Biochemistry, Biophysics, and Structural Biology Commons^™

Aptameric Sensors: In Vitro Selection Of Dna That Binds Bromocresol Purple, Derek B. Miller

Honors Undergraduate Theses

Aptamers being used as sensors is an emerging field that has capabilities of being tomorrow’s diagnostic tools. As aptameric sensors have become more popular, their visualization systems have been limited. The majority of today’s aptameric sensors require expensive machinery such as a fluorometer in order to visualize results. We propose a system that will cut the need for instrumentation and be detected via the naked eye. With the selection of an aptamer to bind the pH indicating dye bromocresol purple (BCP) this may be achieved. When rendered active, the binding towards BCP will facilitate a color change from yellow to …

Variance Of The Amyloid Beta Peptide As A Metric For The Diagnosis Of Alzheimer's Disease, Christina Beckett

Theses and Dissertations--Medical Sciences

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder associated with aging. AD is by far the best understood and most studied neurodegenerative disease. Substantial advances have been made over the last decade, however it is debatable how much closer we are to a clinically useful therapy. A long standing goal in the AD field has been to improve the accuracy of early detection, with the assumption that the ability to intervene earlier in the disease process will lead to a better clinical outcome. Major facets of this effort have been the continued development and improvement of AD biomarkers, with …

Elucidating Proteasome Catalytic Subunit Composition And Its Role In Proteasome Inhibitor Resistance, Kimberly C. Carmony

Theses and Dissertations--Pharmacy

Proteasome inhibitors bortezomib and carfilzomib are FDA-approved anticancer agents that have contributed to significant improvements in treatment outcomes. However, the eventual onset of acquired resistance continues to limit their clinical utility, yet a clear consensus regarding the underlying mechanisms has not been reached.

Bortezomib and carfilzomib are known to target both the constitutive proteasome and the immunoproteasome, two conventional proteasome subtypes comprising distinctive sets of catalytic subunits. While it has become increasingly evident that additional, ‘intermediate’ proteasome subtypes, which harbor non-standard mixtures of constitutive proteasome and immunoproteasome catalytic subunits, represent a considerable proportion of the proteasome population in many cell …

Towards Elucidation Of The Mechanism Of Biological Nanomotors, Zhengyi Zhao

Theses and Dissertations--Pharmacy

Biological functions such as cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging, and cell entry all involve biomotor-driven DNA translocation. In the past, the ubiquitous biological nanomotors were classified into two categories: linear and rotation motors. In 2013, we discovered a third type of biomotor, revolving motor without rotation. The revolving motion is further found to be widespread among many biological systems. In addition, the detailed sequential action mechanism of the ATPase ring in the phi29 dsDNA packaging motor has been elucidated: ATP binding induces a conformational entropy alternation of ATPase …

Molecular And Biochemical Signaling Underlying Arabidopsis-Bacterial/Virus/Fungal Interactions, Mohamed H. El-Shetehy

Theses and Dissertations--Plant Pathology

Systemic acquired resistance (SAR) is a form of inducible defense response triggered upon localized infection that confers broad-spectrum disease resistance against secondary infections. Several factors are known to regulate SAR and these include phenolic phytohormone salicylic acid (SA), phosphorylated sugar glycerol-3-phosphate (G3P), and dicarboxylic acid azelaic acid (AzA). This study evaluated a role for free radicals nitric oxide (NO) and reactive oxygen species (ROS) in SAR. Normal accumulation of both NO and ROS was required for normal SAR and mutations preventing NO/ROS accumulation and/or biosynthesis compromised SAR. A role for NO and ROS was further established using pharmacological approaches. Notably, …

Repositioning Of Drugs Using Open-Access Data Portal Dtome: A Test Case With Probenecid (Review), Mohammad U. Ahmed, Dylan J. Bennett, Tze-Chen Hsieh, Barbara B. Doonan, Saba Ahmed, Joseph M. Wu

NYMC Faculty Publications

The one gene-one enzyme hypothesis, first introduced by Beadle and Tatum in the 1940s and based on their genetic analysis and observation of phenotype changes in Neurospora crassa challenged by various experimental conditions, has witnessed significant advances in recent decades. Much of our understanding of the association between genes and their phenotype expression has benefited from the completion of the human genome project, and has shown continual transformation guided by the effort directed at the annotation and characterization of human genes. Similarly, the idea of one drug‑one primary disease indication that traditionally has been the benchmark for the labeling and …

Synthesis And Biochemical Evaluation Of 3-Phenoxy-1,4-Diarylazetidin-2-Ones As Tubulin-Targeting Antitumor Agents, Thomas F. Greene, Shu Wang, Lisa M. Greene, Seema M. Nathwani, Jade K. Pollock, Azizah M. Malebari, Thomas Mccabe, Brendan Twamley, Niamh O'Boyle, Daniela M. Zisterer, Mary J. Meegan

Articles

Structure-activity relationships for a series of 3-phenoxy-1,4-diarylazetidin-2-ones were investigated leading to the discovery of a number of potent antiproliferative compounds, including trans-4-(3-hydroxy-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (78b) and trans-4-(3-amino-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (90b). X-ray crystallography studies indicate the potential importance of the torsional angle between the 1-phenyl ‘A’ ring and 4-phenyl ‘B’ ring for potent antiproliferative activity, and that a trans configuration between the 3-phenoxy and 4-phenyl rings is generally optimal. These compounds displayed IC₅₀ values of 38 nM and 19 nM respectively in MCF-7 breast cancer cells, inhibited the polymerization of isolated tubulin in vitro, disrupted the microtubular …

Mitochondrial Function Assessed By 31p Mrs And Bold Mri In Non-Obese Type 2 Diabetic Rats, Yuchi Liu, Xunbai Mei, Jielei Li, Nicola Lai, Xin Yu

Electrical & Computer Engineering Faculty Publications

The study aims to characterize age-associated changes in skeletal muscle bioenergetics by evaluating the response to ischemia-reperfusion in the skeletal muscle of the Goto-Kakizaki (GK) rats, a rat model of non-obese type 2 diabetes (T2D). ³¹P magnetic resonance spectroscopy (MRS) and blood oxygen level-dependent (BOLD) MRI was performed on the hindlimb of young (12 weeks) and adult (20 weeks) GK and Wistar (control) rats. ³¹P-MRS and BOLD-MRI data were acquired continuously during an ischemia and reperfusion protocol to quantify changes in phosphate metabolites and muscle oxygenation. The time constant of phosphocreatine recovery, an index of mitochondrial oxidative capacity, …

Computational Studies Of Paradifluorobenzene Cations And Hydrogen Cyanide Molecule, John C. Rowe Iv

Honors Theses

Polycyclic aromatic hydrocarbons (PAHs) are hypothesized to comprise a significant portion of interstellar carbon identified from the Diffuse Interstellar Bands (DIBs). Gas phase cation-molecule reactions between compounds that previously existed as weakly bound species provides the best explanation for the emergence of PAHs in the interstellar medium (ISM). In this work, we use computational methods to characterize one of these weakly-bound systems. We discovered that intramolecular charge distribution (calculated using the Natural Population Analysis) dictate the complexes formed between a paradifluorobenzene cation and either one or two HCN molecules. Additionally, the relative stabilities of the complexes and the binding energies …

Characterization Of Catecholamine Receptors And Transporters In Murine Macrophages, Elizabeth Gonye

Honors Theses

Macrophages are a critical part of the immune response. When circulating monocytes move into tissues they differentiate int macrophages to mount the first line of defense against pathogens.

Calcium (Ii) Catalyzed Nitrone Additions, Elizabeth A. Congdon

Honors Theses

Calcium(II) complexes have be shown to be successful catalysts for nitrone reactions. The addition of n-­‐methyl and n-­‐phenyl nitrones to donor-­‐acceptor cyclopropanes was achieved with calcium triflate (Ca(OTf). Differentially substituted tetrahydro-­‐1,2-­‐oxazines were synthesized in good to excellent yields. Calcium triflate was also found to catalyze the addition of silyl enol ethers to n-­‐phenyl nitrones along the Mukaiyama-­‐Mannich addition pathway. B-­‐amino carbonyls were synthesized from a variety of substituted nitrones. Bulky and cyclic silyl enol ethers were also found to be reactive, the products of which were isolated in good to excellent yields.

Impact Of Vector Range Expansion On Pathogen Transmission Dynamics Of Lyme Disease In Southwestern Virginia, Bishan Bhattarai

Honors Theses

Blacklegged ticks, Ixodes scapularis is the primary vector of Lyme disease in eastern United States. Borrelia Burgdorfei, the etiological agent of Lyme disease is transferred by ticks of Ixodes species. In recent years, its congener, Ixodes affinis has been expanding its range northwards from its southern population. We were interested in studying how the introduction of this new vector affected the interaction between the pathogen genotype and the host. We hypothesized that differential host use by I. affinis and I. scapularis would partly explain observed differences in B. burgdorferi infection prevalence and genotypic structure in southeastern Virginia. The result …

Pluralistic And Stochastic Gene Regulation: Examples, Models And Consistent Theory, Elisa N. Salas, Jiang Shu, Matyas F. Cserhati, Donald P. Weeks, Istvan Ladunga

Department of Biochemistry: Faculty Publications

We present a theory of pluralistic and stochastic gene regulation. To bridge the gap between empirical studies and mathematical models, we integrate pre-existing observations with our meta-analyses of the ENCODE ChIP-Seq experiments. Earlier evidence includes fluctuations in levels, location, activity, and binding of transcription factors, variable DNA motifs, and bursts in gene expression. Stochastic regulation is also indicated by frequently subdued effects of knockout mutants of regulators, their evolutionary losses/gains and massive rewiring of regulatory sites. We report wide-spread pluralistic regulation in ≈800 000 tightly co-expressed pairs of diverse human genes. Typically, half of ≈50 observed regulators bind to both …

The Role Of Oxidative Stress In The Mechanisms Of Ammonia-Induced Brain Swelling And Tolerance In The Goldfish (Carassius Auratus), David F. Jones Lisser Mr.

Theses and Dissertations (Comprehensive)

Toxic build-ups of ammonia can cause potentially fatal brain swelling in mammals, but such swelling is reversible in the anoxia- and ammonia-tolerant goldfish (Carassius auratus). The mechanisms of ammonia-induced brain swelling and tolerance remain elusive, but several studies have suggested a role for reactive oxygen species (ROS), which may damage proteins and lipids in the plasma membrane of astrocytes in the brain. As a result, osmotic gradients across cell membranes may be altered leading to water uptake by astrocytes and swelling. While a role for ROS has been proposed in mammals, no studies have addressed this question in …

Characterization Of A Phosphonate-Specific Cytidylyltransferase, Kissa Batul

Theses and Dissertations (Comprehensive)

Antibiotic resistance is a major global health concern that requires new therapeutic approaches. Furthermore, a lack of narrow spectrum antibiotics on the market produces unintended consequences with respect to changes in our microbial make up. Phosphonates are reduced versions of phosphates that possess a C-P bond which is more resistant to enzymatic and chemical degradation. The role of phosphonate containing macromolecules (e.g. cell surface polysaccharides) remains enigmatic, however their presence suggests that they may confer an advantage. The biosynthesis of phosphonate-containing macromolecules is unknown, but a pathway is proposed involving aLicC type cytidylyltransferase-catalyzed conjugation to a phosphonate followed by a …

Quantitative Comparison Of A Nanoengineered Alumina Coated Cnt Arrays To Sio2 Coated Cnts And Solution Based Delivery System, Shree Aier

Maseeh Summer Undergraduate Research Experience

To meet the growing need for nanoengineered biocompatible materials to serve as drug delivery platforms, in this research, carbon nanotube arrays were fabricated by chemical vapor deposition, followed by an alumina coating by the high yielding, tightly controlled atomic layer deposition. This nanoengineered vertically aligned alumina nanowire array serves as a platform for delivering antigens, which act as cancer adjuvants. The physicochemical characteristics of the nanowires (NWs) can significantly influence the delivery of a biomolecule to immune cells. To investigate the material characteristics, the delivery efficiency of the antigen using NWs was quantitatively assessed by flow cytometry. Further, the mechanism …

Structure And Biological Activity Of A D3g Mutation In The Human Mitochondrial Chaperonin Hsp60, Jihui Li

Open Access Theses & Dissertations

Proteins are essential components in all cellular activities, including signal transduction, gene expression regulation, immune response, structural support and many others. Following their synThesis on the ribosome, proteins need to fold into their precise three-dimensional structure to obtain functionality. If they fold incorrectly they are prone to aggregation and cause a wide range of neurodegenerative diseases. Some proteins are capable of folding without assistance but others need help from protein complexes known as chaperonins. Chaperonins are a family of proteins that assemble into barrel-shaped cages the create a protected chamber for misfolded proteins to refold into their biologically active state. …

Investigation Of Dna Interaction With Bis-Cationic Imidazolium Salts, Kyle Daily 6440928

Williams Honors College, Honors Research Projects

Imidazolium salt compounds have shown promise in vitro against non-small cell lung cancer (NSCLC) lines, but the exact mechanism of activity these compounds is still unknown. Due to their planar aromatic structure, we hypothesized that our bis-cation imidazolium salt series could act through DNA intercalation similar to known intercalators ethidium bromide, acridine orange, and echinomycin. Here, we tested bis-cation imidazolium salts with linkers of 2, 4, 10, and 12 carbons for DNA intercalation via viscosity and fluorescent intercalator displacement (FID) assays. Despite some solubility issues that may have decreased results, we found that our butyl and dodecyl linked compounds had …

Lipid-Protein Interactions Via Fluorescence Quenching In Small Unilamellar Vesicles, Talia Kordahi

Williams Honors College, Honors Research Projects

Studying peptide interactions with model membranes have aided in understanding protein-lipid interactions more clearly. In this report, fluorescent small unilamellar vesicles (SUVs) of a known composition were made. Two peptides, RND1 and MARCKS, were also fluorescently labeled and added to a solution of SUVs. The interactions between these were observed via fluorescence quenching, read out with a spectrofluorimeter. In addition, two environments of buffer were tested: 1X PBS and 0.1X PBS. After obtaining data, it was concluded that MARCKS quenched the SUVs more than RND1. This could be due to the presence of more lysine amino acids in MARCKS, which …

The Role Of Tgfβ1 And Ep Receptors In The Differentiation Of Normal And Diseased Lung Fibroblasts, Madison A. Hexter

Williams Honors College, Honors Research Projects

Fibroblasts, and their differentiation into myofibroblasts, are important in the pathology of several lung diseases including asthma and idiopathic pulmonary fibrosis (IPF). The differentiation process is dependent on several factors, such as soluble factors like transforming growth factor beta 1 (TGFβ1) and mechanical signals such as matrix stiffness. Additionally, the soluble arachidonic acid metabolite Prostaglandin E₂ (PGE₂) prevents TGFβ1 induced differentiation of fibroblasts to myofibroblasts, but the molecular mechanism is not completely understood¹⁵. Transient Receptor Potential Vanilloid 4 (TRPV4), a mechanosensitive ion channel, has previously been implicated in the differentiation of cardiac fibroblasts^12,13. …

Investigation Of Mutations In Nuclear Genes That Affect The Atp Synthase, Russell Dsouza

Wayne State University Dissertations

The F1 domain is the catalytic subunit of the mitochondrial ATP synthase. Studies with respiratory-deficient yeast identified ATP1 and ATP2 as nuclear genes encoding the alpha and beta subunits, respectively, of the mitochondrial F1-ATPase. The mutations in the atp1 and atp2 genes were cloned and sequenced, and they appear to affect the ATP synthase. Most yeast strains with mutations in the or the subunit primarily show an F1 assembly defective phenotype. This feature is similar to the assembly-defective mutants missing the chaperones required for assembly of the F1 oligomer or either the alpha/beta subunits.

Some of the atp2 and atp1 …

Insights Into De Novo Fes-Cluster Biogenesis Via The Eukaryotic Fes-Cluster (Isc) Pathway In Vitro, Stephen Paul Dzul

Wayne State University Dissertations

Fe-S clusters are iron-containing cofactors utilized by numerous proteins within several biological pathways essential to life. In eukaryotes, the primary pathway for Fe-S cluster production is the iron-sulfur cluster (ISC) pathway. The eukaryotic ISC pathway, localized primarily within the mitochondria, has been best characterized within Saccharomyces cerevisiae. In yeast, de novo Fe-S cluster formation is accomplished through coordinated assembly of the substrates iron and sulfur on the primary scaffold assembly protein “Isu1”. The sulfur used for cluster assembly is provided by the cysteine desulfurase “Nfs1”, a protein that works in union with its accessory protein “Isd11”. Frataxin “Yfh1” helps direct …

Calcineurin: From Activation To Inhibition, Erik C. Cook

Theses and Dissertations--Molecular and Cellular Biochemistry

Calcineurin is a Ser/Thr phosphatase whose function is implicated in critical physiological processes such as immune system activation, fetal heart development, and long-term depression in neurons. Calcineurin has been implicated in the progression of Alzheimer’s disease and cardiac hypertrophy. It is not well understood how calcineurin is activated on a molecular level by Ca²⁺ and its activating protein calmodulin. Previous data from our lab show that calmodulin interaction induces the folding of the intrinsically disordered regulatory domain of calcineurin in two discrete and distant regions into α-helical conformations and that this folding is critical for complete activation of calcineurin. …

A Pipeline For Creation Of Genome-Scale Metabolic Reconstructions, Shaun W. Norris

Theses and Dissertations

The decreasing costs of next generation sequencing technologies and the increasing speeds at which they work have lead to an abundance of 'omic datasets. The need for tools and methods to analyze, annotate, and model these datasets to better understand biological systems is growing. Here we present a novel software pipeline to reconstruct the metabolic model of an organism in silico starting from its genome sequence and a novel compilation of biological databases to better serve the generation of metabolic models. We validate these methods using five Gardnerella vaginalis strains and compare the gene annotation results to NCBI and the …

Systems Perturbation Analysis Of A Large-Scale Signal Transduction Model Reveals Potentially Influential Candidates For Cancer Therapeutics, Bhanwar Lal Puniya, Laura Allen, Colleen Hochfelde, Mahbubul Majumder, Tomáš Helikar

Department of Biochemistry: Faculty Publications

Dysregulation in signal transduction pathways can lead to a variety of complex disorders, including cancer. Computational approaches such as network analysis are important tools to understand system dynamics as well as to identify critical components that could be further explored as therapeutic targets. Here, we performed perturbation analysis of a large-scale signal transduction model in extracellular environments that stimulate cell death, growth, motility, and quiescence. Each of the model's components was perturbed under both loss-of-function and gain-of-function mutations. Using 1,300 simulations under both types of perturbations across various extracellular conditions, we identified the most and least influential components based on …

Activation Of Erk And Nf-Κb During Hare-Mediated Heparin Uptake Require Only One Of The Four Endocytic Motifs, Madhu S. Pandey, Colton M. Miller, Edward N. Harris, Paul H. Weigel

Department of Biochemistry: Faculty Publications

Fifteen different ligands, including heparin (Hep), are cleared from lymph and blood by the Hyaluronan (HA) Receptor for Endocytosis (HARE; derived from Stabilin-2 by proteolysis), which contains four endocytic motifs (M1-M4). Endocytosis of HARE•Hep complexes is targeted to coated pits by M1, M2, and M3 (Pandey et al, Int. J. Cell Biol. 2015, article ID 524707), which activates ERK1/2 and NF-κB (Pandey et al J. Biol. Chem. 288, 14068–79, 2013). Here, we used a NF-κB promoter-driven luciferase gene assay and cell lines expressing different HARE cytoplasmic domain mutants to identify motifs needed for Hepmediated signaling. Deletion of M1, M2 or …

Fatty Acid Transport Proteins: Targeting Fatp2 As A Gatekeeper Involved In The Transport Of Exogenous Fatty Acids, Paul N. Black, Constance Ahowesso, David Montefusco, Nipun Saini, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

The fatty acid transport proteins (FATP) are classified as members of the solute carrier 27 (Slc27) family of proteins based on their ability to function in the transport of exogenous fatty acids. These proteins, when localized to the plasma membrane or at intracellular membrane junctions with the endoplasmic reticulum, function as a gate in the regulated transport of fatty acids and thus represent a therapeutic target to delimit the acquisition of fatty acids that contribute to disease as in the case of fatty acid overload. To date, FATP1, FATP2, and FATP4 have been used as targets in the selection of …

Cadmium And Secondary Structure-Dependent Function Of A Degron In The Pca1p Cadmium Exporter, Nathan Smith, Wenzhong Wel, Miaoyun Zhao, Xiaojuan Quin, Javier Seravalli, Heejeong Kim, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Protein turnover is a critical cellular process regulating biochemical pathways and destroying terminally misfolded or damaged proteins. Pca1p, a cadmium exporter in the yeast Saccharomyces cerevisiae, is rapidly degraded by the endoplasmic reticulum-associated degradation (ERAD) system via a cis-acting degron that exists at the 250–350 amino acid region of Pca1p and is transferable to other proteins to serve as a degradation signal. Cadmium stabilizes Pca1p in a manner dependent on the degron. This suggested that cadmium-mediated masking of the degron impedes its interaction with the molecular factors involved in the ERAD. The characteristics and mechanisms of action of the …

Endoplasmic Reticulum-Associated Degradation Of Pca1p, A Polytopic Protein, Via Interaction With The Proteasome At The Membrane, Nathan Smith, David J. Adle, Miaoyun Zhao, Xiaojuan Quin, Heejeong Kim, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Endoplasmic reticulum-associated degradation (ERAD) plays a critical role for destruction of terminally misfolded proteins at the secretory pathway. The system also regulates expression levels of several proteins such as Pca1p, a cadmium exporter in yeast. To gain better insight into the mechanisms underlying ERAD of Pca1p and other polytopic proteins by the proteasome in the cytosol, our study determined the roles for the molecular factors of ERAD in dislodging Pca1p from the ER. Inactivation of the 20S proteasome leads to accumulation of ubiquitinylated Pca1p in the ER membrane, suggesting a role for the proteasome in extraction of Pca1p from the …

Chloroplast Membrane Remodeling During Freezing Stress Is Accompanied By Cytoplasmic Acidification Activating Sensitive To Freezing 2, Allison C. Barnes, Christoph Benning, Rebecca Roston

Department of Biochemistry: Faculty Publications

Low temperature is a seasonal abiotic stress which restricts native plant ranges and crop distributions. Two types of low temperature stress can be distinguished: chilling and freezing. Much work has been done on the mechanisms by which chilling is sensed, but relatively little is known about how plants sense freezing. Recently, SENSITIVE TO FREEZING 2 (SFR2) was identified as a protein which responds in a non-transcriptional manner to freezing. Here, we investigate the cellular conditions which allow SFR2 activation. Using a combination of isolated organelle, whole tissue and whole plant assays, we provide evidence that SFR2 is activated by changes …