A. James Clark School of Engineering
Permanent URI for this communityhttp://hdl.handle.net/1903/1654
The collections in this community comprise faculty research works, as well as graduate theses and dissertations.
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Item 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.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 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.Item 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.