Molecular Biology Commons^™

Investigating The Effects Of Ph On Alphaviral E3-E2 Glycoprotein Association, Organization, And Cellular Tropism, Jason Michael Sequra

Open Access Dissertations

In alphaviruses the role of E3 is required in protecting the fusion peptide region of E1 during intracellular transport. Throughout viral processing, the association of E2 and E3 is required for the successful trafficking and incorporation of E1 into the mature virion. This E3-E2 association has been observed to extend to mature virions in the solved structure for the envelope of Semliki Forest virus (SFV) and supported by the solved structure for the entire Venezuelan equine encephalitis virion (VEEV) with exclusive contacts being made between E3-E2. Immunization with monoclonal antibodies against VEEV E3 provided protection for mice challenged by lethal …

Recombinant Listeria Adhesion Protein Expressing Probiotics Protect Against Listeria Monocytogenes Infection In Animal Models, Valerie E. Ryan

Open Access Theses

Listeria monocytogenes (Lm) is a foodborne pathogen, found ubiquitously in nature, and has a high morbidity rate among immunocompromised individuals, the elderly, and especially pregnant women and their fetuses resulting in abortion, stillbirth, and neonatal infection. There are currently no preventative medical interventions against Lm infection. The Listeria adhesion protein (LAP) is present in both pathogenic and non-pathogenic Listeria (i.e., L. innocua) and has shown to interact with host epithelial proteins causing tight junction dysregulation aiding in pathogen attachment and paracellular translocation across the host intestinal epithelium. Our lab has demonstrated that recombinant probiotics, Lactobacillus casei (LbcWT) expressing LAP …

The Elasmobranch-Microbe Relationship: Trimethylamine N-Oxide Synthesis, Urea Hydrolysis, And Microbe-Osmolyte Interactions In The Atlantic Stingray, Dasyatis Sabina, Kaitlin Kelly Doucette

Master's Theses

The elasmobranch osmoregulatory strategy is predicated on the accumulation of nitrogenous compounds, primarily urea and trimethylamine n¬-oxide (TMAO). Despite the abundance of these plasma osmolytes, it is notable that elasmobranchs appear to lack urease and TMA oxidase (Tmase), enzymes that scavenge urea-nitrogen and synthesize TMAO, respectively. However, urease and Tmase are found in many species of bacteria. Therefore, I hypothesized that intestinal bacteria are responsible for urease and Tmase activity in elasmobranchs. Absent dietary nitrogen sources, I evaluated the effects of reduced intestinal microbiota on osmoregulation in Atlantic stingray (Dasyatis sabina) in vivo. D. sabina were given …

Effects Of Acute Heat And Oxidative Stress On The Hepatic Expression Of Orexin And Its Related Receptors, Stephanie Khaldi

Graduate Theses and Dissertations

It is widely known that orexin A and B peptides as well as their receptors are expressed in the hypothalamus and distributed throughout the central nervous system, but there have been few studies regarding its presences in other parts of the body. There is now evidence that orexin (ORX) and its receptors (ORXR1/2) are present in the avian liver; however, their regulation under different environmental conditions is still unknown. In the current study, we sought to determine the effects of heat and oxidative stress using hydrogen peroxide (H2O2) and 4-hydroxynonenal (4-HNE) on the hepatic expression of ORX and ORXR1/2 in …

Membrane Biophysics Of Listeria Monocytogenes: Analysis Of An Alternate Pathway Of Branched-Chain Fatty Acid Biosynthesis And Elasticity Of Fatty Acid Utilization, Laura E. Kuczek

Theses and Dissertations

The membrane homeostasis of the psychrotolerant foodborne pathogen Listeria monocytogenes involves maintaining fluidity in a range of different conditions by incorporation of specific branched-chain fatty acids (BCFAs). BCFAs are synthesized through a well-characterized pathway from branched-chain amino acids into short branched-chain acyl-CoAs followed by elongation by the FAS II system. Branched-chain alpha-keto acid dehydrogenase (Bkd) is a major player in this pathway. MOR401 is a transposon insertion mutant deficient in Bkd and has decreased membrane BFCAs. Low levels of BCFAs in L. monocytogenes are linked to diminished growth, less resistance to antimicrobials, and a severe reduction in virulence. Rescued growth …

Specific Binding Affinity Of The Non-Catalytic Domain Of Eukaryotic Like Type Ib Topoisomerase Of Vaccinia Virus, Benjamin R. Reed

Dissertations, Theses, and Capstone Projects

Topoisomerases are ubiquitous proteins that alter supercoiling in double stranded DNA (dsDNA) during transcription and replication and. vaccinia and the closely related poxvirus variola virus, at 314 amino acids in length, encode the smallest of the type I topoisomerases(TopIB). TopIB is a two domain protein that recognizes the sequence 5’-T/CCCTT, cleaves at the 3’-end and relaxes supercoiling through rotation. The C-terminal domain (CTD) alone contains the catalytic activity and specificity. Deletion of the N-terminal domain results in a greatly reduced rate of relaxation and rapid dissociation. Biochemical data suggests that the N-terminal domain (NTD) is important for pre-cleavage binding and …

Molecular Analysis Of Ftsz-Ring Assembly In E. Coli Cytokinesis, Kuo-Hsiang Huang

Dissertations, Theses, and Capstone Projects

An essential first step in bacterial division is the assembly of a cytokinetic ring (Z-ring) formed by the tubulin-like FtsZ at midcell. The highly conserved core domain of FtsZ has been reported to mediate assembly of FtsZ polymers in vivo and in vitro. Species-specific differences in the FtsZ C-terminal domain such as the FtsZ CTV region and interactions with several modulatory proteins such as ZapC and ZapD, restricted to certain bacterial classes, also serve as key determinants of FtsZ protofilament bundling. Here, we characterize (i) the roles of the FtsZ CTV region in mediating both longitudinal and lateral interactions …

Development And Performance Evaluation Of An Antibody-Based Technology For Detection Of E. Coli O157 In Meat Samples And Its Potential Evolution Using Antibody Engineering, Yadira Tejeda Saldaña

Electronic Thesis and Dissertation Repository

Escherichia coli O157 is a persistent pathogen linked to food and waterborne infectious outbreaks with severe health consequences such as hemorrhagic colitis and hemolyticuremic syndrome (HUS). Because it is considered one of the major pathogens that contributes to the global burden of foodborne disease, its early detection within the food chain is an important milestone towards reducing foodborne diseases and economic losses due to contaminated food. Herein, the development and validation of a lateral flow immunoassay (LFIA) point-of-care (POC) device is described. Application of the LFIA test kit was focused on detection of E. coli O157 in raw meat products …

Iron Acquisition Strategies Employed By Staphylococcus Lugdunensis, Jeremy R. Brozyna

Electronic Thesis and Dissertation Repository

Iron is crucial for many cellular processes including DNA synthesis and respiration. The majority of iron in mammals is in heme within hemoproteins, inside cells, or transported through circulation by the glycoprotein transferrin, which constitutes the greatest iron source in serum. Limiting iron availability is an important facet of nutritional immunity to help prevent infection.

Staphylococcus lugdunensis is a human skin commensal and opportunistic pathogen capable of causing a variety of infections, including particularly aggressive endocarditis. It is an emerging pathogen with elevated virulence compared to other species of coagulase-negative staphylococci. The versatility of S. lugdunensis to infect multiple niches …

Dynamic Host-Pathogen Interactions Result In Fungal Epitope Unmasking, Alex Hopke

Electronic Theses and Dissertations

Molecular camouflage is used by a diverse set of pathogens to disguise their identity and avoid recognition by protective host receptors. The opportunistic fungal pathogen Candida albicans is a good example, as it masks the inflammatory component β-glucan in its cell wall to evade detection by the immune receptor Dectin-1. Interestingly, it has been seen that β-glucan becomes unmasked during infection in vivo, though the underlying mechanisms remained unclear. Exposure levels of this epitope may be important, as Dectin-1 mediates protection from some strains of C. albicans and alterations in the organization and composition of the Candida cell wall …

Discovery And Characterization Of A New Group Of Is10 Insertion Sequences, Rachel Marie Kinzelman

Theses and Dissertations

Insertion sequences (ISs) are small mobile genetic elements that can have significant impact on the genotype and phenotype of a host organism. Previous work in this laboratory revealed an insertion sequence that disrupted the luxA gene in Vibrio harveyi strain BCB451, knocking out light production. Phylogenetic analysis of this insertion sequence, dubbed IS451, reveals that it is in the IS10 family, but represents a novel variant that is only 79% identical to other known IS10 sequences. Twelve copies of IS451 were isolated from a genomic library and sequenced, and were found to be essentially identical, but located in dispersed chromosomal …

Lateral Replacement Of The Lux Operon In A Vibrio Isolated From The Intestine Of A Coral Reef Fish, Melissa Lee Whyte

Theses and Dissertations

In a screening of bioluminescent bacteria isolated from the intestines of coral reef fish, two strains (designated D6 and M1) were identified that have a luxA gene sequence significantly different from those of other Vibrio species. Phylogenetic analysis of several housekeeping genes, as well as toxR, shows that D6 and M1 branch within a bioluminescent clade (designated the “D1 group,” isolated at the same time and place as D6 and M1) that is a close sister group to Vibrio harveyi. However, whereas the luxA genes of the D1 group are >98% identical to V. harveyi luxA, the luxA genes of …

Characterization Of Histone Mutants Associated With Mitotic Defects In Saccharomyces Cerevisiae, Md Riajul Hossain

Graduate Theses and Dissertations

Nucleosomes, the basic unit and the building blocks of chromatin have an essential role in the tight packaging of DNA into higher order chromatin. Work from our lab and others have provided information on the contributions of different histone proteins and specific domains within the nucleosome made to create the centromeric chromatin structure required for normal chromosome segregation during mitosis. The DNA entry/exit site is a particular region of the nucleosome where histone H2A, H3 and H4 form critical interactions that appear to be essential for the association of Sgo1, a tension sensing protein that monitors kinetochore-microtubule attachment. In our …

Control Of Proteolysis During The Caulobacter Cell Cycle, Joanne Lau

Doctoral Dissertations

Intracellular protein destruction is a carefully coordinated and timed regulatory mechanism that cells utilize to modulate growth, adaptation to environmental cues, and survival. In Caulobacter crescentus, a bacterium known for studies of bacterial cell division cycle, the response regulator CpdR couples phosphorylation events with the AAA+ protease ClpXP to provide punctuated degradation of crucial substrates involved in cell cycle regulation. CpdR functions like an adaptor to alter substrate choice by ClpXP, however it remains unclear how CpdR influences its multiple targets. In this thesis, we show that, unlike canonical ClpXP adaptors, CpdR alone does not strongly bind its substrate. …

Novel Advancements For Improving Sprout Safety, Kyle S. Landry

Doctoral Dissertations

All varieties of bean sprouts (mung bean, alfalfa, broccoli, and radish) are classified as a “super-food” and are common staples for health conscious consumers. Along with the proposed health benefits, there is also an inherent risk of foodborne illness. When sprouts are cooked, there is little risk of illness. The purpose of this dissertation was to explore novel techniques to minimize or prevent the incidence of foodborne illness associated with the consumption of sprouts. Three areas were investigated: 1) the use of a biocontrol organism, 2) the use of a novel spontaneous carvacrol nanoemulsion, and 3) the influence of the …

Role Of Stemloop D In Terminally Deleted Coxsackievirus B3 Replication, Lee K. Jaramillo

Theses & Dissertations

Coxsackievirus B3 (CVB3) is an enterovirus with no known form of latency. However, assays designed to detect enteroviral RNA have shown that CVB3 RNA can persist for weeks beyond the acute infection both naturally and experimentally. Our previous work with coxsackievirus revealed an inhibited version of enteroviral replication where the progeny virus, termed terminally deleted (TD) virus, was missing a maximum of 49 nucleotides from the beginning of the 5’ non-translated region (NTR). The largest terminally deleted virus, TD50, effaced stem a, stemloop b, and stemloop c from the secondary structure, the cloverleaf. We hypothesized that further deletion beyond those …

The Selective Survival Of Escherichia Coli In Freshwater Beach Sand, Natalie Ann Rumball

Theses and Dissertations

The quantification of Escherichia coli or E. coli is the most common method used to detect recent fecal pollution in recreational water, as this species is known for its high abundance in fecal matter and assumed host-associated nature. However, it has been determined that some strains are capable of long-term survival and potential propagation in non-host environments, such as the beach sand. These long-term environmental survivors are host-independent and are not associated with the same health risks as those E. coli from recent fecal pollution. However, they have been shown to impact how water quality is perceived as they are …

Affinity Cryo-Electron Microscopy: Methods Development And Applications, Guimei Yu

Open Access Dissertations

Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies. Although less concentrated and smaller amounts of samples are required for single particle cryo-EM compared to X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques holds great promise to tackle these challenging samples by combining the sample purification and freezing on TEM grids steps in cryo-EM grid preparation into a single step, revolutionize the grid preparation of cryo-EM, and extend single particle cryo-EM to a routine structural biology tool to characterize structures …

Role Of Protein Phosphatase Reg2-Glc7 In The Regulation Of The Yeast Stress Response Kinase, Snf1, Marcin Maziarz

Theses and Dissertations

Kinases of the AMP-activated protein kinase (AMPK) family are conserved in eukaryotes and play central roles in responses to reduced energy availability. AMPK, nicknamed the “fuel gauge” of the cell, monitors cellular energy status via the ratio of AMP to ATP nucleotides. AMPK restores energy homeostasis by reducing energy “spending” and increasing energy “income”. Correspondingly, defects in AMPK signaling have been implicated in diseases including type II diabetes, obesity, and cancer.

In yeast, the AMPK homolog is Snf1 protein kinase. Glucose is the preferred carbon/energy source of yeast, and thus limitation for glucose similarly activates Snf1. Snf1 activation requires phosphorylation …

Oxidative Stress Response In Archaea: Elucidation Of Oxidant Sensing And Tolerance Mechanisms In Methanosarcina Acetivorans, Matthew Edward Jennings

Graduate Theses and Dissertations

Methanogens are archaea possessing a conserved metabolic pathway which produces methane. Many of the enzymes in the methanogenesis pathway are Fe-S proteins, meaning methanogens are sensitive to conditions which disrupt Fe-S clusters. Molecular oxygen is capable of disrupting Fe-S clusters through oxidation of the iron atoms. Furthermore, reduced iron can facilitate the production of reactive oxygen species (ROS), meaning methanogens must possess antioxidant mechanisms. Detection and eradication of ROS is important for all cells, due to the potentially fatal consequences of unchecked oxidation. This dissertation presents two separate projects investigating mechanisms the model methanogen Methanosarcina acetivorans possess for dealing with …

The Ccaat-Binding Factor Dependent Regulation Of The Oxidative Stress Response In Candida Albicans, Ananya Chakravarti

Graduate Theses and Dissertations

The success of Candida albicans as an opportunistic human pathogen has been attributed to several factors, including the ability to survive in limiting iron environments and the ability to evade the respiratory burst of human macrophages and neutrophils. The goal of this research is to elucidate the role of the CCAAT-binding factor in the oxidative stress response of Candida albicans. Prior whole genome microarray studies performed in our lab compared the gene expression of a wild type Candida albicans strain versus a hap5Δ strain under iron-limiting growth conditions. Among the differentially regulated genes, CTA1, encoding catalase, had a four-fold higher …

Engineering A Mutation In The Heparin Binding Pocket Of The Human Fibroblast Growth Factor, Roshni Patel

Chemistry & Biochemistry Undergraduate Honors Theses

Fibroblast growth factors (FGFs) are family of proteins that belong to a group of growth factors that are found in mammals and play an important role in angiogenesis, differentiation, organogenesis, and tissue repair. In summary, their main functionality is involved in cell division and proliferation. Because FGFs plays such a vital role in cell proliferation, they are mainly involved in the process of wound healing and injuries. FGF binds to its ligand, heparin—a heavily sulfated glycosaminoglycan. The binding of heparin to FGF occurs through electrostatic interactions, specifically between the negatively charged sulfate groups on heparin and positively charged residues such …

Protective Effects Of Pigmentation In Pseudomonas Aeruginosa: Insights On Pyomelanin Production And Inhibition By Ntbc, Laura Marie Ketelboeter

Theses and Dissertations

Pyomelanin is an extracellular, reddish-brown pigment produced by several environmental and pathogenic bacteria and fungi. It is derived from the phenylalanine/tyrosine catabolism pathway and is produced when homogentisate (HGA) is secreted from the cell, auto-oxidized, and self-polymerized. Point mutations or chromosomal deletions in hmgA, which encodes homogentisate 1,2-dioxygenase, result in the accumulation of HGA and subsequent pyomelanin production. My work showed that the pyomelanogenic Pseudomonas aeruginosa clinical isolate PA1111 had a chromosomal deletion of hmgA, while a second pyomelanogenic clinical isolate, DKN343, had a loss of function mutation in HmgA. The 4-hydroxyphenylpyruvate dioxygenase (Hpd) inhibiting compound 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) has been …

Modulation Of Cell Death Signaling And Cell Proliferation By The Interaction Of Homoserine Lactones And Paraoxonase 2., Aaron Mackallan Neely

Electronic Theses and Dissertations

Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule that functions to facilitate bacteria-bacteria communication. C12 has also been reported to affect many aspects of human host cell physiology, including evoking cell death in various types of cells. However, the signaling pathway(s) leading to C12-triggerred cell death remains unclear. To clarify cell death signaling induced by C12, we examined mouse embryonic fibroblasts (MEFs) deficient in one or more caspases. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in cells, probably through the direct induction of mitochondrial membrane permeabilization. Previous studies indicate that …

Alternative Pathway For Provision Of Acyl Coa Precursors For Fatty Acid Biosynthesis: Purification And Kinetic Characterization Of Phosphotransbutyrylase And Butyrate Kinase From Listeria Monocytogenes, Sirisha Sirobhushanam

Theses and Dissertations

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a disease characterized by gastroenteritis, meningitis, spontaneous miscarriages and high mortality rate among infected individuals. L. monocytogenes is a major concern in the food industry, due to its ability to grow at refrigeration temperatures and the zero tolerance policy of the FDA, resulting in expensive food recalls. Growth at low temperatures is aided in part by the high membrane content of branched-chain fatty acids (BCFAs) which imparts greater fluidity to the membrane. Mutants of L. monocytogenes impaired in BCFA biosynthesis display diminished growth at normal and low temperatures, exhibit lower tolerance …

Investigation Of Respiratory Syncytial Virus Structural Determinants And Exploitation Of The Host Ubiquitin System, Jillian Nicole Whelan

USF Tampa Graduate Theses and Dissertations

Respiratory syncytial virus (RSV) is a globally circulating, non-segmented, negative sense (NNS) RNA virus that causes severe lower respiratory infections. This study explored several avenues to ultimately expand upon our understanding of RSV pathogenesis at the protein level. Evaluation of RSV intrinsic protein disorder increased the relatively limited description of the RSV structure-function relationship. Global proteomics analysis provided direction for further hypothesis-driven investigation of host pathways altered by RSV infection, specifically the interaction between the RSV NS2 protein and the host ubiquitin system. NS2 primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. The goal …

Structural And Functional Studies Of The Heptose Modifying Enzymes That Play A Role In Campylobacter Jejuni Virulence., Heba Soliman Barnawi

Electronic Thesis and Dissertation Repository

Campylobacter jejuni is a major cause of gastroenteritis in humans. The capsule of some species contains unique modified heptoses. Heptose modification was elucidated for C. jejuni NCTC11168 and 18-176, and novel epimerases and reductases essential for the heptose modification were identified. We hypothesized that heptose modifying enzymes in C. jejuni have specific catalytic residues that allow for substrate and product specificity. Substrate synthesis, structural modeling, point mutations, and enzymatic analysis have been applied to map the active sites. Putative catalytic residues showed substrate and/or product specificity. The epimerases structures were solved by crystallography done by our collaborator. We also hypothesized …

Functional Consequences Of Ama1-Ron2 Interaction During Host Cell Invasion By Toxoplasma., Shruthi Krishnamurthy

Graduate College Dissertations and Theses

T.gondii is a model organism of the phylum Apicomplexa that infects one third of the human population. While the majority of infections are asymptomatic or manifest with mild flu-like symptoms, toxoplasmosis can be fatal in immunocompromised individuals and in the developing fetus. The lytic cycle of tachyzoite-stage parasites causes damage to the host by repeated rounds of host cell invasion, intracellular replication and lysis of the host cell upon egress.

Invasion is a key step for the parasite to maintain its intracellular lifestyle. Apical Membrane Antigen 1 (AMA1) is an adhesin released from a unique set of secretory organelles called …

Defining And Characterizing The Step In The Hiv-1 Viral Life Cycle Affected By Fullerene Derivatives, Zachary Sean Martinez

Open Access Theses & Dissertations

Fullerene derivatives, both C60 and C70, were synthesized and characterized to test their activity against HIV-1 infection. The derivatives tested have previously been reported to inhibit HIV-1 replication and/or in vitro activity of reverse transcriptase. It was found that viruses produced in the presence of low micromolar concentrations of most of these fullerene derivatives exhibit a reduction in viral infectivity greater than 99%. Quantification of virion-associated viral RNA and p24 indicates that RNA packaging and viral production were unremarkable in these viruses. These compounds did not affect infectivity of mature virions, indicating no effect on the early steps of the …

Utility And Origin Of Blood-Based Autoantibodies For Early Detection And Diagnosis Of Neurodegenerative Diseases, Cassandra Demarshall

Graduate School of Biomedical Sciences Theses and Dissertations

Autoantibodies are self-reactive antibodies that have been widely implicated as causal agents of autoimmune diseases. They are found in the blood of all human sera, regardless of age, gender, or the presence or absence of disease. While the underlying reason for their ubiquity remains unknown, it has been hypothesized that they participate in the clearance of blood-borne cell and tissue debris generated in both healthy and diseased individuals on a daily basis. Although much evidence supports this debris clearance role, recent studies also suggest a causal role for autoantibodies in disease. My thesis work has focused on this "cause and/or …