Biochemistry & Molecular Biology Department Theses and Dissertations
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Item Characterizing PDK4 and PKC-Theta in Pancreatic Cancer and Cachexia(2025-11) Gaafer, Omnia Usama Mohammed Shawky; Wells, Clark D.; Zimmers, Teresa A.; Wek, Ronald C.; Brault, Jeff J.; Zhang, Ji; Byrne, Katelyn T.Cachexia is an involuntary wasting syndrome that cannot be reversed by nutritional support. It affects most patients with pancreatic ductal adenocarcinoma (PDAC) and is believed to cause 20% of cancer-associated deaths. Cachectic patients exhibit both metabolic abnormalities and systemic inflammation. Pyruvate Dehydrogenase Kinase 4 (PDK4) is upregulated in the skeletal muscles of many cancer cachexia models and has been shown to drive muscle wasting in some conditions. We show that despite being upregulated in an orthotopic mouse model of PDAC cachexia, whole-body deletion of PDK4 did not prevent the decline in muscle function or the overall wasting. Expression of the other members of the PDK family did not change, ruling out a compensatory mechanism. Deletion of PDK4 did not affect the dysregulated expression of metabolic and catabolic genes in PDAC cachexia. Analysis of publicly available muscle-expression datasets from cachectic cancer patients further revealed that PDK4 is not upregulated in patients' skeletal muscles. Protein Kinase C theta (PKCθ) is a lipid-sensitive kinase expressed in skeletal muscle where it regulates insulin signaling. It is also expressed in T cells, and its deletion alleviates inflammation in several inflammatory conditions. We found that PKCθ expression increases in skeletal muscle but decreases in the spleen in PDAC cachexia as the disease progresses. To delineate its role in PDAC cachexia, we generated mouse models with skeletal muscle-specific, T cell-specific, and whole-body deletion of PKCθ. Deletion of PKCθ did not substantially affect body weight or composition at baseline in any of these models. It also did not affect the wasting or tumor burden in PDAC cachexia. PKCθ-deficient T cells maintained their ability to infiltrate PDAC tumors. Overall, our work shows that despite being upregulated, PDK4 is not required for PDAC cachexia, and PKCθ is dispensable for both wasting and inflammation in this context.Item MAPPING EPITOPES AND RECEPTOR BINDING DETERMINANTS OF LASSA VIRUS GLYCOPROTEIN USING Qβ PHAGE DISPLAY(2025-11) Metangmo, Danielle; Waffo, Alain B.; Tharp, Jeffery M.; Hoang, Quyen Q.Lassa virus (LASV) is a highly infectious pathogen responsible for Lassa fever, a severe hemorrhagic disease endemic to West Africa. Despite its significant health burden, there are currently no widely available vaccines or effective antiviral treatments. The only surface glycoprotein (GP) of LASV is essential for viral entry and immune recognition, making it a prime target for therapeutic and vaccine development. This study involved the engineering and expression of recombinant phages through gene cloning, phage production, and purification, followed by epitope mapping and receptor-binding assays. Qubevirus (Qβ) phage display technology was used to investigate the immunological and receptor-binding properties of LASV GP complex by displaying three novel overlapping fragments, GPF1, GPF2, and GPF3, on the surface of recombinant phages. These engineered phages were screened for their ability to bind anti-GP antibodies and two known host receptors: lysosomal-associated membrane protein 1 (LAMP1) and α-dystroglycan (α-DG). The results demonstrated that GPF2 and GPF3 fragments exhibited strong reactivity with GP-specific antibodies, indicating the presence of accessible and immunodominant epitopes. In contrast, GPF1 showed minimal antibody binding, suggesting that its epitopes are conformational and not well presented on this system. Notably, GPF2 also displayed robust binding to both LAMP1 and α-dystroglycan, identifying it as a key determinant of receptor interaction and immune recognition. The findings highlight GPF2 as a critical region for both host cell entry and immune targeting. These insights were further supported by ELISA that quantified and confirmed the specificity of receptor binding and epitope exposure. Overall, this research provides valuable information on the antigenic landscape and receptor-binding domains of LASV GP. The use of Qβ phage display enabled precise mapping of functional regions, offering a promising platform for the development of subunit vaccines and antiviral drugs, aiming to block LASV infection.Item Perk Protein Kinase Facilities Skin Regeneration via Association with Adhesion Molecules Independent of Its Catalytic Activity(2025-10) Barriera Diaz, Miguel; Wek, Ronald C.; Spandau, Dan F.; Turner, Mathew J.; Maiers, Jessica; Linnemann, AmeliaThe epidermis protects the body from environmental and mechanical insults. When the skin is injured, healing requires re-epithelialization by keratinocytes through a coordinated migratory process known as keratinocyte collective cell migration (KCCM). Diseases such as diabetes often fail to progress to this stage of cutaneous wound healing, resulting in chronic non-healing wounds. Therefore, it is critical to understand the biochemical and molecular mechanisms of KCCM to develop novel therapeutics for treating chronic wounds. Cutaneous wounding triggers the Integrated Stress Response (ISR). The ISR is a cellular pathway that monitors cellular stresses by multiple sensory protein kinases, including PERK (EIF2AK3) and GCN2 (EIF2K4), which phosphorylate the eIF2, thereby mitigating translational control to alleviate stress-induced damage. We previously described that GCN2 facilitates KCCM via sustained phosphorylation of eIF2 and coordinated production of reactive oxygen species and amino acid transport. I have now discovered that PERK plays a crucial role in KCCM via mechanisms that are distinct from GCN2 and the ISR. Deletion of PERK via CRISPR methods resulted in altered keratinocyte morphology, impaired skin differentiation and stratification, and impaired KCCM, indicating the importance of PERK in skin homeostasis. Pharmacological and genetic rescue experiments showed that PERK protein kinase function is dispensable for KCCM, but rather PERK promotes the collective migration by scaffolding or tethering processes requiring the PERK cytoplasmic domain and its association to the ER. To delineate proteins that associate with PERK, I pursued BioID interactome analyses using an UltraID-tagged PERK and identified cytoskeletal and cell adhesion proteins as critical PERK targets. Based on these results, I observed that PERK-KO cells exhibit disrupted F-actin organization and impaired lamellipodia formation at the leading edge during KCCM. PERK-deleted cells showed impaired expression and localization of cell adhesion proteins, concomitant with elevated cell-substrate and cell-cell adhesions. Confocal microscopy and co-immunoprecipitation studies revealed that PERK interacts with the expression of the hemidesmosome proteins ITGA6, ITGB4, and COLXVII, as well as the desmosome proteins JUP, DSG2, and DSG3. These results indicate that PERK participates in multiple scaffolding functions with cell adhesion complexes that are crucial for establishing and maintaining skin homeostasis and keratinocyte migration.Item Characterization of a Novel Small Molecule Inhibitor of Asparagine Synthetase(2025-10) Walda, Nicholas Todd; Staschke, Kirk A.; Wek, Ronald C.; Wells, Clark D.; Takagi, YuichiroAsparagine synthetase (ASNS) is a protein in human cells that is responsible for synthesizing asparagine (ASN), a nonessential amino acid, from glutamine and aspartate. ASNS is frequently overexpressed in cancers, leading to increased growth and resistance to nutrient deprivation. There has been a demand for an effective pharmacological inhibitor of ASNS to starve the tumors by preventing ASN synthesis. In this study, the ASNS specific inhibitor ASX-173 was utilized to determine how human embryonic kidney 293A (HEK293A) cells would respond to inhibition of ASNS in ASN-depleted conditions. Given that amino acid (AA) depletion is a well-documented inducer of GCN2 eIF2 kinase and the integrated stress response (ISR), we utilized multiple cell-based techniques to determine how the cells were responding to the stress of ASNS inhibition. This study showed that biomarkers of ISR, including phosphorylated GCN2 and its substrate eIF2α, ATF4, GADD34, and TRIB3 were all increased in the HEK293A cells treated with ASX-173. The ISR induction by the ASNS inhibitory drug was also supported by the increased expression of ATF4, GADD34, and TRIB3 mRNAs, along with luciferase reporter assays showing that ASX-173 induced ATF4 transcriptional activity. ASX-173 induction of the ISR was dependent on GCN2 activity and was reversed by the addition of exogenous L-Asparagine. It is noteworthy that the levels of ASNS protein and mRNA were not affected by ASX-173. Cell growth was greatly diminished during ASX-173 exposure in ASN-depleted conditions. Emphasizing the importance of GCN2 and the ISR for resistance to amino acid limitation, the growth reduction of HEK293A cells with the combined treatment of ASX-173 was accentuated with the combined addition of GCN2iB, a potent inhibitor of GCN2. These results support the idea that the pharmacological ASNS inhibition could be utilized to treat and eliminate certain types of cancers and other diseases where nutrient starvation is a potent strategy.Item Evaluating Phosphatidylinositide as Lipid Biomarkers for Hyperglycemia-related Triple-negative Breast Cancer Aggressiveness(2025-10) Kile, Aaron; Wells, Clark; Baucum, Anthony J.; Kimble-Hill, Ann; Richardson, AngelaTriple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, marked by limited treatment options and poor patient outcomes. Clinical and epidemiological studies suggest that systemic metabolic conditions, particularly high blood glucose associated with Type 2 Diabetes, may worsen TNBC progression. However, there are no current biomarker for detecting TNBC, especially in hyperglycemic patients. This thesis investigates how elevated glucose levels influence TNBC behavior and whether lipid signaling molecules at the cell membrane, known as phosphatidylinositol lipids (PIs), can serve as biomarkers of disease progression. To address this, we combined functional assays that measure key cancer traits, including cell migration, attachment to the extracellular matrix (ECM), and cell accumulation, with lipidomic analyses of cell membranes under normal and high-glucose conditions. Our findings demonstrate that hyperglycemia enhances aggressive traits in TNBC cells, promoting increased movement and growth, but no effect on ECM attachment. In parallel, lipid profiling revealed alterations in PI molecules that regulate growth and survival pathways, with differences observed across cell models. These results highlight a direct connection between metabolic stress and cancer progression. By linking systemic metabolic dysregulation to cell signaling in TNBC, this work identifies PI4P, PI(4,5)P2 and PIP3 lipids as potential biomarkers and points toward new strategies for risk stratification and therapeutic intervention.Item Development and Application of In Vivo Protein Labeling Methods in Caenorhabditis Elegans(2025-08) Borchers, Christopher Jon; Aoki, Scott T.; Cornett, Evan M.; Hundley, Heather A.; Roh, Hyun CheolThe existence and preservation of life relies on the concerted orchestra of numerous biological processes of which proteins are the primary workhorse. In the highly complex intracellular environment, proteins must be synthesized, properly folded, carry out their specific functions, and be efficiently degraded when no longer required. Over time, we have learned even the most minute mutation in protein can have profound impacts on health and disease. Thus, developing methods to dissect protein dynamics and function are critical to understand the basic molecular processes that govern life. Protein pulse-chase is a versatile tool that can be used to study protein dynamics in cells and multicellular organisms. Original protein pulse-chase methods involved global, non-specific labeling of the entire proteome. While informative, spatial expression and dynamics across tissues are lost. The development of self-labeling enzyme tags such as HaloTag enable protein-specific pulse-chase. Pulse-chase experiments using these tags have been performed in various biological systems. However, their broader application is limited by significant technical and economic challenges. This work outlines the development and application of a HaloTag fluorescent pulse-chase method in the model organism, Caenorhabditis elegans. Strains expressing intestine-specific HaloTag histone reporter proteins were generated and could be efficiently pulse-labeled by soaking animals in the presence of HaloTag fluorescent ligand. Stability of labeled histone reporter protein could be feasibly monitored over time using confocal microscopy and was dependent on the animals’ nutritional state. HaloTag fluorescent labeling was further applied to investigate and characterize aberrant extranuclear divisions in the anterior intestine of C. elegans driven by the exogenous expression of a histone H3.3 protein. Collectively, this work serves as entrée to illustrate the potential of in vivo labeling methodologies in C. elegans. Technical advancements made in C. elegans will serve as a stepping-stone to address a variety of questions in metazoan biology. From pulse-labeling to detection, the method is highly adaptable to cater investigation into a wide variety of biological processes in vivo.Item Thermodynamic and Structural Investigations of Protein Mutations Using λ-Dynamics(2025-08) Barron, Monica Philomena; Vilseck, Jonah Z.; Georgiadis, Millie M.; Johnson, Steven M.; Mosley, Amber L.The λ-dynamics (λD) alchemical free energy method is a powerful tool to investigate the impact that an amino acid change has on the stability of a protein or a protein complex. This method simultaneously calculates thermodynamic relative free energy changes (ΔΔGs) due to protein perturbations and generates simulation trajectories that allow the specific origin of the thermodynamic change to be identified. Historically, alchemical investigations of protein mutations have encountered difficulties when the perturbation involved buried small-to-large changes, changes to backbone flexibility, and/or changes in charge. However, such perturbations frequently arise when studying disease-linked protein mutations. In this thesis, λD was used to model mutations in the 20S proteasome, the insulin receptor, and the RNA exosome complex, demonstrating its ability to model each kind of challenging protein mutations listed above. Using multisite λ-dynamics (MSλD), a series of thermal-sensitive mutants in the proteasome subunit, Pup2 (C76R, T113M, and L204Q) were investigated. The thermodynamic results revealed a large ΔΔGbind of 5 kcal/mol accompanying the C76R mutation. Complimentary stabilization by the T113M and L204Q variants was observed at lower temperatures (30 °C) but disappeared at higher temperatures (50 °C). The insulin ValA3Leu mutation gives the clinically observed insulin Wakayama a 140-500-fold worse binding affinity for the insulin receptor. This loss of binding, along with the binding trends of six other insulin A3 variants, were successfully captured with λD. Structural investigation revealed that the substantially worse impact from LeuA3 stems from a steric clash with Leu’s second Cδ. With λD, a series of challenging EXOSC3 mutations were analyzed, including the neurodegenerative disorder-linked D132A, A139P, G191C, and G191D and the variant of uncertain significance (VUS) G135R. All of these variants were found to destabilize EXOSC3 folding while A139P, G191D, and G135R were also found to negatively impact binding affinity. Structural analysis identified a clear rationale for the thermodynamic impact of all EXOSC3 variants. This study concludes that the VUS G135R is likely pathogenic. This work demonstrates the utility of λD in investigations including extremely challenging protein mutations in macromolecular complexes and applies this tool to explain disease-linked missense mutations and to predict the significance of a VUS mutation.Item Beta Cell Heterogeneity in the Acute Interferon Alpha Response(2025-06) Wagner, Leslie Elaine; Linnemann, Amelia; Elmendorf, Jeffrey; Roh, Hyun Cheol; Spaeth, JasonType 1 diabetes (T1D) is characterized by autoimmune destruction of the insulin-producing β-cells in the pancreatic islet. This autoimmunity is hypothesized to result from a combination of genetic and environmental factors, with early childhood viral infection being a leading hypothesis for the latter. In response to viral infections, the innate immune system produces various cytokines, including interferon alpha (IFN-α), which has long been implicated in T1D pathogenesis. Our study explores how IFN-α influences human β-cell function, particularly its role in reactive oxygen species (ROS) production—signaling molecules essential for normal β-cell function but detrimental in excess. Using intravital microscopy and a β-cell-specific ROS biosensor, we identified a subset of β-cells that rapidly produce ROS in response to IFN-α. Interestingly, phenotyping data from the donors indicated that healthier β-cells were more likely to exhibit this response. In vitro experiments confirmed that IFN-α drives mitochondrial superoxide production in a subset human of β-cells, prompting us to investigate the molecular basis of this phenomenon. RNA sequencing of sorted IFN-α–treated cells revealed an upregulation of immune-related genes, and comparison with single-cell datasets showed that these genes are more highly expressed in β-cells from healthy individuals than those with T1D. These findings suggest that IFN-α–induced ROS production may be a marker of β-cell resilience, highlighting differences in how β-cells respond to stress. Understanding this mechanism could offer new insights into why some β-cells are more vulnerable in T1D and potentially point to novel strategies for preserving β-cell function in diabetes. While our investigation into IFN-α signaling revealed how immune cytokines influence β-cell physiology, we also sought to explore immune landscape changes during disease progression. Using the Akoya Phenocycler, we mapped immune cell populations in the pancreata of human and diabetic mouse models and corresponding controls, providing insight into disease-associated immune changes.Item Impact of Neurodevelopmental Disorder-Associated Clinical Variants on the Catalytic Activity of KMT5B(2025-06) Iyer, Malini; Cornett, Evan M.; Georgiadis, Millie M.; Wells, Clark D.KMT5B is a lysine methyltransferase that is known for its role in catalyzing H4K20 dimethylation. This post-translational modification is involved in DNA repair and heterochromatin formation. Missense variants found in KMT5B cause a related neurodevelopmental disorder in which patients experience neurodevelopmental phenotypes like developmental delay (DD), intellectual deficits (ID), autism spectrum disorder (ASD), seizures, and motor deficits. However, the impact of these variants on enzyme activity is unknown. In this work, we provide qualitative and quantitative evidence showing the differential impacts of four select clinical missense variants of KMT5B on its lysine methylation activity. Recombinant KMT5B was purified and used for in vitro KMT assays to assess enzyme activity on nucleosome and peptide substrates. While three of the four variants tested showed significant decreases in catalytic activity, one variant had a non-significant decrease. This differential KMT5B catalytic activity raises questions about the relationship between levels of catalytic activity and neurodevelopmental phenotypes. The methods established in this work lay the groundwork for testing additional clinically associated KMT5B variants. Understanding the impacts of these variants on catalytic activity is an important first step in determining their underlying mechanisms that contribute to neurodevelopmental dysfunction.Item Role of Polycomb Group Protein Mel18 in Hematopoietic Stem Cell Maintenance(2025-05) Cai, Wenjie; Wek, Ronald; Zhang, Ji; Capitano, Maegan; Wan, Jun; Ren, Hongxia; Liu, YanPolycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1). PRC2 complex deposits the mono-, di-, and tri- methylation on histone 3 lysine 27, whereas PRC1 introduces mono-ubiquitination on histone 2A lysine 119 (H2AK119ub1). PRC1 can also regulate 3D chromatin structure. Bmi1 (PCGF4) and Mel18 (PCGF2) are two major homologs of the PCGF subunit within the canonical PRC1 complex. While Bmi1 is a positive regulator of hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) self-renewal, the role of Mel18 in normal and malignant hematopoiesis is not fully understood. Based upon our previous studies and the literature, I hypothesized that Mel18 inhibits HSC self-renewal and proliferation but promotes HSC senescence. To test my hypothesis, I examined HSC behavior in Mel18 conditional knockout mice (Mel18f/f-Mx1Cre+). I found that acute deletion of Mel18 enhances the repopulating potential of HSCs and increases the number of functional HSCs, without affecting HSC homing. Loss of Mel18 decreased hematopoietic stem and progenitor cell (HSPCs) senescence. In addition, loss of Mel18 promotes cell cycle progression in HSPCs. Therefore, I demonstrated that loss of Mel18 enhances the repopulating potential of HSCs, promotes cell cycle progression in HSCs, but reduces HSC senescence. Mechanistically, loss of Mel18 increases the chromatin accessibility to genes important for HSC self-renewal and ex vivo expansion such as homeobox gene Hoxb4. CUT&RUN sequencing assays revealed that loss of Mel18 reduces the H2AK119ub1 enrichment at the promoter regions of Cdk4 and Cdk6, leading to their enhanced expression in HSPCs. Furthermore, I identified a Mel18-specific chromatin loop at the S100a9 locus, a gene important for senescence and inflammation, using Hi-C assays, Collectively, these findings demonstrated that Mel18 plays an important role in hematopoietic stem cell maintenance. Mel18 inhibits HSC self-renewal and proliferation but promotes HSC senescence through modulating histone modifications, chromatin accessibility, and 3D chromatin structure in HSCs.