Master of Science in Chemistry, Youngstown State University, 2020, Department of Chemistry

Reactive oxygen species (ROS) are chemically reactive oxygen containing molecules and radicals mainly produced from the partial reduction of molecular oxygen. ROS have been associated with aging and several diseases such as atherosclerosis and cancer. Metal-catalyzed oxidation (MCO) systems are systems that produce free radicals using transition metal ions such as copper or iron and hydrogen peroxide. As such, MCO may cause oxidation of proteins. To study the correlation between protein structure and oxidative damage of proteins by MCOs, different mutants of hen egg white lysozyme (HEWL) have been developed. This research focuses on the optimization of the expression of the mutant HEWL H15S. The Pichia pastoris expression system was adapted for the expression of HEWL H15S. The P. pastoris X-33-pPICZαA-hewlH15S strain was subjected to different growth conditions in a glycerol and methanol buffered media under conditions for small scale expression. Both intracellular and extracellular protein expression were analyzed for enzyme activity. Increasing glycerol concentration from 0.5% to 1% did not show significant increase in yeast growth resulting in low protein concentration and enzyme activity at 28 °C. Also, protein expression at three different methanol concentrations at 28 °C: 0.5% (v/v), 1% (v/v), and 2% (v/v) showed an increase in enzyme activity but only small changes in total protein concentration. The addition of calcium chloride showed a significant effect on the expression of H15S to about 1mg/mL compared to the other conditions without CaCl2. Lysing of the cells grown at 28 °C for intracellular analysis by the Bradford assay showed a significant band of protein corresponding to the size of the H15S mutant. A lower temperature of 22 °C at different growth and expression conditions measured high protein concentration and an increase in enzyme activity for extracellular expression. Intracellular analysis on protein expression at 22 °C measured no lysozyme acti (open full item for complete abstract)

Committee: Michael Serra PhD (Advisor); Nina Stourman PhD (Committee Member); John Jackson PhD (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Chemistry; Microbiology

Master of Science in Chemistry, Youngstown State University, 2013, Department of Chemistry

Protein oxidation has been correlated with several chronic diseases including Alzheimer's disease, Parkinson's disease, and cataractogenesis. The purpose of this project was to isolate and characterize the various oxidized forms of hen egg white lysozyme that were produced by a copper(II)/hydrogen peroxide metal-catalyzed oxidation system. Five oxidized protein variants were purified using high performance liquid chromatography on a cation-exchange column. Tandem mass spectrometry determined that several amino acids were oxidized in each variant with histidine 15 being the most readily oxidized residue. Bacteriolytic assays showed decreased activity of Peaks IB, IIB, and III (31.4%, 61.2%, and 86.5%, respectively) relative to native enzyme while the activity for Peaks IV and V was greater than that of native enzyme (215% and 308%, respectively). Crystals of Peaks IB, III, IV, and V were grown, but attempts to determine the crystal structure were unsuccessful.

Committee: Michael Serra PhD (Advisor); Nina Stourman PhD (Committee Member); Gary Walker PhD (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, University of Toledo, 2013, Chemistry

Reactive oxygen species (ROS), which are generated from various sources, are very deleterious in nature. ROS damage biological components like DNA, RNA, proteins and lipids through liberation of free radicals. A lot of research has focused on DNA damage through oxidation. Studies on RNA oxidation have, however, received much less interest until the past decade. RNA oxidation was identified to play a significant role in the etiology of neurological diseases such as Alzheimer’s and Parkinson’s. The goal of this project is to develop the tools required for understanding the mechanisms involved in oxidative damage to RNA at a molecular level. An approach was taken which involved the generation of site specific radicals on nucleosides. Two nucleosides, uridine and pseudouridine, which are specifically found in RNA, were considered for this work. This study focuses on the generation of C5'-nucleosidylradicals from photolabile radical precursors and understanding the mechanisms of formation of radical derived products. For this purpose, syntheses were designed for the preparation of both C5'-pivaloyl substituted uridine and pseudouridine. C5'-pivaloyluridine was successfully synthesized and utilized for the generation of the C5'-uridinylradical through Norrish type I photocleavage. Photolyses were carried out under anaerobic and aerobic conditions in the presence and absence of hydrogen atom donors. Product formation was analyzed by HPLC and mass spectrometry. The formation of the reduction product was observed in the presence of a hydrogen atom donor. Along with the reduction product, base elimination was observed under all photolysis conditions. The synthesis of the C5'-pseudouridinylradical precursor is in progress.

Committee: Amanda Bryant-Friedrich PhD (Advisor); Viranga Tillekeratne PhD (Committee Member); Dragan Isailovic PhD (Committee Member); Timothy Mueser PhD (Committee Member) Subjects: Chemistry

Master of Science in Chemistry, Youngstown State University, 2009, Department of Chemistry

Metal catalyzed oxidation of protein involves a reaction between hydrogen peroxide and protein-bound metal ions. Production of the highly-reactive hydroxyl radical leads to oxidative damage in the immediate vicinity of the bound-metal ion. To examine the relationship between protein structure and oxidative damage a series of site-directed mutants of hen egg white lysozyme (HEWL) were created. This project focuses on generation of the site directed mutant where residue His15 will be altered to a serinyl residue (H15S), also work on the preliminary purification and characterization of the putative mutant would be touched on.

Committee: Michael Serra PhD (Advisor); John Jackson PhD (Committee Member); Peter Norris PhD (Committee Member) Subjects: Biochemistry; Organic Chemistry