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

More information is available at Theses and Dissertations at University of Maryland Libraries.

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    3D ENGINEERING OF VIRUS-BASED PROTEIN NANOTUBES AND RODS: A TOOLKIT FOR GENERATING NOVEL NANOSTRUCTURED MATERIALS
    (2018) Brown, Adam Degen; Culver, James N; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Technological innovation at the nanometer scale has the potential to improve a wide range of applications, including energy storage, sensing of environmental and medical signals, and targeted drug delivery. A key challenge in this area is the ability to create complex structures at the nanometer scale. Difficulties in meeting this challenge using traditional fabrication methods have prompted interest in biological processes, which provide inspiration for complex structural organization at nanometer to micrometer length scales from self-assembling components produced inexpensively from common materials. From that perspective, a system of targeted modifications to the primary amino acid structure of Tobacco mosaic virus (TMV) capsid protein (CP) has been developed that induces new self-assembling behaviors to produce nanometer-scale particles with novel architectures. TMV CPs contain several negatively charged carboxylate residues which interact repulsively with those of adjacent CP subunits to destabilize the assembled TMV particle. Here, the replacement of these negatively charged carboxylate residues with neutrally charged or positively charged residues results in the spontaneous assembly of bacterially expressed CP into TMV virus-like particles (VLPs) with a range of environmental stabilities and morphologies and which can be engineered to attach perpendicularly to surfaces and to display functional molecular patterns such as target-binding peptide chains or chemical groups for attachment of functional targets. In addition, the distinct electrostatic surface charges of these CP variants enable the higher-level coassembly of TMV and VLP into continuous rod-shaped nanoparticles with longitudinally segregated distribution of functionalities and surface properties. Furthermore, the unique, novel, environmentally responsive assembly and disassembly behaviors of the modified CPs are shown to act as simple mechanisms to control the fabrication of these hierarchically structured functional nanoparticles.
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    PROTEIN AND PEPTIDE ENGINEERING FOR IMPROVING THERAPIES FOR APPLICATIONS IN HUMAN HEALTH
    (2018) Moghaddam-Taaheri, Parisa; Karlsson, Amy J; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The work in this dissertation focuses on protein and peptides engineering for improving therapies for applications in human health. First, we describe a directed evolution approach to engineer antibody fragments to bind to intracellular targets. An antibody fragment library was displayed using the twin arginine translocation inner-membrane display pathway, in order to allow only antibodies that are well-folded in the reducing cytoplasmic environment to be screened for binding. Displayed libraries were screened for binding to the apoptosis inhibitor survivin, and scFv cytoplasmic solubility and specificity was characterized. Though the antibodies isolated through this method displayed strong intracellular folding and high binding to survivin, they exhibited non-specific binding as well. We improved the screening approach by using whole-plasmid PCR to recover sequences of isolated antibodies. Additional improvements to the screening process to increase stringency will allow better isolation of antibodies with high affinity and specificity for their target. In a rational design approach, we designed an antimicrobial peptide-based approach for the treatment of candidiasis. Candida albicans is a commensal organism that resides asymptomatically in the body. This opportunistic pathogen can overgrow and cause potentially fatal bloodstream infections. C. albicans biofilms that colonize implanted devices exhibit increased resistance to antimicrobial treatments and current antifungal treatments contribute to the rise of resistant strains of C. albicans or may cause toxicity. Thus, there is a clinical need for new or improved antifungal therapeutics to treat C. albicans infections. Histatin-5 (Hst-5) is an antimicrobial peptide secreted by the salivary glands that exhibits antifungal activity against C. albicans. Hst-5 can, however, be degraded by secreted aspartic proteases (Saps) produced by C. albicans cells, reducing its antifungal activity. Amino acid substitutions made to Hst-5 reduced the likelihood of proteolytic degradation to better maintain antifungal activity. Of these modifications, the K11R-K17R and E16R peptides showed enhanced antifungal activity in preventing C. albicans biofilm formation and eradicating preformed biofilms as compared to parent Hst-5. The improvements to methods and experimental findings in this research contribute to the improvement of therapies to treat human disease.
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    COMPUTATIONAL METHODS IN PROTEIN STRUCTURE, EVOLUTION AND NETWORKS.
    (2013) Cao, Chen; Moult, John; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The advent of new sequencing technology has resulted in the accumulation of a large amount of information on human DNA variation. In order to make sense of these data in the context of biology and medicine, new methods are needed both for analysis and for integration with other resources. In this work: 1) I studied the distribution pattern of human DNA variants across populations using data from the 1000 genomes project and investigated several evolutionary biology questions from the perspective of population genomics. I found population level support for trends previously observed between species, including selection against deleterious variants, and lower frequency of variants in highly expressed genes and highly connected genes. I was also able to show that the correlation between synonymous and non-synonymous variant levels is a consequence of both mutation prevalence variation across the genome and shared selection pressure. 2) I performed a systematic evaluation of the effectiveness of GWAS (Genome Wide Association Studies) for finding potential drug targets and discovered the method is very ineffective for this purpose. I proposed two reasons to explain this finding, selection against variants in drug targets and the relatively short length of drug target genes. I discovered that GWAS genes and drug targets are closely associated in the biological network, and on that basis, developed a machine learning algorithm to leverage the GWAS results for the identification of potential drug targets, making use of biological network information. As a result, I identified some potential drug repurposing opportunities. 3) I developed a method to increase the number of protein structure models available for interpreting the impact of human non-synonymous variants, important for not only the understanding the mechanisms of genetic disease but also in the study of human protein evolution. The method enables the impact of approximately 40% more missense variants to be reliably modeled. In summary, these three projects demonstrate that value of computational methods in addressing a wide range of problems in protein structure, evolution, and networks.
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    THE IMPACT OF CULTURE MEDIA ON THE IN VITRO PRODUCTION OF CAT BLASTOCYSTS AND EXPLANT QUALITY
    (2012) Nestle, Emily; Keefer, Carol L; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Continued improvements in embryo culture media composition allow for the growth of high quality blastocysts, which can be used to derive embryonic stem cells (ESCs). ESCs are capable of becoming any cell type in the body making them a valuable research tool for therapeutic and regenerative research, while furthering our understanding of embryonic development and cell differentiation. The domestic cat is an important model species for both human medicine and wild felids. Cat embryo culture produces blastocysts at a rate far below that of the mouse and initial attempts at deriving cat ESCs have resulted in embryonic stem-like cells, which cannot be maintained indefinitely. In this study we assessed and compared the quality of cat blastocysts produced in vitro using two commercial human blastocyst growth media, and the maintenance of pluripotency markers OCT-4 and NANOG in inner cell mass explants from in vitro produced blastocysts over 14 days.
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    Development and applications of codon scanning mutagenesis: A novel mutagenesis method that facilitates in-frame codon mutations
    (2009) Daggett, Kelly Anne; Cropp, Ashton; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The ability to create protein variants is a very valuable tool in biochemistry. Information about mechanistic roles of amino acid side chains, protein topology and binding can all be obtained. Methodologies to mutate proteins also allow for new catalytic activity to be achieved. While the routinely used methods to alter a protein sequence have proven to be useful, to some degree each of these methods requires some knowledge of protein structure to determine the site of mutation. Further, the routinely used methods also only allow for a specified site to be changed to a pre-determined residue (directed by oligonucleotides) or for multiple random sites to be changed to a non-specified residue. This dissertation focuses on the development of a method that allows for a new defined amino acid to replace a native amino acid at a random location within in the protein. To introduce mutations at random locations within a protein coding sequence, three steps need to be accomplished. First, the coding sequence needs to be randomly digested on both strands; second, three nucleotides (a codon) at the digestion site need to be removed; and last, a new specified codon inserted. This process results in the replacement of a random codon with the new defined codon. To direct a mutation at a random location, the unique properties of a transposase/transposon are used to create both the double strand break and removal of three nucleotides. The insertion of the new defined codon is introduced using a linker sequence that when inserted in the correct reading frame a selectable phenotype is produced. This process has been termed Codon Scanning Mutagenesis (CSM). The advantages of this method over current mutagenesis methods are (1) knowledge of structural information is not required, (2) oligonucleotides are not required to introduce the mutation and (3) the mutagenesis method allows for every amino acid to be mutated regardless of the DNA sequence. Further, this method allows for any natural and unnatural amino acid to be inserted at the mutation site, as well as the ability to create mutational mixtures or introduce multiple user defined mutations.
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    Nutrient regulation by an omnivore and the effects on performance and distribution
    (2009) Pearson, Rachel Estelle Goeriz; Gruner, Daniel S; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Omnivores have a unique interaction with their nutritional environment because they have adapted to consume food from different trophic levels. To successfully navigate their variable resources, omnivores must maintain some level of nutrient regulation. To explore the effects of nutrient regulation by an omnivore, I used a salt marsh katydid, Conocephalus spartinae. To first address the ability of Conocephalus to perform on a wide range of diets and to regulate their nutritional intake, I used artificial diets that differed in relative amounts of protein and carbohydrate (Chapter 1). I found that Conocephalus survival decreased on a high protein diet due in part to a decrease in lipid stores but growth was not affected by diet. In a second experiment Conocephalus showed a degree of nutrient regulation as evidenced by the difference in what they actually ate and the predicted consumption if they had been feeding equally on the diets presented in each treatment. However, I did not find evidence for tight macronutrient regulation. Next I explored capacity of Conocephalus to regulate their nutrient intake (nitrogen and lipid) when fed naturally co-occurring prey (Chapter 2). I first established that the prey differed in their protein and lipid content and that these differences were related to the size of the prey species. When Conocephalus were fed different prey species individuals showed no differences in either growth or survival. In the final experiment, I found that Conocephalus did show evidence of a degree of nitrogen and lipid regulation because they did not feed equally on all of the prey species offered. Lastly, I documented the relationship between the ability of Conocephalus to locate plant and prey resources and the effect that these resources have on omnivore performance (Chapter 3). I found that Conocephalus aggregates in areas of high plant quality but that their numbers do not correspond to areas of high prey density. However, I found that katydid growth and survival was enhanced by prey availability but not plant quality. Overall, I documented how an organism like an omnivore relates to its nutritional environment and how nutrient regulation might affect performance and distribution. Last, I documented the relationship between the ability of katydids to locate plant and prey resources and the effect that these resources have on omnivore performance (Chapter 3). I found that katydids aggregate in areas of high plant quality but that their numbers do not correspond to areas of high prey density.
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    Energetics of Drug Interactions
    (2008-11-26) Todorova, Niya Ancheva; Kelman, Zvi; Schwarz, Frederick P.; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The goal of our research is to determine in terms of thermodynamic change of state functions the effects of experimental factors, such as water, mutagenesis, or the presence of a second substrate on the energetics of drug-inhibitor binding interactions. The binding of non-steroidal anti-inflammatory drugs within the rigid cavities of cyclodextrins was investigated by titration calorimetry and spectrofluorimetry. Loss of bulk water structure upon drug binding in the smaller hydrophobic β-cyclodextrin cavity results in an increase in the binding entropy, while restriction of the configurations of the drug in the cavity decreases the binding entropy. This restriction in the hydrophobic β-cyclodextrin cavity enhances the binding enthalpies so that the β-cyclodextrin binding reactions are enthalpy-driven. In the larger γ-cyclodextrin cavity, water is retained so that, not only are the interactions between the drug and the cavity reduced, there is an increase in the drug configurations resulting in increases in the binding entropies and the binding reactions become entropically-driven. These binding reactions also manifest enthalpy-entropy compensation where changes in the binding enthalpies are compensated by changes in the binding entropies. In drug binding to the more flexible p38α MAP kinase mutants, a single-point C→S mutation distal from the binding site, changes the interaction between the N- and C-terminal structural domains of the kinase as evident in differential scanning calorimetry. Calorimetric results show that drug-inhibitor binding affinities to kinase increase with size of the drugs since the binding reactions are all enthalpically-driven. Drug-inhibitors binding to trimeric human purine nucleoside phosphorylase were investigated by calorimetry in the presence of its second substrate, inorganic phosphate (Pi). Increasing concentrations of Pi modulates the driving-nature of the binding reaction, so that the acyclovir binding almost exclusively to the purine substrate binding site becomes more entropically-driven, while the binding reactions of ganciclovir and 9-benzylguanine interacting also with the adjacent Pi substrate site become more enthalpically-driven. A novel calorimetric enzyme activity assay at the low dissociation concentrations of the phosphorylase show an increase in the enzyme activity at low Pi concentrations, but also a decrease in the 9-benzylguanine binding affinity since this drug also interacts with an adjacent subunit.
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    Molecular Interactions of Ubiquitin and Polyubiquitin with Ubiquitin Binding Domains
    (2007-10-22) Haririnia, Aydin; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Ubiquitin is a small protein that is covalently attached to proteins, either as a single ubiquitin moiety or as polyubiquitin chains. A cascade of enzymatic reactions is required for the isopeptide linkage between the C-terminus of ubiquitin and a lysine residue on a substrate protein or another ubiquitin. Attachment of ubiquitin or polyubiquitin, termed ubiquitination, mediates numerous cellular processes by acting as a versatile signal. The signal transmitted by the tag depends on the nature of the modification, which defines the specificity of the tag for different cellular machinery. This versatility is conferred by the variations in polyubiquitin tags, both in terms of length and lysine-linkage. Polyubiquitin chains can adopt a variety of different conformations based on these variations. The conformational and dynamic properties of the tag may optimize its binding to specific ubiquitin binding domains, therefore committing the target protein to distinct cellular outcomes. A combination of NMR methods are used to study the interaction of several ubiquitin binding domains with Lys48- and Lys63-linked di-ubiquitin, the simplest model of a polyubiquitin chain, to gain insights into polyubiquitin recognition. The di-ubiquitin binding interface with ubiquitin-interacting motifs (UIMs) and ubiquitin-associated domains (UBAs) are mapped. Structural models of the complexes are also presented. The results provide the first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin, which opens the hydrophobic interface. The results also show that the UBA domain of Ede1 preferentially binds to Lys63-linked di-ubiquitin. Structural models of the UBA in complex with Lys48- and Lys63-linked di-ubiquitin are shown. Although ubiquitin is highly conserved in eukaryotes, it is promiscuous with regard to its binding partners, ranging from small molecules to UIM and UBA domains. This study examines the effects of point core leucine to serine mutations on UIM and UBA binding specificity. The results show that these mutations bestow ubiquitin with the ability to discriminate between ubiquitin-receptor proteins. Here, we solved the three-dimensional structure of the L69S Ub mutant in solution by NMR. These mutations have a profound effect on binding specificity while causing subtle changes in the protein's three-dimensional fold and reducing its stability. Modification of a specific lysine located on Ub's hydrophobic surface has been reported to inhibit proteasomal degradation and endocytosis. Here, the effects of mutation to tryptophan at this position are investigated within the context of binding to a proteasomal receptor protein, hHR23A, and an endocytic receptor protein, Ede1.
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    Development of a ZnO/SiOsub2/Si High Sensitivity Interleukin-6 Biosensor
    (2007-04-05) Krishnamoorthy, Soumya; Iliadis, Agis; Electrical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Biosensors offer the opportunity to sense biological material providing valuable information for medical diagnostics and monitoring of pathogens in the environment. Thus the development of high sensitivity, cost effective, real-time and portable biosensors is of primary importance. This thesis presents the development of a ZnO/SiOsub2/Si based CMOS compatible biosensor, for the real-time detection of interleukin-6 (IL-6). In this work, high quality ZnO films were grown on SiOsub2/Si substrates by pulsed laser deposition. A protein immobilization procedure for binding the IL-6 protein to the ZnO active area was developed and the morphology of the bio-molecules was studied using SEM and AFM techniques for the first time. A modified solid-phase Enzyme linked Immunosorbent assay (ELISA) technique was developed to measure the mass of protein bound onto the oxide surface. The study resulted in a mass of 0.364 pg/ml of IL-6 bound onto ZnO for an applied mass of 0.5 ng/ml. A guided shear mode surface acoustic wave (SAW) device in the ZnO/SiOsub2/Si system, with ZnO as the guiding layer on a SiOsub2/Si substrate, was modeled and fabricated. Two devices, operating at 708 MHz (device A) and 1.5 GHz (device B) were developed. The mass sensitivities of these devices were calibrated by applying a known copolymer mass in a window area opened in the SAW devices and by measuring the frequency shift due the application of the mass. The maximum mass sensitivity of devices A and B was 4.162 μm²/pg and 8.687 (μm²/pg) for ZnO guiding layer thickness of 340 nm and 160 nm respectively. A technique to apply IL-6 directly onto the SAW sensor surface was developed. For an applied IL-6 mass ranging from 20ng/ml - 2 μg/ml, applied in a 20x20 μm2 sensing area, the device measures IL-6 masses in the range of 1.2 fg-76.45 fg. A proof-of-concept experiment for the biosensor was setup with normal human serum to detect the presence of IL-6 in trace amounts. The device predicts three times as much IL-6 mass for normal human serum derived from pooled donors under the age of 55 as compared to that from a donor over the age of 55. This is understood to be the result of age related increased IL-6 levels and was independently confirmed through ELISA measurements. The ZnO/SiOsub2/Si sensor system therefore enables highly sensitive mass detection of the IL-6 protein to be realized.
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    Nanopatterning of Recombinant Proteins and Viruses Using Block Copolymer Templates
    (2007-01-19) Cresce, Arthur von Wald; Kofinas, Peter; Material Science and Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The study of interfaces is important in understanding biological interactions, including cellular signaling and virus infection. This thesis is an original effort to examine the interaction between a block copolymer and both a protein and a virus. Block copolymers intrinsically form nanometer-scale structures over large areas without expensive processing, making them ideal for the synthesis of the nanopatterned surfaces used in this study. The geometry of these nanostructures can be easily tuned for different applications by altering the block ratio and composition of the block copolymer. Block copolymers can be used for controlled uptake of metal ions, where one block selectively binds metal ions while the other does not. 5-norbornene-2,3-dicarboxylic acid is synthesized through ringopening metathesis polymerization. It formed spherical domains with spheres approximately 30 nm in diameter, and these spheres were then subsequently loaded with nickel ion. This norbornene block copolymer was tested for its ability to bind histidine-tagged green fluorescent protein (hisGFP), and it was found that the nickel-loaded copolymer was able to retain hisGFP through chelation between the histidine tag and the metal-containing portions of the copolymer surface. Poly(styrene-b-4-vinylpyridine) (PS/P4VP) was also loaded with nickel, forming a cylindrical microstructure. The binding of Tobacco mosaic virus and Tobacco necrosis virus was tested through Tween 20 detergent washes. Electron microscopy allowed for observation of both block copolymer nanostructures and virus particles. Results showed that Tween washes could not remove bound Tobacco mosaic virus from the surface of PS/P4VP. It was also seen that The size and tunability of block copolymers and the lack of processing needed to attain different structures makes them attractive for many applications, including microfluidic devices, surfaces to influence cellular signaling and growth, and as a nanopatterning surface for organized adhesion.