Biochemistry, Biophysics, and Structural Biology Commons^™

Psr Impacts Chromatin Condensation During Spermatogenesis In Nasonia Vitripennis, Jacqueline R. Solomon

CMC Senior Theses

Paternal Sex Ratio (PSR) is a supernumerary, selfish B chromosome found in the jewel wasp (Nasonia vitripennis). PSR is transmitted strictly paternally to progeny, and when present, it causes paternal genome elimination (PGE) just after fertilization. Due to the haplo-diploid reproduction of N. vitripennis, this PGE event results in all-male broods of progeny, ~90% of which carry and transmit PSR. Thus, PGE is beneficial for PSR’s transmission.  A previous study showed that PSR induces PGE by expressing a gene called haploidizer during spermatogenesis. Although how haploidizer functions in PGE is unknown, this finding suggests two basic models: that (i) the …

Fgf Signaling Is Necessary For Newt Lens Regeneration, Leo Alexander Napoleon

Honors Theses and Capstones

The Iberian ribbed newt Pleurodeles waltl can regenerate the lens by transdifferentiating iris pigmented epithelial (IPE) cells into lens epithelial cells (LECs), but the mechanism by which this occurs remains largely uncharacterized. We aim to mechanistically show an early and critical role for FGF signaling by using small molecules to inhibit FGF receptors and observe the histological and intracellular effects. Two inhibitors were found to completely stall lens regeneration. Cell cycle analysis revealed decreased DNA synthesis and uniformly low but unchanged entry to mitosis under signaling inhibition by staining for EdU incorporation and phospho-histone H3 (pH3), respectively. Western blot revealed …

Comparing The Differences Of Hsp90Α And Hsp90Β In Native Protein Complex Incorporation, Daryna Serediuk

Honors Theses and Capstones

Epichaperomes are long-lasting, stable assemblies of chaperones, co-chaperones, and associated factors that facilitate cell survival in maladaptive cellular states. Their inhibition has emerged as an attractive therapeutic strategy for the treatment of cancer and neurodegenerative diseases (Rodina et al., 2016). Epichaperomes are hallmarks of robust proliferation in cancer cell populations across many cancer types and are abundantly present in several stem cell varieties, including induced pluripotent stem cells, human embryonic kidney cells, and cancer stem cells (Kishinevsky et al., 2018).

The core protein of the eukaryotic heat shock protein machinery, Heat Shock Protein 90 (Hsp90), is a central chaperone that …

Generating Concentrated Ring Polymers Through Supercoiled And Open Circle Pdna Isoform Separation, Kelsey E. Hodsdon

Honors Theses and Capstones

DNA can serve as a model polymer due to its high uniformity compared to synthetic polymers. Plasmid DNA (pDNA) in particular offers a well-defined, low-dispersity model for studying topologically uniform systems of a cyclic nature. pDNA has unique isoforms known as open circle (OC) and supercoiled (SC). Using fast protein liquid chromatography (FPLC), specifically anion exchange chromatography (AEX), these OC and SC isoforms can be separated in order to access their unique material properties. Our lab was able to yield the pure SC isoform. However, OC isoform separation has been more challenging. Various difficulties arose during this process, including imaging …

Perinatal Inflammation And Oxidative Stress Alter Adult Rat (Rattus Norvegicus) Motor Behavior, Alexander Young

Honors College Theses

Recent evidence in comparative animal models suggests that early life exposure to elevated inflammation (via LPS exposure) and oxidative stress (via acetaminophen exposure) may synergistically interact to alter typical neurodevelopmental trajectories, resulting in lasting effects on both behavior and gut-brain neurochemistry. The current study observed emotional and motor behaviors and collected fecal-boli to measure Tyrosine (TE) levels in male and female Long-Evans (Rattus norvegicus) rats. Subjects were either treated with [1] saline [2] lipopolysaccharide (LPS) to introduce an inflammatory state, [3] acetaminophen (APAP) to introduce oxidative stress, or [4] a dual-hit of both LPS + APAP on postnatal day (PD) …

Development Of Tunable Rose Bengal Based-Nanogumbos As Potential Selective Chemotherapeutic Agents, William J.A. Russell

Honors College Theses

As cancer remains one of the greatest threats to the global population, the development of chemotherapeutic agents that are selective and tunable is of critical importance. Herein, a series of Rose Bengal (RB)-based GUMBOS, a group of uniform materials based on organic salts, were synthesized via counterion exchange between [Na]₂[RB] and three organic cations: tetrabutylphosphonium bromide [TBP][Br], tetraphenylphosphonium chloride [TPP][Cl], and 1-dodecyl-3-methylimidazolium chloride [C₁₂MIm][Cl]. The resulting GUMBOS were characterized through FT-IR, ESI-MS, NMR, and UV-Vis Spectroscopy. Partition coefficient studies indicated all GUMBOS exhibited hydrophobic physiochemical characteristics. These hydrophobic RB-based GUMBOS …

Mitochondria As Primary Cause Of Neurodegeneration: Evaluating Causality Across Amyotrophic Lateral Sclerosis (Als), Alzheimer's, And Parkinson’S Diseases, Makenzie G. Johnson

Undergraduate Honors Theses

Neurodegenerative diseases like Alzheimer's Disease (AD), Parkinson’s Disease (PD) and Amyotrophic Lateral Sclerosis (ALS) have historically been explained through alternate hypotheses despite shared clinical hallmarks that lead to neuron loss. However, these varying explanations could not account for how disease onset begins among other individual issues. This thesis aims to show that mitochondrial dysfunction is a primary driver of neurodegeneration as well as a unifying mechanism shared between the three diseases. Evidence presented in this thesis will show that many issues can arise in mitochondria including issues with energy production, mitochondrial DNA (mtDNA), apoptosis activation, and calcium regulation. These issues …

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 …

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 …

A Framework For Characterizing The Peripheral Immune Isonome Using Long-Read Single-Cell Rna Sequencing And Its Relevance To Neurological Disease, Patricia Hayes Doyle

Theses and Dissertations--Neuroscience

Long-read single-cell RNA sequencing provides an opportunity to understand human health and disease at isoform resolution, revealing cellular diversity and disease mechanisms difficult to resolve with bulk or short-read methodologies.

Using a modified PIPseq workflow and computational pipeline adapted for Oxford Nanopore (ONT) sequencing, we profiled isoform usage across immune cells, integrating marker expression and isoform discovery, generating the largest long-read single-cell dataset of human immune cells from a single individual to date. We identified non-canonical protein-coding variants of GZMB and CD3G enriched in unexpected cell types. We also discovered novel transcripts from CMC1 and LYAR with cell-type-specific signatures that …

Design And Advancement Of Analytical Frameworks For In Vivo Single-Photon Calcium Imaging To Characterize Temporal- And Treatment-Dependent Neural Adaptations To Opioid Exposure And Withdrawal In The Medial Prefrontal Cortex, Alexia R. Alsum Dr.

Theses and Dissertations--Pharmacy

Despite the availability of several approved medications for opioid use disorder (OUD), it remains a significant public health concern characterized by persistent neurobiological adaptations that drive relapse and impede successful recovery. The medial prefrontal cortex (mPFC), a region critical for executive control and decision-making, undergoes pronounced functional changes during chronic opioid exposure and withdrawal, yet the cellular mechanisms underlying these adaptations remain poorly understood. This dissertation aims to investigate longitudinal alterations in mPFC neural activity across opioid exposure and withdrawal using in vivo single-photon calcium imaging and newly developed computational frameworks.

Following standard preprocessing to extract and normalize calcium traces, …

Exploring The Limitations Of Substrate Scope In Dimethylallyltryptophan Synthases, Evan T. Miller

Theses and Dissertations--Pharmacy

Natural products (NPs), are the largest source of bioactive compounds in Nature, and are essential to the treatment of human disease. Nearly 50% of drugs approved for use from 1981 to 2019 owe some aspect of their development to NPs, either in terms of their structure, pharmacophore, or mechanism of action. However, NPs are difficult to structurally optimize. Chemoenzymatic methods for NP derivatization have been increasingly seen as practical alternatives to traditional synthetic methods. Prenyltransferases (PTs) are involved in the primary and secondary metabolism of plants, bacteria, and fungi, and they are key enzymes in the biosynthesis of many clinically …

Molecular Mechanisms And Metabolic Consequences Of Glucocorticoid Resistance, Genesee J. Martinez

Theses and Dissertations--Pharmacology and Nutritional Sciences

Glucocorticoids are vital steroid hormones that govern metabolism, stress responses, and immune functions through intricate, tissue-specific mechanisms, both genomic and non-genomic. These hormones bind to the glucocorticoid receptor (GR), which then acts as a transcription factor to modulate gene expression. Chronic elevation of glucocorticoid levels may lead to glucocorticoid resistance, resulting in adiposity, inflammation, and insulin resistance, thereby adversely affecting multiple metabolic tissues. Whether produced endogenously or administered exogenously, excessive, chronic glucocorticoid concentrations impair glucocorticoid signaling. Although there are two primary isoforms of the receptor, GR⍺ and GRβ, only GR⍺ interacts with glucocorticoids.Through the research conducted in this dissertation, we …

Exploring Fructose Mimetics As Chemotherapeutic Potentiators: Impact On Glut5-Mediated Cancer Cell Vulnerability, Vedant Buwa

Dissertations, Master's Theses and Master's Reports

Metabolic reprogramming toward increased sugar uptake is a hallmark of cancer, and fructose-specific uptake through the facilitative transporter GLUT5 is selectively upregulated in several adenocarcinomas, including breast and lung cancer. This dissertation develops and characterizes a panel of fluorescent fructose mimetics – the coumarin-based ManCou probes and the quinolinone-based ManQuin probes – as GLUT5-targeted reporters, cytotoxic effectors, and chemosensitization adjuvants, and establishes the boundaries within which each role holds.

The central experimental strategy pairs each GLUT5-targeted glycoconjugate with its sugar-free aglycone control, isolating transporter-mediated cytotoxicity from cytotoxicity intrinsic to the fluorophore itself. In luminal (MCF7) and triple-negative (MDA-MB-231) breast carcinoma, …

Physicochemical Characterization Of Small Extracellular Vesicles: Effects Of Immobilization, Isolation, And Disease State, Bianca M. Mercado Velez

Dissertations, Master's Theses and Master's Reports

Cancer treatments and survival outcomes have improved significantly in the last couple of decades, with survival rates increasing in the U.S. Despite improvements in treatment outcomes, early detection and treatment are crucial for improving cancer prognosis and survival rates. The standard cancer diagnostic procedure of tissue biopsy is highly invasive, leading to the further spread of cancer in some cases. Alternatively, the extraction of biofluids from cancer patients, also called a liquid biopsy, is increasingly popular because it is less invasive and more representative of the tumor microenvironment, which contains cancer cells, tumor genetic material, and extracellular vesicles, including small …

Targeted And Screening Based Approaches To Analyze Drm Transcriptional Repressor Complex Activity In Caenorhabditis Elegans, Emily Washeleski

Dissertations, Master's Theses and Master's Reports

The molecular machinery that control the eukaryotic cell cycle, specifically the processes that mediate the switch between cellular division or quiescence, are essential for cellular and organismal viability. However, how the same machinery integrates with developmental or differentiation programs is less well understood. In Caenorhabditis elegans, the 8-subunit DRM complex (for Dp, Rb and MuvB), whose subunits belong to the Synthetic Mulitivulva (SynMuv) B class genes, is a central regulator of the cell cycle but also prevents inappropriate cell fate specification. The conserved mammalian DREAM complex (for Dp, Rb, E2F, And MuvB) is best known as a transcriptional repressor …

Signaling Scaffold Shoc2 Regulates Lymphangiogenesis By Suppressing Mtorc1-Mediated Ifn Responses, Patricia Wilson, Vishakha Vishwakarma, Rebecca Norcross, Kashmira Khaire, Van N. Pham, Brant M. Weinstein, Hyun Min Jung, Emilia Galperin

Molecular and Cellular Biochemistry Faculty Publications

An interplay of growth factors and signaling pathways governs the development and maintenance of lymphatic vasculature, ensuring proper fluid homeostasis and immune function. Disruption of these regulatory mechanisms can lead to congenital lymphatic disorders and contribute to various pathological conditions. However, the mechanisms underlying the molecular regulation of these processes remain elusive. Here, we reveal a critical and previously unappreciated role for the signaling scaffold protein Shoc2 in lymphangiogenesis. We demonstrate that loss of Shoc2 results in near-complete loss of lymphatic vasculature in vivo and senescence of lymphatic endothelial cells in vitro. Mechanistically, Shoc2 is required for balancing signaling through …

Current And Emerging Therapeutic Strategies For The Treatment Of Duchenne Muscular Dystrophy, Miguel A. Lopez Perez, Noah L. Weisleder

Molecular and Cellular Biochemistry Faculty Publications

Background/Objectives: Duchenne muscular dystrophy (DMD) is a fatal, progressive neuromuscular disorder caused by mutations in the dystrophin gene, leading to the absence of functional dystrophin protein. As the largest gene in the human genome, the DMD locus is highly susceptible to mutations, contributing to a prevalence of approximately 1 in 3800–6300 live male births worldwide. This review aims to provide a comprehensive and critical synthesis of current and emerging therapeutic strategies for DMD.

Methods: We conducted a narrative review of the literature, integrating findings from clinical trials, regulatory approvals, and preclinical studies. We categorized therapeutic approaches into mutation-agnostic and mutation-specific …

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 …

Perfluorooctane Sulfonate (Pfos)-Induced Disruption Of Mitochondrial Activity And Cell Adhesion In Liver Cells, Phuong Dam Nam Tran

Graduate Theses/Dissertations

Perfluorooctane sulfonate (PFOS) is a persistent environmental pollutant associated with potential hepatoxic effects and other health risks. Despite its widespread distribution, the mechanisms underlying its toxicities remain to be fully understood. To investigate PFOS toxicology, my study utilized HepG2 and THLE-2 human hepatic cell models to replicate conditions reflecting PFOS accumulation in the liver. Cell viability, cell stress, and cell death assays were conducted to assess the toxicological influence of the chemical on both cell lines. Total RNA extraction was performed, followed by cDNA sequencing, and RT-qPCR. The XTT viability assay revealed a dose-dependent decrease in number of viable cells …