Thesis--Texas Christian University. / Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 149-168).

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Patman is a fluorescent membrane probe related to Laurdan. The structural distinctions between the two probes are the lengths of the aliphatic tails (eleven carbons in Laurdan and fifteen in Patman) and the presence of a trimethylammonium group on Patman that produces a positively-charged head. Preliminary studies exploring Patman as a probe to detect membrane properties during apoptosis revealed that the fluorescence intensity of two edges of the emission spectrum (435 and 500 nm) stabilizes at different rates as the probe binds to the cell membrane. To test whether these differences represent dissimilarities in probe binding to ordered and disordered domains, experiments were conducted to monitor Patman equilibration with bilayers composed of various mixtures of saturated and unsaturated phosphatidylcholines at temperatures above, at, and below the main thermotropic phase transition. In general, Patman equilibrated more rapidly with bilayers in the liquid-disordered phase than in the solid-ordered phase. With solid phase membranes, the fluorescence stabilized faster at 500 nm than at 435 nm. Similar, yet more subtle, results occurred in the lipid disordered phase. In contrast, the situation was reversed at the phase transition temperature; equilibration was faster at 435 nm than at 500 nm. To determine whether these results reflected specific properties of Patman, the experiments were repeated with Laurdan, and several distinctions were observed. First, equilibration with solid phase lipids was faster than for Patman and not different from equilibration with the fluid phase. Second, differences in rates between the two wavelengths were less than with Patman for solid phase membranes but greater than with Patman for melted bilayers. Third, at the phase transition temperature, the difference in equilibration rates was the opposite of the result obtained with Patman. Computer simulations were used to assist with interpretation of these results. The data suggest that both probes bind superficially to the membrane before incorporating among the lipid molecules. Once within the membrane, Patman localizes to at least two distinct depths within the bilayer. Probe molecules in the shallow, more hydrated position favor 500 nm emission and those occupying a deeper, dehydrated position emit primarily at 435 nm. Laurdan's equilibration additionally represents movement of the probe between leaflets and multiple bilayers.

The mechanism of microparticle shedding from the plasma membrane of calcium-loaded cells has been investigated in erythrocytes and platelets. Recent studies have revealed the physiological and clinical importance of microparticle release from nucleated cells such as lymphocytes and endothelium. The experiments of this study were designed to address whether simple mechanisms discovered in platelets and erythrocytes also apply to the more complex nucleated cells. Four such mechanisms were addressed: potassium efflux, transbilayer phosphatidylserine migration, cytoskeleton degradation, and membrane lipid order. The rate and amount of microparticle release in the presence of a calcium ionophore, ionomycin, was assayed by light scatter at 500 nm. To inhibit the calcium-activated potassium current, cells were exposed to 1 mM quinine or a high-potassium buffer. Both interventions substantially attenuated microparticle shedding induced by ionomycin. Microparticle release was also greatly reduced in a lymphocyte cell line deficient in the expression of scramblase, the enzyme responsible for calcium-stimulated phosphatidylserine migration to the cell surface. This result indicated that such phosphatidylserine exposure is also required for microparticle shedding. The importance of cytoskeletal rearrangement was evaluated through the use of E64-d, a calpain inhibitor, which appeared to have no affect on release. Thus, if cytoskeleton degradation is important for microparticle release, a different enzyme or protein must be involved. Finally, the effect of membrane physical properties was addressed by varying the experimental temperature (32–42 °C). A significant positive trend in the rate of microparticle release as a function of temperature was observed. Fluorescence experiments with trimethylammoniumdiphenylhexatriene and patman revealed significant differences in the level of apparent membrane order along that temperature range. Ionomycin treatment appeared to cause further disordering of the membrane, although the magnitude of this change was minimally temperature-sensitive. Thus, it was concluded that microparticle release depends more on the initial level of membrane order than on the change imposed by calcium uptake. In general, mechanisms involved in particle release from platelets and erythrocytes appeared relevant tolymphocytes with the exception of the hydrolytic enzyme involved in cytoskeletal degradation.

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<p>A historical study on the inflince of private foundations in American political history.</p>