Adipogenesis and ectopic lipid accumulation during aging have a great impact on the aging process and the pathogenesis of chronic diseases with age. However, at present, information on the age-related molecular changes in lipid redistribution patterns and their potential nutritional interventions is sparse. We investigated the mechanism underlying age-related lipid redistribution and its modulation using 5-, 17-, and 24-month-old male Fischer 344 rats fed ad libitum (AL) or a 3-week-long CR (40% less than AL) diet. Results revealed that the activities of adipogenic transcription factors were decreased in the white adipose tissue (WAT) of aged AL rats. In contrast, the skeletal muscle of aged AL rats showed increased fat accumulation through decreased carnitine palmitoyltransferase-1 activity, which was blunted by short-term CR. This study suggests an age-related shift in lipid distribution by reducing the adipogenesis of WAT while increasing intramyocellular lipid accumulation, and that CR can modulate age-related adipogenesis and ectopic lipid accumulation.

No description available.

No description available.

The dystrophin-associated protein complex (DAPC) provides a mechanical link between the intracellular cytoskeleton and extracellular matrix, serving as a mechanosensor and signal transducer across the sarcolemma. Pharmacological stimulation of AMP-activated protein kinase (AMPK) induces the expression of DAPC components in skeletal muscle, whereas physiological reductions in AMPK are associated with DAPC dysfunction. We sought to determine whether AMPK was necessary for the maintenance of DAPC expression in skeletal muscle. Fast glycolytic extensor digitorum longus (EDL) and slow oxidative soleus (SOL) muscles from wild-type (WT) mice, as well as from littermates deficient in both isoforms of the AMPK-β subunit in skeletal muscle (MKO) were analyzed. DAPC mRNA levels, as well as protein expression and localization were similar between genotypes, with the exception of nNOS, which displayed a compensatory sarcolemmal enrichment in MKO muscles. The content of transcriptional and post-transcriptional regulators of the DAPC, such as PGC-1α and KSRP, were also not affected by the loss of AMPK. However, MyoD and myogenin expression was significantly diminished in MKO muscles, which is consistent with previous reports of myopathy in these animals. Furthermore, we observed decrements in extrasynaptic utrophin expression selectively in MKO SOL muscles, despite an adaptive accumulation of PGC-1α at the sarcolemmal compartment. Collectively the evidence indicates that AMPK is sufficient, but not essential for the maintenance of DAPC expression in skeletal muscle. However, AMPK is required for preserving extrasynaptic utrophin levels in slow, oxidative muscles, which underscores the role of AMPK in the gene expression of this disease modifying protein. / Thesis / Master of Science (MSc) / The dystrophin-associated protein complex (DAPC) connects the interior and exterior of muscle cells. Activation of AMP-activated protein kinase (AMPK) increases the expression of the DAPC in skeletal muscle. We sought to determine whether AMPK was necessary for DAPC expression in skeletal muscle. Fast and slow muscles from normal mice, as well as from those deficient in skeletal muscle AMPK (MKO) were analyzed. We found DAPC levels and localization were similar between both groups, with the exception of nNOS, which was enriched at the muscle membrane in MKO muscles. Regulators of the DAPC were also not affected by the loss of AMPK. However, genes important for the production of muscle were significantly diminished in MKO muscles. Furthermore, we observed decrements in utrophin at the muscle membrane selectively in slow MKO muscles. Our work indicates that AMPK is not essential for the DAPC expression in skeletal muscle, however it is required for preserving utrophin levels in slow, oxidative muscles.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25337号 / 農博第2603号 / 新制||農||1106(附属図書館) / 学位論文||R6||N5509 / DGAM / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 矢﨑 一史, 教授 山口 信次郎, 教授 飛松 裕基 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25356号 / 農博第2622号 / 新制||農||1109(附属図書館) / 学位論文||R6||N5528 / DFAM / 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 Agricultural Science / Kyoto University / DFAM

Defeitos peroxissomais estão associados a diversas doenças complexas. O peroxissomo é responsável pela beta-oxidação de ácidos graxos, quando é gerado peróxido de hidrogênio. A catalase A, de ocorrência peroxissomal, é frequentemente considerada a única defesa antioxidante dessa organela, porém, em diversos organismos, a ausência dessa enzima não acarreta uma alteração fenotípica clara. Em Saccharomyces cerevisiae, linhagens mutantes deficientes em catalase A (Δcta1) apresentam viabilidade muito similar à linhagem selvagem correspondente. Trabalhamos com a hipótese de que peroxidases baseadas em cisteína compensam a ausência de catalase A, contribuindo para a detoxificação de peróxidos provenientes do peroxissomo. De fato, linhagens com os genes para as peroxirredoxinas Ahp1 e Tsa1 nocauteados mostraram-se mais sensíveis a hidroperóxido de terc-butila (tBHP) em comparação a linhagem selvagem. A linhagem de levedura deficiente nas cinco peroxirredoxinas (prxΔ) mostrou-se ainda mais sensível a tBHP. Em relação ao estresse induzido por peróxido de hidrogênio, a prxΔ apresentou maior sensibilidade do que as linhagens selvagem e mutantes com deleções simples, apesar da presença de catalases (peroxissomal e citossólica). Esses dados estão de acordo com resultados obtidos no nosso grupo demonstrando um aumento da expressão de genes referentes às peroxirredoxinas Ahp1, Prx1 e Tsa2 em células Δ cta1 crescidas em condições de alta atividade peroxissomal (oleato), indicando uma cooperação entre catalase e peroxirredoxinas na proteção antioxidante. A peroxirredoxina Ahp1 pode apresentar localização organelar (possivelmente mitocondrial ou peroxissomal), o que sugere que Ahp1 pode ser um componente relevante da defesa contra oxidantes provenientes do peroxissomo. No entanto, a linhagem Δ ahp1, normalmente sensível a peróxido orgânico, apresentou ganho de resistência na ausência de atividade de catalase (com a adição de ATZ e na linhagem duplo-mutante Δcta1/ahp1), indicando a existência de uma via antioxidante compensatória induzida pela ausência de catalase A. A construção das linhagens duplo-mutantes Δcta1/ahp1, Δcta1/tsa1, Δcta1/tsa2, Δ cta1/prx1 e Δcta1/dot5 foi realizada com o objetivo de investigar mecanismos compensatórios entre enzimas que podem proteger a levedura contra os oxidantes provenientes do peroxissomo. Para tanto, foram realizados ensaios de viabilidade comparativa em condições de alta atividade peroxissomal. Além disso, os níveis comparativos de proteínas carboniladas foram analisados nessas linhagens. Os resultados indicaram maior sensibilidade a peróxido e maiores níveis de danos oxidativos na linhagem Δcta1/tsa2, apontando a peroxirredoxina Tsa2 como candidata a importante componente da via antioxidante de compensação à ausência de catalase A. Nesses ensaios, também foram utilizadas a linhagem quíntupla mutante (prxΔ) e uma linhagem deficiente nas cinco peroxirredoxinas e três glutationa peroxidases - deficiente em oito tiól-peroxidases baseadas em cisteína (Δ8). A comparação das linhagens prxΔ e Δ8 com as linhagens selvagem, simples-mutantes e duplo-mutantes evidenciou a importância das peroxirredoxinas na defesa antioxidante da célula e o fato das tiól-peroxidases serem imprescindíveis em condições de estresse oxidativo. Ao examinar a expressão gênica de TSA2 em células crescidas em oleato, foi verificada a indução do gene na ausência de catalase A, em condição basal. Os resultados obtidos indicam a existência de uma eficiente via de defesa antioxidante, na qual estão envolvidas tiól-peroxidases, que compensa a ausência de catalase A na célula e que protege leveduras contra estresse induzido tanto por peróxido de hidrogênio como peróxido orgânico. A peroxirredoxina Tsa2 parece estar envolvida na via compensatória à ausência de catalase peroxissomal através de um mecanismo ainda não esclarecido / Defects in peroxisomes are associated with several complex diseases. Beta-oxidation of fatty acids takes place in these organelles, with the concomitant generation of hydrogen peroxide. Generally, it is assumed that peroxisomal catalase is the enzyme responsible for degradation of hydrogen peroxide, but in several organisms, deletion of its gene results in no clear phenotype. In Saccharomyces cerevisiae, catalase A- null (Δcta1) mutant strains exhibit very similar viability levels when compared with the corresponding wild-type strain. We hypothesized here that Cys-based peroxidases compensate the absence of catalase A, contributing to the detoxification of peroxides derived from the peroxisome. Indeed, null mutante strains for the peroxiredoxins Ahp1 and Tsa1 displayed increased sensitivity for tert-butylhydroperoxide (tBHP) in comparison to the wild type strain. Furthermore, a mutant strain whose five genes for peroxiredoxins were interrupted (prxΔ) was even more sensitive to tBHP. In regards to hydrogen peroxide insult, the prxΔ strain was more susceptible to oxidative stress than the single mutant and wild-type strains, despite the activity of catalases. These data are in agreement with previous results from our group demonstrating increased expression of genes encoding the three peroxiredoxin enzymes: Ahp1, Prx1 and Tsa2 in Δcta1 cells at high peroxisomal activity (media containing oleate). Indeed, a yeast strain deleted of all five peroxiredoxin genes is more sensitive to peroxides than the corresponding wild type cells. These results indicated that catalase and peroxiredoxins cooperate to protect yeast in conditions of high fatty acid intake. There are evidences of an organellar location of Ahp1 (possible peroxisomal or mitochondrial), suggesting it could be a relevant component of antioxidant defense relative to the insult derived from the peroxisome. Nonetheless, the ahp1-null strain (Δahp1), which is usually sensitive to organic peroxide, displayed a gain of resistance in the absence of catalase activity (in the presence of ATZ and in the double-mutant strain Δcta1/ahp1), indicating the existance of a compensatory antioxidant pathway induced in the absence of catalase A. The double-mutant strains Δcta1/ahp1, Δcta1/tsa1, Δcta1/tsa2, Δcta1/prx1 and Δcta1/dot5 were developed in order to elucidate the identity of the enzymes that cooperate to protect yeast against oxidative insult derived from the peroxisome. To this end, comparative viability assays in conditions of high peroxisomal activity were realised, as well as assays in comparative total protein carbonyl levels. Among the double-mutant strains, Δcta1/tsa2 displayed higher sensibility to peroxide and higher levels of oxidative damage, suggesting that the peroxiredoxin Tsa2 may be an important component in the antioxidant pathway that compensates the lack of catalase A. In addition, a quintuple mutant strain, lacking all peroxiredoxins, and a mutant strain lacking all eight Cys-based, thiol peroxidases were used in these assays. The comparison of these strains with the wild-type, single-mutant and double-mutant strains demonstrated the importance of peroxiredoxins in the cellular antioxidant defence and that thiol-peroxidases are vital in conditions of oxidative stress. The expression of the TSA2 was induced in the absence of catalase A in cells grown in oleate and with no exogenous oxidants. The results suggest the existence of an efficient pathway of antioxidant defense, involving thiol-peroxidases, which compensates the absence of catalase A in the cell and protects yeast against oxidative stress induced by both hydrogen peroxide and organic peroxide. The peroxiredoxin Tsa2 may be involved in the antioxidant pathway that compensates the absence of peroxisomal catalase through an unknown mechanism.

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

Numerous studies have investigated how bulk lipid metabolism is influenced in obesity and in particular how the composition of triglycerides found in the cytosol change with increased adipocyte expansion. However, in part reflecting the analytical challenge the composition of cell membranes, and in particular glycerophospholipids, an important membrane component, have been seldom investigated. Cell membrane components contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimized environment for numerous proteins and providing important pools for metabolites, such as choline for one-carbon metabolism and S-adenosylmethionine for DNA methylation. Here, I have conducted a comprehensive lipidomic and transcriptomic study of white adipose tissue in mice that become obese either through genetic modification (ob/ob genotype), diet (high-fat diet) or a combination of the two across the life course. Specifically, I demonstrated that the changes in triglyceride metabolism that dominate the overall lipid composition of white adipose tissue were distinct from the compositional changes of glycerophospholipids. These latter lipids became more unsaturated to maintain the fluidity and normal function of the membrane in the initiation of obesity but then turned saturated after long-term administration of HFD and aging. This suggests that while triglycerides within the adipose tissue may be a relatively inert store of lipids, the compositional changes occur in cell membranes with more far-reaching functional consequences in both obesity and aging. The two-phase change of phospholipids can be correlated well with transcriptional and one-carbon metabolic changes within the adipocytes. The transcriptomic study demonstrated that the lipid metabolic pathways regulated by the peroxisome, AMPK, insulin and PPARγ signaling were activated in the initiation of obesity but inhibited in the adipose tissue of old ob/ob mice along with up-regulated inflammation pathways. The brown and white adipose tissue of PPARα-knock-out mice were also studied by lipidomic tools to get a deeper understanding of the effect of the peroxisome and PPAR system on adipose tissue and lipid metabolism during obesity. Most of the lipids were increased and became more saturated and shorter in adipose tissues of PPARα null mice, which is in good accordance with the results of the former animal study. In conclusion, my work using different rodent models and multi-omics techniques demonstrated a protective metabolic mechanism activated in the initiation but impaired at the end of the processes of obesity and aging, which could be an explanation of the similarity of obesity and aging in terms of high incidence of the metabolic syndrome and related diseases.

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