 --- Screening of bacterial artificial chromosome (BAC) clones --- p.61 / Chapter 2.2.1.1 --- Screening of a mouse genomic library --- p.61 / Chapter 2.2.1.2 --- "Purification of BAC DNA by solution I, II,III" --- p.61 / Chapter 2.2.1.2.1 --- Materials --- p.61 / Chapter 2.2.1.2.2 --- Methods --- p.61 / Chapter 2.2.2 --- Confirmation of PPSIG genomic BAC clones --- p.64 / Chapter 2.2.2.1 --- Genomic Southern blot analysis --- p.64 / Chapter 2.2.2.1.1 --- Agarose gel electrophoresis and blotting of BAC DNA --- p.64 / Chapter 2.2.2.1.1.1 --- Materials --- p.64 / Chapter 2.2.2.1.1.2 --- Methods --- p.64 / Chapter 2.2.2.1.2 --- DIG labeling of DNA probe by random priming --- p.65 / Chapter 2.2.2.1.2.1 --- Materials --- p.65 / Chapter 2.2.2.1.2.2 --- Methods --- p.65 / Chapter 2.2.2.1.3 --- Hybridization --- p.66 / Chapter 2.2.2.1.4 --- Post-hybridization washing and colour development --- p.66 / Chapter 2.2.2.2 --- EcoR I digestion --- p.67 / Chapter 2.2.2.2.1 --- Materials --- p.67 / Chapter 2.2.2.2.2 --- Methods --- p.67 / Chapter 2.2.2.3 --- Large scale preparation of BAC DNA --- p.67 / Chapter 2.2.2.3.1 --- Materials --- p.67 / Chapter 2.2.2.3.2 --- Methods --- p.68 / Chapter 2.2.3 --- Determination of PPSIG genomic sequences --- p.68 / Chapter 2.2.3.1 --- Primers design --- p.68 / Chapter 2.2.3.2 --- PCR --- p.73 / Chapter 2.2.3.2.1 --- Materials --- p.73 / Chapter 2.2.3.2.2 --- Methods --- p.73 / Chapter 2.2.3.3 --- Subcloning of the PPSIG genomic fragments --- p.73 / Chapter 2.2.3.3.1 --- Ligation and transformation --- p.73 / Chapter 2.2.3.3.2 --- PCR screening --- p.74 / Chapter 2.2.3.3.2.1 --- Materials --- p.74 / Chapter 2.2.3.3.2.2 --- Methods --- p.74 / Chapter 2.2.3.4 --- DNA sequencing --- p.75 / Chapter 2.3 --- Cloning of PPSIG-promoter reporter constructs --- p.75 / Chapter 2.3.1 --- Amplification of PPSIG 5'-flanking fragment by PCR --- p.75 / Chapter 2.3.1.1 --- Materials --- p.75 / Chapter 2.3.1.2 --- Methods --- p.75 / Chapter 2.3.2 --- Preparation of pGL3-Basic vector DNA --- p.81 / Chapter 2.3.2.1 --- Materials --- p.81 / Chapter 2.3.2.2 --- Methods --- p.81 / Chapter 2.3.3 --- Ligation and transformation --- p.84 / Chapter 2.3.3.1 --- Materials --- p.84 / Chapter 2.3.3.2 --- Methods --- p.84 / Chapter 2.3.4 --- Screening and confirmation of recombinants --- p.85 / Chapter 2.3.4.1 --- Materials --- p.85 / Chapter 2.3.4.2 --- Methods --- p.85 / Chapter 2.4 --- Cloning of PPSIG 5'-deletion promoter constructs --- p.87 / Chapter 2.4.1 --- Deletion of target fragments by restriction enzyme digestion --- p.87 / Chapter 2.4.1.1 --- Materials --- p.87 / Chapter 2.4.1.2 --- Methods --- p.88 / Chapter 2.4.2 --- Ligation and transformation --- p.90 / Chapter 2.4.2.1 --- Materials --- p.90 / Chapter 2.4.2.2 --- Methods --- p.90 / Chapter 2.4.3 --- Screening and confirmation of recombinants --- p.91 / Chapter 2.5 --- Cloning of PPSIG-PPRE reporter constructs --- p.91 / Chapter 2.5.1 --- Amplification of PPSIG-PPRE fragments --- p.91 / Chapter 2.5.1.1 --- Materials --- p.91 / Chapter 2.5.1.2 --- Methods --- p.93 / Chapter 2.5.2 --- Preparation of pGL3-Basic vector DNA --- p.96 / Chapter 2.5.2.1 --- Materials --- p.96 / Chapter 2.5.2.2 --- Methods --- p.96 / Chapter 2.5.3 --- Ligation and transformation --- p.97 / Chapter 2.5.3.1 --- Materials --- p.97 / Chapter 2.5.3.2 --- Methods --- p.97 / Chapter 2.5.4 --- Screening and confirmation of recombinants --- p.97 / Chapter 2.6 --- Cloning of PPSIG-PPRE deletion construct --- p.101 / Chapter 2.6.1 --- Deletion of PPRE fragment by Stu I/Xho I digestion --- p.101 / Chapter 2.6.1.1 --- Materials --- p.101 / Chapter 2.6.1.2 --- Methods --- p.101 / Chapter 2.6.2 --- "Ligation, transformation, screening and confirmation of recombinants" --- p.103 / Chapter 2.7 --- Construction of PPSIG-PPRE-deletion and PPSIG- PPRE-mutation constructs by site-directed mutagenesis --- p.105 / Chapter 2.7.1 --- Primers design --- p.105 / Chapter 2.7.2 --- Amplification of the left and right halves of the PPRE-deletion and PPRE-mutation constructs by PCR --- p.109 / Chapter 2.7.2.1 --- Materials --- p.109 / Chapter 2.7.2.2 --- Methods --- p.109 / Chapter 2.7.3 --- "Ligation, Dpn I digestion and transformation" --- p.110 / Chapter 2.7.3.1 --- Materials --- p.110 / Chapter 2.7.3.2 --- Methods --- p.110 / Chapter 2.7.4 --- Screening and confirmation of recombinants --- p.111 / Chapter 2.7.4.1 --- Materials --- p.111 / Chapter 2.7.4.2 --- Methods --- p.111 / Chapter 2.8 --- Cloning of mouse malonyl-CoA decarboxylase (MCD) and rat acyl-CoA binding protein (ACBP) PPRE reporter constructs --- p.112 / Chapter 2.8.1 --- Preparation of mouse and rat genomic DNA --- p.112 / Chapter 2.8.1.1 --- Materials --- p.112 / Chapter 2.8.1.2 --- Methods --- p.113 / Chapter 2.8.2 --- Amplification of MCD and ACBP PPRE fragments by PCR --- p.113 / Chapter 2.8.2.1 --- Materials --- p.113 / Chapter 2.8.2.2 --- Methods --- p.114 / Chapter 2.8.3 --- Ligation and transformation --- p.117 / Chapter 2.8.4 --- Screening and confirmation of recombinants --- p.117 / Chapter 2.9 --- Cloning of mPPARα and mRXRα expression plasmids --- p.119 / Chapter 2.9.1 --- RT-PCR of mouse PPARα and RXRa cDNAs --- p.119 / Chapter 2.9.1.1 --- Materials --- p.119 / Chapter 2.9.1.2 --- Methods --- p.119 / Chapter 2.9.2 --- Preparation of pSG5 vector DNA --- p.123 / Chapter 2.9.2.1 --- Materials --- p.123 / Chapter 2.9.2.2 --- Methods --- p.123 / Chapter 2.9.3 --- Ligation and transformation --- p.125 / Chapter 2.9.3.1 --- Materials --- p.125 / Chapter 2.9.3.2 --- Methods --- p.125 / Chapter 2.9.4 --- Screening and confirmation of recombinants --- p.125 / Chapter 2.9.4.1 --- Materials --- p.125 / Chapter 2.9.4.2 --- Methods --- p.126 / Chapter 2.10 --- Transient transfection and reporter assays --- p.128 / Chapter 2.10.1 --- Cell culture and transient transfection --- p.128 / Chapter 2.10.1.1 --- Materials --- p.128 / Chapter 2.10.1.2 --- Methods --- p.128 / Chapter 2.10.2 --- Assay for reporter construct luciferase activity --- p.131 / Chapter 2.10.2.1 --- Materials --- p.131 / Chapter 2.10.2.2 --- Methods --- p.131 / Chapter 2.11 --- Electrophoretic mobility-shift assay (EMSA) --- p.133 / Chapter 2.11.1 --- In vitro transcription/translation --- p.133 / Chapter 2.11.1.1 --- Materials --- p.133 / Chapter 2.11.1.2 --- Methods --- p.133 / Chapter 2.11.2 --- Preparation of AML-12 nuclear extract --- p.134 / Chapter 2.11.3 --- Preparation of DIG-labeled PPSIG-PPRE oligonucleotides --- p.136 / Chapter 2.11.3.1 --- Oligonucleotides design --- p.136 / Chapter 2.11.3.2 --- Annealing of single-stranded oligonucleotides to form double- stranded oligonucleotides --- p.136 / Chapter 2.11.3.2.1 --- Materials --- p.136 / Chapter 2.11.3.2.2 --- Methods --- p.138 / Chapter 2.11.3.3 --- 3' end labeling of the double-stranded oligonucleotides --- p.138 / Chapter 2.11.3.3.1 --- Materials --- p.138 / Chapter 2.11.3.3.2 --- Methods --- p.138 / Chapter 2.11.3.4 --- Testing the labeling efficiency of the double-stranded oligonucleoides --- p.139 / Chapter 2.11.3.4.1 --- Materials --- p.139 / Chapter 2.11.3.4.2 --- Methods --- p.139 / Chapter 2.11.4 --- Preparation of unlabeled oligonucleotides as competitors --- p.140 / Chapter 2.11.5 --- Binding reactions --- p.142 / Chapter 2.11.5.1 --- Perform with in vitro transcribed/translated proteins --- p.142 / Chapter 2.11.5.1.1 --- Materials --- p.142 / Chapter 2.11.5.1.2 --- Methods --- p.142 / Chapter 2.11.5.2 --- Perform with AML-12 nuclear extracts --- p.144 / Chapter 2.11.5.2.1 --- Materials --- p.144 / Chapter 2.11.5.2.2 --- Methods --- p.144 / Chapter 2.11.6 --- Detection of shift-up pattern --- p.145 / Chapter 2.11.6.1 --- Materials --- p.145 / Chapter 2.11.6.2 --- Methods --- p.145 / Chapter 2.12 --- Statistical analysis --- p.146 / Chapter Chapter 3 --- Results --- p.147 / Chapter 3.1 --- PPSIG cDNA sequence analysis --- p.147 / Chapter 3.1.1 --- Cloning of PPSIG full-length cDNA sequence --- p.147 / Chapter 3.1.2 --- Northern blot analysis of PPSIG --- p.160 / Chapter 3.1.3 --- "Comparison of PPSIG, Riken cDNA 0610039N19 and all-trans-13'14-dihydroretinol saturase cDNA sequences" --- p.163 / Chapter 3.2 --- PPSIG genomic sequence analysis --- p.166 / Chapter 3.2.1 --- Screening of the PPSIG BAC clone --- p.166 / Chapter 3.2.2 --- Cloning of PPSIG genomic fragments --- p.167 / Chapter 3.2.3 --- Examination of PPSIG genomic organization --- p.170 / Chapter 3.2.3.1 --- "Comparison of PPSIG, Riken cDNA 0610039N19 and all-trans-13'14-dihydroretinol saturase genomic sequence" --- p.177 / Chapter 3.3 --- Characterization of the 5'-flanking region of PPSIG --- p.184 / Chapter 3.4 --- Identification of a functional PPRE in the intron 1 of PPSIG gene --- p.201 / Chapter 3.5 --- Gel shift analysis of PPARa/RXRa heterodimer to PPSIG-PPRE --- p.222 / Chapter Chapter 4 --- Discussion --- p.234 / Chapter Chapter 5 --- Future studies --- p.241 / References --- p.243 / Appendix A Seating plan of transfection experiments (24-wells) / Chapter A1 --- Transfection experiment to study PPSIG-promoter reporter constructs --- p.258 / Chapter A2 --- Transfection experiment to study the PPSIG- promoter deletion constructs --- p.259 / Chapter A3 --- Transfection experiment to study the PPSIG-PPRE reporter constructs --- p.260 / Chapter A4 --- Transfection experiment to study PPSIG-PPRE- deletion and PPSIG-PPRE-mutation constructs --- p.262 / Appendix B Alignment result of RACE clone DNAs --- p.265 / Chapter B1 --- Alignment result of 5´ة#7 --- p.265 / Chapter B2 --- Alignment result of 5'#11 --- p.267 / Chapter B3 --- Alignment result of 5'#12 --- p.269 / Chapter B4 --- Alignment result of 5´ة#16 --- p.271 / Chapter B5 --- Alignment result of 5´ة#20 --- p.274 / Chapter B6 --- Alignment result of 5´ة#31 --- p.276 / Chapter B7 --- Alignment result of 5´ة#32 --- p.278 / Chapter B8 --- Consensus sequence of each 5'RACED clone --- p.280 / Chapter B9 --- Alignment result of all 5'RACE clones consensus sequence --- p.287 / Chapter B10 --- Alignment result of 3´ة#2 --- p.290 / Chapter B11 --- Alignment result of 3´ة#3 --- p.291 / Chapter B12 --- Alignment result of 3´ة#14 --- p.292 / Chapter B13 --- Alignment result of 3´ة#5 --- p.293 / Chapter B14 --- Alignment result of 3´ة#6 --- p.294 / Chapter B15 --- Alignment result of 3´ة#8 --- p.295 / Chapter B16 --- Alignment result of 3´ة#10 --- p.297 / Chapter B17 --- Alignment result of 3´ة#11 --- p.298 / Chapter B18 --- Alignment result of 3´ة#12 --- p.299 / Chapter B19 --- Alignment result of 3´ة#16 --- p.301 / Chapter B20 --- Alignment result of 3´ة#22 --- p.302 / Chapter B21 --- Alignment result of 3´ة#25 --- p.303 / Chapter B22 --- Consensus sequence of each 3'RACED clone --- p.305 / Chapter B23 --- Alignment result of all 3' RACE clones consensus sequence --- p.310 / Appendix C DNA sequencing and alignment result of PPSIG genomic fragments --- p.312 / Chapter C1 --- Exon 1 to exon 2 --- p.312 / Chapter C2 --- Exon 2 to exon 3 --- p.315 / Chapter C3 --- Exon 3 to exon 4 --- p.316 / Chapter C4 --- Exon 4 to exon 5 --- p.318 / Chapter C5 --- Exon 5 to exon 6 --- p.319 / Chapter C6 --- Exon 6 to exon 7 --- p.321 / Chapter C7 --- Exon 7 to exon 8 --- p.322 / Chapter C8 --- Exon 8 to exon 9 --- p.323 / Chapter C9 --- Exon 9 to exon 10 --- p.324 / Chapter C10 --- Exon 10 to exon 11 --- p.325 / Chapter C11 --- Exon 11 to downstream --- p.326 / Chapter C12 --- Consensus sequence of each BAC genomic DNA fragment --- p.328 / Chapter C13 --- The alignment result of all the PPSIG genomic sequence --- p.335 / Appendix D DNA sequencing and alignment result of constructs --- p.347 / Chapter D1 --- "pGL3-PPSIG (-2936/+119), pGL3-PPSIG (-1534/+119), pGL3-PPSIG (-879/+119) and pGL3- PPSIG (-375/+119) reporter constructs DNA sequencing and alignment result" --- p.347 / Chapter D2 --- pSG5-PPARa expression plasmid DNA sequencing and alignment result --- p.351 / Chapter D3 --- pSG5-RXRa expression plasmid DNA sequencing and alignment result --- p.353 / Chapter D4 --- pGL3-MCD reporter constructs DNA sequencing and alignment result --- p.355 / Chapter D5 --- pGL3-PPSIG (-229/+435) reporter construct DNA sequencing and alignment result --- p.356 / Chapter D6 --- pGL3-PPSIG (+94/+435) and pGL3-PPSIG (+94/+190) reporter constructs DNA sequencing and alignment result --- p.357 / Chapter D7 --- pGL3-PPSIG (-229/+3031) reporter construct DNA sequencing and alignment result --- p.358 / Chapter D8 --- pGL3-PPSIG (+94/+3031) reporter construct DNA sequencing and alignment result --- p.360 / Chapter D9 --- pGL3-ACBP reporter construct DNA sequencing and alignment result --- p.362 / Chapter D10 --- PPSIG-PPRE-deletion and PPSIG-PPRE-mutation constructs DNA sequencing and alignment result --- p.363

Os receptores nucleares compreendem uma superfamília de proteínas intracelulares reguladas relacionados estruturalmente, capazes de reconhecer sequências específicas de DNA e regulam a transcrição de genes alvos respondendo a sinais metabólicos, hormônios e outras moléculas regulatórias integrando muitas vias de sinalização. Os receptores ativadores da proliferação de peroxissomos (PPARs) são receptores nucleares que regem a transcrição de vários genes envolvidos principalmente no metabolismo de ácidos graxos e energia. A ativação do PPARY possui um amplo aspecto de funções biológicas, regulando o metabolismo, reduzindo a inflamação, influenciando o equilíbrio das células imunes, inibindo a apoptose e o estresse oxidativo e melhorando a função endotelial. Estes efeitos parecem ser benéficos não apenas em diabetes e aterosclerose, mas também em várias outras condições. Os agonistas do PPARY são utilizados como sensibilizadores de insulina para o tratamento da diabetes II, sendo um alvo molecular dos fármacos tiazolidinadionas. Diversos efeitos colaterais severos associados ao uso dos fármacos desta classe e à importância do PPARY no metabolismo de glicose e na sensibilização da insulina, o presente trabalho justifica-se como um esforço para avançar na compreensão da interação entre ligantes sintéticos com o receptor PPARY e a proposição de moléculas mais seguras e mais eficazes para a manutenção de níveis euglicêmicos. Foi realizada a expressão, a purificação, seguida de estudos cristalográficos em cinco ligantes selecionados a partir de etapas de docking realizados anteriormente pelo nosso grupo de Biotecnologia Molecular do Instituto de Física de São Carlos. Os ensaios de cristalização do PPARY complexado a ligantes sintéticos resultaram em duas estruturas cristalográficas que apresentaram uma conformação em que os ligantes não interagiram diretamente na hélice 12 como descritos para agonistas totais do PPARY, adotando características de agonistas parciais. Esses ligantes apresentaram interações hidrofóbicas que estabilizam as fitas-β. Este conjunto de informações estruturais apresentados neste trabalho para o PPARY proporcionou um entendimento das interações que esse receptor é capaz de fazer na presença de um ligante, além de que poderão ser úteis no desenvolvimento de novos moduladores seletivos do PPARY semelhante ao que já se encontram no mercado, porém com efeitos colaterais reduzidos. / Nuclear receptors comprise a superfamily of structurally-related regulated intracellular proteins capable of recognizing specific DNA sequences and regulating the transcription of target genes responding to metabolic signals, hormones and other regulatory molecules integrating many signaling pathways. Peroxisome proliferator-activating receptors (PPARs) are nuclear receptors that govern the transcription of several genes involved primarily in fatty acid and energy metabolism. Activation of PPARY has a broad aspect of biological functions, regulating metabolism, reducing inflammation, influencing immune cell balance, inhibiting apoptosis and oxidative stress, and improving endothelial function. These effects appear to be beneficial not only in diabetes and atherosclerosis, but also in several other conditions. PPARY agonists are used as insulin sensitizers for the treatment of diabetes II, being a molecular target of the thiazolidinediones drugs. A number of severe side effects associated with the use of drugs of this class and the importance of PPARY in glucose metabolism and insulin sensitization, the present work is justified as an effort to advance the understanding of the interaction between synthetic ligands with the PPARY receptor and proposing safer and more effective molecules for the maintenance of euglycemic levels. The expression, purification, followed by crystallographic studies in five ligands selected from docking steps previously performed by our Molecular Biotechnology group of the Physics Institute of São Carlos. The crystallization assays of PPARY complexed to synthetic ligands resulted in two crystallographic structures that exhibited a conformation in which the ligands did not interact directly in helix 12 as described for total PPARY agonists, adopting characteristics of partial agonists. These ligands showed hydrophobic interactions that stabilize the β-ribbons. This set of structural information presented in this work for the PPARY was of great value for the understanding of the interactions that this receptor is able to make in the presence of a ligand, besides that they could be useful in the development of new selective modulators of the PPARY similar to that are already on the market, but with reduced side effects.

L’adrénoleucodystrophie liée à l’X (X-ALD) est une maladie neurodégénérative sévère caractérisée par une accumulation d’acides gras à très longue chaîne (AGTLC), conséquence d’un défaut de β-oxydation peroxysomale. La maladie est associée à l’absence de la protéine ABCD1, transporteur ABC du peroxysome qui, tout comme son homologue le plus proche, ABCD2, participe à l’import des AGTLC-CoA au sein du peroxysome, l’unique site de leur dégradation par β-oxydation. La compréhension des mécanismes physiopathologiques est aujourd’hui limitée par le manque de modèles expérimentaux pertinents, cellulaires ou animaux. Puisque le défaut peroxysomal dans la microglie apparait comme un événement pathogénique majeur, nous avons généré des lignées de cellules microgliales incapable de transporter et/ou β-oxyder les AGTLC au sein du peroxysome. Quatre lignées cellulaires microgliales BV-2 déficientes en ABCD1, ABCD2, ABCD1 et ABCD2 ou ACOX1 (l’enzyme limitante de la β-oxydation peroxysomale) ont ainsi été générées par édition génique par CRISPR-Cas9. Ces cellules déficientes présentent d’importants défauts biochimiques, une accumulation d’AGTLC mais aussi des changements des contenus en acides gras et cholestérol. Les analyses ultrastructurales effectuées démontrent l’existence d’importantes inclusions lipidiques et indiquent également une augmentation du nombre de peroxysomes et mitochondries dans ces cellules. Les profils transcriptomiques signalent des altérations de la plasticité de ces cellules microgliales et de leur capacité de reprogrammation métabolique en réponse à un stimulus inflammatoire. Les fonctions de phagocytose ou de présentation antigénique des cellules microgliales semblent être affectées par le défaut peroxysomal. Enfin, les résultats obtenus à l’aide de ces modèles suggèrent que l’altération du métabolisme lipidique peroxysomal modifie l’organisation des membranes cellulaires. Ces lignées cellulaires apparaissent donc comme des modèles prometteurs, d’un grand intérêt pour la compréhension de la physiopathologie et l’identification de cibles thérapeutiques de cette maladie neurodégénérative complexe. / X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder characterized by very-long-chain fatty acid (VLCFA) accumulation resulting from a peroxisomal β-oxidation defect. The disease is caused by mutations in the ABCD1 gene, which encodes for a peroxisomal half ABC transporter predicted, like its closest homologue ABCD2, to participate in the entry of VLCFA-CoA into the peroxisome, the unique site of their β-oxidation. Progress in understanding the physiopathogenesis of X-ALD suffers from the lack of appropriate cell and animal models. Since peroxisomal defects in microglia seem to be a key element of the onset of the disease, we generated four microglial cell lines unable to transport and/or β-oxidize VLCFA into the peroxisome. BV-2 microglial cells were engineered with CRISPR-Cas9 to generate four microglial cell lines deficient in ABCD1, ABCD2, both ABCD1 and ABCD2 or ACOX-1 (the first rate-limiting enzyme of the peroxisomal β-oxidation system). Biochemical defects and lipid content changes associated with VLCFA accumulation but also fatty acids and cholesterol changes were identified in deficient microglia. Ultrastructural investigations confirmed cytosolic lipid inclusions and an increased number of peroxisome and mitochondria. Transcriptomic profiles of deficient microglia are indicative of an impaired plasticity and an impaired capacity to operate the metabolic shift required upon an inflammatory stimulation. Peroxisomal defect is likely to affect phagocytosis and antigen presentation capacity of microglia. Peroxisomal lipid metabolism defect is also suggested to modify cell membranes organization. Altogether, these novel mutant cell lines represent a promising model that should permit identification of new therapeutic targets for this complex neurodegenerative disease.

De part leur dimension élevée, les données de puces à ADN nécessitent l'application de méthodes statistiques pour en extraire une information pertinente. Dans le cadre de l'étude des différences entre deux agonistes de PPAR (Peroxisome Proliferator-Activated Receptor), nous avons sélectionné trois méthodes de sélection de variables : T-test, Nearest Shrunken Centroids (NSC) et Support Vector Machine – Recursive Feature Elimination. Ces méthodes ont été testées sur des données simulées et sur les données réelles de l'étude PPAR. En parallèle, une nouvelle méthodologie, MetRob, a été développée afin d'améliorer la robustesse ce ces méthodes vis à vis de la variabilité technique des puces à ADN, ainsi que leur reproductibilité. Cette nouvelle méthodologie permet principalement d'améliorer la valeur prédictive positive, c'est-à-dire la confiance accordée aux résultats. La méthode NSC s'est révélée la plus robuste et ce sont donc les résultats de cette méthode, associée à MetRob, qui ont été étudiés d'un point de vue biologique.

<p>Peroxisome is a ubiquitous eukaryotic organelle with a single-layer membrane. It maintains various functions that differ depending on the species and cell types, as well as the environmental or developmental conditions.</p><p>In the first part of this thesis, the peroxisomal protein content was systematically analyzed in different organs in mouse from different ages using proteomic approaches. Thirty-one peroxisomal proteins were identified and ten putative peroxisomal proteins were suggested. The results indicate that peroxisomal proteins show a tissue-specific functional response to the aging process that is probably dependent on their differential regeneration capacity. Besides, alteration in the fatty acid metabolism could alter membrane protein functions; decrease in catalase expression in kidney may contribute to oxidative stress and isoprenoid biosynthesis could contribute to decline in bile salt synthesis. The ability to detect changes in the peroxisomal proteome associated with organ impairment during the course of aging would provide a conceptual framework to understand the role of peroxisome in aging.</p><p>In the second part, peroxisome proteomics was used as a novel approach in marine pollution assessment. The peroxisomal protein expression profiles were obtained and identified from mussel Mytilus sp. exposed to different pollutants, in both laboratory and field experiments. The identified proteins were involved in α- and β–oxidation pathways, xenobiotics and amino acid metabolism, cell signalling, oxyradical metabolism, peroxisomal assembly, respiration and cytoskeleton pathway, etc. Generally, these findings suggest that protein expression signatures could become a valuable tool to monitor the presence of pollutants in marine environment.</p>

Peroxisome is a ubiquitous eukaryotic organelle with a single-layer membrane. It maintains various functions that differ depending on the species and cell types, as well as the environmental or developmental conditions. In the first part of this thesis, the peroxisomal protein content was systematically analyzed in different organs in mouse from different ages using proteomic approaches. Thirty-one peroxisomal proteins were identified and ten putative peroxisomal proteins were suggested. The results indicate that peroxisomal proteins show a tissue-specific functional response to the aging process that is probably dependent on their differential regeneration capacity. Besides, alteration in the fatty acid metabolism could alter membrane protein functions; decrease in catalase expression in kidney may contribute to oxidative stress and isoprenoid biosynthesis could contribute to decline in bile salt synthesis. The ability to detect changes in the peroxisomal proteome associated with organ impairment during the course of aging would provide a conceptual framework to understand the role of peroxisome in aging. In the second part, peroxisome proteomics was used as a novel approach in marine pollution assessment. The peroxisomal protein expression profiles were obtained and identified from mussel Mytilus sp. exposed to different pollutants, in both laboratory and field experiments. The identified proteins were involved in α- and β–oxidation pathways, xenobiotics and amino acid metabolism, cell signalling, oxyradical metabolism, peroxisomal assembly, respiration and cytoskeleton pathway, etc. Generally, these findings suggest that protein expression signatures could become a valuable tool to monitor the presence of pollutants in marine environment.

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