Cholesterol is critical to ensure proper functioning of a membrane. Despite this, the movement of cholesterol within the cell is not fully understood. The molecular order of binary and ternary mixtures of polyunsaturated fatty acids with varying degrees of hy- drocarbon chain unsaturation with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and/or cholesterol was studied using 2H NMR. The introduction of cholesterol into sam- ples of 18:1PC, 18:2PC (unsaturated lipid/DMPC-d54/CHOL, 75:5:20mol%) increased the C-2H bond order by ∼30%. Similar bond ordering was found for 20:4PC and 22:6PC samples, however, they were temperature dependent. A two-phase region (lo-ld) was found for 22:6PC:DMPC-d54/CHOL (75:5:20mol%) for temperatures below 286.7 K. The reorientation axis formed an angle of 78±4◦ with respect to the C3-2H bond vector re- gardless of the lipid. The order parameter of cholesterol was temperature independent, and ranged from 0.69±0.04 to 0.78±0.04 depending on the lipid unsaturation. The re- orientation axis of cholesterol was oriented at ∼25◦ to the bilayer normal.

Bacterial membranes are dynamic structures, and contain lipid components that are individually simple, but complex as a whole system. The presence of charged functional groups makes them capable of interaction with ubiquitous environmental metals. Physiological responses of bacteria to metals, in preservation of membrane functions and integrity, are unclear. In this study, membrane lipid profiles were characterized for Shewanella putrefaciens CN32. Both fatty acid chemistry and hydrophilic headgroup chemistry were assayed, after growing the cells in a chemically defined medium spiked with Mn, V, or U. Cultures were grown in both aerobic and anaerobic conditions, to examine the effects of O2 and CO2 gases, as well as the combined effects of these gases with metals. The results were compared to scanning transmission X-ray microscopy (STXM) elemental maps and near-edge X-ray absorption fine structure (NEXAFS) spectra of isolated and purified S. putrefaciens CN32 envelopes at V, Mn, Ca, C, N, and O edges. It was found that there were strong correlations between membrane fluidity and fatty acid composition of strain CN32 membranes. The acyl chain chemistry was minimally affected by metal presence in the growth medium, however these subtle changes correlated with significant alterations in the fluid states of the membranes. Uranium seemed to fall outside this relationship, strongly stabilizing cell membranes. Metals in all treatments adsorbed to cell membranes, determined using either NEXAFS or electron microscopy, with the exception of V in aerobic conditions. Permeability effects of metal exposure to Ca(II), Cu(II), Mn(II), U(VI), V(IV), and Zn(II) were also assessed. Bacterial strains for these studies included S. putrefaciens CN32, Escherichia coli AB264 (wildtype K-12), Pseudomonas aeruginosa PAO1 wildtype, and Bacillus subtilis 168, in order to compare published data from the membrane chemistry of those organisms to S. putrefaciens CN32 membranes. Each metal had the same overall impact on each bacterial strain, regardless of variations in cell membrane and surface sugar chemistry, however the strengths of these effects were different for each organism. All metals with the exception of U permeabilized cell walls, while U rendered the membrane much less permeable. These impacts on permeability were concentration dependent from 0.001 mM to 1 mM concentrations. The research demonstrated that growth environment has a significant impact on the physicochemical state of bacterial membranes. Metals in those environments have varying complexation chemistry according to pH and redox conditions, and impact membrane attributes and dynamics depending on cell wall chemical composition. / This research was funded by the National Science and Engineering Research Council of Canada, as well as the Advanced Food and Materials Network.

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

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

The main treatment for myocardial infarction is early reperfusion of ischemic tissue. Ischemia and reperfusion (IR) produces reactive oxygen species that oxidize membrane phospholipids. The production of oxidized lipids and their role on cell death in cardiac IR injury is unknown. Using in vitro model of IR, our goal was to identify oxidized phosphatidylcholines (OxPC) from cardiomyocytes, to determine their bioactivity on cardiomyocyte viability and mitochondrial permeability, and using an OxPC specific EO6 antibody inhibit OxPC activity on cardiomyocytes. Rat cardiomyocytes were exposed to IR and lipid extracts underwent lipidomic analysis with HPLC-MS/MS to quantitate 82 novel OxPC species. Cell viability and mitochondrial permeability were determined in vehicle control, non-oxidized control PC, and fragmented OxPC molecules. EO6 antibody was applied and cell viability was assessed. Cardiomyocytes under IR demonstrated increased relevant OxPCs particularly fragmented species. OxPC treatment resulted in loss of cardiomyocyte viability, increased mitochondrial permeability when compared to control. EO6 antibody blocked the loss of cardiomyocyte viability. We have shown for the first time that OxPCs are generated cardiomyocytes during IR and they have detrimental effects on cardiomyocyte viability. Additionally the EO6 antibody inhibits the bioactivity of the OxPCs on cardiomyocytes and could be part of a future treatment regimen.

Two methods were used to produce exogenous lipid/bR complexes. A detergent method (Huang et al., 1980) reconstituted bR into DMPC or DMPG bilayers, free of all endogenous purple membrane lipids as shown by high resolution ³¹ P-NMR. A novel biological detergent-free method employed bovine liver non-specific lipid transfer protein (nsTP) to mediate addition of DMPC to purple membrane, while retaining 76 - 86% of the endogenous purple membrane phospholipids. The variations with temperature of 2H-NMR quadrupole splittings for the DMPC choline α- and β-methylene CD2 segments were similar to those for protein-free lipid (Gaily et al., 1975) implying that temperature dependent changes in segmental amplitudes of motion within the choline group are preserved in the presence of bR. Incorporation of small quantities of bR increased the amplitudes of segmental motion within the choline headgroup relative to that of pure lipid, but increasing the bR content induced an ordering effect. The choline α- and β-methylene segment quadrupole splittings showed a linear variation with protein content at constant temperature. This is consistent with freeze fracture electron microscopy data, which shows the bR particles to be dispersed at all lipid/protein ratios, when quenched from temperatures above the phase transition. Applying a fast two site exchange model to the ²H-NMR data, values between 12 and 15 were calculated for the number of boundary lipids for bR (26,000 M_r) in DMPC bilayers free of purple membrane lipids. From ESR data, for delipidated bR in DMPC and DMPG bilayers at temperatures above the phase transition, the number of boundary lipids calculated were 18 - 21, which is consistent with the bR being monomeric, as also observed in DMPC bilayers with all the purple membrane lipids retained (Cherry et al., 1978). The purple membrane lipids thus appear to mediate crystallization of the bR particles into a hexagonal lattice at temperatures slightly below the exogenous lipid phase transition.

No description available.

Bibliography: leaves 358-406 / xv, 406 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1985

Offprint of an article by the author inserted / Bibliography: leaves 351-384 / 384 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 1985

Thesis (Ph.D.) -- McMaster University, 1999. / Includes bibliographical references (p. 112-116). Also available via World Wide Web.