The anaerobic metabolism of the intertidal ribbed mussel, Geukensia demissa (= Modiolus demissus), is reported in this dissertation. This mollusc was selected because anaerobic metabolism is believed to be of vital significance to its survival during the air exposure period of the tidal cycle.;Ribbed mussels, Geukensia demissa, were maintained in either aerated or hypoxic water for up to four days. Major metabolites and polysaccharide of each mussel were then analyzed and compared. Succinate, propionate and alanine were found to accumulate hypoxically, but not malate, (alpha)-keto-glutarate and pyruvate. The polysaccharide content of each mussel was quite different and did not show a clear indication of decrease under the hypoxic condition.;Propionate production followed succinate accumulation, which corroborates an earlier report for Mytilus edulis. The linear increase of alanine concentration with hypoxic period observed in this study, has not been reported previously.;The results of this study are consistent with those of Mytilus edulis, Cardium edule, and Anodonta cygnea, bivalves which have recently been studied in Europe. This agreement that bivalves have a similar anaerobic metabolic scheme.;A hypothetical scheme of anaerobic metabolism of bivalves is proposed in which redox balance is achieved in both cytosol mitochondria. This scheme incorporates parts of the Embden-Meyerhof-Parnas glycolytic pathways, carbon dioxide fixation, the Krebs cycle, transamination, and pathways for propionate formation. to date, this is the only bivalve anaerobic scheme which contains all these essential considerations.

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Cathepsin G (CatG), a serine protease found in the azurophil granules of neutrophils, participates in killing engulfed microorganisms. CatG is a poorly understood enzyme, in part because it can only be obtained as mature enzyme purified from human blood, and because it seems to have dual specificity for chymotrypsin-like and trypsin-like substrates. Therefore, yeast Pichia pastoris was used to express immature recombinant human CatG to provide a source of the enzyme free of biohazards and to allow study of its dual specificity and C-terminal processing. To avoid potential cleavage by a yeast kexin protease, the amino sequence was modified without altering CatG’s biological activity to remove one glycosylation site and eight dibasic sites. An N-terminal 6-His-cytochrome B5 (CytB5) heme binding fusion domain was linked to the modified human CatG by an enteropeptidase cleavage site for activation. The DNA for this construct was codon-optimized and placed in the pPICzα secretion vector. After transforming P. pastoris strain X-33, 48 Zeocin-resistant clones were screened for relative levels of CatG activity following activation by recombinant human enteropeptidase. Recombinant human CatG was partially purified from fermentation medium by nickel affinity chromatography and its activity was confirmed by assays using the substrate Succinyl-Ala-Ala-Pro-Phe-SBzl. Supported by a Student Faculty Collaborative Grant from the ETSU Honors College and ETSU Office of Research and Sponsored Programs and by NHLB grant R15HL091770.

Flavonoids are a class of plant metabolites with C6-C3-C6 structure responsible for many biological functions, including coloration and defense. Citrus paradisi, grapefruit, contains a wide variety of flavonoids which are grouped by the extent of modification, examples of which are flavonols, flavones, and flavanones. A major modification is the addition of glucose by glucosyltransferases (GTs) to stabilize the structure and provide ease of transport. Glucosyltransferases can be highly substrate and regiospecific. With Cp3OGT, glucose is added at the 3-hydroxy position. This 3GT only accepts flavonols as its substrate; however, a Vitis vinifera (grape) 3-GT can accept both flavonols and anthocyanidins. Homology modeling using the crystallized structure of the V. vinifera GT predicted sites of amino acids that could influence substrate binding. The 382 position was of particular interest with arginine in C. paradisi and tryptophan in V. vinifera. This research was designed to test the hypothesis that a R382W mutation would result in altered substrate specificity. Site-directed mutagenesis was performed to form the R382W mutant Cp3OGT and the gene was transformed into yeast for protein expression. Western blot determined the optimal protein induction period for the cells, after which the cells were broken to extract the recombinant mutant protein. Purification of the R382W 3GT allowed for enzyme analysis to be performed by measuring the incorporation of radioactive glucose into the reaction product. Docking analysis was performed using AutoDock software with both the wild type and R382W mutant protein with the substrates that showed interesting activity. The results of this study indicate that the point mutation of arginine to tryptophan at position 382 broadened substrate and regiospecificity to include flavanones.

abstract: Glycosaminoglycans (GAGs) are long chains of negatively charged sulfated polysaccharides. They are often found to be covalently attached to proteins and form proteoglycans in the extracellular matrix (ECM). Many proteins bind GAGs through electrostatic interactions. GAG-binding proteins (GBPs) are involved in diverse physiological activities ranging from bacterial infections to cell-cell/cell-ECM contacts. This thesis is devoted to understanding how interactions between GBPs and their receptors modulate biological phenomena. Bacteria express GBPs on surface that facilitate dissemination and colonization by attaching to host ECM. The first GBP investigated in this thesis is decorin binding protein (DBP) found on the surface of Borrelia burgdorferi, causative pathogens in Lyme disease. DBPs bind GAGs of decorin, a proteoglycan in ECM. Of the two isoforms, DBPB is less studied than DBPA. In current work, structure of DBPB from B. burgdorferi and its GAG interactions were investigated using solution NMR techniques. DBPB adopts a five-helical structure, similar to DBPA. Despite similar GAG affinities, DBPB has its primary GAG-binding site on the lysine-rich C terminus, which is different from DBPA. Besides GAGs, GBPs in ECM also interact with cell surface receptors, such as integrins. Integrins belong to a big family of heterodimeric transmembrane proteins that receive extracellular cues and transmit signals bidirectionally to regulate cell adhesion, migration, growth and survival. The second part of this thesis focuses on αM I-domain of the promiscuous integrin αMβ2 (Mac-1 or CD11b/CD18) and explores the structural mechanism of αM I-domain interactions with pleiotrophin (PTN) and platelet factor 4 (PF4), which are cationic proteins with high GAG affinities. After completing the backbone assignment of αM I-domain, paramagnetic relaxation enhancement (PRE) experiments were performed to show that both PTN and PF4 bind αM I-domain using metal ion dependent adhesion site (MIDAS) in an Mg2+ independent way, which differs from the classical Mg2+ dependent mechanism used by all known integrin ligands thus far. In addition, NMR relaxation dispersion analysis revealed unique inherent conformational dynamics in αM I-domain centered around MIDAS and the crucial C-terminal helix. These dynamic motions are potentially functionally relevant and may explain the ligand promiscuity of the receptor, but requires further studies. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

abstract: In my thesis, I characterize multi-nuclear manganese cofactors in modified reaction centers from the bacterium Rhodobacter sphaeroides. I characterized interactions between a variety of secondary electron donors and modified reaction centers. In Chapter 1, I provide the research aims, background, and a summary of the chapters in my thesis. In Chapter 2 and Chapter 3, I present my work with artificial four-helix bundles as secondary electron donors to modified bacterial reaction centers. In Chapter 2, I characterize the binding and energetics of the P1 Mn-protein, as a secondary electron donor to modified reaction centers. In Chapter 3, I present the activity of a suite of four helix bundles behaving as secondary electron donors to modified reaction centers. In Chapter 4, I characterize a suite of modified reaction centers designed to bind and oxidize manganese. I present work that characterizes bound manganese oxides as secondary electron donors to the oxidized bacteriochlorophyll dimer in modified reaction centers. In Chapter 5, I present my conclusions with a short description of future work in characterizing multiple electron transfers from a multi-nuclear manganese cofactor in modified reaction centers. To conclude, my thesis presents a characterization of a variety of secondary electron donors to modified reaction centers that establish the feasibility to characterize multiple turnovers from a multi-nuclear manganese cofactor. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

abstract: Generating amorphous solid dispersions (ASDs) containing active pharmaceutical ingredients has become a favorable technique of emerging prominence to improve drug solubility and overall bioavailability. Cannabidiol (CBD) has now become a major focus in cannabinoid research due to its ability to serve as an anti-inflammatory agent, showing promising results in treating a wide array of debilitating diseases and pathologies. The following work provides evidence for generating homogenous glass phase amorphous solid dispersions containing 50% (w/w) up to 75% (w/w) CBD concentrations in the domain size of 2 – 5 nm. Concentrations up to 85% (w/w) CBD were concluded homogenous in the supercooled liquid phase in domain sizes of 20 – 30 nm. The results were obtained from polarized light microscopy (PLM), differential scanning calorimetry (DSC), as well as solution and solid-state NMR spectroscopy. / Dissertation/Thesis / Masters Thesis Biochemistry 2019

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