Thesis (Ph. D.)--Columbia University, 1936. / Vita. Bibliography: p. 15-16, 18, 21.

Thesis--Wisconsin. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1984. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 136-151).

Thesis (M.A.)--Boston University

A series of cholesterol analogues possessing side chains with either more or less carbon atoms than cholesterol were synthesised from hyodesoxycholic acid and fully characterised by nuclear magnetic resonance, mass spectrometry, and infra red analysis. These analogues were used in (a) physical studies of cholesterol phospholipid interactions and (b) experiments to investigate the substrate specificities of cholesterol 7a"hydroxylase from rat liver and the adrenal mitochondrial cholesterol side chain cleavage enzyme. (a) The physical properties of cholesterol and a series of cholesterol side chain analogues were investigated by incorporating them into liposomes prepared from lipid films containing the sterol and a series of saturated or unsaturated phospholipids in the required molar proportions. A series of spin labels (3-nitroxy octane, Tempo, 25 nitroxy cholestane, and 3 nitroxy cholestane) were incorporated into the sterol phospholipid liposomes and the effect of each analogue on each spin label mobility was observed. In addition the effect of each analogue on water permeability across the liposomal membrane was determined. The results of both sets of experiments showed that the cholesterol molecule with its iso-octane side chain was optimally adapted for maximal interaction with phospholipid. This specificity was not observed in monolayer studies of cholesterol phospholipid interactions. (b) The involvement of the cholesterol side chain in cholesterol oxidation by the liver microsomal enzyme, cholesterol 7a~hydroxylase was determined using an established assay based on the oxidation of added radioactive cholesterol. Using phenobarbitone pretreated rats it was shown that the metabolism of short side chain analogues was stimulated whereas 7a~hydroxylase activity was unaffected. Thus it is postulated that the side chain of cholesterol determines its metabolic fate and that the shorter side chain analogues are metabolised by the inducible hepatic drug metabolising enzyme system. A new G.L.C. assay which measures the production of 7a-hydroxy cholesterol from endogenous microsomal cholesterol has been developed and used to investigate the problem of substrate supply for the 7ahydroxylase enzyme. This involved the addition of cholesterol in detergents in sonicated and unsonicated liposomes containing different molar proportions of sterol and in organic solvents to a cholesterol depleted form of the enzyme. The involvement of a soluble protein isolated from the 100,000 x g supernatant of rat liver by ammonium sulphate fractionation, gel filtration and ion exchange chromatography has also been demonstrated. The molecular weight of the protein has been determined by S.D.S. polyacrylamide gel electrophoresis, and its means of action investigated. The cholesterol side chain cleavage enzyme of rat adrenal mitochondria has been shown to possess an absolute requirement for cholesterol as substrate by use of a radio-immuno-assay that measures pregnenolone production from the exogenous cholesterol side chain analogues. The results of both the physical measurements and the enzyme assays show that the side chain of cholesterol plays a major role in determining the extent of the interaction between cholesterol and phospholipids and the specificity of cholesterol protein interaction.

The effect of bilayer composition, specifically the presence of cholesterol, upon the function of the reconstituted nicotinic acetylcholine receptor (nAcChoR) was investigated using stopped-flow fluorescence. The nAcChoR was purified and reconstituted from the electroplaques of Torpedo nobiliana, using affinity column chromatography, into bilayers of defined composition and the function of each sample assessed and compared with those of the native receptor. Investigation of the effect of bilayer composition upon the kinetics of agonist binding to the nAcChoR, using the fluorescent acetylcholine analogue, Dns-C₆-Cho, established that the receptor pre-existed in equilibrium between the resting and two desensitised states. However, Dns-C₆-Cho inhibited channel gating at high concentrations and another fluorescent probe was sought. The kinetics of carbachol induced nAcChoR conformational changes, reported by ethidium bromide (a non-competitive inhibitor) fluorescence, in native membranes were characterised and an assay developed to investigate whether cholesterol mediated rapid conformational changes in reconstituted samples. It was found that ethidium bromide reported on the carbachol-induced development of the fast desensitised state from the open channel state, and that this conformational change was sensitive to changes in bilayer composition. The onset of fast desensitisation from the open channel state was not observed when the receptor was reconstituted into DOPC or DOPC-DOPA bilayers. However, increasing the cholesterol content in these bilayers increased the amplitude of the component reporting this conformational change, while the observed rate at which it occurred was independent of bilayer cholesterol content. This result agrees with the suggestion that cholesterol facilitates channel opening and the onset of fast desensitisation by binding to specific sites on the nAcChoR and that these must be occupied for a functionally viable receptor (Jones and McNamee, 1988).

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Eliška Šrámová Supervisor: Dr. Georgios Paraskevopoulos, Ph. D Consultant: Mgr. Anna Nováčková Title of Thesis: Study of the effect of cholesterol concentration on monolayer models Skin is composed of 3 major layers: hypodermis, dermis, and epidermis. The uppermost layer, which is called stratum corneum (SC), has a unique structure resembling a well-built wall. Corneocytes represent bricks and the lipid matrix works like a mortar. Ceramides, free fatty acids, and cholesterol (Chol) are the main lipids creating the human SC matrix. In a healthy SC, the ratio of these lipids is equimolar. This ratio is crucial, not only to maintain the barrier function of the skin, but also for the organization of lipids in SC. Chol appears to be required for the correct lamellar organization, and the ordering of lipids inside of the lamellar formation (lateral organization) in SC. Abnormalities in the ratio of the lipids and depleted amount of Chol can lead to a disruption of the skin barrier function resulting in skin disease or multisystemic diseases (e.g. X-linked ichthyosis, Conradi-Hünermann-Happle syndrome, and CHILD syndrome.) The present work deals with a Chol deficiency study on lipid organization on...

Cardiovascular disease is a leading cause of morbidity and mortality worldwide. Lipid biomarkers are frequently used for prediction of cardiovascular disease risk. Triglycerides are routinely checked in blood work, and triglycerides are a key component of lipoproteins that contribute to atherogenic plaques, which cause cardiovascular disease. High triglycerides are a common condition in the general population. The relative effect of high triglycerides on the lipid biomarkers (non-high-density lipoprotein cholesterol, and apolipoprotein B) for cardiovascular disease risk prediction is the focus of this thesis. Using cross-sectional lipid profile data from a large Lipid Clinic, we compared the correlation and concordance between non-high-density lipoprotein cholesterol and apolipoprotein B as cardiovascular disease risk markers among patients with mild, moderate, and severe hypertriglyceridemia. The findings showed that with higher triglycerides, there is lower agreement between the two biomarkers, which raises caution that they are not interchangeable, and further research is needed.

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