published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

No description available.

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

No description available.

Este estudo investigou o efeito da Roziglitazona (RTZ) sobre a perda óssea alveolar induzida pela periodontite (POAIP). Durante 3 semanas, ratos receberam sal puro de RTZ (i.p.) ou a formulaço comercial Avandia<font face=\"Symbol\">&#210; (v.o.); os grupos controles receberam os repectiovos veículos (DMSO ou CMC). Duas semanas após o inicio do tratamento, a periodontite (P) foi induzida. Após 7 dias da indução da P, as mandíbulas foram removidas para mediço da perda óssea alveolar. Amostras de osso alveolar foram analisadas por qPCR para RUNX2, Osterix, TRAF6, TRAF2, RANKL, óxido nítrico sintases (e, n e iNOS) e PPARs (<font face=\"Symbol\">a, <font face=\"Symbol\">b e <font face=\"Symbol\">g). A farmacocinética da RTZ para cada formulaço foi estudada por HPLC-MS/MS. Tanto o sal puro como a formulaço comercial de RTZ resultou no agravamento da POAIP. Apesar dos resultados similares nas concentrações plasmáticas de RTZ os mecanismos de sinalizaço parecem depender da formulaço administrada a qual pode ser devido a interferência do veículo. / This study investigate the effects of rosiglitazone (RTZ) on periodontitis-induced alveolar bone loss (PIABL). Rats received RTZ during 3 weeks, either as the pure maleate salt (i.p.) or the commercial formulation Avandia<font face=\"Symbol\">&#226; (p.o.); control animals received the respective vehicles (DMSO or CMC). Two weeks after the treatments begins, periodontitis (P) were induced. After 7 days after P induction, jaws were removed for ABL measurement. Alveolar bone samples were analyzed by qPCR for RUNX2, Osterix, TRAF6, TRAF2, RANKL, nitric oxide sintase (e, n and iNOS) and PPARs (<font face=\"Symbol\">a, <font face=\"Symbol\">b e <font face=\"Symbol\">g). RTZ pharmacokinetics from each formulation was also studied (HPLC-MS/MS). RTZ, either from the pure maleate salt or the commercial Avandia, resulted in aggravated PIABL. Despite resulting in similar plasma RTZ concentrations, signaling mechanisms seem to depend on the administered formulation which could be due to vehicle related effects interfence.

Tiazolidinadionas (TZDs) são agentes sensibilizadores de insulina que agem por ligação ao receptor gama ativado por proliferador de peroxissomos (PPAR&#947;). Elas têm apresentado efeitos cardioprotetores em humanos e propriedades anti-aterogênicas em modelos animais. Estudos in vitro têm sugerido que esses efeitos anti-aterogênicos da ativação de PPAR&#947; ocorrem por inibição da expressão de genes pro-inflamatórios e por aumentar o efluxo de colesterol via ativação dos receptores LXR-ABCA1. Entretanto, vários efeitos colaterais são associados ao tratamento com as TZDs, tornando necessária a pesquisa por novos compostos desta classe. Neste estudo, 14 novas tiazolidina-2,4- dionas, que são TZDs modificadas por bioisosterismo, foram avaliadas quanto à expressão de fatores aterogênicos e inflamatórios em linhagens de macrófagos J774 e RAW 264.7 e em camundongos com deleção genética para o receptor de LDL (LDLr-/-). Após a avaliação da citotoxicidade em macrófagos, foram eleitas cinco TZDs, denominadas de GQ-11, GQ-97, GQ-177, GQ-145 e LYSO-7. Três destas TZDs (GQ- 145, GQ-177 e LYSO-7) aumentaram significativamente a expressão de RNAm dos fatores de transcrição PPAR&#947;1, PPAR&#947;2 e do receptor CD36, assim como também aumentaram a expressão gênica de ABCA1 em 2.9, 3.5 e 6.7 vezes, respectivamente. Em adição, estas TZDs diminuíram a expressão gênica de iNOS, COX2, VCAM e IL-6 associado a redução na produção de nitritos, mas apenas a LYSO-7 reduziu significativamente a expressão desses genes quando comparada à rosiglitazona (RSG), além de diminuir a expressão da proteína-1 quimiotática para monócitos (MCP-1). No estudo experimental, os camundongos LDLr-/- machos foram alimentados com dieta hipercolesterolêmica por 16 semanas e quatro semanas antes da eutanásia receberam os derivados tiazolidínicos (20 mg/kg/dia) por gavagem. GQ-177 inibiu a progressão da placa aterosclerótica associada à aumento nas concentrações plasmáticas de HDL-C, com elevação na expressão de ABCA1, e redução da via inflamatória CD40-CD40L. LYSO-7 também mostrou inibição da aterogênese associada à redução das concentrações plasmáticas de colesterol total e triacilgliceróis, com diminuição na interação entre CD40-CD40L e expressão de citocinas inflamatórias. A GQ-145 não alterou os níveis plasmáticos dos lipídeos, mas aumentou a expressão de todos os genes pró-aterogênicos e pró-inflamatórios. Adicionalmente, as vias de ativação destas novas TZDs também foram estudadas por ensaio de luciferase, como gene repórter. A GQ-177 induziu ativação de PPAR&#947; e ligação ao seu domínio, enquanto a LYSO-7 estimulou ativação de PPAR&#945; e PPAR&#948;. Portanto, conclui-se que as novas TZDs, especialmente a GQ-177 e a LYSO-7, podem apresentar propriedades ateroprotetoras associadas ao transporte reverso de colesterol e aos efeitos antiinflamatórios, e poderiam ser uma alternativa promissora para o tratamento da aterosclerose. Porém, estudos complementares são requeridos para caracterizar as vias de sinalização intracelular, visto que as duas demonstraram ativar diferentes isotipos do fator de transcrição PPAR. / Thiazolidinediones (TZDs) are insulin-sensitizing agents that act by binding to peroxisome proliferator-activated receptor-&#947; (PPAR&#947;). They have been demonstrated to possess cardioprotective effects in humans and antiatherogenic properties in animal models. In vitro studies have also suggested that these antiatherogenic effects of PPAR&#947; activation occur by inhibiting the inflammatory gene expression and by increasing cholesterol efflux via LXR-ABCA1 activation. However, several side effects are associated with TZDs treatment making necessary the search for new compounds. In this study, 14 new thiazolidine-2,4-diones, modified TZDs by bioisosterism, were tested for aterogenic and inflammtary factors in RAW 264.7 macrophages and in low-density lipoprotein receptor-deficient mice. After the citotoxicity evaluation in RAW 264.7 macrophages the TZDs named GQ-11, GQ-97, GQ-177, GQ-145 e LYSO-7 were selected for this study. Three of these TZDs (GQ-177, GQ-145 and LYSO-7) significantly increased the expression of PPAR&#947;1, PPAR&#947;2 and CD36 mRNA, and enhanced the expression of ABCA1 mRNA in 2.9, 3.5 and 6.7 fold, respectively. Moreover, they also significantly decreased the expression of iNOS, COX2, VCAM and IL-6 mRNA in relation to control, and these results are associated to reduction on nitrits concentration. In addition, LYSO-7 significantly reduced the expression of these genes when compared to rosiglitazone, and decreased expression of MCP1 mRNA. In the experimental study, male LDLr-/- mice were fed an atherogenic diet containing 0.5% cholesterol for 16 weeks, and 4 weeks before euthanasia they received TZDs (20mg/kg/ per day) by gavage. GQ-177 treatment inhibited progression of atherosclerotic plaque associated to increased plasma concentrations of HDL-C, with enhance of ABCA1 expression and reduction on CD40-CD40L interaction. LYSO-7 treatment also showed inhibition of the atherogenesis associated to decreased plasma concentrations of total cholesterol and TAG, with reduction on CD40-CD40L pathway and inflammatory cytokines expression.GQ-145 did not alter the lipid plasma levels and increased the expression of all pro-atherogenic and pro-inflammatory genes. Furthermore, the activation of PPARs has also been studied, by luciferase assay as reporter gene. GQ-177 induced activation of PPAR&#947;, whereas LYSO-7 stimulated activation of PPAR&#945; and PPAR&#946;/&#948;. Altogether, our data suggest that the new TZDs derivatives, specially GQ- 177 and LYSO-7, may have atheroprotective properties associated with the reverse cholesterol transport and anti-inflammatory effects, and could be a promising alternative for the treatment of atherosclerosis. However, further studies are warranted in order to characterize the pathways of intracellular signaling since both have demonstrated to activate different isotypes of PPAR.

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