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 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.Item NEW CHEMICAL TOOLS TO INVESTIGATE RNA FUNCTIONS(2013) Luo, Yiling; Dayie, Theodore K; Sintim, Herman O; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ribonucleic acid (RNA), as one of the essential macromolecules of life, plays an active role in gene regulation, catalysis, and signaling because of its ability to adopt complex 3D structures that can exist in multiple conformations. Until now, RNA preparation methods devised by most investigators utilized partially denatured the RNA. The mis-folding caused by denaturing - renaturing can seriously affect RNA structure and functional activity. To test this hypothesis, in PART I of this dissertation, we presented a simple strategy using `click' chemistry to couple biotin to a `caged' photocleavable guanosine monophosphate to synthesize native RNAs that are properly folded. We demonstrated that RNA ribozymes, ranging in size from 27 to 527 nt, prepared by our non-denaturing method form a homogenous population with superior catalytic activity than those prepared by traditional refolding methods. Having developed a method for in vitro RNA synthesis, we studied the riboswitch family of RNAs that require remolding their structure for function in PART II. C-di-GMP riboswitch, as the only currently known secondary messenger riboswitch, senses c-di-GMP using its aptamer domain to modulate the expression of genes which affects biofilm formation and virulence factors production in bacteria. To specifically target pathogenic bacteria in polymicrobial systems that control the RNA-mediated c-di-GMP signaling pathway through riboswitch regulation, different c-di-GMP analogs have been used as chemical tools to investigate the structure-activity relationship (SAR) of c-di-GMP binding to different c-di-GMP riboswitches. We demonstrated that different 2'-position modified c-di-GMP analogs could differentiate between two different classes of c-di-GMP riboswitches and even bind to one particular riboswitch in class I with different affinities. Specifically, Clostridium tetani (Ct-E88) RNA in class I c-di-GMP riboswitch was used for (structural dynamic) study to understand how ligand binding drives the conformational change to regulate the downstream genes within the expression platform. Our preliminary data obtained via different biochemical and biophysical tools - such as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy - demonstrated that Mg2+ ions accelerate ligand recognition by pre-organizing the RNA, and then rapid ligand binding folds the RNA into a compact structure for likely downstream gene regulation.Item Efforts Toward Synthesis of Novel Analogs of the Bacterial Second-Messenger, c-di-GMP(2009) Shurer, Andrew Joseph; Sintim, Herman O; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The formation of bacterial biofilms is a common mechanism for antibiotic resistance. It has been shown that bis-(3'-5')-cyclic dimeric guanosine monophosphate, c-di-GMP, plays a key role in bacterial biofilm formation; therefore, the proteins that regulate the metabolism or adaptive response of c-di-GMP are favorable targets for novel antimicrobials. We herein describe a solid-support methodology developed in the Sintim Laboratory and efforts toward its application to the synthesis of novel c-di-GMP analogs. Our selected targets are a series of analogs bearing various substitutions at the 2'-position of the ribose backbone. Syntheses of 2'-deoxy and 2'-methoxy analogs were achieved as well as that of key intermediates toward the 2'-fluoro and conformationally flexible analogs.Item Inhibitors of Autoinducer-2 Quorum Sensing and Their Effect on Bacterial Biofilm Formation(2007-07-31) Lennen, Rebecca Melissa; Bentley, William E; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacteria utilize small signaling molecules, or autoinducers, to regulate their gene expression in tandem by a process termed quorum sensing. The gene encoding the synthase for autoinducer-2 (AI-2), luxS, is conserved in dozens of diverse bacteria. Behaviors controlled by AI-2 include virulence, motility, toxin production, and biofilm formation. The development of therapies that interfere with AI-2 quorum sensing are attractive for targeting biofilms, which exhibit inherent resistance to most antibiotics and biocidal agents. In this study, in vitro synthesized AI-2, LuxS inhibitors, and (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone were screened for their effect on biofilm formation in Escherichia coli, Bacillus cereus, and Listeria innocua. The LuxS inhibitors were found to have no influence on biofilm formation in any of the screened species, but reduced exponential phase AI-2 production in Listeria innocua. The brominated furanone significantly inhibited growth in B. cereus and L. innocua, and under certain conditions preferentially inhibited biofilm formation independently from growth.