Life Sciences Commons^™

The Development Of A Dual-Tag Affinity Purification System And Its Application To Elucidate The Interacting Protein Network Surrounding The Human Telomere Binding Proteins Trf1, Trf2, And Pot1, Richard John Giannone

Doctoral Dissertations

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 …

Cis-Acting Determinants Of Coronavirus Genome Translation And Replication, Kortney Michele Gustin

Doctoral Dissertations

Coronaviruses are a family of positive-sense, single-stranded, 5’-capped and 3’- polyadenylated RNA viruses that replicate entirely in the cell cytoplasm. Replication of the viral genome requires translation to produce proteins used for RNA synthesis and virion assembly. The 5’- and 3’- untranslated regions of the coronavirus genome have been found to contain cis-acting elements that are required for replication of the genome and a defective interfering RNA. Presumably, both viral and cellular proteins interact with these elements and serve as trans-acting factors in genome translation and replication. Of interest is the functional significance of a 5’-proximal cis-acting 397-nucleotide …

Comparative Functional Genomics Of The Sabath Family Of Methyltransferases In Plants, Nan Zhao

Doctoral Dissertations

Known members of the plant SABATH family of methyltransferases (MTs) have important biological functions by methylating hormones, signaling molecules and other metabolites. This dissertation aims to systematically investigate the biochemical and biological functions and evolution of SABATH genes in plants. The genomes of rice and poplar have been fully sequenced, which provides unprecedented opportunities for cross-species comparison of the SABATH family. Using a comparative genomic approach, 41 and 33 SABATH genes were identified in rice and poplar, respectively. The expression of these genes in different tissue was analyzed using RT-PCR approach and some genes highly expressed in multiple tissues were …

Plant Improvement For Insect Resistance: Testing Of The Candidate Organism Beauveria Bassiana, Transgenic Tobacco Expressing Protease Inhibitors, And Rapid Screen Of Insect Resistance Genes In An Agroinfiltration Transient Expression System, Brian M. Leckie

Doctoral Dissertations

This study focused on three aspects of plant improvement for insect resistance including: testing of candidate organisms for their production of insecticidal proteins, testing of transgenic plants expressing insect resistance genes, and testing novel systems for the evaluation of insect resistance genes. In the initial part of this study, the candidate fungus Beauveria bassiana was tested for its production of insecticidal proteins through a series of insect bioassays containing fungal protein extracts. These extracts were shown to be orally toxic to Plutella xylostella (diamondback moth) and Spodoptera frugiperda (fall armyworm). Assays involving protease treatments significantly decreased mortality indicating the presence …

Evaluation Of Statistical Correlation And Validation Methods For Construction Of Gene Co-Expression Networks, Suman Duvvuru

Doctoral Dissertations

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 …

Plant Transcriptional Responses To Explosives As Revealed By Arabidopsis Thaliana Microarrays And Its Application In Phytoremediation And Phytosensing, Murali Malavalli Keerthi Narayana Raghavendra Rao

Doctoral Dissertations

This research focused on understanding genetic responses of plants to explosives, which is necessary to produce plants to detect and clean soil and water contaminated with toxic explosive compounds. The first study used microarray technology to reveal transcriptional changes in the model plant Arabidopsis thaliana exposed to the explosive compounds RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine; Royal Demolition Explosive or Research Department Explosive) and TNT (2,4,6-trinitrotoluene). This study yielded a list of genes up- and downregulated by explosive compounds, which can be potentially used for phytoremediation (remediation using plants) or phytosensing (detection using plants) of explosive compounds. The second study presented biotechnology tools to …

Buyer And Grower Perceptions Of Liner Quality And Associated Production Costs Of Nursery Liner Stock, Andrew H. Jeffers

Masters Theses

Liner production is a key segment in the nursery industry. Due to a lack of specific of quality standards by governing industry organizations as well as a lack of general consensus among growers of perceived liner quality, a conjoint analysis study was developed to determine buyer and grower preferences for nursery liner product features during point-of-purchase decisions. The study used a visual survey using six variables (first order lateral roots (FOLR), price, region of production, and height, canopy density and caliper uniformity) with varying levels yielding a 3 x 3 x 3 x 2 x 2 x 2 factorial design. …

Investigating The Oligomerization Of Vitronectin, Yacynth Ruwansara

Masters Theses

Vitronectin is a multi-functional glycoprotein that is present in the plasma and extra-cellular matrix of eukaryotes. It is capable of binding a wide variety of structurally different ligands, including plasminogen activators, plasminogen activator-inhibitors, proteases, cell surface receptors and components of the extra-cellular matrix. Vitronectin exists in two conformations – as a monomer in circulation, and as a multimer in the extracellular matrix. The pathway by which vitronectin undergoes the transition from monomer to multimer is not well characterized, but this laboratory has put forward evidence to suggest that the binding of vitronectin with plasminogen activator inhibitor type-1 (PAI-1), facilitates higher …

Evolution Of The Set Of Signal Transduction Proteins In 10 Species Of Shewanella, Harold Arthur Shanafield

Masters Theses

The recent completion of the sequencing of several species of the Shewanella genus provides a unique opportunity for comparative genomics studies. We chose the first 10 fully sequenced Shewanella genomes to investigate the evolution of signal transduction proteins (ST). ST is a universal and highly regulated system, and as a very well-studied system provides an excellent starting point for investigation. Furthermore, Shewanella have been shown to have a large number of two-component systems and diguanylate cyclases relative to their genome size. In this study we investigate the evolution of signal transduction across several Shewanella strains by utilizing a domainlevel approach …

Characterization Of The Metallohistin Cdna Agnt84 And Pteris Vittata Tissue Culture For Phytoremediation, Blake Lee Joyce

Masters Theses

Contamination of soils with toxic metals such as arsenic and cadmium has become a major environmental and human health risk. Phytoremediation provides a method to remove contaminants from soils that is not only economically viable but also environmentally sound. Metallohistins are proteins that have the capability to bind divalent metal ions such as Ni²⁺, Zn²⁺, Co²⁺, Cu²⁺ and Cd²⁺. In this study, a concatemer sequence was designed to try to increase the presence of metal-binding proteins in transgenic plants. Two methods to increase translational efficiency of the metallohistin protein were used: …

The Role Of Cytomegalovirus Viral Chemokines In Neutrophil Activation And Viral Dissemination, Mindy L. Miller-Kittrell

Doctoral Dissertations

CMV is the leading cause of both non-hereditary mental retardation and hearing loss and CMV infection following transplantation carries a serious risk for complications. The development of a CMV vaccine or better therapeutic treatment is desired but to develop these a more complete understanding of CMV pathogenesis is necessary. Sequence comparisons between attenuated and virulent strains of HCMV map major differences to a 15kb region (ULb’) containing the chemokine homolog, vCXCL-1. The vCXCL-1 protein of the Toledo (Tol) strain was previously shown to function in vitro as a CXC chemokine. Murine CMV (MCMV) also encodes a viral chemokine, MCK2. Prior …

Roles, Perceptions, And Control Of Infant Feeding Among Low-Income Fathers In East Tennessee, Kristen Erika Anderson

Masters Theses

INTRODUCTION: Introduction of solid foods before the recommended age of 4-6 months is a common practice in the United States, and appears to be especially prevalent among infants who are enrolled in the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC). Currently, little is known about how fathers influence early infant feeding decisions, outside of the decision to breast- or formulafeed. The purpose of this study was to explore how fathers perceive the role they play in feeding and caring for their infants. METHODS: Participants were 21 male-caregivers, who were fathers or partners of the mothers …

Tissue Culture And Recombinant Dna Technology: Developing Protocols For Potentially Higher Yielding Switchgrass Cultivars, Stephen Michael Foulk

Masters Theses

The purpose of this thesis project was to, firstly, establish and optimize protocols for high-throughput callus induction and plant regeneration for the new, higher yielding switchgrass (Panicum virgatum L.) cultivars NSL and SL93. Secondly, to subclone the complementary DNA (cDNA) of the anthocyanin transcription factor, C1, from pBECKS.red into pUC18 and pBSL15 for downstream use.

For the first part of this project the cultivars, NF/GA992, NF/GA993, NSL, and SL93, were tested for callus induction by plating whole dehusked caryopses on callus induction media (CIM) containing 8.5uM of the auxin dicamba. NF/GA992 responded best to the treatment with 44% of …

Effect Of First, Second And Third Chromosome On The Promoter Activity Of Cyp6a8 Gene Of Drosophila Melanogaster, Anirban Mukherjee

Masters Theses

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, 2^nd and 3^rd 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 2^nd and 3^rd chromosomes of ry⁵⁰⁶, respectively were used. The X, 2^nd and 3^rd chromosomes of these two transgenic lines were replaced with corresponding chromosomes from DDT-resistant 91-R …

Characterization Of Vitronectin And Plasminogen Activator Inhibitor Type 1: Insights Into Metal Binding, And Production Of Reagents To Facilitate Structural Studies, Cynthia Lee Brown

Masters Theses

Vitronectin is a multifunctional glycoprotein involved in regulation of such processes as hemostasis, cell migration, immunity, and tumor metastasis. Many of its functions require interaction with various binding partners. One such partner, plasminogen activator inhibitor-type 1 (PAI-1), belongs to the serine protease inhibitor (serpin) superfamily of proteins. PAI-1 helps regulate hemostasis by affecting blood clot breakdown, and helps regulate cell migration and tissue remodeling by affecting extracellular matrix digestion. Our laboratory is pursuing structural information on vitronectin and the complex formed between vitronectin and PAI-1. To this end, the work presented in this thesis focuses on two aims: characterizing the …

Novel Method For Recovery Of Escherichia Coli O157:H7 From Beef Surfaces, Matthew Adam Becher

Masters Theses

A study was conducted to compare the efficiency of detection of Escherichia coli O157:H7 EDL 933 on external beef trim by commercial recovery systems based upon of the Microbial-Vac System (M-Vac) and excision sampling. Escherichia coli O157:H7 EDL 933 was cultured in tryptic soy broth for 24 hr at 37°C and transferred for three consecutive days before diluting and spot inoculating onto a 684 cm² piece of beef trim (10 CFU/684 cm²). Beef trim was sampled with the M-Vac by passing the sampling head over the entire surface area in a vertical sampling pattern. The sampling head …

Impacts Of A 4-Lane Highway On The Spatial Ecology Of American Black Bears And The Effectiveness Of Wildlife Underpasses In Eastern North Carolina, Matthew Flanders Mccollister

Masters Theses

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, …

The Status Of Table Mountain Pine (Pinus Pungens) Stands On The Cherokee National Forest, Tennessee, Amy Louise Morgan

Masters Theses

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 …

“Intervention Strategies To Enhance The Safety Of Ready-To-Eat Meat Products By Plant Essential Oils, Chayapa Techathuvanan

Masters Theses

Components of plant essential oil (PEO) extracts are known to have antimicrobial properties. However, their antimicrobial efficacy in food systems is low due to their hydrophobic nature and association with other food components. Incorporation of PEO components into an appropriate carrier may offer a potential solution to improve their activity in food systems. This study was conducted to determine the effect of PEO components (thymol, eugenol, linalool, carvacrol, and cinnamaldehyde) incorporated into zein coating on inactivation of Listeria monocytogenes on a ready-to-eat meat model, frankfurters (hot dogs). Hot dogs were inoculated with 7 log CFU/sample and dipped into prepared PEO-zein …

An Experimental Study Of Juvenile Competition And Habitat Niche Partitioning Between A Native Lizard (Anolis Carolinensis) And An Introduced Congener (Anolis Sagrei) In The Southeastern United States, Justin W. Walguarnery

Doctoral Dissertations

Since its introduction to Florida, the brown anole, Anolis sagrei, has steadily expanded its range into that of its native congener in the southeastern United States, the green anole, A. carolinensis. Anolis sagrei achieves very high densities both in its native and invaded range and appears to impose population declines and shifts in the realized habitat niche of A. carolinensis. In order to investigate whether these effects arise prior to the adult age class in which they have previously been described, I studied the behavior of juvenile anoles at the individual, dyadic, and neighborhood levels. Contrary to some …

The Identification, Functional Characterization And Phylogeny Of The Nodulin-Like Anion Transporter (Nlat) Family In Plants, Eric Daniel Vincill

Doctoral Dissertations

A cDNA was isolated from soybean (Glycine max) nodules that encodes a putative transporter (GmN70). GmN70 is expressed predominantly in mature nitrogenfixing root nodules. By western-blot and immunocytochemical analyses, GmN70 was localized to the symbiosome membrane of infected root nodule cells, suggesting a transport role in symbiosis. To investigate its transport function, cRNA encoding GmN70 was expressed in Xenopus laevis oocytes, and two-electrode voltage clamp analysis was performed. Ooctyes expressing GmN70 showed outward currents that are carried by anions with a selectivity of nitrate > nitrite >> chloride. These currents showed little sensitivity to pH or the nature …

Distribution Of Mycobacterium Ulcerans In Aquatic Environments In Ghana, Heather Williamson

Doctoral Dissertations

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a necrotizing skin disease prevalent in 30 countries of West Africa and Australia. The disease begins as a painless nodule or papule that, if left untreated, can lead to ulceration that can cover much of the body. Though not usually fatal, morbidity is high, especially in rural areas where the disease is prevalent.

Epidemiological evidence has linked incidence of Buruli ulcer to slow moving or stagnant waters, but despite this, the mode of transmission is not known. Evidence for M. ulcerans in aquatic environments has relied on detection based upon PCR …

Forest Responses To Rising Atmospheric Co2: Causes And Consequences Of Increased Fine-Root Production In A Co2-Enriched Sweetgum Plantation, Colleen Marie Iversen

Doctoral Dissertations

Increased forest growth in response to rising atmospheric concentrations of CO₂ may mitigate a portion of fossil fuel emissions, especially if carbon is sequestered in longlived biomass or soil pools. Greater carbon uptake under elevated atmospheric [CO₂] in forested ecosystems may facilitate the production of small diameter (i.e. “fine”) roots used for nutrient acquisition. Increased fine-root production in forested ecosystems may affect soil carbon storage and nitrogen cycling because fine roots live and die in the span of a year. My dissertation research took advantage of a long-term, on-going Free-Air CO₂-Enrichment experiment in a sweetgum …

Phenotypic And Genetic Correlations Among Mating Traits In Three Species Of Nasonia (Hymenoptera: Pteromalidae, Jason E. Leonard

Doctoral Dissertations

Phenotypic correlations between traits are thought to reflect genetic correlations. However, traits related to a central function, like reproduction, need not all be genetically correlated. Identifying genetic correlations between behavioral traits can help identify the evolutionary relationship between different behaviors, as well as provide initial information on the number of genes involved in behavioral variation and the rates and direction of evolution. This body of work describes the phenotypic and genetic relationship between mating traits in the parasitoid wasp genus Nasonia. Chapters 2 and 3 describe behavioral differences among the three species of Nasonia. Two of the species, …

Characterization And Performance Improvement Of Chitosan Films As Affected By Preparation Method, Synthetic Polymers, And Blend Ratios, Jiajie Li

Doctoral Dissertations

Chitosan films prepared with addition of other polymers have been widely studied for their modified properties. In this dissertation, poly (ethylene oxide) (PEO) and poly (N-vinyl-2- pyrrolidone) (PVP) were blended with chitosan. The objectives of the study were (1) to investigate the the effects of film thickness, blend ratios, and preparation methods on the physical, and mechanical properties and functional performance of chitosan/PEO films, and (2) to compare characteristics and functional properties of chitosan/PVP and chitosan/PEO films.

The results demonstrated that regular cast chitosan/PEO films have altered properties than films produced from either polymer alone. Regardless of molecular weight, chitosan …

Studies Of The Di/Tripeptide Transporter In Saccharomyces Cerevisiae: The N-Terminal Cytoplasmic Domain Of Ptr2p Is Involved In Post-Translational Regulation, Steven Clinton Minkin Jr.

Doctoral Dissertations

Throughout nature cells use peptides as a source of nutrition. For microbes, an ability to utilize peptides is especially important in nitrogen-poor environments, as peptides can be catabolized for their use as a nitrogen source. The yeast Saccharomyces cerevisiae imports di/tripeptides from the environment using the peptide transporter Ptr2p. Cellular levels of Ptr2p are highest under poor-nitrogen conditions. Here we report that the addition of a rich nitrogen source to the growth medium results in a down-regulation of Ptr2p, wherein plasma membrane Ptr2p is ubiquitinated, endocytosed, and delivered to the vacuole for destruction. We report evidence that the N-terminal portion …

A Proteomic Investigation Of Phytophthora Species Using Mass Spectrometry And Reverse Genetics, Alon Savidor

Doctoral Dissertations

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 …

Quantum Mechanical/Molecular Mechanical Molecular Dynamics Simulations On Enzymes, Qin Xu

Doctoral Dissertations

The dynamic nature of proteins in solution is often an indispensable factor in biological function such as enzymatic catalysis. Complementary to the conventional structural analysis, computational simulations have the advantage to reflect the dynamic nature of proteins or enzymes. One of the computational simulation methods, the quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations, has been widely applied to the research in structural analysis, ligand-receptor binding and enzymatic catalysis.

In this dissertation, QM/MM MD simulations were applied to the studies on cytidine deaminase (CDA), yeast cytosine deaminase (yCD), and kumamolisin-As, as well as two protein lysine methyltransferases (PKMTs), DIM-5 and …

Community Invasibility: From Pattern To Process Across Multiple Spatial Scales In An Old-Field Ecosystem, Lara A. Souza

Doctoral Dissertations

Biological invasions threaten biodiversity, and understanding the factors that influence a community’s susceptibility to invasion informs both management of invasive species and conservation of biodiversity. The biotic resistance hypothesis postulates that communities with greater number of competitors, predators and/or pathogens will resist biological invasions. The underlying mechanism of biotic resistance, in the realm of competition, is that in species-rich communities harbor fewer open niches for introduced species to colonize therefore decreasing the probability of invasion. My dissertation research evaluated the role of native species diversity, as well as other biotic, abiotic and landscape factors shaping exotic species richness at multiple …

Distribution And Dynamics Of Pyrene-Degrading Mycobacteria In Freshwater Sediments Contaminated With Polycyclic Aromatic Hydrocarbons, Jennifer M. Debruyn

Doctoral Dissertations

Microbial biodegradation of polycyclic aromatic hydrocarbons (PAHs) is the primary means of attenuation of these toxic and carcinogenic compounds from contaminated soils and sediments. The documented toxicity and carcinogenicity of many PAHs demands remedial action for PAH-contaminated soils and sediments. This is especially important for historically contaminated sites, where higher molecular weight PAHs (HMW) are recalcitrant. Recently, fast-growing Mycobacteria have been identified that can degrade HMW PAHs, such as pyrene and benzo[a]pyrene. These bacteria have been isolated from a variety of geographical locations, indicating a cosmopolitan distribution.

This dissertation work was driven by the need for a better understanding of …