Chan Wing Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 142-153). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract --- p.II / Abstract 摘要 --- p.IV / List of Abbreviations --- p.VI / List of Figures --- p.VIII / List of Tables --- p.X / Contents --- p.XII / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Apolipoprotein and Lipoprotein Metabolism --- p.1 / Chapter 1.2 --- Molecular Information of Apolipoprotein E --- p.2 / Chapter 1.3 --- Protein Structure of Apolipoprotein E --- p.4 / Chapter 1.4 --- Polymorphism of Apolipoprotein E --- p.5 / Chapter 1.4.1 --- Genetic Polymorphism --- p.5 / Chapter 1.4.2. --- Posttranslational Glycosylation --- p.8 / Chapter 1.5 --- Tissue Distribution of Apolipoprotein E --- p.8 / Chapter 1.6 --- Functions of Apolipoprotein E --- p.10 / Chapter 1.6.1 --- Role of Apolipoprotein E in Lipid Metabolism --- p.10 / Chapter 1.6.1.1 --- Chylomicron Metabolism and Apolipoprotein E --- p.11 / Chapter 1.6.1.2 --- VLDL Metabolism and Apolipoprotein E --- p.11 / Chapter 1.6.1.3 --- HDL Metabolism and Apolipoprotein E --- p.11 / Chapter 1.6.2 --- Lipid-independent Role of Apolipoprotein E --- p.12 / Chapter 1.6.2.1 --- Stimulation on Neurite Extension --- p.13 / Chapter 1.6.2.2 --- Inhibition of Agonist-induced Platelet Aggregation --- p.13 / Chapter 1.6.2.3 --- Immunoregulation --- p.13 / Chapter 1.6.2.4 --- Regulation of Androgen Synthesis by Ovary --- p.14 / Chapter 1.7 --- Association Between Various Disorders and Apolipoprotein E --- p.15 / Chapter 1.7.1 --- Type III Hyperlipoproteinemia and Apolipoprotein E --- p.15 / Chapter 1.7.2 --- Alzheimer's Disease and Apolipoprotein E --- p.15 / Chapter 1.7.3 --- Atherosclerosis and Apolipoprotein E --- p.16 / Chapter 1.8 --- Cell Proliferation and Cell Cycle Progression --- p.18 / Chapter 1.8.1 --- Cyclin-dependent Kinases and Cyclins --- p.19 / Chapter 1.8.2 --- Cyclin-dependent Kinases and Their Inhibitors --- p.21 / Chapter 1.8.3 --- Restriction Point in Cell Cycle --- p.22 / Chapter 1.9 --- Cell Proliferation and Mitogen-activated Protein Kinase (MAPK) --- p.22 / Chapter 1.9.1 --- Organization of MAPK Signaling Pathway --- p.22 / Chapter 1.9.2 --- Regulation of Cell Proliferation via Activator Protein-1 (AP-1) --- p.24 / Chapter 1.9.3 --- Regulation of Cell Proliferation via c-Myc --- p.25 / Chapter 1.10 --- Cell Proliferation and Apolipoprotein E --- p.26 / Chapter 1.10.1 --- Effects of Exogenous Apolipoprotein E on Cell Proliferation --- p.26 / Chapter 1.10.2 --- Effects of Endogenous Apolipoprotein E on Cell Proliferation --- p.27 / Chapter 1.11 --- Aims and Scopes of Study --- p.27 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Materials --- p.29 / Chapter 2.1.1 --- Construction of ApoE Isoform-encoding Plasmids --- p.29 / Chapter 2.1.1.1 --- Human ApoE3 cDNA --- p.29 / Chapter 2.1.1.2 --- Mammalian Expression Vector --- p.29 / Chapter 2.1.1.3 --- Reagents for Agarose Gel Electrophoresis --- p.29 / Chapter 2.1.1.4 --- Reagents for Ligation Reaction --- p.31 / Chapter 2.1.1.5 --- Bacterial Culture Medium --- p.31 / Chapter 2.1.1.6 --- Reagents for Polymerase Chain Reaction (PCR) --- p.32 / Chapter 2.1.1.7 --- Site-directed Mutagenesis --- p.33 / Chapter 2.1.2 --- Establishment of ApoE Isoform-expressing Cell Lines --- p.34 / Chapter 2.1.2.1 --- F111 Rat Embryonic Fibroblasts --- p.34 / Chapter 2.1.2.2 --- Reagents for Cell Culture --- p.34 / Chapter 2.1.2.3 --- Cell Culture Wares --- p.36 / Chapter 2.1.2.4 --- Reagents for Transfection --- p.36 / Chapter 2.1.2.5 --- Reagents for Protein Assay --- p.37 / Chapter 2.1.2.6 --- Reagents and Buffers for Western Blot Analysis --- p.37 / Chapter 2.1.2.7 --- Reagents for Enzyme-linked Immunosorbent Assay (ELISA) --- p.42 / Chapter 2.1.3 --- Study on Growth Properties Mediated by ApoE Isoforms --- p.44 / Chapter 2.1.3.1 --- Reagents for Growth Curve Construction --- p.44 / Chapter 2.1.3.2 --- Reagents for 3H-thymidine Incorporation Assay --- p.45 / Chapter 2.1.3.3 --- Reagents for Flow Cytometry --- p.45 / Chapter 2.1.4 --- Study on Signaling Mechanisms Mediated by ApoE Isoforms --- p.46 / Chapter 2.1.4.1 --- RNA Extraction --- p.46 / Chapter 2.1.4.2 --- Reagents for Gene Expression Profiles using Commercial Gene Array --- p.47 / Chapter 2.1.4.3 --- Reagents for Protein Quantification and Western Blot Analysis --- p.48 / Chapter 2.1.4.4 --- Reagents for Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.49 / Chapter 2.1.4.5 --- Reagents for Real Time PCR --- p.50 / Chapter 2.2 --- Methods --- p.52 / Chapter 2.2.1 --- Construction of ApoE Isoform-encoding Plasmids --- p.52 / Chapter 2.2.1.1 --- Subcloning --- p.52 / Chapter 2.2.1.2 --- Site-directed Mutagenesis --- p.53 / Chapter 2.2.2 --- Establishment of ApoE Isoform-expressing Cell Lines --- p.55 / Chapter 2.2.2.1 --- Transfection --- p.55 / Chapter 2.2.2.2 --- Generation of Polyclonal Stable Cell Lines --- p.56 / Chapter 2.2.2.3 --- Protein Extraction --- p.57 / Chapter 2.2.2.4 --- Concentration of Media --- p.57 / Chapter 2.2.2.5 --- Quantification of Proteins --- p.58 / Chapter 2.2.2.6 --- Detection of ApoE Protein by Western Blot Analysis --- p.58 / Chapter 2.2.2.7 --- Enhanced Chemiluminescent (ECL´ёØ) Assay --- p.60 / Chapter 2.2.2.8 --- Quantification of ApoE Protein Level by ELISA --- p.61 / Chapter 2.2.2.9 --- Cell Morphology Study --- p.63 / Chapter 2.2.3 --- Characterization of Growth Properties of ApoE Isoform- expressing Cells --- p.63 / Chapter 2.2.3.1 --- Cell Proliferation Rates --- p.63 / Chapter 2.2.3.2 --- Measurement of DNA Synthesis --- p.63 / Chapter 2.2.3.3 --- Flow Cytometry Analysis of Cell Cycle Profile --- p.64 / Chapter 2.2.4 --- Study on Signaling Mechanisms mediated by ApoE Isoforms --- p.65 / Chapter 2.2.4.1 --- Isolation of Total RNA --- p.65 / Chapter 2.2.4.2 --- Study of Gene Expression Profile by Commercial Gene Array --- p.67 / Chapter 2.2.4.3 --- Determination of Activation Status of MAPK and Transcription Factors by Western Blot --- p.68 / Chapter 2.2.4.4 --- Quantification of Gene Expression Level by Real Time Polymerase Chain Reaction (PCR) --- p.70 / Chapter 2.2.5 --- Statistical Analysis --- p.75 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Construction of ApoE Isoform-encoding Plasmids --- p.76 / Chapter 3.2 --- Establishment of ApoE Isoform-expressing Cell Lines --- p.78 / Chapter 3.2.1 --- Cell Morphology Study --- p.81 / Chapter 3.3 --- Characterization of Growth Properties of ApoE Isoform- expressing Cells --- p.83 / Chapter 3.3.1 --- Cell Proliferation Rates --- p.83 / Chapter 3.3.2 --- DNA Synthesis --- p.86 / Chapter 3.3.3 --- Flow Cytometry Analysis of Cell Cycle Progression --- p.88 / Chapter 3.4 --- Study on Signaling Mechanisms Mediated by ApoE Isoforms --- p.92 / Chapter 3.4.1 --- Study of Gene Expression Profile by Commercial Gene Array --- p.92 / Chapter 3.4.2 --- Study of Activation Status of MAPKs and MEK1/2 --- p.95 / Chapter 3.4.3 --- Study of the Effects of ApoE Isoforms on Transcription Factors --- p.101 / Chapter 3.4.4 --- Real Time PCR Reaction Verification of Primers --- p.105 / Chapter 3.4.5 --- Quantification of Gene Expression Level by Real Time PCR --- p.107 / Chapter Chapter 4: --- Discussions / Chapter 4.1 --- Establishment of Stable Cell Lines Expressing ApoE Isoforms --- p.110 / Chapter 4.1.1 --- Rat Embryonic Fibroblasts as Cell Model --- p.110 / Chapter 4.1.2 --- PCR Amplification of ApoE cDNA --- p.111 / Chapter 4.1.3 --- Expression Level of ApoE Isoform Transgenesin F111Rat Fibroblasts --- p.112 / Chapter 4.2 --- Growth Properties Mediated by ApoE Isoforms --- p.115 / Chapter 4.2.1 --- Effects of ApoE Isoforms on Cellular Morphology --- p.115 / Chapter 4.2.2 --- Serum as the Stimulator of Cell Proliferation --- p.116 / Chapter 4.2.3 --- Isoform-specific Effects of Endogenous ApoE on Cell Proliferation --- p.118 / Chapter 4.2.4 --- Comparison of Effects Mediated by Endogenous and Exogenous ApoE --- p.119 / Chapter 4.3 --- Signaling Mechanisms Mediated by ApoE Isoforms --- p.121 / Chapter 4.3.1 --- Effects of ApoE Isoforms on MAPK Activities --- p.121 / Chapter 4.3.1.1 --- Effects of ApoE Isoforms on ERK1/2 MAPK --- p.122 / Chapter 4.3.1.1.1 --- Upstream Effectors of ERK1/2 --- p.123 / Chapter 4.3.1.1.2 --- Downstream Effectors of ERK1/2 --- p.124 / Chapter 4.3.1.2 --- Effects of ApoE Isoforms on ERK5 MAPK --- p.125 / Chapter 4.3.1.2.1 --- Upstream Effectors of ERK5 --- p.126 / Chapter 4.3.1.2.2 --- Downstream Effectors of ERK5 --- p.127 / Chapter 4.3.2 --- Effects of ApoE Isoforms on Cell Cycle Regulatory Molecules --- p.130 / Chapter 4.3.2.1 --- Effects of ApoE Isoforms on Cyclin Expression --- p.130 / Chapter 4.3.2.2 --- Effects of ApoE Isoforms on CKIs Expression --- p.132 / Chapter 4.3.3 --- Summary of the Signaling Mechanisms Underlying ApoE-mediated Responses --- p.135 / Chapter 4.3.4 --- Other Pathways Involved in ApoE-mediated Responses --- p.137 / Chapter 4.3.4.1 --- Synthesis of Nitric Oxide --- p.137 / Chapter 4.3.4.2 --- Synthesis of Perlecan --- p.138 / Chapter 4.4 --- Conclusions and Future Prospects --- p.139 / Chapter 4.4.1 --- Conclusions --- p.140 / Chapter 4.4.2 --- Future Prospects --- p.141 / Chapter Chapter 5: --- References --- p.142

Thesis (M.S.)--Eastern Illinois University, 2008. / Includes bibliographical references (leaves 69-79).

Hannover, Universiẗat, Diss., 1999.

ApoE is a polymorphic protein that has been found to play many different roles in biological processes including lipid transport, neurobiology and immunoregulation. ApoE occurs in the human population in three major isoforms; apoE2, apoE3 and apoE4. The apoE4 isoform has been identified as a major risk factor for several diseases including atherosclerosis and Alzheimer's disease, therefore a greater understanding of apoE biology is highly sought after. In my thesis, I have investigated several novel aspects of apoE biology. I have identified an association between increased apoE expression and apoptosis in a neuronal cell type and demonstrated that apoE becomes enriched within the neuronal apoptotic debris, consistent with a possible role for apoE in facilitating apoptotic debris clearance. A possible anti-apoptotic role of apoE in macrophages was assessed by reducing or eliminating apoE expression using siRNA and cells isolated from apoE knockout animals, respectively. The removal of apoE did not alter overall sensitivity to apoptosis, however, it did significantly increase staurosporine-induced caspase-3 activation. In other studies, the poorly understood accumulation of apoE within the nucleus was found to be enhanced during serum starvation and to localise in intra-nuclear structures that are distinct from inter-chromatin granule clusters. Analysis of apoE within the human brain revealed a correlation between fragmentation and the apoE3 isoform which was independent from AD status and brain region examined. Additionally, a portion of brain apoE3 was found to be present in the form of disulphide-linked dimers. Collectively, these studies have further expanded the current knowledge of apoE biology in terms of its association with apoptosis, nuclear localization and structural differences between the apoE3 and apoE4 isoforms in the human brain.

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are a risk factor for a variety of atherosclerotic disorders including coronary heart disease. In the current study, the investigators report that incubation of cultured human umbilical vein endothelial cells (HUVECs) with high concentrations of apolipoprotein(a)(apo(a)/Lp(a)) induces apoptosis and endothelial dysfunction in a dose dependent manner. Apo(a), the component of Lp(a) mediates these effects by inducing externalization of Annexin V, DNA condensation and fragmentation which are the hallmarks of death by apoptosis. The pathway of apo(a)-induced apoptosis is associated with overexpression of Bax, caspase-9, p53 phosphorylation, decreased in Bcl-2 expression and activation of caspase-3. Taken together, the data suggest that elevated concentration of apo(a) induces apoptosis in endothelial cells probably by activating the intrinsic pathway. The data also showed that apo(a) induces increased expression of the growth arrest protein (Gas1), which has been known to induce apoptosis and growth arrest in vitro. In addition the data showed that elevated apo(a)/Lp(a) attenuates endothelial nitric oxide (eNOS) activity and endothelin-1 (ET-1) in a dose and time-dependent manner, particularly with small apo(a) isoforms. In summary, the authors proposed a new signaling pathway by which apo(a)/Lp(a) induce apoptosis and this finding could help explain how apo(a)/Lp(a) mediate atherosclerosis related diseases. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-06-20 13:59:48.473

ApoE is a polymorphic protein that has been found to play many different roles in biological processes including lipid transport, neurobiology and immunoregulation. ApoE occurs in the human population in three major isoforms; apoE2, apoE3 and apoE4. The apoE4 isoform has been identified as a major risk factor for several diseases including atherosclerosis and Alzheimer's disease, therefore a greater understanding of apoE biology is highly sought after. In my thesis, I have investigated several novel aspects of apoE biology. I have identified an association between increased apoE expression and apoptosis in a neuronal cell type and demonstrated that apoE becomes enriched within the neuronal apoptotic debris, consistent with a possible role for apoE in facilitating apoptotic debris clearance. A possible anti-apoptotic role of apoE in macrophages was assessed by reducing or eliminating apoE expression using siRNA and cells isolated from apoE knockout animals, respectively. The removal of apoE did not alter overall sensitivity to apoptosis, however, it did significantly increase staurosporine-induced caspase-3 activation. In other studies, the poorly understood accumulation of apoE within the nucleus was found to be enhanced during serum starvation and to localise in intra-nuclear structures that are distinct from inter-chromatin granule clusters. Analysis of apoE within the human brain revealed a correlation between fragmentation and the apoE3 isoform which was independent from AD status and brain region examined. Additionally, a portion of brain apoE3 was found to be present in the form of disulphide-linked dimers. Collectively, these studies have further expanded the current knowledge of apoE biology in terms of its association with apoptosis, nuclear localization and structural differences between the apoE3 and apoE4 isoforms in the human brain.

Thesis (M.S.)--Eastern Illinois University, 2009. / Includes bibliographical references (leaves 80-84).

Regensburg, Univ., Diss., 2009.

Regensburg, Univ., Diss., 2009.

No description available.