Biochemistry Commons^™

Exploring Phosphoregulation Of Myo3a Using Quantitative Fluorescence Image Analysis In Cos7 Cells, Vu Hao Minh Nguyen Phan

Honors Theses

Myosin 3A (MYO3A) is an unconventional myosin involved in the formation and maintenance of hair-cell stereocilia of the sensory epithelia in the inner ear. The kinase domain has been implicated in phosphoregulation of MYO3A activity through intermolecular autophosphorylation. Previous studies using mass spectrometry identified two potential phosphorylation sites in the motor domain. To investigate the regulatory roles of these sites, we generated glutamic acid point mutations in our mchr-MYO3AΔK construct to mimic phosphorylation and assayed the constructs for their ability to tip-localize and influence filopodial density via transfection into COS7 cells. The phosphomimic constructs were less able to generate filopodia …

Bariatric Surgery Impacts Immune Cell Metabolism And Function Dependent On Metabolic Status, Samantha N. Hart

University of Kentucky Doctoral Dissertations

Type 2 Diabetes (T2D), one of the top ten causes of death worldwide, is fueled by chronic inflammation. T2D is considered a metabolic disease, and there is a great push to target metabolic and associated inflammatory pathways to ameliorate the disease & its comorbidities i.e., obesity and cardiovascular disease. Thus far, clinical trials of anti-inflammatory drugs have had modest impacts on T2D and have not led to changes in clinical practice. This may in part be due to a gap in knowledge in the mechanism(s) driving obesity-associated chronic inflammation. I posit that metabolic abnormalities in immune cells in those with …

Effects Of Rnai Of Ribophorin 1, Eukaryotic Translation Initiation Factor 2 Beta, And Peptidylprolyl Isomerase A In Acyrthosiphon Pisum, Brandon L. Kennemer

Master's Theses or Doctor of Nursing Practice

Three genes found in the Unfolded protein response, Ribophorin 1, Eukaryotic translation initiation factor 2 beta, and Peptidylprolyl isomerase A are thought to be potential targets for RNA Interference (RNAi) in Acyrthosiphon pisum.  Ribophorin 1 (RPN1) is a transmembrane glycoprotein that assists in anchoring ribosomes to the rough endoplasmic reticulum membrane.  It acts as a substrate-specific chaperone.  It facilitates N-glycosylation by delivering newly synthesized proteins to the oligosaccharyltransferase (OST) complex.  Eukaryotic translation initiation factor 2 beta (eIF2B) is a key component of the eIF2 heterotrimer that facilitates protein synthesis initiation by binding GTP and recruiting a specific transfer RNA to …

Absolute Quantification And Identification Of Rna From Rna-Lipid Nanoparticles Using High Resolution Mass Spectrometry, Jason C. Funderburk, Yasir A. Alshehry, Matthew S. Halquist Phd, Sandro R.P. Da Rocha Phd

Graduate Research Posters

Background

RNA therapeutics are a rising drug category with potential use for a range of conditions encompassing infectious diseases to therapies for cancer, diseases, and genetic disorders. RNA-lipid nanoparticles (RNA-LNPs) are the prominent delivery method for these therapeutics approved products include mRNA vaccines and polyneuropathy treatments. The emergency use authorizations and orphan drug status of current RNA-LNP drugs has allowed approval without finalization of the regulatory analytical procedures for quality monitoring. The objective of the study was to develop an LC-MS assay to simultaneously measure identity and concentration of two therapeutically relevant intact RNA constructs extracted from RNA-LNPs to enhance …

Characterization Of Phytoplankton-Excreted Metabolites Mediating Carbon Flux Through The Surface Ocean, Yuting Zhu, Hanna S. Anderson, Eli Salcedo, Samuel E. Miller, Krista Longnecker, Melissa C. Kido Soule, Sheean T. Haley, Gretchen J. Swarr, Rogier Braakman, Sonya T. Dyhrman, Elizabeth B. Kujawinski

Chemistry & Biochemistry Faculty Publications

The marine labile dissolved organic carbon (DOC) pool is a dynamic reservoir of thousands of molecules that cycles approximately one-quarter of Earth’s primary production within days to weeks. After excretion by phytoplankton and other microbes, metabolites are rapidly consumed, resulting in low standing concentrations (picomolar to low nanomolar). Despite the decades-long search for labile DOC sources and molecular identities, marine phytoplankton exometabolomes are not well characterized, largely due to difficulties in measuring small polar molecules in saline water. Here, we profiled the exometabolomes of six axenic phytoplankton species representing key functional groups including a diatom (Thalassiosira pseudonana CCMP1335), a …

Seeing Through The Fuzz: Understanding Conformational Diversity In The Fuzzy Coat Of Amyloids Across Fibril Polymorphs Using Solid-State Nmr, Carah K. Allen

Scripps Senior Theses

The aggregation of α-synuclein (α-syn) into amyloid fibrils is a central pathological hallmark of Parkinson’s disease and other related synucleinopathies. Although monomeric α-syn is intrinsically disordered, the protein assembles into multiple fibril polymorphs that differ in their β-sheet core structures. Surrounding each polymorph is the “fuzzy coat,” which is a highly dynamic, intrinsically disordered region (IDR) that is not resolved by high-resolution structural techniques, such as Cryo-EM, but is thought to influence fibril interactions, propagation, and toxicity. Understanding whether different polymorphs maintain distinct fuzzy-coat conformational ensembles is therefore essential for connecting amyloid structure to biological function. To address this question, …

Structural Determination And Regulatory Mechanisms Of Rice Lipoxygenase-1, Lillie Ballanco, Nathaniel Gilbert, Adam Offenbacher

Distinguished Undergraduate Researcher Program

Lipoxygenases (LOXs) are non-heme iron containing enzymes that catalyze the oxygenation of polyunsaturated fatty acids (PUFAs) such as linoleic acid (LA). These lipoxygenase enzymes are found in both plants and animals. In humans, LOXs have been linked to inflammatory diseases such as asthma, atheroscelerosis, and allergic rhinitis. Plant LOXs have been primarily linked to the growth, development, and stress response of the plant. This paper focuses on Oryza sativa lipoxygenase-1 (OsLOX1), an enzyme found in rice (Oryza sativa). OsLOX1 has been associated with enhanced drought tolerance in rice and may play a role in regulating growth under water-limited conditions. Understanding …

An Investigation Into The Potential Role Of Thioester Bonds In Prebiotic Atp Synthesis, Adam J. Ruf

Williams Honors College, Honors Research Projects

In this project I will attempt to show a potential prebiotic pathway for the synthesis of ATP. This pathway involves the use of thioester bond containing molecules, as derivatives of these bonds have been shown to exhibit enzyme-like catalytic properties. Nuclear magnetic resonance spectroscopy will be utilized to characterize each step of the pathway to ensure it is successful.

C. Difficile Detection Method For First Responder Glove Application, Alli N. Senedak

Williams Honors College, Honors Research Projects

Clostridioides Difficile (C. diff) is a Anaerobic Gram-positive bacillus that is capable of spore formation, making it difficult to control its spread and duration in clinical environments. This phenomenon can provide danger to first responders, healthcare workers, and patients. The goal of this project is to create a biosensor capable of detecting C. diff in a clinical setting that can be applied to a glove apparatus. The project will involve the use of C. diff aptamers activated on the surface of an electrode. Once surface activation has been verified via surface analysis, the electrodes will be exposed to C. diff …

Multicomponent Deoxyribozyme Probes For Targeted Detection Of Cancer Associated Gene Insertions, Samuel R. Veleke

Honors Undergraduate Theses

Insertions and deletions (indels) in DNA sequences of genes are linked with cancers such as glioblastoma (GB). Thus, indels may be used as potential cancer biomarkers. In the NANOG gene, its pseudogene NANOGP8 has a 22 base pair insertion sequence for GB patients. In this work, the NANOG gene and the insertion sequence were detected in one sample using a dual-fluorescent assay containing two multicomponent split deoxyribozyme (Dz) probe complexes. One complex used TXR fluorescence to detect the gene regardless of the insertion’s presence. The other complex used fluorescein (FAM) fluorophore to detect the insertion. The probes were able to …

Disrupted Circadian Rhythms Affect Hallmark Pathologies In Alzheimer’S Disease-Related Mouse Models, Valeria Buzinova

Theses and Dissertations--Molecular and Cellular Biochemistry

Alzheimer’s Disease (AD) is a complex neurodegenerative disease with two hallmark pathologies: extracellular amyloid-b (Ab) and intracellular neurofibrillary tangles (NFTs). Ab is proteolytically processed from amyloid precursor protein (APP) by b-secretase and g-secretase as a monomeric peptide prone to aggregation. Eventually, the aggregate-prone monomers will form dense plaques that are difficult to break down and remove. These plaques begin to deposit into the cortex decades prior to the formation of NFTs and the onset of cognitive decline. NFTs are comprised of hyper-phosphorylated tau. Tau is a protein that serves to promote and stabilize the formation of microtubules. The formation of …

Characterizing The Human Metapneumovirus Matrix Protein And Its Role In Infection, Chase J. Heim

Theses and Dissertations--Molecular and Cellular Biochemistry

Human metapneumovirus (HMPV) causes severe respiratory tract infections in all cohorts, but especially in vulnerable groups such as children, older adults, and the immunocompromised. The matrix (M) protein of HMPV, like the M proteins of other members of the Mononegavirales order, is involved in virus assembly and budding. However, other functions of the HMPV M protein have yet to be elucidated. To investigate these various functions of the M protein, we used a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) antisense agent designed to block translation of the M gene to reduce M expression during infection. Treatment with varying concentrations of an …

Absolute Quantification And Identification Of Rna From Rna-Lipid Nanoparticles Using High Resolution Mass Spectrometry, Jason C. Funderburk

Theses and Dissertations

RNA therapeutics are a rising drug category with potential use for a range of conditions encompassing infectious diseases to therapies for cancer, diseases, and genetic disorders. RNA-lipid nanoparticles (RNA-LNPs) are the prominent delivery method for these therapeutics approved products include mRNA vaccines and polyneuropathy treatments. The emergency use authorizations and orphan drug status of current RNA-LNP drugs has allowed approval without finalization of the regulatory analytical procedures for quality monitoring. The objective of the study was to develop an LC-MS assay to simultaneously measure identity and concentration of two therapeutically relevant intact RNA constructs extracted from RNA-LNPs to enhance quality …

Interaction Of The Dap-10 Fluorescent Light-Up Aptamer With An Environment-Sensitive Dye, Crystal Violet, Marina Tsukanova

Honors Undergraduate Theses

Crystal violet (CV) is an environment-sensitive dye whose fluorescence is strongly influenced by microenvironment polarity and viscosity, increasing markedly when rotational and vibrational motions are restricted. While the fluorescence enhancement of CV by fluorescent light-up aptamers (FLAPs) has been noted in literature, the specific binding interactions between CV and aptamers in the DAP-10 family have remained under-investigated.  The objective of this work is to elucidate the binding site interactions between CV and the DAP-10-42 FLAP. It is hypothesized that positions 34 and 35 of the aptamer’s sequence reside in a proposed quadruplex-duplex junction that form the dye-binding pocket of DAP-10 …

Development Of A Split Deoxyribozyme-Based Assay For Detecting Hepatitis B Virus, Dalila Zovko

Honors Undergraduate Theses

Rapid, accessible nucleic acid-based detection methods are needed for hepatitis B virus (HBV). A split deoxyribozyme assay was developed for sequence-specific fluorescence detection. The assay was evaluated using synthetic target and plasmid-derived material. The assay showed target-dependent fluorescence and low-concentration linear range with a LOD of 1.9 nM. The assay supports detection and semi-quantification of HBV genetic material as shown using plasmid-derived transcript samples from HBV genotypes B, C, and D. The selected target corresponded to nt 363–406 of the HBV genome and was chosen based on sequence alignment showing conservation across most genotypes. Genotype-specific detection remains a future application …

Dna Half-Adder Based Calculator Driven By 4j Molecular Switches, Andrea Gissele Canizalez Covaleda

Honors Undergraduate Theses

DNA nanostructures have computing functions due to the predictable base pairing of nucleic acids (Watson–Crick). This property allows the programming of DNA circuits that integrate DNA logic gates capable of recognizing oligonucleotides. This molecular device offers the advantages of biocompatibility and potential applications in personalized medicine for diagnosis and therapies.

Our lab has developed DNA logic gates formed by the connection of YES strands via DNA four-way junctions (4J), which can function as molecular switches to develop a DNA-based calculator. Our DNA half-adder calculator nanostructure is designed to recognize up to three combinations of DNA inputs using three DNA strands …

The Role Of Small Extracellular Vesicles In Modulating Radiation Resistance In H3k27m-Pediatric Diffuse Midline Glioma, Viral Oza

Theses and Dissertations--Molecular and Cellular Biochemistry

Pediatric diffuse midline gliomas with H3K27M alteration (H3K27M-pDMG) are the leading cause of pediatric brain tumor-associated deaths. All H3K27M-pDMG are initially treated with fractionated radiotherapy, the standard of care, and most children succumb to their disease within two years of diagnosis. There are no universally effective chemotherapies and full resection is impossible due to the diffuse nature of the tumor and sensitive location. There has not been a significant clinical advancement in more than 40 years.

Tumors become completely resistant to radiation within the first six months of treatment. The mechanism of radiation resistance is unknown but is thought to …

Patterns Of Ovarian And Gonadotropin Hormones In Emerging Adult Oral Contraceptive Users, Summer Mcormond

Undergraduate Research Posters

Oral contraceptives (OCs) are often initiated during adolescence and emerging adulthood, a period of continued maturation of the hypothalamic–pituitary–gonadal (HPG) axis. Because hormonal exposure during this window may shape emotional, biological, and reproductive outcomes, it is important to understand the specific formulations young women take and the hormonal profiles these formulations produce. This study characterizes OC formulation features and measured hormone levels in young adult OC users (n = 30).

Ethinyl estradiol (EE) doses ranged from 0.01–0.035 mg (mean = 0.022 mg), with 70% using ≤0.02 mg. Pill regimens included 21/7 packs (83%), 24/4 packs (10%), and 84/7 regimens (3%). …

The Chemistry Of Red Lipstick: The Impact Red Lipstick Has On Society, Lilliana Weldeslassie

Electronic Theses & Dissertations (2024 - present)

Cosmetic chemistry is deeply embedded in everyday life, shaping the products people use from morning to night. Among these, red lipstick stands out as a formulation that blends complex chemistry with a cultural meaning. This thesis examines the scientific foundations of red lipstick through its chemical composition, pigment structure, toxicological history, analytical methods, and evolving sustainability practices. Historically, red lipsticks relied on high-risk ingredients such as lead, mercury, and other toxic metals, reflecting an era before chemical safety and regulatory oversight were incorporated.²¹ Modern formulations have established safer synthetic dyes, natural waxes, plant-based oils, and stabilizers created to optimize …

Bio-Orthogonal Chemistry-Based Strategy To Turn-On And Turn-Off Crispr-Cas9 Gene Editing In Solution And In Live Cells, Bhoomika Pandit

Electronic Theses & Dissertations (2024 - present)

The CRISPR–Cas9 system is a widely popular tool for genome engineering. There is a strong interest in developing tools for temporal control of CRISPR-Cas9 activity to address some of the challenges and to broaden the scope of potential applications. In this thesis I describe two biorthogonal based approaches to either Turn-ON and Turn-OFF CRISPR-Cas9 gene editing.

In first project work I describe a bio-orthogonal chemistry-based approach to Turn-ON Cas9 nuclease activity with temporal precision. We report a TCO-acylimidazole reagent that acylates 2′-OH groups of RNA. Poly-acylation (“cloaking”) of RNA was optimized in vitro using a model 18-nt oligonucleotide, as well …