Search results

Title: X-ray crystallographic studies of SNAP190RcRd (Small Nuclear RNA Activating Protein) complex and E. Coli glycogen synthase
Creator: Sheng, Fang
Date: 2008
Collection: Electronic Theses & Dissertations

Title: Ligand effects in the chemistry of gas phase metal ions
Creator: Stepnowski, Richard Martin
Date: 1988
Collection: Electronic Theses & Dissertations

Title: Metabolic changes in cellular components of the vasculature : examining the link between diabetes and cancer
Creator: Giebink, Adam
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Currently, cancer and diabetes are two of the more devastating diseases worldwide, both in the number of people affected as well as the financial cost it takes to treat those with said conditions. While these two diseases are significantly different in their pathologies and physiological impacts, they both have characteristics of altered mechanisms by which they metabolize glucose when compared to healthy individuals. In fact, there is an increased risk that has been identified for people...
Show moreCurrently, cancer and diabetes are two of the more devastating diseases worldwide, both in the number of people affected as well as the financial cost it takes to treat those with said conditions. While these two diseases are significantly different in their pathologies and physiological impacts, they both have characteristics of altered mechanisms by which they metabolize glucose when compared to healthy individuals. In fact, there is an increased risk that has been identified for people with diabetes to develop certain types of cancer. Motivated by the finding observed 90 years ago by Otto Warburg regarding the `aerobic glycolytic' conditions exhibited by cancer cells and a study indicating a risk of developing cancer in non-obese persons with elevated fasting glucose levels, the presented work examines how the glucose metabolism of healthy cells is altered following exposure to biomolecules that are elevated in diabetes, drugs for the treatment of diabetes, and elevated glucose levels. The altered metabolism can then be compared to that of cancerous cells.First, certain metabolic alterations in cultured bovine pulmonary artery endothelial cells following treatment with metal-activated C-peptide were monitored using liquid scintillation, platereader, and microfluidic techniques. As recent reports from the Spence group have identified C-peptide as a stimulator of glucose uptake and adenosine triphosphate (ATP) release from the red blood cell (RBC), similar in vitro experiments were performed on the endothelial cells lining the inner walls of the vasculature. Results indicate that C-peptide not only has a similar influence on the glucose uptake in endothelial cells as it does on the RBCs, but also stimulates the production of a significant vasodilator in endothelial nitric oxide (NO) by a RBC mediated mechanism.C-peptide is then administered to the RBCs and endothelial cells in the presence of the type 2 diabetes drug, metformin. The metabolism of the two cell types is monitored by measuring the glucose uptake and lactate release. The studies provide a hypothesis behind metformin's action by increasing the interaction of C-peptide with the RBCs while also increasing glucose uptake. In the endothelial cells, metformin alone causes a similar increase in glucose uptake. However, this uptake increase in both cell types results in the production of elevated levels of lactate, especially in hyperglycemic conditions, which can potentially lead to lactic acidosis. This increased lactate production from the endothelial cells following metformin administration led to an overall decrease in the intracellular pH hypothesized to be caused from the release of lactate from the cell through the monocarboxylate transporter (MCT1).Lastly, the metabolic changes that occur in healthy endothelial cells in normal and elevated glucose environments are compared to those same changes in cancerous HeLa cells. The endothelial cells had an increased lactate release when they were incubated in the hyperglycemic buffers while the HeLa cells' metabolism remained relatively constant. This suggests a change in the healthy cells' metabolism depending on the sources of energy, namely glucose and pyruvate, available to it while the cancer cells' metabolism remains the same in any condition.This research demonstrates some of the factors that alter the metabolism of normal, healthy cells. While the findings do not show that high glucose causes cancer directly, it does show that the metabolism can be altered by changes brought about by hyperglycemia, sometimes resembling many metabolic characteristics observed in cancer cells.
Show less

Title: Functional membranes for limited protein digestion and phosphopeptide enrichment prior to mass spectrometry
Creator: Tan, Yujing
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Mass spectrometry (MS) is the leading technique for rapid identification of proteins and characterization of their posttranslational modifications. However, as with most analyses, the success of MS methods depends critically on sample purification and pretreatment. Microporous membranes are attractive as a platform for enrichment and modification of proteins and peptides because convection and short radial diffusion distances in pores provide rapid analyte mass transport to binding or...
Show moreMass spectrometry (MS) is the leading technique for rapid identification of proteins and characterization of their posttranslational modifications. However, as with most analyses, the success of MS methods depends critically on sample purification and pretreatment. Microporous membranes are attractive as a platform for enrichment and modification of proteins and peptides because convection and short radial diffusion distances in pores provide rapid analyte mass transport to binding or catalytic sites. Additionally, variation of the transmembrane flow rate can control the extent of reactions such as digestion. This research exploits facile layer-by-layer adsorption to incorporate nanoparticles or enzymes in micropores and create functional membranes. As tools for sample preparation prior to MS analysis, TiO2-modified membranes enrich phosphopeptides and protease-containing membranes afford control over protein digestion.Posttranslational protein phosphorylation is one of the most important mechanisms for creating protein diversity in eukaryotic cells. However due to the relatively low abundance of most phosphopeptides in proteolytic digests, phosphopeptide enrichment is a prerequisite step for effective and efficient MS analysis of phosphorylation. Alternating adsorption of poly(styrene sulfonate) (PSS) and TiO2 nanoparticles in porous nylon substrates creates TiO2-modified membranes for rapid enriching of phosphopeptides from small-volume (10's of &muL) samples. These TiO2-modified membranes bind 540 nmols of phosphopeptides in a 22-mm diameter disc and selectively isolate the phosphopeptides from -casein, -casein and ovalbumin digests even in a 100-fold excess of non-phosphorylated BSA digests if 2 % TFA is the loading and washing buffer. The combination of membrane enrichment and tandem MS reveals seven phosphorylation sites from in vivo phosphorylated tau protein, which is associated with Alzheimer's disease, and identification of such sites might aid the design of therapeutic drugs that inhibit phosphorylation.Using a similar approach to membrane modification, sequential adsorption of PSS and proteases in porous nylon substrates yields enzymatic membrane reactors for limited protein digestion. Although a high local enzyme density (~30 mg/cm3) and small pore diameters in the membrane lead to digestion in < 1 s, the low membrane thickness (170 &mum) affords control over residence times at the ms level to limit digestion. Most importantly, pepsin-containing membranes afford control of peptides sizes by altering the flow rate. Apomyoglobin digestion demonstrates that peptide lengths increase as the residence time in the membrane decreases. Under denaturing conditions, limited membrane digestion of bovine serum albumin and subsequent ESI-Orbitrap MS analysis reveal large peptides (3-10 kD) that increase the sequence coverage from 53 % (2-s digestion) to 82 % (0.05-s digestion). Moreover, electron transfer dissociation tandem MS on a large myoglobin proteolytic peptide (8 kD) provides a resolution of 1-2 amino acids, which suggests that this protease-modified membrane might find applications in hydrogen-deuterium exchange or middle-down proteomics studies. In addition, using trypsin- and &alpha-chymotrypsin-modified membranes, a limited proteolysis study of a large Arabidopsis GTPase, ROOT HAIR DEFECTIVE 3, shows suitable probing for labile regions near the C-terminus to suggest what protein reconstruction might make RHD3 more suitable for crystallization. These studies clearly demonstrate the potential of functional membranes for rapid and controlled purification or pretreatment prior to MS analysis.
Show less

Title: Electrocatalytic oxidation of alcohols using platinum and palladium nanoporous solids
Creator: Dimos, Margaretta Mary
Date: 2011
Collection: Electronic Theses & Dissertations
Description: As the world's supply of fossil fuels continues to diminish, there is an increasing need for the development of alternative energy sources. Fuel cell technology, coupled with renewable, biomass-derived fuel stocks such as methanol and ethanol, is one such energy source that will find increasing use in both the near- and long-term future. The present generation of catalysts for fuel cells is characterized by limited efficiency and durability, and as such there is a great need to improve upon...
Show moreAs the world's supply of fossil fuels continues to diminish, there is an increasing need for the development of alternative energy sources. Fuel cell technology, coupled with renewable, biomass-derived fuel stocks such as methanol and ethanol, is one such energy source that will find increasing use in both the near- and long-term future. The present generation of catalysts for fuel cells is characterized by limited efficiency and durability, and as such there is a great need to improve upon and develop new catalytic materials. Nanoporous solids, also known as inverse opals, are potentially useful materials, due in part to their relatively regular structure and physical integrity. It is the purpose of this work to initiate and investigation of nanoporous catalytic materials.We have developed nanoporous solids made from the electrodeposition of platinum and palladium metal around a silica nanosphere template. We used these materials to probe and interpret the catalysis of selected alcohols using cyclic voltammetry and chronoamperometry measurements. Initially we examined the electrocatalytic oxidation of methanol and ethanol, under acidic and basic conditions, using Pt and Pd nanoporous solid electrodes. Our results were then compared to planar Pt and Pd substrates, prepared using the same electrodeposition process as the nanoporous materials. The current density enhancement seen for the Pt nanoporous solids was more pronounced under basic conditions than acidic conditions. It was also determined that the nanoporous Pd was more catalytically active for ethanol than for methanol, with the nanoporous Pt producing higher catalytic efficiency for methanol. The enhancement that was observed with the nanoporous solids over their planar counterparts could be the result of either geometric factors or the catalyst surface morphology. The effect of morphology of the nanoporous Pt substrates was examined by studying the electrocatalytic oxidation of 1,2-propanediol under basic conditions. Our work was performed in alkaline media because, for this reactant, electrocatalytic oxidation is more efficient under these conditions. Our data for 1,2-propanediol point to metal morphology as the primary explanation for the enhanced current density relative to the planar solid electrode observed for the reaction with the nanoporous Pt.Our work with 1,2-propanediol proved to be informative, and we continued to examine the geometric vs. morphological enhancement issue by examining the electro-catalytic oxidation of 1,3-propanediol and four butanediols at nanoporous Pt and planar solid Pt electrodes under basic conditions. These reactants were chosen based on the positions of the hydroxyl moieties, so that the role of functional group position could be studied. The electro-catalysis of the aforementioned diols was studied both in terms of their reaction mechanism(s) and their kinetics. Our data indicate that the dominant factor in mediating the electrocatalytic oxidation pathway is the proximity of the hydroxyl groups on the diol and that there is a difference in the morphology of the nanoporous Pt and the planar solid Pt. This work leads to the conclusion that it is the electrode morphology rather than the geometric considerations that appears to be the dominant factor in the enhancement observed for the nanoporous solids and that this enhancement also depends upon the reactants used.
Show less

Title: 'Fixed charge' chemical derivatization and data dependent multistage tandem mass spectrometry for protein structural analysis
Creator: Zhou, Xiao
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Protein surface accessible residues play an important role in protein folding, protein-protein interactions and protein-ligand binding. With the advantages in sensitivity, speed, and the capability of analyzing large/complex protein systems, mass spectrometry combined with protein labeling has found increasing utility for the characterization of protein surface. However, a common problem associated with the use of chemical labeling methods for mapping protein solvent accessible residues is...
Show moreProtein surface accessible residues play an important role in protein folding, protein-protein interactions and protein-ligand binding. With the advantages in sensitivity, speed, and the capability of analyzing large/complex protein systems, mass spectrometry combined with protein labeling has found increasing utility for the characterization of protein surface. However, a common problem associated with the use of chemical labeling methods for mapping protein solvent accessible residues is that when a complicated peptide mixture resulting from a large protein or protein complex is analyzed, the modified peptides may be difficult to identify and characterize amongst the largely unmodified peptide population (i.e., the `needle in a haystack' problem). To address this challenge, an experimental strategy was developed involving the synthesis and application of a novel `fixed charge' sulfonium ion containing amine-specific protein modification reagent, S,S'-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), coupled with capillary HPLC-electrospray (ESI)-MS, automated collision induced dissociation (CID)-MS/MS, and data dependent neutral loss mode MS3 in an ion trap mass spectrometer, to map the surface accessible lysine residues in a small model protein, Cellular Retinoic Acid Binding Protein II (CRABP II). After reaction with different reagent : protein ratios and digestion with Glu-C, modified peptides were selectively identified and the number of modifications within each peptide were determined by CID-MS/MS, via the exclusive neutral loss(es) of dimethylsulfide, independently of the amino acid composition and precursor ion charge state (i.e. proton mobility) of the peptide. The observation of these characteristic neutral losses were then used to automatically `trigger' the acquisition of an MS3 spectrum to allow the peptide sequence and the site(s) of modification to be characterized. Using this approach, the experimentally determined relative solvent accessibilities of the lysine residues were found to show good agreement with the known solution structure of CRABP II. With the initial success demonstrated on a model protein, the experimental strategy was extended to reveal the mechanisms corresponding to oxidation induced inactivation of calcineurin (CN), from a structural perspective. CN is a Ca2+/calmodulin (CaM) activated phosphatase that participates in a wide variety of physiological processes. CaN is also reported to be inactivated by H2O2- or superoxide-induced oxidation both in vivo and in vitro. However, the mechanism is still under debate. Here, the relative rates of H2O2 induced oxidation of methionine residues within CN were first determined using a multi enzyme digestion strategy coupled with analysis using capillary HPLC-ESI-MS and CID/ETD-MS/MS. Then the developed experimental strategy, i.e., combining protein modification by DMBNHS with data dependent multistage tandem mass spectrometry, was applied to characterize changes in CN conformation before and after oxidation. In addition, targeted ETD-MS/MS was used to characterize and quantify individual unmodified lysine residues from isomers of DMBNHS modified peptides containing multiple modifiable sites. The extent of DMBNHS modification of observed CN lysine residues was found to increase upon oxidation. More importantly, the methionine residues that were highly susceptible towards oxidation and lysine residues exhibited large increase in solvent accessibility upon oxidation all locate in CN functional domains that are involved in Ca2+/CaM binding regulated activation, thus indicating of a role for oxidation induced conformation change in CN as a possible cause of CN inactivation by inhibiting Ca2+/CaM regulated CN activations.
Show less

Title: Design and construction of a multichannel detector for flow injection analysis
Creator: Castellanos, Edwin Josué
Date: 1992
Collection: Electronic Theses & Dissertations

Title: The Favorskii ring contraction of steroidal epoxy ketones
Creator: Mouk, Robert Watts
Date: 1970
Collection: Electronic Theses & Dissertations

Title: Catalytic asymmetric aza-Diels-Alder reaction : regulation of orthogonal functions in a duel chiral/non-chiral catalyst system
Creator: Newman, Cory Allan
Date: 2007
Collection: Electronic Theses & Dissertations

Title: Ion-exchange membranes prepared using layer-by-layer polyelectrolyte deposition
Creator: Liu, Guanqing
Date: 2009
Collection: Electronic Theses & Dissertations

Title: Fundamental study of the ion permeability of ultrathin, layered polyelectrolyte films
Creator: Harris, Jeremy James
Date: 2001
Collection: Electronic Theses & Dissertations

Title: Motional and excitation transport dynamics in restricted environments
Creator: Stevenson, Sarah Ann
Date: 2006
Collection: Electronic Theses & Dissertations

Title: Energy migration and morphology control in multilayers
Creator: Mehrens, Shawn Marie
Date: 2001
Collection: Electronic Theses & Dissertations

Title: A kinetic study of the electron exchange reaction between antimony (III) and antimony (V)
Creator: Sincius, Joseph Anthony
Date: 1960
Collection: Electronic Theses & Dissertations

Title: Parallel and perpendicular momentum distributions in projectile fragmentation reactions
Creator: Meierbachtol, Krista C.
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Projectile fragmentation has been used for decades to produce rare isotope beams for use in advancing nuclear science. Multiple observables are available for studying the underlying reaction mechanism including measurement of the linear momentum of final fragmentation products. Furthermore, the two components of the linear momentum, the parallel momentum distribution and the perpendicular momentum distribution, have been studied very disparately with more measurements of the parallel momentum...
Show moreProjectile fragmentation has been used for decades to produce rare isotope beams for use in advancing nuclear science. Multiple observables are available for studying the underlying reaction mechanism including measurement of the linear momentum of final fragmentation products. Furthermore, the two components of the linear momentum, the parallel momentum distribution and the perpendicular momentum distribution, have been studied very disparately with more measurements of the parallel momentum distribution of fragmentation products. The full parallel and perpendicular momentum distributions have been measured as a function of fragment mass loss for a wide range of fragments (3776Ge beam at 130 MeV/nucleon on either a 9Be or 197Au target. The parallel momentum distributions were found to be independent of target species and agree with both previous measured distributions and models of distribution width by Goldhaber and Morrissey. The perpendicular momentum distributions were found to agree with models of the distribution width by Van Bibber for fragments produced with the light beryllium target or fragments with a mass loss of <&Delta>A>20 produced with the heavy gold target. The distribution widths of the heaviest fragments produced with the gold target had scattering angles that could be described by a calculation of the classical deflection function using a repulsive Coulomb plus an attractive nuclear scattering potential between the fragment and the gold target.The particle identification procedure used with the S800 spectrometer at the National Superconducting Cyclotron Laboratory has been improved by the addition of the identification of the atomic charge-state of incoming particles. The total kinetic energy of incoming particles can now be measured with a new CsI(Na) hodoscope array, which has been characterized as a function of particle energy, mass, and nuclear charge. The energy resolution was deduced to be approximately 3% in the 100 GeV total kinetic energy regime.
Show less