Density gradient ultracentrifugation (DGU) is a powerful method for analyzing lipoprotein particles in great detail. It yields considerable amounts of information regarding the density distribution of these particles when coupled with fluorometric analysis and is an invaluable tool in determining their relative abundance. This union allows relationships between subclasses of lipoproteins to be established that gives researchers a more focused path to aid them in developing methods to predict the early onset of coronary artery disease (CAD). The research presented here focuses on the pairing of DGU with post-separatory techniques including matrix-assisted laser desorption mass spectrometry (MALDI-MS), liquid chromatography mass spectrometry (LC-MS), capillary electrophoresis (CE), isoelectric focusing (IEF) and apoptosis studies involving cell cultures. It is becoming clearer that cholesterol concentrations themselves do not provide sufficient data to assess the quality of cardiovascular health. As a result, research is becoming more focused on identifying better markers that may be indicative of development of CAD in a patient. Of specific interest is group of particles known as high density lipoproteins (HDL). Classically, this molecule is considered the “good cholesterol”, but literature from the last decade suggests that there may be atherogenic variants to this group. By utilizing DGU as a preparatory method for secondary analyses, new dimensions can be added to the density distribution analysis to allow a better determination of markers of cardiovascular health. The aim of this work is to utilize the principles involved with these various techniques to develop a comprehensive set of methods to aid in the detection of potential risk markers. In this study, the properties of metal ion complexes of EDTA as solute systems for analysis of lipoproteins by DGU are analyzed. We show that by varying the complexing ion and counter-ion of these metal-ion complexes, we gain the ability to control the separation of lipoprotein subclasses for subsequent analyses. Qualitative and quantitative data is presented that describes the analysis of different density regions of HDL for apolipoprotein content. Trends between control and atherogenic samples are also described and a clinical link between the biological activity of these regions and the chemical analysis is discussed.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Following a mild head injury (MHI), a person may report a variety of symptoms such as headache, memory disturbance, dizziness, and concentration difficulties. For most persons the symptoms are transient, but some suffer persistent symptoms that can have a major impact on everyday life. It remains poorly understood why some but not others have full recovery after MHI. The aim of this thesis was to investigate outcomes after MHI, with particular focus on neuropsychological functioning, fatigue, and risk of dementia. A related objective was to examine the potential association of a genetic factor, Apolipoprotein (APOE), with MHI outcome. The APOE є4 allele has been associated with unfavorable outcomes after moderate or severe head injury, but little is known about its influence on outcome after MHI. In Study I and II, data from a population-based longitudinal study were used to compare neuropsychological functioning and fatigue before and after MHI. The results from Study I showed a post-injury decline in neuropsychological performance for є4-carriers, whereas the performance remained unchanged for non-carriers. Study II showed an increase in self-reported fatigue after MHI for both є4-carriers and non-carriers, with a more pronounced increase for є4-carriers. In Study III, a case-control study was conducted to examine whether a history of MHI increased the risk of developing dementia later in life. It was found that MHI alone did not increase the risk, but the combination of MHI and APOE є4 was associated with increased risk of dementia. Taken together, the studies generally indicate a positive outcome after MHI, but in combination with APOE є4 even mild head injury may lead to long-lasting negative outcomes. Consideration of pre-injury level of functioning and genetic factors seems critical for a complete understanding of the impact of MHI.

Atherosclerosis is a complex disease which develops silently over decades and can lead to acute myocardial infarction or stroke, the main cause of death worldwide. Apoliporotein E (apo E) is a glycoprotein known for its major role in lipid metabolism and its pro-atherogenic effects. Swine make a unique and viable research model as it shares most of the anatomic and physiologic characteristics with humans, notably for the the cardiovascular system. In addition, it is the only animal species, other than nonhuman primates, that develops atherosclerosis spontaneously. In this study we examined the feasibility for creating an apo E-deficient pig model of atherosclerosis using RNA interference (RNAi) and somatic cell nuclear transfer (SCNT). The knockdown efficiency was tested in porcine granulosa cells. It varied from 45% to 82% compared to control cells, as revealed by real-time PCR analysis. Accordingly, short hairpin RNA-expressing vectors were constructed and used to transfect porcine fetal fibroblast cells. Cell lines with stable chromosomal integration were established and used to produce embryos by SCNT. Development of SCNT embryos to the blastocyst stage (33%) was comparable to non-transgenic embryos. The integration of the shRNA into the genome of GFP-expressing embryos was revealed by PCR and gel electrophoresis. These findings indicate that porcine embryos harboring shRNA-specific to apo E created by SCNT may lead to the production of apo E-deficient pigs. These pigs would be a promising new animal model for advancing atherosclerosis research.

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are associated with increased risk of atherothrombotic disease. Lp(a) is a unique lipoprotein consisting of a low density lipoprotein-like moiety covalently linked to apolipoprotein(a) (apo(a)), a homologue of the fibrinolytic proenzyme plasminogen. Apo(a) is extremely heterogeneous in size with small isoforms being independently associated with increased cardiovascular risk. Several in vitro and in vivo studies have shown that Lp(a)/apo(a) can inhibit tissue-type plasminogen activator (tPA)-mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apo(a) remains controversial. Essential to fibrin clot lysis are a number of plasmin-dependent positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin-mediated conversion of Glu1-plasminogen to Lys78-plasminogen. Additionally, abnormal fibrin clot structures have been associated with both an increased risk of cardiovascular disease and elevated Lp(a) levels. Similarly, oxidized phospholipids have been implicated in the development of cardiovascular disease, and are not only preferentially carried by Lp(a) in the plasma but have also been shown to covalently-modify both apo(a) and plasminogen. In this thesis, we built upon the understanding of the role of apo(a) in plasminogen activation on the fibrin/degraded fibrin surface by determining that: (i) apo(a) inhibits plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion and identifying the critical domains in apo(a) responsible for this effect, (ii) apo(a) isoform size does not affect either the inhibition of tPA-mediated plasminogen activation or the inhibition of plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion, (iii) apo(a) modifies fibrin clot structure to form more dense clots with thinner fibers and reduced permeability, modifications that enhance the ability of apo(a) to inhibit tPA-mediated plasminogen activation and (iv) the phosphorus content of apo(a) affects its ability to inhibit tPA-mediated plasminogen activation and the phosphorus content of plasminogen affects its ability to be activated by tPA. By understanding these individual reactions, each of which has the potential to affect the broader fibrin clot lysis process, we have expanded our understanding of the overall effect of Lp(a)/apo(a) in the inhibition of plasminogen activation on the fibrin/degraded fibrin surface and thus broadened our understanding of how Lp(a)/apo(a) may mediate the inhibition of thrombolysis in vivo. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-12-14 08:26:54.99

Mémoire (M.Med.Exp.)-- Université du Québec à Chicoutimi, 1994. / Ce mémoire a été réalisé à l'Université du Québec à Chicoutimi dans le cadre du programme de maîtrise en médecine expérimentale de l'Université Laval extensionné à l'UQAC. CaQCU Document électronique également accessible en format PDF. CaQCU

Thesis (Ph. D. in Neuroscience)--Vanderbilt University, Aug. 2009. / Title from title screen. Includes bibliographical references.

Thesis (Ph.D.)--Georgetown University, 2009. / Includes bibliographical references.

Atherosclerosis is a disease of both lipids and inflammatory immune cells. More specifically, elevated plasma levels of low-density lipoproteins (LDL) ultimately lead to migration of circulating monocytes into the artery wall. Lipid-loaded monocytes proliferate and become macrophage foam cells, the hallmark of atherosclerotic lesions. A proposed mechanism for the protective effects of high-density lipoprotein (HDL) is apolipoprotein A-I (apoA-I) acting as a mediator of cholesterol efflux from cells and subsequent foam cell regression. To better understand the biological changes stimulated by apoA-I treatment, differential gene expression analysis of microarray data was performed on spleen cells from mice treated with recombinant HDL (rHDL). LDL receptor null (LDLr-/-) and LDL receptor and apoA-I null (LDLr-/-, apoA-I-/-) mice were fed a Western diet consisting of 0.2% cholesterol and 42% calories as fat (HF) for a total of 12 weeks. After six weeks of diet, a subset of mice for each genotype was subcutaneously injected with 200 micrograms of rHDL (protein weight) three times a week for the remaining six weeks. The control group of mice was subcutaneously injected with 200 micrograms of bovine serum albumin (BSA). Spleen cell RNA was isolated, purified, and analyzed via Illumina BeadArray Microarray Technology. Individual differential gene expression analysis that contrasted treated to non-treated groups for each genotype was performed. LDLr-/-, apoA-I-/- rHDL treated mice showed 281 significantly differentially expressed genes compared to non-treated mice while LDLr-/- mice had 1502 such genes. Of the significant genes, 189 intersected across both genotypes. In LDLr-/-, apoA-I-/-, 73 of these were up-regulated and 116 were down-regulated. LDLr-/- similarly showed 71 of the intersected genes to be up-regulated and 118 to be down-regulated. One-directional gene set pathway analysis was also performed. LDLr-/-, apoA-I-/- treated mice revealed 49 significant pathways while LDLr-/- showed a total of 63. Of these, 21 were up-regulated and 14 were down-regulated in both genotypes. Of the overrepresented, up-regulated pathways, eight of the top ten most significant ones were related to immune cells. Major functions involved receptor, adhesion, and chemokine signaling. Overall, preliminary analysis suggests apoA-I treatment induces similar gene expression changes across different genotypes in mouse spleen cells.

Apolipoproteins have important roles in the transport of lipids and the regulation of lipoprotein metabolism as cofactors for enzymes and ligands for receptor-binding. Their function and metabolism are closely related to the development of many diseases. This dissertation describes the investigation of the distribution and metabolism of apoE and glycated apoE in diabetes, obesity and hyperlipidaemia in comparison with healthy people. In order to carry out the research, I developed several robust laboratory methods and techniques for the isolation and measurement of apoE and glycated apoE. These included (1) a modified in-house ultracentrifugation for isolation of lipoprotein fractions (2) high sensitivity sandwich enzyme-linked immunosorbent assay (ELISA) for apoE and (3) m-aminophenylboronate affinity chromatography for the separation of glycated and non- glycated apoE.In healthy people the apoE concentration in different lipoprotein fractions is influenced by age, gender and apoE genotype. The effect of atorvastatin on serum apoE concentration in patients with type 2 diabetes with nephropathy was dependent on the dose of atorvastatin and apoE genotype and was strongly correlated with the reduction in triglycerides (TG) in very low density lipoprotein (VLDL).The effect of bariatric surgery on obese patients with and without diabetes demonstrated that after bariatric surgery, VLDL-apoE increased and apoE in low density lipoprotein (LDL), high density lipoprotein (HDL) and d>1.21g/ml fractions decreased; both glycated LDL-apoE and glycated HDL-apoE decreased. Total apoE and glycated apoE concentrations in plasma decreased to levels comparable to those of healthy controls. However, the distribution within the lipoprotein fractions was very different. The effect of niacin/laropiprant (LRPT) on lipoproteins in hyperlipidaemia patients was assessed in a blind crossover trial. Niacin/LRPT slightly decreased VLDL-apoE and LDL-apoE. It had no effect on apoE in HDL. Glycated apoE did not change in hyperlipidaemia. These results show that, compared with healthy people, the apoE distribution in obese and hyperlipidaemia patients is abnormal despite no change in total apoE concentration in some cases. The results also demonstrate that glycated apoE originates preferentially from VLDL. Various mechanisms for these results and relationships with other lipids are discussed. Furthermore, I suggest several potential directions, especially in vitro, for further research on apoE function and metabolism.