Biochemistry Commons^™

Electrogenerated Chemiluminescence Study Of Semiconductor Nanoparticles Towards Sensitive Detection Of Biomolecules, Yiliyasi Wusimanjiang

Dissertations

The main focus of this dissertation is to unfold the fundamental aspects of electrogenerated chemiluminescence (ECL) generation from semiconductor nanoparticles (also known as quantum dots or QDs) within different ECL systems. The ECL and photo-physical interactions between the CdTe QDs (λ_emission= ~760 nm) and the CdSe QDs (λ_emission= ~550 nm), as well as the effects of carbon nanotubes on ECL of QDs were separately investigated. Optimum experimental conditions for peptide bond formation on an electrode surface through EDC (1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride)/NHS (N-Hydroxysulfosuccinimide) coupling were also revealed using cyclic voltammetry technique. Based on the information …

A Novel Role Of Human Dna Damage Checkpoint Protein Atr In Suppressing Ca2+ Overload-Induced Parp1-Mediated Necrosis, Hui Wang-Heaton

Electronic Theses and Dissertations

Ataxia telangiectasia and Rad3-related (ATR) is well known for its regulatory role in DNA damage responses (DDR) as a checkpoint kinase that phosphorylates hundreds of protein substrates. However, its role in cellular non-DNA damage stress responses (NDDR) is unknown. Necrosis is one form of cell death and traditionally has been regarded as a passive and uncontrolled cell death. Recently, evidence has emerged to support the concept that necrosis also may occur in a programmed manner and that PARP1 can be a mediator. Active poly (ADP-ribose) polymerase 1 (PARP1) hydrolyzes nicotinamide adenine dinucleotide (NAD⁺) to produce poly (ADP-ribose) (PAR) …

The Role Of Phosphorylation In Pam2 Motif-Containing Proteins Mediated Messenger Rna Deadenylation, Kai-Lieh Huang

Dissertations and Theses (Open Access)

Phosphorylation regulates many cellular processes. However, its role in mRNA deadenylation, a process to remove poly adenosines from the mature mRNA 3’ end tail, is unclear. The length of poly(A) tail determines mRNA stability and translation efficiency. Poly(A)-binding protein (PABP), which binds to newly synthesized poly(A) tails homogeneously and is known as a scaffold protein for PAM2 motif-containing proteins, plays a pivotal role in the shortening of poly (A) tails. This study is to examine the role of phosphorylation of PAM2 motif–containing proteins in regulating their interactions with PABP and mRNA deadenylation function.

The PAM2 motif, a region required for …

Membrane Chromatography For Bioseparations: Ligand Design And Optimization, Zizhao Liu

Graduate Theses and Dissertations

Membrane chromatography, or membrane adsorber, represents an attractive alternative to conventional packed bed chromatography used in downstream processing. Membrane chromatography has many advantages, including high productivity, low buffer consumption and ease to scale up. This doctoral dissertation focuses on developing novel polymeric ligands for protein separations using membrane chromatography. Atom transfer radical polymerization (ATRP), known as a controlled radical polymerization technique, has been used to control the architecture of grafted polymeric ligands. The center theme of this dissertation is to develop new polymeric ligands and investigate how the polymer’s property (e.g. flexibility, hydrophobicity) and architecture (e.g. chain density, chain length) …

Interactions In The Cpsrp Dependent Targeting Of Light Harvesting Chlorophyll Binding Protein To The Thylakoid Membrane, Rory Henderson

Graduate Theses and Dissertations

Targeting of proteins is a critical component of cellular function. A universally conserved targeting system of the cytosol utilizes a signal recognition particle (SRP) to target many proteins contranslationally to the endoplasmic reticulum in eukaryotes or the inner membrane in prokaryotes. A homologous SRP system exists in the chloroplast that delivers light harvesting chlorophyll binding proteins (LHCP) to they thylakoid membrane. The chloroplast SRP (cpSRP) is a heterodimer composed of a novel 43 kDa subunit and a 54 kDa subunit homologous to a component of the SRP system, SRP54. Many details regarding the interactions between the proteins of the cpSRP …

Influence Of Ph And Acidic Side Chain Charges On The Behavior Of Designed Model Peptides In Lipid Bilayer Membranes, Venkatesan Rajagopalan

Graduate Theses and Dissertations

The molecular properties of transmembrane proteins and their interactions with lipids regulate biological function. Of particular interest are interfacial aromatic residues and charged residues in the core helix whose functions range from stabilizing the native structure to regulating ion channels. This dissertation addresses the pH dependence and influence of potentially negatively charged tyrosine, glutamic acid or aspartic acid side chains. We have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) as favorable host peptide framework. We have substituted W5 with Tyr (Y5GWALP23) and Leu residues with Glu (L12E, L14E or L16E) or Asp (L14D or L16D), and have incorporated specific 2H-labeled alanine residues within the …

Bioorthogonal Reactions: Synthesis And Evaluation Of Different Ligands In Copper Catalyzed Azide-Alkyne1,3-Dipolar Cycloaddition (Cuaac), Zainab Abdullah Almansaf

Graduate Theses and Dissertations

The Copper CatalyzedAzide-Alkyne1,3-Dipolar Cycloaddition (CuAAC) reaction has unique features that qualify it to be one of the best click reactions. Its applications have been shown in different aspects and for multiple purposes. The oxidative degradation of biological systems (labile proteins and live cells) is, however, generally recognized as the major problem when using this reaction in living systems. Reactive oxidation species can be easily produced in the presence of copper(II), ascorbate and air, and this is the main cause of toxicity. However, the uses of ligands have shown a major impact on reducing copper toxicity, protecting Cu(I) from the redox …

Characterization Of The Substrate Interactions And Regulation Of Protein Arginine Methyltransferase, Yalemi Morales

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Protein arginine methylation is a posttranslational modification catalyzed by the family of proteins known as the protein arginine methyltransferases (PRMTs). Thousands of methylated arginines have been found in mammalian cells. Many targets of arginine regulation are involved in important cellular processes like transcription, RNA transport and processing, translation, cellular signaling, and DNA repair. Since PRMT dysregulation has been linked to a variety of disease states, understanding how the activity of the PRMTs is regulated is of paramount importance. PRMT1 is the predominant PRMT, responsible for about 85% of all arginine methylation in cells, but very little is known about how …

Conformationally Superarmed S-Ethyl Glycosyl Donors As Effective Building Blocks For Chemoselective Oligosaccharide Synthesis In One Pot, Mithila Bandara, Jagodige Yasomanee, Nigam Rath, Christian Pedersen, Mikael Bols, Alexei Demchenko

Chemistry & Biochemistry Faculty Works

A new series of superarmed glycosyl donors has been investigated. It was demonstrated that the S-ethyl leaving group allows for high reactivity, which is much higher than that of equally equipped S-phenyl glycosyl donors that were previously investigated by our groups. The superarmed S-ethyl glycosyl donors equipped with a 2-O-benzoyl group gave complete β-stereoselectivity. Utility of the new glycosyl donors has been demonstrated in a one-pot one-addition oligosaccharide synthesis with all of the reaction components present from the beginning.

The Complex Evolutionary History Of Aminoacyl-Trna Synthetases, Anargyros Chaliotis, Panayotis Vlastaridis, Dimitris Mossialos, Michael Ibba, Hubert D. Becker, Constantinos Stathopoulos, Grigorios D. Amoutzias

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNA synthetases (AARSs) are a superfamily of enzymes responsible for the faithful translation of the genetic code and have lately become a prominent target for synthetic biologists. Our large-scale analysis of >2500 prokaryotic genomes reveals the complex evolutionary history of these enzymes and their paralogs, in which horizontal gene transfer played an important role. These results show that a widespread belief in the evolutionary stability of this superfamily is misconceived. Although AlaRS, GlyRS, LeuRS, IleRS, ValRS are the most stable members of the family, GluRS, LysRS and CysRS often have paralogs, whereas AsnRS, GlnRS, PylRS and SepRS are often absent …

Nucleoside-Diphosphate-Kinase Of P. Gingivalis Is Secreted From Epithelial Cells In The Absence Of A Leader Sequence Through A Pannexin-1 Interactome, Kalina Atanasova, Jungnam Lee, Joann Roberts, Kyulim Lee, David M. Ojcius, Özlem Yilmaz

All Dugoni School of Dentistry Faculty Articles

Nucleoside-diphosphate-kinases (NDKs) are leaderless, multifunctional enzymes. The mode(s) of NDK secretion is currently undefined, while extracellular translocation of bacterial NDKs is critical for avoidance of host pathogen clearance by opportunistic pathogens such as Porphyromonas gingivalis. P. gingivalis-NDK during infection inhibits extracellular-ATP (eATP)/P2X7-receptor mediated cell death in gingival epithelial cells (GECs) via eATP hydrolysis. Furthermore, depletion of pannexin-1-hemichannel (PNX1) coupled with P2X7-receptor blocks the infection-induced eATP release in GECs, and P. gingivalis-NDK impacts this pathway. Ultrastructural and confocal microscopy of P. gingivalis-co-cultured GECs or green-fluorescent-protein (GFP)-P. gingivalis-NDK transfected GECs revealed a perinuclear/cytoplasmic localization of NDK. eATP stimulation induced NDK recruitment to …

The Intestinal Copper Exporter Cua-1 Is Required For Systemic Copper Homeostasis In Caenorhabditis Elegans, Haarin Chun, Anuj Kumar Sharma, Jaekwon Lee, Jefferson Chan, Shang Jia, Byung-Eun Kim

Department of Biochemistry: Faculty Publications

Copper plays key catalytic and regulatory roles in biochemical processes essential for normal growth, development, and health. Defects in copper metabolism cause Menkes and Wilson’s disease, myeloneuropathy, and cardiovascular disease and are associated with other pathophysiological states. Consequently, it is critical to understand the mechanisms by which organisms control the acquisition, distribution, and utilization of copper. The intestinal enterocyte is a key regulatory point for copper absorption into the body; however, the mechanisms by which intestinal cells transport copper to maintain organismal copper homeostasis are poorly understood. Here, we identify a mechanism by which organismal copper homeostasis is maintained by …

The Effects Of Phytohormones And Isoprenoids In Dihydroartemisinin-Induced Dormancy In The Erythrocytic Stages Of Plasmodium Falciparum, Marvin Duvalsaint Duvalsaint

USF Tampa Graduate Theses and Dissertations

Our ability to control malaria has been challenged by increasing antimalarial resistance. Plasmodium falciparum undergoes dormancy in the blood stages which is hypothesized to be a means by which they are able to survive under drug pressure. This helps select for resistant parasites which grow following removal of drug. The mechanisms behind dormancy and the subsequent recrudescence are not fully understood but translating knowledge from related organisms which undergo a similar phenomenon might shed some light. Higher plants utilize dormancy during the early development stages to survive under unfavorable conditions, increasing fitness of the seedling and ensuring viability when this …

Phytochrome B Integrates Light And Temperature Signals In Arabidopsis, Martina Legris, Cornelia Klose, E Sethe Burgie, Cecilia Costigliolo Rojas Rojas, Maximiliano Neme, Andreas Hiltbrunner, Philip A. Wigge, Eberhard Schäfer, Richard D. Vierstra, Jorge J. Casal

Biology Faculty Research

Ambient temperature regulates many aspects of plant growth and development, but its sensors are unknown. Here, we demonstrate that the phytochrome B (phyB) photoreceptor participates in temperature perception through its temperature-dependent reversion from the active Pfr state to the inactive Pr state. Increased rates of thermal reversion upon exposing Arabidopsis seedlings to warm environments reduce both the abundance of the biologically active Pfr-Pfr dimer pool of phyB and the size of the associated nuclear bodies, even in daylight. Mathematical analysis of stem growth for seedlings expressing wild-type phyB or thermally stable variants under various combinations of light and temperature revealed …

Quantitative Proteomic Investigation Of Disease Models Of Type 2 Diabetes, Mark Gabriel Athanason

USF Tampa Graduate Theses and Dissertations

PANcreatic DERived factor (PANDER, FAM3B) is a member of a superfamily of FAM3 proteins that are uniquely structured and strongly expressed from the endocrine pancreas and co-secreted with insulin. Unique animal models available to our lab have indicated that PANDER can induce a selective hepatic insulin resistant (SHIR) phenotype whereby insulin signaling is blunted yet lipogenesis is increased. The complexity of the biological networks involved with this process warranted the logical approach of employing quantitative mass spectrometry based proteomic analysis using stable isotope labeling of amino acids in cell culture (SILAC) to identify the global proteome differences between the PANDER …

Significance Of Pten Phosphorylation And Its Nuclear Function In Lung Cancer, Prerna Malaney

USF Tampa Graduate Theses and Dissertations

Phosphorylation mediated inactivation of PTEN leads to multiple malignancies with increased severity. However, the consequence of such inactivation on downstream functions of PTEN are poorly understood. Therefore, the objective of my thesis is to ascertain the molecular mechanisms by which PTEN phosphorylation drives lung cancer. PTEN phosphorylation at the C-terminal serine/threonine cluster abrogates its tumor suppressor function. Despite the critical role of the PTEN C-tail in regulating its function, the crystal structure of the C-tail remains unknown. Using bioinformatics and structural analysis, I determined that the PTEN C-tail is an intrinsically disordered region and is a hot spot for post-translational …

An Iron Detection System Determines Bacterial Swarming Initiation And Biofilm Formation, Chuan-Sheng Lin, Yu-Huan Tsai, Chih-Jung Chang, Shun-Fu Tseng, Tseng-Ru Wu, Chia-Chen Lu, Ting-Shu Wu, Jang-Jih Lu, Jim-Tong Horng, Jan Martel, David M. Ojcius, Hsin-Chih Lai, John Ding-E. Young

All Dugoni School of Dentistry Faculty Articles

Iron availability affects swarming and biofilm formation in various bacterial species. However, how bacteria sense iron and coordinate swarming and biofilm formation remains unclear. Using Serratia marcescens as a model organism, we identify here a stage-specific iron-regulatory machinery comprising a two-component system (TCS) and the TCS-regulated iron chelator 2-isocyano-6,7-dihydroxycoumarin (ICDH-Coumarin) that directly senses and modulates environmental ferric iron (Fe3+) availability to determine swarming initiation and biofilm formation. We demonstrate that the two-component system RssA-RssB (RssAB) directly senses environmental ferric iron (Fe³⁺) and transcriptionally modulates biosynthesis of flagella and the iron chelator ICDH-Coumarin whose production requires the pvc cluster. …

Development Of In Vivo Systems For Detecting And Studying Ribosome Inhibition By Small Molecules, Shijie Huang

Chemistry and Chemical Biology ETDs

The ribosome is the quintessential antibacterial drug target, with many structurally and mechanistically distinct classes of antibacterial agents acting by inhibiting ribosome function. Detecting and quantifying ribosome inhibition by small molecules and investigating their binding modes and mechanisms of action are critical to antibacterial drug discovery and development efforts. To develop a ribosome inhibition assay that is operationally simple, yet provides direct information on the drug target and the mechanism of action, we have developed engineered E. coli strains harboring an orthogonal ribosome controlled green fluorescent protein reporter that produce fluorescent signal when the O-ribosome is inhibited. As a proof …

Regulation Of Palmitoylation Enzymes And Substrates By Intrinsically Disordered Regions, Krishna D. Reddy

USF Tampa Graduate Theses and Dissertations

Protein palmitoylation refers to the process of adding a 16-carbon saturated fatty acid to the cysteine of a substrate protein, and this can in turn affect the substrate’s localization, stability, folding, and several other processes. This process is catalyzed by a family of 23 mammalian protein acyltransferases (PATs), a family of transmembrane enzymes that modify an estimated 10% of the proteome. At this point in time, no structure of a protein in this family has been solved, and therefore there is poor understanding about the regulation of the enzymes and their substrates. Most proteins, including palmitoylation enzymes and substrates, have …

Engineering Small Molecule Based Dimerization To Induce Translation And Provide Optogenetic Control, Catherine Wright

Chemistry and Chemical Biology ETDs

We wanted to develop a system that combines the spatial control of photoactivation and control of translation to build a tool to spatially control translation in neurons. This kind of tool could be used to investigate the role of spatially controlled translation of any protein in neural behavior. In this way the development and growth of neural processes could be studied to elucidate the mechanisms for spatially sensitive events such as pathfinding, repair, or long-term potentiation.

Chemically induced dimerization was used to install a switch into the activation of translation for specific genes. An abscisic acid (ABA) dependent dimerization of …

Design Of Novel Ion Channel Modulators, Vladimir Yarov-Yarovoy

Science Seminar Series

Function and modulation of neuronal sodium channels are critical for the neuromodulation of electrical excitability and synaptic transmission in neurons - the basis for many aspects of signal transduction, learning, memory and physiological regulation. Mutations in neuronal voltage-gated sodium channel genes are responsible for various human neurological disorders. Furthermore, human neuronal voltage-gated sodium channels are primary targets of therapeutic drugs used as local anesthetics and for treatment of neurological and cardiac disorders. Yarov-Yarovoy's lab is working on rational design of novel therapeutically useful blockers of voltage-gated sodium channels for treatment of pain and epilepsy. Serious, chronic pain affects at least …

A Pipette-Based Calibration System For Fast-Scan Cyclic Voltammetry With Fast Response Times, Eric S. Ramsson

Peer Reviewed Articles

Fast-scan cyclic voltammetry (FSCV) is an electrochemical technique that utilizes the oxidation and/or reduction of an analyte of interest to infer rapid changes in concentrations. In order to calibrate the resulting oxidative or reductive current, known concentrations of an analyte must be introduced under controlled settings. Here, I describe a simple and cost-effective method, using a Petri dish and pipettes, for the calibration of carbon fiber microelectrodes (CFMs) using FSCV.

Polymorphisms In Mthfr, Ms And Cbs Genes And Premature Acute Myocardial Infarction In A Pakistani Population, Mohammad Perwaiz Iqbal, Khalida Iqbal, Asal Khan Tareen, Siddiqa Parveen, Naseema Mehboobali, Ghulam Haider, Saleem Perwaiz Iqbal

Department of Biological & Biomedical Sciences

High prevalence of premature coronary heart disease in Pakistanis compared to other populations points towards the genetic predisposition of this population to develop this disease. Since no investigations have been carried out in Pakistan to study the relationship of polymorphisms in genes involved in homocysteine cycle, the objective of the present study was to find out if there is any association of methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C; methionine synthase (MS) A2756G; cystathionine-beta-synthase (CBS) 844ins68, G919A polymorphisms with premature acute myocardial infarction (AMI) in a population of Pakistani patients with this disease. In a cross-sectional study, DNA samples of 143 AMI …

The Biological Function Of Hammerhead Rana Ribozyme In Human Genome, Ibtehal M. A. Awad

Theses

Hammerhead RNA [HHR] sequence originally found in viroids is also detected in many species. HHR are often found in introns of different genes ranging from prokaryotes to

eukaryotes. HHR can form specific tertiary structure that make specific region susceptible to cleavage. However, the role of the HHR is not clear in other life forms. This study aims to

Investigate the role of HHR in the intron 6 of (RECK) gene. Our study shows that when blocking the HHR sequence in the intron6, the production of RECK protein declines which may be due to the effect of HHR in splicing and …

A Polyaniline-Based Sensor Of Nucleic Acids, Partha Sengupta, Jared Gloria, Marcus K. Parker, Alex S. Flynt

Faculty Publications

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies rely on enzymatic modification that can introduce bias and artifacts. A critical element of next generation detection platforms will be direct molecular sensing, thereby avoiding a need for amplification or labels. Advanced nanomaterials may provide the suitable chemical modalities to realize label-free sensors. Conjugated polymers are ideal for biological sensing, possessing properties compatible with biomolecules and exhibit high sensitivity to localized environmental changes. In this article, a method is presented for detecting nucleic acids using the electroconductive …

Expression, Purification, And Crystallization Of An Endoxylanase From Bacteroides Vulgatus, Jesslyn Park, Jason Hurlbert

The Winthrop McNair Research Bulletin

Sustainable sources of energy are growing in demand as fossil fuels are rapidly expended. One such energy source is fuel ethanol generated from the fermentation of plant biomass by engineered bacterial biocatalysts. The creation of a biocatalyst capable of converting nearly any plant matter to fuel ethanol requires the identification of novel enzymes capable of degrading specific carbohydrate polymers and cloning these enzymes into a bacterial host. This study seeks to structurally characterize a novel xylanase of glycosyl hydrolase family 30 (GH30) from Bacteroides vulgatus, a bacterium found in the human gut microbiome, via x-ray crystallography. The gene for …

Pyk2 Activates The Nlrp3 Inflammasome By Directly Phosphorylating Asc And Contributes To Inflammasome-Dependent Peritonitis, I-Che Chung, Chun-Nan Ouyang, Sheng-Ning Yuan, Hsin-Pai P. Li, Jeng-Ting Chen, Hui-Ru Shieh, Yu-Jen Chen, David M. Ojcius, Ching-Liang Chu, Jau-Song Yu, Yu-Sun Chang, Lih-Chyang Chen

All Dugoni School of Dentistry Faculty Articles

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1β secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, …

Infrared Spectroscopy Of The Nitrogenase Mofe Protein Under Electrochemical Control: Potential-Triggered Co Binding, P. Paengnakorn, Philip A. Ash, Sudipta K. Shaw, Karamatullah Danyal, T. Chen, Dennis R. Dean, Lance C. Seefeldt, Kylie A. Vincent

Chemistry and Biochemistry Faculty Publications

We demonstrate electrochemical control of the nitrogenase MoFe protein, in the absence of Fe protein or ATP, using europium(III/II) polyaminocarboxylate complexes as electron transfer mediators. This allows the potential dependence of proton reduction and inhibitor (CO) binding to the active site FeMo-cofactor to be established. Reduction of protons to H2 is catalyzed by the wild type MoFe protein and Β-98^Tyr→His and Β-^99Phe→His variants of the MoFe protein at potentials more negative than -800 mV (vs. SHE), with greater electrocatalytic proton reduction rates observed for the variants compared to the wild type protein. Electrocatalytic proton reduction is strongly …

Purification Of 26s Proteasomes And Their Subcomplexes From Plants, Richard S. Marshall, David C. Gemperline, Richard D. Vierstra

Biology Faculty Research

The 26S proteasome is a highly dynamic, multisubunit, ATP-dependent protease that plays a central role in cellular housekeeping and many aspects of plant growth and development by degrading aberrant polypeptides and key cellular regulators that are first modified by ubiquitin. Although the 26S proteasome was originally enriched from plants over 30 years ago, only recently have significant advances been made in our ability to isolate and study the plant particle. Here, we describe two robust methods for purifying the 26S proteasome and its subcomplexes from Arabidopsis thaliana; one that involves conventional chromatography techniques to isolate the complex from wild-type …

Atomic Tiles: Manipulative Resources For Exploring Bonding And Molecular Structure, Alan L. Kiste, Rebecca G. Hooper, Gregory E. Scott, Seth Bush

Chemistry and Biochemistry

A simple manipulative resource, Atomic Tiles, is described for scaffolding the learning of Lewis structures without using algorithmic, rule-based methods of drawing. Students use Atomic Tiles to (1) create models of bonding that lead to drawing Lewis structures, (2) use the structures they create to infer patterns required for rational structures and common organic functional groups, (3) translate between Lewis structures and molecular models, and (4) use molecular models to identify isomers.