This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

No description available.

No description available.

No description available.

by Frankie Kar-Ming Wong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 101-107). / Acknowledgements --- p.IV / Abstract --- p.V / Abbreviations --- p.VI / Chapter Chapter 1 --- Introduction to Lipoprotein and Apolipoprotein --- p.1 / Chapter 1.1 --- Lipoprotein structure and classification --- p.1 / Chapter 1.2 --- Apolipoprotein A-I and B100 --- p.1 / Chapter 1.2.1 --- Apolipoprotein A-I (apoA-I) --- p.1 / Chapter 1.2.2 --- Apolipoprotein B100 (apoB100) --- p.3 / Chapter 1.2.3 --- Biological functions of apolipoprotein --- p.4 / Chapter 1.3 --- Evidence linking apoA-I and B100 with atherosclerosis --- p.4 / Chapter 1.4 --- The roles of apoA-I and B100 in the development of atherosclerosis --- p.6 / Chapter 1.5 --- Measurement of human serum lipoproteins as an assessment of risk for coronary heart disease (CHD) --- p.8 / Chapter 1.6 --- Aims of this study --- p.10 / Chapter Chapter 2 --- Purification of ApoA-I and B100 and Production of Polyclonal Antibodies --- p.12 / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.1.1 --- Purification of apoA-I and B100 from human serum --- p.12 / Chapter 2.1.2 --- Immunization for polyclonal antibodies production against apoA-I and B100 --- p.14 / Chapter 2.1.3 --- Antibody purification --- p.15 / Chapter 2.1.3.1 --- Ammonium sulfate precipitation --- p.17 / Chapter 2.1.3.2 --- DEAE and QEAE Sepharose --- p.17 / Chapter 2.1.3.3 --- Protein A and Protein G --- p.17 / Chapter 2.1.3.4 --- Affinity chromatography --- p.18 / Chapter 2.2 --- Methods --- p.20 / Chapter 2.2.1 --- Purification of HDL and LDL --- p.20 / Chapter 2.2.2 --- Purification of apolipoproteins --- p.22 / Chapter 2.2.3 --- Immunization of rabbit with apoA-I and B100 --- p.23 / Chapter 2.2.4 --- Enzyme-linked immunosorbent assay (ELISA) --- p.24 / Chapter 2.2.5 --- Purification of lipoprotein specific immunoglobulin from antisera --- p.25 / Chapter 2.2.5.1 --- Salt fractionation --- p.25 / Chapter 2.2.5.2 --- Purification of immunoglobulin by Protein A affinity chromatography --- p.25 / Chapter 2.2.5.3 --- Isolation of specific antibody by lipoprotein-coupled affinity chromatography --- p.26 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Purification of apoA-I and B100 --- p.27 / Chapter 2.3.2 --- Purification of immunoglobulins from rabbit anti-apolipoprotein sera --- p.32 / Chapter 2.4 --- Discussion --- p.38 / Chapter Chapter 3 --- Production of monoclonal antibodies against apoA-I and B100 --- p.48 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.1.1 --- What is monoclonal antibody? --- p.48 / Chapter 3.1.2 --- The basic methodology --- p.49 / Chapter 3.1.2.1 --- Immunization of host --- p.49 / Chapter 3.1.2.2 --- Cell lines required for fusion --- p.49 / Chapter 3.1.2.3 --- Fusion --- p.51 / Chapter 3.1.2.4 --- Selection of hybrids --- p.52 / Chapter 3.1.2.5 --- Screening assay --- p.54 / Chapter 3.1.2.6 --- Cloning --- p.54 / Chapter 3.1.2.7 --- Bulk production of monoclonal antibody --- p.55 / Chapter 3.1.2.8 --- Monoclonal antibody purification --- p.55 / Chapter 3.2 --- Methods --- p.55 / Chapter 3.2.1 --- Immunization of mice with apoA-I and apoB100 --- p.55 / Chapter 3.2.2 --- Preparation before fusion --- p.58 / Chapter 3.2.2.1 --- Preparation of tissue culture working solutions --- p.58 / Chapter 3.2.2.2 --- Preparation of spleen cells --- p.59 / Chapter 3.2.2.3 --- Preparation of myeloma cells --- p.60 / Chapter 3.2.3 --- Fusion --- p.60 / Chapter 3.2.4 --- Screening assay for positive clones --- p.61 / Chapter 3.2.5 --- Limiting dilution cloning --- p.61 / Chapter 3.2.6 --- Determination of isotype --- p.62 / Chapter 3.2.7 --- Cryopreservation of myeloma and established hybridoma cell lines --- p.62 / Chapter 3.2.7.1 --- Freezing cells --- p.62 / Chapter 3.2.7.2 --- Thawing cells --- p.63 / Chapter 3.2.8 --- Bulk production of monoclonal antibodies from ascites --- p.63 / Chapter 3.2.9 --- Purification of monoclonal antibodies from ascites --- p.63 / Chapter 3.2.10 --- Western blot analyses of the monoclonal antibodies --- p.64 / Chapter 3.2.11 --- Iodination of apolipoproteins --- p.64 / Chapter 3.2.12 --- Binding of the monoclonal antibody to iodinated apolipoprotein --- p.65 / Chapter 3.2.13 --- Competitive displacement analyses --- p.65 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Development of monoclonal antibodies --- p.66 / Chapter 3.3.2 --- Purification of monoclonal antibody from ascites --- p.69 / Chapter 3.3.3 --- Western blotting analyses of AB6 and BE8 --- p.69 / Chapter 3.3.4 --- Monoclonal antibody titration curve for apolipoproteins by radioimmunoassays --- p.75 / Chapter 3.3.5 --- Competitive displacement analysis of AB6 and BE8 --- p.75 / Chapter 3.4 --- Discussion --- p.79 / Chapter Chapter 4 --- Enzyme-linked immunosorbent assay (ELISA) for ApoA-I --- p.84 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.1.1 --- Alkaline phosphatase (ALP) --- p.84 / Chapter 4.1.2 --- Conjugation methods --- p.85 / Chapter 4.1.3 --- Design of the immunoassay format --- p.87 / Chapter 4.1.4 --- Modified solid-phase: Protein A antibody-capture ELISA (PACE) --- p.87 / Chapter 4.2 --- Materials and Methods --- p.90 / Chapter 4.2.1 --- Conjugation of AB6 with maleimide activated alkaline phosphatase --- p.90 / Chapter 4.2.2 --- Titration curve of AB6-ALP conjugate --- p.90 / Chapter 4.2.3 --- Calibration curve of apoA-I sandwich ELISA --- p.91 / Chapter 4.2.4 --- Measurement of apoA-I by Protein A antibody-capture ELISA --- p.91 / Chapter 4.3 --- Results --- p.92 / Chapter 4.3.1 --- Characterization of AB6-ALP conjugate --- p.92 / Chapter 4.3.2 --- Calibration curve for the measurement of apoA-I --- p.92 / Chapter 4.4 --- Discussion --- p.95 / Chapter Chapter 5 --- General Conclusions --- p.99 / References --- p.101

dsc-4(qm182), a mutation that suppresses the lengthened defecation cycle of clk-1 also suppresses the delay in germline development. dsc-4 encodes a putative orthologue of microsomal triglyceride transfer protein (MTP), a protein essential for the assembly and secretion of apo-B-containing low density lipoproteins (LDL). The effect of dsc-4 on clk-1(qm30), coupled to studies of apoB homologues in worms led to a model suggesting the possibility of using C. elegans in the study of LDL-like lipoprotein particles. The impact of the level of lipoproteins is particularly evident in the germline developmental rate of the worms. / We report here a further elucidation of clk-1 mutants in the study of the biology of LDL-like particles. In particular, we investigated the effect of targeting LDL receptor-like genes by RNA interference (RNAi) on the egg laying rate of clk-1(qm30). We find positive modulating effects by disruption of these putative LDL receptors. In confirmation of our model of lipoprotein metabolism in clk-1 mutants, we find that disruption of these putative LDL receptors produces strikingly different effects in wild-type, clk-1(qm30) or clk-1(qm30); dsc-4(qm182) animals. / In addition, we report unexpected effects of various clk-1 alleles on the phenotypes of animals in which lrp-1 and rme-2 are disrupted. Specifically, we observe an allele specific amelioration of the phenotypes associated with disruption of these genes (abnormal molting and sterility, respectively). We discuss the possible significance of these findings. (Abstract shortened by UMI.)

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Western Australia, 2007.

The cellular degradation of the low density lipoprotein (LDL) receptor, and its ligand, LDL, were investigated in order to clarify certain mechanistic aspects of these important processes. Long-term lymphoblastoid cell lines and cultured human skin fibroblasts were used to examine the fate of ¹²⁵I-LDL subsequent to its uptake via receptor-mediated endocytosis. In both cases, binding activity was saturable, depended on the presence of calcium ions in the medium, and was calculated to have an equilibrium dissociation constant at 4ᵒC of 2 μg ¹²⁵I-LDL/ml. No high-affinity binding was detected when the ligand was modified by acetylation. After incubating the monolayers at 37°C LDL/LDL receptor complexes were internalized, and the receptors were recycled back to the surface within about 10 minutes. Apolipo-protein B in the LDL particles was largely degraded to the amino acid level: chloroquine, a lysosomotropic agent, inhibited the formation of the ¹²⁵I-LDL degradation products. Cells obtained from a number of heterozygous and homozygous familial hypercholesterolemic patients, as expected, bound markedly reduced amounts of ligand. The half-life of ¹²⁵I-LDL was measured after it had been introduced into cultured fibroblasts by one of the following processes: (i) uptake via receptor-mediated endocytosis in human skin fibroblasts with normal LDL receptors, or (ii) incorporation via scrape-loading into fibroblasts defective in LDL receptor content. The half-lives obtained were about 1 hour and 50 hours, respectively, indicating that efficient degradation of LDL occurred only when it was deIivered to lysosomes via receptor-mediated endocytosis.

No description available.