UMD Theses and Dissertations
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Item UNRAVELING THE ROLE OF LASSA VIRUS TRANSMEMBRANE DOMAIN IN VIRAL FUSION MECHANISM(2024) Keating, Patrick Marcellus; Lee, Jinwoo; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lassa virus (LASV) is the most prevalent member of the arenavirus family and the causative agent of Lassa fever, a viral hemorrhagic fever. Although there are annual outbreaks in West Africa and recently isolated cases worldwide, no current therapeutics or vaccines pose LASV as a significant global public health threat. One of the key steps in LASV infection is the delivery of its genetic material by fusing its viral membrane with the host cell membrane. This process is facilitated by significant conformational changes within glycoprotein 2 (GP2), yielding distinct prefusion and postfusion structural states. However, structural information is missing to understand the changes that occur in the transmembrane domain (TM) during the fusion process. Investigating how the TM participates in membrane fusion will provide new insights into the LASV fusion mechanism and uncover a new therapeutic target sight to combat the dangerous infection. Here, we describe our protocols for expressing and purifying the isolated TM and our GP2 constructs which we use to probe the relationship between the structure of the TM and its influence on the function of GP2.We express TM as a fusion protein with a Hisx9 tag and a TrpLE tag using E. coli bacterial cells. We purify the TM using Nickel affinity chromatography and enzymatic cleavage to remove the tags. Since the TM is prone to aggregation, we must use a strong denaturant, trichloroacetic acid (TCA), to remove the TM from the resin. The isolated TM is then buffer exchanged to a detergent solution for structural studies. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, our structural studies revealed a pH-dependent structural change resulting in an N-terminal extension of the alpha helix in the postfusion state. To test the importance of this structural change, we used the GP2 construct to perform a modified lipid mixing fusion assay. Our results from the fusion assay and a combined mutational study revealed that this structural change is important for the fusion efficiency of GP2. Loss of this extension resulted in lower fusion activity. To further understand these structural changes and to probe the TM’s environmental interactions, we turned to fluorine NMR. This method gives us a unique and highly sensitive probe to monitor changes in the structure and membrane environment. We describe our incorporation protocol of fluorine into the TM and our method for incorporating the TM into a lipid bilayer system. We describe preliminary results showing sensitive changes in the structure of the TM and the implications this method has to enhance our understanding of the LASV membrane fusion mechanism.Item Probing the 3D Structure and Function of a Cation/H+ Exchanger in Plant Reproduction(2015) Czerny, Daniel; Sze, Heven; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Maintaining intracellular pH and K+ homeostasis are necessary for a cell to divide, grow, and communicate with other cell types. How a cell responds to stimuli and subsequently regulates intracellular pH and K+ content are largely unknown. Ion transporters, including cation/H+ exchangers are one potential determinant of intracellular pH and K+ content. A novel family of CHX transporters, predicted to exchange a cation for a H+, is found in all land plants, though their functions in the plant and the mode of transport are mostly unknown. What is the mode of transport of Arabidopsis thaliana CHX17? Model structures of the CHX17 transmembrane domain were built from two crystallized bacterial Na+/H+ antiporters. Based on protein architecture and homology, residues were selected for mutagenesis and CHX17 activity was tested in yeast. Thr170 and Lys383 in the discontinuous α-helices of transmembrane 4 and 11, and Asp201 and Lys355 in the middle of transmembranes 5 and 10 are necessary for CHX17 activity. Results suggest these are core residues that participate in cation binding and/or catalysis. Glu111 near the cytosolic surface of CHX17 was necessary for activity, suggesting CHX17 could be regulated by cytosolic pH. Thus the protein fold and mode of transport of Arabidopsis CHX17 resemble a K+/H+ exchanger. What roles do K+/H+ exchangers play in plant reproduction? chx17/18/19 mutant plants showed a 56%-77% reduction in seed set though the biological basis was unknown. Reciprocal crosses showed reduced seed set was primarily caused by defects in the male gametophyte. Mutant chx17/18/19 pollen grains developed normally and pollen tubes grew and reached most ovules. However, half the ovules receiving a mutant pollen tube failed to develop. Wild-type pistils that received chx17/18/19 pollen showed unfertilized ovules, ovules with single fertilizations, and some embryos that developed similarly to wild-type. Thus, some triple mutant pollen showed failure to complete fertilization. When fertilization was successful, embryos from self-fertilized chx17/18/19 pods showed delays in development. Our findings suggest maintenance of pH and K+ homeostasis in endomembrane compartments by CHX17 and its homologs could regulate membrane trafficking events necessary for pollen tube growth, male gamete fusion, and embryo development.Item Effect of Borohydride Reduction and pH on the Optical Properties of Humic Substances(2014) Schendorf, Tara Marie; Blough, Neil V; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Despite decades of research, the structural basis for the optical properties of chromophoric dissolved organic matter (CDOM) and humic substances (HS) are still not clear. Through several analytical techniques, it is known that CDOM contains carbonyls (aromatic ketones, aldehydes, and quinones), carboxylic acids, and phenols. The charge-transfer model proposed to explain the optical properties of these materials assigns the short-wavelength absorption (<350 nm) and fluorescence emission to electron donors (phenols) and acceptors (carbonyls), while the long-wavelength absorption is attributed to charge-transfer interactions among these species. Because carbonyls are reducible species a method was developed to eliminate them and to investigate its effects on the optical properties of HS in relation to their structure. In addition, the effect of pH on the optical absorption spectra for both untreated and borohydride reduced HS was examined and related to the deprotonation of carboxylic acids and phenolic moieties.Item THE DYNAMICS OF DIUBIQUITIN REVEALED BY NMR: WHAT IS THE DRIVING FORCE BETWEEN THE OPEN AND CLOSED STATES?(2012) Lai, Ming-Yih; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The K48-linked polyubiquitin chains are important signals for proteasomal degradation and other biological processes. Their recognition of ubiquitin binding partners such as the UBA2 (ubiquitin associated domain 2) domain of hHR23a is via the canonical hydrophobic patch formed by L8, I44, and V70. In near physiological pH (pH 6.8), the K48-linked diubiquitin predominantly adopts the closed conformation in which the binding sites for ubiquitin-binding partners are buried in the inter-domain interface, and therefore are not available for binding. The K48-linked diubiquitin also can adopt an open conformation at acidic pH. However, the mechanism of the transition between the open and closed states is poorly understood. This study is aimed at elucidating the driving force for the exchange between the open and closed conformations of K48-linked diubiquitin. Using different mutations of H68 in diubiquitin and NMR methods, I found that the protonation state of the histidine side chain is crucial for controlling the equilibrium between open and closed conformations. I also found that H68 is essential for maintaining the integrity of the inter-domain interface. I concluded that there are at least four interactions involved in controlling the transitions between open and closed states. These are point-to-point repulsion (strongest), point-to-bulk repulsion (medium), bulk-to-bulk repulsion (weakest), and hydrophobic interaction. Based on these results, I also proposed a pre-open state model for K48-linked diubiquitin which assumes that the closed conformation of Ub2 opens by twisting instead of directly pulling two domains away from each other.Item Characterization of Trace Metal Leaching from Maryland Coal Fly Ashes(2011) Ozkok, Enes; Aydilek, Ahmet H; Davis, Allen P; Civil Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Three coal fly ashes with different acid-base characteristics and their mixtures with an embankment soil were analyzed for arsenic, copper, and chromium leaching as function of pH using batch-type water leach tests (pH 4-10). Leach tests results showed that significant Cu release occurred only at pH ~4.3 and dissolved Cr concentrations typically increased with increasing pH. Cr(VI) was determined as the predominant oxidation state in leachates and results from WLTs spiked with Cr(VI) suggest that Cr was strongly sorbed below pH 7. Sorption affinity of fly ashes for Cr(VI) seemed to be to correlated to their oxalate-extractable Fe content, which is presumably a surrogate for amorphous iron (hydr)oxide content. Arsenic release typically followed a similar leaching pattern observed for Cr, with the exception of 100% alkaline fly ash; decreased As release above pH 9 for this sample was attributed to precipitation of Ca-As phases due to its high CaO content.Item MODELING OF MINERAL TRAPPING FOR CO2 SEQUESTRATION(2011) Alizadeh Nomeli, Mohammad; Riaz, Amir; Torrents, Alba; Civil Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In order to prevent CO2 concentrations in the atmosphere from rising to unacceptable levels, carbon dioxide is sequestered beneath the ground surface. CO2 can be trapped as a gas under a low-permeable cap rock (structural trapping) or can dissolve into the ground water (hydrodynamic trapping); it can also react with minerals and organic matter that are dissolved in the brine to form precipitates (mineral trapping). From the perspective of secure, long term storage, mineral trapping has been identified as the most effective mechanism related to subsurface sequestration. Temperature, pressure and salinity are among the primary parameters governing the overall behavior of the process of mineral trapping. In this study, the primary goal is to simulate the behavior of carbon dioxide with an improved model under the conditions of temperature and pressure typical of saline aquifers, i.e. 50 to 100oC and 1-500 bar, respectively. The objective is to determine how the related quantities of molar volume as well as CO2 fugacity change in response to changes in pressure and temperature so that the associated changes in the solubility and the precipitation of carbonates, indicating the rate of CO2 consumption, can be quantified. This study finds that the dissolution rate of anorthite and the rate of precipitation of calcite both rise with the increase in pressure and temperature. The dissolution rate of anorthite has been found to be the rate-limiting process in the sequestration of CO2 and governs the consumption rate of CO2 in the aqueous phase. These results show good agreement with those obtained from experimental work reported in other studies. This study also agrees earlier findings based on relatively less precise models, with respect to the increase in CO2 solubility at higher pressures and a decrease in solubility associated with increasing values of temperature and salinity.Item An empirical re-evaluation of the boron isotope/pH proxy in marine carbonates(2009) Klochko, Kateryna; Kaufman, Alan J; Geology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The boron isotopic composition measured in marine carbonates is considered to be a tracer of seawater pH. However, an accurate application of this proxy has been hampered by our lack of intimate understanding of chemical kinetics and thermodynamic isotope exchange between the two dominant boron-bearing species in seawater: boric acid B(OH)3o and borate ions B(OH)4-, as well as their subsequent partitioning into a carbonate lattice. In this dissertation I have taken on a task of a systematic empirical re-evaluation of the fundamental parameters and assumptions on which the boron isotope paleo-pH proxy is based. As a result of this research strikingly different values of the boron isotope exchange constant in solution (Klochko et al., 2006) and boron speciation and partitioning in carbonates (Klochko et al., 2009) were determined, suggesting that the most parameters and assumptions that were believed to be previously constrained and have been widely applied to the 11B-pH reconstructions were incorrect. Recognizing that both biological and inorganic processes may potentially affect boron speciation and isotopic composition in carbonates, to isolate purely inorganic effects on the boron isotope co-precipitation with carbonates, we have designed a series of pH-controlled 11B calibration experiments of inorganic calcite and inorganic aragonite. Results to date reveal that precipitates from our experiments at pH = 8.7 fall exactly along the borate ion 11B curve predicted by our empirically determined boron isotope fractionation factor (Byrne et al., 2005; Klochko et al., 2006). Extending these experiments to wider range of pH conditions will provide the necessary inorganic baseline for paleo-studies of inorganic carbonate and future investigations of the purely biological effects on the boron isotope distributions in carbonates.Item Effect of pH on phytoplankton and bacteria production(2009) Johns, Desmond Justine; Stoecker, Diane K; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In recent decades eutrophication has fueled intense phytoplankton blooms that frequently negatively impact ecosystems. Dramatic pH elevations are commonly overlooked during such blooms, which may also be harmful. Cyanobacteria blooms in the Sassafras River, MD were sampled during Autumn 2008 and measured for primary and bacterial production (PP and BP), and differences in bacteria communities composition were examined. pH elevations above 8.8 in the field corresponded to decreased PP, but had no effect on BP. Laboratory experiments demonstrated that negative effects of pH were dependant on light intensity; PP increased with pH at moderate light intensities, but decreased at low irradiance. There was some evidence that BP is affected by high pH, although bacteria community differences as determined by DGGE were not. Negative effects of high pH are probably most important during spring and summer in low salinity environments when pH fluctuations are more common and last longer.Item Protein folding and amyloid formation in various environments(2008-11-21) O'Brien, Edward Patrick; Thirumalai, Devarajan; Brooks, Bernard; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Understanding and predicting the effect of various environments that differ in terms of pH and the presence of cosolutes and macromolecules on protein properties is a formidable challenge. Yet this knowledge is crucial in understanding the effect of cellular environments on a protein. By combining thermodynamic theories of solution condition effects with statistical mechanics and computer simulations we develop a molecular perspective of protein folding and amyloid formation that was previously unobtainable. The resulting Molecular Transfer Model offers, in some instances, quantitatively accurate predictions of cosolute and pH effects on various protein properties. We show that protein denatured state properties can change significantly with osmolyte concentration, and that residual structure can persist at high denaturant concentrations. We study the single molecule mechanical unfolding of proteins at various pH values and varying osmolyte and denaturant concentrations. We find that the the effect of varying solution conditions on a protein under tension can be understood and qualitatively predicted based on knowledge of that protein's behavior in the absence of force. We test the accuracy of FRET inferred denatured state properties and find that currently, only qualitative estimates of denatured state properties can be obtained with these experimental methods. We also explore the factors governing helix formation in peptides confined to carbon nanotubes. We find that the interplay of the peptide's sequence and dimensions, the nanotube's diameter, hydrophobicity and chemical heterogeneity, lead to a rich diversity of behavior in helix formation. We determine the structural and thermodynamic basis for the dock-lock mechanism of peptide deposition to a mature amyloid fibril. We find multiple basins of attraction on the free energy surface associated with structural transitions of the adding monomer. The models we introduce offer a better understanding of protein folding and amyloid formation in various environments and take us closer to understanding and predicting how the complex environment of the cell can effect protein properties.Item Efficiency and Ecological Risks of Reducing Soil pH during Thlaspi caerulescens Phytoextraction of Cadmium and Zinc(2004-11-29) Wang, Shengchun; Angle, Jay S; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The major aims of this research were to determine whether reducing soil pH can enhance phytoextraction and to examine the ecological risks of reducing pH. Two soils differing in Cd and Zn concentrations were used and adjusted to 5 or 6 different pH levels ranging from 7.27 to 4.74 and seeded with a hyperaccumulator of Cd and Zn, Thlaspi caerulescens. Plants were harvested after six months, the pH were restored to above 6.5, incubated for 6 months. Soils were analyzed for biological activities and microbial population changes after both pH adjustments. Reducing pH significantly (p=0.05) enhanced plant metal uptake. For the high metal soil, plant grew best at the lowest pH treatment (4.74) and the highest metal concentration was at the second lowest pH treatment (5.27). For the low metal soil, due to low pH induced Al and Mn toxicity, plant growth and metal uptake were highest at the intermediate pH level (6.07). Metal sequential extraction results further verified that reducing pH redistributed Cd and Zn among five fractions. The most soluble metal form (F1) was greatly increased. In addition, T. caerulescens was able to differentially utilize Cd in all 5 fractions while it could only access Zn from the F1 and F2 pools. Reducing soil pH significantly reduced a number of soil biological activities and shifted the community structure at different levels. Generally, soil biological activities were more sensitive than soil microbial populations to pH change. Good indicators of soil pH status were acid phosphatase activity, alkaline phosphatase activity, acid to alkaline phosphatase activity ratio, arylsulphatase, nitrification potential, soil microbial biomass C and N, and population of rhizobium. After raising pH to > 6.5, negatively impacted soil parameters were partially restored to original levels. Soil biological activities showed lower recovery than soil microbial populations. The threshold pHs were 6.1 and 5.3 for low and high metal soils, respectively. Above this value, most soil biological activities and all microbial populations returned to background levels within a short period.