Biochemistry Commons^™

Structural Analysis Of Neutralizing Epitopes Of The Sars-Cov-2 Spike To Guide Therapy And Vaccine Design Strategies, Maxwell T. Finkelstein , '22, Adam G. Mermelstein , '21, Emma Parker Miller , '22, Paul C. Seth , '22, Erik-Stephane D. Stancofski , '21, Daniela Fera

Chemistry & Biochemistry Faculty Works

Coronavirus research has gained tremendous attention because of the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (nCoV or SARS-CoV-2). In this review, we highlight recent studies that provide atomic-resolution structural details important for the development of monoclonal antibodies (mAbs) that can be used therapeutically and prophylactically and for vaccines against SARS-CoV-2. Structural studies with SARS-CoV-2 neutralizing mAbs have revealed a diverse set of binding modes on the spike’s receptor-binding domain and N-terminal domain and highlight alternative targets on the spike. We consider this structural work together with mAb effects in vivo to suggest correlations between structure …

Purification And Activity Analysis Of A Highly Active Recombinant Catalase From Psychrophilic Bacterium Psychrobacter Cryohaloentis, Andrew Stewart

Honors Program Theses

To date, the genus Psychrobacter includes 37 recognized bacterial species isolated from different sources ranging from Siberian permafrost (P. cryohalolentis) to seal feces (P. lutiphocae). Due to its diverse ecological niches, several species belonging to the genus have different enzymatic activities with potential applications in bioremediation and the food industry. In this study, a bioprospecting approach was used to perform a in depth analysis of a Psychrobacter-like bacterial strain isolated from goose feces (designated as Psychrobacter unknown). The phylogenetic analysis of 16S rRNA gene and homologous sequence studies suggest that the strain may represent a novel species of the Psychrobacter …

High Throughput Analysis To Study Emerging Pollutants And Nanoparticle Fate In Biological Systems, Xiaolong He

Doctoral Dissertations

”The increasing applications of emerging and fugitive contaminants (EFCs) and engineered nanoparticles (ENPs) attract significant research interest for their potential risks to human health and the environment. In order to assess the health risks of these emerging contaminants, rapid and reliable analytical methods to measure the concentrations and fates of these contaminants are imperative. This dissertation focuses on the developments of advanced analytical methods and their applications to study those emerging contaminants in crop plant and simulated gastric fluid (SGF). Three types of mass spectrometry based methodologies have been developed, one is freeze-thaw/centrifugation extraction followed by high performance liquid chromatography …

Lysosomal Slc46a3 Modulates Hepatic Cytosolic Copper Homeostasis, Jung-Hwan Kim, Tsutomu Matsubara, Jaekwom Lee, Cristina Fenollar-Ferrer, Kyungreem Han, Donghwan Kim, Shang Jia, Christopher J. Chang, Heejung Yang, Tomokazu Nagano, Kristopher W. Krausz, Sun Hee Yim, Frank J. Gonzalez

Department of Biochemistry: Faculty Publications

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly …

The Quantitative Assessment Of Pond Scum: An Examination Of The Biogeochemistry Of Phosphorus Cycling In The Belgrade Lakes, Abbey M. Sykes

Honors Theses

The internal recycling phosphorus in freshwater lake bottom sediments represents a significant source of hypolimnetic phosphorus (P) release for many of Maine’s lakes. In summer months, Maine lakes often thermally stratify and the lake hypolimnion develops anoxia, leading to a reduction in redox potential at the sediment-water interface. These reducing conditions facilitate the reductive dissolution of ferric iron, and, since phosphorus is often present in freshwater lake sediments as solid FeOOH-PO₄ complexes, results in release of soluble phosphorus into the water column. Our current study presents field and laboratory data from sediment fractionation extractions designed to quantify concentrations of …

Photoreceptor Phosphodiesterase (Pde6): Activation And Inactivation Mechanisms During Visual Transduction In Rods And Cones, Rick H. Cote

Faculty Publications

Rod and cone photoreceptors of the vertebrate retina utilize cGMP as the primary

intracellular messenger for the visual signaling pathway that converts a light stimulus into an electrical response. cGMP metabolism in the signal-transducing photoreceptor outer segment reflects the balance of cGMP synthesis (catalyzed by guanylyl cyclase) and degradation (catalyzed by the photoreceptor phosphodiesterase, PDE6). Upon light stimulation, rapid activation of PDE6 by the heterotrimeric G-protein (transducin) triggers a dramatic drop in cGMP levels that lead to cell hyperpolarization. Following cessation of the light stimulus, the lifetime of activated PDE6 is also precisely regulated by additional processes. This review summarizes …

Photoreceptor Phosphodiesterase (Pde6): Structure, Regulatory Mechanisms, And Implications For Treatment Of Retinal Diseases, Rick H. Cote, Richa Gupta, Michael J. Irwin, Xin Wang

Faculty Publications

The photoreceptor phosphodiesterase (PDE6) is a member of large family of Class I phosphodiesterases responsible for hydrolyzing the second messengers cAMP and cGMP. PDE6 consists of two catalytic subunits and two inhibitory subunits that form a tetrameric protein. PDE6 is a peripheral membrane protein that is localized to the signaling-transducing compartment of rod and cone photoreceptors. As the central effector enzyme of the G-protein coupled visual transduction pathway, activation of PDE6 catalysis causes in a rapid decrease in cGMP levels that results in closure of cGMP-gated ion channels in the photoreceptor plasma membrane. Because of its importance in the phototransduction …

Use Of Small Molecule Fanconi Anemia Pathway Inhibitors As Sensitizing Agents To Laromustine., Sam W. Marchant

Honors Theses

Laromustine is an experimental chemotherapeutic sulfonyl hydrazine prodrug shown in clinical trials to be effective against acute myeloid leukemia. The mechanism of action of laromustine involves interstrand crosslinking, via chloroethylation, and enzyme inhibition, caused by carbamoylation. The work described herein aims to investigate whether inhibition of the replication-dependent interstrand crosslink repair Fanconi Anemia pathway further sensitizes cells to laromustine. By measuring metabolic activity immediately after drug exposure, we find laromustine to be equally as cytotoxic towards Fanconi Anemia deficient and wild type cells. However, through clonogenic assays we show Fanconi Anemia mutations sensitize cells to laromustine’s anti-proliferative effect. Furthermore, we …

Marine Phytoplankton Functional Types Exhibit Diverse Responses To Thermal Change, S. I. Anderson, A. D. Barton, Sophie Clayton, S. Dutkiewicz, T. A. Rynearson

OES Faculty Publications

Marine phytoplankton generate half of global primary production, making them essential to ecosystem functioning and biogeochemical cycling. Though phytoplankton are phylogenetically diverse, studies rarely designate unique thermal traits to different taxa, resulting in coarse representations of phytoplankton thermal responses. Here we assessed phytoplankton functional responses to temperature using empirically derived thermal growth rates from four principal contributors to marine productivity: diatoms, dinoflagellates, cyanobacteria, and coccolithophores. Using modeled sea surface temperatures for 1950-1970 and 2080-2100, we explored potential alterations to each group's growth rates and geographical distribution under a future climate change scenario. Contrary to the commonly applied Eppley formulation, our …

Detrital Neodymium And (Radio)Carbon As Complementary Sedimentary Bedfellows? The Western Arctic Ocean As A Testbed, Melissa S. Schwab, Jörg D. Rickli, Robie W. Macdonald, H. Rodger Harvey, Negar Haghipour, Timothy I. Eglinton

OES Faculty Publications

Interactions between organic and detrital mineral phases strongly influence both the dispersal and accumulation of terrestrial organic carbon (OC) in continental margin sediments. Yet the complex interplay among biological, chemical, and physical processes limits our understanding of how organo-mineral interactions evolve during sediment transfer and burial. In particular, diverse OC sources and complex hydrodynamic processes hinder the assessment of how the partnership of organic matter and its mineral host evolves during supply and dispersal over continental margins. In this study, we integrate new and compiled sedimentological (grain size, surface area), organic (%OC, OC-δ¹³C, OC-F¹⁴C), and inorganic …

Expression And Characterization Of Dextransucrase From Leuconostoc Citreum Abk-1, Kakuttapun Bulan

Chulalongkorn University Theses and Dissertations (Chula ETD)

Dextransucrase is an enzyme in the glycoside hydrolase 70 family that catalyze the hydrolysis of sucrose to fructose and glucose, the glucose residues are transferred to synthesize dextran via transglycosylation reaction. Dextransucrase from Leuconostoc citreum ABK-1 (Lcdextransucrase ), which has 99.5% homology with dextransucrase in the NCBI database, was cloned, expressed, and purified. The size of the Lcdextransucrase was predicted by ExPASy and the result showed that its size was 168 kDa with a pI of 5.15. Protein expression was confirmed by western blot analysis. Lcdextransucrase was purified through Ni-sepharose coupled with DEAE anion exchange column chromatography to approximately 80% …

Influence Of Metal Sleeves In The Accuracy Of Dental Implant Placement Using Guided Implant Surgery, Coleman Adams

Theses and Dissertations

The fabrication of implant surgical guides through stereolithographic 3D printing has become a staple in dental implant guided surgery over the last couple decades. These surgical guides have typically utilized metal sleeves to assist in guidance of the drills during osteotome preparation. The metal sleeves can be costly and potentially cause deviations if improperly placed during post-processing of the guide. This research explored a novel method for the utilization of sleeve-free surgical guides by comparing the dimensional and angulational deviations between the implant guides with and without a metal sleeve. To achieve this goal, two separate aims were pursued. Our …

Computational Analysis And Prediction Of Intrinsic Disorder And Intrinsic Disorder Functions In Proteins, Akila I. Katuwawala

Theses and Dissertations

COMPUTATIONAL ANALYSIS AND PREDICTION OF INTRINSIC DISORDER AND INTRINSIC DISORDER FUNCTIONS IN PROTEINS

By Akila Imesha Katuwawala

A dissertation submitted in partial fulfillment of the requirements for the degree of Engineering, Doctor of Philosophy with a concentration in Computer Science at Virginia Commonwealth University.

Virginia Commonwealth University, 2021

Director: Lukasz Kurgan, Professor, Department of Computer Science

Proteins, as a fundamental class of biomolecules, have been studied from various perspectives over the past two centuries. The traditional notion is that proteins require fixed and stable three-dimensional structures to carry out biological functions. However, there is mounting evidence regarding a “special” class …

Single Molecule Investigations Of Holliday Junction Binding Protein Ruva, Dalton Reed Gibbs

Theses and Dissertations

DNA breaks are inevitable as they mainly occur due to cells’ own reactive oxygen species (ROS). While DNA breaks can be single-stranded or double-stranded, the double-stranded DNA (dsDNA) breaks are more dangerous. If such damage is not repaired, it can lead to genetic instability and serious health issues including cancers. One way dsDNA breaks can be repaired is via a process called homologous recombination (HR), which involves several DNA-binding proteins. Therefore, to have a better insight into the repair mechanism and origin of repair defects, we need a better understanding of how these proteins interact with DNA itself and DNA …

Novel Roles Of Yy1 In Astrocytes, Karli A. Mockenhaupt

Theses and Dissertations

Astrocytes tile the entire central nervous system, are diverse across brain regions, and perform specialized functions fine-tuned for their neuronal circuit. Initially, astrocyte heterogeneity is generated through intrinsic programs specified by their progenitors. Then, as astrocyte mature, they are specialized by region-specific communication with surrounding cells. The molecular programs that regulate this diversity and allow for the accommodation to the needs of the local environment remains elusive. We explored a ubiquitously expressed zinc finger transcription factor, Yin Yang 1 (YY1), that both activates and represses transcription by regulating loops of active chromatin. This report describes the diverse functions of astrocytic …

Identification And Characterization Of Modulators Of Human Mrp1 (Abcc1) And Human Mrp2 (Abcc2) Expression, Vivian Osei Poku

Electronic Theses and Dissertations

ATP-binding cassette (ABC) transporters are known to play a critical role in conferring multidrug resistance (MDR) in various cancers. Several retrospective analyses of chemotherapy results have reported high expression of Multidrug Resistance Protein 1 (MRP1) and Multidrug Resistance Protein 2 (MRP2) in tumor cells exhibiting the MDR phenotype. High MRP1 and MRP2 expression in cancer patients predict a higher risk of treatment failure, resulting in relapse and disease recurrence as well as shortened survival rates. The key role of MRP1 and MRP2 play in the development of MDR makes them important therapeutic targets that hold a great promise for addressing …

Characterization Of Cucurbitacin-Inspired Estrone Analogues As Novel Inhibitors Of Human Atp- Binding Cassette Proteins (Abcb1 And Abcc1), Jennifer Kyeremateng

Electronic Theses and Dissertations

ATP-binding cassette (ABC) transporters are a large class of integral membrane proteins that contribute to key physiological functions in all organisms by utilizing ATP binding and hydrolysis to transport diverse substrates across membrane barriers. P-glycoprotein (P-gp/ ABCB1) and Multidrug Resistance protein 1 (MRP1/ABCC1) are widely reported ABC transporters associated with multidrug resistance in cancer. Multidrug resistance (MDR) mediated by P-gp and MRP1 is responsible for treatment failures of many metastatic cancers as a result of reduced accumulation, bioavailability and diminished potency of anticancer drugs. Currently, known P-gp and MRP1 inhibitors are limited due to toxicity, lack of selectivity and low …

Host Factors Affecting Group Ii Intron Retrotransposition, Justin Michael Waldern

Legacy Theses & Dissertations (2009 - 2024)

Group II introns are self-splicing mobile elements that are thought to be the evolutionary ancestors of eukaryotic retrotransposons, the spliceosome, and spliceosomal introns. Yet, little is known about how group II introns have coevolved within their native hosts or about the corresponding host biology.Using the Ll.LtrB intron in its native host, Lactococcus lactis, I first sought to identify host factors that influence retrotransposition. Since retrotransposition can be costly to the host organism, group II introns must achieve a delicate balance between self-proliferation and host preservation. By utilizing the native host organism, my goal was to unearth retrotransposition-related mechanisms that have …

Pre-Steady-State Kinetic Characterization Of An Antibiotic-Resistant Mutation Of Staphylococcus Aureus Polc, Rachel Alice Nelson

Legacy Theses & Dissertations (2009 - 2024)

In this dissertation I provide a pre-steady-state kinetic characterization of an antibiotic-resistant mutant of a Staphylococcus aureus DNA polymerase. Staphylococcus aureus is one of the most common causes of infections in humans, and is widely known for its ability to acquire resistance to most antibiotics. Staphylococci infections pose a significant burden to the healthcare system and increase mortality, as more than 95% of Methicillin-Resistant S. aureus (MRSA) infections do not respond to first-line antibiotics. The limited treatment options for Staphylococci infections underscores the need for novel, alternative strategies. In this thesis, I discuss 6-anilinouracils (6-AU), a family of potent dGTP …

Development Of Chemical Methods For Oligonucleotide Purification, Paramagnetic Labeling And Synthesis Of Dna-Based Advanced Materials, Muhan He

Legacy Theses & Dissertations (2009 - 2024)

This thesis describes a chemical method for alternative oligonucleotide purification that is non-chromatographic and gel-free and allows to routinely synthesize and purify long functional RNA strands. The purification of long RNAs is based on the bio-orthogonal inverse electron demand Diels-Alder (IEDDA) chemistry between trans-cyclooctene (TCO) and tetrazine (Tz). Target oligonucleotide strands are selectively tagged with Tz and can be captured and purified from the failure sequences with immobilized TCO. RNA strands are synthesized on solid support through a photolabile linker to avoid the loss of Tz tag. Purity of the isolated oligonucleotides was evaluated using gel electrophoresis, HPLC and mass …

Missense Mutations In The Gamma Crystallins And Mechanisms Of Lens Opacity, Wenjuan Hou

Legacy Theses & Dissertations (2009 - 2024)

Cataract, or clouding of the ocular lens, among the most common types of eye diseases, is the leading cause of blindness worldwide. With the opacity or clouding of the lens, light incident on the lens is scattered rather than being transmitted and is thus prevented from focusing on the retina. The lens becomes cataractous due to a large number of reasons, among which aging and genetic mutations are two of the most common factors. Clouding of the center of the lens or nuclear opacity, is the most frequently observed type of age-onset cataract, as well as inherited, congenital cataract [1, …

Kinetic Characterization Of Two C-Family Polymerases From The Gram-Positive Bacterium Staphylococcus Aureus, Sean P. Fagan

Legacy Theses & Dissertations (2009 - 2024)

In this dissertation, I review the fundamental processes and mechanisms for bacterial DNA replication, especially the mechanisms employed by high-fidelity DNA polymerases to replicate the genome. Unlike the prototypical bacterial system from Escherichia coli which uses a single C-family polymerase, DNA polymerase IIIα (Pol IIIα), to replicate the genome, low-GC content Gram-positive bacteria utilize two essential C-family polymerases, PolC and DnaE. PolC and DnaE work cooperatively to replicate the genome, with DnaE initiating synthesis from RNA-primers and PolC performing the bulk synthesis. Although atomic structures of both PolC and Pol IIIα are available, detailed pre-steady state kinetic analysis of the …

Functional Characterization Of Threonine 49 Phosphorylation Of Cytochrome C, Antoine Khobeir

Wayne State University Theses

Cytochrome c (Cytc) is a pivotal multifunctional mitochondrial protein that serves as a single electron carrier between complexes III and IV of the electron transport chain. It has important roles in both cellular respiration and apoptosis. The novel Thr49 (T49) phosphorylation of Cytc likely affects mitochondrial respiration, membrane potential, ROS production, ATP production, and apoptosis. Based on the functional characterization of previously mapped phosphorylation sites (Tyr97, Tyr48, Thr28, Ser47, Thr58) of the lab, we hypothesize that T49 phosphorylation will lead to controlled respiration, optimal intermediate mitochondrial membrane potential, lower ROS production, and inhibition of apoptosis compared to unphosphorylated Cytc. Here …

Metabolite Profile And Biological Activities Of Crude Early Immature Durian Durio Zibethinus Fruit Extracts, Putthamas Pewlong

Chulalongkorn University Theses and Dissertations (Chula ETD)

Durian (Durio zibethinus) is a popularly consumed fruit in Thailand. To maintain the quality of fruit including size and taste, lots of early immature durian fruit (30-45 days after anthesis) were thinned. Therefore, the fruit at this stage is considered as an agricultural waste and there is no study focusing on this waste yet. The aim of the present study was to investigate the metabolite profile and biological activities related to human skin and hair dermal papilla cells. Four groups of primary metabolites (sugars, organic acids, amino acids, and miscellaneous) were annotated using GC-MS, meanwhile HPLC-Q-TOF-MS/MS analysis was performed to …

Lonidamine Is A Novel Inhibitor Of Fatty Acid Oxidation, Tianhai He

Theses and Dissertations

In this thesis project, we examined the hypothesis that lonidamine is an effective inhibitor of FAO in mammalian cells, underling its inhibitory effects on bioenergetic production and anti-cancer action. We compared its effects on FAO and glycolysis in multiple human cancer cell lines of different lineages.

Interactions Of Post-Pks Enzymes Of The Mithramycin Biosynthetic Pathway, Ryan Wheeler

Theses and Dissertations--Pharmacy

Combinatorial biosynthesis is a powerful tool for generating new, more active drug analogues to combat disease. But in order for combinatorial biosynthesis to be employed to its full potential, a deep understanding of the enzymes that produce the parent molecule must be had. The goals of the work presented in this thesis are to characterize the reaction catalyzed by MtmW, the final enzyme in the mithramycin (MTM) biosynthetic pathway, and to discover the interaction between MtmW and MtmOIV.

MtmW is an aldol-ketoreductase responsible for reducing the most distal carbonyl on the MTM pentyl side chain. It forms an octamer that …

An Integrative Analysis On The Role Of Notch1 And Notch3 In Musculoskeletal Disease, Haydee Magdalena Torres

Electronic Theses and Dissertations

Notch signaling is an evolutionarily conserved signaling pathway involved in cell fate decisions and cell renewal of multiple systems, including skeletal and muscle homeostasis. As the multifunctional nuclear complex of Notch1 is essential for its pathological function, identifying these interacting partners represents a potentially broad range of druggable vulnerabilities. We have completed a BioID proteomics study and found a large set of nuclear proteins associated with NOTCH1, including transcriptional regulators and epigenetic modulators, of which Histone Deacetylase 1 (HDAC1) is a promising interacting partner of Notch1. Our Cytoscape analysis predicted HDAC1 to be a top critical node in an activated …

Sars-Cov-2: An Investigation On Mutagenicity And Its Effects On Infectivity And Mortality, Tyler Elliott Silverwood

Honors Theses and Capstones

SARS-CoV-2, the etiological agent of the COVID-19 pandemic, has rapidly become a worldwide public health concern. Classified as a betacoronavirus, it is the third human coronavirus (HCoV) to emerge in the 21st century that causes severe disease, alongside SARS-CoV and MERS-CoV. The genome consists of open reading frames encoding accessory proteins and four structural proteins, including the spike protein which is a key determinant of host cell tropism. Mutations within the genome, particularly the spike gene, have been linked in-vitro to increased binding affinity to the human receptor angiotensin-converting enzyme 2 (hACE2), increased fitness in human hosts, and immune evasion. …

Biocompatible And Multifunctional Trityl Spin Probes For Electron Paramagnetic Resonance Spectroscopy, Teresa D. Gluth

Graduate Theses, Dissertations, and Problem Reports (ETD)

The primary objective of my thesis was to develop and utilize a biocompatible multifunctional trityl spin probe for concurrent measurement of pO₂, pH_e, and [P_i] in vivo by electron paramagnetic resonance (EPR) spectroscopy (Chapter 2). My first goal was to synthesize the proposed probe we are terming HOPE71. Secondly, HOPE71 was characterized by X-band and L-band EPR spectroscopy. Next, the biocompatibility of HOPE71 was assessed through an albumin binding test, cytotoxicity assays, and in vivo intravenous tolerance. Then, the use of HOPE71 to measure the target parameters was demonstrated in a breast cancer …

An Investigation On The Regulation Of The Tweak–Fn14–Nf-Kb Pathway, Jawahar Khetan

Doctoral Dissertations

“Dysregulation of inflammatory pathways is strongly implicated in cancers and autoimmune diseases. The most consequential of these pathways involves the nuclear translocation of NF-kB, a transcription factor that induces the transcription of multiple proteins associated with cell survival, inflammation, proliferation and death. It is activated when the fibroblast growth factor-inducible 14 kDa protein (Fn14), a trimeric receptor recruits its ligand, TWEAK. Studies have shown that Fn14 is over-expressed in many tumors, the aggressiveness of which is often correlated with the degree of upregulation. Furthermore, TWEAK-Fn14 activation has been shown to result in persistent NF-kB activation. Using a mechanistic model of …