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

慢性乙型肝炎病毒（HBV）感染是肝癌發生的重要因素，其中乙肝病毒X蛋白（HBx）在這一過程起著關鍵作用。研究發現，一些HBV變體和HBx突變具有更高致癌風險，而且這些變體和突變存在地區差異。香港是HBV感染高發地帶，因此本研究目的是從這一地區120個肝癌組織標本中篩查出HBx突變位點。我們用巢式PCR從84.16% (101/120)的標本中提取和擴增了HBx，並進行基因測序。三種HBx突變被檢測出，包括點突變，遠端羧基端截斷和缺失突變。其中點突變位點有39個，特別的是在50%的標本中檢測出A1630G/G1721A 和 A1762T/G1764A雙突變。在31.68% (32/101)的標本中發現遠端羧基端截斷，以及在2.97% (3/101)的標本中檢測出缺失突變。總之，大多數突變集中在HBx轉錄啟動域，表明這些突變在肝癌發生中可能起著重要作用。 / 缺氧誘導因數-1α（HIF-1α）在肝癌的發生和發展中也起著重要作用。研究發現，野生型HBx可以啟動HIF-1α，但是變異型HBx和HIF-1α的關係還沒有研究清楚。我們研究表明HBx轉錄啟動域是必需而且足夠啟動HIF-1α的。在這個區域的突變中，雙突變K130M/V131Z增強HBx對HIF-1α的活性，但遠端羧基端截斷和缺失突變削弱其功能。進一步研究發現，羧基端特別是119-140氨基酸對HBx的穩定和功能非常重要。肝癌標本中，我們也發現HBx和HIF-1α的表達呈正相關。因此，雖然不同的突變對於HBx的功能有不同的影響，但總的來說這些突變可以促進HIF-1α的表達和啟動，進而導致肝癌患者的預後不良。 / 靶向治療在肝癌綜合治療中扮演重要角色。索拉菲尼（Sorafenib）是一種多激酶抑制劑，臨床實驗發現它對晚期肝癌治療有效，但其抑制腫瘤血管生成機制還不完全清楚。我們研究發現Sorafenib明顯而且劑量依賴性地降低HIF-1α的表達和活化，進而抑制血管內皮生長因數（VEGF）的表達。Sorafenib抑制mTOR, ERK, p70S6K, RP-S6, eIF4E和4E-BP1等翻譯起始因數的磷酸化，從而抑制HIF-1α的合成而不影響其降解。體外實驗進一步發現Sorafenib降低HIF-1α和VEGF的表達，從而抑制腫瘤的血管形成和生長。總之，我們的研究表明sorafenib可能通過阻斷mTOR/p70S6K/4E-BP1 和 ERK 信號通路來抑制HIF-1α的合成，從而發揮其抗腫瘤血管生成作用。 / Chronic HBV infection is the leading cause of hepatocellular carcinoma (HCC) and HBx plays a crucial role in the molecular pathogenesis of HBV-related HCC. Previous investigations have indicated that some variations of HBV or mutations of HBx are associated with higher risk of HCC development, whereas the mutations profiles may be disparate in different regions. In the present studies, we thus aim to screen and identify the HBx mutation hotspots in 120 HCC tissues from Hong Kong, a region with HBV hyper-endemic. HBV DNAs were successfully isolated and amplified in 84.16% (101/120) HCC specimens via nest-PCR, and then subjected to gene sequencing. Three types of HBx mutations, including point mutations, distal carboxyl-terminal truncations and deletion mutations, were discovered. Among the point mutations, 39 mutation hotspots were indentified, with two double mutations (A1630G/G1721A and A1762T/G1764A) occurring in approximate 50% of 101 HCC cases. Distal C-terminal truncated mutations were discovered in 31.68% (32/101) of HCC cases, whereas deletion mutations were detected in 2.97% (3/101) of them. Overall, majority of identified mutations were located at the transactivation domain of HBx, suggesting the crucial roles of these mutations in HCC development. / Hypoxia-inducible factor-1α (HIF-1α) also closely involves in the development and progression of HCC. Wild-type HBx has been shown to activate HIF-1α. But the relationship between HBx mutants and activation of HIF-1α has not been fully elucidated. We here revealed that the transactivaiton domain of HBx was necessary and sufficient to activate HIF-1α. Double mutations K130M/V131Z in this domain enhanced the functionality of HBx in upregulating the expression and the activation of HIF-1α, whereas C-terminal truncations and deletion mutations weakened this prosperity of HBx. We further uncovered that the C-terminus, especially the region of amino acids 119-140, was essential for the stability and transactivation of HBx. The positive association between the HBx mutants and HIF-1α was found in the HCC tissue samples. Therefore, although mutations exerted different effects on the functionality of HBx, the overall activity of HBx mutants was suggested to upregulate HIF-1α, whose level is related to poor prognosis of HCC patients. / The therapy targeting a critical molecule in the development of HCC such as HIF-1α may be a potential and effective treatment regimen for HCC patients. Sorafenib, a multikinase inhibitor, has demonstrated promising results for the treatment of advanced HCC in clinical trials, but the mechanism that accounts for the anti-angiogenic efficiency of this agent has not been fully elucidated. We here revealed that sorafenib remarkably and dose-dependently decreased the expression and the transcriptional activity of HIF-1α, and its target gene, vascular endothelial grow factor (VEGF). Further analysis revealed that this reduction of HIF-1α by sorafenib was caused by the inhibition of HIF-1α protein synthesis rather than by the promotion of HIF-1α protein degradation. Moreover, the phosphorylated levels of mTOR, ERK, p70S6K, RP-S6, eIF4E and 4E-BP1 were significantly suppressed by sorafenib. In vivo studies further confirmed the inhibitory effect of sorafenib on the expression of HIF-1α and VEGF proteins, leading to a decrease of tumor vascularisation and growth. Collectively, our data suggest that sorafenib may exhibit anti-angiogenic activity by inhibiting HIF-1α synthesis, which is likely to be achieved through suppressing the phosphorylation of mTOR/p70S6K/4E-BP1 and ERK. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Liping. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 133-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.I / 摘要 --- p.IV / Publications --- p.VI / Acknowledgements --- p.VII / Abbreviations --- p.IX / List of Figures --- p.XI / List of Tables --- p.XIII / Table of Contents --- p.XIV / Chapter Chapter I --- General Introduction --- p.1 / Chapter 1.1 --- Overview of Hepatocellular Carcinoma --- p.1 / Chapter 1.2 --- HBV Infection and HCC Development --- p.6 / Chapter 1.3 --- Overview on Hepatitis B virus X Protein --- p.10 / Chapter 1.4 --- Roles of Hypoxia-inducible Factors in HCC --- p.17 / Chapter 1.5 --- Targeted Therapies and Sorafenib --- p.27 / Chapter Chapter II --- Identification of HBx Mutation Hotspots in HCC Tissues --- p.31 / Chapter 2.1 --- Abstract --- p.31 / Chapter 2.2 --- Introduction --- p.32 / Chapter 2.3 --- Materials and Methods --- p.35 / Chapter 2.4 --- Results --- p.40 / Chapter 2.5 --- Discussion --- p.53 / Chapter Chapter III --- The Relationship between HBx Mutants and HIF-1α --- p.59 / Chapter 3.1 --- Abstract --- p.59 / Chapter 3.2 --- Introduction --- p.60 / Chapter 3.3 --- Materials and Methods --- p.63 / Chapter 3.4 --- Results --- p.70 / Chapter 3.5 --- Discussion --- p.91 / Chapter Chapter IV --- The Effects of Sorafenib on Hypoxia-inducible Factor-1α --- p.96 / Chapter 4.1 --- Abstract --- p.96 / Chapter 4.2 --- Introduction --- p.98 / Chapter 4.3 --- Materials and Methods --- p.101 / Chapter 4.4 --- Results --- p.108 / Chapter 4.5 --- Discussion --- p.124 / Chapter Chapter V --- Conclusion and Future Plans --- p.129 / Chapter 5.1 --- Conclusion --- p.129 / Chapter 5.2 --- Future Plans --- p.131 / References --- p.133