Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2024, College of Arts and Sciences

The meiotic cell division is highly sensitive to heat stress across many sexually reproducing organisms. Reasons for this sensitivity remain unclear. With increasing global temperatures, understanding the mechanisms underlying meiotic heat sensitivity is crucial. In my thesis, I have explored the genetic factors of heat sensitivity in budding yeast. In this model organism, temperatures compatible with mitotic cell division cause complete prophase I arrest during meiosis. My findings indicate that this meiotic progression block is mediated by a widely conserved cell cycle checkpoint involving components of the DNA damage response network. Accordingly, cells resume meiotic divisions upon return to permissive conditions, indicating a retention of meiotic functionality. Moreover, elimination of recombination restores meiotic cell divisions, suggesting that heat-induced arrest is triggered by defects in the pathway that generates crossovers from programmed double strand breaks. Importantly, methylation of histone H3 at lysine (K)79 by the conserved histone methyltransferase Dot1 is necessary for meiotic arrest and for cell viability under heat stress conditions. Histone H3K79 methylation via Dot1 is also part of the DNA damage response in vegetative cells. My analysis has identified two steps of meiotic recombination that are particularly sensitive to heat stress during wild-type meiosis: These steps comprise the formation of programmed meiotic double-strand breaks and the resolution of double Holliday junctions into crossovers. By contrast, processing of DSBs into non-crossovers is mostly unaffected by heat stress. Elimination of Dot1 or H3K79 methylation significantly restores DSBs and resolution of double Holliday junctions into crossovers. Yeast, cells lacking Dot1-mediated meiotic arrest show significant loss of viability demonstrating the key role of this checkpoint in preserving gamete functionality. Together, these find (open full item for complete abstract)

Doctor of Philosophy, Miami University, 2025, Chemistry and Biochemistry

The purpose of this dissertation is to observe physical properties of molecules in solution. Structural dynamics information is provided for three systems: a cobalt-centered complex converting between three coordination states at low temperature, a lanthanide complex adopting two NMR-active enantiomers according to identity of the metal center, and a chaperone protein interaction determining binding symmetry. These systems are investigated using a variety of analytical methods – including crystallography, NMR spectroscopy, Density Functional Theory calculations, and Molecular Dynamics simulations. Chapter 2 examines the dynamics of TpPh,Me Cobalt (II) NO3 (TpPh,MeCoNO3) [TpPh,Me = tris-3-phenyl-5-methylpyrazolylborate]. Solid-state XRD structure of TpPh,MeCoNO3 is presented for the first time, showing a five-coordinate Co (II) complex with TpPh,Me with NO3 bound as a bidentate ligand. Variable temperature NMR spectra are complicated at low temperature, with signals coalescing as temperature is increased. The high temperature NMR spectra indicate a four-coordinate structure above room temperature. Spectral analysis demonstrates the TpPh,MeCoNO3 complex occupies three concurrent structures at low temperatures. These three structures are analyzed using Density Functional Theory (DFT) calculations of four- and five-coordinate structures generated in silica from the crystal structure. In Chapter 3, the conformational interconversion of two NMR-active LnDOTAM structures (Ln=La-Lu; DOTAM=1,4,7,10-tetrakis(acetamido)-1,4,7,10-tetraaza-cyclododecane) are examined using a series of 13 lanthanide ions. Variable-temperature 1H NMR spectra demonstrate the concentration of the two identifiable conformations in solution depends on the identity of the metal ion. At low temperature, early LnDOTAM (Ce-Nd) have a high concentration of the twisted square antiprismatic geometry (TSAP), and later LnDOTAM (Sm, Eu, Tb-Yb) have a higher concentration of the square antiprismatic geomet (open full item for complete abstract)

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2024, College of Arts and Sciences

Trypanosoma brucei is a parasite that causes human African trypanosomiasis and regularly switches its major surface antigen, Variant Surface Glycoproteins (VSGs), to evade the host immune response and to establish a long-term infection. VSG genes are located at subtelomeric regions, and the active VSG is transcribed by RNA polymerase I. Antigenic variation is a critical pathogenesis mechanism in T. brucei that has two key aspects: monoallelic VSG expression and VSG switching. Monoallelic VSG expression ensures that at any moment, T. brucei presents a single type of VSG on its cell surface. Telomeres are nucleoprotein complexes located at the chromosome ends. RAP1 is a protein that is conserved from protozoa to mammals and associates with the telomere chromatin. TbRAP1 is essential for T. brucei cell proliferation, telomere/sub-telomere integrity, monoallelic VSG expression, and suppressing VSG switching. To better understand TbRAP1's essential functions, we attempted to identify TbRAP1's interaction factors focusing on its conserved functional domains: BRCT, Myb, MybLike (including RRM and DB), and RCT. We identified several proteins that interact with TbRAP1 using TbRAP1 MybLike, Myb to RRM, and RCT domains separately as bait in yeast 2-hybrid (Y2H) screens. First, we identified Importin α (Tb427.06.2640) as a TbRAP1 Myblike domain (aa 633-766)-interacting factor. We also confirmed that TbRAP1 MybLike contains a bipartite nuclear localization signal (NLS) necessary for importing TbRAP1 into the nucleus through interaction with Importin α. Second, we identified many proteins as TbRAP1-Myb-RRM (aa 427-733) interacting candidates, including TbCactin (Tb927.11.11610). Lastly, we also identified many proteins interacting with the TbRAP1-RCT (aa 742-855) domain, including recombinase RAD51 (Tb927.11.2550). Subsequently, we validated the interaction between TbRAP1 and TbCactin by Co-IP and showed that TbCactin is essential for T. brucei proliferation. Cactin, a conserved (open full item for complete abstract)

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2024, College of Arts and Sciences

Eukaryotic initiation factor 2A (eIF2A) is a highly conserved 65 kDa eukaryotic protein that functions in minor initiation pathways, which affect the translation of only a subset of messenger ribonucleic acid (mRNAs), such as internal ribosome entry site (IRES)-containing mRNAs and/or mRNAs harboring upstream near cognate/non-AUG start codons. These non-canonical initiation events are important for regulation of protein synthesis during cellular development, differentiation and/or the integrated stress response. Selective eIF2A knockdown in cellular systems was shown to inhibit translation of such mRNAs, which rely on alternative initiation mechanisms for their translation. However, there existed a gap in our understanding of how eIF2A functions in mammalian systems in vivo (on the organismal level) and ex vivo (in cells). To address this question we have developed the eIF2A-total body knockout (KO) mouse model. Using this model, we presented evidence implicating eIF2A in the biology of aging and metabolic syndrome. We discovered that eIF2A-KO mice have reduced life span and that eIF2A plays an important role in maintenance of lipid homeostasis, the control of glucose tolerance and insulin resistance. We also showed the eIF2A KO affects male and female mice differently, suggesting that eIF2A may affect sex-specific pathways. The metabolic syndrome phenotype has three main etiological categories: obesity and disorders of adipose tissue (particularly increased size of adipocytes); glucose intolerance and insulin resistance; and a constellation of independent factors of hepatic, vascular, and immunologic origin and all of these features were observed in the eIF2A-KO mice. Increased adipocyte size is, in particular, well known to be positively correlated with impaired insulin sensitivity and glucose tolerance leading to metabolic syndrome. To specifically check whether the absence of eIF2A in adipose tissue is responsible for metabolic abnormalities observed in the tota (open full item for complete abstract)

PHD, Kent State University, 2025, College of Arts and Sciences / School of Biomedical Sciences

New insights into cellular interactions and key biomolecules involved in LC bone metastasis will have remarkable therapeutic benefits. Using a panel of four LC cells, we investigated the interaction between LC and bone (LC-OC interaction) by exposing differentiating OCs to LC cells directly in a co-incubation setting or indirectly via treatment with LC secretomes (CM or exosomes). LC-OC interaction facilitated the production of large-sized OCs (nuclei >10) coupled with extensive bone resorption pits on bovine bone slices. Proteomics and western blotting analysis identified Gal3bp as a potential biomarker which was primarily released by LC exosomes. The enhancement of OC differentiation and function by LC-exosomal Gal3bp was supported by studies where recombinant Gal3bp and anti-Gal3bp were applied. Our results indicated that dysregulation of crucial OC markers, Gal3bp and Gal3 during LC-OC interaction possibly contributed to the stimulation of osteoclastogenesis. Overall, this work implicated LC-exosomal Gal3bp in osteolytic metastasis of LC which warrants further studies to assess its potential prognostic and therapeutic relevance. BM is a significant complication of solid malignancies such as LC, frequently manifesting in advanced stages and affecting 30-40% of patients with LC, resulting in poor prognoses. Research on the LC-exosomal marker Gal3bp indicates its potential as a predictive biomarker due to its role in promoting osteoclastogenesis, although its in vivo implications remain to be elucidated. To investigate Gal3bp in vivo, we utilized murine BM models with intratibial and intracardiac administration of A549-Luc2 cells. Skeletal samples from athymic (nu/nu) mice were analyzed for structural deformities using 3D reconstruction and quantitative parameters (cortical and trabecular). The results demonstrated that intratibial rGal3bp supplementation of LC cells significantly increased bone resorption and compromised bone structure due to enhanced osteoclast (open full item for complete abstract)

Doctor of Philosophy (PhD), Wright State University, 2024, Biomedical Sciences PhD

DNA replication can be perturbed by various agents that slow or stall the replication forks, causing replication stress. If undetected, stressed forks may collapse, causing mutagenic DNA damage or cell death. In response to replication stress and DNA damage, the eukaryotic cell activates the DNA replication checkpoint (DRC) and DNA damage checkpoint (DDC) pathways to promote DNA synthesis, repair, and cell survival. The two cell cycle checkpoint pathways are controlled by the protein sensor kinases Rad3 (hATR/scMec1) and Tel1 (hATM/scTel1) in fission yeast, although Tel1 plays a minimal role in checkpoint functions. Rad3 and Tel1 belong to a family of phosphatidylinositol-3-kinase-related kinases (PIKKs), whose stability is regulated by the heterotrimeric TTT (Tel2-Tti1-Tti2) complex. The current model suggests that the TTT complex works with Hsp90 and R2TP complex in the co-translational maturation of all PIKKs for their proper folding and stability. We have previously reported a tel2-C307Y mutant with a moderately reduced Rad3 protein level (~60% of wild-type cells). This mutation eliminates Rad3 mediated signaling in the DRC pathway but moderately reduces signaling in the DDC pathway. This result suggests that Tel2 of the TTT complex may specifically regulate the DRC pathway. In this study, we investigated this possibility by taking a genetic approach to analyze the functions of Tti1, the largest subunit of the TTT complex. We randomly mutated the tti1 gene and integrated the mutations at the genomic locus by pop-in and pop-out recombination strategy. As a result, 100 primary tti1 mutants were successfully screened, based on their increased sensitivities to hydroxyurea (HU) which depletes cellular dNTPs and/or the DNA damaging agent methyl methanesulfonate (MMS). Preliminary characterization of the primary Tti1 mutants, based on their relative sensitivities to HU, MMS or both agents, led us to focus on a collection of 24 mutants. Among the 24 mutants, DNA seq (open full item for complete abstract)

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2024, College of Arts and Sciences

Bacterial genomes encode multiple 16S-23S rRNA operons whose sequences are used for prokaryotic taxonomy. Cyanobacteria have ribosomal operons (RO) that encode three enzymatic RNA genes (16S, 23S, 5S) and two Internal Transcribes Spacer (ITS) regions. The first ITS region may also contain two tRNA genes (tRNAIle and tRNAAla). Cyanobacterial genomes have one to six RO copies. The monophyletic species concept, standard in cyanobacterial systematics, requires the description of an autapomorphy to test phylogenetic hypotheses. Increasingly, ITS sequence comparisons replace morphological autapomorphies in taxonomic studies. However, few researchers determine the number of genomic RO copies and no methodology for identifying orthologs and paralogs among multiple ROs has been developed. This research analyzed 111 unique consensus 16S-23S ITS sequences isolated from 69 strains of the cyanobacterial genus Brasilonema. An equation for defining orthologous and paralogous operon copies was developed using sequences patterns and secondary structures of a helical ITS domain. Four unique RO paralogs were identified in Brasilonema, with all but one being recovered with and without accompanying tRNA genes (tRNA+ and tRNA- forms). Orthologs with variable tRNA content displayed consistent changes to spacer regions upstream and downstream of tRNA gene loci. Spacer sequence dissimilarities inflated average distance estimates (percent difference) from 0.76% to 18.03%, especially among interspecific comparisons. Intragenomic p-distance estimates increased on average 3.43% when otherwise orthologous operons with variable tRNA content were compared, solely from the presence of divergent spacer regions. Thus, taxonomic comparisons of orthologous ROs with divergent tRNA content are inappropriate from an evolutionary standpoint. Differences in adjacent spacer sequences among orthologs with variable tRNA content were hypothesized to result from a now quiescent Short Interspersed Element (SINE-l (open full item for complete abstract)

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2024, College of Arts and Sciences

Ulcerative colitis disease can progress into cancer over many stages of dysplasia if not treated at early stages. Inflammatory responses are the main factors in the progression of ulcerative colitis. They are triggered by epithelial lesions as well as microbiota, which infiltrate into the interstitium layer and augment inflammatory responses. The inflammatory responses are mediated by cytokines and chemokines, which are induced by colon fibroblasts and epithelial cells, immune cells, as well as the microbiome. This study focuses on one potent, pro-inflammatory chemokine, CXCL8, which attracts immune cell to the lesion areas and augments disease. The first part of the dissertation focuses on the signaling pathways that regulate upregulation of CXCL8 levels in colitis-associated and colitis-associated cancer (CAC) fibroblasts. Using unbiased mRNA sequencing analysis, we identified that the progression of the fibroblasts is associated with an inflammatory status and involves the injury-induced senescence pathway. Testing three immune-derived cytokines (TNF, IL-1 and IFN) as well as two bacterial signals (LPS and Flagellin/FliC) demonstrated that the NFB signaling pathway is the major signaling pathway associated with upregulation of CXCL8 expression levels in colon fibroblasts in vitro and in vivo. In addition, investigating mycoplasma as a representative in vivo stimulus also resulted in upregulated CXCL8 expression. Moreover, normal colon fibroblasts, colitis-associated fibroblasts and colon epithelial cell demonstrate overlapping, but also cell type-specific ways to activate CXCL8 expression. Finally, reprogramming colon fibroblasts demonstrates an epigenetic memory in colon fibroblasts from ulcerative colitis that is associated with elevation of CXCL8 expression. The second part of the dissertation focuses on the regulation of microRNAs (miRNAs) in downregulating CXCL8 expression levels. Investigating E2F7/E2F8, cMyc, and miRNAs of the miR-17 family demonstra (open full item for complete abstract)

Doctor of Philosophy (Ph.D.), University of Dayton, 2024, Biology

The Drosophila model is an extremely useful tool to study development and disease. It offers several advantages with its short lifespan, high number of genetically identical offsprings, repertoire of genetic tools and conservation in developmental pathways and processes. With high genetic conservation and ease of screening phenotypes, the Drosophila eye offers an excellent model for studying development and disease. In this study, we have used the Drosophila eye to study the regulation achieved by a transcription factor, and transcriptional pausing factor in eye development with implications in human craniofacial development. In addition, we also assayed the impact of various proteins of SARs-CoV2 using the Drosophila eye model. In the first study, we focused on the role of a dorsal selector gene - defective proventriculus, that encodes a K-50 homeodomain containing transcription factor. Axial patterning is required to establish antero-posterior, dorso-ventral and proximo-distal axes and is crucial for the formation of a 3D organ. In the eye, dorso-ventral axis is the first to form. Previously, we characterized dve as a dorsal selector gene based on (1) its expression in the dorsal head vertex region of the eye disc, (2) its gain-of-function phenotype showing complete eye suppression and (3) its loss-of-function phenotype showing dorsal eye enlargements, as in other dorsal eye genes. We also reported that differences in Dve expression impact the placement of eyes on the head of an organism resulting in diversity across animal groups. The human ortholog of Dve is SATB1 and it is highly conserved. Misexpression of SATB1 in the developing eye results in eye suppression and ectopic Wg expression, as in dve gain-of-function. SATB1 is highly expressed in cancers and metastasis. As part of this thesis, I have performed structure-function analysis of Dve protein to dissect its diverse roles in development and growth. We identified N-terminal+ULD+Hox1 as the minimal domain (open full item for complete abstract)

Master of Science, Miami University, 2024, Biology

This study aims to identify the precise position of the methylation site for the Lmna gene in reprogrammed brown adipose tissue (BAT)-like cells. Results previously obtained from our lab have shown that by the co-expression of HB-EGF and ADAM12s, cellular reprogramming into brown adipose tissue (BAT)-like cells led to Lmna mRNA downregulation. Experiments were designed to identify whether the Lmna gene downregulation was due to methylation. To achieve this, primers were specifically designed to target the promoter gene regulatory region and investigate potential epigenetic modifications responsible for transcription and regulation of gene expression. This approach will allow for a focused analysis of key regions that may play a role in regulating downregulated genes in reprogrammed BAT-like cells. This study showed that Lmna may be downregulated by methylation and responsible for decreased expression.

Master of Science (M.S.), University of Dayton, 2024, Materials Engineering

Rare earth elements (REEs) are essential to many modern-day technological applications. Due to their difficult and environmentally harmful refining methods, many of these REEs are imported to the U.S. from various other countries. With countries like China dominating the market, the U.S. supply chain is at risk. A potential solution to this issue would involve the use of proteins to extract these REEs in an environmentally sustainable manner. Custom proteins would be designed to extract specific REEs from their mixed metal ores through computer simulations, namely molecular dynamics. Currently the design process is stymied by the lack of working force fields for REEs within many molecular dynamics programs. This work seeks to address this issue by creating custom force fields designed around replicating basic experimental properties the REE ions have with water, counterions, and REE binding proteins. This is done utilizing a Bayesian optimization algorithm which can efficiently and accurately choose new parameters to test and verify for a wide variety of systems.

Doctor of Philosophy, Case Western Reserve University, 2025, Pathology

The COVID-19 pandemic claimed the lives of millions of people and affected communities worldwide. SARS-CoV-2, the virus that causes COVID-19, continues to be a global health concern, as does the inevitable threat of new viral outbreaks. We must therefore learn from this virus in the hope of better preparing for future pandemics. We investigated the SARS-CoV-2 accessory protein ORF3a and its roles in modulating host antiviral strategies, namely inflammatory signaling and autophagy regulation. ORF3a activates NF-κB signaling, which induces an inflammatory response in infected cells and can also prime certain cells for inflammasome assembly and subsequent cell death. We found that, unlike the homologous protein SARS-CoV ORF3a, SARS-CoV-2 ORF3a does not depend on its N-terminal TRAF-binding sequence to activate NF-κB. The ORF3a homologs thus affect NF-κB signaling through different mechanisms. Second, SARS-CoV- 2 ORF3a blocks autophagy by binding to the human protein VPS39, a member of the complex that facilitates membrane fusion between autophagic compartments. We discovered that the predicted β-propeller domain of VPS39 is critical to its interaction with ORF3a. Regulating autophagy is important for productive SARS-CoV-2 infection; disrupting the ORF3a:VPS39 interaction could therefore be a future strategy to hinder SARS-CoV-2 propagation.

Doctor of Philosophy, Case Western Reserve University, 2024, Genetics

Background: Cystic Fibrosis (CF) is a progressive, systemic disease caused by mutations in the CFTR gene. Despite the availability of highly effective CFTR modulators, there is continued need for the development of CF therapeutics because there is a portion of people with CF not eligible for modulator therapy due to their mutations. CF mouse models are powerful tools to study mutation-specific CF biology and to screen novel therapeutics. This dissertation describes the creation of several novel mouse models using different genetic approaches. Methods & Results: Human-exon (hEx) replacement mouse strains in which a mouse (m)Cftr exon-of-interest was replaced with the corresponding human exon's sequence were designed. hEx replacement strains carrying either human exon 3, 11, 12, or 26 expressed chimeric Cftr from the endogenous mCftr locus. WT hEx3, hEx11, hEx12, and hEx26 strains displayed phenotypes similar to non-chimeric WT controls and highly dissimilar to CF mice. Additionally, a mouse model containing the W1282X mutation in the mCftr gene was created. W1282X mice recapitulated common CF manifestations including low Cftr mRNA expression, poor survival and growth, and altered electrophysiology in various tissues. Forskolin induced swelling (FIS) was performed on intestinal organoids derived from the W1282X mouse and an existing G542X mouse treated with various pharmacologics. Both organoids demonstrated FIS upon treatment with readthrough agents, nonsense-mediated decay inhibitors, and CFTR modulators; however, the specific drug combinations that elucidated the most robust rescue differed in W1282X and G542X organoids. Discussion: The hEx replacement mice provide a novel genetic approach for creating humanized mouse models. hEx 3, 11, 12, and 26 strains displayed WT phenotypes with no evidence of CFTR dysfunction therefore validating the hEx approach for modeling CF mutations. The W1282X mouse is the first W1282X-specific in vivo CF model. It (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2024, Molecular Medicine

Glomerular podocytes are integral to the filtration barrier needed for kidney function. Their dysfunction is a major contributor to chronic kidney disease, affecting 14% of adults in the U.S. There are no treatment options, and those who have progressed into end-stage kidney (ESKD) disease must resort to dialysis or undergo a kidney transplant to survive. Individuals with recent African ancestry have a 3.5-fold increased risk for ESKD compared with European ancestry. This discrepancy is partly due to two variants (G1 and G2) in the apolipoprotein L1 (APOL1) gene. One high-risk allele provides resistance to African Sleeping Sickness, but two significantly increase the risk of kidney disease in an undetermined manner. We generated induced pluripotent stem cell (iPSC) lines containing low (G0) or high-risk (G1, G2) APOL1 variants. APOL1 podocyte (G0, G1, G2) populations were generated using our directed differentiation protocol and had comparable interferon-gamma-stimulated APOL1 and podocyte marker expression. Cytotoxicity assays and transcriptome analysis demonstrated no variant-dependent cell death or differential gene signatures, regardless of genotype. Further analysis revealed that interferon-gamma treatment alone drove the most significant transcriptome shift. A Design of Experiment (DoE) approach was employed based on developmental and kidney biology to identify factors that induce APOL1 expression without IFN-γ stimulation. A synergistic combination of four signaling pathways was identified, providing a novel framework for studying podocyte and APOL1 biology in a human-based model.

Doctor of Philosophy, Case Western Reserve University, 2025, Biomedical Engineering

The sex determining region Y-box (Sox) gene family is important for stem cells and early development in metazoans. Sox expression is associated with pluripotency, neuronal differentiation, developmental processes, and cancer. Platyhelminthes (flatworms) are flattened, bilaterally symmetrical invertebrates that lack body cavity. While there are many free-living species of flatworms, the most well-known flatworms are parasitic. Schistosome helminths infect over 200 million people across 78 countries and are responsible for nearly 300,000 deaths annually. We have characterized and a Sox-like gene SmSOXS1. SmSoxS1 is a developmentally regulated activator that localizes to the anterior and posterior ends of the schistosomula and binds to Sox-specific DNA elements. We also identified an additional six Sox genes in schistosomes, two Sox B, one SoxC, and three Sox genes that may establish a flatworm- specific class of Sox genes. To further explore this flatworm-specific group of Sox genes, we made a comparative analysis to identify Sox proteins in nine flatworm species, including S mansoni and S mediterenea. We identified 62 flatworm Sox proteins across the 9 species, 49 of them were unannotated. Using phylogenetic analysis, we found all of the flatworm species share a SoxD and SoxB homologs. In addition, we identified a clear flatworm-only group of sox proteins, observed in each flatworm species. Overall, we have identified novel Sox genes in schistosomes, and other flatworms. This thesis expands the potential functional roles for the Sox family and may provide interesting insights into early multicellular development of flatworms.

Master of Science, Miami University, 2024, Cell, Molecular and Structural Biology (CMSB)

This study aims to determine the effect of novel bioactive compounds from the Zingiberaceae family on inflammatory mediators, including pro-inflammatory cytokines and enzymes. To investigate the molecular mechanisms through which the Zingiberaceae bioactive components reduce inflammation, we employed a mouse model of 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce skin inflammation. TPA-induced skin inflammation and weight increase is a widely recognized model for assessing the protective effects of dietary compounds against skin inflammation. In this study, there was a significant reduction in the average TPA-induced weight increase of ear punches treated with 6-paradol (3 μmol), yakuchione-A (2 μmol), and yakuchione-B (2 μmol). We measured ear thickness, topical application of 6-paradol (1.5 and 3μmol), 6-shogaol (3 μmol), oxyphyllacinol (2 μmol), yakuchione-A (2 μmol), and yakuchione-B (2 μmol) significantly inhibited ear thickening and edema. To explore the underlying mechanism of action of the bioactive compounds, we employed ELISA, qPCR, and IHC to determine the expression of pro-inflammatory cytokines and pro-inflammatory enzymes. The results from this study suggest that 6-paradol, 6-shogaol, oxyphyllacinol, yakuchione-A, and yakuchione-B inhibit TPA-induced skin inflammation.

Doctor of Philosophy, The Ohio State University, 2024, Molecular Genetics

Pre-mRNA splicing is one of the earliest regulatory steps in the mRNA processing pathway and is directly tied to the regulatory and quality control pathway nonsense mediated mRNA decay (NMD). My graduate research has focused on understanding how disruption of the spliceosome affects the downstream NMD pathway. Pre-mRNA splicing produces NMD targets via alternate splicing and deposits a crucial NMD sensor, the exon junction complex (EJC), on mRNA. Despite the known relationship between pre-mRNA splicing and NMD, relatively little is known about how disruption of the spliceosome affects the efficiency of NMD, particularly in the case of disease-causing mutations in spliceosome components. I have used a combination of bioinformatic approaches to identify how NMD targets change in response to different types of spliceosome disruption. The core of my analysis relies on transcript quantification and differential expression analysis of RNA-seq datasets to determine how individual transcripts change in abundance following depletion of spliceosome components (Chapter 2). Through this analysis I determined that spliceosome component depletion alters splicing, which reduces the canonical isoform abundance and increases the abundance of non-canonical isoforms, including NMD-targeted isoforms. This increase in abundance of NMD-targets occurs in all three modes of spliceosome disruption tested: spliceosome component depletion, splice altering drugs, and spliceosome component mutations. However, some spliceosome component depletions result in the increase of abundance of NMD targets greater than other non-canonical isoforms, suggesting they may play a more direct role in NMD than other spliceosome components tested. In addition to altered splicing and gene expression, the increase in abundance of NMD targets are likely due to a combination of other reasons, including reduction of the levels of some key NMD factors and over-production of novel NMD substrates that may over-burde (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2024, Molecular, Cellular and Developmental Biology

Cardiovascular disease in the leading cause of death worldwide. While the etiology of heart disease is diverse, a well-documented unifying theme in the progression of heart disease is the development of cardiac fibrosis. Cardiac fibrosis, defined as excess collagen and extracellular matrix deposition in the heart, is mediated by the activation and differentiation of resident quiescent fibroblasts into myofibroblasts. Pathologic fibrotic remodeling in the heart occurs in response to persistent stress stimuli, as well as injury. Fibrotic remodeling leads to stiffening of the ventricles, impaired relaxation, and progressive impairment in cardiac function. While current standard-of-care medications have been vital in the treatment of heart diseases over the last several decades, there are currently there are currently no effective therapies in clinical use specifically targeting fibrosis in the heart. This highlights a significant need to identify novel therapeutics that target fibroblasts and fibrotic remodeling. Our group recently identified Sertad4 (Serta Domain Containing Protein 4) as a potential regulator of fibroblast activation in the heart. However, the function of Sertad4 is unknown. In this dissertation, I sought to investigate the role of Sertad4 in pathologic cardiac remodeling through a combination of in vitro and in vivo studies. To study the role of Sertad4 in vivo we utilized a reporter mouse and a global loss of function mouse. We also utilized three models of cardiac dysfunction: 1) myocardial infarction through coronary LAD ligation; 2) chronic angiotensin II/phenylephrine infusion through osmotic minipumps and 3) chronological aging as a model of diastolic dysfunction. We observed that Sertad4 is predominantly expressed in activated fibroblasts and its expression is significantly elevated in failing human and mouse hearts. Loss of Sertad4 in mice led to preserved cardiac function post-MI and decrease in cardiac hypertrophy and fibrosis. Using is (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2024, Molecular, Cellular and Developmental Biology

Modulating immune responses can be an effective approach to achieve favorable disease outcomes. Acute graft-versus-host disease (GVHD) is a frequent and serious complication arising in patients after allogeneic hematopoietic stem cell transplant (allo-HCT), often leading to non-relapse-related deaths in recipients. In acute GVHD, donor-derived alloreactive T cells recognize and attack host tissues as foreign, particularly targeting organs like the liver, skin, and gastrointestinal tract. This underscores the need for an immune modulation strategy that specifically targets T cells to reduce their inflammatory activity. MicroRNA-155 (miR-155) has a key role in T cell-driven acute GVHD responses. Therefore, we used CRISPR/Cas9 to target MIR155HG, the gene encoding miR-155, as a strategy to mitigate GVHD. Importantly, the donor T cells driving GVHD also contribute to graft-versus-leukemia (GVL) effects by eliminating residual leukemic cells in the host. We show that our approach not only reduces the severity of acute GVHD but also preserves the beneficial GVL response. Similarly, T cell dysregulation is central to pancreatic ductal adenocarcinoma (PDAC), a highly lethal cancer where T cells are suppressed within the tumor microenvironment, impeding anti-tumor immunity. In PDAC, the formation of immune cell aggregates known as tertiary lymphoid structures (TLS) correlates with a higher neoantigen burden and improved survival. To explore the influence of TLS in anti-tumor immunity, we used an immune modulation strategy to induce TLS in a mouse PDAC model. We found that TLS-positive tumors support stem-like T (T-stem) cells and effector memory phenotypes, suggesting their role in anti-tumor immune responses. Although acute GVHD and PDAC may initially appear unrelated-with GVHD arising from transplant-related immune dysregulation and PDAC being a malignancy-they are both linked by T cell dysfunction. Therefore, our dual immune modulation strategy to enhance appropriate T ce (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2025, Biochemistry

Appropriate levels of gene expression are required for cellular fitness and survival. Messenger RNAs (mRNAs) play a central role in gene expression and thus must be tightly regulated. Despite most mRNAs undergoing degradation through the same decay pathway, mRNA half-lives are highly disparate and can vary by orders of magnitude. Some transcript-specific features, such as stable structures in the 5' UTR or regulatory sequences in the 3' UTR, can affect mRNA expression. However, these elements are not sufficient to explain the variation in mRNA stability observed transcriptome-wide. Our lab originally identified codon optimality as a global determinant of mRNA stability. Codon optimality refers to how efficiently each codon is translated and is dependent on the relative availability of charged cognate tRNAs. The most likely explanation for the observed link between codon optimality and mRNA stability is that ribosome decoding rates influence mRNA stability. However, a transcriptome-wide link between the contribution of codons to translation rate and to mRNA stability has not been previously established. Using ribosome profiling in Saccharomyces cerevisiae, we demonstrate that codon-level and transcript-level elongation rates indeed globally correlate with mRNA stability. Chemical modification of mRNA nucleosides provides an additional layer of regulation. Prior to our work, N4-acetylcytidine (ac4C) was an understudied modification solely found to exist in polyA-selected mRNA by HPLC/MS-MS. Through a collaborative effort, we identified more than 2,000 human mRNAs that are modified with ac4C, and our studies revealed novel roles for ac4C in promoting mRNA stability and translation. We also find that the acetylating enzyme N-acetyltransferase 10 (NAT10) associates with translating ribosomes, suggesting that it may monitor translation and co-translationally acetylate mRNA. Furthermore, ac4C displayed stronger base-pairing interactions with guanosine (open full item for complete abstract)