Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Jeanne Freeland-Graves. Vita. Includes bibliographical references.

Thesis (Ph. D.)--Drexel University, 2006. / Includes abstract and vita. Includes bibliographical references.

The rates of plasma clearance of human native low density lipoproteins (LDL) and acetylated human low density lipoproteins (acetyl-LDL) were compared between copper-deficient (CuD) and copper-adequate (CuA) rats. Purified human LDL (d 1.02-1.063) were labeled with 125I and injected to fasted recipient rats intravenously. At different time intervals plasma clearance of 125I radioactivity was measured. The percent of clearance was calculated based on the total plasma volume, as determined by a radioisotopic dilution method. Native human 125I-LDL were cleared at a faster rate in CuD, compared with CuA rats. The half-times (t 1 2) of 125I-LDL clearance are 4.90 ± 0.20 and 5.80 ± 0.30 hours in CuD and CuA rats, respectively. The plasma trichloroacetic acid-soluble 125I-radioactivity was significantly and steadily increased in CuD rats at each interval, reflecting the faster clearance and degradation of LDL in those rats. The plasma removal of 125I-acetyl-LDL was faster compared with that of 125I-LDL. The half-times (t 1 2) of acetyl-LDL in CuD and CuA rats are 5.20 ± 0.06 and 5.16 ± 0.08 minutes, respectively, with no significant difference between the groups. The data indicates that the uptake of LDL via the "scavenger" receptor remains unaffected in copper-deficient rats. The faster removal of the unmodified (native) LDL in CuD group suggests that the apoB,E receptor is up-regulated in copper-deficient rats and that the hypercholesterolemia observed in copper deficiency is not associated with the defective uptake of LDL by the apoB,E-receptor dependent mechanism.

The vesicle-mediated degradation of low-density lipoprotein (LDL) is an essential cellular function due to its role in cellular biosynthesis of membranes and steroids. Using multi-color single particle tracking fluorescence microscopy, the intracellular degradation of LDL was probed in live, intact cells. Unique to these experiments is the direct observation of LDL degradation using an LDL-based probe that increases fluorescence intensity upon degradation. Specifically, individual LDL particles were labeled with multiple fluorophores resulting in a quenched fluorescent signal. The characteristics of the vesicle responsible for degradation were determined and the vesicle dynamics involved in LDL degradation were quantified. Visualization of early endosomes, late endosomes and lysosomes was accomplished by fluorescently labeling vesicles with variants of GFP. Transient colocalization of LDL with specific vesicles and the intensity of the LDL particle were measured simultaneously. These studies, which are the first to directly observe the degradation of LDL within a cell, strive to completely describe the endo-lysosomal pathway and quantify the dynamics of LDL degradation in cells.

Elevated plasma concentrations of lipoprotein(a) [Lp(a)] have been identified as an independent risk factor for vascular diseases including coronary heart disease and stroke. In the current study, we have examined the binding and degradation of recombinant forms of apolipoprotein(a) [r-apo(a)], the unique kringle-containing moiety of Lp(a), using a cultured cell model. We found that the incubation of human hepatoma (HepG2) cells with an iodinated 17 kringle-containing (17K) recombinant form of apo(a) resulted in a two-component binding system characterized by a high affinity (Kd = 12 nM), low capacity binding site, and a low affinity (Kd = 249 nM), high capacity binding site. We subsequently determined that the high affinity binding site on HepG2 cells corresponds to the LDL receptor. In the HepG2 cell model, association of apo(a) with the LDL receptor was shown to be dependent on the formation of Lp(a) particles from endogenous LDL. Using an apo(a) mutant incapable of binding to the high affinity site through its inability to form Lp(a) particles (17KΔLBS7,8), we further demonstrated that the LDL receptor does not participate in Lp(a) catabolism. The low affinity binding component observed on HepG2 cells, familial hypercholesterolemia (FH) fibroblasts and human embryonic kidney (HEK) 293 cells may correspond to a member(s) of the plasminogen receptor family, as binding to this site(s) was decreased by the addition of the lysine analogue epsilon-aminocaproic acid. The lysine-dependent nature of the low affinity binding site was further confirmed in HepG2 binding studies utilizing r-apo(a) species with impaired lysine binding ability. We observed a reduction maximum binding capacity for 17K r-apo(a) variants lacking the strong lysine binding site (LBS) in kringle IV type 10 (17KΔAsp) and the very weak LBS in kringle V (17KΔV). Degradation of Lp(a)/apo(a) was found to be mediated exclusively by the low affinity component on both HepG2 cells and FH fibroblasts. Fluorescence confocal microscopy, using the 17K r-apo(a) variant fused to green fluorescent protein, further confirmed that degradation by the low affinity component on HepG2 cells does not proceed by the activity of cellular lysosomes. Taken together, these data suggest a potentially significant route for Lp(a)/apo(a) clearance in vivo. / Thesis (Master, Biochemistry) -- Queen's University, 2009-09-26 02:15:50.754

Thesis (Ph. D.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 233-271). Also available via the World Wide Web.

Background Elevated concentrations of low-density lipoprotein cholesterol (LDL-c) are a major risk factor for the development of coronary artery disease (CAD) because of their role in the progression of atherosclerosis. The black South African population is known to have had historically low LDL-c and in the past there was almost no CAD in the population. However, as this population moves through the nutrition transition, LDL-c levels are increasing. LDL-c levels are regulated by the LDL receptors, which is the major protein involved with transporting cholesterol across cell membranes in humans. Proprotein convertase subtilisinlike/kexin type 9 (PCSK9) is another protein involved with the regulation of LDL-c through its role in assisting with the degradation of the LDL receptor. Variants in both genes can cause elevated or lowered LDL-c levels. Very little information is available on the frequency or presence of variants in the low-density lipoprotein receptor (LDLR) and PCSK9 gene in the black South African population and on how these variants associate with LDL-c. The main aim of the study was thus to determine novel and existing genetic variants in these two genes and to describe the manner in which they associate with plasma LDL-c levels in a black South African population undergoing an epidemiological transition. Methods The 2005 baseline data from the Prospective Urban and Rural (PURE) study population were used in this study. The study population consisted of apparently healthy black volunteers form the North West province of South Africa, aged 35 to 60 years. Thirty individuals were randomly chosen from the 1860 volunteers to determine the presence of known and novel variants in these genes by automated bidirectional sequencing. The promoter region, exons and flanking regions were sequenced and variants were identified utilising CLC DNA Workbench. Deoxyribonucleic acid (DNA) samples for 1500 individuals of the PURE study population were genotyped by means of a Golden Gate Genotyping Assay. Analyses of covariance (ANCOVA) were used to test for associations between the different genotypes in both the LDLR and PCSK9 genes and LDL-c levels. Haplotypes were generated by using the confidence intervals on the software programme, HaploView. A genetic risk score (GRS) was determined by including variants which associated significantly with LDL-c. The GRS, the haplotypes and the variants that associated significantly with LDL-c were used in separate linear regression models with variants which correlated with LDL-c to determine how all these variables contribute to the differences in LDL-c levels. Results and discussion Novel and known variants were identified in both the genes and in total 52 variants were genotyped. Rare variants such as rs17249141 and rs28362286 were detected in the study population and are associated with low levels of LDL-c. The variants identified in the LDLR gene were situated largely in regulatory regions such as the promoter, intron and 3‟untranslated regions. Haplotypes in the LDLR gene with the highest frequency associated with lower LDL-c levels, which could contribute to the study population‟s low mean LDL-c level. Haplotypes identified in the PCSK9 gene had a weaker association with LDL-c levels. The minor allele frequencies of many of the variants differed from those of the European population and therefore the importance of population-specific research cannot be sufficiently emphasised. The GRS, haplotypes and variants used in the regression models to determine whether they contributed to predicting the variance in LDL-c in the study population made a small contribution to explaining this. BMI best explained the variance in LDL-c levels. Older women with a body mass index (BMI)>25kg/m2 were identified as being at greater risk of developing elevated LDL-c levels than the rest of the study population. Heterozygote carriers of variant, rs28362286, had 0.787 mmol/L lower LDL-c than carriers of the wild type and this is associated with a reduced risk of developing CAD. Conclusion and recommendation When considering the results mentioned above, adding genetic analysis to explaining the variance in LDL-c levels seems to have its limitations, but the study included only two of many genes that play a role in the metabolism and regulation of LDL-c levels. Incorporating more genes and more variants into analyses and prediction models will add greater value to defining LDL-c levels. Rarer variants with a large impact on protein function, such as rs28362286, have a greater effect on LDL-c levels and could predict the variance better than the common variants. Risk factors such as BMI can also still be trusted to indicate which individuals or groups are at risk of developing elevated LDL-c levels. Health advice should be given to appropriate target groups such as older women with a BMI >25kg/m2 in order to prevent CAD from becoming a burden in this population. / PhD (Dietetics), North-West University, Potchefstroom Campus, 2014

Background Elevated concentrations of low-density lipoprotein cholesterol (LDL-c) are a major risk factor for the development of coronary artery disease (CAD) because of their role in the progression of atherosclerosis. The black South African population is known to have had historically low LDL-c and in the past there was almost no CAD in the population. However, as this population moves through the nutrition transition, LDL-c levels are increasing. LDL-c levels are regulated by the LDL receptors, which is the major protein involved with transporting cholesterol across cell membranes in humans. Proprotein convertase subtilisinlike/kexin type 9 (PCSK9) is another protein involved with the regulation of LDL-c through its role in assisting with the degradation of the LDL receptor. Variants in both genes can cause elevated or lowered LDL-c levels. Very little information is available on the frequency or presence of variants in the low-density lipoprotein receptor (LDLR) and PCSK9 gene in the black South African population and on how these variants associate with LDL-c. The main aim of the study was thus to determine novel and existing genetic variants in these two genes and to describe the manner in which they associate with plasma LDL-c levels in a black South African population undergoing an epidemiological transition. Methods The 2005 baseline data from the Prospective Urban and Rural (PURE) study population were used in this study. The study population consisted of apparently healthy black volunteers form the North West province of South Africa, aged 35 to 60 years. Thirty individuals were randomly chosen from the 1860 volunteers to determine the presence of known and novel variants in these genes by automated bidirectional sequencing. The promoter region, exons and flanking regions were sequenced and variants were identified utilising CLC DNA Workbench. Deoxyribonucleic acid (DNA) samples for 1500 individuals of the PURE study population were genotyped by means of a Golden Gate Genotyping Assay. Analyses of covariance (ANCOVA) were used to test for associations between the different genotypes in both the LDLR and PCSK9 genes and LDL-c levels. Haplotypes were generated by using the confidence intervals on the software programme, HaploView. A genetic risk score (GRS) was determined by including variants which associated significantly with LDL-c. The GRS, the haplotypes and the variants that associated significantly with LDL-c were used in separate linear regression models with variants which correlated with LDL-c to determine how all these variables contribute to the differences in LDL-c levels. Results and discussion Novel and known variants were identified in both the genes and in total 52 variants were genotyped. Rare variants such as rs17249141 and rs28362286 were detected in the study population and are associated with low levels of LDL-c. The variants identified in the LDLR gene were situated largely in regulatory regions such as the promoter, intron and 3‟untranslated regions. Haplotypes in the LDLR gene with the highest frequency associated with lower LDL-c levels, which could contribute to the study population‟s low mean LDL-c level. Haplotypes identified in the PCSK9 gene had a weaker association with LDL-c levels. The minor allele frequencies of many of the variants differed from those of the European population and therefore the importance of population-specific research cannot be sufficiently emphasised. The GRS, haplotypes and variants used in the regression models to determine whether they contributed to predicting the variance in LDL-c in the study population made a small contribution to explaining this. BMI best explained the variance in LDL-c levels. Older women with a body mass index (BMI)>25kg/m2 were identified as being at greater risk of developing elevated LDL-c levels than the rest of the study population. Heterozygote carriers of variant, rs28362286, had 0.787 mmol/L lower LDL-c than carriers of the wild type and this is associated with a reduced risk of developing CAD. Conclusion and recommendation When considering the results mentioned above, adding genetic analysis to explaining the variance in LDL-c levels seems to have its limitations, but the study included only two of many genes that play a role in the metabolism and regulation of LDL-c levels. Incorporating more genes and more variants into analyses and prediction models will add greater value to defining LDL-c levels. Rarer variants with a large impact on protein function, such as rs28362286, have a greater effect on LDL-c levels and could predict the variance better than the common variants. Risk factors such as BMI can also still be trusted to indicate which individuals or groups are at risk of developing elevated LDL-c levels. Health advice should be given to appropriate target groups such as older women with a BMI >25kg/m2 in order to prevent CAD from becoming a burden in this population. / PhD (Dietetics), North-West University, Potchefstroom Campus, 2014

Master of Science / Department of Physics / Christopher M. Sorensen / This work emphasizes advancements in Aerosol Gelation. We have attempted to expand the available materials used to synthesize Aerosol Gels by moving away from gas phase precursors toward liquid phase precursors and eventually reactants in the solid phase. The primary challenge was to efficiently administer the liquid fuels into the detonation chamber. After several attempts, it was concluded that the most efficient delivery technique was to heat the liquid fuel past the vapor point and evaporate it into the oxidizing gas for combustion. This method consistently yields soot with a density of 3.2 mg/cc approximately 10 minutes after the combustion. It was concluded that four criterion must be met to create an Aerosol Gel from a liquid: 1. The liquid must be as finely divided as possible 2. The energy of the spark must be large enough to cause a sustainable combustion 3. The fuel must have a Lower Explosive Limit above the necessary concentration to meet a volume fraction of 10[superscript]4 4. The fuel must have a relatively low boiling point

The apoC-III proteoform containing two sialic acid residues (apoC-III2) has different in vitro effects on lipid metabolism compared with asialylated (apoC-III0) or the most abundant monosialylated (apoC-III1) proteoforms. Cross-sectional and longitudinal associations between plasma apoC-III proteoforms (by mass spectrometric immunoassay) and plasma lipids were tested in two randomized clinical trials: ACT NOW, a study of pioglitazone in subjects with impaired glucose tolerance (n = 531), and RACED (n = 296), a study of intensive glycemic control and atherosclerosis in type 2 diabetes patients. At baseline, higher relative apoC-(I)II2 and apoC-III2/apoC-III1 ratios were associated with lower triglycerides and total cholesterol in both cohorts, and with lower small dense LDL in the RACED. Longitudinally, changes in apoC-III2/apoC-III1 were inversely associated with changes in triglycerides in both cohorts, and with total and small dense LDL in the RACED. apoC-III2/apoC-III1 was also higher in patients treated with PPAR-gamma agonists and was associated with reduced cardiovascular events in the RACED control group. Ex vivo studies of apoC-III complexes with higher apoC-III2/apoC-III1 showed attenuated inhibition of VLDL uptake by HepG2 cells and LPL-mediated lipolysis, providing possible functional explanations for the inverse association between a higher apoC-III2/apoC-III1 and hypertriglyceridemia, proatherogenic plasma lipid profiles, and cardiovascular risk.