Biochemistry, Biophysics, and Structural Biology Commons^™

Role Of Syk In The Regulation Of Cytoskeleton And Stress Granules In Breast Cancer, Mariya Krisenko

Open Access Dissertations

The Syk protein-tyrosine kinase, a well-characterized modulator of immune recognition receptor signaling, also plays important, but poorly characterized, roles in tumor progression, acting as an inhibitor of cellular motility and metastasis in highly invasive cancer cells. Multiharmonic atomic force microscopy (AFM) was used to map nanomechanical properties of live MDA-MB-231 breast cancer cells either lacking or expressing Syk. The expression of Syk dramatically altered the cellular topography, reduced cell height, increased elasticity, increased viscosity, and allowed visualization of a more substantial microtubule network. The microtubules of Syk-expressing cells were more stable to nocodazole-induced depolymerization and were more highly acetylated than …

A Novel In Vivo Tumor Oxygen Profiling Assay: Combining Functional And Molecular Imaging With Multivariate Mathematical Modeling, Chung-Wein Lee

Open Access Dissertations

Purpose: The objective of this study is to develop and test a novel high spatio-temporal in vivo assay to quantify tumor oxygenation and hypoxia. The assay implements a biophysical model of oxygen transport to fuse parameters acquired from in vivo functional and molecular imaging modalities. ^ Introduction: Tumor hypoxia plays an important role in carcinogenesis. It triggers pathological angiogenesis to supply more oxygen to the tumor cells and promotes cancer cell metastasis. Preclinical and clinical evidence show that anti-angiogenic treatment is capable of normalizing the tumor vasculature both structurally and functionally. The resulting normalized vasculature provides a more efficient and …

Quantitative Mrna Detection With Advanced Nonlinear Microscopy, Jing Liu

Open Access Dissertations

Cell-specific information on quantity and localization of key mRNA transcripts in single-cell level are critical to the assessment of cancer risk, therapy efficacy, and effective prevention strategies. While current techniques are not capable to visualize single mRNA transcript beyond the diffraction limit. In this thesis, two nonlinear technologies, second harmonic super-resolution microscopy (SHaSM) and transient absorption microscopy (TAM), are developed to detect and quantify single Human edimer receptor 2 (Her2) mRNA transcripts. The SHaSM is used to detect single mRNA transcript beyond the diffraction limit, while the TAM is employed to detect mRNA without the interference of fluorescence background. The …

Structural And Biophysical Analysis Of The Proteasomal Deubiquitinase, Uch37, Marie Elizabeth Morrow

Open Access Dissertations

Ubiquitin carboxyl-terminal hydrolase 37, or UCH37, is a deubiquitinating enzyme associated with the 26S proteasome, the primary protein degradation machinery in eukaryotic cells. UCH37 is responsible for the disassembly of polymeric ubiquitin chains, or polyubiquitin, which have been ligated onto proteins in order to target them for degradation. The 26S utilizes two associated deubiquitinating enzymes, UCH37 and USP14, and one intrinsic, Rpn11, to remove polyubiquitin chains from substrate proteins as they are unfolded and translocated into the proteolytic core of the proteasome, where proteins are cleaved into small peptides and then released for recycling by the cell. UCH37 associates with …

The Effect Of Macromolecular Crowding On The Structure Of The Protein Complex Superoxide Dismutase, Ajith Rathnaweera Rajapaksha Mudalige

Open Access Dissertations

Biological environments contain between 7 - 40% macromolecules by volume. This reduces the available volume for macromolecules and elevates the osmotic pressure relative to pure water. Consequently, biological macromolecules in their native environments tend to adopt more compact and dehydrated conformations than those in vitro. This effect is referred to as macromolecular crowding and constitutes an important physical difference between native biological environments and the simple solutions in which biomolecules are usually studied.^ We used small angle scattering (SAS) to measure the effects of macromolecular crowding on the size of a protein complex, superoxide dismutase (SOD). Crowding was induced using …

Paralogous Genes In Arabidopsis Thaliana Contribute To Diversified Phenylpropanoid Metabolism, Li Yi

Open Access Dissertations

Significant evidence supports the idea that gene duplication drives the evolution of new gene function. Besides being silenced, duplicated genes can either neofunctionalize or subfunctionalize under selective pressure or neutral drift. Understanding the trajectory of how each gene is fixed and presumably provides added fitness remains difficult. Plant specialized metabolism provides an attractive platform to study the fixation of genes post duplication and how this process leads to the chemical diversity seen today. Specialized metabolites by definition are thought to be dispensable under normal growth conditions. Thus deleterious mutations occurring in paralogous genes that otherwise would be selected against in …

Analysis Of Chd Remodelers During Development: A Tale In Two Organisms, Brett Bishop

Open Access Dissertations

The correct development of different organisms requires the precise timing of genes important for development transitions. Organisms have recruited ATP-dependent chromatin remodelers to ensure the correct timing of gene expression during developmental transitions. Here I show how different CHD ATP-dependent chromatin remodelers regulate developmental transitions of different organisms. I show that PICKLE not only promotes H3K27me3 during development to repress developmental genes but also is targeted to these genes. The association of PICKLE to these genes suggests that both repression and H3K27me3 levels is a direct action of PICKLE on these loci. Using zebrafish as a model system, I show …

Biochemical Investigation Of The Ubiquitin Carboxyl-Terminal Hydrolase Family, Joseph Rashon Chaney

Open Access Dissertations

The proteasome is the machinery in eukaryotic cells that degrades protein and recycles the amino acids. Protein degradation is a highly regulated process which starts by the attachment of chains of ubiquitin, which serves as a tag that marks a protein for degradation. This function involves the work of several proteins at the proteasome that work either as ubiquitin chaperones, ubiquitin binders or cleave ubiquitin from the protein that is to be degraded. As this is a highly regulated process, various irregularities can have deleterious effects including the onset of disease, including cardiovascular, cancer, and neurological. ^ The focus of …

Novel Methods For Manipulating Ion Types In The Solution And Gas Phases For The Structural Analysis Of Biomolecules Using Mass Spectrometry, Christine M Fisher

Open Access Dissertations

Mass Spectrometry has become a valuable tool for the analysis of a variety of molecules, making it applicable to many fields. The advent of nanoelectrospray ionization (nESI) as a soft/low energy ionization technique has enabled the analysis of large, intact biomolecules. Most mass spectrometry experiments consist of three main steps: ionization, probe step(s), and mass analysis. The present work focuses on a variety of methods for altering ion types at various stages of the mass spectrometry experiment to affect ion fragmentation. Ion types can be manipulated in the solution/droplet phases using novel nESI emitters, generated from borosilicate theta capillaries. These …

Identification Of Perilipin 5 Interaction Partners, Emily E. Burton

Undergraduate Honors Thesis Projects

Perilipin 5 is one of the more recently discovered in the PAT protein family. Several studies on this protein have shown that it resides in multiple intracellular locations such as the endoplasmic reticulum, the cytosol, the mitochondria, and most interestingly on lipid droplets. These lipid droplets have proven not to be just clumps of fat, but to be very complex intracellular organelles, with a core of lipids surrounded by a phospholipid monolayer coated by specific proteins, one of those being perilipin 5. Some proteomic studies have revealed these lipid droplets to be involved in intracellular trafficking, signaling, cytoskeletal organization and …

Functional Genomic Analyses Of Switchgrass Developmental Processes, Nathan Palmer

Department of Biochemistry: Dissertations, Theses, and Student Research

Switchgrass (Panicum virgatum L.), a C_4-perennial grass species, is being developed as a bioenergy crop. Although much is known from a breeding perspective, there is limited information on the functional genomics of this crop, specifically regarding molecular mechanisms controlling aerial senescence, winter dormancy, and traits that confer winter hardiness. Using functional genomics to generate a transcriptional roadmap underpinning senescence and winter dormancy will provide researchers with a molecular understanding that can be applied to improve switchgrass germplasm.

In an initial study, a de novo assembly of the crown and rhizome transcriptome from an upland cultivar Summer was …

In Vivo Near Ir Fluorescent Imaging Of Hollow Polymeric Nanocapsules, Venkata Suresh Patthipati

Biological Sciences Theses & Dissertations

Medical Imaging has been an integral component in medical diagnosis and therapeutics. It is frequently used in conjunction with other treatment modalities and has been used for developing applications that range from cancer management to clinical practice. Nanotechnology and its applications are foreseen to have a profound impact on life sciences. Nanomaterial based imaging has a significant potential in addressing medical challenges such as tumor localization, targeted delivery of drugs and real time diagnosis and management of circulation pathology. In particular, polymer based nanoparticles are increasingly being developed as they can be customized according to the diagnostic and/or therapeutic requirements. …

Aggregatibacter Actinomycetemcomitans Cytolethal Distending Toxin Activates The Nlrp3 Inflammasome In Human Macrophages Leading To The Release Of Pro-Inflammatory Cytokines, Bruce J. Shenker, David M. Ojcius, Lisa P. Walker, Ali Zekavat, Monika Damek Scuron, Kathleen Boesze-Battaglia

All Dugoni School of Dentistry Faculty Articles

The cytolethal distending toxin (Cdt) is produced from a number of bacteria capable of causing infection and inflammatory disease. Our previous studies with Actinobacillus actinomycetemcomitans Cdt demonstrate not only that the active toxin subunit functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase but also that macrophages exposed to the toxin were stimulated to produce proinflammatory cytokines. We now demonstrate that the Cdt-induced proinflammatory response involves the activation of the NLRP3 inflammasome. Specific inhibitors and short hairpin RNA (shRNA) were employed to demonstrate requirements for NLRP3 and ASC as well as caspase-1. Furthermore, Cdt-mediated inflammasome activation is dependent upon upstream signals, including reactive …

Immunoassays Of Potential Cancer Biomarkers In Microfluidic Devices, Jayson Virola Pagaduan

Theses and Dissertations

Laboratory test results are important in making decisions regarding a patient's diagnosis and response to treatment. These tests often measure the biomarkers found in biological fluids such blood, urine, and saliva. Immunoassay is one type of laboratory test used to measure the level of biomarkers using specific antibodies. Microfluidics offer several advantages such as speed, small sample volume requirement, portability, integration, and automation. These advantages are motivating to develop microfluidic platforms of conventional laboratory tests. I have fabricated polymer microfluidic devices and developed immunoassays on-chip for potential cancer markers. Silicon template devices were fabricated using standard photolithographic techniques. The template …

Assessing Glucose Uptake Through The Yeast Hexose Transporter 1 (Hxt1), Adhiraj Roy, Angela D. Dement, Kyu Hong Cho, Jeong-Ho Kim

Biochemistry and Molecular Medicine Faculty Publications

The transport of glucose across the plasma membrane is mediated by members of the glucose transporter family. In this study, we investigated glucose uptake through the yeast hexose transporter 1 (Hxt1) by measuring incorporation of 2-NBDG, a non-metabolizable, fluorescent glucose analog, into the yeast Saccharomyces cerevisiae. We find that 2-NBDG is not incorporated into the hxt null strain lacking all glucose transporter genes and that this defect is rescued by expression of wild type Hxt1, but not of Hxt1 with mutations at the putative glucose-binding residues, inferred from the alignment of yeast and human glucose transporter sequences. Similarly, the …

Cis-Encoded Non-Coding Antisense Rnas In Streptococci And Other Low Gc Gram (+) Bacterial Pathogens, Kyu Hong Cho, Jeong-Ho Kim

Biochemistry and Molecular Medicine Faculty Publications

Due to recent advances of bioinformatics and high throughput sequencing technology, discovery of regulatory non-coding RNAs in bacteria has been increased to a great extent. Based on this bandwagon, many studies searching for trans-acting small non-coding RNAs in streptococci have been performed intensively, especially in the important human pathogen, group A and B streptococci. However, studies for cis-encoded non-coding antisense RNAs in streptococci have been scarce. A recent study shows antisense RNAs are involved in virulence gene regulation in group B streptococcus, S. agalactiae. This suggests antisense RNAs could have important roles in the pathogenesis of streptococcal …

Inorganic Arsenic-Induced Cellular Transformation Is Coupled With Genome Wide Changes In Chromatin Structure, Transcriptome And Splicing Patterns, Caitlyn Riedmann, Ye Ma, Manana Melikishvili, Steven Grason Godfrey, Zhuo Zhang, Kuey-Chu Chen, Eric C. Rouchka, Yvonne N. Fondufe-Mittendorf

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Arsenic (As) exposure is a significant worldwide environmental health concern. Low dose, chronic arsenic exposure has been associated with a higher than normal risk of skin, lung, and bladder cancer, as well as cardiovascular disease and diabetes. While arsenic-induced biological changes play a role in disease pathology, little is known about the dynamic cellular changes resulting from arsenic exposure and withdrawal.

RESULTS: In these studies, we sought to understand the molecular mechanisms behind the biological changes induced by arsenic exposure. A comprehensive global approach was employed to determine genome-wide changes to chromatin structure, transcriptome patterns and splicing patterns in …

Conditional Regulation Of Puf1p, Puf4p, And Puf5p Activity Alters Yhb1 Mrna Stability For A Rapid Response To Toxic Nitric Oxide Stress In Yeast, Joseph Russo, Wendy Olivas

Biology Department Faculty Works

Puf proteins regulate mRNA degradation and translation through interactions with 3' untranslated regions (UTRs). Such regulation provides an efficient method to rapidly alter protein production during cellular stress. YHB1 encodes the only protein to detoxify nitric oxide in yeast. Here we show that YHB1 mRNA is destabilized by Puf1p, Puf4p, and Puf5p through two overlapping Puf recognition elements (PREs) in the YHB1 3' UTR. Overexpression of any of the three Pufs is sufficient to fully rescue wild-type decay in the absence of other Pufs, and overexpression of Puf4p or Puf5p can enhance the rate of wild-type decay. YHB1 mRNA decay …

Adenovirus Entry From The Apical Surface Of Polarized Epithelia Is Facilitated By The Host Innate Immune Response, Poornima L.N. Kotha, Priyanka Sharma, Abimbola O. Kolawole, Ran Yan, Mahmoud Alghamri, Trisha L. Brockman, Julian Gomez-Cambronero, Julian Cambronero

Biological Sciences Faculty Publications

Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR), a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAR^Ex8 may be to facilitate host innate immunity. We show that IL-8, …

Relationship Between Heat-Labile Enterotoxin Secretion Capacity And Virulence In Wild Type Porcine-Origin Enterotoxigenic Escherichia Coli Strains, Prageeth Wijemanne, Jun Xing, Emil M. Berberov, David B. Marx, David H. Francis, Rodney A. Moxley

School of Veterinary and Biomedical Sciences: Faculty Publications

Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system(T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme- linked immunosorbent assay tomeasure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had …

Comparative Genomics Of Cluster O Mycobacteriophages, Steven G. Cresawn, Welkin H. Pope, Deborah Jacobs-Sera, Charles A. Bowman, Daniel A. Russell, Rebekah M. Dedrick, Tamarah Adair, Kirk R. Anders, Sarah Ball, David Bollivar, Caroline Breitenberger, Sandra H. Burnett, Kristen Butela, Deanna Byrnes, Sarah Carzo, Dr. Kathleen Cornely, Trevor Cross, Richard L. Daniels, David Dunbar, Ann M. Findley, Chris R. Gissendanner, Urszula P. Golebiewska, Grant A. Hartzog, J. Robert Hatherill, Lee E. Hughes, Chernoh S. Jalloh, Carla De Los Santos, Kevin Ekanem, Sphindile L. Khambule, Rodney A. King, Christina King-Smith, Karen Klyczek, Greg P. Krukonis, Christian Laing, Jonathan S. Lapin, A. Javier Lopez, Sipho M. Mkhwanazi, Sally D. Molloy, Deborah Moran, Vanisha Munsamy, Eddie Pacey, Ruth Plymale, Marianne Poxleitner, Nathan Reyna, Joel F. Schildbach, Joseph Stukey, Sarah E. Taylor, Vassie C. Ware, Amanda L. Wellmann, Daniel Westholm, Donna Wodarski, Michelle Zajko, Thabiso S. Zikalala, Roger W. Hendrix, Graham F. Hatfull

Chemistry & Biochemistry Faculty Publications

Mycobacteriophages – viruses of mycobacterial hosts – are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages – Corndog, Catdawg, Dylan, Firecracker, and YungJamal – designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8–9 strongly predicted SigA promoters and all five Cluster O …

The Hosoya Entropy Of A Graph, Abbe Mowshowitz, Matthias Dehmer

Publications and Research

This paper demonstrates properties of Hosoya entropy, a quantitative measure of graph complexity based on a decomposition of the vertices linked to partial Hosoya polynomials. Connections between the information content of a graph and Hosoya entropy are established, and the special case of Hosoya entropy of trees is investigated.

The Fragile Fiber1 Kinesin Contributes To Cortical Microtubule-Mediated Trafficking Of Cell Wall Components, Chuanmei Zhu, Anindya Ganguly, Tobias I. Baskin, Daniel D. Mcclosky, Charles T. Anderson, Cliff Foster, Kristoffer A. Meunier, Ruth Okamoto, Howard Berg, Ram Dixit

Biology Faculty Research

The cell wall consists of cellulose microfibrils embedded within a matrix of hemicellulose and pectin. Cellulose microfibrils are synthesized at the plasma membrane, whereas matrix polysaccharides are synthesized in the Golgi apparatus and secreted. The trafficking of vesicles containing cell wall components is thought to depend on actin-myosin. Here, we implicate microtubules in this process through studies of the kinesin-4 family member, Fragile Fiber1 (FRA1). In an fra1-5 knockout mutant, the expansion rate of the inflorescence stem is halved compared with the wild type along with the thickness of both primary and secondary cell walls. Nevertheless, cell walls in fra1-5 …

Environmental Microbes And Uveitis: Is Microbial Exposure Always Bad?, Chandirasegaran Massilamany, A. Gangaplara, J. Reddy

School of Veterinary and Biomedical Sciences: Faculty Publications

The eye generally is considered to be an immune-privileged organ, but this notion is being increasingly challenged as ocular antigens can be expressed in the generative lymphoid organs, resulting in attainment of self-tolerance. What triggers a break in this tolerant state is a fundamental question in autoimmunity research. The general belief is that exposure to environmental microbes can break self-tolerance in genetically susceptible individuals, leading to the induction of autoimmune responses. The molecular mimicry hypothesis has been proposed as one major mechanistic, pathway through which microbes, by generating crossreactive immune responses, can induce ocular damage of the kind that might …

The Effects Of Tetracycline And Ibuprofen On Common Duckweed, Lemna Minor, John Von Drasek

2015 Honors Council of Illinois Region Student Symposium

Pharmaceuticals and chemicals are finding their way into drinking water by means of household and industrial sewage. This study explores the effects of two common pharmaceuticals—tetracycline and ibuprofen—on the growth of Common Duckweed, Lemna minor. The number of duckweed per test tube for each treatment was compared after 37 days. Tetracycline was not found to effect clonal growth of duckweed. There was significant difference in duckweed count between the ibuprofen treatment and the control treatment with ibuprofen inhibiting the growth of the duckweed. It is possible that the tetracycline prevented the protein synthesis of any harmful bacteria growing with the …

Protein Sectors: Statistical Coupling Analysis Versus Conservation, Tiberiu Teşileanu, Lucy J. Colwell, Stanislas Leibler

Publications and Research

Statistical coupling analysis (SCA) is a method for analyzing multiple sequence alignments that was used to identify groups of coevolving residues termed “sectors”. The method applies spectral analysis to a matrix obtained by combining correlation information with sequence conservation. It has been asserted that the protein sectors identified by SCA are functionally significant, with different sectors controlling different biochemical properties of the protein. Here we reconsider the available experimental data and note that it involves almost exclusively proteins with a single sector. We show that in this case sequence conservation is the dominating factor in SCA, and can alone be …

The Infrared Spectrum Of The He-C2d2 Complex, N. Moazzen-Ahmadi, A. R. W. Mckellar, Berta Fernandez, David Farrelly

Chemistry and Biochemistry Faculty Publications

Spectra of the helium-acetylene complex are elusive because this weakly bound system lies close to the free rotor limit. Previously, limited assignments of He–C2D2 transitions in the R(0) region of the ν3 fundamental band (≈2440 cm⁻¹) were published. Here, new He–C2D2 infrared spectra of this band are obtained using a tunable optical parametric oscillator laser source to probe a pulsed supersonic slit jet expansion from a cooled nozzle, and the analysis is extended to the weaker and more difficult P(1) and R(1) regions. A term value approach is used to obtain a consistent set of “experimental” energy levels. …

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 …

Metabolome Searcher: A High Throughput Tool For Metabolite Identification And Metabolic Pathway Mapping Directly From Mass Spectrometry And Using Genome Restriction, A. Ranjitha Dhanasekaran, Jon L. Pearson, Balasubramanian Ganesan, Bart C. Weimer

Nutrition, Dietetics, and Food Sciences Faculty Publications

Background

Mass spectrometric analysis of microbial metabolism provides a long list of possible compounds. Restricting the identification of the possible compounds to those produced by the specific organism would benefit the identification process. Currently, identification of mass spectrometry (MS) data is commonly done using empirically derived compound databases. Unfortunately, most databases contain relatively few compounds, leaving long lists of unidentified molecules. Incorporating genome-encoded metabolism enables MS output identification that may not be included in databases. Using an organism’s genome as a database restricts metabolite identification to only those compounds that the organism can produce.

Results

To address the challenge of …

A Functional Chlorophyll Biosynthesis Pathway Identified In The Kleptoplastic Sea Slug, Elysia Chlorotica, Julie A. Schwartz

USF Tampa Graduate Theses and Dissertations

The sacoglossan sea slug, Elysia chlorotica, feeds upon and sequesters plastids from the heterokont alga, Vaucheria litorea, and maintains the metabolically active organelles for up to nine months under starvation conditions while utilizing the photosynthate to survive and reproduce. The photosynthetic pigment, chlorophyll a (Chla), is found in all oxygenic photosynthetic organisms and is responsible for capturing photons of light and converting them into chemical energy. Chlorophyll and its associated proteins involved in the light capturing process are subject to photo oxidative damage and must be continually replaced for ongoing photosynthesis to continue; however, genes encoding these proteins are present …