Biochemistry, Biophysics, and Structural Biology Commons^™

Nicotinamide Riboside And Beta-Hydroxybutyrate Activate Parallel Pathways For C. Elegans Lifespan Extension, Mckenzie Peters

Undergraduate Honors Theses

Supplementation with nicotinamide riboside (NR), a form of vitamin B3 and a precursor of nicotinamide adenine dinucleotide (NAD⁺) extends lifespan in the nematode C. elegans and delays aging-related pathologies in mammals. During aging, levels of NAD⁺ decline causing metabolic dysfunction and oxidative damage. Studies in C. elegans found that when NR was administered during larval development it induced the mitochondrial unfolded protein response (UPR^mt), which is frequently associated with lifespan extension. Both calorie restriction (CR) and ketogenic diets (KD) have been shown to extend lifespan, in part through increasing NAD⁺ and through increasing levels …

In Vitro Characterization Of Fluorogenic Chemical Tools To Study Human Carboxylesterases (Cess), Taylor Parish Spidle

Undergraduate Honors Theses

Human carboxylesterases (CESs) are enzymes that are responsible for the metabolism of many important pharmaceuticals. Although CESs are key players in the hydrolysis of many ester-containing drugs, they remain understudied. Our group hypothesizes that this is primarily because there are few methods capable of reporting activity in live cells. Here, I report a new series of fluorogenic chemical tools to study the CES activity of one of the two major CESs in humans, CES1, in live cells. MCP-Me, MCP-Et, and MCP-iPr utilize the same carbonate group of a previously developed chemical tool in our group, FCP-1, and work to mimic …

Synthesis And Biological Testing Of Small-Molecule Mitochondrial Complex I Inhibitors, Willough Sloan

Undergraduate Honors Theses

This thesis delineates two main projects: the first outlines the structure elucidation efforts toward a Diels-Alder adduct of a novel reaction for the synthesis of chimaphilin, a naphthoquinone-based natural product with apoptotic or antiproliferative activity in certain cancer cells^1,2. The structure elucidation extends to derivatives of chimaphilin synthesized by the same cyclization reaction. While Diels-Alder reactions are usually regioselective, ¹H-NMR and ¹³C-NMR of the adducts was inconclusive and indicated the possibility of regioisomer presence, with one regioisomer being chimaphilin (or derivatives). A multitude of crystallization methods were carried out in order to be able to analyze …

From The Headlines: A Textual Analysis Of Social Polarization And Discord In Times Of Pandemic Across A Century In The United States, Alexa Demarco

Undergraduate Honors Theses

Within the past century, three major pandemics have affected the United States – the Spanish Flu, AIDS, and COVID-19. Each of these pandemics has tested the capabilities of the public health sector and the social resilience of the population. Scientists have studied the viruses and implemented public health measures to limit viral transmission, but the social responses to these preventative measures proved to be difficult to predict and control. The dissonance and polarization between the public health initiatives and the response of the general public in the most recent pandemic was apparent. Was this a pattern in other pandemics? Was …

Qualitative Proteomic And Genomic Analysis Of Klebsiella Pneumoniae Bacteriophage Kaomega, Emilee Carr

Undergraduate Honors Theses

Klebsiella pneumoniae is a Gram-negative Enterobacteriaceae that is a common nosocomial pathogen causing pneumonia, infections in the bloodstream, wound infections, and meningitis. It has developed natural resistance to multiple antibiotics, most notably carbapenems which are often seen as the last line of defense against multi-drug resistant pathogens. Bacteriophages are being investigated as a promising alternative treatment to antibiotics in fighting these resistant pathogens. KaOmega, a Klebsiella pneumoniae bacteriophage, was isolated, sequenced, and annotated to characterize and understand its potential for use in a phage therapy. Characterization included Phyre2 analysis to predict putative protein functions based on structural homology, burst size …

Exploring Melatonin As A Treatment For Oral Ulcers, William H. Sutherland

Undergraduate Honors Theses

The hormone melatonin is best known for its role in the sleep-wake cycle, but its anti-inflammatory and antioxidant effects have significant implications that have not been fully explored in oral health. Some studies use melatonin to treat gastrointestinal ulcers, including duodenal ulcers and oral mucositis, but we found no study reporting its effects on more common oral ulcers, like aphthous stomatitis. We hypothesize that the anti-inflammatory and antioxidant characteristics of melatonin could effectively prevent and heal oral ulcers. In this paper, we review the literature on melatonin to propose its use as a treatment for oral ulcers. We also include …

The Regulation Of Atg9a-Mediated Aggrephagy By An Ulk1-Independent Atg13-Atg101 Complex, Joshua Youngs

Undergraduate Honors Theses

Aggrephagy, a type of autophagy, is an essential cellular process by which protein aggregates are collected and broken down in the lysosome. Protein aggregates are implicated in several diseases including Alzheimer’s disease, diabetes, and cancer. Here, we investigate the ATG13-ATG101 protein complex, a sub-complex of the canonical ULK1 complex whose regulatory role in aggrephagy is not completely understood. We also develop a protein fragment complementation (PFC) assay using the biotin ligase TurboID to study the functions of the ATG13-ATG101 complex with increased specificity. We demonstrate that ATG13 is required for optimal degradation of p62-ubiquitin condensates. We also show that a …

The Synthesis Of 3-Formyltyrosine For The Purpose Of In Virto Fluorescent Visualization Of Microtubules, Angelina Bonacasa

Undergraduate Honors Theses

Microtubules and the desire to visualize them has been of utmost importance in understanding the behavior of the cytoskeleton. Even further, scientists are using this knowledge to elucidate cancerous activities as they occur within cells. In order to acquire this knowledge, the following methods have been established: the expression of tubulin after it has been fused with a fluorescent protein, the insertion of tubulin that has been exogenously labeled, and the use of fluorescent-probe conjugated microtubule ligands, such as paclitaxel. These methods, however, tend to distort the protein’s native function, thereby preventing an accurate analysis. This concern drives the need …

Computational Investigation Of Calmodulin Photocontrol With The Help Of An Azobenzene Derivative, Jeremy Wells

Undergraduate Honors Theses

The ability to control the activity and binding capability of enzymes in a reversible manner offers tremendous control over biological processes. Photocontrol, in particular, is promising in that electromagnetic radiation can be fine-tuned in terms of its strength, location, and duration. Photosensitive compounds, such as the azobenzene family, experience an isomerization at certain wavelengths, and attaching these compounds to enzymes has the potential to alter their structure and activity. Calmodulin (CaM) is a Ca2+ -sensitive signaling protein that has the ability to affect several downstream processes in eukaryotes and is an excellent target for photocontrol due to its small size …

Severe Hypoxia Up-Regulates Gluconeogenesis In Daphnia, Morad C. Malek

Undergraduate Honors Theses

Hypoxia is a significant low oxygen state that has complex and diverse impacts on organisms. In aerobes, various adaptive responses to hypoxia are observed that vary depending on the level of oxygen depletion and previous adaptation, hence the continued attention to hypoxia as an important abiotic stressor. Adaptive responses to hypoxia are primarily governed by the hypoxia-inducible factors (HIFs), which activate downstream genetic pathways responsible for oxygen transport and metabolic plasticity. In aquatic habitats, oxygen availability can vary greatly over time and space. Therefore, aquatic organisms’ adaptation to hypoxia is likely pervasive, especially in genotypes originating from waterbodies prone to …

Vitamin D And Its In Vitro Therapeutic Action Mediated Through Vdr Rather Than Pdia3, Jaeden Pyburn

Undergraduate Honors Theses

Brain calcification is a common occurrence in the aging process, with >20% of individuals over the age of 65 showing hardened plaques in the basal ganglia. Loss of the vitamin D receptor (VDR) in transgenic mice leads to formation of calcified plaques in the basal ganglia and thalamus within the mice. Vitamin D signals through two known vitamin D responsive proteins, protein disulfide isomerase A3 (PDIA3) and VDR. In vitro, vitamin D has been demonstrated to suppress calcification in osteoblast-like cells. Here, we aim to elucidate which of either PDIA3 or VDR transduce vitamin D mediated suppression of calcification in …

Glypro As A Self-Immolative Spacer For The Release Of Payloads From Antibody-Drug Conjugates, Justin Michael Howe

Undergraduate Honors Theses

Antibody-drug conjugates (ADCs) are an increasingly popular modality for targeted drug delivery in many oncological and immunological applications. The paratope of a monoclonal antibody (mAb) directs the delivery of a conjugated therapeutic payload to antigen expressing cells, resulting in a controlled transport of payload to a desired cell type. Internalization of the ADC followed by lysosomal degradation results in the release of a payload to perform its biochemical function. A chemical linker between the drug and the antibody is responsible for the stability of the conjugate in circulation alongside mediating the release of an unmodified payload under lysosomal conditions. Currently, …

Honey As A Biomonitor For Air Pollutant Deposition In The Eastern United States Using Ion Chromatography And Scanning Electron Microscopy, Cole Cochran

Undergraduate Honors Theses

Anthropogenic activities generate metal, acid, and particulate air pollutants which negatively impact human and ecological health. In the United States, power plant, industrial, and vehicle emissions are leading causes of air pollution, however, the measurement of air pollution at high-resolution spatial regimes remains a challenge. Honey has emerged as a powerful biomonitoring tool to effectively quantify contaminants without the need for a large array of monitoring instruments. I hypothesized that honey could be used to effectively measure and map modern air pollutant spatiotemporal relationships over the Eastern U.S. Using ion chromatography with sulfate as an indicator for air pollution and …

On The Relationship Of Diabetes And Sleep Apnea: Evolution And Epigenetics, Nancy Wilson

Undergraduate Honors Theses

This thesis gives an overview of the relationship between diabetes, sleep apnea, obesity, and heart disease. It then addresses evidence that the traditional understanding of this relationship is incomplete or misleading. In the process, there is a brief discussion of the evolutionary rationale for the development and retention of sleep apnea in light of blood sugar dysregulation, as an adaptive mechanism in response to environmental stressors, followed by a brief overview of the general concepts of epigenetics. Finally, this paper presents the results of a literature search on the epigenetic marks and changes in gene expression found in sleep apnea …

A Fret Flow Cytometry-Based Screening Assay For Multiplex Analysis Of Metabolites In T. Brucei, Ronald A. Zegarra

Undergraduate Honors Theses

Kinetoplastid parasites are a significant public health issue in some tropical and subtropical regions of the world. Kinetoplastid parasites all require glycolysis for survival, with host glucose key for ATP production. One such parasite, Trypanosoma brucei, exclusively metabolizes glucose in its bloodstream form. Trypanosomal glycolysis is unique because it displays unconventional structural features. Hence, glucose metabolism has been studied extensively in T. brucei and is a therapeutic target in kinetoplastid parasites.The lack of in vivo analytical techniques for measuring vital glycolytic metabolites in situ has restricted the ability of researchers to test, with high sensitivity and specificity, the essential roles …

Exploration Of Fluorinated Α,Β-Dehydroamino Acids And Their Structure, Austin Lesueur

Undergraduate Honors Theses

This thesis explores the synthesis of fluorinated α,β-dehydroamino acids, specifically a fluorinated dehydrovaline derivative. Previous work has been done on the equivalent dehydrovaline derivative without fluorine present and this work builds toward the fluorinated version with the goal of comparing the two structurally. The synthesis presented here pulls from previous synthetic strategies employed for dehydrovaline while also exploring the synthetic impact of the electronegative fluorine atoms.

Proteomic Analysis Of Mycobacteriophage Crimd, William Moeller

Undergraduate Honors Theses

Bacteriophages represent a large portion of the biomatter on our planet, and many of them have yet to be fully characterized. Here we discuss the proteomic analysis of a particular Bacteriophage, Mycobacteriophage CrimD. This phage was discovered on the Campus of William & Mary and has had its genome characterized. We took the next logical step of proteomic analysis.

In our analyses we made use high pressure liquid chromatography paired with linear ion trap mass spectrometry to analyze the proteome of CrimD at specific time points after the infection of its host, Mycobacterium smegmatis. Additionally, we used nanospray ionization with …

Reaction Pathways Initiated By One-Electron Oxidation Of Guanine: Reaction Pathways Leading To Stable End Products, Zach Cutright

Undergraduate Honors Theses

8-hydroxyguanine (8-oxoG) is one of the most important products resulting from the oxidation of guanine in DNA. 8-oxoG is known as a biomarker of oxidative stress such as lung cancer. 2,5-diamino-4H-imidazol-4-one (Iz) and its hydrolysis product 2,2-diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)-amino]-2,5-dihydrooxazol-5-one (Oz) are also important products of oxidation of guanine in DNA. Recently, novel types of dimer intermediates related to 8-oxoG and Iz have been identified in our research lab. Using high performance liquid chromatography (HPLC), LC-MS, and NMR coupled with educated hypotheses, it is possible to identify the products, X1 and X2, resulting from the oxidation of guanine and decipher the mechanisms leading …

Telsam-Target Protein Fusions Can Form Diffraction-Quality Crystals Without Direct Inter-Polymer Contacts, Moriah Longhurst

Undergraduate Honors Theses

X-ray diffraction is a robust method for determining the detailed 3D structures of specific proteins. However, this requires the formation of well-ordered protein crystals, a process that is time-consuming, expensive, and only has about a 10-30% success rate. New methods are needed to enable the efficient crystallization of challenging proteins. One such technique is explored here, which utilizes a protein polymer (the sterile alpha motif domain of the human protein translocation Ets leukemia, or TELSAM) as a crystallization chaperone to form a more ordered crystal lattice of target proteins and drive crystallization. This method was successfully used to crystallize, collect …

The Role Of Inflammasome In Vascular Remodeling Following Injury, Ted Amadi

Undergraduate Honors Theses

The purpose of the project presented in this thesis is to assess the contribution of inflammasome activation in mediating vascular wall remodeling that occurs following carotid artery injury due to decreased blood flow. To examine this, we used a mouse model of carotid artery ligation in mice lacking the expression of a key mediator of the inflammasome signaling pathway. Following ligation, we completed a histological assessment to compare the degree of the neointima formation and expression of inflammasome related proteins in both injured and the non-injured contralateral carotid artery in both wild-type (WT) and Caspase 1 knockout mice (Cas1 -/-).

Characterization Of Bpa Sensitive Strains In C. Elegans, Giulianna I. Escobar

Undergraduate Honors Theses

Bisphenol A (BPA) has been present in our environment since 1891 when it was first synthesized by Aleksandr Dianin. BPA has since been a topic of interest due to the possibility of diseases associated in human consumption of the chemical. Interestingly, Caenorhabditiselegans, commonly known as C.eleganshave shown to be able to tolerate the chemical. Thisorganism is an advantageous model organism for research since they are simple organisms to study, cheap, produce a large brood size, and have a short life cycle. Previously a forward genetic screen was done to isolate 2000 healthy strains in order to determine which strains showed …

Building An Ins-1 Cdna Library For A Genome-Wide Crispr-Cas9 Screen, Idongesit Ekpo

Undergraduate Honors Theses

By the year 2040, an estimated 642 million people are expected to have diabetes globally. Diabetes results from an elevation of metabolic stressors, such as glucotoxicity, lipotoxicity, oxidative stress and apoptosis. In type 2 diabetes, these stressful conditions contribute to the malfunction and loss of functional insulin-producing β-cells. Current treatment methods for diabetes include insulin therapy, islet transplant and anti-diabetes medication. These treatments are not curative and ignore other factors that contribute to the pathogenesis of diabetes beyond insulin resistance and islet β-cell failure. Previous research on β-cells has focused on ways to replace functional β-cell mass, trigger β-cell proliferation, …

Electrophysiological Characterization Of The Inhibitory Effects Of 2-Amino-4-Bis (Aryloxy Benzyl) Amino Butanoic Acids On Alanine Serine Cysteine Transporter 2 And Sodium Coupled Neutral Amino Acid Transporter 2, Paul Serguei Zakrepine

Undergraduate Honors Theses

Alanine Serine Cysteine Transporter 2 (ASCT2) is a member of the solute carrier 1A (SLC1A) family of transport proteins. It is a Na+ dependent, obligatory neutral amino acid exchanger with the capability to transport glutamine and it is a primary glutamine transport protein in cancer cells. This glutamine transport capability confers an important physiological role for ASCT2 in the maintenance of intracellular amino acid pools for various metabolic and cell signaling pathways. Because of this, ASCT2 is found to be overexpressed in many cancers, which rely heavily on glutamine as an essential nutrient for cell survival and growth. A wealth …

Therapeutic Drug Monitoring Of Apixaban Using Chromogenic Kits, Brooke Vogel

Undergraduate Honors Theses

Apixaban is a novel oral anticoagulant that prevents clotting by directly inhibiting Factor Xa in the coagulation cascade. Due to its different pharmacokinetics, previous standards for testing anticoagulant concentrations are ineffective at measuring apixaban. In this study, Hyphen Biomed Biophen Direct Xa Inhibitor and Biophen Heparin chromogenic kits from Aniara Diagnostica were used along with a NanoDrop™ One/One^C Microvolume UV-Vis Spectrophotometer to see if either of these kits provide acceptable precision and accuracy for the quantification of apixaban in plasma samples, as well as if there is a significant difference in these two kits at varying concentrations of apixaban. …

Modeling Hybrid Novel Traits: A Case Study In Complex Petal Pigment Patterning In Hybrid Mimulus, Xingyu Zheng

Undergraduate Honors Theses

Hybridization between species, by introducing dramatic trait variation into the population and creating viable, transgressive offsprings with novel phenotypes, can have huge evolutionary implications. Some hybrid traits have been studied in the classical genetics or population genetics context, but most complex traits are determined by multiple causes, e.g. the number of loci involved, the rewiring of the genetic circuitries, and the changes in gene expression pattern. Using the hybrid monkeyflower petal pigment patterning as an example, we present a case study to investigate complex hybrid traits in a systematic manner that includes empirical data analysis and quantitative mathematical modeling of …

Effects Of Nicotinamide Riboside And Beta-Hydroxybutyrate On C. Elegans Lifespan, Jeffery Peters

Undergraduate Honors Theses

The nicotinamide riboside (NR) form of vitamin B₃and the ketone body ß-hydroxybutyrate (BHB) are two of the most promising natural compounds yet identified for the treatment of aging and aging-related diseases. Forms of vitamin B₃are precursors for the synthesis of the coenzymes nicotinamide adenine dinucleotide (NAD(H)) and nicotinamide adenine dinucleotide phosphate (NADP(H)). In aged cells levels of NAD⁺decline, decreasing metabolism and decreasing activity of protective sirtuin protein deacetylases. In aged cells NR, but not more common forms of vitamin B3, boost NAD⁺levels. BHB is naturally produced by the body when individuals fast …

Modification, Verification Of Sequence And Optimization Of Expression Of P297f An Inactive Mutant Of Flavonol Specific Glucosyltransferase From Grapefruit (Cp3gt), Sarah Fox

Undergraduate Honors Theses

Citrus fruits are widely consumed and can offer various health benefits. One enzyme found in grapefruits, Citrus paradisi flavonol specific 3-O-glucosyltransferase (CP3GT), catalyzes the addition of glucose to one specific flavonoid class and at only one site. These flavonoids are plant secondary metabolites that can be used in a variety of plant functions including signaling and protection. The only class of flavonoids that CP3GT glucosylates is flavonols, and this specificity is of interest to study for potential benefits in biotechnology and enzyme modeling. In order to study this enzyme and its structure, a variety of mutants were created using site-directed …

Subcellular Localization Of Tobacco Sabp2 Under Normal And Stress Conditions, Sanjeev Das

Undergraduate Honors Theses

Subcellular Localization of Tobacco SABP2 under Normal and Stress Conditions

Salicylic acid (SA), a phytohormone, plays an important role in plant physiology. SA mediated innate immune pathway is an important pathway for plant immunity against pathogens. Plants resisting pathogen infection synthesize higher levels of Methyl Salicylate (MeSA), which is then converted to SA by the esterase activity of Salicylic Acid Binding Protein 2 (SABP2). The high level of the converted SA leads to enhanced pathogen resistance. The study of subcellular localization of a protein is critical in explaining its potential biochemical functions. SABP2 tagged with eGFP was expressed transiently in …

Optimization Of Adc Linkers: Design And Evaluation Of A Fret-Based Adc Linker-Library, Jared T. Miller

Undergraduate Honors Theses

Success of Antibody-drug conjugates (ADCs) relies on the maintained integrity of a chemical linker fusing a therapeutic payload to a monoclonal antibody. Recent reports have revealed the lead linker ValCitPABC has poor stability in rodent models compared to mammalian counterparts, resulting in many clinical investigations predicting poorer ADC efficacy due to premature payload release in these model systems. Optimization of this chemical linker to be resistant both in mouse and human models would streamline ADC progression into clinical trials. Herein we describe the synthesis and development of a FRET-based assay for evaluating linker stability in vitro. Evaluation of lysosomal release …

Sunflower Seedlings Fail To Remove Uranium Pollution In The Navajo Nation: Participatory Science As A Path To Build Community And Address Environmental Injustice, Zak R. Webber

Undergraduate Honors Theses

Mid-20th century mining on Naabeehó Bináhásdzo (Navajo Nation) polluted groundwater with high concentrations of uranium and arsenic. The Navajo Nation and other rural residents of this region use groundwater for drinking, livestock, and irrigation. However, many individuals and communities must purchase and transport treated water from locations that are often hours away. Sunflowers have been shown to preferentially take up heavy metals, including uranium and arsenic, potentially representing a tool to improve water quality through on-site, low-cost phytoremediation. We carried out a collaborative research project with a high school class on the Navajo Nation in 2018 and 2019. The students …