The research outlined in this dissertation involves the development and demonstration of a mass spectrometry-based proteomics approach to characterize the global level molecular response of Shewanella oneidensis MR-1 to chromate exposure. The proteomics approach is centered on a high performance technique of multidimensional on-line liquid chromatographic separations with subsequent tandem mass spectrometric detection. Since very complex proteome samples are digested into peptides and then directly measured by MS, this technique is termed shotgun proteomics. This approach affords the identification and quantification of complex mixtures by directly analyzing their proteolytic peptides and then using computational techniques to reassemble the protein information. The research goals for this dissertation project were two-fold: (1) enhancement of the experimental and computational methodologies to permit deeper and more confident proteome characterizations, and (2) demonstration of this optimized approach for the comprehensive investigation of the molecular level response of the bacterium S. oneidensis to chromate insult. To address research needs, we developed a single-tube lysis method for cell lysis-proteome digestion to enable investigations of small amounts of cellular biomass, and identified suitable bioinformatic approaches to mine post-translational modifications from proteome datasets. These advancements were then utilized to examine the molecular level response of S. oneidensis to chromate insult, which was accomplished by varying chromate concentrations, dosages, and time points. These measurements provided the first global proteome-level observation of the dynamic changes of S. oneidensis in response to chromate insult.

Organisms in the genus Phytophthora are important plant pathogens, although understudied. Phytophthora was first brought into human awareness with the identification of P. infestans as the culprit for the Irish potato famine in the mid 1800s. Since then, over 80 Phytophthora species have been identified, many of which infect a wide variety of crops worldwide with devastating results. Traditionally, much of the work aimed at controlling Phytophthora diseases involved applied research. In recent years there has been a marked increase in molecular work on Phytophthora. This increase is evident not only from increased funding by agencies such as the National Science Foundation (NSF), but also from the type of research applied to Phytophthora for the first time. The first Phytophthora species to have their genomes sequenced were P. sojae and P. ramorum at 2004. Since then the genomes of two more Phytophthora species- P. capsici and P. infestans, were also sequenced. Availability of Phytophthora genome sequences provided us with the basis necessary for a proteomic investigation of these organisms. The study presented here represents the first large scale proteomic study of any Phytophthora species. Using mass spectrometry and available or newly developed bioinformatic tools we measured the proteomes of different asexual Phytophthora life stages. We also measured the protein complement of P. capsici infected tomato plants, the so called “interactome”, in order to gain an insight into the biological processes occurring in the pathogen during infection, and in the plant in response to the pathogen. We also used data from these proteomic experiments as a part of a novel approach aimed at improving the genome annotation of those Phytophthora species. Finally, we used different molecular techniques, including a reverse genetic technique called Targeted Induced Local Lesions in Genome (TILLING), to begin characterization of a few protein targets identified in those experiments. The accumulated data from all our experiments identified certain molecular processes, metabolic and others, that may explain the success of Phytophthora as a plant pathogen. The data from these experiments provides a platform on which future experiments can be based on to further characterize these interesting organisms.

Protein-protein interactions (PPI) play a vital role in almost every cellular process. Although many methodologies exist to probe PPIs, one of the most successful and widely employed is tandem affinity purification coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS). Although best demonstrated in yeast, TAP has encountered significant hurdles in its application to mammalian systems, especially the observed low yield of bait protein and its interacting partners after two consecutive purifications. To address these issues, a novel dual-tag affinity purification (DAP) system was developed that not only enhances bait protein recovery, but also allows for rapid evaluation of dual-tag compatibility with a protein of interest based on its known subcellular localization. In addition, several tags of varying composition were constructed to allow for maximal bait protein compatibility. With this system, mammalian bait protein yield was improved by more than 200% relative to previously published results. Capitalizing on this success, DAP was applied to human telomere binding proteins TRF1, TRF2, and POT1 to garner a greater understanding of the protein networks that involve the telomere. Expectedly, all the members of the telosome complex were identified at frequencies that lend evidence towards the currently accepted architecture. Also identified were several other novel proteins and subcomplexes that may enhance our understanding of telomere maintenance / length regulation. For instance, members of the classical nuclear import system co-purified with both TRF1 and TRF2. Although previously documented for TRF1, TRF2’s association with importin alpha (KPNA2) and beta (KPNB1) has not been demonstrated till now. Interestingly, further study revealed that KPNA2 acts as a negative regulator of TRF2 nuclear localization. This observation could have far-reaching implications as TRF2 is thought to be also heavily involved in the DNA damage response. Along these lines, a more indepth MS analysis revealed several putative phosphorylation sites along TRF2’s sequence. One site, pS380, seems to be phosphorylated by the DNA-damage kinase ATM and plays a role in a cell’s proliferative capacity, possibly affecting telomere length regulation. The studies contained here within demonstrate the efficacy of DAP-LC-MS/MS to provide useful leads with regards to the study of PPIs.

High-throughput technologies such as microarrays have led to the rapid accumulation of large scale genomic data providing opportunities to systematically infer gene function and co-expression networks. Typical steps of co-expression network analysis using microarray data consist of estimation of pair-wise gene co-expression using some similarity measure, construction of co-expression networks, identification of clusters of co-expressed genes and post-cluster analyses such as cluster validation. This dissertation is primarily concerned with development and evaluation of approaches for the first and the last steps – estimation of gene co-expression matrices and validation of network clusters. Since clustering methods are not a focus, only a paraclique clustering algorithm will be used in this evaluation. First, a novel Bayesian approach is presented for combining the Pearson correlation with prior biological information from Gene Ontology, yielding a biologically relevant estimate of gene co-expression. The addition of biological information by the Bayesian approach reduced noise in the paraclique gene clusters as indicated by high silhouette and increased homogeneity of clusters in terms of molecular function. Standard similarity measures including correlation coefficients from Pearson, Spearman, Kendall’s Tau, Shrinkage, Partial, and Mutual information, and Euclidean and Manhattan distance measures were evaluated. Based on quality metrics such as cluster homogeneity and stability with respect to ontological categories, clusters resulting from partial correlation and mutual information were more biologically relevant than those from any other correlation measures. Second, statistical quality of clusters was evaluated using approaches based on permutation tests and Mantel correlation to identify significant and informative clusters that capture most of the covariance in the dataset. Third, the utility of statistical contrasts was studied for classification of temporal patterns of gene expression. Specifically, polynomial and Helmert contrast analyses were shown to provide a means of labeling the co-expressed gene sets because they showed similar temporal profiles.

This dissertation is a compilation of four studies that were conducted in the laboratory of Dr. C. Neal Stewart, Jr. at the University of Tennessee, Knoxville. The first study describes an investigation into arsenate metabolism in Arabidopsis thaliana using microarray technology. The second study summarizes progress made to date towards the development of an As-specific phytosensor, or a plant genetically engineered to detect the presence of As in the environment. The third study describes efforts towards genetic transformation of Pteris cretica and Pteris vittata, both As-hyperaccumulating ferns that have been recently demonstrated as effective in the removal of As from contaminated areas. This paper demonstrates the development of a modified tissue culture protocol that was effective in callus generation from both Pteris vittata and Pteris cretica gametophytes as well as regeneration of plantlets from that callus. Attempts towards genetic transformation were made via biolistic bombardment and Agrobacterium-mediated transient expression using leaf infiltration. Optimization of the Pteris tissue culture protocol will facilitate continued efforts towards the genetic transformation of this unique plant, thereby enabling means of more effectively exploring the underlying mechanisms of As hyperaccumulation. The final study reports a field-scale investigation of plant metal uptake at a local contaminated site in Knoxville, TN. The Smokey Mountain Smelters Site is an abandoned secondary aluminum smelter where waste product from the smelting process (slag) was illegally dumped in large piles over much of the property. Interestingly, wild vegetation was found growing on the slag piles without any obvious symptoms of toxicity. Therefore, a study was conducted to quantify the v metal uptake of these plants, characterize the metal profile of the slag material, and investigate the capacity of Pteris cretica in extracting arsenic from slag on-site. As a result, these studies have provided new insights into arsenate metabolism in plants, and generated many testable hypotheses to enhance our understanding of plant genetic responses to metal stress. The following introduction serves to provide a background on phytoremediation, arsenic, and plant responses to the toxic metalloid.

Polyacrylamide was studied for its efficacy in reducing turbidity. The purpose of this research was to determine optimum conditions for 10 blends of PAM to reduce turbidity from construction site runoff. The research was based on 10 different PAM blends, researching concentrations (0, 1, 5, 10 ppm), PAM forms (dry granular or solution), mixing powers (80 and 150 rpm), and mixing times (1 and 2 minutes). The research was conducted in a laboratory setting using a PB-700TM Standard Jar Tester (Phipps and Bird, Richmond, Virginia) with a clay texture sediment source. The claytextured sediment source was researched under two sediment-water concentrations of 2,000 and 10,000 parts per million (ppm). For non-polymer control, each sediment concentration was mixed for five minutes to ensure complete mixing. One minute after the cessation of mixing, turbidity measurements were taken on time intervals of 0.5, 2, 5, and 10 minutes. Using these same samples, polyacrylamide was then added to the sediment-water solution. Three replicates of the various treatment conditions, and similar turbidity measurements were then taken. The data showed the most key aspects, on the average at reducing turbidity the greatest, were the dry forms of PAM for the 2,000 ppm sediment-water concentration and the solution forms of PAM for the 10,000 ppm sediment-water concentrations. Turbidity reductions were not as dramatic with the dry from of PAM, at the lower mixing power, with the shorter mixing time, at low PAM concentrations. The data also provided evidence that all PAM blends demonstrate different turbidity reduction under different treatment combinations. This information shows the important issue that PAM is not exactly 100% PAM and each PAM product (blend) are very soil dependent. This key issue needs to be considered when a PAM product is applied in any form or fashion.

Table Mountain pine (Pinus pungens Lamb.)(TMP) is a threatened species, endemic to the southern Appalachian Mountains. The status of TMP following the southern pine beetle (Dendroctonus frontalis Zimmerman) outbreak of 1999–2001 is unknown. This study focuses on stands of the Cherokee National Forest (CNF) in eastern Tennessee that had a TMP component in the 1994 Continuous Inventory of Stand Condition (CISC) data. This project has two parts: an inventory of the 1994 stands as well as a case study of cost comparison of release treatments for a young overstocked stand. The objective of the inventory was to visit the TMP stands designated in the 1994 CISC data on the CNF to determine whether these stands still contain a significant component of TMP and to document the present stand condition and successional status. The objective of the case study was to produce a cost analysis/comparison of releasing young TMP that are in the stem exclusion stage of stand development by several silvicultural methods: strip thinning, crop tree release, and prescribed burning. TMP is declining across the CNF with less that 900 acres dominated by the species. TMP was a major component on more than 7400 acres from the 1994 data, but many have also succeeded to hardwoods because of the absence of fire and SPB infestations. Management actions should be taken to maintain the health of remaining TMP stands on the CNF. Reintroduction of a controlled burning regime to create seedbed conditions favorable to TMP regeneration and to control hardwoods in existing stands is suggested. If TMP is to remain in Southern Appalachian ecosystems, more direct, cost-effective, and positive management approaches are necessary. Initial cost effectiveness of release treatments were analyzed. Regardless of treatment, costs ranged from $18 to $45 per acre. In this study, prescribed burning, generally considered more cost effective than mechanical treatments, was most expensive because of the small tract size and the labor involved to monitor the burn. The crop tree release treatment had the least cost because small trees were cut and cost of equipment is minimal

From 2001 through 2005, the North Carolina Department of Transportation rerouted and upgraded a section of U.S. Highway 64 in Washington County to a 4-lane divided highway. This new roadway included 3 wildlife underpasses with adjacent wildlife fencing to mitigate the effects of the highway on wildlife, in particular American black bears (Ursus americanus). From 2000 to 2001, the University of Tennessee conducted research on the spatial ecology and population demographics of the black bear population at the new highway site and on a nearby control area of similar habitat composition. From 2006 to 2007, after highway construction, data collection was repeated and additional data were collected to document use of the 3 wildlife underpasses and wildlife mortalities from vehicle collisions. I tested several hypotheses to determine if the new highway caused changes in home-range characteristics, spatial responses, habitat use, movement characteristics, and activity patterns of black bears. Using a dataset of 5,775 hourly locations and 4,998 daily locations from 57 bears, I detected no changes in home-range characteristics or movement characteristics of bears because of the new highway. However, the power for several of these analyses was relatively low and my research focused on females. I did detect changes in bear habitat use and activity patterns resulting from the new road. In particular, bears from the new highway area were closer to the road and became more active in morning when highway traffic was low. Underpass monitoring yielded 2,053 photographs of wildlife and 3,622 wildlife crossings based on track counts. The highway surveys recorded 196 animal mortalities from vehicle collisions. I observed an increase in wildlife crossings at the underpass sites, but no difference in roadkill frequency between protected sections of the highway (underpasses and fencing) compared with unprotected sections. However, substantially fewer animal-vehicle collisions (primarily deer [Odocoileus virginianus]) were reported in the study section of U.S. 64 compared with adjacent sections of this highway. Overall, I found few impacts on black bear spatial ecology resulting from the highway and that the 3 wildlife underpasses were effective.

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as a cofactor. Since THF is an essential factor for nucleotide biosynthesis, inhibition of this enzyme in bacteria with folate analogs such as trimethoprim results in bacterial cell death. Plasmid encoded R67 DHFR confers resistance to trimethoprim and is both sequentially and structurally unrelated to any known chromosomal version of the enzyme. R67 DHFR is a 34,000 Da. homo-tetramer containing a rare 222 axis of symmetry in the center of its active site pore. The active site pore is contacted by residues belonging to each of the four subunits. The enzyme can nonspecifically bind 2 NADPH’s, 2 DHF’s, or, in the case of the productive complex, 1 NADPH and 1 DHF. R67 DHFR employs the endo transition state as opposed to the exo transition state used by E. coli DHFR. In this study molecular dynamics approach using the CHARMM program is employed to study the dynamics of the enzyme and energetics of the hydride transfer step catalyzed by R67 DHFR in silico. Structural and dynamic properties of four different mutants are also examined.

The mechanism of insect CYP gene regulation is largely unknown. In the present investigation, I used Drosophila as a model insect to understand the role of X, 2nd and 3rd chromosomes on the promoter activity of Cyp6a8 gene. Two reporter transgenic strains, 0.8luc110 H-ry and 0.8luc14, carrying 0.8luc-A8 reporter transgene (chimera of 0.8-kb upstream DNA of Cyp6a8 and the firefly luciferase gene) on the 2nd and 3rd chromosomes of ry506, respectively were used. The X, 2nd and 3rd chromosomes of these two transgenic lines were replaced with corresponding chromosomes from DDT-resistant 91-R strain (overproducer of Cyp6a8). The effect was determined by measuring luciferase activity. Results showed that the 3rd chromosome of the 91-R strain had a strong and the 2nd chromosome had a weak stimulatory effect on Cyp6a8 expression. The effect of the 1st chromosome was strongly inhibitory only if the 3rd chromosome from 91-R and the 2nd chromosome from the ry506 strains were present in the genome. Chromosomal effect on the Inducibility of 0.8-kb Cyp6a8 promoter DNA by phenobarbital and barbital was also examined. It was found that these compounds could induce the promoter significantly if the genome had X or second chromosome from the ry506 or 91-R strain. However, no induction was observed when the third chromosome of 91-R was present in the genome but the third chromosome of the ry506 strain supported barbiturate inducibility of the Cyp6a8 promoter. To further investigate if Cyp6a8 is regulated by other genes, I studied the effect of third chromosome-linked DHR96 (mammalian CAR/RXR homolog) mutation on the promoter activity of the 0.8Kb upstream DNA of Cy6a8. I found that the DHR96 mutation gave 7-12 fold higher constitutive expression of Cyp6a8 compared to the wild type strain in all developmental stages. This investigation concludes that Cyp6a8 expression in Drosophila is influenced by all three major chromosomes and the third chromosome-linked DHR96 gene has a negative effect on Cyp6a8 expression.