Fischell Department of Bioengineering Theses and Dissertations

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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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    Mechanical, Structural and Biological Properties of Biopolymer-Based Hydrogels
    (2012) Hyland, Laura; Yu, Yihua B.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The aim of this dissertation was to begin to understand how biopolymer interactions affect mechanical and structural properties of biomaterials, and how those properties affect stem cell behavior. Polysaccharide, oligopeptide and oligopeptide-polysaccharide composite materials were made and then characterized using a range of techniques. It was found that chondroitin addition to chitosan-alginate networks improved both tensile and compressive strength. Increasing polysaccharide concentration also improved mechanical properties. Also, polysaccharide incorporation into peptide hydrogels increased biomaterial resistance to strain break. Structural analysis supported mechanical data, showing that incorporation of the peptides dramatically changed the morphology of the polysaccharide networks. Biopolymer chirality was also explored in this work. By incorporating polysaccharides and oligosaccharides into both L- and D-forms of peptide hydrogels, we observed differences in mechanical properties not seen in L- and D-oligopeptide hydrogels alone. Greater interactions between L-oligopeptides and D-saccharides lead to stronger materials with distinctively different structural characteristics from hydrogels made from D-oligopeptides and D-saccharides. This phenomenon, known as chiral selectivity, has previously only been seen at the molecular level. Here, we showed that chiral selectivity is another unique property of biopolymers that can be exploited to tune mechanical and structural properties of materials. Chiral selectivity was also observed in terms of stem cell behavior in this work. However another property, hydrogel charge, was used to diminish the effects of chiral selectivity in order to enhance the biocompatibility of D-oligopeptide hydrogels. It was found that negative charges significantly improved human mesenchymal stem cell attachment and proliferation in D-oligopeptide gels but had little effect on their interactions with L-oligopeptide gels. These results suggest that it is possible to use charge and other properties of biopolymers to engineer biomaterials whose chirality is distinct from that of natural biomaterials but whose performance is close to that of natural biomaterials. These oligopeptide-based biomaterials also offer new tools to investigate biohomochirality, an important and unresolved question in biology.