Biochemistry Commons^™

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 …

Exploring The Effect Of Climate Change On Biological Systems, Nardos Sori

Chemistry & Biochemistry Theses & Dissertations

The present and potential future effect of global warming on the ecosystem has brought climate change to the forefront of scientific inquiry and discussion. For our investigation, we selected two organisms, one from cyanobacteria and one from a cereal plant to determine how climate change may impact these biological systems. The study involved understanding the physiological and adaptive responses at both the genetic and protein function levels to counteract environmental stresses. An increase in atmospheric carbon dioxide is a key factor in global climate change and can lead to alterations in ocean chemistry. Cyanobacteria are important, ancient and ubiquitous organisms …

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 …

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, …

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 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 …

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. …

Identification And Quantification Of Gaseous Organic Compounds Emitted From Biomass Burning Using Two-Dimensional Gas Chromatography–Time-Of-Flight Mass Spectrometry, L. E. Hatch, W. Luo, J. F. Pankow, Robert Yokelson, C. Stockwell, K. C. Barsanti

Chemistry and Biochemistry Faculty Publications

The current understanding of secondary organic aerosol (SOA) formation within biomass burning (BB) plumes is limited by the incomplete identification and quantification of the non-methane organic compounds (NMOCs) emitted from such fires. Gaseous organic compounds were collected on sorbent cartridges during laboratory burns as part of the fourth Fire Lab at Missoula Experiment (FLAME-4) and analyzed by two-dimensional gas chromatography-time-of-flight mass spectrometry (GC x GC-ToFMS). The sensitivity and resolving power of GC x GC-ToFMS allowed the acquisition of the most extensive data set of BB NMOCs to date, with measurements from 708 positively or tentatively identified compounds. Estimated emission factors …

The Exchanged Ef-Hands In Calmodulin And Troponin C Chimeras Impair The Ca2+-Induced Hydrophobicity And Alter The Interaction With Orai1: A Spectroscopic, Thermodynamic And Kinetic Study, Drake Jensen, Nicole Reynolds, Ya-Ping Yang, Shubha Shakya, Zhi-Qiang Wang, Dennis J. Stuehr, Chin-Chuan Wei

Chemistry Faculty Research, Scholarship, and Creative Activity

Background

Calmodulin (CaM) plays an important role in Ca²⁺-dependent signal transduction. Ca²⁺ binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. CaM regulates a Ca²⁺-dependent inactivation process in store-operated Ca²⁺entry, by interacting Orai1. To understand the relationship between Ca²⁺-induced hydrophobicity and CaM/Orai interaction, chimera proteins constructed by exchanging EF-hands of CaM with those of Troponin C (TnC) are used as an informative probe to better understand the functionality of each EF-hand.

Results

ANS was used to assess the context of the induced …

Gamma-Aapeptides As A New Class Of Peptidomimetics: Synthesis, Structures, And Functions, Haifan Wu

USF Tampa Graduate Theses and Dissertations

Peptidomimetics are synthetic oligomers that resemble the activities of peptides. Their advantages over peptides include high stability towards proteolysis and enormous chemical diversity. Over the past two decades, there have been extensive efforts to develop peptide mimics, such as beta-peptides, peptoids, D-peptides, etc. The research on peptidomimetics have led to many important applications in both medicinal and material science. In order to explore new functions, the discovery of peptidomimetics with novel frameworks is essential. We reported the synthesis and evaluation of a new class of peptidomimetics, termed as gamma-AApeptides. Previous studies of gamma-AApeptides have revealed that gamma-AApeptides are highly resistant …

Functional Expression And Characterization Of A Truncated Candida Antarctica Lipase B In Yeast, Samantha Robinson

Theses and Dissertations

Candida antarctica lipase B is an enzyme capable of hydrolyzing ester bonds in a lipid-water interface with potential to become a useful biocatalyst for production of biodiesel fuel. Enzymatically catalyzing the transesterification of triacylglycerides to produce fatty acid alkyl esters (biodiesel) is an environmentally cleaner alternative to the current process which utilizes chemical reagents as catalysts. Optimization and development of strains to express highly active, stable, large quantities of these biocatalysts is essential for inexpensive production of biodiesel. Additionally, lipase enzymes can be immobilized onto a resin, eliminating the need to recover the catalyst from the product and allowing multiple …

Intramolecular S···O Chalcogen Bond As Stabilizing Factor In Geometry Of Substituted Phenyl-Sf3 Molecules, Vincent De Paul Nziko, Steve Scheiner

Chemistry and Biochemistry Faculty Publications

Density functional methods are used to examine the geometries and energetics of molecules containing a phenyl ring joined to the trigonal bipyramidal SF3 framework. The phenyl ring has a strong preference for an equatorial position. This preference remains when one or two ether -CH2OCH3 groups are added to the phenyl ring, ortho to SF3, wherein an apical structure lies nearly 30 kcal/mol higher in energy. Whether equatorial or apical, the molecule is stabilized by a S···O chalcogen bond, sometimes augmented by CH···F or CH···O H-bonds. The strength of the intramolecular S···O bond is estimated to lie in the range between …

Characterization Of Ranbpm Molecular Determinants That Control Its Subcellular Localization, Louisa M Salemi, Sandra O Loureiro, Caroline Schild-Poulter

Biochemistry Publications

RanBPM/RanBP9 is a ubiquitous, nucleocytoplasmic protein that is part of an evolutionary conserved E3 ubiquitin ligase complex whose function and targets in mammals are still unknown. RanBPM itself has been implicated in various cellular processes that involve both nuclear and cytoplasmic functions. However, to date, little is known about how RanBPM subcellular localization is regulated. We have conducted a systematic analysis of RanBPM regions that control its subcellular localization using RanBPM shRNA cells to examine ectopic RanBPM mutant subcellular localization without interference from the endogenously expressed protein. We show that several domains and motifs regulate RanBPM nuclear and cytoplasmic localization. …

The Artificial Divide Between Biomedical Sciences And Clinical Medicine In Undergraduate Medical Education, Abdul Rehman, Noor Ul-Ain Baloch, Muhammad Awais

Department of Biological & Biomedical Sciences

No abstract provided.

Proline Metabolism Increases Katg Expression And Oxidative Stress Resistance In Escherichia Coli, Lu Zhang, James R. Alfano, Donald F. Becker

Department of Biochemistry: Faculty Publications

The oxidation of L-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and _1-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the …

Conformational Heterogeneity And Exchange Within The Junction Of The Human U2-U6 Snrna Complex Measured By 19f Nmr Spectroscopy, Caijie Zhao

Dissertations, Theses, and Capstone Projects

Pre-mRNA splicing, the process by which non-coding sequences are removed and coding sequences are ligated, plays an important role in the maturation of precursor messenger RNA molecules prior to their translation into proteins. In eukaryotes, splicing is catalyzed by the spliceosome, a large and dynamic nuclear macromolecular "machine" that comprises five small nuclear (sn)RNA molecules and more than one hundred proteins. Among the RNA components, the complex formed between the U2 and U6 snRNA molecules is implicated in catalytic activity. In this dissertation, I have used ¹⁹F NMR techniques to characterize the conformation ("fold") and dynamics of a protein-free …

Synthesis Of Bioinspired Carbohydrate Amphiphiles That Promote And Inhibit Biofilms, Eric L. Dane, Alicia E. Ballok, George A. O'Toole, Mark W. Grinstaff

Dartmouth Scholarship

The synthesis and characterization of a new class of bioinspired carbohydrate amphiphiles that modulate Pseudomonas aeruginosa biofilm formation are reported. The carbohydrate head is an enantiopure poly-amido-saccharide (PAS) prepared by a controlled anionic polymerization of β-lactam monomers derived from either glucose or galactose. The supramolecular assemblies formed by PAS amphiphiles are investigated in solution using fluorescence assays and dynamic light scattering. Dried samples are investigated using X-ray, infrared spectroscopy, and transmission electron microscopy. Additionally, the amphiphiles are evaluated for their ability to modulate biofilm formation by the Gram-negative bacterium Pseudomonas aeruginosa. Remarkably, from a library of eight amphiphiles, we identify …

Protein Motions Control Activity In Biologically Important Phosphatases, Ryan Hirschi, Gwen Moise

Research on Capitol Hill

No abstract provided.

Characterization Of Biomass Burning Emissions From Cooking Fires, Peat, Crop Residue, And Other Fuels With High-Resolution Proton-Transfer-Reaction Time-Of-Flight Mass Spectrometry, C. Stockwell, P. Veres, J. Williams

Chemistry and Biochemistry Faculty Publications

We deployed a high-resolution proton-transferreaction time-of-flight mass spectrometer (PTR-TOF-MS) to measure biomass-burning emissions from peat, crop residue, cooking fires, and many other fire types during the fourth Fire Lab at Missoula Experiment (FLAME-4) laboratory campaign. A combination of gas standard calibrations and composition sensitive, mass-dependent calibration curves was applied to quantify gas-phase non-methane organic compounds (NMOCs) observed in the complex mixture of fire emissions. We used several approaches to assign the best identities to most major “exact masses”, including many high molecular mass species. Using these methods, approximately 80–96% of the total NMOC mass detected by the PTR-TOFMS and Fourier …

Observations And Analysis Of Organic Aerosol Evolution In Some Prescribed Fire Smoke Plumes, A. A. May, T. Lee, G. R. Mcmeeking, Sheryl Kashi Akagi, A. P. Sullivan, S. P. Urbanski, Robert Yokelson, S. M. Kreidenweis

Chemistry and Biochemistry Faculty Publications

Open biomass burning is a significant source of primary air pollutants such as particulate matter and non-methane organic gases. However, the physical and chemical atmospheric processing of these emissions during transport is poorly understood. Atmospheric 5 transformations of biomass burning emissions have been investigated in environmental chambers, but there have been limited opportunities to investigate these transformations in the atmosphere. In this study, we deployed a suite of real-time instrumentation on a Twin Otter aircraft to sample smoke from prescribed fires in South Carolina, conducting measurements at both the source and downwind to character10 ize smoke evolution with atmospheric aging. …

Identification And Characterization Of N-Acyltransferase Enzymes That Are Involved In The Biosynthesis Of Fatty Acid Amides, Daniel Robert Dempsey

USF Tampa Graduate Theses and Dissertations

Fatty acid amides are an emerging family of bioactive lipids that consists of N-acylethanolamines, N-acylarylalkylamides, N-acylglycines, N-acyl amino acids, N-monoacylpolyamides, and primary fatty acid amides. Short chain fatty acid amides are products of inactivated biogenic amines such as dopamine, histamine, octopamine, and serotonin, whereas long chain fatty acid amides have been implicated in a number of physiological process such as the perception and inhibition of chronic pain through binding to their specific receptors. The most famous; therefore, the most studied long chain fatty acid amide is anandamide or also known as N-arachidonylethanolamine. The biosynthesis …

Accidental Poison: Analysis Of 1,4-Butanediol In A Popular Children's Arts And Crafts Toy, Christopher R. Dockery, John H. Shugart, Jonathan D. Parker, William J. Lawson

Faculty Articles

A popular children's arts and crafts toy made international headlines when it was discovered that 1,4-butanediol had been substituted into some of its formulations. The chemical 1,4-butanediol rapidly converts to the date-rape drug g-hydroxybutyric acid (GHB) upon ingestion and resulted in the accidental poisoning of some children. In this experiment, students extract 1,4-butanediol from commercially available toy beads (or prepared simulations) using their knowledge of pharmaceutical chemistry, solubility, etc. Using case studies available in the literature, students develop a quantitative analysis experiment for 1,4-butanediol extracted from the toy beads and, through approximation, relate the extractable amount of 1,4-butanediol to dose-response …

An Investigation Of The Effect Of Mn2+ On The Fth1 Ire-Irp Complex, Eric Thomas Mendenhall

Theses, Dissertations and Capstones

Iron is a widely distributed metal found in almost every physiological system in nature, thus being an essential part of life. Iron homeostasis in the human body must be tightly regulated as iron is both beneficial and harmful to different cellular processes. Ferritin is a protein that has a prominent role in maintaining iron homeostasis via iron sequestration. Additionally, this protein itself is regulated by the Iron Responsive Element (IRE) and its interaction with an apoaconitase protein or the Iron Regulatory Protein (IRP). The binding of the IRP to this IRE, located on the 5’ untranslated stem loop region of …

Glycation Of Lysozyme, Wisam Talib Joudah

Theses, Dissertations and Capstones

For this research project, I propose a method for the analysis of glycated lysozyme using Matrix Associated Laser Desorption / Ionization-Time of Flight Mass spectrometry (MALDI-TOF MS). Glycation or "Maillard reaction" is a non-enzymatic reaction that occurs between amino acid residues, usually primary and secondary amines, of proteins and the carbonyl group of reducing sugars to yield Schiff bases. The glycation mechanism occurs between the carbonyl group of the reducing sugars and a primary amine by nucleophilic addition, resulting in the formation of a Schiff base. The Schiff base can then rearrange into the more stable Amadori product which is …

Investigation Into The Biological Importance And Function Of Proinsulin C-Peptide, Christina L. Newsome

Theses, Dissertations and Capstones

The C-peptide of insulin was thought to be biologically inactive, but recent studies have shown that the C-peptide causes multiple molecular and physiological effects. Evidence has shown that C-peptide binds to a cell surface receptor, probably a G-protein coupled receptor, and that the COOH-terminal pentapeptide is essential for binding and constitutes an active site. For a further understanding of the detailed nature of the physiological effects of C-peptide, the receptor structure needs to be determined. We designed an affinity column using C-peptide to try and gain a better understanding of the biological effects by examining what proteins the affinity column …

Atrial Fibrillation: Biophysics, Molecular Mechanisms, And Novel Therapies., Alexey V. Glukhov, Leonid V. Rosenshtraukh, Anamika Bhargava, Michele Miragoli, Bas J. D. Boukens

Anatomy and Regenerative Biology Faculty Publications

No abstract provided.