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 …

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 …

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 …

Spermatogonia Stem Cell Dynamics Following Hormonal Induction, Ischemic Disturbance In Vivo Or Proliferation Under In Vitro Culture In Pre- And Postpubertal Bulls, Gustavo M. Schuenemann

Doctoral Dissertations

The overall aim of the studies described herein was to evaluate bovine spermatogonial cell dynamics under various conditions. Results from these experiments will provide the basis for potential production of offspring following spermatogonial stem cell transfer. Experiment 1 evaluated gonadotropin administration effects at initiation of inhibin passive immunization in Jersey bull calves on testicular morphology and development. Primary treatments consisted of control (KLH) or immunization (INH) plus a combination of saline, FSH, or GnRH. Administration of FSH at the time of initial immunization against inhibin significantly increased number of germ cells (92.2 ± 9 x 106 cells) compared to INH-Saline …

Analysis And Application Of Key Modeling Concepts Utilized In Predictive Microbiology For Food Processing, Darryl G. Black

Doctoral Dissertations

The use of modeling techniques for safety and risk prediction in the food supply is a common practice. Factors affecting microbial heat resistance include those inherent to the organism, environmental conditions and the intrinsic properties of the heating menstruum. Varying physiological states of microorganisms could affect the measured response and add uncertainty to results from predictive models. Inactivation tests were performed using Escherichia coli strain K12 and E. coli O157:H7 for various growth conditions: traditionally or statically grown cells, chemostat‐grown cells, and chemostatgrown cells with buffered feed media. Heating menstruum was non‐buffered 0.1% peptone, 0.1 M phosphate buffer (pH 7.0), …

Contribution Of Water And Energetics Of Ligand Binding In The Catalytic Mechanism Of R67 Dihydrofolate Reductase, Shaileja Chopra

Doctoral Dissertations

R67 dihydrofolate reductase (DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate (DHF) to produce tetrahydrofolate (THF). The enzyme is a homotetramer and its 222 symmetry allows for binding of both ligands to a single active site pore. A productive ternary complex is formed by the binding of one molecule of DHF and NADPH and inter-ligand cooperativity has been suggested to be essential for binding and catalysis. To gain further insight into the thermodynamics involved in the ground state and the transition state, temperature dependent studies on DHF binding and catalysis were performed. It was observed that …

Application And Optimization Of Bioluminescence Resonance Energy Transfer (Bret) For Real Time Detection Of Protein-Protein Interactions In Transgenic Arabidopsis As Well As Structure-Based Functional Studies On The Active Site Of Coelenterazine-Dependent Luciferase From Renilla And Its Improvement By Protein Engineering, Jongchan Woo

Doctoral Dissertations

Bioluminescence resonance energy transfer (BRET) is a biological phenomenon in some marine organisms such as Renilla reniformis and Aequorea victoria. In BRET, resonance energy from decarboxylation of coelenterazine, a substrate of Renilla luciferase (RLUC), is transferred to its acceptor such as green fluorescent protein (GFP) or yellow fluorescent protein (YFP), dependent on a distance of around 5 nm between the energy donor (RLUC) and its acceptor. The activation of the energy acceptor results in a spectral change in luminescence emission. The BRET system allows investigation of in vivo protein-protein interactions in real time. This was demonstrated with two heterodimeric …

Vertically Aligned Carbon Nanofiber Arrays As A Platform For Gene Delivery And Expression Analysis In Mammalian Cells, David George James Mann

Doctoral Dissertations

Vertically aligned carbon nanofiber (VACNF) arrays have been developed as a novel tool for direct physical introduction and expression of DNA in mammalian cells (termed impalefection). This study describes the optimization of impalefection, the quantification of immobilized DNA on VACNFs, and the application of VACNFs in analysing gene expression in mammalian cells. Mechanical, chemical and biological parameters were optimized for impalefection. Alterations in a majority of the parameters resulted in no significant difference in impalefection efficiency, including nanofiber composition, DNA precipitation, cell confluency, cell concentration and sodium butyrate. The optimal DNA concentration ranged between 100 nanograms and 1 microgram, and …

Enterobacter Sakazakii Growth Profile And Tolerance To Chlorine Sanitizers, Diana Carolina Naar Cifuentes

Doctoral Dissertations

Enterobacter sakazakii is considered an emerging opportunistic pathogen associated with sporadic life-threatening bacterial infections in neonates linked to the compsumption of contaminated infant formula [Stoll et al., 2004]. In 2001 a neonate fatal infection associated with the presence of E. sakazakii in infant formula occurred in the neonatal intensive care unit (NICU) of the University of Tennessee Hospital [Himelright et al., 2002], as a result of this outbreak, the hospital made several policy changes and requested the Food Safety Center of Excellence of University of Tennessee to analyze the growth pattern of this microorganism at the conditions maintained in the …

Experiments On Multiple Factors Affecting Pinaceae Invasions On Isla Victoria, Nahuel Huapi National Park, Argentina, Martin Andres Nuñez

Doctoral Dissertations

Invasive species are one of the greatest threats to global biodiversity. Therefore, to conserve biodiversity and manage exotic species effectively, it is crucial to determine the factors that regulate biological invasions. Historically, the study of invasions has focused on the dynamics and characteristics of successful invasive species. While studying successful invaders has enhanced our understanding of biological invasions, studying the failures can also illuminate the factors that limit invasions. To further understand the factors limiting the spread of exotic species, I studied invasion dynamics of several species in the family Pinaceae on Isla Victoria, Argentina. Approximately 80 years ago, thousands …

Facilitating Private Forestland Management: Relating Landowners’ Experience Of Their Forestland And Their Conceptualization Of Forest Management To Their Management Behavior, Miriam L.E. Steiner Davis

Doctoral Dissertations

Privately owned forestland accounts for the majority of forested land in the US and provides numerous ecological, economic and social benefits to its owners and society at large. However, numerous issues ranging from fragmentation and parcelization, to pressure from the forest products industry, to increasing land values for development and real estate interests threaten to “unravel” the forest landscape. Active management of forestland is seen as one way to combat such threats. Active management of private forestland has been linked to numerous factors such as private forest landowners’ (PFLs’) general education level and familiarity with forest management, their goals, interests, …

Systematics Of Gratiola (Plantaginaceae), Larry D. Estes

Doctoral Dissertations

Gratiola (Plantaginaceae tribe Gratioleae) is a genus of ca. 33 wetland-adapted herbaceous species found mostly in temperate or tropical-montane regions of the Americas, Eurasia, and Australasia. The only worldwide taxonomic treatment of Gratiola was published more than 160 years ago and only a few representatives of the genus have been included in published molecular phylogenetic studies. The overall aim of this investigation was to provide a modern systematic study of Gratiola by determining the phylogenetic placement of Gratiola within the Gratioleae using chloroplast DNA sequence data (Chapter 2); examining the phylogenetic relationships, morphological character evolution, and biogeographical patterns within Gratiola …

Biological Invasions, Global Climate Change And Species Distribution Models: An Investigation Of Species-Climate Relationships Across Space And Time, Matthew C. Fitzpatrick

Doctoral Dissertations

Species distribution models are increasingly being applied to questions in ecology, biogeography and evolution, and in particular to the problem of predicting the potential spread of invasive species and the potential impacts of climatic change on biodiversity. However, despite their broad application, several conceptual limitations still preclude the use of species distribution models in many theoretical and practical applications. Chief among these is the assumption that climate alone determines the geographic ranges of species, as opposed to biotic interactions and dispersal limitations, and that such species-climate relationships remain largely unchanged across space and time. In this context, I explore the …

Wildfire Risk Assessment And Community Wildfire Protection In The Chilhowee Mountain Area Of Blount County, East Tennessee, Piyarat Chimchome

Doctoral Dissertations

The growing Wildland Urban Interface community in the Chilhowee Mountain area of Blount County, Tennessee, like many other forested areas in the mountains and hills of East Tennessee, was identified by officials of the Tennessee Division of Forestry as being a high risk area for wildfire. The purpose of this research was to help wildfire managers better understand how to facilitate wildfire protection and mitigation in the Chilhowee Mountain area. A mail survey was sent to the 474 property owners in the communities of Campers Paradise, Happy Valley, and Top of the World. The survey contained 33 questions related to …

Prevalence Of Class 1 Integrons And Antibiotic Resistance Patterns In Bacteria Of Swine And Chicken In The Us And Thailand, Sumalee Liamthong

Doctoral Dissertations

Studies were conducted to investigate the prevalence and potential for transfer of class 1 integrons and antimicrobial resistance in bacteria of broiler chickens and swine from the US and Thailand. Antibiograms were characterized and integron sequences were detected using standard methods. To determine if transfer of integrons occurred between bacterial species the location of the integrons (plasmid versus chromosome) was determined, and when integron-positive E. coli and Salmonella isolates possessed identical amplicon patterns, PCR products were sequenced to determine homology. Class 1 integrons were detected in 1,732 of 3,824 isolates from broiler chickens and 1,782 of 4,253 isolates from swine. …