▼ To be a successful scientist, one must understand all aspects of the research project at hand. Attention to minute detail is paramount when trouble shooting must be done. Research is 99% failure and 1% success. It is the joy of these successful moments that brings us back to the lab each day. This work seeks to explain the KCNE1 project from a beginner’s perspective and impart upon the reader the basic knowledge needed to work on the project and realize these successes. The following is not only a cook book of recipes and protocols but also an explanation of how and why each protocol works. It follows the philosophy that the stronger the foundation one has in the basics, the more successful they will be in their work. Advisors/Committee Members: Lorigan, Gary.

▼ A thesis presented on the potential of unique metal complexes, namely Platinum based heterobimetallic paddlewheel complexes, to act as antitumor drugs. The goal of this work is first to present a journal review of currently present platinum and rhodium based antitumor drugs, including cisplatin and di-rhodium complexes which were instrumental in the design of our new proposed antitumor drugs. This will be followed by an introduction to the research I am currently performing in synthesizing these Pt-based heterobimettalic complexes. Finally, results and discussion of my current research findings will be presented. I hope that this work will be both informative and enjoyable to the reader. Advisors/Committee Members: Zhou, Hong-Cai.

▼ Tannins have two main properties; they readily bind to protein and they can scavenge radicals for days. When tannin is bound to protein, we hypothesize that the complex will provide persistent antioxidant activity in the gastrointestinal tract, and aid in treatment of inflammatory bowel disease. The interaction between proteins and tannins is inadequately understood, so steps were taken to better understand the interaction. First, a method was developed to analyze the complex, and its formation. Two proteins, bovine serum albumin (BSA) and the C-terminus of the SV-40 peptide were tested for complex formation with the tea polyphenolic epigallocatechin gallate (EGCG), and BSA was selected as the best model. Two reaction conditions, covalent crosslinking with formaldehyde and gentle oxidation at basic pH, were examined and the oxidation condition was selected. Anion exchange HPLC was successful in separating native BSA from the BSA-EGCG complex, but size exclusion HPLC was not successful. Kinetics were run using anion exchange HPLC to determine the rate of formation of the complex. When protein is in excess, the product peak increases in size as a function of time over 24 hours. Work was initiated to determine if protein-tannin complexes formed between tannin and protein in cell culture media or if the complex needed to be formed before it was added to the media. Free EGCG interfered with BSA-EGCG in our analysis methods, so ammonium sulfate precipitation and Sephadex-G10 packed columns were tested as methods for removing the free EGCG from the complex. These steps are preliminary for eventually forming complex and analyzing its antioxidant activity in a cell system. Advisors/Committee Members: Hagerman, Ann.

▼ NMR analysis of the structural characteristics of macromolecules has become an important technique in recent years. NMR allows for the observation of protein-protein interactions, ligand-binding reactions, and various other types of interplay between proteins and other biological molecules. This technology has been especially useful in analyzing the relationships between the various proteins in the Troponin complex. This procedure was performed to compare the overproduction of a fragment of cardiac Troponin T, cTnT(171-233), produced in BL21(codon plus) DE3-RP competent cells using a pET23d plasmid vector, in various experimental media. In the past, it was necessary to overproduce the desired protein in minimal media that had been prepared using either 13C-labeled glucose or 15N-labeled NH4Cl, depending on the NMR experiment to be performed. This is problematic at best, and I have encountered many protein fragments which are relatively easy to overproduce in Luria-Bertani (LB), but which yield little to no protein when grown in minimal media. One such peptide is cTnT(171-233). While overproduction in minimal media is possible with this protein, yields are very low when compared to those obtained in LB broth. In order to obtain high yields of recombinant protein, with the necessary isotopic enrichment, I have explored the feasibility of using ISOGROÔ Powder as a supplement to the minimal media. It was hypothesized that adding varying percentages of ISOGROÔ, containing 13C-carbon and 15N-nitrogen, would improve the growth rate of the bacterial cells. To this end, I performed the following comparison between the levels of protein production in the various growth media, using cultures derived from the same transformed bacterial colony. I also examined the relative growth rates and quality of overproduction for the bacterial transformants in minimal media that had been enriched with varying percentages of ISOGROÔ medium, ranging from 10% to 30% enrichment. Protein overproduction was found to be enhanced by the addition of ISOGROÔ to traditional minimal media mixture. Following protein overproduction, NMR analysis was performed on the cTnT(171-233) troponin fragment. Despite extensive study, currently there is no known high resolution structure of cTnT, free or bound to other members of the troponin complex. To this end, the objective of this experiment was to determine the structure of cTnT(171-233) using multidimensional heteronuclear NMR. Advisors/Committee Members: Hagerman, Ann E.

▼ Glyoxalase II catalyzes the breakdown of S-D-lactoylglutathione into glutathione and lactic acid. The activity of the enzyme is typically analyzed by a continuous spectrophotometric assay, which follows the decomposition of S-D-lactoylglutathione through its absorbance at 240 nm. The new method described here uses an alternative method, high performace liquid chromatography, to monitor S-D-lactoylglutathione concentrations and thus to to analyze the activity of glyoxalase II. Samples of S-D-lactoylglutathione were reacted with the enzyme for varying periods of time, and the enzyme was then denatured with acid. The S-D-lactoylglutathione concentration of these samples was then analyzed by high performance liquid chromatography, and the results were then used to determine the activity of glyoxalase II. This method resulted in kinetic data for glyoxalase II that was similar to that obtained spectrophotometrically. Advisors/Committee Members: Hagerman, Ann E.

▼ If you are thinking about a career in veterinary medicine, it is very important for you to know what is involved in your journey. Acceptance into veterinary colleges is very competitive, so I generated a website that will provide interested pre-veterinary students with almost all of the information they will need to know. This website addresses information about relevant coursework, veterinary and animal experiences, personality qualities of veterinarians, admission information (i.e. entrance exams, the application process, and interviewing techniques), and frequently asked questions. The accompanying document describes my vision for the website and the process I took to achieve it. Advisors/Committee Members: Hoffman, Susan.