京都大学 / 新制・課程博士 / 博士(農学) / 甲第25356号 / 農博第2622号 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 井上 和生, 教授 佐々木 努, 准教授 後藤 剛 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Leung Wan-chi. / Thesis submitted in: November 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 276-284). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese Version) --- p.v / Acknowledgements --- p.viii / Tables of Contents --- p.ix / List of Abbreviations --- p.xxx / List of Figures --- p.xxxiii / List of Tables --- p.xlii / Chapter Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Peroxisome proliferator activator receptors --- p.1 / Chapter 1.2 --- Peroxisome proliferators --- p.6 / Chapter 1.2.1 --- Hepatomegaly --- p.9 / Chapter 1.2.2 --- Peroxisome proliferation --- p.11 / Chapter 1.2.3 --- Target genes regulation --- p.12 / Chapter 1.2.4 --- Hypolipidemic effect --- p.16 / Chapter 1.2.5 --- Hepatocarcinogenesis --- p.18 / Chapter 1.3 --- Mode of actions --- p.20 / Chapter 1.3.1 --- Oxidative stress --- p.21 / Chapter 1.3.2 --- Inhibition of apoptosis --- p.22 / Chapter 1.3.2 --- Increase in cell replication --- p.22 / Chapter 1.3.4 --- Alterations in cell cycle control --- p.23 / Chapter 1.4 --- Objectives --- p.23 / Chapter Chapter 2 --- Materials and Methods --- p.25 / Chapter 2.1 --- Animal tail-genotyping --- p.25 / Chapter 2.1.1 --- Materials --- p.25 / Chapter 2.1.2 --- Methods --- p.28 / Chapter 2.2 --- Animal treatment --- p.29 / Chapter 2.2.1 --- Materials --- p.29 / Chapter 2.2.2 --- Methods --- p.29 / Chapter 2.3 --- Serum cholesterol and tryiglyceride analysis --- p.30 / Chapter 2.3.1 --- Materials --- p.31 / Chapter 2.3.2 --- Methods --- p.31 / Chapter 2.3.2.1 --- Serum preparation --- p.31 / Chapter 2.3.2.2 --- Serum cholesterol analysis --- p.31 / Chapter 2.3.2.3 --- Serum triglyceride analysis --- p.32 / Chapter 2.4 --- Histological analysis --- p.32 / Chapter 2.4.1 --- Materials --- p.32 / Chapter 2.4.2 --- Methods --- p.33 / Chapter 2.5 --- Total RNA isolation --- p.34 / Chapter 2.5.1 --- Materials --- p.34 / Chapter 2.5.2 --- Methods --- p.34 / Chapter 2.6 --- DNase I treatment of total liver RNA --- p.37 / Chapter 2.6.1 --- Materials --- p.37 / Chapter 2.6.2 --- Methods --- p.37 / Chapter 2.7 --- Reverse transcription (RT) of mRNA and non- fluorescent PCR (non-fluoroDD PCR) --- p.38 / Chapter 2.7.1 --- Materials --- p.43 / Chapter 2.7.2 --- Methods --- p.43 / Chapter 2.8 --- Reverse transcription (RT) of mRNA and fluorescent PCR (fluoroDD PCR) --- p.44 / Chapter 2.8.1 --- Materials --- p.44 / Chapter 2.8.2 --- Method --- p.44 / Chapter 2.9 --- Fluorescent differential display (fluoroDD) --- p.45 / Chapter 2.9.1 --- Materials --- p.45 / Chapter 2.9.2 --- Methods --- p.45 / Chapter 2.9.2.1 --- FluoroDD gel preparation --- p.45 / Chapter 2.9.2.2 --- Sample preparation and electrophoresis --- p.45 / Chapter 2.10 --- Excision of differentially expressed cDNA fragments --- p.46 / Chapter 2.10.1 --- Materials --- p.46 / Chapter 2.10.2 --- Methods --- p.46 / Chapter 2.11 --- Reamplification of differentally expressed cDNA fragments --- p.48 / Chapter 2.11.1 --- Materials --- p.48 / Chapter 2.11.2 --- Methods --- p.50 / Chapter 2.12 --- Subcloning of reamplified cDNA fragmens --- p.50 / Chapter 2.12.1 --- Materials --- p.53 / Chapter 2.12.2 --- Methods --- p.53 / Chapter 2.12.2.1 --- Ligation --- p.53 / Chapter 2.12.2.2 --- Transformation --- p.53 / Chapter 2.12.2.3 --- Phenol-choloroform extraction --- p.54 / Chapter 2.12.2.4 --- Confirmation of insert size by EcoRI digestion --- p.54 / Chapter 2.12.2.5 --- Mini-preparation of plasmid DNA from recombinant clones --- p.55 / Chapter 2.13 --- Sequencing of subcloned cDNA fragments --- p.55 / Chapter 2.13.1 --- Materials --- p.56 / Chapter 2.13.2 --- Methods --- p.56 / Chapter 2.13.2.1 --- Sequencing of fluoroDD cDNA fragments --- p.56 / Chapter 2.13.2.2 --- Blast search against computer database --- p.57 / Chapter 2.14 --- Northern blot analysis of sequenced cDNA fragments --- p.57 / Chapter 2.14.1 --- Materials --- p.58 / Chapter 2.14.2 --- Methods --- p.58 / Chapter 2.14.2.1 --- Formaldehyde agarose gel electrophoresis of total RNA --- p.58 / Chapter 2.14.2.2 --- Preparation of DIG-labeled RNA probes for hybridization --- p.59 / Chapter 2.14.2.3 --- Preparation of PCR DIG-labeled cDNA probes for hybridization --- p.60 / Chapter 2.14.2.4 --- Hybridization and colour development --- p.60 / Chapter Chapter 3 --- Results --- p.62 / Chapter 3.1 --- Confirmation of genotypes by PCR --- p.62 / Chapter 3.2 --- Body weight changes --- p.62 / Chapter 3.3 --- Organ weight changes --- p.67 / Chapter 3.4 --- Serum cholesterol and triglyceride levels --- p.70 / Chapter 3.5 --- Liver histology --- p.78 / Chapter 3.6 --- Reverse transcription (RT) of mRNA and non-fluorescent PCR (non-flurroDD PCR) --- p.114 / Chapter 3.7 --- Reverse transcription (RT) of mRNA and fluorescent PCR (fluoroDD PCR) --- p.125 / Chapter 3.8 --- Reamplification of fluorescent differential display (FDD) fragments --- p.138 / Chapter 3.9 --- Subcloning of reamplifled FDD fragments --- p.162 / Chapter 3.10 --- Sequencing of subcloned cDNA fragments --- p.176 / Chapter 3.11 --- Northern blot analysis of sequenced cDNA fragments --- p.195 / Chapter Chapter 4 --- Discussion --- p.250 / Chapter 4.1 --- Body weight changes --- p.250 / Chapter 4.2 --- Organ weight changes --- p.251 / Chapter 4.3 --- Serum cholesterol and triglyceride levels --- p.253 / Chapter 4.4 --- Liver histology --- p.254 / Chapter 4.5 --- "Functions and roles of identified PPARa-dependent and Wy-14,643- responsive genes" --- p.255 / Chapter 4.6 --- Mechanism of PP-induced hepatocarcinogeneis --- p.270 / Chapter Chapter 5 --- Conclusions --- p.274 / References --- p.276 / Appendix A Tables of preparation of reaction mix --- p.285 / Table A1. Preparation of animal tail genotyping PCR reaction --- p.285 / Table A2. Preparation of DNase I treatment --- p.285 / Table A3. Preparation of reverse transcription of non-fluoroDD and fluoroDD --- p.285 / Table A4. Preparation of non-fluoroDD and fluoroDD RT-PCR --- p.286 / Table A5. Preparation of reamplification of differentially expressed cDNA fragments --- p.286 / Table A6. Preparation of PCR reaction for DNA sequencing --- p.286 / Table A7. Preparation of PCR reaction for RNA probe --- p.287 / Table A8. Preparation of PCR reaction for cDNA probe --- p.287 / Appendix B DNA sequences and sequencing alignments of FluoroDD Fragments --- p.288 / Chapter B 1.1: --- DNA sequence of cDNA subclone AA1#2 (AP1 & ARP2) using M13 forward (-20) primer --- p.288 / Chapter B 1.2: --- "Sequencing alignment of cDNA subclone AA1#2 with mouse peroxisomal delta 3, delta 2-enoyl-Coenzyme A isomerase (Peci) by BLAST searching against the National Center for Biotechnology Information database" --- p.288 / Chapter B 1.3: --- Summary of sequence alignment of cDNA subclone AA1#2 with mouse Peci --- p.288 / Chapter B 2.1: --- DNA sequence of cDNA subclone AA1#3 (AP1 & ARP2) using M13 forward (-20) primer --- p.289 / Chapter B 2.2: --- "Sequencing alignment of cDNA subclone AA1#3 with mouse peroxisomal delta 3, delta 2-enoyl-Coenzyme A isomerase (Peci) by BLAST searching against the National Center for Biotechnology Information database" --- p.289 / Chapter B 2.3: --- Summary of sequence alignment of cDNA subclone AA1#3 with mouse Peci --- p.289 / Chapter B 3.1: --- DNA sequence of cDNA subclone AA1#4 (AP 1 & ARP2) using Ml3 reverse primer --- p.290 / Chapter B 3.2: --- "Sequencing alignment of cDNA subclone AA1#4 with mouse peroxisomal delta 3, delta 2-enoyl-Coenzyme A isomerase (Peci) by BLAST searching against the National Center for Biotechnology Information database" --- p.290 / Chapter B 3.3: --- Summary of sequence alignment of cDNA subclone AA1#4 with mouse Peci --- p.290 / Chapter B 4.1: --- DNA sequence of cDNA subclone AA1#20 (AP 1 & ARP2) using Ml3 forward (-20) primer --- p.291 / Chapter B 4.2: --- "Sequencing alignment of cDNA subclone AA1#20 with mouse peroxisomal delta 3, delta 2- enoyl-Coenzyme A isomerase (Peci) by BLAST searching against the National Center for Biotechnology Information database" --- p.291 / Chapter B 4.3: --- Summary of sequence alignment of cDNA subclone AA1#20 with mouse Peci --- p.291 / Chapter B 5.1: --- DNA sequence of cDNA subclone AA4#1 (AP 1 & ARP2) using Ml3 forward (-20) primer --- p.292 / Chapter B 5.2: --- Sequencing alignment of cDNA subclone AA4#1 with mouse apolipoprotein A-V (Apoa5) by BLAST searching against the National Center for Biotechnology Information database --- p.292 / Chapter B 5.3: --- Summary of sequence alignment of cDNA subclone AA4#1 with mouse Apoa5 --- p.292 / Chapter B 6.1: --- DNA sequence of cDNA subclone AA4#9 (AP 1 & ARP2) using Ml3 reverse primer --- p.293 / Chapter B 6.2: --- Sequencing alignment of cDNA subclone AA4#9 with mouse apolipoprotein A-V (Apoa5) by BLAST searching against the National Center for Biotechnology Information database --- p.293 / Chapter B 6.3: --- Summary of sequence alignment of cDNA subclone AA4#9 with mouse Apoa5 --- p.293 / Chapter B 7.1: --- DNA sequence of cDNA subclone AA5#5 (AP 1 & ARP2) using Ml3 forward (-20) primer --- p.294 / Chapter B 7.2: --- Sequencing alignment of cDNA subclone AA5#5 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.294 / Chapter B 7.3: --- Summary of sequence alignment of cDNA subclone AA5#5 with mouse mitochondrion --- p.294 / Chapter B 8.1: --- DNA sequence of cDNA subclone AA6#1 (AP1 & ARP2) using Ml3 forward (-20) primer --- p.295 / Chapter B 8.2: --- Sequencing alignment of cDNA subclone AA6#1 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.295 / Chapter B 8.3: --- Summary of sequence alignment of cDNA subclone AA6#1 with mouse mitochondion --- p.295 / Chapter B 9.1: --- DNA sequence of cDNA subclone AA6#9 (AP 1 & ARP2) using Ml3 reverse primer --- p.296 / Chapter B 9.2: --- Sequencing alignment of cDNA subclone AA6#9 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.296 / Chapter B 9.3: --- Summary of sequence alignment of cDNA subclone AA6#9 with mouse mitochondrion --- p.296 / Chapter B 10.1: --- DNA sequence of cDNA subclone AA7#3 (AP 1 & ARP2) using Ml3 forward (-20) primer --- p.297 / Chapter B 10.2: --- Sequencing alignment of cDNA subclone AA7#3 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.297 / Chapter B 10.3: --- Summary of sequence alignment of cDNA subclone AA7#3 with mouse mitochondrion --- p.297 / Chapter B 11.1: --- DNA sequence of cDNA subclone AA7#5 (AP 1 & ARP2) using Ml3 reverse primer --- p.298 / Chapter B 11.2: --- Sequencing alignment of cDNA subclone AA7#5 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.298 / Chapter B 11.3: --- Summary of sequence alignment of cDNA subclone AA7#5 with mouse mitochondrion --- p.298 / Chapter B 12.1: --- DNA sequence of cDNA subclone AA10#1 (AP1 & ARP2) using M l3 forward (-20) primer --- p.299 / Chapter B 12.2: --- Sequencing alignment of cDNA subclone AA10#1 with mouse cysteine sulfinic acid decarboxylase (Csad) by BLAST searching against the National Center for Biotechnology Information database --- p.299 / Chapter B 12.3: --- Summary of sequence alignment of cDNA subclone AA10#1 with mouse Csad --- p.299 / Chapter B 13.1: --- DNA sequence of cDNA subclone AA10#1 (AP 1 & ARP2) using M13 reverse primer --- p.300 / Chapter B 13.2: --- Sequencing alignment of cDNA subclone AA10#1 with mouse cysteine sulfinic acid decarboxylase (Csad) by BLAST searching against the National Center for Biotechnology Information database --- p.300 / Chapter B 13.3: --- Summary of sequence alignment of cDNA subclone AA10#1 with mouse Csad --- p.300 / Chapter B 14.1: --- DNA sequence of cDNA subclone AA12#4 (AP1 & ARP2) using Ml3 forward (-20) primer --- p.301 / Chapter B 14.2: --- "Sequencing alignment of cDNA subclone AA12#4 with mouse acetyl-coenzyme A dehydrogenase, medium chain (MCAD) by BLAST searching against the National Center for Biotechnology Information database" --- p.301 / Chapter B 14.3: --- Summary of sequence alignment of cDNA subclone AA12#4 with mouse MCAD --- p.301 / Chapter B 15.1: --- DNA sequence of cDNA subclone AA12#4 (AP 1 & ARP2) using Ml3 reverse primer --- p.302 / Chapter B 15.2: --- "Sequencing alignment of cDNA subclone AA12#4 with mouse acetyl-coenzyme A dehydrogenase, medium chain (MCAD) by BLAST searching against the National Center for Biotechnology Information database" --- p.302 / Chapter B 15.3: --- Summary of sequence alignment of cDNA subclone AA12#4 with mouse MCAD --- p.302 / Chapter B 16.1: --- DNA sequence of cDNA subclone AB7#2 (AP3 & ARP3) using Ml3 forward (-20) primer --- p.303 / Chapter B 16.2: --- "Sequencing alignment of cDNA subclone AB7#2 with mouse UDP-glucuronosyltransferase 2 family, member 5 (UGT2b5) by BLAST searching against the National Center for Biotechnology Information database" --- p.303 / Chapter B 16.3: --- Summary of sequence alignment of cDNA subclone AB7#2 with mouse UGT2b5 --- p.303 / Chapter B 17.1: --- DNA sequence of cDNA subclone AB7#8 (AP3 & ARP3) using M13 reverse primer --- p.304 / Chapter B 17.2: --- "Sequencing alignment of cDNA subclone AB7#8 with mouse UDP-glucuronosyltransferase 2 family, member 5 (UGT2b5) by BLAST searching against the National Center for Biotechnology Information database" --- p.304 / Chapter B 17.3: --- Summary of sequence alignment of cDNA subclone AB7#8 with mouse UGT2b5 --- p.304 / Chapter B 18.1: --- DNA sequence of cDNA subclone AB17#16 (AP3 & ARP3) using M13 reverse primer --- p.305 / Chapter B 18.2: --- Sequencing alignment of cDNA subclone AB17#16 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.305 / Chapter B 18.3: --- Summary of sequence alignment of cDNA subclone AB17#16 with mouse mitochondrion --- p.305 / Chapter B 19.1: --- DNA sequence of cDNA subclone AB18#4 (AP3 & ARP3) using M13 forward (-20) primer --- p.306 / Chapter B 19.2: --- Sequencing alignment of cDNA subclone AB18#4 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.306 / Chapter B 20.1: --- DNA sequence of cDNA subclone AB18#4 (AP3 & ARP3) using M13 reverse primer --- p.307 / Chapter B 20.2: --- Sequencing alignment of cDNA subclone AB18#4 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.307 / Chapter B 20.3: --- Summary of sequence alignment of cDNA subclone AB 18#4 with mouse mitochondrion --- p.307 / Chapter B 21.1: --- DNA sequence of cDNA subclone AB19#2 (AP3 & ARP3) using M13 forward (-20) primer --- p.308 / Chapter B 21.2: --- Sequencing alignment of cDNA subclone AB 19#2 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.308 / Chapter B 21.3: --- Summary of sequence alignment of cDNA subclone AB19#2 with mouse mitochondrion --- p.308 / Chapter B 22.1: --- DNA sequence of cDNA subclone AB19#10 (AP3 & ARP3) using Ml3 reverse primer --- p.309 / Chapter B 22.2: --- Sequencing alignment of cDNA subclone AB 19#10 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.309 / Chapter B 22.3: --- Summary of sequence alignment of cDNA subclone AB19#10 with mouse mitochondrion --- p.309 / Chapter B 23.1: --- DNA sequence ofcDNA subclone AB22#9 (AP3 & ARP3) using M13 forward (-20) primer --- p.310 / Chapter B 23.2: --- Sequencing alignment of cDNA subclone AB22#9 with mouse peroxisome biogenesis factor 16 (Pexl6) by BLAST searching against the National Center for Biotechnology Information database --- p.310 / Chapter B 23.3: --- Summary of sequence alignment of cDNA subclone AB22#9 with mouse Pexl6 --- p.310 / Chapter B 24.1: --- DNA sequence of cDNA subclone AB22#9 (AP3 & ARP3) using Ml3 reverse primer --- p.311 / Chapter B 24.2: --- Sequencing alignment of cDNA subclone AB22#9 with mouse peroxisome biogenesis factor 16 (Pexl6) by BLAST searching against the National Center for Biotechnology Information database --- p.311 / Chapter B 24.3: --- Summary of sequence alignment of cDNA subclone AB22#9 with mouse Pexl6 --- p.311 / Chapter B 25.1: --- DNA sequence ofcDNA subclone AB24#9 (AP3 & ARP3) using Ml3 forward (-20) primer --- p.312 / Chapter B 25.2: --- Sequencing alignment of cDNA subclone AB24#9 with mouse Cyp4al4 by BLAST searching against the National Center for Biotechnology Information database --- p.312 / Chapter B 25.3: --- Summary of sequence alignment of cDNA subclone AB24#9 with mouse Cyp4al4 --- p.312 / Chapter B 26.1: --- DNA sequence of cDNA subclone AB24#9 (AP3 & ARP3) using M13 reverse primer --- p.313 / Chapter B 26.2: --- Sequencing alignment of cDNA subclone AB24#9 with mouse Cyp4al4 by BLAST searching against the National Center for Biotechnology Information database --- p.313 / Chapter B 26.3: --- Summary of sequence alignment of cDNA subclone AB24#9 with mouse Cyp4al4 --- p.313 / Chapter B 27.1: --- DNA sequence of cDNA subclone AB25#6 (AP3 & ARP3) using Ml3 forward (-20) primer --- p.314 / Chapter B 27.2: --- Sequencing alignment of cDNA subclone AB25#6 with mouse Cyp4a l4 by BLAST searching against the National Center for Biotechnology Information database --- p.314 / Chapter B 27.3: --- Summary of sequence alignment of cDNA subclone AB25#6 with mouse Cyp4al4 --- p.314 / Chapter B 28.1: --- DNA sequence of cDNA subclone AB26#17 (AP3 & ARP3) using Ml3 forward (-20) primer --- p.315 / Chapter B 28.2: --- Sequencing alignment of cDNA subclone AB26#17 with mouse Cyp4al4 by BLAST searching against the National Center for Biotechnology Information database --- p.315 / Chapter B 28.3: --- Summary of sequence alignment of cDNA subclone AB26#17 with mouse Cyp4al4 --- p.315 / Chapter B 29.1: --- DNA sequence of cDNA subclone AB26#3Q (AP3 & ARP3) using M13 reverse primer --- p.316 / Chapter B 29.2: --- Sequencing alignment of cDNA subclone AB26#30 with mouse Cyp4al4 by BLAST searching against the National Center for Biotechnology Information database --- p.316 / Chapter B 29.3: --- Summary of sequence alignment of cDNA subclone AB26#30 with mouse Cyp4al4 --- p.316 / Chapter B 30.1: --- DNA sequence of cDNA subclone AB29#7 (AP3 & ARP3) using Ml3 forward (-20) primer --- p.317 / Chapter B 30.2: --- Sequencing alignment of cDNA subclone AB29#7 with mouse catalase by BLAST searching against the National Center for Biotechnology Information database --- p.317 / Chapter B 30.3: --- Summary of sequence alignment of cDNA subclone AB29#7 with mouse catalase --- p.317 / Chapter B 31.1: --- DNA sequence of cDNA subclone AC1#1 (AP2 & ARP19) using Ml3 forward (-20) primer --- p.318 / Chapter B 31.2: --- Sequencing alignment of cDNA subclone AC1#1 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.318 / Chapter B 31.3: --- Summary of sequence alignment of cDNA subclone AC1#1 with mouse SPI --- p.318 / Chapter B 32.1: --- DNA sequence of cDNA subclone AC1#1 (AP2 & ARP 19) using Ml3 reverse primer --- p.319 / Chapter B 32.2: --- Sequencing alignment of cDNA subclone AC 1# 1 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.319 / Chapter B 32.3: --- Summary of sequence alignment of cDNA subclone AC1#1 with mouse SPI --- p.319 / Chapter B 33.1: --- DNA sequence of cDNA subclone AC1#2 (AP2& ARP 19) using M13 forward (-20) primer --- p.320 / Chapter B 33.2: --- Sequencing alignment of cDNA subclone AC 1#2 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.320 / Chapter B 33.3: --- Summary of sequence alignment of cDNA subclone AC1#2 with mouse SPI --- p.320 / Chapter B 34.1: --- DNA sequence of cDNA subclone AC1#2 (AP2& ARP 19) using M13 reverse primer --- p.321 / Chapter B 34.2: --- Sequencing alignment of cDNA subclone AC1#2 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.321 / Chapter B 34.3: --- Summary of sequence alignment of cDNA subclone AC1#2 with mouse SPI --- p.321 / Chapter B 35.1: --- DNA sequence ofcDNA subclone AC2#2 (AP2 & ARP19) using Ml3 reverse primer --- p.322 / Chapter B 35.2: --- Sequencing alignment of cDNA subclone AC2#2 with mouse bifunctional enzyme (PBFE) by BLAST searching against the National Center for Biotechnology Information database --- p.322 / Chapter B 35.3: --- Summary of sequence alignment of cDNA subclone AC2#2 with mouse PBFE --- p.322 / Chapter B 36.1: --- DNA sequence of cDNA subclone AC2#5 (AP2 & ARP19) using Ml3 reverse primer --- p.323 / Chapter B 36.2: --- Sequencing alignment of cDNA subclone AC2#5 with mouse catalase by BLAST searching against the National Center for Biotechnology Information database --- p.323 / Chapter B 36.3: --- Summary of sequence alignment of cDNA subclone AC2#5 with mouse catalase --- p.323 / Chapter B 37.1: --- DNA sequence of cDNA subclone AC2#6 (AP2 & ARP19) using Ml3 forward (-20) primer --- p.324 / Chapter B 37.2: --- Sequencing alignment of cDNA subclone AC2#6 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.324 / Chapter B 37.3: --- Summary of sequence alignment of cDNA subclone AC2#6 with mouse SPI --- p.324 / Chapter B 38.1: --- DNA sequence ofcDNA subclone AC4#3 (AP2 & ARP19) using Ml3 forward (-20) primer --- p.325 / Chapter B 38.2: --- Sequencing alignment of cDNA subclone AC4#3 with mouse Cyp2a5 by BLAST searching against the National Center for Biotechnology Information database --- p.325 / Chapter B 38.3: --- Summary of sequence alignment of cDNA subclone AC4#3 with mouse Cyp2a5 --- p.325 / Chapter B 39.1: --- DNA sequence ofcDNA subclone AC4#3 (AP2 & ARP 19) using M13 reverse primer --- p.326 / Chapter B 39.2: --- Sequencing alignment of cDNA subclone AC4#3 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.326 / Chapter B 39.3: --- Summary of sequence alignment of cDNA subclone AC4#3 with mouse SPI --- p.326 / Chapter B 40.1: --- DNA sequence of cDNA subclone AC7#5 (AP2& ARP 19) using M13 forward (-20) primer --- p.327 / Chapter B 40.2: --- Sequencing alignment of cDNA subclone AC7#5 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.327 / Chapter B 40.3: --- Summary of sequence alignment of cDNA subclone AC7#5 with mouse SPI --- p.327 / Chapter B 41.1: --- DNA sequence of cDNA subclone AD6#4 (AP2 & ARP 18) using Ml3 reverse primer --- p.328 / Chapter B 41.2: --- Sequencing alignment of cDNA subclone AD6#4 with mouse N-terminal Asn amidase (Ntanl) by BLAST searching against the National Center for Biotechnology Information database --- p.328 / Chapter B 41.3: --- Summary of sequence alignment of cDNA subclone AD6#4 with mouse Ntanl --- p.328 / Chapter B 42.1: --- DNA sequence of cDNA subclone AD6#10 (AP2 & ARP 18) using Ml3 forward (-20) primer --- p.329 / Chapter B 42.2: --- Sequencing alignment of cDNA subclone AD6#10 with mouse Cyp4al0 by BLAST searching against the National Center for Biotechnology Information database --- p.329 / Chapter B 42.3: --- Summary of sequence alignment of cDNA subclone AD6#10 with mouse Cvp4al0 --- p.329 / Chapter B 43.1: --- DNA sequence of cDNA subclone AD6#10 (AP2 & ARP18) using M13 reverse primer --- p.330 / Chapter B 43.2: --- Sequencing alignment of cDNA subclone AD6#10 with mouse Cyp4al0 by BLAST searching against the National Center for Biotechnology Information database --- p.330 / Chapter B 43.3: --- Summary of sequence alignment of cDNA subclone AD6#10 with mouse Cyp4al0 --- p.330 / Chapter B 44.1: --- DNA sequence of cDNA subclone AD8#2 (AP2 & ARP 18) using M13 forward (-20) primer --- p.331 / Chapter B 44.2: --- Sequencing alignment of cDNA subclone AD8#2with mouse Cyp4a l0 by BLAST searching against the National Center for Biotechnology Information database --- p.331 / Chapter B 44.3: --- Summary of sequence alignment of cDNA subclone AD8#2 with mouse Cvp4a10 --- p.331 / Chapter B 45.1: --- DNA sequence ofcDNA subclone AD8#7 (AP2 & ARP18) using Ml3 reverse primer --- p.332 / Chapter B 45.2: --- Sequencing alignment of cDNA subclone AD8#7 with mouse Cyp4al0 by BLAST searching against the National Center for Biotechnology Information database --- p.332 / Chapter B 45.3: --- Summary of sequence alignment of cDNA subclone AD8#7 with mouse Cyp4a10 --- p.332 / Chapter B 46.1: --- DNA sequence of cDNA subclone AD9#2 (AP2 & ARP 18) using Ml3 forward (-20) primer --- p.333 / Chapter B 46.2: --- Sequencing alignment of cDNA subclone AD9#2 with mouse Cyp4al0 by BLAST searching against the National Center for Biotechnology Information database --- p.333 / Chapter B 46.3: --- Summary of sequence alignment of cDNA subclone AD9#2 with mouse Cyp4al0 --- p.333 / Chapter B 47.1: --- DNA sequence of cDNA subclone AD9#3 (AP2 & ARP 18) using M13 reverse primer --- p.334 / Chapter B 47.2: --- Sequencing alignment of cDNA subclone AD9#3 with mouse Cyp4al0 by BLAST searching against the National Center for Biotechnology Information database --- p.334 / Chapter B 47.3: --- Summary of sequence alignment of cDNA subclone AD9#3 with mouse Cvp4a10 --- p.334 / Chapter B 48.1: --- DNA sequence ofcDNA subclone AF1#8 (AP10 & ARP13) using M13 forward (-20) primer --- p.335 / Chapter B 48.2: --- Sequencing alignment of cDNA subclone AF1#8 with mouse very-long-chain acyl-coA synthetase (VLACS) by BLAST searching against the National Center for Biotechnology Information database --- p.335 / Chapter B 48.3: --- Summary of sequence alignment of cDNA subclone AF1#8 with mouse VLACS --- p.335 / Chapter B 49.1: --- DNA sequence of cDNA subclone AF1#8 (AP 10 & ARP 13) using Ml3 reverse primer --- p.336 / Chapter B 49.2: --- Sequencing alignment of cDNA subclone AF1#8 with mouse very-long-chain acyl-coA synthetase (VLACS) by BLAST searching against the National Center for Biotechnology Information database --- p.336 / Chapter B 49.3: --- Summary of sequence alignment of cDNA subclone AF1#8 with mouse VLACS --- p.336 / Chapter B 50.1: --- DNA sequence of cDNA subclone AF21#5 (AP 10 & ARP 13) using M13 reverse primer --- p.337 / Chapter B 50.2: --- "Sequencing alignment ofcDNA subclone AF21#5 with mouse cell death-inducing DNA fragmentation factor, alpha subunit-like effector B (Cideb) by BLAST searching against the National Center for Biotechnology Information database" --- p.337 / Chapter B 50.3: --- Summary of sequence alignment of cDNA subclone AF21#5 with mouse Cideb --- p.337 / Chapter B 51.1: --- DNA sequence ofcDNA subclone AF25#6 (AP10 & ARP13) using M13 forward (-20) primer --- p.338 / Chapter B 51.2: --- Sequencing alignment of cDNA subclone AF25#6 with mouse major urinary protein 2 (MUPII) by BLAST searching against the National Center for Biotechnology Information database --- p.338 / Chapter B 51.3: --- Summary of sequence alignment of cDNA subclone AF25#6 with mouse MUP II --- p.338 / Chapter B 52.1: --- DNA sequence of cDNA subclone AF25#7 (AP 10 & ARP 13) using Ml3 reverse primer --- p.339 / Chapter B 52.2: --- Sequencing alignment of cDNA subclone AF25#7 with mouse major urinary protein 2 (MUP II) by BLAST searching against the National Center for Biotechnology Information database --- p.339 / Chapter B 52.3: --- Summary of sequence alignment of cDNA subclone AF25#7 with mouse MUPII --- p.339 / Chapter B 53.1: --- DNA sequence ofcDNA subclone AF30#4 (AP10 & ARP13) using M13 forward (-20) primer --- p.340 / Chapter B 53.2: --- Sequencing alignment of cDNA subclone AF30#4 with mouse mRNA for suppressor of actin mutations (SAC1 gene) by BLAST searching against the National Center for Biotechnology Information database --- p.340 / Chapter B 53.3: --- Summary of sequence alignment of cDNA subclone AF3Q#4 with mouse SAC1 --- p.340 / Chapter B 54.1: --- DNA sequence of cDNA subclone AF30#5 (AP 10 & ARP 13) using Ml3 reverse primer --- p.341 / Chapter B 54.2: --- Sequencing alignment of cDNA subclone AF30#5 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.341 / Chapter B 54.3: --- Summary of sequence alignment of cDNA subclone AF30#5 with mouse mitochondrion --- p.341 / Chapter B 55.1: --- DNA sequence ofcDNA subclone AH1#6 (AP11 & ARP19) using M13 forward (-20) primer --- p.342 / Chapter B 55.2: --- Sequencing alignment of cDNA subclone AH1#6 with mouse EST by BLAST searching against the National Center for Biotechnology Information database --- p.342 / Chapter B 55.3: --- Summary of sequence alignment of cDNA subclone AH1#6 with mouse EST --- p.342 / Chapter B 56.1: --- DNA sequence of cDNA subclone AIl#5 (AP6 & ARP4) using Ml3 forward (-20) primer --- p.343 / Chapter B 56.2: --- Sequencing alignment of cDNA subclone AIl#5 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.343 / Chapter B 56.3: --- Summary of sequence alignment of cDNA subclone All#5 with mouse SPI --- p.343 / Chapter B 57.1: --- DNA sequence of cDNA subclone AI1#5 (AP6 & ARP4) using Ml3 reverse primer --- p.344 / Chapter B 57.2: --- Sequencing alignment of cDNA subclone AIl#5 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST --- p.344 / Chapter B 57.3: --- Summary of sequence alignment of cDNA subclone AIl #5 with mouse SPI --- p.344 / Chapter B 58.1: --- DNA sequence of cDNA subclone AI18#6 (AP6 & ARP4) using Ml3 forward (-20) primer --- p.345 / Chapter B 58.2: --- Sequencing alignment of cDNA subclone AI18#6 with mouse argininosuccinate lyase (Asl) by BLAST searching against the National Center for Biotechnology Information database --- p.345 / Chapter B 58.3: --- Summary of sequence alignment of cDNA subclone AI18#6 with mouse Asl --- p.345 / Chapter B 59.1: --- DNA sequence of cDNA subclone AI18#6 (AP6 & ARP4) using M13 reverse primer --- p.346 / Chapter B 59.2: --- Sequencing alignment of cDNA subclone AI18#6 with mouse argininosuccinate lyase (Asl) by BLAST searching against the National Center for Biotechnology Information database --- p.346 / Chapter B 59.3: --- Summary of sequence alignment of cDNA subclone AI18#6 with mouse Asl --- p.346 / Chapter B 60.1: --- DNA sequence ofcDNA subclone AJ1#4 (AP6 & ARP14) using Ml3 forward (-20) primer --- p.347 / Chapter B 60.2: --- Sequencing alignment of cDNA subclone AJ1#4 with mouse carboxylesterase by BLAST searching against the National Center for Biotechnology Information database --- p.347 / Chapter B 60.3: --- Summary of sequence alignment of cDNA subclone AJ1#4 with mouse carboxylesterase --- p.347 / Chapter B 61.1: --- DNA sequence ofcDNA subclone AJ1#5 (AP6 & ARP14) using Ml3 reverse primer --- p.348 / Chapter B 61.2: --- Sequencing alignment of cDNA subclone AJ1#5 with mouse carboxylesterase by BLAST searching against the National Center for Biotechnology Information database --- p.348 / Chapter B 61.3: --- Summary of sequence alignment of cDNA subclone AJ1#5 with mouse carboxylesterase --- p.348 / Chapter B 62.1: --- DNA sequence ofcDNA subclone AJ2#10 (AP6 & ARP14) using M13 forward (-20) primer --- p.349 / Chapter B 62.2: --- Sequencing alignment of cDNA subclone AJ2#10 with peroxisomal acyl-coA oxidase (AOX) by BLAST searching against the National Center for Biotechnology Information database --- p.349 / Chapter B 62.3: --- Summary of sequence alignment of cDNA subclone AJ2#10 with mouse AOX --- p.349 / Chapter B 63.1: --- DNA sequence ofcDNA subclone AJ2#10 (AP6 & ARP14) using Ml3 reverse primer --- p.350 / Chapter B 63.2: --- Sequencing alignment of cDNA subclone AJ2#10 with peroxisomal acyl-coA oxidase (AOX) by BLAST searching against the National Center for Biotechnology Information database --- p.350 / Chapter B 63.3: --- Summary of sequence alignment of cDNA subclone AJ2#10 with mouse AOX --- p.350 / Chapter B 64.1: --- DNA sequence ofcDNA subclone AJ9#1 (AP6 & ARP 14) using Ml3 forward (-20) primer --- p.351 / Chapter B 64.2: --- Sequencing alignment of cDNA subclone AJ9#1 with mouse catalase by BLAST searching against the National Center for Biotechnology Information database --- p.351 / Chapter B 64.3: --- Summary of sequence alignment of cDNA subclone AJ9#1 with mouse catalase --- p.351 / Chapter B 65.1: --- DNA sequence ofcDNA subclone AJ9#1 (AP6 & ARP14) using Ml3 reverse primer --- p.352 / Chapter B 65.2: --- Sequencing alignment of cDNA subclone AJ9#1 with mouse suppressor of actin mutations (SAC1 gene) by BLAST searching against the National Center for Biotechnology Information database --- p.352 / Chapter B 65.3: --- Summary of sequence alignment of cDNA subclone AJ9#1 with mouse SAC1 --- p.352 / Chapter B 66.1: --- DNA sequence ofcDNA subclone AL2#8 (AP7 & ARP15) using M13 forward (-20) primer --- p.353 / Chapter B 66.2: --- Sequencing alignment of cDNA subclone AL2#8 with mouse hydroxy steroid (17-beta) dehydrogenase 11 (Hsdl7pil) by BLAST searching against the National Center for Biotechnology Information database --- p.353 / Chapter B 66.3: --- Summary of sequence alignment of cDNA subclone AL2#8 with mouse HSD17β11 --- p.353 / Chapter B 67.1: --- DNA sequence of cDNA subclone AL3#3 (AP7& ARP 15) using Ml3 forward (-20) primer --- p.354 / Chapter B 67.2: --- Sequencing alignment of cDNA subclone AL3#3 with mouse hydroxy steroid (17-beta) dehydrogenase 11 (Hsdl7pll) by BLAST searching against the National Center for Biotechnology Information database --- p.354 / Chapter B 67.3: --- Summary of sequence alignment of cDNA subclone AL3#3 with mouse HSD17β11 --- p.354 / Chapter B 68.1: --- DNA sequence of cDNA subclone AL3#3 (AP7& ARP 15) using M13 reverse primer --- p.355 / Chapter B 68.2: --- Sequencing alignment of cDNA subclone AL3#3 with mouse hydroxysteroid (17-beta) dehydrogenase 11 (Hsdl7β1l) by BLAST searching against the National Center for Biotechnology Information database --- p.355 / Chapter B 68.3: --- Summary of sequence alignment of cDNA subclone AL3#3 with mouse HSD17β11 --- p.355 / Chapter B 69.1: --- DNA sequence of cDNA subclone AO1#2 (AP5 & ARP 10) 356 using Ml3 forward (-20) primer --- p.356 / Chapter B 69.2: --- Sequencing alignment of cDNA subclone AO1#2 with mouse 356 adipose differentiation related protein (ADFP) by BLAST searching against the National Center for Biotechnology Information database --- p.356 / Chapter B 69.3: --- Summary of sequence alignment of cDNA subclone AO1 #2 with 356 mouse ADFP --- p.356 / Chapter B 70.1: --- DNA sequence ofcDNA subclone AO1#5 (AP5 & ARP10) 357 using M13 reverse primer --- p.357 / Chapter B 70.2: --- Sequencing alignment of cDNA subclone AO1#5 with mouse 357 carnitine O-octanoyltransferase (Crot) by BLAST searching against the National Center for Biotechnology Information database --- p.357 / Chapter B 70.3: --- Summary of sequence alignment of cDNA subclone AO1 #5 with 357 mouse Crot --- p.357 / Chapter B 71.1: --- DNA sequence ofcDNA subclone AO2#6 (AP5 & ARP10) 358 using Ml3 forward (-20) primer --- p.358 / Chapter B 71.2: --- Sequencing alignment of cDNA subclone A02#6 with mouse 358 RNase A family 4 (Rnase4) by BLAST searching against the National Center for Biotechnology Information database --- p.358 / Chapter B 71.3: --- Summary of sequence alignment of cDNA subclone AO2#6 358 with mouse Rnase4 --- p.358 / Chapter B 72.1: --- DNA sequence of cDNA subclone AO2#6 (AP5 & ARP 10) 359 using Ml3 reverse primer --- p.359 / Chapter B 72.2: --- Sequencing alignment of cDNA subclone A02#6 with mouse 359 RNase A family 4 (Rnase4) by BLAST searching against the National Center for Biotechnology Information database --- p.359 / Chapter B 72.3: --- Summary of sequence alignment of cDNA subclone A02#6 359 with mouse Rnase4 --- p.359 / Chapter B 73.1: --- DNA sequence ofcDNA subclone AO2#8 (AP5 & ARP10) 360 using Ml3 reverse primer --- p.360 / Chapter B 73.2: --- Sequencing alignment of cDNA subclone A02#8 with mouse 360 carnitine O-octanoyltransferase (Crot) by BLAST searching against the National Center for Biotechnology Information database --- p.360 / Chapter B 73.3: --- Summary of sequence alignment of cDNA subclone AO2#8 with 360 mouse Crot --- p.360 / Chapter B 74.1: --- DNA sequence ofcDNA subclone AO8#2 (AP5 & ARP10) 361 using M13 forward (-20) primer --- p.361 / Chapter B 74.2: --- Sequencing alignment of cDNA subclone A08#2 with mouse 361 RNase A family 4 (Rnase4) by BLAST searching against the National Center for Biotechnology Information database --- p.361 / Chapter B 74.3: --- Summary of sequence alignment of cDNA subclone AO8#2 with 361 mouse Rnase4 --- p.361 / Chapter B 75.1: --- DNA sequence of cDNA subclone AP4#4 (AP12 & ARP2) 362 using Ml3 forward (-20) primer --- p.362 / Chapter B 75.2: --- Sequencing alignment of cDNA subclone AP4#4 with mouse 362 mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.362 / Chapter B 75.3: --- Summary of sequence alignment of cDNA subclone AP4#4 with 362 mouse mitochondrion --- p.362 / Chapter B 76.1: --- DNA sequence ofcDNA subclone AP4#4 (AP12 & ARP2) 363 using Ml3 reverse primer --- p.363 / Chapter B 76.2: --- Sequencing alignment of cDNA subclone AP4#4 with mouse 363 mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.363 / Chapter B 76.3: --- Summary of sequence alignment of cDNA subclone AP4#4 with 363 mouse mitochondrion --- p.363

Lui, Ming Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 117-129). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract --- p.III / Abstract in Chinese --- p.V / List of Abbreviations --- p.VII / List of Figures --- p.X / List of Tables --- p.XII / Table of Contents --- p.XIII / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Overview on type 2 diabetes --- p.1 / Chapter 1.1.1 --- Definition of diabetes --- p.1 / Chapter 1.1.2 --- Diagnostic criteria --- p.2 / Chapter 1.1.3 --- Prevalence and societal impact --- p.2 / Chapter 1.1.4 --- Risks factors for type 2 diabetes --- p.4 / Chapter 1.1.4.1 --- Metabolic syndrome --- p.4 / Chapter 1.1.4.2 --- Genetics of type 2 diabetes --- p.6 / Chapter 1.1.4.3 --- "Environmental risk factors, lifestyle and energy imbalance" --- p.8 / Chapter 1.1.5 --- Pathophysiology of type 2 diabetes --- p.9 / Chapter 1.1.5.1 --- Insulin secretion and signaling --- p.9 / Chapter 1.1.5.1.1 --- Insulin Secretion --- p.9 / Chapter 1.1.5.1.2 --- Insulin signaling --- p.11 / Chapter 1.1.5.2 --- Natural history of type 2 diabetes --- p.12 / Chapter 1.1.5.3 --- Insulin resistance --- p.13 / Chapter 1.1.5.4 --- Impairment in insulin secretion --- p.15 / Chapter 1.1.5.5 --- Endocannabinoid system: A new target for energy balance and metabolism --- p.16 / Chapter 1.1.5.6 --- Effects of diabetes mellitus and its complications --- p.16 / Chapter 1.2 --- Biology of prostacyclin synthase (PTGIS) --- p.18 / Chapter 1.2.1 --- Molecular information of PTGIS --- p.18 / Chapter 1.2.2 --- Transcriptional control of PTGIS --- p.19 / Chapter 1.2.3 --- Protein structure of PGIS --- p.21 / Chapter 1.2.4 --- Sub-cellular localization and tissue distribution --- p.22 / Chapter 1.2.5 --- Function of PGIS --- p.25 / Chapter 1.2.5.1 --- Function of PGI2 in blood vessels --- p.26 / Chapter 1.2.5.2 --- Role of PGh in embryo development --- p.26 / Chapter 1.2.5.3 --- Role of PGI2 in apoptosis --- p.27 / Chapter 1.2.5.4 --- Targeted knock-out mice phenotype --- p.27 / Chapter 1.2.6 --- Relationship between PTGIS and diseases --- p.28 / Chapter 1.2.6.1 --- Genetic association --- p.28 / Chapter 1.2.6.2 --- Inactivation and tyrosine nitration of PGIS by peroxynitrite --- p.29 / Chapter 1.3 --- Biology of peroxisome proliferator-activated receptor delta (PPARD) --- p.30 / Chapter 1.3.1 --- Molecular information of PPARD --- p.30 / Chapter 1.3.2 --- Transcriptional control of PPARD --- p.31 / Chapter 1.3.3 --- Translational control and protein structure --- p.32 / Chapter 1.3.4 --- Sub-cellular localization and tissue expression --- p.35 / Chapter 1.3.5 --- Function of PPARδ --- p.37 / Chapter 1.3.5.1 --- Mechanisms of action --- p.37 / Chapter 1.3.5.2 --- Ligands for PPARδ --- p.38 / Chapter 1.3.5.3 --- PPARδ in lipoprotein metabolism --- p.39 / Chapter 1.3.5.4 --- PPARδ action in adipose tissue --- p.39 / Chapter 1.3.5.5 --- PPARδ action in skeletal and cardiac muscle --- p.40 / Chapter 1.3.5.6 --- PPARδ action in liver --- p.42 / Chapter 1.3.5.7 --- PPARδ and endocannabinoid system --- p.42 / Chapter 1.3.5.8 --- PPARδ action in inflammation --- p.43 / Chapter 1.3.5.9 --- Targeted knock-out mice phenotype --- p.44 / Chapter 1.3.5.10 --- Disease association --- p.44 / Chapter 1.4 --- Functional relationship of PGIS and PPARδ: possible role in energy metabolism --- p.46 / Chapter 1.5 --- Methods for studying genetics of type 2 diabetes and linkage analysis results --- p.47 / Chapter 1.5.1 --- Genome-wide scan --- p.47 / Chapter 1.5.2 --- Candidate gene approach --- p.48 / Chapter 1.6 --- Hypothesis and objectives --- p.49 / Chapter 1.7 --- Long-term significance --- p.49 / Chapter Chapter 2: --- Association Study of Prostacyclin Synthase and Peroxisome Proliferator-Activated Receptor Delta Gene Polymorphisms with Type2 Diabetes and Related Metabolic Traits / Chapter 2.1 --- Introduction and research design --- p.50 / Chapter 2.2 --- Study population --- p.52 / Chapter 2.2.1 --- Ethics approval --- p.52 / Chapter 2.2.2 --- Subjects --- p.52 / Chapter 2.2.3 --- Clinical assessments --- p.52 / Chapter 2.3 --- Materials and methods --- p.55 / Chapter 2.3.1 --- DNA samples --- p.55 / Chapter 2.3.2 --- Marker selection --- p.55 / Chapter 2.3.3 --- Genotyping --- p.57 / Chapter 2.3.4 --- Statistical analysis --- p.59 / Chapter 2.4 --- Results and Discussion --- p.60 / Chapter 2.4.1 --- Clinical characteristics of the study population --- p.60 / Chapter 2.4.2 --- Genotyping and LD analysis --- p.60 / Chapter 2.4.3 --- Association with type 2 diabetes and related metabolic traits --- p.61 / Chapter 2.4.3.1 --- Single SNP association with type 2 diabetes --- p.61 / Chapter 2.4.3.2 --- Single SNP association with metabolic traits --- p.64 / Chapter 2.4.3.3 --- Gene-gene interaction on type 2 diabetes --- p.74 / Chapter 2.4.3.4 --- Gene-gene interaction on metabolic traits --- p.74 / Chapter 2.5 --- Limitation and improvement --- p.79 / Chapter 2.6 --- Conclusions --- p.79 / Chapter Chapter 3: --- Functional Studies of Prostacyclin Synthase rs508757-A/G Intronic Polymorphism / Chapter 3.1 --- Introduction and research design --- p.80 / Chapter 3.2 --- Materials and methods --- p.81 / Chapter 3.2.1 --- Bioinformatics --- p.81 / Chapter 3.2.1.1 --- Cross-species alignment --- p.81 / Chapter 3.2.1.2 --- BLAST search and open reading frame prediction --- p.81 / Chapter 3.2.1.3 --- Transcription factor binding sites prediction --- p.82 / Chapter 3.2.2 --- PCR amplification from cDNA --- p.82 / Chapter 3.2.3 --- Culture of mammalian cell --- p.83 / Chapter 3.2.3.1 --- Cell line --- p.83 / Chapter 3.2.3.2 --- Medium and supplement --- p.83 / Chapter 3.2.3.3 --- Cell culture wares --- p.83 / Chapter 3.2.3.4 --- Cell culture conditions --- p.84 / Chapter 3.2.4 --- Construction of reporter vectors with rs508757 flanking sequence --- p.84 / Chapter 3.2.4.1 --- Cloning and vector preparation --- p.84 / Chapter 3.2.4.2 --- Site-directed mutagenesis --- p.84 / Chapter 3.2.5 --- Dual-luciferase reporter assay --- p.85 / Chapter 3.2.5.1 --- Transfection of VSMC --- p.85 / Chapter 3.2.5.2 --- Cell lysis and luminescence measurement --- p.86 / Chapter 3.2.6 --- Circular Dichroism --- p.87 / Chapter 3.2.6.1 --- Introduction to DNA quardruplex structure and circular dichroism --- p.87 / Chapter 3.2.6.1.1 --- DNA quardruplex --- p.87 / Chapter 3.2.6.1.2 --- Circular dichroism --- p.88 / Chapter 3.2.6.2 --- Circular dichroism measurement --- p.89 / Chapter 3.2.6.2.1 --- DNA samples --- p.89 / Chapter 3.2.6.2.2 --- CD spectroscopy --- p.89 / Chapter 3.2.7 --- Statistical analysis --- p.90 / Chapter 3.3 --- Results and Discussion --- p.91 / Chapter 3.3.1 --- Cross-species alignment --- p.91 / Chapter 3.3.2 --- BLAST search and ORF prediction --- p.92 / Chapter 3.3.3 --- PCR results on testing the presence of a new transcript --- p.93 / Chapter 3.3.4 --- Effect of rs508757 flanking sequence on SV40 promoter activity --- p.94 / Chapter 3.3.5 --- Circular dichroism experiment on rs508757 flanking sequence --- p.96 / Chapter 3.3.6 --- DNA slipping model --- p.98 / Chapter 3.3.7 --- Transcription factor binding site prediction --- p.99 / Chapter 3.4 --- Limitation and improvement --- p.107 / Chapter 3.5 --- Conclusions --- p.107 / Chapter Chapter 4: --- "General Discussion, Conclusion and Future Perspectives" / Chapter 4.1 --- General discussion --- p.108 / Chapter 4.2 --- Future perspectives --- p.115 / Chapter 4.2.1 --- "Association on type 2 diabetes and molecular interaction between transcription factors, PTGIS and PPARD" --- p.115 / Chapter 4.2.2 --- Association with diabetic nephropathy --- p.115 / Chapter 4.2.3 --- Study tissue or cell type specific actions of PGIS and PPARδ --- p.116 / Chapter 4.3 --- Conclusions to my project --- p.116 / Chapter Chapter 5: --- Bibliography --- p.117 / Appendix --- p.130

Os receptores ativadores da proliferação de peroxissomos (PPARs) pertencem à superfamília de receptores nucleares que funcionam como fatores transcricionais. Eles exercem um papel fundamental em processos que envolvem, principalmente, o metabolismo lipídico, em resposta à ativação por ligantes naturais e sintéticos como os ácidos graxos e os fibratos, respectivamente. A crescente descoberta de importantes funções fisiológicas, coordenadas pelos PPARs, e a necessidade de se conhecer como os agonistas, atualmente disponíveis, atuam nesses receptores, têm incitado pesquisas que vislumbram sua melhor exploração nos tratamentos de doenças metabólicas e inflamatórias, minimizando os efeitos adversos de ativações suprafisiológicas. Nesse cenário, o presente trabalho buscou compreender melhor as bases estruturais envolvidas nas funções atribuídas aos PPARs e explicar como as interações com seus ligantes ocorrem. Para isso, foram realizadas a subclonagem do domínio de ligação ao ligante do PPARα, sua expressão e purificação, seguidas de ensaios cristalográficos e biofísicos, além da abordagem de testes funcionais. Uma vez que a formação de oligômeros está relacionada à funcionalidade desses receptores, foram abordados estudos de oligomerização dos PPARs α e γ, compreendendo tanto o processo de homo- quanto o de heterodimerização. Os ensaios de cristalização do hPPARα LBD complexado a ligantes naturais e sintéticos, resultaram em estruturas cristalográficas que permitiram a identificação dos resíduos envolvidos no reconhecimento dos ligantes e a caracterização de sítios de ligação nunca antes descritos. A presença de ligantes nessas regiões afeta a conformação da proteína e, consequentemente, a modulação de sua função e o recrutamento da maquinaria transcricional. Adicionalmente, as estruturas cristalográficas da proteína complexada a ácidos graxos auxiliaram na compreensão de como essa importante classe de ligantes naturais possui efeitos farmacológicos similares aos de ligantes sintéticos. Esses resultados têm imediato impacto na procura racional de agonistas para esses receptores e se inserem em uma perspectiva de promoção do desenvolvimento científico-tecnológico na área de endocrinologia molecular. / The peroxisome proliferation-activated receptors (PPARs) belong to the nuclear receptors superfamily, acting as transcriptional factors. They play a key role in processes involving essentially lipid metabolism in response to activation by natural and synthetic ligands such as fatty acids and fibrates, respectively. The rising discovery of important physiological functions coordinated by PPARs and the necessity to know how the currently available agonists act on these receptors, have encouraged researches envisioning a better receptor exploration in the treatment of metabolic and inflammatory diseases, minimizing the adverse effects of supraphysiological activations. In this scenario, the present study aimed to better understand the structural basis involved in PPARs functions and elucidates how the interactions with their ligands takes place. For this, the ligand-binding domain of PPARα was subjected to subcloning, expression and purification steps, followed by crystallographical and biophysical assays, in addition to functional testing approaches. Since the degree of oligomerization is related to the functionality of these receptors, oligomeric studies of PPARs α and γ oligomerization were also achieved, comprising both homo- and hetero-dimerization. The co-crystallization assays of hPPARα LBD complexed with natural and synthetic ligands resulted in crystallographic structures that allowed the identification of residues involved in ligand recognition and the characterization of novel binding sites. The presence of ligands in these regions affects the conformation of the protein and thereby modulates their function and transcriptional machinery recruitment. Additionally, the crystallographic structures of the protein complexed to fatty acids were valuable for the understanding of how this important class of natural ligands has similar pharmacological effects to those of synthetic ligands. These results have direct impact on rational agonists design to these receptors and are inserted in a perspective of scientifical promotion and technological development in the field of molecular endocrinology.

Lipoprotein lipase (LPL) is the main enzyme hydrolyzing triglyceride-rich lipoproteins in plasma. Proteoglycan-bound LPL on the vascular endothelium represent the functional pool of active enzyme. LPL is regulated in a tissue specific manner according to metabolic demands. Rapid regulation of LPL activity is necessary to provide free fatty acids for storage or energy production. This regulatory mechanism appears to be post-translational and requires synthesis of other protein/proteins. Recently it was demonstrated that angiopoietin-like protein 4 (ANGPTL4) is involved in the metabolism of plasma triglycerides and that it is able to inhibit LPL activity in vitro. These properties were linked to the N-terminal coiled-coil domain of ANGPTL4 (ccd-ANGPTL4), but the mechanism for the inhibition was not known. The aim of this thesis was to investigate the molecular mechanism for inhibition of LPL by ccd-ANGPTL4, to characterize regions in ccd-ANGPTL4 that are important for inactivation of LPL and to study the role of ANGPTL4 for regulation of LPL in vivo. Binding of ccd-ANGPTL4 to LPL was demonstrated by several methods, including surface plasmon resonance. The interaction was transient and resulted in conversion of the enzyme from catalytically active dimers to inactive monomers with decreased affinity for heparin. We have shown that ANGPTL4 mRNA in rat adipose tissue turns over rapidly and that changes in the ANGPTL4 mRNA abundance were inversely correlated to LPL activity, both during the fed to fasted and the fasted to fed transitions. We conclude that ANGPTL4 is a fasting-induced controller of LPL in adipose tissue, acting extracellularly on the native conformation of LPL in an unusual fashion, like an unfolding molecular chaperone. Site directed mutagenesis was used to explore regions in ccd-ANGPTL4 important for inactivation of LPL, and for binding of ANGPTL4 to heparin. Others had shown that ccd-ANGPTL4 forms higher oligomers. Structure prediction analyses demonstrated that the coiled-coil domain of ccd-ANGPTL4 probably forms three consecutive α-helices with strong hydrophobic faces, and that there are clusters of positively charged residues both on the helices and in intervening sequences. We made replacements of hydrophobic residues, positively charged residues, cysteine residues and negatively charged residues in ccd-ANGPTL4. In addition, helix-breaking proline residues were introduced in all three helices. We found that hydrophobic residues are important for oligomer formation. The higher oligomers appeared to be stabilized by disulfide bonds, but cysteines are not crucial for oligomerization. Introduction of Pro-residues in the first and second helix prevented formation of higher oligomers and reduced the ability of ccd-ANGPTL4 to inactivate LPL. We found that negatively charged residues in ccd-ANGPTL4 are important for inactivation of LPL. A heparin binding site was localized in the C-terminal end of ccd-ANGPTL4 (amino acid residues 114-140). To investigate whether LPL is differently processed in different depots of adipose tissue we measured the levels of LPL mRNA, protein and activity in omental and subcutaneous adipose tissue in human subjects undergoing elective surgery. Our results show that, although the expression level of LPL was higher in subcutaneous adipose tissue, the specific LPL activity (ratio of activity over the LPL protein mass) was higher in omental adipose tissue. Interestingly, the levels of ANGPTL4 mRNA were lower in omental compared to subcutaneous adipose tissue in most of the studied subjects. This difference can possibly explain the higher specific activity of LPL in omental adipose tissue and indicated that ANGPTL4 is involved in regulation of LPL activity also in humans. LPL produced by macrophages in the artery wall promotes local accumulation of lipids in these cells, and thereby plays an important role in development of atherosclerosis. The known association between type 2 diabetes and atherosclerosis forwarded us to study production of LPL by THP-1 macrophages under hyperglycemic conditions and under treatment with a peroxisome proliferator-activated receptor delta (PPARδ) agonist (GW501516). We found that LPL activity (but not LPL mass) produced by macrophages was decreased by GW501516. The loss of LPL activity coincided with increased level of ANGPTL4 mRNA, indicating that the agonist regulates LPL activity through expression of ANGPTL4. This effect was even more pronounced in cells grown under hyperglycemic conditions. Our data suggest that a suitable PPARδ agonist, like GW501516, may have protective effects against development of atherosclerosis in subjects with diabetes type 2.

[Truncated abstract] Glucocorticoids are critical for the maturation of the fetus late in pregnancy. Indeed, clinical administration of glucocorticoids is used to accelerate fetal lung maturation in mothers at risk of pre-term delivery. Increased glucocorticoid exposure, however, can have detrimental effects on fetal and placental growth and increase the risk of disease in later life. Many studies have focused on the effect of an increase in the transplacental passage of glucocorticoids on both fetal growth and subsequent postnatal development. But there is a growing body of evidence to suggest that the impact of glucocorticoids on fetal growth is mediated, in part, via their direct effects on the placenta . . . Overall, these studies quantify the labyrinth zone-specific increases in placental expression of PPARG and VEGF in association with a marked increase in vascularisation observed near term. Furthermore, this study demonstrates for the first time that these increases in gene expression are prevented by maternal dexamethasone treatment which also inhibits growth of the fetal capillary network. Elevated expression of SFRP4 in the regressing basal zone late in gestation and in both placental zones after dexamethasone-induced placental growth restriction is consistent with a role for SFRP4 in glucocorticoid-mediated inhibition of wnt signalling. Collectively, the data presented in this thesis show that glucocorticoid inhibition of fetal growth is mediated in large part via effects on the placenta, specifically through inhibition of signals that promote proliferation and vascularisation.

Os receptores ativadores da proliferação de peroxissomos (PPARs) pertencem à superfamília de receptores nucleares que funcionam como fatores transcricionais. Eles exercem um papel fundamental em processos que envolvem, principalmente, o metabolismo lipídico, em resposta à ativação por ligantes naturais e sintéticos como os ácidos graxos e os fibratos, respectivamente. A crescente descoberta de importantes funções fisiológicas, coordenadas pelos PPARs, e a necessidade de se conhecer como os agonistas, atualmente disponíveis, atuam nesses receptores, têm incitado pesquisas que vislumbram sua melhor exploração nos tratamentos de doenças metabólicas e inflamatórias, minimizando os efeitos adversos de ativações suprafisiológicas. Nesse cenário, o presente trabalho buscou compreender melhor as bases estruturais envolvidas nas funções atribuídas aos PPARs e explicar como as interações com seus ligantes ocorrem. Para isso, foram realizadas a subclonagem do domínio de ligação ao ligante do PPARα, sua expressão e purificação, seguidas de ensaios cristalográficos e biofísicos, além da abordagem de testes funcionais. Uma vez que a formação de oligômeros está relacionada à funcionalidade desses receptores, foram abordados estudos de oligomerização dos PPARs α e γ, compreendendo tanto o processo de homo- quanto o de heterodimerização. Os ensaios de cristalização do hPPARα LBD complexado a ligantes naturais e sintéticos, resultaram em estruturas cristalográficas que permitiram a identificação dos resíduos envolvidos no reconhecimento dos ligantes e a caracterização de sítios de ligação nunca antes descritos. A presença de ligantes nessas regiões afeta a conformação da proteína e, consequentemente, a modulação de sua função e o recrutamento da maquinaria transcricional. Adicionalmente, as estruturas cristalográficas da proteína complexada a ácidos graxos auxiliaram na compreensão de como essa importante classe de ligantes naturais possui efeitos farmacológicos similares aos de ligantes sintéticos. Esses resultados têm imediato impacto na procura racional de agonistas para esses receptores e se inserem em uma perspectiva de promoção do desenvolvimento científico-tecnológico na área de endocrinologia molecular. / The peroxisome proliferation-activated receptors (PPARs) belong to the nuclear receptors superfamily, acting as transcriptional factors. They play a key role in processes involving essentially lipid metabolism in response to activation by natural and synthetic ligands such as fatty acids and fibrates, respectively. The rising discovery of important physiological functions coordinated by PPARs and the necessity to know how the currently available agonists act on these receptors, have encouraged researches envisioning a better receptor exploration in the treatment of metabolic and inflammatory diseases, minimizing the adverse effects of supraphysiological activations. In this scenario, the present study aimed to better understand the structural basis involved in PPARs functions and elucidates how the interactions with their ligands takes place. For this, the ligand-binding domain of PPARα was subjected to subcloning, expression and purification steps, followed by crystallographical and biophysical assays, in addition to functional testing approaches. Since the degree of oligomerization is related to the functionality of these receptors, oligomeric studies of PPARs α and γ oligomerization were also achieved, comprising both homo- and hetero-dimerization. The co-crystallization assays of hPPARα LBD complexed with natural and synthetic ligands resulted in crystallographic structures that allowed the identification of residues involved in ligand recognition and the characterization of novel binding sites. The presence of ligands in these regions affects the conformation of the protein and thereby modulates their function and transcriptional machinery recruitment. Additionally, the crystallographic structures of the protein complexed to fatty acids were valuable for the understanding of how this important class of natural ligands has similar pharmacological effects to those of synthetic ligands. These results have direct impact on rational agonists design to these receptors and are inserted in a perspective of scientifical promotion and technological development in the field of molecular endocrinology.

Marine environments are under constant pressure from anthropogenic pollution. Chemical pollutants are introduced into the aquatic environment through waste disposal, sewage, land runoff and environmental exploitation (harbours, fisheries, tourism) leading to disastrous effects on the marine wildlife. Developmental malformations, reproduction failure including sex changes and high death rates are commonly observed in aquatic animal populations around the world. Unfortunately, the underlying molecular mechanisms of these pollution effects, in particular for marine invertebrate species, are often unknown. One proposed mechanism through which environmental pollution affects wildlife, is the disruption of nuclear receptors (NRs), ligand-binding transcription factors in animals. Environmental pollutants can directly interact with nuclear receptors, inducing incorrect signals for gene expression and subsequently disrupt developmental and physiological processes. Elucidation of the exact mechanism in invertebrates, however, is sparse due to limited understanding of invertebrate endocrinology and molecular regulatory mechanisms. Here, I have investigated the presence, expression and function of NRs in the Pacific oyster, Crassostrea gigas, and explored their interrelation with known environmental pollutants. Using a suite of molecular techniques and bioinformatics tools I demonstrate that the Pacific oyster possesses a large variety of NR homologs (43 NRs), which display individual expression profiles during embryo/larval development and supposedly fulfil distinct functions in developmental and physiological processes. Functional studies on a small subset of oyster NRs provided evidence for their ability to regulate gene expression, including interactions with DNA, other NRs or small molecules (ligand-binding). Oyster receptors also show a high likeliness to be disrupted by environmental pollutants. Computational docking showed that the retinoid X receptor ortholog, CgRXR, is able to bind and be activated by 9-cis retinoic acid and by the well-known environmental contaminant tributyltin. A potential interaction between tributyltin and the peroxisome proliferator-activated receptor ortholog CgPPAR has also been found. In addition, exposure of oyster embryos to retinoic acids and tributyltin resulted in shell deformations and developmental failure. In contrast, computer modelling of another putative target for pollutants, the retinoic acid receptor ortholog CgRAR, did not indicate interactions with common retinoic acids, supporting a recently developed theory of loss of retinoid binding in molluscan RARs. Sequence analyses revealed six residues in the receptor sequence, which prevent the successful interaction with retinoid ligands. In conclusion, this investigative work aids the understanding of fundamental processes in invertebrates, such as gene expression and endocrinology, as well as further understanding and prediction of effects of environmental pollutants on marine invertebrates.

La stéatohépatite non alcoolique (NASH) est une maladie du foie à évolution clinique grave, dont la prévalence est en constante progression. La stéatohépatite non alcoolique est caractérisée par un dépôt excessif de lipides dans les hépatocytes (stéatose) associé à une inflammation chronique, au contraire de la stéatose hépatique (NAFLD), manifestation initiale mais bénigne d'un dérèglement métabolique. Le NASH augmente le risque de progression vers la fibrose, la cirrhose et le carcinome hépatocellulaire et ne peut être soigné que par une greffe hépatique. Le risque de développer un diabète de type est aussi significativement augmenté chez les patients atteints de NASH. PPAR⍺ est un récepteur nucléaire connu pour réguler l'utilisation des acides gras dans le foie et réprimer les voies de signalisation pro-inflammatoires. [...]Nous avons conçu mutant de PPAR⍺ dont l'activité de liaison à l'ADN est abolie. La comparaison de ses activités transcriptionnelles in vitro avec le PPAR⍺ non muté démontre que les activités de contrôle du métabolisme sont abolies pour ce mutant, alors que les activités anti-inflammatoires restent intactes. [...] Dans cette étude, nous montrons donc pour la première fois que PPAR⍺ inhibe la progression de la stéatose vers le NASH et la fibrose par un mécanisme anti-inflammatoire direct, indépendant de son effet sur le métabolisme lipidique hépatique. / Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent liver condition characterized by excessive lipid deposition in the hepatocytes steatohepatitis (NASH) is hallamarked by chronic inflammation. NASH markedly increases the risk of progression towards liver fibrosis, cirrhosis ans hepatocellular carcinoma. The nuclear peroxisome proliferator-activated receptor alpha (PPAR⍺) regulates hepatic fatty acid utilization and represses pro-inflammatory signaling pathways. [...]Liver-specific expression of wild type or DNA binding-deficient PPAR⍺ in acute and chronic models of inflammation demonstrated that PPAR's anti-inflammatory, but not metabolic activities, result from DNA binding-independent mechanisms in vivo. We futher show that PPAR⍺ inhits the transition from steatosis toward NASH and fibrosis through a direct, anti-inflammatory mechanism independent of its effetc on hepatic lipid metabolism.

Although once considered a simple energy storage depot, the adipose tissue is now known to be a powerful regulator of whole body insulin sensitivity and energy metabolism. This metabolically dynamic organ functions to safely store excess fatty acid as triglyceride, thereby preventing lipotoxicity in peripheral tissues and the development of insulin resistance. In addition, the adipose tissue acts as an endocrine organ and secretes factors, called adipokines, which influence whole body insulin sensitivity and glucose homeostasis. Therefore, understanding adipose tissue development and biology is essential to understanding whole body energy metabolism. A master regulator of adipose tissue development and whole body insulin sensitivity is the nuclear receptor, PPARγ. Due to the importance of this nuclear receptor in maintaining adipocyte function, disruptions in PPARγ activity result in severe metabolic abnormalities, such as insulin resistance and type 2 diabetes. Conversely, PPARγ activation by synthetic agonists ameliorates these conditions, demonstrating the potent control this nuclear receptor has on whole body metabolism. Therefore, understanding how PPARγ expression and activity are regulated, particularly in the adipose tissue, is paramount to understanding the pathogenesis of type 2 diabetes. While there are several synthetic PPARγ agonists available, identifying the endogenous ligand or ligands is still an area of intense investigation. Since fatty acids can induce PPARγ activation, in the first part of this thesis, I screened several fatty acid metabolizing enzymes present in the adipocyte to identify novel modulators of PPARγ activity. These studies revealed that the fatty acid Δ9 desaturase, Stearoyl CoA Desaturase 2 (SCD2), is absolutely required for 3T3-L1 adipogenesis and to maintain adipocyte-specific gene expression in fully differentiated cells. Although SCD2 does not appear to regulate PPARγ ligand production, it does potently regulate PPARγ activity by maintaining the synthesis of PPARγ protein. Surprisingly, this effect was found only with SCD2 and not with the highly homologous protein, SCD1. Therefore, these findings identify separate cellular functions for these SCD isoforms and reveal a novel and essential role for fatty acid desaturation in the adipocyte. Equally important to understanding PPARγ regulation is identifying the downstream mechanisms by which PPARγ activation improves insulin sensitivity. Evidence suggests that the PPARγ target gene, Cidea, is involved in mediating insulin sensitivity by binding to lipid droplets and promoting lipid storage in the adipocyte. Therefore, the second part of thesis provides mechanistic detail into Cidea function by showing that the carboxy terminal 104 amino acids is necessary and sufficient for lipid droplet targeting and the stimulation of triglyceride storage. However, these studies also identified a novel function for Cidea, which requires both the carboxy and amino termini: to induce larger and fewer droplets from smaller dispersed droplets, indicating the possible fusion of droplets. Perhaps this striking change in lipid droplet morphology allows tighter packing and more efficient storage of triglyceride and identifies a novel role for Cidea in lipid metabolism. The results presented in this thesis elucidate key aspects of lipid metabolism that maintain adipocyte function: SCD2 is required to maintain PPARγ protein expression in the mouse; Cidea is a downstream effector of PPARγ activity by promoting efficient triglyceride storage. Therefore, these findings enhance our understanding of adipocyte biology.