Biochemistry, Biophysics, and Structural Biology Commons^™

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 …

Inhibition Of P53 Dna Binding Function By The Mdm2 Acidic Domain, Brittany Lynne Cross

USF Tampa Graduate Theses and Dissertations

MDM2 regulates p53 predominantly by promoting p53 ubiquitination. However, ubiquitination-independent mechanisms of MDM2 have also been implicated. Here we show that MDM2 inhibits p53 DNA binding activity in vitro and in vivo. MDM2 binding promotes p53 to adopt a mutant-like conformation, losing reactivity to antibody Pab1620, while exposing the Pab240 epitope. The acidic domain of MDM2 is required to induce p53 conformational change and inhibit p53 DNA binding. ARF binding to the MDM2 acidic domain restores p53 wild type conformation and rescues DNA binding activity. Furthermore, histone methyl transferase SUV39H1 binding to the MDM2 acidic domain also restores p53 wild …

Analysis Of Ahr Expression And Stability In A Recombinant Yeast Model System, Sarah Elizabeth Cuccinello

USF Tampa Graduate Theses and Dissertations

The aryl hydrocarbon receptor (Ahr) and the aryl hydrocarbon receptor nuclear translocator (Arnt) are well characterized bHLH-PAS transcription factors shown to regulate expression of xenobiotic metabolism genes. Extensive study has shown that upon treatment with certain aromatic hydrocarbons, mammalian cells rapidly activate the Ahr signaling pathway in order to stimulate gene expression and attempt to metabolize the xenobiotic compounds. It has been shown that after DNA-binding, the Ahr but not the Arnt protein, is quickly eliminated from the nuclear compartment thereby attenuating the dose of gene regulation administered by the Ahr*Arnt transcription factor complex. Previous studies have implicated involvement of …

Lead Discovery And Optimization Strategies Towards The Development Of 4(1h)-Quinolones And 1,2,3,4-Tetrahydroacridone Analogs With Antimalarial Activity, Richard Matthew Cross

USF Tampa Graduate Theses and Dissertations

The goal of our research endeavor was to successfully employ modern lead discovery and optimization strategies towards the development and identification of compounds possessing antimalarial activity. Preliminary data from in vitro screening at the Walter Reed Army Institute of Research identified several chemotypes including 4(1H)-quinolones and 1,2,3,4-tetrahydroacridones to have potent antimalarial activities. Multiple synthetic routes were devised and implemented which enabled the rapid preparation and isolation of over 400 structurally diverse 4(1H)-quinolones and 1,2,3,4-tetrahydroacridones.

Our research towards discovering and optimizing antimalarials was inspired from the severe impact malaria has had on our planet especially on impoverished countries. There are over …

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 …

Role Of Protein Kinase C-Iota In Glioblastoma, Shraddha R. Desai

USF Tampa Graduate Theses and Dissertations

The focus of this research was to investigate the role of protein kinase C-iota (PKC-é) in the regulation of Bad function, a pro-apoptotic member of the Bcl-2 family and Cdk7 function, a master cell cycle regulator in glioblastoma.

The results were obtained from the human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-é co-localized and directly associated with Bad as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-é directly phosphorylated Bad at phospho specific residues, S112, S136 and S155 which in turn induced inactivation of Bad and disruption of …

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 …

Role Of Protein Kinase C-Iota In Neuroblastoma And The Effect Of Ica-1, A Novel Protein Kinase C-Iota Inhibitor On The Proliferation And Apoptosis Of Neuroblastoma Cells, Prajit P. Pillai

USF Tampa Graduate Theses and Dissertations

Protein Kinase C-iota (PKC-é), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-é confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of the dissertation was three-fold: First to study the role of PKC-é in the proliferation of neuroblastoma. Secondly, to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-é inhibitor in neuroblastoma cell proliferation and apoptosis. Finally, to analyze whether PKC-é could self-regulate …

Multivariate Anti-Inflammatory Approaches To Rescue Neurogenesis And Cognitive Function In Aged Animals, Sandra Antonieta Acosta

USF Tampa Graduate Theses and Dissertations

Studies have shown that there is a strong correlation between aging and neurodegenerative diseases. Aging is considered the number one risk factor to develop neuropathologies such as memory loss, senile dementia, Alzheimer's disease (AD), and Parkinson's disease. Neurodegenerative diseases tend to start during adulthood, and aggravate over time, making them difficult to prevent and to treat. In the Unites States, demographic studies by U.S. Bureau of the Census have determined that our aging population of >65 years is expected to increase from the present 35 million to 78 million in 2030. This would result, not only to an increase of …

Characterization Of Pregnancy Induced Cardiac Hypertrophy In Rats, Patricia Holden

Online Theses and Dissertations

During pregnancy, an increase in blood volume occurs to compensate for fetal development, resulting in cardiac hypertrophy. For the majority of women, this physiological cardiac hypertrophy resolves following pregnancy. Prolonged cardiac hypertrophy can lead to heart failure. We propose that by studying the biochemical mechanisms that mediate healthy cardiac remodeling associated with pregnancy, we will gain a better understanding of mechanisms involved in pathological cardiac hypertrophy. To determine the biochemical changes that occur during pregnancy induced cardiac remodeling, we subjected rats to timed matings and collected morphological and biochemical data from not pregnant, 19 days pregnant, and 24 hours postpartum. …

Quantitative Proteomics Analysis Of Differentially Expressed Proteins In Aβ(17-42) Treated Synaptosomes, Jaffer Mohammed

Online Theses and Dissertations

Oxidative stress has been associated in the pathogenesis of numerous diseases such as neurodegenerative disorders, ischemia, and cancer. The brain is susceptible to oxidative stress due to its high content of peroxidizable unsaturated fatty acids, high consumption of oxygen per unit weight, high levels of free radicals, and comparatively low levels of antioxidant defense systems. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) can react with biomolecules such as proteins, lipids, carbohydrates, DNA, and RNA, which can lead to oxidative damage, cellular dysfunction, and can ultimately cause cell death. Down syndrome (DS) is the most common form of chromosomal …

The Incidence Of Type-1 Diabetes In Nod Mice Is Modulated By Restricted Flora Not Germ-Free Conditions., Cecile King, Nora Sarvetnick

Journal Articles: Regenerative Medicine

In the NOD mouse, the incidence of type-1 diabetes is thought to be influenced by the degree of cleanliness of the mouse colony. Studies collectively demonstrate that exposure to bacterial antigen or infection in the neonatal period prevents diabetes [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], supporting the notion that immunostimulation can benefit the maturation of the postnatal immune system [11]. A widely accepted extrapolation from this data has been the notion that NOD mice maintained under germ-free conditions have an increased incidence of diabetes. However, evidence supporting this influential concept is surprisingly limited [12]. In this …

Conformation And Ion Transport Of Neuronal Uncoupling Proteins: Ucp2, Ucp4, And Ucp5, Tuan Hoang

Theses and Dissertations (Comprehensive)

Located in the inner mitochondrial membrane of brown adipose tissue, uncoupling protein -1 (UCP1) dissipates the proton electrochemical gradient, causing reduction in the rate of ATP synthesis, and generates heat by non-shivering thermogenesis. Three other UCP homologs (UCP2, UCP4 and UCP5), expressed in neurons, are suggested to have potential roles in the function and protection of the central nervous system (CNS). Up to date, structural information for UCPs still remains limited. Extensive biochemical studies on UCP2 have provided adequate evidence for its participation in proton and anion transport. So far, no functional studies in proteoliposome systems have been performed on …

A Microfluidic Device For Impedance Spectroscopy, Ahmet Can Sabuncu

Mechanical & Aerospace Engineering Theses & Dissertations

Recently, microfluidics has become a versatile tool to investigate cellular biology and to build novel biomedical devices. Dielectric spectroscopy, on the other hand, allows non-invasive probing of biological cells. Information on the cell membrane, cytoplasm, and nucleus can be obtained by dielectric spectroscopy provided that appropriate tools are used in specific frequency ranges. This dissertation includes fabrication, characterization, and testing of a simple microfluidic device to measure cell dielectric properties. The dielectric measurements are performed on human T-cell leukemia (Jurkat), mouse melanoma (B16), mouse hepatoma (Hepa), and human costal chondrocyte cells. Dielectric measurements consist of measuring the complex impedance of …

An Upper Limit For Macromolecular Crowding Effects, Andrew C. Miklos, Congang Li, Courtney D. Sorell, L. Andrew Lyon, Gary J. Pielak

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Background: Solutions containing high macromolecule concentrations are predicted to affect a number of protein properties compared to those properties in dilute solution. In cells, these macromolecular crowders have a large range of sizes and can occupy 30% or more of the available volume. We chose to study the stability and ps-ns internal dynamics of a globular protein whose radius is similar to 2 nm when crowded by a synthetic microgel composed of poly(N-isopropylacrylamide-co-acrylic acid) with particle radii of similar to 300 nm.

Results: Our studies revealed no change in protein rotational or ps-ns backbone dynamics and only mild …

Preventing Mother-To-Child Transmission Of Human Immunodeficiency Virus-1 (Hiv-1): Effects Of Intrapartum And Neonatal Single-Dose Nevirapine Prophylaxis And Subsequent Hiv-1 Drug Resistance At Antiretroviral Treatment Initiation, Amanda L. Harmon

CMC Senior Theses

The prevention of mother-to-child transmission is one of the most powerful tools in human immunodeficiency virus type 1 (HIV-1) prevention and has huge potential to improve both maternal and child health. In the absence of any preventative measures, infants born to and breastfed by their HIV-positive mothers have roughly a one-in-three chance of acquiring the infection themselves. HIV can be passed on from mother-to-child during pregnancy, during labor and delivery, and even after during breastfeeding.

Intrapartum and neonatal single-dose nevirapine (sd-NVP) is the foundation of preventing mother-to-child transmission in lower resource settings where it has been used alone or as …

Enhancement Of Reactive Oxygen Species Production And Chlamydial Infection By The Mitochondrial Nod-Like Family Member Nlrx1, Ali A. Abdul-Sater, Najwene Saïd-Sadier, Verissa M. Lam, Bhavni Singh, Matthew A. Pettengill, Fraser Soares, Ivan Tattoli, Simone Lipinski, Stephen E. Girardin, Philip Rosenstiel, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

Chlamydia trachomatis infections cause severe and irreversible damage that can lead to infertility and blindness in both males and females. Following infection of epithelial cells, Chlamydia induces production of reactive oxygen species (ROS). Unconventionally, Chlamydiae use ROS to their advantage by activating caspase-1, which contributes to chlamydial growth. NLRX1, a member of the Nod-like receptor family that translocates to the mitochondria, can augment ROS production from the mitochondria following Shigella flexneri infections. However, in general, ROS can also be produced by membrane-bound NADPH oxidases. Given the importance of ROS-induced caspase-1 activation in growth of the chlamydial vacuole, we investigated the …

Strategies To Resolve The Three-Dimensional Structure Of The Genome Of Small Single-Stranded Icosahedral Viruses, Eduardo Sanz Garcia

Theses and Dissertations

The aim of this study is the three-dimensional structural characterization of the genome packaging inside viral capsids via cryo-electron microscopy and three-dimensional reconstruction. The genome of some single-stranded viruses can be densely packaged within their capsid shells. Several stretches of the genome are known to adopt stable secondary structures, however, to date, little is known about the three-dimensional organization of the genome inside their capsid shells. Two techniques have been developed to facilitate the structural elucidation of genome packaging: the asymmetric random-model method, and the symmetry-mismatch, random model method. Both techniques were successfully tested with model and experimental data. The …

Chemical Vapor Deposition Of Silanes And Patterning On Silicon, Feng Zhang

Theses and Dissertations

Self assembled monolayers (SAMs) are widely used for surface modification. Alkylsilane monolayers are one of the most widely deposited and studied SAMs. My work focuses on the preparation, patterning, and application of alkysilane monolayers. 3-aminopropyltriethoxysilane (APTES) is one of the most popular silanes used to make active surfaces for surface modification. To possibly improve the surface physical properties and increase options for processing this material, I prepared and studied a series of amino silane surfaces on silicon/silicon dioxide from APTES and two other related silanes by chemical vapor deposition (CVD). I also explored CVD of 3-mercaptopropyltrimethoxysilane on silicon and quartz. …

Dual Inhibition Of Sodium-Mediated Proton And Calcium Efflux Triggers Non-Apoptotic Cell Death In Malignant Gliomas, William Harley, Candace Floyd, Tamara Dunn, Xiao-Dong Zhang, Tsung-Yu Chen, Manu Hegde, Hasan Palandoken, Michael H. Nantz, Leonardo Leon, K.L. Carraway Iii, Bruce Lyeth, Fredric A. Gorin

Chemistry and Biochemistry

Malignant glioma cells maintain an elevated intracellular pH (pH_i) within hypoxic–ischemic tumormicroenvironments through persistent activation of sodium–proton transport (McLean et al., 2000). Amiloride has been reported to selectively kill human malignant glioma cell lines but not primary astrocytes (Hegde et al., 2004). While amiloride reduces pH_i of malignant gliomas by inhibiting isoform 1 of sodium–proton exchange (NHE1), direct acidification was shown to be cytostatic rather than cytotoxic. At cytotoxic concentrations, amiloride has multiple drug targets including inhibition of NHE1 and sodium–calciumexchange. Amiloride's glioma cytotoxicity can be explained, at least in part, by dual inhibition of NHE1 and …

Campylobacter Ureolyticus: An Emerging Gastrointestinal Pathogen?, Susan Bullman, Daniel Corcoran, James O'Leary, Brigid Lucey, Deirdre Byrne, Roy D. Sleator

Department of Biological Sciences Publications

A total of 7194 faecal samples collected over a 1-year period from patients presenting with diarrhoea were screened for Campylobacter spp. using EntericBios, a multiplex-PCR system. Of 349 Campylobacter-positive samples, 23.8% were shown to be Campylobacter ureolyticus, using a combination of 16S rRNA gene analysis and highly specific primers targeting the HSP60 gene of this organism. This is, to the best of our knowledge, the first report of C. ureolyticus in the faeces of patients presenting with gastroenteritis and may suggest a role for this organism as an emerging enteric pathogen.

Measurements Of Gas‐Phase Inorganic And Organic Acids From Biomass Fires By Negative‐Ion Proton‐Transfer Chemical‐Ionization Mass Spectrometry, P. Veres, James M. Roberts, I. R. Burling, C. Warneke, Joost De Gouw, Robert J. Yokelson

Chemistry and Biochemistry Faculty Publications

[1] Emissions from 34 laboratory biomass fires were investigated at the combustion facility of the U.S. Department of Agriculture Fire Sciences Laboratory in Missoula, Montana. Gas-phase organic and inorganic acids were quantified using negative-ion proton-transfer chemical-ionization mass spectrometry (NI-PT-CIMS), open-path Fourier transform infrared spectroscopy (OP-FTIR), and proton-transfer-reaction mass spectrometry (PTR-MS). NI-PT-CIMS is a novel technique that measures the mass-to-charge ratio (m/z) of ions generated from reactions of acetate (CH₃C(O)O⁻) ions with inorganic and organic acids. The emission ratios for various important reactive acids with respect to CO were determined. Emission ratios for isocyanic acid (HNCO), 1,2 …

Genotype And Breed Trend Influences On Citric Acid And Coagulation Times Of Raw Milk, Melissa Looney

Dairy Science

The objective of the study was to determine if citric acid levels measured in milk was related to genetic variants identified in Holstein and Jersey cows. The data used were milk samples collected from both Holstein and Jersey cows at Cal Poly, San Luis Obispo. Citric acid levels and other constituents were measured using FTIR methods with the FOSS MilkoscanTM FT2 on each sample. Genotypes were obtained for the DGAT 1 locus using polymerase chain reaction and an enzymatic digestion using the MWO I restriction enzyme. Observations were obtained on 13 Holsteins and 12 Jersey cows. Results indicated that citric …

Lateral Blood Flow Velocity Estimation Based On Ultrasound Speckle Size Change With Scan Velocity, Tiantian Xu, Gregory R. Bashford

Biomedical Imaging and Biosignal Analysis Laboratory

Conventional (Doppler-based) blood flow velocity measurement methods using ultrasound are capable of resolving the axial component (i.e., that aligned with the ultrasound propagation direction) of the blood flow velocity vector. However, these methods are incapable of detecting blood flow in the direction normal to the ultrasound beam. In addition, these methods require repeated pulse-echo interrogation at the same spatial location. A new method has been introduced which estimates the lateral component of blood flow within a single image frame using the observation that the speckle pattern corresponding to blood reflectors (typically red blood cells) stretches (i.e., is smeared) if the …

Development Of A Genetic Modification System In Clostridium Scatologenes Atcc 25775 For Generation Of Mutants, Prasanna Tamarapu Parthasarathy

Masters Theses & Specialist Projects

3-Methyl indole (3-MI) is a malodorant in food and animal waste and Clostridium scatologenes ATCC 25775 is the model organism for the study of 3-MI production. 3-MI is an anaerobic degradation product of L-tryptophan and can cause pulmonary disorders and death in cattle and goats. To elucidate the 3-MI biosynthesis pathway and the underlying genes, it is necessary to develop a system to allow genetic modification in Clostridium scatologenes ATCC 25775. Bacteriophages and transposons are useful tools to achieve this goal. Isolation of Clostridium scatologenes ATCC 25775 bacteriophage was attempted by prophage induction and enrichments using environmental sources. To induce …

[Accepted Article Manuscript Version (Postprint)] Pex5 Mutants That Differentially Disrupt Pts1 And Pts2 Peroxisomal Matrix Protein Import In Arabidopsis, Bibi Khan, Bethany Zolman

Biology Department Faculty Works

PEX5 and PEX7 are receptors required for the import of peroxisome-bound proteins containing one of two peroxisomal targeting signals (PTS1 or PTS2). To better understand the role of PEX5 in plant peroxisomal import, we characterized the Arabidopsis (Arabidopsis thaliana) pex5-10 mutant, which has a T-DNA insertion in exon 5 of the PEX5 gene. Sequencing results revealed that exon 5, along with the T-DNA, is removed in this mutant, resulting in a truncated pex5 protein. The pex5-10 mutant has germination defects and is completely dependent on exogenous Suc for early seedling establishment, based on poor utilization of seed-storage fatty acids. This …

Expression Of Functional Bacterial Undecaprenyl Pyrophosphate Synthase In The Yeast Rer2{Delta} Mutant And Cho Cells., Jeffrey S. Rush, Sergey Matveev, Ziqiang Guan, Christian R H Raetz, C J Waechter

Molecular and Cellular Biochemistry Faculty Publications

During evolution the average chain length of polyisoprenoid glycosyl carrier lipids increased from C55 (prokaryotes) to C75 (yeast) to C95 (mammalian cells). In this study, the ability of the E. coli enzyme, undecaprenyl pyrophosphate synthase (UPPS), to complement the loss of the yeast cis-isoprenyltransferase in the rer2Δ mutant was tested to determine if (55)dolichyl phosphate (Dol-P) could functionally substitute in the protein N-glycosylation pathway for (75)Dol-P, the normal isoprenologue synthesized in S. cerevisiae. First, expression of UPPS in the yeast mutant was found to complement the growth and the hypoglycosylation of carboxypeptidase Y defects suggesting that the (55)polyprenyl-P-P intermediate was …

Applications Of Site-Specific Recombination Systems In Transgene Expression And Marker Gene Removal, Mehmet Aydin Akbudak

Graduate Theses and Dissertations

Site Specific Recombination systems, such as FLP-FRT and Cre-lox, have been successfully used for site-specific gene integration and marker-gene deletion in plant systems. They are very useful tools in the integration of single-copy full-length transgene cassettes into the genome because the transgene integration via conventional methods often generate multi-copy locus. Such complex locus containing direct and inverted repeats of full-length and truncated copies of the transgene cassette generate aberrant RNA resulting in gene silencing. Therefore, for stable gene expression, a single copy transgene locus is preferred. However, even single copy locus sometimes succumbs to gene silencing. Although the mechanism is …

Kinetic, Mechanistic, And Structural Investigation Of Features Controlling Stereoselectivity Of (R)- And (S)-Hydroxypropyl Com Dehydrogenases From Xanthobacter Autrophicus Strain Py2, Dariusz Adam Sliwa

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Enantiopure alcohols are valuable intermediates in fine organic synthesis, in particular for preparation of biologically active compounds. The necessity of preparing single enantiomer drugs in an optically pure form has triggered much research, especially in the pharmaceutical industry. The biocatalytical production of chiral alcohols by alcohol dehydrogenase enzymes is characterized by the asymmetric reduction of the corresponding ketones, usually with high degree of stereoselectivity. The commercial value of the enzymes as stereoselective biocatalysts has been a significant driving force in understanding features that control their mechanism of catalysis and stereoselectivity. This work focuses on two enantiocomplementary dehydrogenase enzymes ((R …

Production, Purification And Crystallization Of Membrane Integrated Multimeric Bax, Adelbert Mark Villoso

UNLV Theses, Dissertations, Professional Papers, and Capstones

Apoptosis, or programmed cell death, is a vital process intimately involved in the embryonic development and homeostatic maintenance of all multicellular organisms. The committing step to apoptosis is regulated by a key protein, Bax, and its ability to integrate and form a pore structure at the outer mitochondrial membrane.

Unfortunately, the molecular details of apoptosis remain largely unclear due to the lack of structural data of integral membrane (IM) Bax. Experimental limitations of membrane protein production have slowed the pursuit of an IM-Bax structure simply because standard protocols for producing recombinant IM-Bax are inefficient in producing adequate quantities of IM-Bax …