Biochemistry, Biophysics, and Structural Biology Commons^™

Response To "Sodium Current Inhibition By Nanosecond Pulsed Electric Field (Nspef) - Fact Or Artifact?" By Verkerk Et Al, Andrei G. Pakhomov

Bioelectrics Publications

It was nice to learn that our studies of nanosecond pulsed electric field (nsPEF) effects on membrane currents [Nesin et al., 2012; Nesin and Pakhomov, 2012] gained the attention of scientists outside the immediate field of bioelectromagnetics.

Y-Family Dna Polymerases : Mechanism Of Single-Base Deletion And Effect Of Pcna Interaction, Yifeng Wu

Legacy Theses & Dissertations (2009 - 2024)

Translesional synthesis (TLS) by specialized Y-family DNA polymerases is a DNA damage tolerance pathway to bypass DNA lesions that have not been repaired by other DNA repair mechanisms. Despite their valuable lesion bypass ability, the Y-family DNA polymerases display a much lower fidelity upon replicating undamaged DNA. Their activity therefore needs to be well regulated in the cell. In our first project, we examined how an archaeal Y-family DNA polymerase, Dpo4, makes single-base deletions during replication. Dpo4 belongs to the DinB subfamily which is known to make single-base deletions. Although previous studies suggested that it uses a dNTP-stabilized misalignment mechanism, …

Characterizing The Effects Of Glutaraldehyde On The Ryanodine Receptor Calcium Release Channel, Joshua Dov Strauss

Legacy Theses & Dissertations (2009 - 2024)

Ryanodine receptors (RyR) are large intracellular calcium release channels, which

Structure-Activity Relationship Of 2,3-Benzodiazepin-4-Ones As Noncompetitive Ampa Receptor Antagonists, Mohammad Shaban Qneibi

Legacy Theses & Dissertations (2009 - 2024)

2,3-Benzodiazepin-4-one (BDZ-11) derivatives are a special group of 2,3-benzodiazepine compounds. These compounds have been previously synthesized as AMPA receptor inhibitors, and they are also potential drugs for the treatment of various neurological diseases involving excessive AMPA receptor activity. AMPA receptors are a subtype of glutamate ion channel proteins and are responsible for the majority of excitatory neurotransmission in the mammalian central nervous system. Specifically, they are critically involved in neuronal development and brain activities, including learning and memory. Overstimulation of AMPA receptors is associated with some neurological diseases such as ALS, stroke and Alzheimer's disease. Despite the fact that hundreds …

Real-Time In Vivo Imaging Of Size-Dependent Transport And Toxicity Of Gold Nanoparticles In Zebrafish Embryos Using Single Nanoparticle Plasmonic Spectroscopy, Lauren M. Browning, Tao Huang, Xiao-Hong Nancy Xu

Chemistry & Biochemistry Faculty Publications

Noble metal nanoparticles (NPs) show distinctive plasmonic optical properties and superior photostability, enabling them to serve as photostable multicoloured optical molecular probes and sensors for real-time in vivo imaging. To effectively study biological functions in vivo, it is essential that the NP probes are biocompatible and can be delivered into living organisms non-invasively. In this study, we have synthesized, purified and characterized stable (non-aggregated) gold (Au) NPs (86.2 +/- 10.8 nm). We have developed dark-field single NP plasmonic microscopy and spectroscopy to study their transport into early developing zebrafish embryos (cleavage stage) and their effects on embryonic development …

Elucidation Of The Functional Role Of Pcbp-The Cytosolic Iron Chaperone Protein Family, In Cellular Iron Homeostasis, Poorna Subramanian

Wayne State University Dissertations

Biological Role of Ferritin - Iron is essential for life and often utilized as a cofactor in many proteins. In humans, iron accumulation causes cirrhosis, arthritis, cardiomyopathy and diabetes mellitus, and it is associated with increased risk of cancer and heart disease. In contrast, decreased brain iron content results in permanent neurocognitive and motor impairment. Intracellular iron content must be maintained within a narrow range to avoid the adverse effects of iron depletion or excess, and this function is performed by the protein ferritin. Ferritins are iron storage proteins that are ubiquitously expressed in animals, plants and bacteria. They serve …

Single-Stranded Dna Scanning And Deamination With Single-Molecule Resolution, Padhuk Gamarala Senavirathne

Wayne State University Dissertations

SINGLE-STRANDED DNA SCANNING AND DEAMINATION WITH SINGLE-MOLECULE RESOLUTION

by

GAYAN SENAVIRATHNE

December 2013

Advisor: Dr. David Rueda & Dr.Ashok Bhagwat

Major: Chemistry (Analytical)

Degree: Doctor of Philosophy

APOBEC3G (Apo3G) and Activation-Induced cytidine Deaminase (AID) are the most notable members of APOBEC enzymes, a family of cytidine (C) deaminases with crucial functions in the immune system. In T cells, Apo3G deaminates C in viral cDNA to halt the replication of HIV 1 strains lacking viral infectivity factor (vif). In B cells, AID is required for the diversification of antibodies through initiation of somatic hypermutation (SHM), and class switch recombination (CSR), by …

Interaction Between Maltose Binding Protein And Escherichia Coli Maltose Transporter, Yan Huang

Open Access Theses

The ATP-binding cassette (ABC) transporter superfamily is one of the largest families of transport proteins. The ABC transporters are responsible for selective permeability of solute across membranes energized by ATP hydrolysis, which occurs in all domains of life. Maltose transporter is an ABC importer that mediates maltose/maltodextrin uptake in bacteria and archaea. It is identified as an essential virulence factor in pathogenic species of Streptococcis pyogenes and Vibrio cholera (1, 2). Escherichia coli maltose transporter is a well-characterized system with crystal structures and exclusive biochemical studies available. Knowledge of the E. coli maltose transport mechanism will lead to a better …

The Disordered Regulation Of Calcineurin: How Calmodulin-Induced Regulatory Domain Structural Changes Lead To The Activation Of Calcineurin, Victoria B. Dunlap

Theses and Dissertations--Molecular and Cellular Biochemistry

Calcineurin (CaN) is a highly regulated Ser/Thr protein phosphatase that plays critical roles in learning and memory, cardiac development and function, and immune system activation. Alterations in CaN regulation contribute to multiple disease states such as Down syndrome, cardiac hypertrophy, Alzheimer’s disease, and autoimmune disease. In addition, CaN is the target of the immunosuppressant drugs FK506 and cyclosporin A. Despite its importance, CaN regulation is not well understood on a molecular level. Full CaN activation requires binding of calcium-loaded calmodulin (CaM), however little is known about how CaM binding releases CaN’s autoinhibitory domain from the active site. Previous work has …

Reaction Enthalpy And Volume Profiles For Excited State Reactions Involving Electron Transfer And Proton-Coupled Electron Transfer, William Antonio Maza

USF Tampa Graduate Theses and Dissertations

Electron transfer, ET, and proton-coupled electron transfer, PCET, reactions are central to biological reactions involving catalysis, energy conversion and energy storage. The movement of electrons and protons in either a sequential or concerted manner are coupled in a series of elementary reaction steps in respiration and photosynthesis to harvest and convert energy consumed in foodstuffs or by absorption of light into high energy chemi-cal bonds in the form of ATP. These electron transfer processes may be modulated by conformational dynamics within the protein matrix or at the protein-protein interface, the energetics of which are still not well understood. Photoacoustic calorimetry …

Structure, Dynamics, And Evolution Of The Intrinsically Disordered P53 Transactivation Domain, Wade Michael Borcherds

USF Tampa Graduate Theses and Dissertations

in numerous disease states, including cancers and neurodegenerative diseases. All proteins are dynamic in nature, occupying a range of conformational flexibilities. This inherent flexibility is required for their function, with ordered proteins and IDPs representing the least flexible, and most flexible, respectively. As such IDPs possess little to no stable tertiary or secondary structure, they instead form broad ensembles of heterogeneous structures, which fluctuate over multiple time scales. Although IDPs often lack stable secondary structure they can assume a more stable structure in the presence of their binding partners in a coupled folding binding reaction.

The phenomenon of the dynamic …

Fabricating And Characterizing Physical Properties Of Electrospun Polypeptide-Based Nanofibers, Dhan Bahadur Khadka

USF Tampa Graduate Theses and Dissertations

This dissertation has aimed to fabricate polypeptide based biomaterial and characterize physical properties. Electrospinning is used as a tool for the sample fabrication. Project focused on determining the feasibility of electrospinning of certain synthetic polypeptides and certain elastin-like peptides from aqueous feedstocks and to characterize physical properties of polymer aqueous solution, cast film and spun fibers and fiber mats. The research involves peptide design, polymer electrospinning, fibers crosslinking, determining the extent of crosslinking, fibers protease degradation study, fibers stability and self-organization analysis, structure and composition determination by various spectroscopy and microscopy techniques and characterization of mechanical properties of individual suspended …

Structural Basis For Ternary Complex Formation Between Tau, Hsp90, And Fkbp51, Alexander Steven Barrett

USF Tampa Graduate Theses and Dissertations

The accumulation of the microtubule associated protein tau has been implicated in several neurological disorders; however, its interaction with chaperones along its normal degradation pathway remains largely uncharacterized at single residue resolution. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to probe the interaction between tau, the molecular chaperone Hsp90, and the immunophilin FKBP51. Resonance intensity changes were observed for specific residues in the heteronuclear single quantum coherence (HSQC) spectra of 15N-labeled tau in the presence of Hsp90 and/or FKBP51. Analysis of the HSQC spectra identified the two hydrophobic hexapeptide motifs located at residues V275 - K280 and …

Optical Investigations Of Neurohypophysial Excitability And Amyloid Fibril Formation, Joseph Leo Foley

USF Tampa Graduate Theses and Dissertations

This dissertation describes the work done on two distinct projects. In the first part I sought to unravel the mechanisms that underlie the activity-dependent modulation of response in the excitation-secretion coupling of the neurohypophysis. In the second part, I optically monitored and analyzed the secondary structure changes accompanying amyloid fibril formation along multiple pathways, under both denaturing and near-physiological conditions.

Neuronal plasticity plays an important role in regulating various biological systems by modulating release of hormones or neurotransmitters. The changing response to the same stimulus, depending on the context and previous stimulation events, is also the basis of learning and …

Estimating Loop Length From Cryoem Images At Medium Resolutions, Andrew Mcknight, Dong Si, Kamal Al Nasr, Andrey Chemikov, Nikos Chrisochoides, Jing He

Computer Science Faculty Publications

Background: De novo protein modeling approaches utilize 3-dimensional (3D) images derived from electron cryomicroscopy (CryoEM) experiments. The skeleton connecting two secondary structures such as α-helices represent the loop in the 3D image. The accuracy of the skeleton and of the detected secondary structures are critical in De novo modeling. It is important to measure the length along the skeleton accurately since the length can be used as a constraint in modeling the protein.

Results: We have developed a novel computational geometric approach to derive a simplified curve in order to estimate the loop length along the skeleton. The method was …

Nonthermal Plasma Jet Efficacy On Candida Kefyr, Amber Marie Mattson Jackson

Electrical & Computer Engineering Theses & Dissertations

The inactivation abilities of a nonthermal plasma jet were investigated with the yeast C. kefyr, a pathogen with increasing importance in healthcare. The plasma is generated using a microhollow cathode discharge (MHCD) geometry operated with a DC voltage of 2 kV and a current of 30 mA using air as the operating gas at an airflow rate of 8 SLM. The plasma discharge approaches room temperature at a few mm away from the plasma jet allowing for the use on heat sensitive surfaces. Dry air and humid air were used as the operating gas to see if humidity plays …

Size And Shape Characterization Of Salt Dependent Thermoreversible Micelles Synthesized From Elastin-Like Polypeptides, Kaitlin W. Vandemark

Undergraduate Research Posters 2012

Environmentally responsive nanoparticles synthesized from Elastin-Like Polypeptides (ELP) present a promising system for applications such as biosensors, drug delivery vehicles, and viscosity modifiers.

Investigations Of Protein-Lipid Interactions In Model Membranes: Influence Of Aromatic Anchoring Residues And Buried Polar Residues, Nick Gleason

Graduate Theses and Dissertations

To investigate in detail the interactions between transmembrane proteins and the lipid bilayers in which they are constituted, designed model peptides with selective isotopic labels were synthesized and analyzed by means of solid-state deuterium NMR spectroscopy. Starting from the well-characterized model peptide GWALP23, acetyl-GGALW(LA)6LWLAGA-amide, several Trp to Tyr mutations were compared to evaluate their respective interfacial anchoring abilities. It was found that Tyr, substituted on either or both termini, can effectively anchor the transmembrane alpha-helix, which then adopts a similar transmembrane topology in a range of bilayer thicknesses. Nevertheless, a consistent ~10° shift in tilt direction (helix rotation) is observed …

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

Symposium 2012

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

Structural Analysis Of The Drosophila Innexin Shakb: Role Of The N-Terminus In Rectifying Electrical Synapses, William Marks

Biology Theses

Gap junction channels mediate direct intercellular communication in all multicellular animals. They are comprised of the connexin family of proteins in vertebrates and the innexin family in prechordates. Connexins and innexins share many functional and structural similarities as orthologous proteins. Both types are capable of forming electrical synapses. Rectifying junctions are specialized electrical synapses found in neural systems that control escape responses. It has been shown that heterotypic gap junction channels mediate asymmetric properties of rectifying junctions. Shaking B N+16 and Shaking B lethal are variants of the ShakB locus in Drosophila and the organization of these innexins into heterotypic …

Determining The Genotype-Phenotype Connection In Synthetic Inducible Gene Expression Systems, Rhys M. Adams

Dissertations and Theses (Open Access)

Introduction Gene expression is an important process whereby the genotype controls an individual cell’s phenotype. However, even genetically identical cells display a variety of phenotypes, which may be attributed to differences in their environment. Yet, even after controlling for these two factors, individual phenotypes still diverge due to noisy gene expression. Synthetic gene expression systems allow investigators to isolate, control, and measure the effects of noise on cell phenotypes. I used mathematical and computational methods to design, study, and predict the behavior of synthetic gene expression systems in S. cerevisiae, which were affected by noise.

Methods I created probabilistic …

Nano- And Micro-Second Electrical Pulsing Of B16-F10 Mouse Melanoma Cells: Plasma Membrane And Sub-Cellular Organelle Changes, Yiling Chen

Theses and Dissertations in Biomedical Sciences

High electric field-treated cells are permeable to molecular dye through either opening of pores in the plasma membrane or other unknown processes which can disturb the membrane in an organized way. However, direct morphological evidence is lacking and responses of intracellular organelles are not clear. We used traditional chemical fixatives and biochemical techniques to capture cell membrane and organelle changes immediately after pulsing with high voltage electric field application. Different pulse durations, nanosecond (ns) and microsecond (µs), and field magnitudes, 60 kV/cm and 1.2 kV/cm, were applied to mouse melanoma B16-F10 cells. Two different ns durations (60 and 300 ns) …

Nonosecond Pulsed Electric Field Induced Changes In Dielectric Properties Of Biological Cells, Jie Zhuang

Electrical & Computer Engineering Theses & Dissertations

Nanosecond pulsed electric field induced biological effects have been a focus of research interests since the new millennium. Promising biomedical applications, e.g. tumor treatment and wound healing, are emerging based on this principle. Although the exact mechanisms behind the nanosecond pulse-cell interactions are not completely understood yet, it is generally believed that charging along the cell membranes (including intracellular membranes) and formation of membrane pores trigger subsequent biological responses, and the number and quality of pores are responsible for the cell fate. The immediate charging response of a biological cell to a nanosecond pulsed electric field exposure relies on the …

Critical Heat Stress Evaluation Of Two-Layer Clothing Ensembles And The Contributionof A Full-Face Negative Pressure Respirator, Oclla Michele Fletcher

USF Tampa Graduate Theses and Dissertations

Protective clothing ensembles are worn by workers as a barrier to chemical and physical hazards, but can restrict heat loss and increase worker heat stress. The question of whether a respirator adds to heat stress or strain burden is a continuing concern among occupational health professionals. The purpose of this study was to determine if there are differences in heat stress or strain among the current Toxicological Agent Protective (TAP) ensemble and two ensemble variations used in demilitarization of chemical weapons. Four acclimatized adult males wore five ensembles in a balanced design while walking in a climatic chamber at a …

Single-Molecule Analysis Of The Microtubule Cross-Linking Protein Map65-1 Reveals A Molecular Mechanism For Contact-Angle-Dependent Microtubule Bundling, Amanda Tulin, Sheri Mcclerklin, Yue Huang, Ram Dixit

Biology Faculty Research

Bundling of microtubules (MTs) is critical for the formation of complex MT arrays. In land plants, the interphase cortical MTs form bundles specifically following shallow-angle encounters between them. To investigate how cells select particular MT contact angles for bundling, we used an in vitro reconstitution approach consisting of dynamic MTs and the MT-cross-linking protein MAP65-1. We found that MAP65-1 binds to MTs as monomers and inherently targets antiparallel MTs for bundling. Dwell-time analysis showed that the affinity of MAP65-1 for antiparallel overlapping MTs is about three times higher than its affinity for single MTs and parallel overlapping MTs. We also …

"Fine-Tuning" Of Ribosomal Structure And Functions By Pseudouridylation And Rna-Protein Interactions, Jun Jiang

Wayne State University Dissertations

ABSTRACT

"Fine-tuning" of ribosomal structure and functions by pseudouridylation and RNA-protein interactions

by

JUN JIANG

AUGUST 2012

Advisor: Prof. John SantaLucia Jr.

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

Ribosomal structure and functions appear to be "fine-tuned" by pseudouridylation and RNA-protein interactions. Pseudouridylation may promote base stacking interactions by mediating the base stacking between residues on both sides. In the RNA duplex region, this enhanced stacking interaction contributes to stabilization of duplex folding. In the loop region, enhanced stacking in one structural motif may destabilize the conformation of adjacent structural residues. This hypothesis is supported by both UV-melting experiments, where …

Cell Permeabilization And Inhibition Of Voltage-Gated Ca²+ And Na+ Channel Currents By Nanosecond Pulsed Electric Fields, Vasyl Nesin, Angela M. Bowman, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Previous studies have found that nanosecond pulsed electric field (nsPEF) exposure causes long-term permeabilization of the cell plasma membrane. In this study, we utilized the whole-cell patch-clamp method to study the nsPEF effect on currents of voltage-gated (VG) Ca²⁺ and Na⁺ channels (I_Ca and I_Na) in cultured GH3 and NG108 cells. We found that a single 300 or 600 ns pulse at or above 1.5-2 kV/cm caused prolonged inhibition of I_Ca and I_Na. Concurrently, nsPEF increased a non-inactivating leak current (I_leak), presumably due to the formation of nanoelectropores or larger …

Inhibition Of Voltage-Gated Na+ Current By Nanosecond Pulsed Electric Field (Nspef) Is Not Mediated By Na+ Influx Or Ca²+ Signaling, Vasyl Nesin, Andrei G. Pakhomov

Bioelectrics Publications

In earlier studies, we found that permeabilization of mammalian cells with nsPEF was accompanied by prolonged inhibition of voltage-gated (VG) currents through the plasma membrane. This study explored if the inhibition of VG Na⁺ current (I_Na) resulted from (i) reduction of the transmembrane Na⁺ gradient due to its influx via nsPEF-opened pores, and/or (ii) downregulation of the VG channels by a Ca²⁺ -dependent mechanism. We found that a single 300?ns electric pulse at 1.65.3?kV/cm triggered sustained Na⁺ influx in exposed NG108 cells and in primary chromaffin cells, as detected by increased fluorescence of a …

The Spliceosomal Protein Prp8 Stabilizes A Compact Conformation Of The U2-U6 Complex, Subasinghe Appuhamilage Lemintha Imali Subasinghe

Wayne State University Theses

The spliceosome is a large, RNA-protein complex that catalyzes pre-mRNA splicing during mRNA maturation. The RNA components (small nuclear RNA; snRNAs) of the spliceosome have been well studied and are believed to be involved in the splicing catalysis. Although proteins are essential for splicing, they may not be directly involved in catalysis. Among hundreds of proteins, Prp8 is the only protein that interacts with all of the catalytically important snRNAs. Therefore, it is hypothesized that Prp8 may catalyze splicing either by directly participating in catalysis or by stabilizing the conformation of the catalytically active spliceosome. In order to test whether …

Structural And Functional Characterization Of Dna Polymerase Ss Mutator Mutants, Sneha Rangarajan

Legacy Theses & Dissertations (2009 - 2024)

DNA Polymerase ß (polß) plays a crucial role in repairing damaged DNA in a process called Base Excision Repair (BER). BER is a major pathway of DNA repair, making this system absolutely vital for maintaining genomic integrity. Recent studies estimate 30% of human tumors to contain polß variants that led us to believe that there is a high degree of association between mutations in polß and cancer. In this pathway, after recognition and excision of the damaged base, the DNA is cleaved at an apurinic (AP) site by AP endonuclease leaving behind a 3' hydroxyl and 5' deoxyribose phosphate (dRP). …