Doctoral Dissertations

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Browsing Doctoral Dissertations by Department "Biology"
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    A STUDY OF ANTICANCER AGENTS DERIVED FROM PLANTS UTILIZED IN TRADITIONAL CHINESE MEDICINE (TCM)
    (Middle Tennessee State University, 2018-03-27) Almosnid, Nadin ; Altman, Elliot ; Gao, Ying ; Altman, Elliot ; Gao, Ying ; Farone, Anthony ; Farone, Mary ; Wang, Chengshan ; Biology
    ABSTRACT
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    Biochemical and bioinformatics approach to the study of lipids and their biosynthetic pathways in Chromera velia and Vitrella brassicaformis
    (Middle Tennessee State University, 2014-07-11) Khadka, Manoj ; Leblond, Jeffrey ; Farone, Anthony ; Robertson, James ; Walck, Jeffrey ; Seipelt-Thiemann, Rebecca ; Biology
    Vitrella brassicaformis and Chromera velia are marine microalgae that photosynthetic plastids of red algal origin, are closely related to non-photosynthetic apicomplexan parasites and are classified as the phylum Chromerida and familes Vitrellaceae and Chromeraceae, respectively. To dates, only one study has been performed on the galactolipid of Chromera velia and few other studies have been based on the ultrastructural and phylogenetic studies in Chromerida. We used a combination of mass spectrometry techniques and RNA-seq data to elucidate the sterol and galactolipid composition of the Chromerida, and to identify their biosynthetic genes, respectively. The identified genes were further utilized to perform the gene phylogenies of the Chromerida with red algae, dinoflagellates and apicomplexans.
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    Characterization of the Human Inflammatory Response to Gardnerella vaginalis
    (Middle Tennessee State University, 2014-11-12) Vick, Eric ; Farone, Anthony ; Farone, Mary ; Elrod-Erickson, Matthew ; Seipelt-Thiemann, Rebecca ; Kline, Paul ; Biology
    Gardnerella vaginalis is a Gram-positive bacterium associated with BV (BV), pelvic inflammatory disease, and preterm birth. BV is the most prevalent vaginal infection in women, characterized by the absence of normal Lactobacilli and overgrowth of G. vaginalis and other bacterial species. This study tested the hypothesis that G. vaginalis induces an inflammatory response in the human cell line, THP-1. The objectives of the study were to 1) determine whether different strains of G. vaginalis cause proinflammatory cytokinesproduction in THP-1 cells, 2) characterize intracellular pathways by which these cytokines are produced, and 3) determine molecular mechanisms involved in death of cells treated with strains of G. vaginalis.
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    Development and Characterization of a Salt-Tolerant Luciferase to Investigate Genetic Regulation in Haloferax volcanii
    (Middle Tennessee State University, 2018-05-14) Davis, Christopher Ray ; Biology
    Haloarchaea, especially Haloferax volcanii, are of particular interest for genetics research due to the wide array of tools and procedures available. However, few reporters exist for Archaea that provide a measurable output in vivo. Haloarchaeal proteins evolved characteristics that promote proper folding and function at high salt concentrations, but many mesohalic proteins lack these characteristics. However, proteins with salt-stabilizing mutations can lead to proper function in haloarchaea. Using laboratory directed evolution, a salt tolerant luciferase was developed and applied for use in genetic studies involving a previously described inducible promoter and genetic translation inhibitor in H. volcanii. A luciferase assay offers several advantages over other reporter systems: capturing data is done in real time due to a high temporal fidelity; data can be captured continuously using solid or liquid media with proper equipment; and the reporter gene itself is innocuous to the organism. Lastly, the individual mutations of the newly developed luciferase were characterized in order to identify changes in the protein leading to improved haline environment luminescence.
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    Evaluation and Development of Aquatic Toxicity Test Methodologies
    (Middle Tennessee State University, 2015-03-26) Beasley, Amy ; Otter, Ryan ; Bailey, Frank ; Belanger, Scott ; Van Genderen, Eric ; Jessen, Jason ; Biology
    International chemical management legislation such as the European Union's REACH (Registration, Evaluation, Authorisation, and Restriction of Chemicals) has increased the need for more and better toxicity data while recognizing the 3R's of animal use (reduction, replacement, and refinement). To address this need, the Organisation for Economic Cooperation and Development's (OECD) Fish Toxicity Framework guidance document recommended improvements to test guidelines and integrated testing strategies. This study contributes to recommendations outlined in the Fish Toxicity Framework and similar guidelines.
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    EVOLUTIONARY MAINTENANCE OF GEOGRAPHIC VARIATION FOR FLOWER COLOR IN LEAVENWORTHIA STYLOSA
    (Middle Tennessee State University, 2017-06-23) Fernando, Mahaguruge Thilina R. ; Walck, Jeffrey ; Herlihy, Chris ; Morris, Ashley ; Howard, Steve ; Leblod, Jeffrey ; Biology
    Flower color polymorphism is a striking feature of some angiosperm species, especially when it is geographically structured. Geographic patterns of flower color can be maintained by either non-adaptive processes, like genetic drift, or by natural selection, acting directly or indirectly. Leavenworthia stylosa, an endemic to the cedar glades of middle Tennessee, occurs mostly in monomorphic populations of yellow or white flowered morphs. The overall objective of my study was to understand why most of the L. stylosa populations are monomorphic and what maintains the geographic pattern of flower color variation of the species. I studied the pollinator assemblages, flower color preferences, and their constancy in foraging and seed predation across the species range. To check the differences in abiotic factors in different sites, I studied the soil chemistry, water holding capacity and water loss in white and yellow sites. Additionally, I conducted a series of reciprocal transplant experiments at different life history stages and evaluated the performance of the early life history stages of the two morphs across a water-related stress gradient. In both white and yellow sites, pollinators favored the white morph over the yellow morph across the species range. Seed predation was lower on the white morph compared to the yellow morph. Bombylius showed exclusive visits to the white morph in white sites favoring the white morph and preventing the yellow morph from increasing in frequency. In yellow sites, the fitness-enhancing pollinators preferred yellow morph and fitness-reducing seed predators showed no preference. There was no difference between white and yellow sites in soil chemistry. But yellow sites had higher water holding capacity and higher water loss rate than white sites. During the early life history stages the white morph was more successful over yellow morph while during late life history stages yellow morph was more successful over white morph. The yellow morph showed limited evidence for local adaptation in flower number and fruit number survival through reproductive stage. Stress experiments showed conflicting results. Thus, it is likely that the maintenance of geographic variation for flower colors of L. stylosa is influenced by multiple selective agents including pollinators, seed predators and abiotic conditions.
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    GENOME ANNOTATION AND ROLE OF NON-CODING RNAS IN DISEASE RESISTANCE, GROWTH AND MUSCLE QUALITY TRAITS IN RAINBOW TROUT
    (Middle Tennessee State University, 2017-06-22) Paneru, Bam Dev ; Salem, Mohamed ; Farone, Anthony ; Farone, Mary ; Bergemann, Sarah ; Jessen, Jason ; Biology
    In Rainbow trout, effort to annotate the genome reference is ongoing. While recently published trout genome has discovered large number of protein coding genes, many protein coding genes appear to be missing. In addition, non-coding RNAs, which occupy vast majority of the transcribed portion of genome, are not investigated. In the present study, we have sequenced and assembled the transcriptomes of lncRNA (long non-coding RNA) and mRNA to facilitate gene discovery, and have investigated the role of non-coding RNAs in disease resistance, muscle atrophy, growth and muscle quality traits.
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    GENOME-WIDE ANALYSES OF GENES AFFECTING GROWTH, MUSCLE ACCRETION AND FILLET QUALITY TRAITS IN RAINBOW TROUT
    (Middle Tennessee State University, 2019) Ali, Ali Reda Eid ; Salem, Mohamed ; Farone, Anthony ; Jessen, Jason ; Farone, Mary ; Altman, Elliot ; Biology
    Growth performance is one of the most economic important traits for the aquaculture industry. In addition, fillet quality attributes are among the primary determinants of consumer acceptability. To study the genetic architecture of these traits, phenotypic characterization of whole body weight (WBW), muscle yield, fat content, shear force, moisture, protein content, and whiteness were measured in ~500 fish representing 98 families from a growth-selected line. RNA-Seq was used to sequence the muscle transcriptome of different families exhibiting divergent phenotypes for each trait. In total, 240 and 1,280 differentially expressed (DE) protein-coding genes and long noncoding RNAs (lncRNAs), respectively, were identified in fish families exhibiting contrasting phenotypes. Expression of many DE lncRNAs (n = 229) was positively correlated with overlapping, neighboring or distantly located protein-coding genes (n = 1,030), resulting in 3,392 interactions. Three DE antisense lncRNAs were co-expressed with sense genes known to impact muscle quality traits. A 50K SNP chip has been developed and used across four genome-wide association (GWA) studies to identify quantitative trait loci (QTL) explaining variations in fish growth and fillet quality attributes. In the first study, QTL explaining up to 28.40% of the additive genetic variance for muscle yield were identified; particularly on chromosomes 14 and 16. In the second study, 247 QTL associated with bodyweight gain were identified. Most SNPs affecting muscle yield and bodyweight gain exist in genes that act as major regulators of developmental processes. In the third study, the additive genetic variance for fillet firmness and protein content was investigated where RYR3 harbored most SNPs affecting the two traits. In the fourth study, sixty-one common SNPs on chromosomes 19 and 29 affecting the muscle fat and moisture content were identified. SNP-harboring genes, in the common QTL, were mainly involved in lipid metabolic process and cytoskeleton remodeling. Presence of common QTL associated with multiple phenotypes suggests common mechanisms underlying those phenotypes in fish. The present work identified DE genes and genetic markers explaining variations in growth and fillet quality phenotypes in selectively bred trout populations. Such markers could be used for marker-assisted and genomic selection in breeding programs of rainbow trout.
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    Improving biological detoxification of furfural and acetate in lignocellulosic hydrolysates using metabolic engineering
    (Middle Tennessee State University, 2018-04-13) Crigler, Jacob ; Altman, Elliot ; Farone, Mary ; Kline, Paul ; Robertson, James ; Salem, Mohamed ; Biology
    Cellulosic ethanol biofuel, made from plant waste products or perennial energy crops like switchgrass, offers many advantages over corn starch-derived ethanol, including less land competition and a lower carbon footprint. However, the efficiency of conversion is currently lower and the cost higher due to the recalcitrance of lignocellulosic biomass. A chemical and/or physical pretreatment step is required to overcome recalcitrance, and common pretreatment methods (e.g., acid, steam, and/or depressurization) release microbial inhibitors, including furfural and acetate, into the hydrolysate, lowering the yield of ethanol via fermentation. Furfural is generated via sugar dehydration, and acetate derives from acetylated xylan in hemicellulose. Utilizing a detoxifying strain is one strategy to overcome the inhibitor dilemma. Biological detoxification potentially allows lower process costs compared to chemical or enzymatic detoxification or alternative pretreatment methods aimed at reducing inhibitor generation. One drawback is time. Thus increasing the rate of furfural and acetate detoxification is desirable. While most microbial species can catabolize acetate, most do not possess the furfural catabolic pathway. A novel Pseudomonas putida isolate ALS1267, with a growth rate of 0.25/h in 10 mM minimal furfural medium, was characterized. The genome was sequenced and the furfural pathway cloned into wild-type P. putida KT2400, which cannot metabolize furfural, creating a novel strain with an improved growth rate of 0.34/h in 10 mM minimal furfural medium. Genomic library screening was used to find targets for engineering increased acetate consumption in Escherichia coli. Sixteen plasmid clones were generated, with growth rate increases of 42.6 to 76.9 percent in 10 g/l minimal acetate medium, which is a highly inhibitory concentration for this strain. Clones included an uncharacterized oxidoreductase, transporters and other outer membrane proteins, carbon scavengers, and stress defense mechanisms. Genomic mutants with improved acetate consumption were also generated during selection, with mutations in the gluconeogenesis gene pck promoter, the '5 UTR of the poorly characterized cold-shock gene ynaE, and the global regulator of secondary carbon sources CRP. The second major drawback to biological detoxification is consumption of the sugars to be used to produce ethanol by the detoxifying strain. Elimination of glucose metabolism in E. coli was studied by characterizing fast-growing revertants in strains engineered to be glucose minus. All of the revertants either altered or overproduced the N-acetylglucosamine phosphotransferase system. Deletion of the N-acetylglucosamine transporter stabilized the glucose minus phenotype and prevented the occurrence of fast-growing revertants.
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    Stress Induced Nuclear Granules form within the Nucleus in Response to Environmental Stress in C. elegans
    (Middle Tennessee State University, 2017-04-07) Sampuda, Katherine Marie ; Boyd, Lynn ; Elrod-Erickson, Matthew ; Nelson, David ; Jessen, Jason ; Tansey, William ; Biology
    Protein misfolding caused by environmental stress can be damaging to a cell and it has been linked to many human diseases. Cells contend with misfolded proteins by employing an arsenal of protein quality control mechanisms (PQC). One PQC mechanism that aids in destroying misfolded and damaged proteins is the ubiquitin proteasome system (UPS). PQC mechanisms have been observed in the cytosol, endoplasmic reticulum, and mitochondria. However, little information is known about the PQC mechanisms in the nucleus. The nucleus has been suggested to be a site of protein degradation based on ubiquitin and proteasome localization in the nucleus under normal conditions. The research in this dissertation aims to understand how nuclear UPS is involved in environmental stress in C. elegans. Results from this study have shown that ubiquitin, proteasome, and TIA-1/TIAL RNA binding protein homolog (TIAR-2) localize into distinct structures termed stress induced nuclear granules (SINGs) in response to osmotic stress, oxidative stress, and starvation. SINGs were found to be enriched in K48 polyubiquitin chains and their formation was inhibited by proteasome inhibitors, which indicates SINGs being sites of protein degradation. Knockdown of ubiquitin conjugating enzymes ubc-18, ubc-20, and ubc-22 decreased the appearance of SINGs during stress, which indicates they are part of the pathway leading to SING formation. Similar results were seen with knockdown of the ubiquitin ligase chn-1 and nuclear import factors ima-1, ran-1, and smo-1. The formation of SINGs can be inhibited by a brief exposure to heat shock, which was found to be HSF-1 dependent. This finding suggests that increased chaperone expression is able to prevent SINGs. In addition, this experiment indicates that the accumulation of misfolded proteins is required to induce SING formation. Embryos containing SINGs are unable to complete cell division suggesting that SINGs interfere with cell cycle progression. These results suggest a model in which the UPS is triggered in response to misfolded proteins in the nucleus.
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    Taking the Heat: The Effect of Growth Temperature on Lipids of the Diatoms
    (Middle Tennessee State University, 2014-08-25) Dodson, Vernon Joshua ; Leblond, Jeffrey ; Farone, Anthony ; Farone, Mary ; Seipelt-Thiemann, Rebecca ; Robertson, Brian ; Biology
    Diatoms are one of the largest groups of primary producers in the oceans, and because of their environmental and increasing economic importance, more information is being revealed about their lipid biochemistry. Mass spectrometry techniques were used to examine the lipids of diatoms, with particular focus on how growth temperature would affect different lipid classes. Galactolipid-associated fatty acid composition and regiochemistry was examined in a selection of diatoms. Two centric diatoms, Skeletonema marinoi and Thalassiosira weissflogii, and one pennate diatom, Phaeodactylum tricornutum, contained primarily C20/C16 (sn-1/sn-2) and C18/C16 forms of the galactolipids. The other pennate diatoms, Haslea ostrearia and Navicula perminuta, contained primarily C18/C16 or C18/C18 forms of these lipids. This shows a split in galactolipid profiles that does not correspond with the phylogeny of these organisms and their plastids.
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    THE EFFECTS OF THE CIS- AND TRANS-GNETIN H ISOLATED FROM PAEONIA SUFFRUTICOSA AND A SYNTHETIC AURONE, (Z)-2-((5-(HYDROXYLMETHYL) FURAN-2-YL) METHYLENE) BENZOFURAN-3(2H)-ONE ON NF-κB AND MAPK PATHWAYS IN LPS-STIMULATED MACROPHAGES
    (Middle Tennessee State University, 2017-03-21) Park, Hyo Sim ; Farone, Anthony ; Farone, Mary ; Altman, Elliot ; Dunlap, Norma ; Newsome, Anthony ; Biology
    Inflammation is central to many disease processes such as autoimmunity and chronic inflammatory diseases, yet there are relatively few classes of compounds used as anti-inflammatory drugs. Nonsteroidal anti-inflammatory drugs (NSAIDS) and steroids have been used for many years and recently, biotherapeutic modifiers such as monoclonal antibodies and fusion proteins were developed to treat chronic inflammation, however, many patients experience serious side effects, and are thus reserved for devastating inflammatory diseases such as rheumatoid arthritis and ankylosing spondylitis. Recently, more attention has been given to herbal ingredients that have been used to treat inflammatory diseases. This is due to safety and efficacy of the herbal medicine, lower risk of side effects, lower costs, and potential use as adjunct treatments to Western medicines. In this study, cis- and trans-gnetin H and a synthetic aurone, (Z)-2-((5-(hydroxymethyl) furan-2-yl) methylene) benzofuran-3(2H)-one (aurone 1) were evaluated for their mechanism of action in suppressing the intracellular proinflammatory pathways that regulate inflammatory cytokines in activated macrophages. All three compounds were found to suppress the cytokines, TNF-α, IL-1β, and IL-8, through inhibition of IKK-β which is a key regulator of the NF-κB pathway. The effects of cis- and trans-gnetin H on MAPK pathway was also investigated in THP-1 cells and showed both gnetin H compounds were able to suppress SAPK/JNK but ERK 1/2 were suppressed only by cis-gnetin H. Neither gnetin H were able to suppress p38 phosphorylation. Further studies using a murine cell line confirmed the effects of aurone 1 in suppressing the NF-κB pathway by concentration-dependent suppression of iNOS as well as the production of NO which are regulated by the NF-κB pathway. The investigation of the effects of aurone 1 on MAPK mediators from human and murine macrophages suggested selective inhibition NF-κB signaling without affecting the MAPK pathway. Together, these findings suggest the gnetin H compounds and aurone 1 are anti-inflammatory compounds with therapeutic potential for the possible treatment of chronic inflammatory diseases.
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    Using a collection of nonfunctional missense mutants in the β-galactosidase and catechol 2,3-dioxygenase enzymes to better understand the complexity of protein folding
    (Middle Tennessee State University, 2017-07-16) Cole, Ashley Elliott ; Altman, Dr. Elliot ; Farone, Dr. Mary ; Farone, Dr. Anthony ; Kline, Dr. Paul ; MacDougall, Dr. Preston ; Biology
    Missense mutants can have devastating effects on proteins and usually act by causing perturbations in the folding of the protein that affects their final tertiary or quaternary forms. In order to better understand how missense mutants affect protein structure, a set of randomly generated nonfunctional missense mutants were isolated in two well-characterized enzymes, β-galactosidase and catechol 2,3-dioxygenase. This collection of missense mutants yielded important information regarding the key structural elements within correctly folded proteins and the ability of protein predictive tools to predict the effects of missense mutants. Many of the missense mutants were found to be salt-correctible and the rescuability of the missense mutants by various salts correlated with the Hofmeister series of ions that affect protein stability. In an additional study the promiscuous or broad-acting sumA glyV tRNA Gly3(GAU/C) missense suppressor that can recognize GAU or GAC aspartic acid codons and insert a glycine amino acid instead of aspartic acid was characterized.