Doctor of Philosophy / Department of Biochemistry / Thomas E. Roche / Specific mutants were developed to evaluate the roles of several residues in [Alpha]8 helix of the regulatory (R) domain of human pyruvate dehydrogenase kinase 2 (PDHK2) in the linkage between the Regulatory (R) and catalytic (Cat) domain (Q144A), dichloroacetate (DCA)/pyruvate inhibition (R154C, R158A, I157F) and stimulation by reductive acetylation (L160A, R154C/L160A). All mutants, with the exception of L160A, were active, and were bound to and had their activities enhanced by dihydrolipoyl acetyltransferase (E2). The cross arms between subunits are anchored by W383. Based on the studies on the W383F mutant, W383 provided majority of the intrinsic Trp fluorescence; and ligand(s) binding quenched primarily (pyruvate) or exclusively (ADP or ATP) the fluorescence of W383. The Q144 mutation in the R domain caused 14-fold weaker K[superscript]+ binding with ATP in the Cat domain but did not alter the weaker K[superscript]+ binding with ADP unless Pi was included. Similarly, with 100 mM K[superscript]+, the Q144A mutant had weaker ATP binding but the affinity for ADP was not changed even in the presence of Pi, which enhanced the binding of ADP to kinase by 2-fold. R154 and R158 were shown to be important residues in the inhibition by pyruvate, DCA and Cl[superscript]-. The R154C, R154C/L160A and R158A mutations reduced the inhibition by DCA or pyruvate using E1 or E1[dot in middle of line]E2 as the substrates. Pyruvate plus ADP did not significantly hinder the binding of GST-L2 to these mutants in AUC studies. Cl[superscript]- appears to bind to kinase at the same site as DCA/pyruvate based on lack of Cl[superscript]- effects with above mutants and evidence that Cl[superscript]- weakened the inhibition by DCA or pyruvate of native PDHK2. Q144 and L160 may play important roles in the signal transmission from the lipoyl group-binding site to the active site. Q144A and R154C/L160A mutants were less stimulated by reductive acetylation than other mutants and native PDHK2. Nov3r binds to where lipoyl goup binds PDHK2. Using E1 alone as substrate, Nov3r binding caused a 20% increase of kinase activity at low levels. Nov3r binding also reduced the inhibition of DCA/pyruvate with elevated K[superscript]+ plus Pi.

Doctor of Philosophy / Department of Biochemistry / Dolores J. Takemoto / Exposure to oxidative stress leads to accumulation of reactive oxygen species and this stimulates protective cellular functions as a compensatory response to prevent the spread of apoptotic signal and prevent cell death. The purpose of this dissertation is to understand the importance of PKCγ activation and regulation of the retinal gap junction protein Cx50, and what role PKCγ plays in this neuro-protective effect. Through electron microscopy we were able to show that PKCγ knockout mice retinas had incomplete cellular organization in the outer plexiform layer (OPL) of the retina, the layer of retina where Cx50 plays an important role in retinal cellular synapses. Electroretinograms confirmed that this structural disorganization also led to loss of functional response to light stimuli in PKCγ knockout mice retinas. In vivo exposure to 100% hyperbaric oxygen (HBO) caused significant degradation of the retina in knockout mice compared to control mice. Thicknesses of the inner and nuclear and ganglion cell layers were increased, with complete disruption of OPL in PKCγ KO mice retinas. Damage to the outer segments of the photoreceptor layer and ganglion cell layer was significantly more apparent in the central retinas of HBO-treated knockout mice. Cx50 immunolabeling showed significant reduction to HBO treatment of PKCγ control mice retinas, HBO treatment failed to produce reduction of Cx50 immunolabeling in KO mice retinas. In the R28 retinal cell line, PKCγ enzyme was shown to be activated by phorbol ester (TPA) and hydrogen peroxide. This resulted in translocation to the cellular membrane as confirmed by western blot and confocal microscopy. Suppression of PKCγ by siRNA rendered R28 cells more sensitive to oxidative stress-induced cell apoptosis, the process of apoptosis started earlier, and this resulted in cell death. R28 treatment with phorbol esters and hydrogen peroxide led to reduction in gap junction activity and Cx50 gap junction cell disassembly. This dissertation shows that PKCγ plays an important role in structural organization of retina and has a neuro-protective effect in response to oxidative stress, in part because of its control of Cx50.

Doctor of Philosophy / Department of Biochemistry / Lawrence C. Davis / Halogenated organic compounds have had widespread and massive applications in industry, agriculture, and private households, for example, as degreasing solvents, flame retardants and in polymer production. They are released to the environment through both anthropogenic and natural sources. The most common chlorinated solvents present as contaminants include tetrachloroethylene (PCE, perchloroethene), trichloroethene (TCE), trichloroethane (TCA), and carbon tetrachloride (CT). These chlorinated solvents are problematic because of their health hazards and persistence in the environment, threatening human and environmental health. This contribution provides insight on PCE degradation at laboratory and field scale at a former dry cleaning site in Manhattan, KS. Biostimulation experiments included combinations and concentrations of the following nutrients: soy oil methyl esters (SOME), yeast extract (YE), glucose, lactate, methanol and cheese whey. Bioaugmentation studies used KB-1 bacterial consortium (commercially available culture containing Dehalococcoides). This culture is known to complete the degradation of PCE to a safe end product, ethene. Concentrations of PCE and its degradation intermediates were monitored in the gas phase of the microcosm vials. Biostimulation of the natural ground water and soil microflora did not completely degrade PCE as cis-DCE (c-DCE) accumulated in the sample. Bioaugmented microcosms containing YE and SOME created reducing conditions for KB-1 culture, resulting in ~ 90% dechlorination of PCE to methane and c-DCE. Cheese whey microcosms containing 0.05% cheese whey inhibited the KB-1 culture. This inhibition was due to a drop of pH that inhibited the culture activity. Lower concentrations of cheese whey (e.g. 0.01% to 0.025%) reduced PCE and generated methane in KB-1 augmented microcosms. Based on microcosm results, a pilot bioremediation field study was conducted for a dry cleaning site contaminated with PCE. Ground water flow threatened public water wells located 1.5 miles from the source. Concentrations of PCE in the aquifer was 15 mg/L above the maximum contaminant level of 5 µg/L. Tracer studies with potassium bromide (KBr) were conducted before, during and after the bioremediation study. Nutrient solutions prepared with YE, SOME, lactate and glucose were used for biostimulation and preconditioning of ground water prior to KB-1 injection. Nutrients were provided twice during the pilot study to supplement microbial growth and cheese whey was used. During biostimulation no degradation beyond DCE was evident. The addition of KB-1 reduced PCE and DCE concentrations in the monitoring wells of the pilot study area. Total chlorinated ethene concentrations did not reach background levels 2 years after the last nutrient addition. Tracer results showed that microbial growth decreased ground water velocity during the study, but returned to normal conditions 1 year after the last nutrient addition. In this study we were able to show that native microbial population was not able to degrade PCE to final end products. Therefore, it was necessary to introduce KB-1 culture a long with nutrients to support complete reductive dechlorination of PCE.

Master of Science / Department of Grain Science and Industry / Yong Cheng Shi / In vitro digestibility of starch in sorghum grains differing in endosperm hardness and flour particle size was assayed by an Englyst resistant starch (RS) method. The starch digestibility increased as the particle size of flour decreased, but no significant difference in starch digestibility was observed among sorghum flours milled from grains with different hardness. To further understand the digestion of starch in sorghum, the effects of protein on starch digestion and amylose content in starch were determined. pH value was a factor affecting protein digestion since protein digestibility was higher at pH 2.0 than that at pH 1.3. Protein hydrolysis increased with time of pepsin treatment, leading to an increased starch digestion. RS content was 10.61-29.54% in native sorghum flours and 8.47-26.28% in isolated sorghum starch. The amounts of [Gamma]-kafirins extracted increased with time of pepsin treatment while [Alpha]- and [Beta]-kafirins decreased. The starch in sorghum flour with median hardness had a higher amylose content (23.9%) than the starch in hard and soft flours (~21%), which gave lower starch digestibility. Protein digestibility decreased after cooking while starch digestibility increased. Sulfhydryl groups decreased after cooking, indicating that disulfide bonds formed between protein molecules and may have formed a barrier for enzymes to access and digest starch. Confocal laser scanning microscopy (CLSM) showed that the protein matrix was less evident after pepsin treatment. As a result, starch digestion increased after protein matrix was removed.

Master of Science / Department of Biochemistry / Michael R. Kanost / Laccases are multicopper oxidases that catalyze the oxidation of a broad range of substrates, typically phenols and anilines. Research on laccases in fungi, plants, and bacteria has indicated that they have roles in detoxification, pigmentation, wound healing, morphogenesis and lignin synthesis and degradation. However, there has been relatively little investigation on laccases that exist in insects or other invertebrates. Insects have multiple laccase genes, but the function of just one type is known; laccase-2 (Lac2) orthologs are required for tanning of newly synthesized exoskeleton. In the mosquito Anopheles gambiae and other insect species whose genomes have been sequenced, alternative exon splicing may generate two isoforms of Lac2. The objective of this study was to characterize the two isoforms of AgLac2. They are identical in their first 500 residues, but the carboxyl-terminal 262 residues derived from alternative exons are 81% identical. Recombinant Lac2A and Lac2B were expressed and purified. They are both glycoproteins of ~81 kDa, and both can oxidize the laccase substrate ABTS as well as the catechols, N-β-alanyldopamine (NBAD) and N-acetyldopamine (NADA). Lac2A and Lac2B with ABTS have pH optima of 5.0-5.5 and 4.5-5.0, respectively. The pH optima with NBAD and NADA are 5.5-6.5. The Km values (mM) for Lac2A and Lac2B with NBAD are 5.4 ± 2.1 and 5.0 ± 2.6, respectively. The Km values (mM) for Lac2A and Lac2B with NADA are 0.7 ± 0.2 and 1.4 ± 0.5, respectively. Thus, there is little difference between the isoforms in K[subscript]m for these two substrates. The K[subscript]m values do indicate that both isoforms have a greater affinity for the substrate NADA. The kcat values (s[superscript]-1) for Lac2A and Lac2B with NBAD are 14.2 ± 3.5 and 6.0 ± 1.8, respectively. The k[subscript]cat values (s[superscript]-1) for Lac2A and Lac2B with NADA are 2.4 ± 0.2 and 0.5 ± 0.04, respectively. The most apparent difference between the two isoforms detected in the study is that Lac2A was four-fold more active than Lac2B when NADA was used as a substrate. Although the two isoforms are very similar in their amino acid sequences, the differences in catalytic properties may indicate different roles in insect physiology.

Master of Science / Department of Biochemistry / Bruce D. Schultz / John M. Tomich / Peptides based on the second transmembrane segment of the glycine receptor (M2GlyR) were made to provide a potential therapeutic treatment for cystic fibrosis (CF) and a latent absorption enhancer for drug delivery. For similarity of presentation, unique synthetic peptide sequences have been given alpha-numeric designations. Results are presented from studies focusing on four peptides. In the first study, the contributions of synthetic peptides p1171, p1172 and p1173 to net transepithelial ion transport were measured as a first step toward the goal of testing whether pore length or electrostatics of pore lining residues will affect anion transport. Peptide p1130 exhibits many attributes that make it an ideal synthetic peptide for CF treatment, but has low permselectivity for anions. Therefore, it is used as a platform for modification. Peptide p1171 is doubly substituted with diaminopropionic acid at positions T13 and T17. Peptide p1172 and p1173 are separately one and two helical turn(s) inserted into the p1130 backbone. Apical exposure of MDCK monolayers to these peptides caused a rapid increase in short circuit current (Isc), an indicator of net ion transport. The increase in Isc caused by p1172 or p1173 was accompanied by increase in transepithelial electrical conductance (gte). The electrophysiological results suggested that these modified peptides can assemble in the apical membrane of epithelial cells to form functional ion-conducting pores. Peptide NC-1059, which provides for ion transport across epithelial cells derived from many sources, was studied further to assess cellular changes that account for increased gte. NC-1059 increased Isc, gte and enhanced permeation of dextrans in a concentration dependent manner. Results from previous and current studies show that NC-1059 modulated the epithelial paracellular pathway by altering the distribution and abundance of junctional proteins. Immunoblotting and immunolabeling with confocal microscopy showed that NC-1059 induces reorganization of actin and causes a reduction in F-actin abundance in epithelial cells. The distributions were changed and cellular abundances were reduced of tight junction proteins occludin and ZO-1 and adherens junction proteins E-cadherin and β-catenin by NC-1059. These effects were largely reversed in 24 hr and fully recovered in 48 hr. Therefore, NC-1059 has the therapeutic potential to increase the efficiency of drug delivery across barrier membranes.

Master of Science / Department of Chemistry / Duy H. Hua / (+)-myriceric acid A is known as a non-peptide ETA receptor antagonist. It is isolated from the natural plant Myrica cerfera with 0.01% yield which is very low. The total synthesis of (+)-myriceric acid A is being pursued in Hua’s lab. (+)-myriceric acid A specifically blocks the vasoconstriction caused by endothelin-1 (ET-1). Because some derivatives of (+)-myriceric acid A were shown to have ET-1 receptor antagonistic effect, the tetracyclic terpenoid intermediates toward the total synthesis of (+)-myriceric acid A are postulated to have the similar antagonistic activities. The objective of this project is to study the release of vasoconstriction of these synthetic intermediates and compare their antagonistic potency. The ET-1 receptor antagonistic bioactivity of six (+)-myriceric acid A intermediates as well as (+)-myriceric acid A were evaluated by the in vitro spiral modiolar artery (SMA) bioassay. The synthetic intermediates which have not been reported in the literature were previously synthesized in Hua’s laboratory by Dr. Angelo Aguilar and Dr. Aibin Shi. Their synthesis was described in Dr. Aguilar’s PhD thesis. All the antagonistic effect evaluations were based on the SMA’s diameter changes. SMA’s diameter changes were induced by the superfusion of different extracellular solutions. The dose-response curves and straight lines were plotted to compare the antagonistic potency of these compounds. Based on the EC50 value of (+)-myriceric acid A intermediates (0.090 µM ~ 0.582 µM for the curves and 0.095 µM ~ 0.385 µM for the straight lines), all of the compounds have ET-1 receptor antagonistic activity, therefore the synthesis and screening of (+)-myriceric acid A intermediates is probably a promising route to develop new non-peptide ETA receptor antagonists.

Master of Science / Department of Biochemistry / Dolores J. Takemoto / Spinocerebellar ataxia type 14 (SCA14) is a rare, autosomal dominant neurodegenerative disease caused by mutations in the gene encoding for protein kinase Cγ (PKCγ). These mutations affect the translocation and activation of the protein and are particularly damaging to the Purkinje cells of the cerebellum. This translocation and activation leads to the down regulation of gap junction activity by direct phosphorylation on the C-terminal tail of connexin proteins. This process is necessary in terminating the propagation of apoptotic signaling and is disrupted by SCA14-type mutations. Gap junctions allow the passive diffusion of small molecules from one adjoining cell to another. Gap junctions function as electrical synapses in neuronal tissue and are formed from connexin proteins. The connexin family of proteins contains approximately 20 members, each of which is expressed in a tissue dependent manner. One of the dominant connexin proteins expressed in Purkinje cells is connexin 57 (Cx57). Here, I have tested if Cx57 is regulated by PKCγ. This thesis shows that activation of PKC and PKCγ caused internalization of Cx57 gap junction plaques in HT-22 cell culture. PKC and PKCγ activation led to the phosphorylation of Cx57 primarily on serine residues. Furthermore, the expression of SCA14-type PKCγ led to increased sensitivity to oxidative stress, resulting decreased cell viability.

Doctor of Philosophy / Department of Chemistry / Paul E. Smith / The force fields used in computer simulations play an important role in describing a particular system. In order to estimate the accuracy of a force field, physical or thermodynamic properties are usually compared with simulation results. Recently, we have been developing a force field which is called the Kirkwood-Buff Force Field (KBFF). This force field is established by transforming experimental data into Kirkwood-Buff (KB) integrals and then attempting to reproduce those KBIs with molecular dynamic (MD) simulations. Here we investigate a variety of intermolecular interactions in aqueous solutions through KB theory and molecular simulations. First, we describe a force field for the simulation of alkali halide aqueous solutions. These models are developed specifically to reproduce the experimentally determined Kirkwood-Buff integrals and solution activities as a function of molality. Additionally, other experimentally known properties including ion diffusion constants, relative permittivities, the densities and heats of mixing are reproduced by these models. Second, In an effort to understand the interactions which occur between amino acids in solution we have developed new force fields for simple amino acids and their analogs including glycine, betaine, β-alanine, dl-alanine, NH4Cl, NH4Br, N(CH3)4Cl, N(CH3)4Br, CH3NH3Cl, and CH3COONa. The new force fields reproduce the experimental Kirkwood-Buff integrals which describe the relative distribution of all the species in a solution mixture. Furthermore, it is shown that these simple amino acids can be understood in terms of the interactions of their functional groups and that, to a very good approximation, the transferability and additivity usually assumed in the development of biomolecular force fields appear to hold true. Third, an analysis of the effect of a cosolvent on the association of a solute in solution is presented by using the Kirkwood-Buff theory of solutions. The derived expressions provide a foundation for the investigation of cosolvent effects on molecular and biomolecular equilibria, including protein association, aggregation, and cellular crowding. Finally, in an effort to understand peptide aggregation at the atomic level we have performed simulations of polyglycine ((gly)n) using our recently developed force fields. Experimentally, the association of glycine polypeptides increases with n. Our force fields reproduce this behavior, and we investigated the reasons behind this trend. In addition to studying closed ensembles, we also simulate these systems in a semi-open ensemble that was designed to mimic cellular environments typically open to water, using a simple direct approach. The differences between the two ensembles are investigated and compared with our recent theoretical descriptions of aggregating systems using Kirkwood-Buff theory.

Doctor of Philosophy / Department of Biochemistry / Michal Zolkiewski / ClpB is a bacterial heat-shock protein that disaggregates and reactivates strongly aggregated proteins in cooperation with the DnaK chaperone system. ClpB contains two ATP-binding AAA+ modules, a linker coiled-coil domain, and a highly mobile N-terminal domain. It forms ring-shaped hexamers in a nucleotide-dependent manner. The unique aggregation reversing chaperone activity of ClpB involves ATP-dependent translocation of substrates through the central channel in the ClpB ring. The initial events of aggregate recognition and the events preceding the translocation step are poorly understood. In addition to the full-length ClpB95, a truncated isoform ClpB80, that is missing the whole N-terminal domain, is also produced in vivo. Various aspects of the structure and function of ClpB were addressed in this work. The thermodynamic stability of ClpB in its monomeric and oligomeric forms, as well as the nucleotide-induced conformational changes in ClpB were investigated by fluorescence spectroscopy. Equilibrium urea-induced unfolding showed that two structural domains-the small domain of the C-terminal AAA+ module and the coiled-coil domain-were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation or interactions during formation of the ClpB rings. Several locations of Trp-fluorescence probes were also found to respond to nucleotide binding. The biological role of the two naturally-occurring ClpB isoforms was also investigated. We discovered that ClpB achieves optimum chaperone activity by synergistic cooperation of the two isoforms that form hetero-oligomers. We found that ClpB95/ClpB80 hetero-oligomers form preferentially at low protein concentration with higher affinity than homo-oligomers of ClpB95. Moreover, hetero-oligomers bind to aggregated substrates with a similar efficiency as homo-oligomers of ClpB95, do not show enhanced ATPase activity over that of the homo-oligomers, but display a strongly stimulated chaperone activity during the reactivation of aggregated proteins. We propose that extraction of single polypeptides from aggregates and their delivery to the ClpB channel for translocation is the rate-limiting step in aggregate reactivation and that step is supported by the mobility of the N-terminal domain of ClpB. We conclude that the enhancement of the chaperone activity of the hetero-oligomers is linked to an enhancement of mobility of the N-terminal domains.