In conclusion, our study has demonstrated that TGZ, a synthetic PPARgamma ligand, inhibits lung cancer cells growth through cell-cycle arrest, increased cell differentiation and induction of apoptosis. In this pathway, the activation of ERK by TGZ plays a central role in promoting apoptosis, which appears to be mediated via a mitochondria-related mechanism and functions in a PPARgamma-dependent manner. The interaction between PPARgamma and ERK may create an auto-regulatory and positive feedback loop to enhance the effect of ERK whereas the activation of Akt may generate a negative regulation to control the degree of apoptosis occurred in lung cancer cells. TGZ may counteract NNK function to inhibit lung cancer cell growth in the PPARgamma-dependent manner. / Lung cancer is the world's leading cause of cancer death. Currently there is not an acceptable adjuvant or palliative treatment modalities that have been conclusively shown to prolong survival in lung cancer. Therefore, translational research to improve outcomes with this disease is critical. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear hormone receptor superfamily of ligand-activated transcription. PPARgamma ligands have been demonstrated to inhibit growth of cancer cells. The role of the PPARgamma in cell differentiation, cell cycle arrest and apoptosis has attracted increasing attention. Our study focused on the role of PPARgamma and its ligand troglitazone (TGZ) in the cell death of human lung cancer and the interaction between PPARgamma system and 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a major tobacco-specific carcinogen. / The epidemic of lung cancer is directly attributable to cigarette. However, it is still not completely known the molecular pathway of cigarette smoking in the pathogenesis of lung cancer. Among the carcinogenoic chemicals of cigarette smoking, 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent, which induces lung cancer in all animal species tested. Unlike PPARgamma ligands, NNK can promote cell proliferationa and growth. It is interesting to know whether PPARgamma ligands can inhibit the growth-promoting function of NNK. To address this question, we used NCI-H23 lung cancer cells as the model to study how TGZ influenced the function of NNK. Results showed that NNK stimulated cell proliferation, induced the DNA binding activity of nuclear factor-kappaB (NF-kappaB), down-regulated Bad expression, and up-regulated PPARgamma protein expressions. Inhibition of NF-kappaB nuclear translocation led to the suppression of NNK-mediated Bad expression, indicating that NNK may regulate Bad expression through the activation of NF-kappaB. TGZ significantly inhibited cell proliferation induced by NNK. Though TGZ did not affect nuclear factor-kappaB (NF-kappaB) activity, it up-regulated Bad expression. Taken together, TGZ can efficiently inhibit the proliferation of lung cancer cells induced by NNK via Bad- and PPARgamma- related pathways, which may not be directly relevant to the activity of NF-kappaB. / To elucidate the mechanism responsible for the effect of PPARgamma and TGZ on lung cancer cells, we further studied the PPARgamma molecular pathway in NCIH23 treated by TGZ. The result demonstrated that TGZ induced PPARgamma and ERK1/2 accumulation in the nucleus, where the co-localization of both proteins was found. It showed that the activation of ERK1/2 resulted in apoptosis via the mitochondrial pathway, reflecting by reduction of mitochondria membrane potential, change in Bcl-2 family members, release of cytochrome c into cytosol, and activation of caspase 9. Both PPARgamma siRNA and U0126, a specific inhibitor of ERK1/2, were able to block these effects of TGZ, suggesting that apoptosis induced by TGZ was PPARgamma- and ERK1/2-dependent. Inhibition of ERK1/2 by U0126 also led to a significant decrease in the level of PPARgamma, indicating that there was probably a positive cross-talk between PPARgamma and ERK 1/2 or an auto-regulatory feedback mechanism to amplify the effect of ERK1/2 on cell growth arrest and apoptosis. In addition to ERK1/2, TGZ also activated Akt. Interestingly, inhibition of ERK1/2 prevented the activation of Akt whereas suppression of Akt had no effect on ERK1/2, suggesting that Akt was not necessary for TGZ-PPARgamma-ERK pathway. However, the inhibition of Akt promoted the release of cytochrome c. Thus, the activation of Akt may have a negative effect on apoptosis induced by TGZ. Wortmannin, a PI3K inhibitor, inhibited TGZ-induced ERK1/2 and Akt activation, indicating that PI3K may function at the up-stream of ERK and Akt. In conclusion, our study has demonstrated that TGZ induced apoptosis in NCI-H23 lung cancer cells via a mitochondrial pathway and this pathway was PPARgamma-and ERK1/2-dependent. / We first investigated the effect of PPARgamma ligand TGZ on two human lung cancer cells (NCI-H23 and CRL-2066) and one human lung normal cell (CCL-202). The results showed that in consistence with the loss of cell viability, TGZ induced apoptosis in CRL-2066 and NCI-H23 cells but not in CCL-202 cells. TGZ up-regulated PPARgamma expression in all these three lung cell lines, especially in the cancer cells. In association of the time-dependent inhibition of the cell proliferation, TGZ down-regulated the expression of Bcl-w and Bcl-2 but activated ERK1/2 and p38, suggesting that the growth-inhibitory effect of TGZ is associated with the reduction of Bcl-w and Bcl-2 and the increase of ERK1/2 and p38 activation. SAPK/JNK activation assay showed a decreased activity in all these three cell lines treated by TGZ. It was also demonstrated that TGZ was able to activate PPARgamma transcriptionally. We conclude that TGZ inhibits the growth of human lung cancer cells via the induction of apoptosis, at least in part, in a PPARgamma-relevant manner. / Li Mingyue. / "June 2006." / Advisers: George Gong Chen; Anthony Ping Chuen Yim. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6202. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 174-207). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Peroxisomes compartmentalize a variety of important metabolic reactions including fatty acid β-oxidation and the related process of IBA β-oxidation. Peroxisomal proteins are encoded by nuclear genes and must be post-translationally imported. A dynamic import process is vital for proper matrix protein localization and is dependent on the family of peroxin (PEX) proteins. The delivery and peroxisomal import of cargo from a loaded receptor, PEX5 or PEX7, is carried out by the early-acting peroxins, including PEX13 and PEX14, and receptor recycling is carried out by the late-acting peroxins, including PEX4 and PEX6. In this thesis, I describe the use of double mutant analysis to differentiate early-acting and late-acting pex mutants by phenotypic and molecular analysis. I found that double mutants made with two early-acting or two late-acting pex mutants showed enhanced phenotypes in β-oxidation and import defects. In contrast, defects of double mutants made with a weak early-acting mutant and a late-acting mutant were suppressed. Additionally, I found that receptor localization is central to proper peroxisomal function. My results suggest that when the receptor is not removed from the peroxisome, stabilized peroxisomal pores may be formed, perhaps impairing peroxisomal function due to leaching of peroxisomal contents. Together my data suggest that balance between import and receptor recycling is fundamental for peroxisomal function. In humans, peroxisomal biogenesis disorders are most often caused by defects in late-acting peroxins. Peroxisomal defects occur in plants and humans as a result of the same lesions in PEX proteins. The understanding of how these late-acting defects can be ameliorated in plants, may inspire new approaches to human therapeutics.

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

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

No description available.

Na fase ativa da leishmaniose visceral (LV) ocorrem alterações no metabolismo de lipoproteínas com redução dos níveis de HDL e aumento de triglicérides. A partir desses dados, focamos neste projeto essas alterações na progressão da infecção e apontamos alguns elementos como seus possíveis desencadeantes. Como essas alterações poderiam resultar de redução de atividade e expressão da lipoproteína lipase (LPL), do receptor alfa do proliferador ativado de peroxissoma (PPAR?) e da proteína transferidora de ésteres de colesteril (CETP), a sua expressão foi avaliada durante a progressão da LV em hamster. Em hamsteres infectados com 2 x 107 amastigotas de L. (L.) infantum observamos aumento de triglicérides nos hamsteres com 55 dias (mediana = 294,0 mg/dL) e 90 dias (303,0 mg/dL ) de infecção comparados aos controles de 55 dias (119,0 mg/dL) e de 90 dias (117,0 mg/dL) (p <= 0,05). Os níveis de colesterol total e de HDL não apresentaram diferença significante entre controles e infectados com 30, 55 e 90 dias de infecção. A expressão de mRNA de PPAR? no fígado com 55 e 90 dias de infecção apresentou tendência de redução nos infectados. Já de CETP no fígado dos hamsteres com 55 dias de infecção, a expressão relativa (CT) estava reduzida nos infectados (0,08) comparados aos controles (1,69) (p <= 0,05) e de LPL no coração dos hamsteres com 90 dias de infecção também estava reduzida (1,43) com relação aos controles (2,61) (p <= 0,05). Há dados na literatura sugerindo a importância de lipídios para o desenvolvimento de amastigotas no hospedeiro vertebrado e é possível que as alterações dos níveis de lipoproteínas contribuam na progressão da infecção. Assim, avaliamos neste estudo o efeito da droga hipolipemiante ciprofibrato no controle do parasitismo na LV em hamster, sabendo-se que ciprofibratos atuam aumentando a expressão de PPAR? e a produção e atividade de LPL. O tratamento com ciprofibrato nos hamsteres com 55 dias de infecção gerou redução de triglicérides (123,0 mg/dL) em relação aos infectados não tratados (294,0 g/dL) (p <= 0,05), além dos níveis de triglicérides nos animais infectados não tratados terem aumentado quando comparados aos controles não tratados (119,0 mg/dL) (p <= 0,05). Houve também, redução de triglicérides nos animais não infectados tratados com ciprofibrato (89,0 mg/dL) comparando-se aos infectados não tratados (p <= 0,05). Os níveis de colesterol nos hamsteres não infectados tratados com ciprofibrato reduziram (53,5 mg/dL) em comparação aos infectados não tratados (93,0 mg/dL) (p <= 0,05). Já naqueles que foram infectados e tratados com ciprofibrato, constatamos redução de colesterol (53,5 mg/dL) quando comparados aos infectados não tratados (p <= 0,05). Os níveis de HDL não aumentaram com ciprofibrato e foram similares entre os hamsteres infectados não tratados e os controles não tratados. A carga parasitária no baço e no fígado não foi reduzida com ciprofibrato. Na leishmaniose visceral em hamster ocorrem alterações do metabolismo lipídico com aumento de triglicérides e redução da expressão da mRNA de LPL e CETP. O tratamento com ciprofibrato foi eficaz no controle das alterações de níveis de lipoproteínas. / In the active phase of visceral leishmaniasis (VL) changes occur in lipoprotein me-tabolism with reduction in HDL and increase in triglyceride (TG) levels. From these data, in this project we focused these changes during the progression of the infection and we approached some elements as their underlying factors. Since these changes may result from the reduction of the activity and the expression of the lipoprotein lipase (LPL), of the peroxisome proliferator-activated receptor alpha (PPAR?) and of the cholesteryl ester transfer protein (CETP), their expression were evaluated during VL progression in hamster. In 2 x 107 L. (L.) infantum amastigote-infected hamsters we observed an increase in the triglycerides in hamsters with 55 days (median = 294.0 mg/dL) and 90 days (303.0 mg/dL) of infection compared with controls of 55 days (119.0 mg/dL) and of 90 days (117.0 mg/dL) (p <= 0.05). The total cholesterol and the HDL levels did not present significant differences between control and in-fected groups at 30, 55 and 90 days of infection. The expression of mRNA of the PPAR in the liver with 55 and 90 days of infection tended to be reduced in infected animals. However the relative expression (CT) of CETP in the liver of hamsters with 55 days of infection was signicantly reduced in infected (0.08) compared with control animals (1.69) (p <= 0.05). The relative expression (CT) of LPL in the heart of hamsters with 90 days of infection was also reduced (1.43) in relation to controls (2.61) (p <= 0.05). There are data in the literature suggesting the importance of lipids for the development of amastigotes in vertebrate host and it is possible that the changes in the lipoprotein levels contribute for the infection progression. Therefore, we evaluated in this study the effect of the lipid-lowering drug ciprofibrate in the control of parasitism in VL in the hamster, knowing that ciprofibrate acts increasing the expression of the PPAR? and of the LPL production and activity. The treatment with ciprofibrate in infected hamsters at 55 days lead to the reduction of triglyceride level (123.0 mg/dL) in relation to non-treated infected animals (294.0 g/dL) (p <= 0.05). Further the triglyceride levels in the non-treated infected animals were in-creased when compared with untreated controls (119.0 mg/dL) (p <= 0.05). There was also reduction of triglyceride in ciprofibrate treated-non infected animals (89.0 mg/dL) compared with non-treated infected animals (p <= 0.05). The cholesterol lev-els were reduced in the ciprofibrate-treated non-infected hamsters (53.5 mg/dL) in comparison to the non-treated infected ones (93.0 mg/dL) (p <= 0.05). In the ciprofibrate-treated infected ones we found a reduction of cholesterol level (53.5 mg/dL) when compared with non treated infected animals (p <= 0.05). The HDL lev-els did not increase with ciprofibrate and they were similar between the non-treated infected hamsters and non-treated controls. The parasite load in the spleen and liver were not reduced with ciprofibrate. In the visceral leishmaniasis in hamster changes occur in the lipid metabolism with increase in the triglyceride level and the reduction of expression of mRNA of LPL and CETP. The treatment with ciprofibrate was ef-fective in the control of changes in the lipoprotein levels.

Lee Wing Sum. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 206-226). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese Version) --- p.iv / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xxiv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.3 / Chapter 2.1 --- Peroxisomes --- p.3 / Chapter 2.2 --- Peroxisome proliferators --- p.5 / Chapter 2.3 --- Human exposure pathways to peroxisome proliferators --- p.5 / Chapter 2.4 --- Peroxisome proliferator-induced pleiotropic effects in rodents --- p.7 / Chapter 2.4.1 --- Short-term effects --- p.7 / Chapter 2.4.1.1 --- Hepatomegaly --- p.7 / Chapter 2.4.2.1 --- Peroxisome proliferation --- p.8 / Chapter 2.4.1.3 --- Alteration of gene transcriptions --- p.8 / Chapter 2.4.2 --- Long-term effect --- p.9 / Chapter 2.5 --- Mechanisms of actions of peroxisome proliferators --- p.9 / Chapter 2.5.1 --- Substrate overload --- p.9 / Chapter 2.5.2 --- Receptor-mediated --- p.11 / Chapter 2.6 --- Peroxisome proliferator-activated receptors (PPARs) --- p.11 / Chapter 2.6.1 --- Structure of PPARs --- p.11 / Chapter 2.6.2 --- Tissue-specific expression of PPARs --- p.15 / Chapter 2.6.3 --- Physiological functions of PPARs --- p.19 / Chapter 2.6.3.1 --- PPARα --- p.19 / Chapter 2.6.3.2 --- PPARγ --- p.21 / Chapter 2.6.3.3 --- PPARδ --- p.23 / Chapter 2.7 --- Role of PPARα involved in peroxisome proliferator-induced pleiotropic responses --- p.24 / Chapter 2.7.1 --- Short-term effects --- p.24 / Chapter 2.7.2 --- Long-term effect --- p.24 / Chapter 2.8 --- Mechanisms of peroxisome proliferator-induced hepatocarcinogenesis --- p.25 / Chapter 2.8.1 --- Oxidative stress --- p.25 / Chapter 2.8.2 --- Suppression of apoptosis --- p.26 / Chapter 2.8.3 --- Increased cell proliferation --- p.27 / Chapter 2.9 --- Species difference to peroxisome proliferator-induced pleiotropic effects --- p.28 / Chapter 2.10 --- Fluorescent differential display (FDD) --- p.32 / Chapter Chapter 3 --- Objectives --- p.35 / Chapter Chapter 4 --- Materials and methods --- p.37 / Chapter 4.1 --- Animals and treatments --- p.37 / Chapter 4.1.1 --- Materials --- p.37 / Chapter 4.1.2 --- Methods --- p.37 / Chapter 4.2 --- Serum triglyceride and cholesterol analyses --- p.39 / Chapter 4.2.1 --- Materials --- p.41 / Chapter 4.2.2 --- Methods --- p.41 / Chapter 4.2.2.1 --- Serum preparation --- p.41 / Chapter 4.2.2.2 --- Triglyceride determination --- p.41 / Chapter 4.2.2.3 --- Cholesterol determination --- p.42 / Chapter 4.3 --- Statistical analysis --- p.42 / Chapter 4.4 --- Tail-genotyping --- p.42 / Chapter 4.4.1 --- Materials --- p.44 / Chapter 4.4.2 --- Methods. --- p.44 / Chapter 4.4.2.1 --- Preparation of genomic tail DNA --- p.44 / Chapter 4.4.2.2 --- PCR reaction --- p.45 / Chapter 4.5 --- Total RNA isolation --- p.45 / Chapter 4.5.1 --- Materials --- p.48 / Chapter 4.5.2 --- Methods --- p.48 / Chapter 4.6 --- DNase I treatment --- p.48 / Chapter 4.6.1 --- Materials --- p.49 / Chapter 4.6.2 --- Methods --- p.49 / Chapter 4.7 --- Reverse transcription of mRNA and fluorescent PCR amplification --- p.50 / Chapter 4.7.1 --- Materials --- p.50 / Chapter 4.7.2 --- Methods --- p.53 / Chapter 4.8 --- Fluorescent differential display (FDD) --- p.53 / Chapter 4.8.1 --- Materials --- p.53 / Chapter 4.8.2 --- Methods --- p.54 / Chapter 4.9 --- Excision of differentially expressed cDNA fragments --- p.54 / Chapter 4.9.1 --- Materials --- p.57 / Chapter 4.9.2 --- Methods --- p.57 / Chapter 4.10 --- Reamplification of differentially expressed fragments --- p.57 / Chapter 4.10.1 --- Materials --- p.60 / Chapter 4.10.2 --- Methods --- p.60 / Chapter 4.11 --- Subcloning of reamplified cDNA fragments --- p.62 / Chapter 4.11.1 --- PCR-TRAP® cloning system --- p.62 / Chapter 4.11.1.1 --- Materials --- p.63 / Chapter 4.11.1.2 --- Methods --- p.63 / Chapter 4.11.2 --- AdvaTage´ёØ PCR cloning system --- p.65 / Chapter 4.11.2.1 --- Materials --- p.65 / Chapter 4.11.2.2 --- Methods --- p.66 / Chapter 4.12 --- Purification of plasmid DNA from recombinant clones --- p.69 / Chapter 4.12.1 --- Materials --- p.69 / Chapter 4.12.2 --- Methods --- p.69 / Chapter 4.13 --- DNA sequencing of differentially expressed cDNA fragments --- p.70 / Chapter 4.13.1 --- CEQ 2000 Dye Terminator Cycle Sequence system --- p.71 / Chapter 4.13.1.1 --- Materials --- p.71 / Chapter 4.13.1.2 --- Methods --- p.71 / Chapter 4.13.2 --- ABI PRISM´ёØ dRhodamine Terminator Cycle Sequencing system --- p.72 / Chapter 4.13.2.1 --- Materials --- p.72 / Chapter 4.13.2.2 --- Methods --- p.72 / Chapter 4.13.3 --- Homology search against computer databases --- p.73 / Chapter 4.14 --- Northern analysis of differentially expressed cDNA fragments --- p.73 / Chapter 4.14.1 --- Formaldehyde gel electrophoresis of total RNA --- p.74 / Chapter 4.14.1.1 --- Materials --- p.74 / Chapter 4.14.1.2 --- Methods --- p.74 / Chapter 4.14.2 --- Preparation of cDNA probes for hybridization --- p.74 / Chapter 4.14.2.1 --- PCR DIG labeling --- p.75 / Chapter 4.14.2.1.1 --- Materials --- p.75 / Chapter 4.14.2.1.2 --- Methods --- p.75 / Chapter 4.14.2.2 --- Random Prime cDNA DIG labeling --- p.75 / Chapter 4.14.2.2.1 --- Materials --- p.75 / Chapter 4.14.2.2.2 --- Methods --- p.76 / Chapter 4.14.3 --- Purification of DNA from agarose gel --- p.77 / Chapter 4.14.3.1 --- Materials --- p.77 / Chapter 4.14.3.2 --- Methods --- p.78 / Chapter 4.14.4 --- Hybridization --- p.78 / Chapter 4.14.4.1 --- Materials --- p.78 / Chapter 4.14.4.2 --- Methods --- p.73 / Chapter 4.14.5 --- Synthesis of mouse GAPDH probe from normalization --- p.80 / Chapter 4.14.5.1 --- Materials --- p.80 / Chapter 4.14.5.2 --- Methods --- p.80 / Chapter Chapter 5 --- Results --- p.82 / Chapter 5.1 --- Liver morphology --- p.82 / Chapter 5.2 --- Liver weight --- p.82 / Chapter 5.3 --- Serum triglyceride and cholesterol levels --- p.88 / Chapter 5.4 --- Confirmation of genotypes --- p.91 / Chapter 5.5 --- DNase I treatment --- p.91 / Chapter 5.6 --- FDD RT-PCR and band excision --- p.98 / Chapter 5.7 --- Reamplification of excised cDNA fragments --- p.111 / Chapter 5.8 --- Subcloning of reamplified cDNA fragments --- p.121 / Chapter 5.9 --- DNA sequencing of subcloned cDNA fragments --- p.124 / Chapter 5.10 --- Confirmation of the differentially expressed cDNA fragments by Northern blot analysis --- p.132 / Chapter 5.11 --- Temporal expression pattern of differentially expressed genes --- p.157 / Chapter 5.12 --- Tissue distribution pattern of differentially expressed genes --- p.171 / Chapter Chapter 6 --- Discussions --- p.183 / Chapter 6.1 --- "Lack of hepatomegaly, hypotriglyceridemia and hepatic nodule formation in PPARα (-/-) mice" --- p.184 / Chapter 6.2 --- "Identification of PPARα-dependent and Wy-14,643 responsive genes" --- p.185 / Chapter 6.3 --- Functional roles of the isolated cDNA fragments --- p.186 / Chapter 6.3.1 --- Fragments B14 and H4 --- p.187 / Chapter 6.3.2 --- Fragment H1 --- p.189 / Chapter 6.3.3 --- Fragment H5 --- p.192 / Chapter 6.3.4 --- Fragment H8 --- p.194 / Chapter 6.4 --- Temporal expression patterns of the isolated cDNA fragments --- p.196 / Chapter 6.5 --- Tissue distribution patterns of the isolated cDNA fragments --- p.197 / Chapter Chapter 7 --- Conclusions --- p.200 / Chapter Chapter 8 --- Future studies --- p.204 / Chapter 8.1 --- Subcloning and characterization of the other differentially expressed genes --- p.204 / Chapter 8.2 --- Overexpression and inhibition expression of specific genes --- p.204 / Chapter 8.3 --- Generating transgenic mice with target disruption of specific gene --- p.205 / References --- p.206

Yuen, Yee Lok. / On t.p. "alpha" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 165-177). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiii / List of Figures --- p.xv / List of Tables --- p.xx / Chapter Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Peroxisome proliferator-activated receptor (PPAR) --- p.1 / Chapter 1.1.1 --- PPARα isoforms --- p.1 / Chapter 1.2 --- PPARα ligands --- p.2 / Chapter 1.3 --- Biological roles of PPARα --- p.3 / Chapter 1.3.1 --- Lipid metabolism --- p.3 / Chapter 1.3.2 --- Bile acid metabolism --- p.4 / Chapter 1.3.3 --- Biotransformation --- p.6 / Chapter 1.4 --- Roles of PPARα in hepatocarcinogenesis --- p.7 / Chapter 1.4.1 --- Cell proliferation and apoptosis --- p.7 / Chapter 1.4.2 --- Oxidative stress --- p.8 / Chapter 1.5 --- Discovery of novel PPARα target genes --- p.9 / Chapter 1.5.1 --- Identification of a novel PPARα-regulated gene L5#55 by fluorescent differential mRNA display (FDD) analysis --- p.9 / Chapter 1.6 --- Sulfotransferase (SULT) --- p.15 / Chapter 1.7 --- Objective of the present study --- p.16 / Chapter Chapter 2 --- Molecular cloning and characterization of mouse liver sulfotransferase-like (mL-STL) gene --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and methods --- p.17 / Chapter 2.2.1 --- Animals --- p.17 / Chapter 2.2.2 --- Treatments --- p.18 / Chapter 2.2.3 --- Total RNA extraction --- p.18 / Chapter 2.2.3.1 --- Materials --- p.18 / Chapter 2.2.3.2 --- Methods --- p.19 / Chapter 2.2.4 --- Rapid amplification of cDNA ends (RACE) --- p.19 / Chapter 2.2.4.1 --- Materials --- p.19 / Chapter 2.2.4.2 --- Methods --- p.20 / Chapter 2.2.4.2.1 --- Primer design --- p.20 / Chapter 2.2.4.2.2 --- Rapid amplification of 5'- and 3'-cDNA ends --- p.20 / Chapter 2.2.5 --- Cloning of the 5'- and 3' RACE products --- p.25 / Chapter 2.2.5.1 --- Materials --- p.25 / Chapter 2.2.5.2 --- Methods --- p.25 / Chapter 2.2.6 --- Northern blot analysis --- p.28 / Chapter 2.2.6.1 --- Materials --- p.28 / Chapter 2.2.6.2 --- Methods --- p.28 / Chapter 2.2.6.2.1 --- Formaldehyde-agarose gel electrophoresis and blotting of RNA --- p.31 / Chapter 2.2.6.2.2 --- PCR DIG-labeling --- p.31 / Chapter 2.2.6.2.3 --- Hybridization and signal detection --- p.32 / Chapter 2.2.7 --- Reverse transcription (RT)-PCR --- p.34 / Chapter 2.2.7.1 --- Materials --- p.34 / Chapter 2.2.7.2 --- Methods --- p.34 / Chapter 2.3 --- Results and discussion --- p.37 / Chapter 2.3.1 --- Cloning of the full-length mL-STL cDNA --- p.37 / Chapter 2.3.2 --- In silico analysis of the mL-STL cDNAs --- p.50 / Chapter 2.3.3 --- Genomic organization of the mL-STL gene --- p.61 / Chapter 2.3.4 --- Tissue distribution of mL-STL mRNA transcript --- p.68 / Chapter 2.3.5 --- "PPARα-dependent regulation of mL-STL mRNA expression by fasting and Wy-14,643 treatment" --- p.74 / Chapter Chapter 3 --- Identification of the native mL-STL protein in mouse liver --- p.86 / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.2 --- Materials and methods --- p.87 / Chapter 3.2.1 --- Animal and treatments --- p.87 / Chapter 3.2.2 --- Cloning of the mL-STL cDNA into a modified pRSET (mpRSET) expression vector --- p.88 / Chapter 3.2.2.1 --- Materials --- p.88 / Chapter 3.2.2.2 --- Methods --- p.88 / Chapter 3.2.2.2.1 --- Amplification of mL-STL cDNA fragments --- p.88 / Chapter 3.2.2.2.2 --- Preparation of mpRSET expression vector --- p.92 / Chapter 3.2.2.2.3 --- "Ligation, transformation, and screening of recombinants" --- p.92 / Chapter 3.2.3 --- Over-expression of the mL-STL recombinant proteins in E coli strains --- p.94 / Chapter 3.2.3.1 --- Materials --- p.94 / Chapter 3.2.3.2 --- Methods --- p.94 / Chapter 3.2.4 --- Mass spectrometry analysis of the mL-STL recombinant proteins --- p.95 / Chapter 3.2.4.1 --- Materials --- p.96 / Chapter 3.2.4.2 --- Methods --- p.96 / Chapter 3.2.4.2.1 --- Trypsin digestion and peptide extraction --- p.96 / Chapter 3.2.4.2.2 --- Matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) mass spectrometry --- p.97 / Chapter 3.2.5 --- Purification of the mL-STL recombinant proteins --- p.98 / Chapter 3.2.5.1 --- Materials --- p.98 / Chapter 3.2.5.2 --- Methods --- p.98 / Chapter 3.2.5.2.1 --- Semi-purification of the mL-STL recombinant proteins by preparative SDS-PAGE --- p.98 / Chapter 3.2.5.2.2 --- Purification of mL-STL recombinant proteins by column chromatography --- p.99 / Chapter 3.2.6 --- Rabbit immunization using purified mL-STL recombinant proteins --- p.101 / Chapter 3.2.7 --- Subcellular fractionation of mouse liver by ultracentrifugation --- p.101 / Chapter 3.2.7.1 --- Materials --- p.101 / Chapter 3.2.7.2 --- Methods --- p.102 / Chapter 3.2.8 --- Western blot analysis of the native mL-STL protein --- p.104 / Chapter 3.2.8.1 --- Materials --- p.104 / Chapter 3.2.8.2 --- Methods --- p.104 / Chapter 3.2.8.2.1 --- SDS-PAGE and electro-blotting of proteins --- p.104 / Chapter 3.2.8.2.2 --- Immunostaining and signal detection --- p.105 / Chapter 3.3 --- Results and discussion --- p.106 / Chapter 3.3.1 --- Cloning of the mL-STLl and mL-STL2 cDNAs into a modified pRSET (mpRSET) vector --- p.106 / Chapter 3.3.2 --- IPTG induction of the mpRSET-mL-STL protein expression --- p.106 / Chapter 3.3.3 --- Confirmation of mL-STL recombinant proteins by mass spectrometry --- p.118 / Chapter 3.3.4 --- Purification of mL-STL recombinant proteins for rabbit immunization and polyclonal antisera production --- p.130 / Chapter 3.3.5 --- Antigenicity of mL-STL antisera --- p.134 / Chapter 3.3.6 --- Identification of mL-STL native protein and its induction pattern in mouse liver --- p.139 / Chapter 3.3.7 --- "Time-course of fasting and Wy-14,643 treatment on the mL- STLl native protein expression" --- p.147 / Chapter Chapter 4 --- Overall discussion --- p.153 / Future study --- p.163 / References --- p.165 / "Appendix A. Alignment of nucleotide sequences of mouse chromosome 7，Riken2810007J24, mL-STLl, and mL-STL2 cDNA sequences" --- p.178 / Appendix Bl. Theoretical tryptic peptide masses of mpRSET- mL-STLl protein --- p.217 / Appendix B2. Raw data from mass spectrometry analysis of mpRSET-mL-STLl protein --- p.218 / Appendix C1. Residue molecular mass of amino acids --- p.219 / Appendix C2. Di-peptide table --- p.220 / Appendix D1. Theoretical tryptic peptide masses of mpRSET- mL-STL2 protein --- p.221 / Appendix D2. Raw data from mass spectrometry analysis of mpRSET-mL-STL2 protein --- p.222

Xu, Jian. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 238-255). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016).