Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item Protein permeability pathways in the mitochondrial outer membrane during apoptosis(2012) Vidyaramanan, Ganesan; Colombini, Marco; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Apoptosis, a form of programmed cell death is a physiological, homeostatic process that guides the systematic removal of unwanted, dying or damaged cells from the body. A key step in apoptosis is the irreversible release of mitochondrial intermembrane space (IMS) proteins into the cytosol by a process called Mitochondrial Outer Membrane Permeabilization (MOMP). MOMP is regulated by a special class of proteins called Bcl-2 family proteins and a sphingolipid called ceramide. The pro-apoptotic Bcl-2 proteins, especially Bax and Bak can cooperate with ceramide to form channels in mitochondria that cause protein efflux during MOMP. The ability of ceramide to form protein-permeable channels in MOM is established. Bax and ceramide enhanced MOMP synergistically. The ability of Bax to stimulate ceramide channels was investigated. It was found that the apparent affinity of Bax for a ceramide channel increases with the ceramide channel size. The results indicate that Bax binds a small ceramide channel and drives its growth until the Bax molecule finds the best fit to the channel. This interaction between Bax and a ceramide channel does not require of the presence of other Bcl-2 proteins or mitochondrion-specific factors. The critical structural features of ceramide were investigated for their role in ceramide channel formation. Analogs of ceramide bearing modifications in the functional groups were analyzed for their ability to form channels to assess stability and also to interact with native ceramide to form channels to assess compatibility between interacting groups. The C1-hydroxyl was found to be indispensable for channel formation while the C3-hydroxyl was inconsequential to channel formation. The amide nitrogen with its ability to donate hydrogen was important for stability as methylating the nitrogen diminished the channel forming ability. Similarly, converting the carbonyl oxygen to a urea group, now more polar and a stronger hydrogen bond former resulted in more stable permeabilization. Changes to the hydrocarbon tails did not affect the ability to form channels. Phytoceramide, which has a C4 hydroxyl instead of the C4-C5 trans double bond formed stable channels but phytoceramide inhibited channel formation by ceramide suggesting incompatibility in structure. Bax activation involves translocation of Bax from the cytosol to the MOM, conformational changes and subsequent channel formation. All steps involved in Bax activation are not well-understood. We have used ionic strength as a modulating tool to dissect the different steps in Bax mediated MOMP. Increasing the ionic strength was found to delay formation of real-time permeability by Bax. Increasing the ionic strength resulted in smaller channels that grew in size slowly. The high permeability induced by low ionic strength was not reversed by raising the ionic strength suggesting that Bax channels are not in dynamic equilibrium with Bax monomers. Ionic strength also altered the sensitivity of Bax mediated MOMP to inhibition by Bcl-xL. Ionic strength, however did not affect Bax insertion into membranes. Thus, ionic strength presents a good diagnostic tool to modulate Bax mediated channel formation downstream of Bax insertion into membranes.Item The role of charge in solvation at liquid/liquid interfaces(2005-05-27) Huffman, Carmen Louise; Walker, Robert A.; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation describes the development and characterization of new surfactants, dubbed "molecular rulers," that provide an upper limit to the dipolar width in aqueous/organic systems. Here, dipolar width describes the distance required for the dielectric properties of one phase to converge to those of the adjacent phase. Molecular rulers consist of a hydrophobic, solvatochromic chromophore and a charged headgroup connected via a variable length methylene chain. These surfactants are anchored to the aqueous phase by the ionic headgroup while the solvatochromic probe "floats" into the organic phase. The length of the alkyl chain controls the position of the chromophore within the interfacial region. Resonance-enhanced second harmonic generation (SHG) is used to profile the electronic excitation energy of the chromophore as a function of alkyl chain length. Since the solute's excitation energy depends on solvent polarity, we can infer interfacial dipolar width. In previous work anionic molecular rulers were used to characterize the water/cyclohexane interface. Anionic rulers having two carbon alkyl chains sample a polarity between that of bulk water and bulk cyclohexane. Analogous cationic rulers described in this dissertation sample an environment equivalent to that of bulk cyclohexane. These results suggest that interfacial polarity may depend on surface charges having a close proximity to the adsorbed solute. This idea was tested using cationic rulers adsorbed to the water/vapor surface of an electrolyte solution saturated with 1-octanol (a mimic of the water/alkane interface). As ionic strength increases, cationic ruler SHG behavior approaches that of the anionic species, suggesting that the ions in solution shield the cationic charge from a probe-headgroup interaction that was observed with NMR experiments for bulk aqueous solution samples. A neutral organic molecule at the electrolyte solution/cyclohexane interface was employed to elucidate the role of charge in interfacial solvation. Observed shifts in SHG spectra from salt-free limits are similar to those of absorbance spectra for the solute in bulk electrolyte solutions. We conclude that, in the absence of direct charge-probe correlation, charges have a similar influence on interfacial solvation of neutral species as they do in bulk solution.