Biochemistry, Biophysics, and Structural Biology Commons^™

Comprehensive Long- And Short- Read Sequencing Approaches To Screen The Mutational Landscape After Therapeutic Crispr-Editing For Duchenne Muscular Dystrophy, Mary Jia, Made Harumi Padmaswari, Landon Burcham, Shilpi Agrawal, Alexis Ivy, Abbey Bryan, Christopher E. Nelson

Biomedical Engineering Undergraduate Honors Theses

Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disease caused by mutations in the Dmd gene resulting in non-functional dystrophin. Adeno-associated virus (AAV)-mediated delivery of classical CRISPR systems resulting in double-stranded breaks (DSBs) has yielded promise as a curative DMD therapeutic. However, the consequences of AAV-CRISPR require more thorough characterization. PCR-enriched short-read amplicon sequencing at cut sites is the most common method of sequencing CRISPR editing, but PCR bias results in unreliable quantification of on-target edits. We analyze orthogonal long- and short-read sequencing approaches to screen the outcomes of CRISPR technologies at the DNA and RNA-level for DMD. We …

Nanosecond Pulsed Electric Field Modulates Electron Transport And Mitochondrial Structure And Function, Lucas Nelson Potter

Biomedical Engineering Theses & Dissertations

Pulsed power treatment has been used to induce regulated cell death (RCD) in cells or ablate tumors in animals. A subset of pulsed power as electroporation with high voltage and pulse duration of milliseconds is used for biomedical treatment to induce pores in the plasma membrane of cells. Nanosecond Pulsed Electric Fields (nsPEFs)– an extension of electroporation, uses waveforms with pulse durations on the order of 10-900 nanoseconds. nsPEF treatment has demonstrated intracellular effects for potential biomedical applications. In this work, nsPEF treatment is used to demonstrate changes that affect viability, plasma membrane permeability ROS (Reactive Oxygen Species) in the …

E-Cadherin Force Transmission And Stiffness Sensing, Mazen Mezher

Mechanical & Aerospace Engineering Theses & Dissertations

E-cadherin is the chief mediator of cell-cell adhesion between epithelial cells and is a known mechanosensor. Force transmission and stiffness sensing are two crucial aspects of E-cadherin mechanobiology.

E-cadherin has an extracellular adhesive region, a transmembrane region and an intracellular region that binds to adhesion-associated proteins. Here, we assessed how different factors affect the level of force transmission (i) from inside the cell such as adhesion-associated proteins, (ii) on the cell membrane, such as growth factor receptors and (iii) outside the cell, such as different binding partners in adhesion. To study the level of force transmission inside the cell, we …

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 …

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 …

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) …

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 …