Digital Commons Network^™

Biochemical Characterization Of Fsa1572 From Fervidibacter Sacchari, The First Hyperthermophilic Gh50 With Β-1, 4-Glucanase Activity, Jonathan Covington

UNLV Theses, Dissertations, Professional Papers, and Capstones

The bacterium Fervidibacter sacchari is an aerobic hyperthermophile that catabolizes various polysaccharides and is the only cultivated member of the class Fervidibacteria within the phylum Armatimonadota. Among its glycoside hydrolase (GH) cache is an enzyme from GH family 50 (GH50), an understudied family with only 25 characterized representatives and two known activities from 1,518 predicted members in the Carbohydrate-Active EnZyme (CAZy) Database. Here, we expressed, purified, and functionally characterized an extracellular GH50 from F. sacchari called Fsa1572. Using colorimetric assays, we show it has novel β-1,4-glucanase activity and only weak agarose activity that is typical for GH50 enzymes. The purified …

Long-Range Regulation By The Shigella Virulence Gene Regulator, Virb, Cody Cris

UNLV Theses, Dissertations, Professional Papers, and Capstones

Nucleoid-structuring proteins (NSPs) in bacterial cells can exert gene regulatory effects over great distances on DNA. This contrasts with the traditional view of bacterial transcriptional regulation, where transcription factors bind cis-acting regulatory sites proximal (<250 bp) to promoters to influence gene expression. In Shigella species, the histone-like nucleoid-structuring protein (H-NS) transcriptionally silences many genes on the large (~220 kb) virulence plasmid. During host colonization, however, silencing by H-NS is alleviated by the anti-silencer protein, VirB, which remodels the H-NS:DNA complex. This transcriptional anti-silencing is mediated by VirB:DNA interactions and the modulation of DNA supercoiling, which lead to the up-regulation of virulence genes. VirB is a central regulator of Shigella virulence, but the mechanistic basis for transcriptional regulation is incompletely characterized. Here, I focus on the potential for VirB to regulate transcription from remotely located DNA binding sites. Employing complementary in vivo approaches, I assessed the spatial stringency of the VirB binding site and elucidated how varying cellular VirB levels impacts regulation over different distances. I also evaluated how a molecular roadblock on DNA influences remote regulation by VirB. Additionally, I developed control tools to validate the ability of VirB to spread along DNA, an activity of VirB that putatively drives transcriptional regulation. These data are consistent with transcriptional anti-silencing being driven by VirB spreading along DNA. Overall, these findings highlight how VirB is not a conventional transcriptional regulator and broaden our perspective on transcriptional control in bacteria. Furthermore, this work contributes to our understanding of long-range gene regulatory effects, which are frequently overlooked and poorly characterized in bacteria.

Poop! There It Is! Anti-Germinants And Biological Variables As Modulators Of Clostridioides Difficile Infection (Cdi), Jacqueline Renee Phan

UNLV Theses, Dissertations, Professional Papers, and Capstones

Clostridioides difficile infection (CDI) is a major cause of antibiotic-associated diarrhea. Due to its insidious nature, the Centers for Disease Control and Prevention (CDC) has declared CDI an urgent threat. A key characteristic of C. difficile is its ability to form dormant spores that act as the infectious vehicles for disease. In the gut, spores recognize bile salts to germinate into toxin-producing cells.Dysbiosis of the gut microbiome is a key factor in allowing the C. difficile spores to germinate. Normal gut microbiota naturally protects from CDI. However, biological variables such as diet and sex have been found to modulate to …

Gonads Without Glp-1: Silencing Glp-1 In The Male Somatic Gonad In Caenorhabditis Elegans, Matthew Titus

Undergraduate Theses

In C. elegans, the gene glp-1 encodes for a Notch receptor called GLP-1, one of two found in C. elegans’ genome. The gene has been previously implicated in the development of the hermaphroditic germline as well as playing a role in the mitosis/meiosis decision. Genetic screening has further identified it as potentially playing a role in the development of the male somatic gonad, making it an ideal candidate for a reverse genetic. We did this by silencing glp-1 and observing if any alterations to the gonad’s phenotype occur.

Normally this could be done by performing a gene knockout. …

Electrochemical Reduction Of Carbon Dioxide Using Ruthenium And Amines, Sydney Moise

Honors Theses

With an increased concern for climate change in the recent years, a significant area of research has been devoted to the reduction of greenhouse gases. Carbon dioxide (CO2) resides in the atmosphere between 300 – 1000 years, making the reduction of the molecule a substantial field of study.1 Amines have been used as CO2 scrubbing agents in literature historically, due to their ability to form bonds to carbon.2 Although studies involving metal catalysts and amines have been reported numerous times, research involving chemical reduction of CO2 using purely amines is scarce. In this paper, amines, in addition to hydride donors …

Role Of Cdx4 And Sp5l In Zebrafish Development, Wesley Tsai

Honors Theses

The Caudal Type Homeobox transcription factors cdx are a family of genes found in vertebrates that regulates body regionalization and anterior-posterior patterning. They are also responsible for regulating axial elongation, but the mechanisms behind this behavior are not known. Previous studies in mouse embryonic stem cells have shown that the cdx genes are necessary for upregulating the gene sp5 which may be linked to axial elongation. Sp5 is a zinc-finger transcription factor belonging to the specificity protein (sp) family. Our group has used in-situ hybridization experiments on zebrafish embryos to show that sp5-like (sp5l) is transcribed within tailbud tissues that …

Bryophytes Of Goochland County, Virginia, Mikayla Quinn

Honors Theses

Bryophytes are non-vascular land plants that include mosses, liverworts, and hornworts. Although easier to overlook because of their smaller size, bryophytes are a fundamental part of the ecosystem. As such, maintaining record of their biodiversity is important. Yet, records of bryophyte species in Goochland County, VA were low compared to more thoroughly documented counties such as Prince Edward County. This study expands the documentation of bryophyte flora and presents a checklist of bryophyte species found Virginia’s Goochland County from 2020-2023. Fieldwork conducted at public and privately-owned properties throughout the county between January 2020 and March 2023 yielded 702 specimens that …

Potential Use Of The Gel Extract Of Butterfly Pea Flower As Topical Therapy To Prevent Photodamage By Downregulating Tnf-Α And Caspase-3 Expression Levels In Uvb-Exposed Rats, Elvana Cahyani, Agung Putra, Prasetyowati Subchan

Makara Journal of Health Research

Background: Prolonged exposure to UVB radiation causes DNA damage in skin cells by raising the levels of reactive oxygen species, resulting in the production of inflammatory factors and skin issues. Plant extracts are frequently used to counteract photodamage due to their antioxidant properties. One example is the floral extract of the butterfly pea plant, which contains flavonoid antioxidants. However, the effect of the extract on inflammatory factors is unknown. This study investigated how tumor necrosis factor-alpha (TNF-α) and caspase-3 expression changed when a butterfly pea flower extract gel was applied topically to UVB-exposed animals.

Methods: Experimental and control …

Investigating The Helicase Activity Of Methylated Vs Unmethylated Ded1, Hannah Lukow

Honors Theses

Ded1 is an RNA helicase protein of the DEAD-box subfamily in eukaryotic organisms (Sharma & Jankowsky, 2014) which can act as an activator or initiation factor, during translation (Hilliker et al., 2011). Ded1 has several functions in yeast including assembly of translational initiation factors, scanning the mRNA for the start codon, and unwinding any double stranded segments of mRNA with its helicase ability. Ded1 was discovered to be methylated at four arginine sites in vivo (Low et al., 2013), with a fifth methylation site being discovered recently (Low et al., 2020), however the purpose of such post-translational modifications is still …

The Role Of Sars-Cov-2 Orf8 Protein Arks Motif On Novobiocin Binding, Si Chun Chiu, My T. Nguyen

Celebrating Scholarship and Creativity Day (2018-)

The discovery of the SARS-CoV-2 virus during the COVID-19 pandemic required scientists to develop medical solutions to reduce infectivity, severity of symptoms, and death. Although vaccines and drugs provided urgent assistance, the need to continue developing better drugs is necessary long term, and understanding the structure of the virus and finding potential inhibitors would prove vital to discovering solutions to this worldwide health problem. This experimental project focuses on targeting the unique accessory protein, Open Reading Frame 8 (ORF8) in SARS-CoV-2 through studying its interactions with a repurposable drug, novobiocin. Importantly, ORF8 specializes in helping evade immune system checks by …

Genome Announcement For E Cluster Phage Tarkin, Katherine Cleary

Chemistry & Biochemistry Student Scholarship

Katherine Cleary ’23
Major: Biochemistry
Faculty Mentor: Dr. Kathleen Cornely, Chemistry and Biochemistry

The Role Of Cerium(Iii) In Bacterial Growth And The Microbial Transformation Of Aromatic Hydrocarbons, Shruti Sathish

Honors Theses

Biofilms are communities of surface-attached bacterial cells encased in an exopolymeric matrix. In this state, they are more resistant to antimicrobial treatment and can have adverse effects in medical, agricultural, and industrial settings. Whereas, as biocatalysts, biofilms from nonpathogenic bacteria enhance their performance and stability in catalysis. Unfortunately, there are several challenges when using bacteria in organic transformations due to their complex cellular chemistry. Trivalent lanthanide metals were discovered to serve regulatory roles in some bacterial catalytic processes, including those of Pseudomonas putida KT2440 (P. putida), a non-infectious Gram-negative bacterium. The main goal of our research is to use cerium(III) …

Testing Of Indazole Inhibitors Of Kasa, A Vital Enzyme Of M. Tuberculosis, Karissa Highlander

Departmental Honors & Graduate Capstone Projects

Tuberculosis is a disease that affects the lungs caused by Mycobacterium tuberculosis (M. tuberculosis). Although drug treatment options exist, increased rates of antibiotic resistant strains have become more prevalent in recent years, driving a need for new treatment approaches. KasA, a β-ketoacyl synthase, has been found to synthesize parts of the cell wall and been identified as an attractive drug target. Previous medicinal chemistry research has been completed to synthesize six effective competitive inhibitors of KasA that would potentially block the enzyme from binding the substrate, preventing elongation of the backbone and creation of the mycolic fatty acids that …

Toward The Design, Synthesis, And Characterization Of Abiotic Coiled-Coil Peptides Via Solid-Phase Copper-Catalyzed Azide-Alkyne Cycloaddition (Sp-Cuaac) Click Reaction For The Preparation Of Controlled Self-Assembly Molecular Building Block, Liaquat Ali

Chemistry Theses

The production of cutting-edge materials, the development of novel medications, drug delivery systems, technological advancements, and biosynthesizing all depend on molecular building blocks. Proteins are required for the creation of intricate, well-organized structures, and coiled-coil protein domains are vital subunits for the oligomerization of protein complexes, gene expression, and the structural components of biological materials. The numerous interactions between a wide variety of amino acids make it difficult to assemble protein complexes with a particular shape. In the current study, we successfully designed and synthesized four different 32-residue peptides, each of which had two modified amino azide residues. These peptides …

Analysis Of Adsorbed Polyphosphate Changes On Milled Titanium Dioxide, Using Low-Field Relaxation Nmr And Photoelectron Spectroscopy, Laura N Elliott, David Austin, Richard A Bourne, Ali Hassanpour, John Robb, John L Edwards, Stephen Sutcliffe, Timothy N Hunter

Faculty, Staff and Students Publications

In this study, changes in the adsorbed amount and surface structure of sodium hexametaphosphate (SHMP) were investigated for aluminum-doped TiO2 pigment undergoing milling. Relaxation NMR was utilized as a potential at-line technique to monitor the effect of milling on surface area and surface chemistry, while XPS was used primarily to consider the dispersant structure. Results showed that considerable amounts of weakly adsorbed SHMP could be removed with washing, and the level of dispersant removal increased with time, highlighting destructive effects of sustained high-energy milling. Nonetheless, there were no significant chemical changes to the dispersant, although increases to the bridging oxygen …

Apoptosis Induction In Jurkat T-Lymphocytes By Proton Pump Inhibitors (Ppis), Shreya Murali, Randall Reif

Departmental Honors & Graduate Capstone Projects

Apoptosis, commonly known as programmed cell death, constantly occurs in humans. As a cancer cell increases in acidity, apoptosis is induced. In healthy cells, proton pump proteins allow for H⁺ ions to permeate cellular membranes, regulating pH. However, proton pump inhibitors (PPIs), such as omeprazole, prevent proton movement. In previous studies, omeprazole induced cell death in Jurkat T lymphocytes; however, there was no confirmation of whether the cells died through apoptosis, or through necrosis, where the cell bursts. By using Annexin-V staining, the effects of omeprazole, dexlansoprazole, and esomeprazole on apoptosis induction can be measured. Cell death was observed …

From Deep Mutational Mapping Of Allosteric Protein Landscapes To Deep Learning Of Allostery And Hidden Allosteric Sites: Zooming In On “Allosteric Intersection” Of Biochemical And Big Data Approaches, Gennady M. Verkhivker, Mohammed Alshahrani, Grace Gupta, Sian Xiao, Peng Tao

Mathematics, Physics, and Computer Science Faculty Articles and Research

The recent advances in artificial intelligence (AI) and machine learning have driven the design of new expert systems and automated workflows that are able to model complex chemical and biological phenomena. In recent years, machine learning approaches have been developed and actively deployed to facilitate computational and experimental studies of protein dynamics and allosteric mechanisms. In this review, we discuss in detail new developments along two major directions of allosteric research through the lens of data-intensive biochemical approaches and AI-based computational methods. Despite considerable progress in applications of AI methods for protein structure and dynamics studies, the intersection between allosteric …

Functional Domains Of The Hsv-1 Ul37 Protein In Virus Infectivity And Retrograde Transport In Neurons, Carolyn Clark

LSU Doctoral Dissertations

Herpes simplex virus type 1 is large alphaherpesvirus with 20 envelope proteins and 23 tegument proteins. Virus entry is via fusion of the viral envelope with cellular membranes mediated by a complex of viral glycoproteins. Glycoprotein B (gB) is the only fusogenic glycoprotein, while other viral glycoproteins and membrane proteins including gD, gH, gL, gK and UL20 membrane protein modulate gB’s fusogenicity. Previously, Our laboratory developed the HSV-1 (VC2) vaccines strain with partial deletions in the amino termini of gK (ΔgK(31–68)) and the UL20 membrane protein (ΔUL20(4–22)). VC2 enters epithelial cells only via endocytosis followed by release of the capsid …

A Potential Role Of Gsk-3beta In The Development Of Pain Post-Spinal Cord Injury, Emily Ernst

Undergraduate Theses

Chronic neuropathic pain is a very common consequence following spinal cord injury (SCI) and poses significant clinical challenge. Current treatments are largely ineffective and as a result, patients are often left with debilitating pain at and below the level of the spinal cord injury. After spinal injury, a cascade of events occurs within the nervous system. One important aspect of this cascade for investigation is the anatomical changes that occur post-SCI. There is evidence of maladaptive sprouting/growth in sensory nerve fibers that is thought to play a role in the development and amplification of pain signals. Sprouting of primary afferents …

Ppgpp And Rna-Polymerase Backtracking Guide Antibiotic-Induced Mutable Gambler Cells, Yin Zhai, P J Minnick, John P Pribis, Libertad Garcia-Villada, P J Hastings, Christophe Herman, Susan M Rosenberg

Faculty, Staff and Students Publications

Antibiotic resistance is a global health threat and often results from new mutations. Antibiotics can induce mutations via mechanisms activated by stress responses, which both reveal environmental cues of mutagenesis and are weak links in mutagenesis networks. Network inhibition could slow the evolution of resistance during antibiotic therapies. Despite its pivotal importance, few identities and fewer functions of stress responses in mutagenesis are clear. Here, we identify the Escherichia coli stringent starvation response in fluoroquinolone-antibiotic ciprofloxacin-induced mutagenesis. Binding of response-activator ppGpp to RNA polymerase (RNAP) at two sites leads to an antibiotic-induced mutable gambler-cell subpopulation. Each activates a stress response …

Assessment Of Histone Acetyltransferase Homologs In Cryptococcus Neoformans, Phoebe Bridy, Felice Kho, Yuqi Wang

Liberty University Research Week

Undergraduate

Basic

Rescue Of Glutaric Aciduria Type I In Mice By Liver-Directed Therapies, Mercedes Barzi, Collin G Johnson, Tong Chen, Ramona M Rodriguiz, Madeline Hemmingsen, Trevor J Gonzalez, Alan Rosales, James Beasley, Cheryl K Peck, Yunhan Ma, Ashlee R Stiles, Timothy C Wood, Raquel Maeso-Diaz, Anna Mae Diehl, Sarah P Young, Jeffrey I Everitt, William C Wetsel, William R Lagor, Beatrice Bissig-Choisat, Aravind Asokan, Areeg El-Gharbawy, Karl-Dimiter Bissig

Faculty, Staff and Students Publications

Glutaric aciduria type I (GA-1) is an inborn error of metabolism with a severe neurological phenotype caused by the deficiency of glutaryl-coenzyme A dehydrogenase (GCDH), the last enzyme of lysine catabolism. Current literature suggests that toxic catabolites in the brain are produced locally and do not cross the blood-brain barrier. In a series of experiments using knockout mice of the lysine catabolic pathway and liver cell transplantation, we uncovered that toxic GA-1 catabolites in the brain originated from the liver. Moreover, the characteristic brain and lethal phenotype of the GA-1 mouse model was rescued by two different liver-directed gene therapy …

Study Of Protein Extraction Techniques For M. Foliorum Phages, Lola Sibaud, Anna Elgersma

Student Academic Conference

Characterizing phages is becoming increasingly important in biochemistry and biotechnology due to their potential to treat bacterial infections without need for antibiotics. Last year, students attempted to isolate proteins from Microbacterium foliorum infecting phages Babydotz and Rosadiaz. Proteins were successfully extracted; however, after further analysis it was shown that the obtained proteins belonged to the host bacteria and not the phages. The purpose of this study was to find a method to separate phage from host bacterial debris to successfully isolate phage proteins and analyze them later. Isolating phage proteins could help us characterize more phages and know more about …

Novel Plant Peroxidase Investigation For Use In Various Biochemical And Practical Applications, Sally Hess

Liberty University Research Week

Undergraduate

Basic

Activation Of Nrf2 Pathway By Natural Products, Genesis Schat, Madelyn Sedlachek

Liberty University Research Week

Undergraduate

Basic

Characterization Of A Sas3 Homolog In The Human Pathogenic Yeast Cryptococcus Neoformans, Lillianne Bartone

Liberty University Research Week

Undergraduate

Basic

Characterization Of Select Lysine Mutations Of The Cystine/Glutamate Transporter, System XC-, Anna Koppin, Claire Buck, Amanda Gibson

22nd Annual Celebration of Undergraduate Research and Creative Activity (2023)

System xc- is a membrane transport system that plays a critical role in mitigating oxidative stress. As such, its regulation is critical for proper brain functioning. Recent work in our lab has shown that System xc- activity increases immediately during an oxidative insult by undergoing a change in localization to the plasma membrane, but we have yet to identify the specific mechanism for the redistribution of the transporter. Previous studies have demonstrated that post-translational modifications of proteins can lead to differential protein distribution within cells. Therefore, in this study, we sought to determine if post-translational modification (PTM) of the transporter …

Does Phosphorylation On Serine 26 Of System Xc- Lead To Changes In Cell Surface Expression?, Katherine Lane

22nd Annual Celebration of Undergraduate Research and Creative Activity (2023)

System x_c^- is involved in transporting cystine into cells and glutamate out of cells, and ultimately in production of the antioxidant glutathione. Antioxidants are important in protecting cells from oxidative stress which can occur when waste products like H₂O₂ build up in the cell. Previous studies have shown that a critical regulator mTORC regulates system xc- by phosphorylating serine 26 (S26) on the N-terminus of the cystine/glutamate antiporter xCT, leading to a reduction in transport activity. The specific objective of this study is to determine the mechanism by which phosphorylation of S26 affects activity. To …

Creation Of An N-Terminal Xct Mutant Lacking Lysines For Use In Protein Turnover Studies, Alexandria Switzer

22nd Annual Celebration of Undergraduate Research and Creative Activity (2023)

xCT plays a role in protecting cells from oxidative stress as well as intracellular glutathione synthesis. If this process is impeded, reactive oxygen species accumulate in the cell, leading to cellular damage, and if left unchecked, it can lead to neuronal loss. Ubiquitin is a small protein that some evidence suggests can negatively affect the stress response pathway modulated by xCT, as well as induce cell death. It is hypothesized that ubiquitin binds to xCT on the N-terminal lysine residues, and there are six conserved lysines in the N-terminal domain at positions K4R, K12R, K30R, K37R, K41R, and K43R. Previous …

Early Life Δ9-Tetrahydrocannabinol Exposure Of F0 Zebrafish Causes Hyperactivity In F1 Offspring, Jenna Cripe

Honors Theses

As cannabis increases in its accessibility, potency, and acceptance across the United States, investigation into the multigenerational effects of Δ9-tetrahydrocannabinol (THC) exposure during key stages of development is critical. The aim of this study was to evaluate whether there would be behavioral impacts in the F1 offspring following a dose-response of THC exposure (0.08, 0.4, or 1 µM) during development in the F0 generation. Zebrafish (Danio rerio) were utilized in this study. Behaviors (locomotive activity and anxiety-like behavior) in the F1 generation were evaluated at 120 hours post-fertilization (hpf) with the larval photometer response (LPR) assay, and at 3, 11, …