Biochemistry, Biophysics, and Structural Biology Commons^™

Caehnorhabditis Elegans: A Low-Cost In Vivo Animal Model For Efficacy Studies Of Novel Antibiotics, Rylee Ann Gregory

Undergraduate Honors Capstone Projects

Since the 1940s, antibiotics have greatly reduced the adverse effects of infectious diseases caused by microbes. However, due to excessive, and often incorrect, use of known antibiotics, many organisms have adapted antibiotic resistance. Currently, over 70% of known infectious bacteria are resistant to at least one antibiotic. In the U.S. , 90,000 deaths occur each year due to infection by bacteria resistant to antibiotics. This number has increased by nearly 75,000 in the last 20 years. It is necessary, therefore, to continue developing new antibiotics in an effort to keep up with increasing antibiotic resistance. Traditional in vitro and whole …

Screening Synthetic Combinatorial Protein Libraries For Changes In Calmodulin Binding Specificity, Donald E. Dick

Kaleidoscope

No abstract provided.

Starch Phosphates: A Means To Manipulate Biofuel Production, Travis M. Bridges

Kaleidoscope

No abstract provided.

The Role Of Argininosuccinate Synthase Serine 328 Phosphorylation In Nitric Oxide Production, Ricci Haines

USF Tampa Graduate Theses and Dissertations

Until recently, the main mechanism of argininosuccinate synthase (AS) regulation was described to exist mainly at the level of transcription. Transcriptional regulation of AS has been shown to be coordinate with eNOS in response to shear stress, hypoxia, tumor necrosis factor á (TNF-á), and PPAR ã agonist troglitizone. However, it is now understood that one level of NO regulation is cellular control of arginine availability to eNOS via post-translational modifications of AS such as phosphorylation. The purpose of this investigation was to determine under what conditions AS is phosphorylated at S328, identify the pathway that AS phosphorylation at S328 plays …

Live-Cell Monitoring Of Periodic Gene Expression In Synchronous Human Cells Identifies Forkhead Genes Involved In Cell Cycle Control, Gavin D. Grant, Joshua Gamsby, Viktor Martyanov, Lionel Brooks, Lacy K. George, J. Matthew Mahoney, Jennifer J. Loros, Jay C. Dunlap, Michael L. Whitfield

Dartmouth Scholarship

We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle …

Mda-7/Il-24; A Promising Cancer Therapeutic Agent, Hossein Hamed

Theses and Dissertations

Glioblastoma multiforme (GBM) is an aggressive cancer that affects millions of patients per year. Conventional therapies combining chemotherapeutic agents with radiation can only extend survival by a few months; therefore, there is a dire need for an effective means of treating this deadly disease. Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), currently in the early stages of FDA pre-IND drug trials, has proven to be an effective cancer specific cytokine, able to trigger the onset of mitochondrial dysfunction and/or autophagy. GBM’s have mutations that often result in the activation of cytoprotective cell signaling pathways, preventing cancer therapeutics and even MDA-7/IL-24 treatments from being …

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 …

(1)H, (15)N And (13)C Backbone Resonance Assignments Of The Kelch Domain Of Mouse Keap1., Elio Cino, Jingsong Fan, Daiwen Yang, Wing-Yiu Choy

Biochemistry Publications

Kelch-like ECH-associated Protein 1 (Keap1) is a multi-domain protein that functions as an inhibitor of the transcription factor nuclear factor E2-related factor 2 (Nrf2) in the cellular response to oxidative stress. Under normal conditions, Keap1 binds to Nrf2 via its C-terminal Kelch domain and the interaction ultimately leads to the ubiquitin-dependent degradation of Nrf2. It has been proposed that designing molecules to selectively disrupt the Keap1-Nrf2 interaction can be a potential therapeutic approach for enhancing the expression of cytoprotective genes. Here, we reported the (1)H, (13)C, and (15)N backbone chemical shift assignments of the Kelch domain of mouse Keap1. Further, …

Systematics Of Harrisia (Cactaceae), Alan R. Franck

USF Tampa Graduate Theses and Dissertations

The genus Harrisia Britton (Cactaceae) comprises species of columnar cacti that are united by a unique seed morphology. The species range in form from prostrate shrubs to large trees and are native to South America and the Caribbean region. Harrisia is placed in an unresolved position within subtribe Trichocereinae of tribe Cereeae of subfamily Cactoideae. Relationships among the species within Harrisia are also poorly understood. In this study, several species of Harrisia were sequenced for as many as seven different regions of nuclear and plastid DNA. Species in the Caribbean were also examined with amplified fragment length polymorphisms. The morphology …

Association Of A Multi-Synthetase Complex With Translating Ribosomes In The Archaeon Thermococcus Kodakarensis, Medha Raina, Sara Elgamal, Thomas J. Santangelo, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

In archaea and eukaryotes aminoacyl-tRNA synthetases (aaRSs) associate in multi-synthetase complexes (MSCs), however the role of such MSCs in translation is unknown. MSC function was investigated in vivo in the archaeon Thermococcus kodakarensis, wherein six aaRSs were affinity co-purified together with several other factors involved in protein synthesis, suggesting that MSCs may interact directly with translating ribosomes. In support of this hypothesis, the aminoacyltRNA synthetase (aaRS) activities of the MSC were enriched in isolated T. kodakarensis polysome fractions. These data indicate that components of the archaeal protein synthesis machinery associate into macromolecular assemblies in vivo and provide the potential …

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 …

Aspirin But Not Ibuprofen Use Is Associated With Reduced Risk Of Prostate Cancer: A Plco Study., F. M. Shebl, L. C. Sakoda, A. Black, J. Koshiol, G. L. Andriole, R. Grubb, T. R. Church, D. Chia, C. Zhou, L. W. Chu, W. Y. Huang, U. Peters, V. A. Kirsh, N. Chatterjee, M. F. Leitzmann, R. B. Hayes, A. W. Hsing

Biochemistry and Molecular Medicine Faculty Publications

Background:

Although most epidemiological studies suggest that non-steroidal anti-inflammatory drug use is inversely associated with prostate cancer risk, the magnitude and specificity of this association remain unclear.

Methods:

We examined self-reported aspirin and ibuprofen use in relation to prostate cancer risk among 29 450 men ages 55–74 who were initially screened for prostate cancer from 1993 to 2001 in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Men were followed from their first screening exam until 31 December 2009, during which 3575 cases of prostate cancer were identified.

Results:

After adjusting for potential confounders, the hazard ratios (HRs) of …

Role Of The Nla6s And Nla28s Histidine Kinases In Fruiting Body Development Of Myxococcus Xanthus, Zaara Sarwar

Biology - Dissertations

The complex life cycle of Myxococcus xanthus makes it a model organism for studying multicellular developmental processes in bacteria. In response to adverse environmental conditions, M. xanthus aggregates and forms multicellular structures known as fruiting bodies. Two component signal transduction systems (TCS) are widely used by bacteria to detect and respond to environmental cues by regulating large-scale changes in gene expression. They contain a histidine kinase sensor that detects environmental cues and a response regulator that modulates cellular processes. Two key regulators of the early stages of fruiting body development are the Nla6S/Nla6 and Nla28S/Nla28 TCSs. The response regulators of …

Minor Fibrillar Collagens, Variable Regions Alternative Splicing, Intrinsic Disorder, And Tyrosine Sulfation, Ming Fang, Reed B. Jacob, Owen Mcdougal, Julia Oxford

Biomedical Research Institute Publications and Presentations

Minor fibrillar collagen types V and XI, are those less abundant than the fibrillar collagens types I, II and III. The alpha chains share a high degree of similarity with respect to protein sequence in all domains except the variable region. Genomic variation and, in some cases, extensive alternative splicing contribute to the unique sequence characteristics of the variable region. While unique expression patterns in tissues exist, the functions and biological relevance of the variable regions have not been elucidated. In this review, we summarize the existing knowledge about expression patterns and biological functions of the collagen types V and …

Development Of A Protocol To Measure Gene Expression In The Mouse Tibia, Daniel Hoover

Biomedical Engineering

Numerous molecular factors active in bone tissue direct fracture repair and remodeling which can be altered by disease conditions such as Peripheral Arterial Disease (PAD) and Osteoporosis. Methods of molecular biology are commonly applied to investigate the expression and role of these molecular factors. This project presents a robust three-step protocol for examining gene expression in the mouse tibia. The protocol begins with isolating RNA from a flash frozen tibia sample. The isolated RNA is reverse transcribed into cDNA. Finally, PCR is performed to indentify expressed genes. Establishing this protocol will allow further research into the mechanisms of bone remodeling …

A Study Of Histone Deacetylase Inhibitors: Determination Of The Binding Energetics Of Suberoylanilide Hydroxamic Acid With Zinc And Cobalt And Preliminary Screening Of Nitric Oxide Donors, Erin Gallagher

College of Science and Health Theses and Dissertations

Inhibiting histone deacetylases (HDACs) can suppress tumor cell growth eventually leading to their death, makeing HDACs an important drug target. Therefore, understanding the mechanism behind inhibiting HDACs is imperative. To determine the thermodynamic parameters for the interaction of a known competitive HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), with zinc(II), the metal ion in the HDAC active site, and cobalt(II), isothermal titration calorimetry (ITC) was used. The results showed that SAHA binds to zinc(II) more strongly than it does to cobalt(II) as the equilibrium binding constants were 4.3 ± 0.5 x 10² M^-1 and 2.0 ± 0.5 x 10 …

Urinary Copper Elevation In A Mouse Model Of Wilson’S Disease Is A Regulated Process To Specifically Decrease The Hepatic Copper Load, Lawrence W. Gray, Fangyu Peng, Shannon A. Molloy, Venkata S. Pendyala, Abigael Muchenditsi, Otto Muzik, Jaekwon Lee, Jack H. Kaplan, Svetlana Lutsenko

Department of Biochemistry: Faculty Publications

Body copper homeostasis is regulated by the liver, which removes excess copper via bile. In Wilson’s disease (WD), this function is disrupted due to inactivation of the copper transporter ATP7B resulting in hepatic copper overload. High urinary copper is a diagnostic feature of WD linked to liver malfunction; the mechanism behind urinary copper elevation is not fully understood. Using Positron Emission Tomography-Computed Tomography (PET-CT) imaging of live Atp7b^‒/^‒ mice at different stages of disease, a longitudinal metal analysis, and characterization of copper-binding molecules, we show that urinary copper elevation is a specific regulatory process mediated …

Roles Of Resveratrol And Genistein In Invasion And Metastasis Of Breast Cancer, Brittany Wolfe

College of Science and Health Theses and Dissertations

Breast cancer is a cellular disease characterized by the exploitation of several cellular and molecular mechanisms involved in cell proliferation, apoptosis, motility, and invasion. Effective treatment is available for non-invasive breast cancer at diagnosis, leading to a very high survival rate compared to the low survival rate for those where the breast cancer has spread. Thus, identifying effective therapies and preventative agents is imperative to successfully treat breast cancer. Since diet compromises a large component of the risk factors for breast cancer, it would be beneficial to examine dietary compounds that could potentially play a beneficial role in inhibiting cancer …

A Proteomic Analysis Of Differentiation In The Mammary Epithelium, Laura Therese Strand

Master's Theses

While a great deal is known about the changing hormonal environment and the structural development of the mammary gland from pregnancy to lactation, very little is known about the molecular mechanisms governing differentiation of the mammary epithelium into a milk-secreting phenotype. It is important to acknowledge the diversity among the mammary glands of different species in order to better understand applications in human health and the dairy industry. In this study, we examined global protein expression during two states of differentiation in mammary epithelial cells from two species: in vitro proliferating and differentiated MAC-T cells (a bovine immortal cell-line), and …

Host Pathogen Interactions: Is Arabidopsis Thaliana Remembered By Its Nemesis Pseudomonas Syringae?, Daniel Z. Kreiser

Lawrence University Honors Projects

Plants contain innate immune systems that deter pathogen infection. Pattern recognition receptors bind microbe-associated molecular patterns (MAMPs), triggering immunity. MAMPs are proteins exclusive to pathogens that are typically indispensable for their survival. For this reason, MAMPs cannot be mutated or removed without causing pathogen death. However, this does not necessitate constitutive expression of MAMPs. In this study, the MAMP response of Arabidopsis thaliana was utilized to determine differential detection of MAMPs expressed by Pseudomonas syringe pv. tomato DC3000 when pretreated with A. thaliana. Results demonstrated that more MAMPs are detected when P. syringae had previously encountered A. thaliana, …

Fret-Based Identification Of Mrnas Undergoing Translation, Benjamin Stevens, Chunlai Chen, Ian Farrell, Haibo Zhang, Jaskiran Kaur, Steven L. Broitman, Zeev Smilansky, Barry S. Cooperman, Yale E. Goldman

Biology Faculty Publications

No abstract provided.

Energy-Saving Hypothermia Reduces Flight Ability In Mourning Doves, Jennie M. Carr, Steven L. Lima

Symposium 2012

Overwintering birds are frequently exposed to thermal challenges that can quickly decrease energy reserves, thus leading to an increase in the risk of starvation. During these periods of energetic hardship, many avian species use nocturnal hypothermia to conserve energy that would otherwise be lost remaining warm throughout the night (McKechnie & Lovegrove 2002). However, a cool body temperature (Tb) may limit a bird’s ability to monitor the environment and may slow their response to a potential threat. Thus, birds likely trade-off between the benefits of energy-saving hypothermia and the potential costs of reduced behavioral responsiveness to predators (Welton et al. …

Synthesis Of S-Ribosyl-L-Homocysteine And Analogs Modified At The Homocysteine-C3 Position, Ruoyi Liu

Master's Theses

Quorum sensing (QS) is a process of bacterial cell-to-cell communication that conveys population density information in order to coordinate gene expression to produce synchronized behaviors. QS regulates the expression of virulence genes in many species of bacteria; hence, the manipulation of QS pathways may lead to treatment options against many bacterial diseases. The LuxS enzyme converts S-ribosyl-L-homocysteine (SRH) into homocysteine (HCys) and 4(S),5-dihydroxypentane-2,3-dione (DPD), which is the precursor of autoinducer-2 (AI-2). Thus, inhibitors of LuxS could prevent QS by halting the conversion of SRH to AI-2 rendering the cell “uncommunicative”. This work shows the successful chemical synthesis …

Elevating Ascorbate Content In Tomato And Studying The Role Of Jasmonates In Modulating Ascorbate In Arabidopsis, Shashank S. Kulkarni

Student Theses and Dissertations

Vitamin C (ascorbate, AsA) is the most abundant water soluble antioxidant in plants and is essential for plant and human health. Ascorbate plays a key role in protecting plants from reactive oxygen species generated during normal metabolic processes and under stress conditions including wounding, pathogen attack, salt, heat, light, and ozone exposure. Jasmonates (JAs), a group of plants hormones involved in stress responses also protect plants from these stresses. There is evidence indicating that JAs modulate AsA content in plants. The recent discovery of the inositol pathway has opened possibilities for AsA enhancement via metabolic engineering in plants. Over-expression of …

Shoc2 Is Targeted To Late Endosomes And Required For Erk1/2 Activation In Egf-Stimulated Cells, Emilia Galperin, Lina Abdelmoti, Alexander Sorkin

Molecular and Cellular Biochemistry Faculty Publications

Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF) receptor (EGFR), we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S) mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and …

Synthesis Of Fluorogenic Substrates For The Enzymatic Characterization Of Rv0045c From M. Tuberculosis, Kelly Jo Mckenna

Undergraduate Honors Thesis Collection

Mycobacterium tuberculosis is the pathogenic bacterial agent commonly responsible for tuberculosis, or TB. Although treatment exists for the active form of tuberculosis, no method has been developed for eliminating M tuberculosis in its dormant state. One hypothesized method for the elimination of dormant TB is to develop an inhibitor specific for M tuberculosis esterases and lipases, as these esterases and lipases are essential to the survival of dormant TB infection. In this research, the substrate specificity of the Rv0045c esterase from M tuberculosis was studied due to the essential role of Rv0045c in TB metabolism and its dissimilarity to other …

Exploring The Interaction Between Human Granulin-A (Hgrn-A) And Amyloid-Β (Aβ) Peptide, Michael Samel

Honors Theses

Alzheimer’s disease (AD) is the most common form of dementia affecting people over the age of 60, yet a detailed understanding of the molecular changes involved in AD remains a significant challenge to modern medicine. The hallmarks of an AD brain include the presence of senile plaques formed by a protein called amyloid-β (Aβ), along with inflammation of the brain. Aβ self-associates to form clumps of protein called “aggregates” which are known to be the primary toxic agents in AD pathology; however, several other proteins have also been implicated in this pathology. One such protein called progranulin (PGRN), which under …

Refining Vibrationally-Resonant Sum Frequency Generation Spectroscopy For Studies Of Interfacial Interactions, Alexander D. Curtis

Theses and Dissertations

Many phenomena of interest to science and engineering occur at interfaces; however, access to, or discrimination of, interfacial interactions has been challenging, especially at buried interfaces. Vibrationally resonant sum-frequency generation (VR-SFG) spectroscopy is a powerful tool for investigating the molecular structure of free or buried interfaces, but spectral analysis has relied on many assumptions. To claim accurate new insights, practitioners must be able to make unique determinations from the data without experimental artifacts affecting the final results. For example, two independent and overlapping studies for the polystyrene/air interface were carried out, but reported different surface structures. Initially, this difference was …

Conservation Of Oxidative Protein Stabilization In An Insect Homologue Of The Parkinsonism-Associated Protein Dj-1, Jiusheng Lin, Janani Prahlad, Mark A. Wilson

Department of Biochemistry: Faculty Publications

DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well conserved in other DJ-1 homologues, including two (DJ-1α and DJ-1β) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1β. The structure of D. melanogaster DJ-1β is similar to that of human DJ-1, although two important residues in …

Szilard Engine Reversibility As Quantum Gate Function, F. Matthew Mihelic

Faculty Publications

A quantum gate is a logically and thermodynamically reversible situation that effects a unitary transformation of qubits of superimposed information, and essentially constitutes a situation for a reversible quantum decision. A quantum decision is a symmetry break, and the effect of the function of a Szilard engine is a symmetry break. A quantum gate is a situation in which a reversible quantum decision can be made, and so if a logically and thermodynamically reversible Szilard engine can be theoretically constructed then it would function as a quantum gate. While the traditionally theorized Szilard engine is not thermodynamically reversible, if one …