Biotechnology Commons^™

Flavonol Glucosylation: A Structural Investigation Of The Flavonol Specific 3-O Glucosyltransferase Cp3gt, Aaron S. Birchfield

Electronic Theses and Dissertations

Flavonoid glycosyltransferases (GTs), enzymes integral to plant ecological responses and human pharmacology, necessitate rigorous structural elucidation to decipher their mechanistic function and substrate specificity, particularly given their role in the biotransformation of diverse pharmacological agents and natural products. This investigation delved into a comprehensive exploration of the flavonol 3-O GT from Citrus paradisi (Cp3GT), scrutinizing the impact of a c-terminal c-myc/6x histidine tag on its enzymatic activity and substrate specificity, and successfully achieving its purification to apparent homogeneity. This established a strong foundation for potential future crystallographic and other structure/function analyses. Through the strategic implementation of site-directed mutagenesis, a thrombin …

The Role Of Foxd1 In Clear Cell Renal Cell Carcinoma, Kyle H. Bond

Electronic Theses and Dissertations

Renal cell carcinoma (RCC) is the 8^th most common cancer in the United States, with the clear cell variant (ccRCC) being the most prevalent. Over 14,000 people die every year to RCC, with rates continuing to increase with an aging general population. Patients suffering from metastatic RCC (mRCC) have extremely poor prognoses, with a 5-year survival of only 11.2%. Current treatment options include resection of primary lesions, tyrosine kinase inhibition (Sunitinib, Pazopanib), mTOR inhibition (Temsirolimus, Everolimus), and immune checkpoint inhibition (Nivolumab, Atezolizumab). Recent attention has been drawn to inhibition of transcription factors like HIF2α (Belzutifan). There is a need …

Expression And Functional Characterization Of Avocado Dgat1 And Pdat1 In Arabidopsis And Camelina, Josphat Kiunga

Electronic Theses and Dissertations

The study is aimed to determine the role of avocado DGAT1 and PDAT1 in seed oil synthesis. Triacylglycerol (TAG) has a nutritional and industrial value and is essential for plant growth. DGAT1 and PDAT1 catalyze the final step of TAG Assembly. We hypothesized that both PaPDAT1 and PaDGAT1, although predominantly expressed in non-seed tissues, could contribute to oil accumulation in seeds. Agrobacterium transformants with PaPDAT1 and PaDGAT1 cloned in pCAMBIA were generated to test this. Subsequently, the Agrobacterium-mediated transformation of Arabidopsis mutant lines and camelina was carried out by floral dipping. The T₁ camelina seeds …

Functional Role Of Ppal And Potential For Moss In Industrial Applications., Susana Perez Martinez

Electronic Theses and Dissertations

This dissertation is an examination and characterization of the functional roles of PPAL. PROTEIN PRENYLTRANSFERASE ALPHA SUBUNIT-LIKE (PPAL) is a recently discovered gene. PPAL homologs are present in all plants and many animals, where its function is largely unknown. It is possible that PPAL could participate in prenylation processes since it shares similarity to the α subunits of known prenylation enzymes. Prenylation is a post-translational modification of proteins that involves the addition of a lipid moiety to proteins to facilitate membrane targeting and association and promote protein-protein interactions. Prenylation has important roles in plant growth and development, including …

Real-Time Monitoring Of Cell Death Progress Using Capacitance Spectroscopy, Suyang Wu

Electronic Theses and Dissertations

Biologics, including the monoclonal antibody (mAb), has experienced rapid development in the last decade. However, the price of biologics is often prohibitively high because of the low process efficiency. Delaying the inevitable cell death improves the productivity of upstream bioprocessing, whose success relies on monitoring the cell death onset that indicates the timing for preventive actions.

This study proposes to develop a real-time monitoring model that quantifies the dying cell percentage in lab-scale bioreactors using capacitance spectroscopy. The capacitance spectroscopy contains cell death-related information due to various physical properties changes during the cell death process, e.g., cytoplasmic conductivity change. The …

Understanding The Adhesion Mechanism In Mycelium-Assisted Wood Bonding, Wenjing Sun

Electronic Theses and Dissertations

The increasing environmental awareness has led to an increased interest in developing more sustainable materials as alternatives to petroleum-derived products. Among different nature-based products, fungal-mycelium-based bio-composites have gained considerable attention in various applications. Multiple materials with different densities and structures and potential applications can be fabricated by inoculating filamentous white-rot fungi in lignocellulosic materials and other substrates. Different from lower-density as-grown foam-like mycelium composites, higher-density mycelium-lignocellulosic panels have the potential to replace commercial particleboard and fiberboard bonded by petroleum-based resins. This kind of composite can be produced by directly adding heat and pressure to the low-density foams or by assembling …

Characterization Of Sabp2-Interacting Proteins (Sip) 428: An Nad+-Dependent Deacetylase Enzyme In Plant Abiotic Stress Signaling, Oviavo Nohoesu

Electronic Theses and Dissertations

Abiotic stress leads to a change in the water content of plants. Salinity and osmotic stress affect both the morphology and physiology of plants. Plants have therefore responded to these environmental changes by adapting and tolerating them. The SABP2-interacting proteins (SIP) 428-silenced RNAi transgenic tobacco lines were subjected to various abiotic stresses (salinity, osmotic, and drought). The effect of SIP428-silencing on the tobacco plants subjected to these abiotic stresses was monitored. The results from the root growth data show that the sip428-silenced lines exhibit enhanced tolerance to the stressors compared to the wild-type plants. Interestingly, results of the relative chlorophyll …

Molecular Dynamics Simulations Provide Insight Into Stability Of Hyperthermophilic Endoglucanases, Logan E. Sheffield

Electronic Theses and Dissertations

Endoglucanases play a key role in the industrial production of bioethanol, but the most efficient method requires the utilization of high temperatures and is currently limited by the thermostability of endoglucanases. For this reason, it would be beneficial to discover more high-efficiency, thermostable enzymes to utilize in the hydrolytic process. In this study molecular dynamics simulations were performed on structurally similar endoglucanases with varying levels of thermostability to gain insight on what factors contribute to thermostability in endoglucanases. RMSD, RMSF, PCA, hydrogen bonding and salt bridges were analyzed. Finally, protein energy networks were constructed from nonbonded interaction potentials and analysis …

Preclinincal Development Of Avaren-Fc: A Novel Lectin-Fc Fusion Protein Targeting Cancer-Associated High-Mannose Glycans., Matthew William Dent

Electronic Theses and Dissertations

This dissertation explores the anticancer activity of Avaren-Fc (AvFc), a novel lectin-Fc fusion protein or “lectibody” targeting cancer and virus-associated high-mannose glycans. Previously, we have shown that AvFc recognizes a broad selection of established cancer cell lines from a wide array of tissue types, can potently induce antibody-dependent cell-mediated cytotoxicity (ADCC) against them, and exhibits anti-cancer activity in vivo. However, the exact mechanism of action remains elusive. We hypothesized that the primary mechanism of action is through Fc-mediated effector functions, and the purpose of this dissertation is to explore this question through the use of Fc variants that either …

Assessing The Utility Of Environmental Dna Techniques To Monitor White Shrimp (Litopenaeus Setiferus) Abundance On The Georgia Coast, Raven Hurt

Electronic Theses and Dissertations

Environmental DNA (eDNA) surveys have developed over the last decade from once being a novel tool to now acting as an effective technology often used in complement to traditional capture surveys for assessing the distribution of organisms in freshwater and marine environments. However, many uncertainties on how to properly develop, operate, and analyze eDNA based techniques still hinder this technology effectiveness in the field. The white shrimp, Litopenaeus setiferus, is a common, commercially and recreationally important species in the United States, with landings exceeding $254 million in 2020. L. setiferus is also used as a key indicator species to changes …

Zika Virus-Induced Lysis Of Cervical Cancer Cells, Harini Krishnapura

Electronic Theses and Dissertations

Cervical cancer is the fourth most frequent cancer in women with an estimated 570,000 new cases globally in 2018. Treatment of advanced cervical cancer is often unsuccessful leading to high cancer-related mortality rates, especially in under-resourced countries. Recently, a possible role for the cell surface glycoprotein CD24 in host cell specificity of Zika virus was reported. As an extension of this work, Zika viruses have been proposed as oncolytic therapy for the treatment of neuroblastoma and other CD24 positive tumors. To determine the permissiveness of cervical cancer cells to Zika virus infection and its association with CD24, we assessed cytopathic …

Development Of A Lectin-Fc Fusion Protein With Antiviral And Anti-Cancer Activity., Matthew William Dent

Electronic Theses and Dissertations

This thesis describes the development of a novel lectin-Fc fusion protein and its antiviral and anti-cancer activity. The molecule, Avaren-Fc (AvFc), is a fusion of a variant of the actinomycete lectin actinohivin (Avaren) and the Fc region of human IgG1, and is selective for the terminal α1,2-mannose residues found at the ends of high-mannose-type glycans that can be found on the surface of certain heavily glycosylated viruses and cancer cells. Here, AvFc was found to be able to neutralize simian immunodeficiency virus as well as Hepatitis C virus with nanomolar IC₅₀ values. Furthermore, AvFc recognizes a number of cell …

Biochemical Characterization Of Rv2633c From Mycobacterium Tuberculosis And The Effects Of Mutagenesis On Iron Binding, Kyle Strickland

Electronic Theses and Dissertations

Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that is the causative agent of the disease Tuberculosis (TB). TB kills an estimated 1.8 million people annually and roughly one third of the world's population carries Mtb in a dormant state. Drug resistant Mtb strains are on the rise, thus a new method of combating this disease is paramount. Mtb survival inside of macrophages requires overcoming various stressors such as; iron restriction, reactive oxygen species, and hypoxic conditions. Mtb employs the use of catalases, nitric oxide reductase, superoxide dismutase, and siderophores to aid in survival. These functions have also been found in …

Selenium Vs. Sulfur: Investigating The Substrate Specificity Of A Selenocysteine Lyase, Michael Johnstone

Electronic Theses and Dissertations

Selenium is a vital micronutrient in many organisms. While traces are required for survival, excess amounts are toxic; thus, selenium can be regarded as a biological "double-edged sword". Selenium is chemically similar to the essential element sulfur, but curiously, evolution has selected the former over the latter for a subset of oxidoreductases. Enzymes involved in sulfur metabolism are less discriminate in terms of preventing selenium incorporation; however, its specific incorporation into selenoproteins reveals a highly discriminate process that is not completely understood. In this work, we add knowledge to the mechanism for selenium-over-sulfur specificity in hopes of further understanding the …

Bone Morphogenetic Protein 7 Inhibits Pyroptotic Cell Death In Vascular Smooth Muscle Cells Of Atherosclerotic Apolipoprotein E -/- Mice, Kaley Garner

Electronic Theses and Dissertations

Atherosclerosis (ATH) is an inflammation-mediated disease in which cell death underlies the formation of lesions along the intima layer of vascular walls resulting in vessel narrowing, decreased blood flow, and increased risk of lesion rupture leading to myocardial infarction and stroke. The current study was undertaken to investigate whether inflammation in ATH can induce pyroptosis in vascular smooth muscle cells (SMC's). We therefore hypothesized that pyroptosis occurs and is inhibited by bone morphogenetic protein 7 (BMP7). We examined SMC pyroptosis at acute (D5) and midstage (D28) following disturbed flow-induced hemodynamic injury to the vascular wall using our partial left carotid …

Metabolic Effects Of 17a-Estradiol Are Growth Hormone Independent And Sex Specific, Silvana Sidhom

Electronic Theses and Dissertations

Aging is a major risk factor for metabolic syndromes and type two diabetes. With growing elderly populations worldwide and increasing incidence of age-related diseases there is a great need to develop pharmacological interventions that would delay aging and protect from age-related diseases. 17-alpha estradiol (17α-E2) is an epimer of the primary female sex hormone estradiol and has been shown to extend lifespan and downregulate markers of age-related metabolic dysfunction in male mice. Because 17α-E2 does not induce feminization in males it holds potential as a novel therapeutic in humans for age-related metabolic dysfunction. Importantly, we have previously shown that 17α-E2 …

Atp Induced Molecular Disassembly Of Cytolethal Distending Toxin's B/C Heterodimer, George Huhn

Electronic Theses and Dissertations

Cytolethal distending toxin (CDT) is a virulence factor produced by many Gram-negative bacteria, including Haemophilus ducreyi. This fastidious pathogen is the causative agent of genital cancroid. CDT is a heterotrimeric toxin with an AB2 structure consisting of a cell-binding "B" domain (CdtA + CdtC) and a catalytic "A" domain (CdtB) that has DNase activity. This toxin assembles in the bacterial periplasm that lacks ATP and is secreted into the extracellular environment. After cell binding, CDT is internalized by endocytosis and travels through the endosomes and Golgi before arriving in the endoplasmic reticulum (ER). CdtA is lost from the holotoxin before …

Amelioration Of Alzheimer's Disease Pathology In 12-Month-Old Happ(Sweind) Transgenic Mice After Treatment With A Cysteine Rich Whey Supplement, Immunocal®, Srivalli Puttagunta

Electronic Theses and Dissertations

Deficits in Reelin expression and signaling play a pathogenic role in Alzheimer’s disease (AD). Thus, strategies aimed at correcting Reelin deficits may provide a novel therapeutic approach to treating AD. The cysteine-rich, whey protein supplement, Immunocal®, has recently been shown to rescue Reelin expression in a mouse model of Schizophrenia. Given that Reelin-expressing neurons of the entorhinal cortex region are a highly vulnerable population of cells that are lost early in AD, we examined the effects of Immunocal® in the hippocampal-entorhinal cortex formation in a mouse model of AD. Glutathione levels and Reelin expression in the hippocampal-entorhinal cortex formation (entorhinal …

Characterization Of Vps41 And Its Role In The Regulated Secretory Pathway, Christian Henry Burns

Electronic Theses and Dissertations

Insulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific membrane proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determines this molecular composition, remain poorly understood. VPS41, a component of the endolysosomal tethering HOPS complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine/neuronal granules. We now find that a stable pool of VPS41 exists outside of …

Determining Differential Effects Of Interleukin-2 On Innate And Adaptive Immune Cells In Lymphoid Organs And The Gastrointestinal Tract, Ayushi Singh

Electronic Theses and Dissertations

Interleukin-2 (IL-2) is a pleiotropic cytokine demonstrated to be effective in treating cancer. However, the clinical use of IL-2 can be associated with severe side effects including gastrointestinal toxicity (GT). Similar GT symptoms are observed in inflammatory diseases such as CD (CD). Interestingly mounting evidence indicates a role for IL-2 in CD, but the underlying mechanisms are unknown. Indeed, studies on the in-vivo activities of IL-2 have mostly focused on secondary lymphoid organs and immune cells associated with them. Very few studies have addressed how IL-2 signals impact populations of immune cells in the gut. Here, we aim to identify …

The Comparison Of Effects Of Synthetic And Natural Arachidin-3 On Rotavirus Infected Cells, Rebekah Napier-Jameson

Electronic Theses and Dissertations

Rotavirus (RV) causes severe, life-threatening diarrhea, in infants, young children and immunocompromised adults. There are several effective vaccines for young children, however they are strain specific and are not protective against many RV strains in developing countries. Therefore, it is important to investigate anti-RV therapeutic agents. Our laboratory has shown arachidin-1 (A1) and arachadin-3 (A3) significantly inhibit RV replication in two cell lines, however the molecular mechanism(s) of action are not known. A synthetic molecule of A3 (sA3) has been produced, but its’ antiviral effects have not been examined. Our hypothesis is that sA3 produces the same effects on RV-infected …

The Effect Of Compound L19 On Human Colorectal Cells (Dld-1), Sepideh Mohammadhosseinpour

Electronic Theses and Dissertations

Colorectal cancer is the third most common type of cancer in the world and the second leading cause of death among humans. Extracts of water soluble compounds from the roots and leaves of Rumex crispus were screened for compounds that induced apoptosis in DLD-1 cells. A compound referred to as L19 was isolated using Accelerated Solvent Extraction (ASE) followed by High Performance Liquid Chromatography (HPLC). Gas chromatography coupled with mass spectrometry was used to identify the L19 as a tetrahydrofuran with a molecular weight of 72.11 g/mole. Each HPLC fraction resulted in 94.4% purity. In the present research, specific genes …

Development Of A Bacteriophage-Based Portable Biosensor For The Detection Of Shiga-Toxin Producing Escherichia Coli (Stec) Strains In Food And Environmental Matrices, Irwin A. Quintela

Electronic Theses and Dissertations

A fast and reliable on-site foodborne pathogens screening can reduce the incidence of foodborne illnesses, hospitalizations and economic loss. It can also circumvent conventional laboratory-based tests with minimal sample treatments and shorter turnaround time. Rapid detection of biological hazards has been largely dependent on immunological agents (ie antibodies). Antibodies are expensive to manufacture and experience cross-reactivity, instability with shorter shelf life. Our aim was to improve the screening process of Shiga-toxin producing Escherichia coli (STEC) strains in food and environmental matrices by developing a novel, inexpensive handheld bacteriophage-based amperometric biosensor that can directly detect live STEC cells.This biosensor development began …

Role Of Single Nucleotide Polymorphisms (Snps) In Ptpn2/22 And Mycobacterium Avium Subspecies Paratuberculosis (Map) In Rheumatoid Arthritis And Crohn's Disease, Robert Sharp

Electronic Theses and Dissertations

Both genetic pre-disposition and potential environmental triggers are shared between Rheumatoid arthritis (RA) and Crohn's disease (CD). We hypothesized that single nucleotide polymorphisms (SNPs) in the negative T-cell regulators Protein Tyrosine Phosphatase Nonreceptor type 2 and 22 (PTPN2/22) lead to a dysregulated immune response as seen in RA and CD. To test the hypothesis, peripheral leukocytes samples from 204 consented subjects were TaqMan genotyped for 9 SNPs in PTPN2/22. The SNPs effect on PTPN2/22 and IFN-y expression was determined using RT-PCR. Blood samples were analyzed for the Mycobacterium avium subspecies paratuberculosis (MAP) IS900 gene by nPCR. T-cell proliferation and response …

Role Of Lipid Peroxide Derived Dicarboxylic Acids In Atherosclerotic Calcification, Aladdin Riad

Electronic Theses and Dissertations

Cardiovascular diseases, including atherosclerosis, are the leading cause of death in the United States. Atherosclerotic lesions are formed by deposition of lipids in the intima of arteries. Upon exposure to oxidative stresses, low-density lipoprotein (LDL) is converted to highly atherogenic oxidized LDL (ox-LDL) particles, contributing to disease development and progression. Advanced disease stages may result in calcification of lesions. This calcification process is important, as it has been shown to be associated with stable plaques that are less prone to rupture. Calcification is present in lipid rich domains of lesions, however neither the composition of the mineralized calcium deposits nor …

Deciphering The Role Of Adrenergic Hormones In Embryonic Cardiac Calcium Signaling And Metabolism, Jessica Peoples

Electronic Theses and Dissertations

The adrenergic hormones norepinephrine (NE) and epinephrine (EPI) are critical regulators of mammalian cardiovascular physiology. NE and EPI mediate stress responses to enhance cardiovascular function, however dysregulation of adrenergic signaling leads to heart failure, congenital heart malformations, and sudden cardiac death. Adrenergic hormone-expressing cells were found in the early embryonic heart, and NE has been determined essential for embryonic cardiac development. Despite extensive work in adults, the regulatory roles and adrenergic targets of these hormones during embryonic cardiac development have not yet been fully determined. Prior transcriptomic studies from our lab showed that expression of signal transduction and metabolic genes …

Pg-Vtv Biogenesis Requires Atp To Facilitate Phosphorylation Of Syntaxin 17, Anika Saxena

Electronic Theses and Dissertations

The uptake of cytotoxic free fatty acids (FFA) and their conversion to physiologically expedient triglycerides (TAG) which are later on assimilated to very low density lipoproteins (VLDL) is of utmost value among the multifarious tasks performed by the liver. Inflated concentration of VLDL in the blood stream directly correlates with the reinforcement of atherosclerosis. VLDL is synthesized in the hepatic endoplasmic reticulum (ER) and transported to the Golgi where it encounters several alterations. It is then enclosed in distinct post-Golgi VLDL transport vesicles (PG-VTVs) and released into the blood. The data generated in our lab has proved the requirement of …

Characterization Of Neural Cells Derived From Reelin-Deficient Schizophrenic Patient Ips Cells, Nicole Roberts

Electronic Theses and Dissertations

Reelin is a large, extracellular glycoprotein that binds to several membrane receptors on neural stem cells (HNSCs), neural progenitor cells (NPCs), and neuroblasts of mammals to direct their migration. Previously, our lab established the presence of Reelin increased migration of wild-type fetal-derived HNSC's, both in vitro and in vivo. In addition, we demonstrated that Reelin protein treatment also increases the formation of radial glia via Notch-1 signaling, in vitro. Radial glia are precursors to NPCs, as well as a scaffold for neuroblast migration during cortical lamination. Reelin has long been associated with Schizophrenia (SZ). Because post-mortem brains are limited to …

Knowledge And Attitudes Regarding The Recent Zika Outbreak Among A Sample Of South Florida Residents, Mohamed Elakkari

Electronic Theses and Dissertations

South Florida has had the largest number of U.S. Zika infection cases during the recent outbreak. This study aimed at assessing South Floridians' basic knowledge, perceptions of the seriousness and susceptibility to Zika infection, their information sources and needs, as well as their attitudes towards the protective measures proposed by the CDC. We also wanted to assess whether any of those factors have affected the frequency of participants' undertaking of the protective behaviors. To this end, we designed an online questionnaire and surveyed the responses of five hundred South Floridians (Age=18-78 years). We found significant gaps in participants' knowledge about …

Embryonic Stem Cell-Derived Exosomes Inhibit Doxorubicin-Induced Pyroptosis In Cell Culture Models, Zahra Tavakoli Dargani

Electronic Theses and Dissertations

Doxorubicin (Dox) is a potent chemotherapeutic drug used for the treatment of various cancers. Unfortunately, its use is limited as Dox induces adverse cardiotoxicity (DIC) and muscle toxicity (DIMT), which are mediated through oxidative stress, ER stress, and inflammation. However, it remains unknown whether Dox induces an inflammation mediated cell death, called “pyroptosis”. The current study is designed to determine whether Dox induces pyroptosis in cardiac and muscle cell culture models. Moreover, the protective effects of embryonic stem cell-derived exosomes (ES-Exos) in inhibiting pyroptosis will also be determined. For this purpose, we designed two different cell culture models using H9c2 …