Theses and Dissertations from UMD
Permanent URI for this communityhttp://hdl.handle.net/1903/2
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
More information is available at Theses and Dissertations at University of Maryland Libraries.
Browse
4 results
Search Results
Item Radiation-Induced Modification of Aramid Fibers: Optimizing Crosslinking Reactions and Indirect Grafting of Nanocellulose for Body Armor Applications(2022) Gonzalez Lopez, Lorelis; Al-Sheikhly, Mohamad; Material Science and Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The goal of this dissertation was to design, synthesize, and analyze novel aramid fibers by covalently grafting nanocellulose through electron beam irradiation. These nanocellulose functionalized fibers showed enhanced strength and larger surface areas, which improves their performance and applicability in fiber-reinforced composites. Unmodified aramid fibers have smooth and chemically inert surfaces, which results in poor adhesion to many types of resins. Nanocellulose was chosen as the ideal filler to functionalize the fibers due to its reactive surface and high strength-to-weight ratio. Aramid fibers were further modified by radiation-induced crosslinking reactions as a means to avoid scission of the polymeric backbone and to further increase the fiber strength.An indirect radiation-induced grafting approach was used for synthesizing these novel nanocellulose-grafted aramid fibers while avoiding the irradiation of nanocellulose. The fibers were irradiated using the e-beam linear accelerator (LINAC) at the Medical Industrial Radiation Facility (MIRF) at the National Institute of Standards and Technology (NIST). After the irradiation, the fibers were kept in an inert atmosphere and then mixed with a nanocellulose solution for grafting. The grafted fibers were evaluated by gravimetric analysis, Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and nuclear magnetic resonance (NMR) spectroscopy. The mechanical properties of the synthesized fibers were studied by single fiber tensile tests. Aramid fibers were also irradiated at the MIRF in the presence of acetylene gas and triacrylate solution as a means to induce crosslinking reactions. These fibers were irradiated at both low doses and high dose rates at room temperature. A mechanism for the crosslinking of aramid fibers was proposed in this dissertation. Mechanical testing of the fibers after crosslinking showed an increase in the strength of the fibers of up to 15%. Ultra-high molecular weight polyethylene (UHMWPE) fibers were also studied, but due to an issue of entanglement of the fibers during the grafting process, their mechanical properties could not be analyzed. Future work will focus on using a better set up to avoid entanglement of these fibers. To complete the study of the radiation effects on polymers, this thesis explored the radiation-induced degradation of aromatic polyester-based resins. The composition of the resins studied included phenyl groups and epoxies, which complicate radiation-induced grafting and crosslinking reactions. Unlike aramid and polyethylene fibers, polyester-based resins have a C-O-C bond that is susceptible to degradation. The resins were irradiated at high doses in the presence of oxygen. The scission of the polymeric backbone of the polymers was studied using Electron Paramagnetic Resonance (EPR) analysis. EPR showed the formation of alkoxyl radicals and C-centered radicals as the primary intermediate products of the C-O-C scissions. The degradation mechanisms of the resins in the presence of different solvents were proposed. Changes in the Tg of the polymers after irradiation, as an indication of degradation, were studied by Dynamic Mechanical Analysis (DMA). The results obtained from this work show that irradiation of these resins results in continuous free radical-chain reactions that lead to the formation of recyclable oligomers.Item EVALUATING IN VITRO AND IN VIVO LOOPING EFFICIENCIES OF ARTIFICIAL DNA-BINDING PROTEINS(2014) Sucayan, Sarah; Kahn, Jason D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)DNA looping plays an important role in gene regulation by increasing the local concentration of a transcriptional activator or repressor at its primary binding site. Several in vitro and in vivo studies on DNA looping showed that the stability of protein-mediated DNA loops depends on the flexibility of both the looping protein and the DNA that contains the binding sites. We designed two types of short and rigid DNA looping proteins, based on a coiled-coil motif, in order to probe DNA flexibility on the thermodynamics of protein-mediated DNA loop formation. In vitro characterization of the putatively tetrameric DNA binding protein lzee by electrophoretic mobility shift assays (EMSA) did not show evidence of a sandwich complex, which is a necessary precursor to DNA looping. A quantitative in vivo looping assay, adapted from the reporter gene assay developed by Becker, Kahn, and Maher (2005), showed relatively weak enhancement of repression on GCN4 operators spaced >300 bp apart by lzee and the dimeric looping proteins LZD73, LZD80 and LZD87. However, lzee and LZD87 expression triggers cell toxicity orhighly decreased reporter protein expression on reporter strains containing GCN4 sites <240 bp apart. We proposed recombination events to explain the unexpected behavior in this distance range. Results from in vitro Plasmid Conformation Capture (PCC) revealed a very weak increase in crosslinking efficiency on 450- and 900-bp DNA loops. The apparent failure in capturing DNA loops by in vivo and in vitro PCC was attributed to the LZD proteins not being able to crosslink to DNA. Lastly, we introduced two kinds of modifications to our DNA binding proteins. The first modification sought to improve the linker sequence in lzee in order to select for better tetrameric looping proteins. The other modification introduced lysine residues at the DNA binding domains in the dimeric GCN4 looping protein LZD87 to enhance their ability to crosslink DNA. The in vivo repression assay failed to select for lzee mutants that are better repressors than lzee, while the crosslinking assays on the LZD single mutants did not show clear evidence that the new proteins can crosslink DNA. Taking all of these results together, we have concluded that the inability of the LZD proteins to stabilize DNA loops in vivo support the model that DNA plays a more passive role in the thermodynamics of DNA looping. However, the unique ability of lzee to trigger recombination in our repression assays can be utilized to design assays that detect recombination as a consequence of DNA looping, although further studies are required to understand this behavior. The molecular tools presented in this work would serve not only in providing a deeper understanding the thermodynamics of DNA looping, but could also be used as a starting point to develop better systems for modulating gene expression.Item DEVELOPMENT OF TOOLS TO CHARACTERIZE PROTEIN-PROTEIN INTERACTIONS(2010) Jiang, Jiangsong; Li, Shuwei; Stewart, Richard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Protein-protein interactions (PPIs) are crucial to most biological processes and activities. Large-scale PPI screening has been applied to model organisms as well as to human cells. Two approaches have been used extensively in high-throughput PPI studies: (i) the Yeast Two-Hybrid (Y2H) assay (a bottom-up method), and (ii) the tandem affinity purification (TAP) (a top-down method). However, a close examination of both techniques revealed issues that limit their effectiveness. Thus, it is important to develop new methods that can bridge the gap between the Y2H and the TAP. In this thesis, two approaches were developed to meet this need. The first approach was a photoaffinity labeling tool, which was based on a photo-caged reactive intermediate para-quinone methide (pQM) to study protein-peptide associations. This system was developed and optimized by using the interaction between catPTP1Bm and the EGFR peptide as a test case. Highly specific protein labeling was achieved, and mass spectrometry (MS) was used to identify the crosslinked site on the target protein. Interestingly, two peptides from catPTP1Bm detected by MS were found close to the enzyme-substrate binding interface in the three-dimensional structure of the complex, which demonstrated this method might be useful for the analysis of protein complex conformation. The second approach, named "PCA plus", took advantage of a technique referred to as "Protein-fragment Complementation Assay (PCA)". A hydrolysis-deficient mutant β-lactamase (E166N) was used, which enabled interacting protein labeling in live cells. With this modification, the PCA plus method realized live cell imaging with subcellular resolution. Fluorescent microscopy and flow cytometry analysis demonstrated its potential applications. In addition, a new β-lactamase substrate was developed for the PCA plus method and was applied to enable purification, from living cells, of prey protein interacting with a bait protein. The observed enrichment of interacting partners suggested the system could be used for high-throughput PPI screening. Moreover, this method could also be useful for the characterization of low affinity and transient PPIs because of its capacity on labeling interacting protein inside cells.Item Kinetic Characterization and Domain Analysis of the helicase RecD2 from Deinococcus radiodurans(2010) Shadrick, William Robert; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The gram positive bacterium D. radiodurans is known for its extreme resistance to radiation and an extraordinary ability to reconstitute its genome after sustaining large numbers of double strand breaks (DSB's). Genome analysis does not immediately reveal a biochemical basis for this incredible DNA repair ability. In E. coli, DSB's are mainly repaired through the RecBCD pathway via homologous recombination. The D. radiodurans genome contains no known homologues of RecB or RecC, but sequence analysis has identified a homologue of RecD, termed RecD2. The function of RecD2 in D. radiodurans is unknown, as RecD elsewhere has only been found as a component of the RecBCD complex. Our research has focused on biochemical characterization of RecD2. Previous work in our lab established that RecD2 is a DNA helicase with limited processivity and a preference for forked substrates. We have studied the unwinding mechanism of the enzyme, as measured by rates of DNA unwinding and behavior on various substrates. Reactions conducted under single turnover conditions have allowed us to determine the processivity and the step size of RecD2. RecD2 pre-bound to dsDNA substrate is capable of unwinding 12 bp, but not 20 bp, when excess ssDNA is added to prevent rebinding of enzyme to substrate. Unwinding of the 12 bp substrate under single turnover conditions could be modeled using a two step mechanism, with kunw = 5.5 s-1 and dissociation from partially unwound substrate koff = 1.9 s-1. Results derived from these rate constants indicate an unwinding rate of 15-20 bp/ sec, with relatively low processivity (P = 0.74). Glutaraldehyde cross-linking showed formation of multimers of RecD2 in the absence of DNA, but this was not detectable by size exclusion chromatography. We were able to separate the N-terminal region from the helicase core of RecD2 using limited proteolysis. It was not possible to characterize the C-terminal helicase domain due to its low solubility upon overexpression in E. coli.