Biochemistry, Biophysics, and Structural Biology Commons^™

Unraveling The Role Of Novel G5 Peptidase Family Proteins In Virulence And Cell Envelope Biogenesis Of Staphylococcus Aureus, Stephanie M. Marroquin

USF Tampa Graduate Theses and Dissertations

Virulence factors and the bacterial cell envelope are two important components in S. aureus pathogenesis and survival. More importantly, understanding the regulation of these cellular processes is crucial to further understanding and combating this successful pathogen. To date, numerous factors have been identified as playing a role in the regulation of Agr activity in S. aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigate the product of SAUSA300_1984 (termed MroQ), a transmembrane G5 peptidase family protein, as a novel effector of this system. Using a USA300 mroQ mutant we observed a drastic reduction in proteolysis, hemolysis, and …

Investigation Of An Alternative Protocol For The Production Of Sars-Cov-2 Antigenic Proteins, Nichole Ninaltowski

USF Tampa Graduate Theses and Dissertations

With the COVID-19 pandemic showing no signs of slowing down, large-scale antigenic protein production is still needed for surveillance using serologic assays. From screening to vaccines to biotherapeutics, being able to produce the proteins for these assays is essential; however, the current gold standard method for producing SARS-CoV-2 spike proteins is prohibitively expensive for most research groups.

Alternative methods of transfecting mammalian cells to produce recombinant proteins that are relatively inexpensive have been used for years. Unlike the expensive, commercially available lipid-based methods, other established methods such as polyethyleneimine (PEI), are considerably easier, and cheaper to meet the needs of …

Transcriptomic And Functional Investigation Of Bacterial Biofilm Formation, Brooke R. Nemec

USF Tampa Graduate Theses and Dissertations

Staphylococcus aureus and Acinetobacter baumannii are two highly successful human pathogens, which have adopted very different, but effective survival strategies. The success of S. aureus is attributed to the tight regulation of an arsenal of virulence factors. Conversely, A. baumannii lacks what would be considered traditional virulence factors and, instead, has developed a high tolerance for environmental stress, which allows it to persist in unforgiving environments, including nosocomial settings and the human body. One common characteristic of these two organisms is their proclivity for biofilm formation. Herein, we discuss the diverse mechanisms governing biofilm formation for A. baumannii and S. …

Posttranslational Modification And Protein Disorder Regulate Protein-Protein Interactions And Dna Binding Specificity Of P53, Robin Levy

USF Tampa Graduate Theses and Dissertations

p53 is an intrinsically disordered transcription factor that suppresses tumor development by arresting the cell cycle and promoting DNA repair. p53 deletions or mutations can lead to cancer due to the inability of cells to respond to stress. The protein levels and post-translational modification state of p53 changes in response to cellular stress like DNA damage. Previous studies have shown that p53 can undergo coupled folding and binding with the E3 ubiquitin ligase, Mdm2, and the histone deacetylase, p300. In normal cells, p53 is kept at a low level by Mdm2, which marks it with ubiquitin, targeting p53 for proteasome …

To Mid-Cell And Beyond: Characterizing The Roles Of Gpsb And Ypsa In Cell Division Regulation In Gram-Positive Bacteria, Robert S. Brzozowski

USF Tampa Graduate Theses and Dissertations

The bacterial cell division protein FtsZ is a tubulin homolog that forms a ring-like structure at the site of cell division in most bacterial species. There it acts as a scaffold, aiding in the recruitment of other divisome proteins to the site of cell division. Furthermore, studies focusing on the role of FtsZ treadmilling and septal peptidoglycan synthesis implicates that FtsZ plays a direct role in the ultimate closure of the division septum. Thus, many studies in the field of bacterial cell division have focused on FtsZ in terms of its spatial and temporal regulation as well as its ability …

The Role Of Secreted Proteases In Regulating Disease Progression In Staphylococcus Aureus, Brittney D. Gimza

USF Tampa Graduate Theses and Dissertations

Staphylococcus aureus is a highly successful pathogen capable of producing a wealth of virulence factors in the human host. Of note, ten extracellular proteases are produced alongside these virulence factors and play a multifaceted role during infection. They not only cleave host proteins to promote bacterial invasion, immune evasion and survival, but also control disease progression by modulating the stability of self-derived pathogenic determinants. The importance of the secreted proteases modulating virulence factor stability is evidenced by our groups previous finding that a protease-null strain has a substantially increased infectious capacity in a murine model of sepsis; resulting from the …

Targeting Bacterial Resistance And Cancer Metastasis: A Structure Based Approach, Kyle Galen Kroeck

USF Tampa Graduate Theses and Dissertations

Current research in pharmaceutical development commonly utilizes a profusion of methods in molecular modeling in order to probe intricate biological problems. Many original and promising compounds have been identified and developed by integrating experimental and computational methods. Structural biology utilizes many different research techniques including x-ray crystallography, NMR, and electron microscopy in order to develop molecular models of macromolecules that are of biological interest. Such techniques can be used in conjunction with molecular docking, which utilizes those molecular models in order to target macromolecules of therapeutic interest by computationally analyzing the conformations adopted by ligands upon interaction with a desired …

A Small Rna And Dna Binding Protein Contribute To Biofilm Development In Bartonella Henselae, Udoka Okaro

USF Tampa Graduate Theses and Dissertations

A biofilm, which is associated with 80% of chronic infections in humans, is formed when bacteria aggregate, attach to a substrate and secrete a matrix protecting the bacteria from host cell defenses and antibiotics. Bartonella henselae (B. henselae) is the causative agent of cat scratch disease, persistent bacteremia, and one of the most frequently reported causes of blood-culture negative endocarditis (BCNE) in patients. The ability of B. henselae to adhere to the heart valve, form a biofilm and vegetation to cause endocarditis increases the morbidity and mortality rate in infected patients. The presence of a trimeric autotransporter adhesin (TAA) called …

Mechanism Elucidation And Inhibitor Discovery Against Serine And Metallo-Beta-Lactamases Involved In Bacterial Antibiotic Resistance, Orville A. Pemberton

USF Tampa Graduate Theses and Dissertations

The emergence and proliferation of Gram-negative bacteria expressing β-lactamases is a significant threat to human health. β-Lactamases are enzymes that degrade the β-lactam antibiotics (e.g., penicillins, cephalosporins, monobactams, and carbapenems) that we use to treat a diverse range of bacterial infections. Specifically, β-lactamases catalyze a hydrolysis reaction where the β-lactam ring common to all β-lactam antibiotics and responsible for their antibacterial activity, is opened, leaving an inactive drug. There are two groups of β-lactamases: serine enzymes that use an active site serine residue for β-lactam hydrolysis and metalloenzymes that use either one or two zinc ions for catalysis. Serine enzymes …

Cell Division Regulation In Staphylococcus Aureus, Catherine M. Spanoudis

USF Tampa Graduate Theses and Dissertations

Cell division is a fundamental biological process that occurs in all kingdoms of life. Our understanding of cell division in bacteria stems from studies in the rod-shaped model organisms: Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. The molecular underpinnings of cell division regulation in non-rod-shaped bacteria remain to be studied in detail. Rod-shaped bacteria possess many positive and negative regulatory proteins that are essential to the proper placement of the division septa and ultimately the production of two identical daughter cells, many of which are absent in cocci. Given that essential cell division proteins are attractive antibacterial drug targets, it …

Discovering Antibacterial And Anti-Resistance Agents Targeting Multi-Drug Resistant Eskape Pathogens, Renee Fleeman

USF Tampa Graduate Theses and Dissertations

Antibiotic resistance has been a developing problem for mankind in recent decades and multi-drug resistant bacteria are now encountered that are resistant to all treatment options available. In 2014, the World Health Organization announced that this problem is driving us towards a “post-antibiotic era” that will change the face of modern medicine as we know it. If lack of novel antibiotic development and FDA approval continues, by the year 2050, 10 million people will die each year to an antimicrobial resistant bacterial infection. With lack of pharmaceutical industry involvement in developing novel antibiotics, the responsibility now lies within the academic …

A Novel Abi-Domain Protein Controls Virulence Determinant Production In Staphylococcus Aureus, Stephanie Michelle Marroquin

USF Tampa Graduate Theses and Dissertations

A major factor in the success of Staphylococcus aureus as a pathogen is its vast arsenal of virulence determinants and, more importantly, the tight and precisely- timed regulation of these factors. Here we investigate the product of the S. aureus gene, SAUSA300_1984, encoding a putative transmembrane protein. This as yet uncharacterized protein belongs to the Abi (abortive infection) family, which are commonly annotated as CAAX-proteases, and are significantly understudied in prokaryotes. In S. aureus the disruption of SAUSA300_1984 results in a drastic reduction of proteolytic and hemolytic activity, as well as diminished pigmentation. This phenotype appears to be mediated through …

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 …

Characterization Of Two Novel Gene Regulatory Systems In The Zoonotic Bacterium Bartonella Henselae, Nhan Tu

USF Tampa Graduate Theses and Dissertations

The genus Bartonella contains Gram-negative arthropod-borne bacteria that are found in many small animal reservoirs and are capable of causing human disease. Bacteria utilize a general stress response system to combat stresses from their surrounding environments. In α-proteobacteria, the general stress response system uses an alternate σ factor as the main regulator and incorporates it with a two-component system into a unique system. Our study identifies the general stress response system in the α-proteobacterium, Bartonella henselae, where the gene synteny is conserved and both the PhyR and alternate σ factor have similar sequence and domain structures with other α-proteobacteria. …

Promoting Genome Stability Via Multiple Dna Repair Pathways, Scott Cukras

USF Tampa Graduate Theses and Dissertations

Maintaining genome integrity is indispensible for cells to prevent and limit accruement of deleterious mutations and to promote viable cell growth and proliferation. Cells possess a myriad of mechanisms to detect, prevent and repair incurred cellular damage. Here we discuss various proteins and their accompanying cellular pathways that promote genome stability. We first investigate the NEDD8 protein and its role in promoting homologous recombination repair via multiple Cullin E3 ubiquitin ligases. We provide specific mechanisms through which, UBE2M, an E2 conjugating enzyme, neddylates various Cullin ligases to render them catalytically active to degrade their substrates by the proteasome. We show …

Exploring The Role Of Intracellular Aminopeptidases In Staphylococcus Aureus Pathogenesis, Devon Nicole Marking

USF Tampa Graduate Theses and Dissertations

Staphylococcus aureus is a remarkably pathogenic bacterium that is widely prevalent among the human population. It is the leading agent of skin and soft tissue infections, and is also responsible for causing an array of severe and life threatening diseases. The invasiveness of the pathogen, coupled with increasing antibiotic resistance seen for S. aureus infections, makes this bacterium a prominent public health concern. The extended pathogenicity of S. aureus is largely due to its repertoire of virulence factors, which are typically characterized by being bound to the cell wall, or secreted into the extracellular environment. Previously, our lab identified a …

Mechanisms And Molecular Biology Of Major Tumor Suppressors, Brienne E. Engel

USF Tampa Graduate Theses and Dissertations

This dissertation is devoted to the study of the molecular biology of major tumor suppressors, defined as those that prevent the cellular processes identified as the hallmarks of cancer. Specifically, the major tumor suppressors pRb and STK11 are explored in the context of osteosarcoma and lung cancer, respectively.

RB1 was the first tumor suppressor gene discovered. Over four decades of work have revealed that the Rb protein (pRb) is a master regulator of biological pathways influencing virtually every aspect of intrinsic cell fate including cell growth, cell-cycle checkpoints, differentiation, senescence, self-renewal, replication, genomic stability and apoptosis. While these many processes …

Role And Regulation Of Snon/Skil And Plscr1 Located At 3q26.2 And 3q23, Respectively, In Ovarian Cancer Pathophysiology, Madhav Karthik Kodigepalli

USF Tampa Graduate Theses and Dissertations

Ovarian cancer is one of the most common causes of gynecological cancer related deaths in women. In 2014, the estimated number of deaths due to ovarian cancer is 14,270 with occurrence of over 22, 240 new cases (National Cancer Institute, http://seer.cancer.gov/statfacts/html/ovary.html). Despite improvement in treatment strategies, the 5-year survival rate is still below 50% mainly due to chemoresistance and relapse. Amplification of chromosomal region 3q26 is a common characteristic in various epithelial cancers including ovarian cancer. This region harbors various oncogenes including the TGFβ signaling mediators EVI1 and SnoN/SkiL, PKCι and PIK3CA amplified at 3q26.2 and 3q26.3, respectively, in ovarian …

Genetic Basis For The Virulence Of Enterohemorrhagic Escherichia Coli Strain Tw14359, Jason Kyle Morgan

USF Tampa Graduate Theses and Dissertations

Enterohemorrhagic Escherichia coli (EHEC) is a virulent pathotype of E. coli that is associated with major outbreaks of hemorrhagic colitis and the life-threatening kidney disease hemolytic uremic syndrome. For successful host colonization and attachment to the intestinal mucosa, EHEC requires the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (TTSS) responsible for secreting and translocating effector proteins into host colonocytes. Regulation of the LEE is primarily directed through the first operon, LEE1, encoding the locus encoded regulator (Ler), and occurs through the direct and indirect action of several regulators. The 2006 U.S. spinach outbreak …

Structure-Based Design Of Novel Inhibitors And Ultra High Resolution Analysis Of Ctx-M Beta-Lactamase, Derek Allen Nichols

USF Tampa Graduate Theses and Dissertations

The emergence of CTX-M class-A extended-spectrum β-lactamases, which confer resistance to second and third-generation cephalosporins, poses a serious health threat to the public. CTX-M β-lactamases use a catalytic serine to hydrolyze the β-lactam ring. Specifically, the hydrolysis reaction catalyzed by CTX-M β-lactamase proceeds through a pre-covalent complex, a high-energy tetrahedral acylation intermediate, a low-energy acyl-enzyme complex, a high-energy tetrahedral deacylation intermediate after attack via a catalytic water, and lastly, the hydrolyzed β-lactam ring product which is released from the enzyme complex. The crystallographic structure of CTX-M at sub-angstrom resolution has enabled us to study enzyme catalysis as well as perform …

Sigma Factor N (Σn): A Novel Regulator Of Extreme Acid Resistance In Enterohemorrhagic E. Coli O157:H7, Pamela Ann Fay

USF Tampa Graduate Theses and Dissertations

Extreme acid resistance contributes to the successful transmission of enterohemorrhagic E. coli (EHEC) through acidic food matrices and the stomach, allowing it to gain access to the intestine and elicit disease in humans. Alternative sigma factor N (σ^N, encoded by rpoN) was previously identified as a novel regulator of extreme acid resistance in EHEC. This study investigated the role for σ^N and co-expressed products of the rpoN operon in the acid resistance phenotype of EHEC. The results revealed that σ^N primarily controls acid resistance through repression of the glutamate-dependent acid resistance (GDAR) system through control …

Investigation Into The Mechanism Of Salicylate-Associated Genotypic Antibiotic Resistance In Staphylococcus Aureus, Nada Salah Helal

USF Tampa Graduate Theses and Dissertations

Growth of Staphylococcus aureus with the NSAID salicylate increases phenotypic resistance (SAPAR), and the frequency at which heritable resistance occurs to various antibiotics (SAGAR). This study describes the effect of salicylate on heritable and phenotypic resistance to a set of antibiotics for laboratory and multi-drug resistant strains of S. aureus and investigates the link between resistance and SAGAR. Drug gradient plates were used to determine phenotypic resistance to antibiotics targeting DNA replication, transcription, translation and the cell wall in the presence or absence of salicylate. To measure heritable resistance, mutation frequencies were determined for each antibiotic in the presence and …

Stable Carbon Isotope Discrimination By Rubisco Enzymes Relevant To The Global Carbon Cycle, Amanda J. Boller

USF Tampa Graduate Theses and Dissertations

Five different forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO; IA, IB, IC, ID, II), the carboxylase of the Calvin-Benson-Bassham cycle (CBB), are utilized by plants, algae and autotrophic bacteria for carbon fixation. Discrimination against ¹³C by RubisCO is a major factor dictating the stable carbon isotopic composition (δ¹³C = {[¹³C/¹²C sample/¹³C/¹²C standard] - 1} X 1000) of biomass. To date, isotope discrimination, expressed as ε values (={[¹²k/¹³k] - 1} X 1000; ¹²k and ¹³k …

Biochemical And Pharmacological Characterization Of Cytochrome B5 Reductase As A Potential Novel Therapeutic Target In Candida Albicans, Mary Jolene Patricia Holloway

USF Tampa Graduate Theses and Dissertations

The opportunistic fungus Candida albicans is a commensal member of the human microflora and is the most common causative agent of fungal-related disease with particular significance in immunocompromised individuals. Emerging drug resistance is a major problem in Candida, contributed by enzymes involved in the detoxification of xenobiotics and pharmacological agents. One such enzyme, cytochrome b5 reductase (cb5r), has a high pharmacological significance owing to its role in fatty acid elongation, ergosterol (or cholesterol in mammals) biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics.

We have compared the kinetic, biochemical, and pharmacological characteristics of C. albicans cb5r isoforms, Cbr1 and Mcr1, as …

Identification And Characterization Of The Human Herpesviruses 6a And 6b Genome Integration Into Telomeres Of Human Chromosomes During Latency, Jesse Herbert Arbuckle

USF Tampa Graduate Theses and Dissertations

While the latent genome of most Herpesviruses persists as a nuclear circular episome, previous research has suggested that Human Herpesvirus 6 (HHV-6) may integrate into host cell chromosomes, and be vertically transmitted in the germ-line. Because the HHV-6 genome encodes a perfect TTAGGG telomere repeat array at the right end direct repeat (DR_R) and an imperfect TTAGGG repeat at the end of the left end direct repeat (DR_L), we established a hypothesis that during latency, the HHV-6A and HHV-6B genome integrates into the telomeres of human chromosomes through homologous recombination with the n(TTAGGG) viral repeats, and …

Development Of An Elisa For Eastern Equine Encephalitis Virus That Can Differentiate Infected From Vaccinated Horses, Andrea Bingham

USF Tampa Graduate Theses and Dissertations

Eastern Equine Encephalitis virus (EEEV) causes a fatal mosquito-borne virus that is vaccine preventable for horses. The conventional serological tests measure antibodies to the structural proteins of EEEV which are also found in the vaccine. This makes it difficult to differentiate infected and vaccinated animals (DIVA). Detection of antibodies to non-structural proteins (NSPs) is a theoretical strategy that would allow you to survey natural infections among vaccinated populations. This test would also allow for more accurate representations of the natural infection rate, vaccination rate, and help identify vaccine failures. The potential uses of the NSPs of Eastern Equine Encephalitis virus …

Microbial Ecology And Functional Genomics Of Deep-Water Coral-Associated Microbes, Julia Parker Galkiewicz

USF Tampa Graduate Theses and Dissertations

Lophelia pertusa is a cosmopolitan cold-water coral, often found in aphotic waters (>200m). Aggregations of L. pertusa (reefs) provide important habitat to many invertebrate and fish species and act as biodiversity hotspots in the deep sea. The health and diversity of these reefs is of vital importance to deep-sea ecosystems, and the microbial consortia associated with L. pertusa form the most basic ecological level. Deciphering the diversity and function of these microbes provides insight into the roles they play in maintaining reef health. This dissertation takes microbiological techniques that are used in shallow-water coral microbial research and applies them …

Microbial Landscapes Of Corals And Ctenophores, Camille Arian Daniels

USF Tampa Graduate Theses and Dissertations

As technology and engineering allow mankind to survey nature at finer scales, the importance of bacteria has been elucidated in their metabolic diversity, ability to transfer genetic information, involvement in biogeochemical cycling, and sheer abundance. With an individual domain of life unto themselves, this diverse group of microorganisms plays an integral role in facilitating life on land and in the oceans, and is second only to viruses in abundance on Earth. They carve niches in a wide range of environments, including those inhospitable to other life forms, and reside in concert or to the detriment of other microbes and/or hosts …

Development Of A Non-Invasive Electrode For Intradermal Electrically Mediated Dna Vaccination, Amy Lynn Donate

USF Tampa Graduate Theses and Dissertations

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use against a wide variety of diseases. Alone, this vaccine technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity against the expressed antigen. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our …