PhD, University of Cincinnati, 2024, Medicine: Systems Biology and Physiology

While there is a lack of viable treatment solutions for regenerating lost motor function following traumatic injury, there is a plethora of research substantiating the role of propriospinal neurons in recovery of function. However, it remains unclear whether these neurons exhibit largely similar responses to injury or if their diversity dictates distinct alterations in gene expression. Using single nuclei gene expression analyses, we show that excitatory V2a propriospinal neurons display substantial molecular and cellular heterogeneity in adult mice. In addition to providing evidence for conserved fetal V2a subtypes (early and late born), we also demonstrate additional subtypes that show distinct patterns of localization. Notably, we show that Rbms3+/Nav3+ expressing V2a neurons, enriched for synaptic transmission and cell-cell adhesion, are nearly absent following injury. Finally, we present that remaining V2a populations exhibited distinctive transcriptional alterations, indicating a potential rewiring of cellular interactions within the spinal cord microenvironment. Thus, our study reveals the diverse responses of propriospinal neurons to injury. These analyses provide a framework for understanding changes in propriospinal neurons that may lead to adaptive (or maladaptive) changes in circuits following injury.

Committee: Steven Crone Ph.D. (Committee Chair); Steven Kleene Ph.D. (Committee Member); Nathan Salomonis Ph.D. (Committee Member); Warren Alilain Ph.D. (Committee Member); Mark Baccei Ph.D. (Committee Member); Sarah Pixley Ph.D. (Committee Member) Subjects: Neurosciences

PhD, University of Cincinnati, 2023, Medicine: Biomedical Informatics

Single-cell sequencing technologies have significantly advanced our understanding of complex biological systems. Researchers and initiatives like the Human Cell Atlas (HCA) and the Brain Research through Advancing Innovative Neurotechnologies Initiative (BICCN) have successfully created Cell Atlases for different tissues and developmental stages. The growing volume of single-cell data has created a demand for tools that offer comprehensive capabilities, encompassing gene signature visualization, exploration, and harmonization. The widespread adoption of single-cell technologies and the decreasing cost of single-cell experiments have led researchers to utilize these techniques in studies involving perturbations, including diseases, treatments, genetic mutations, time-series analyses, and more. This application has accelerated the exploration of transcriptional profiles across different perturbation states, enabling comparisons with control conditions. However, the analysis of single-cell data presents several emerging computational challenges that need to be addressed. Firstly, there is a need for a deeper understanding of diverse biological covariates and technical effects that may impact the data. Secondly, it is crucial to develop user-friendly visualization and interaction methods capable of handling large-scale datasets, particularly at the atlas-level. Lastly, there is a pressing need to comprehend the effects of perturbations across multiple dimensions, with a particular emphasis on temporal dynamics. These computational hurdles demand innovative solutions to effectively tackle the complexities associated with single-cell data analysis. To address these challenges, we have developed two tools, namely ToppCell and CellDrift, that facilitate comprehensive exploration of large-scale single-cell data and enhance our understanding of temporal perturbation effects. To demonstrate the capabilities of our tools, we utilized the context of COVID-19 and uncove (open full item for complete abstract)

Committee: Bruce Aronow Ph.D. (Committee Chair); Nathan Salomonis Ph.D. (Committee Chair); Rhonda Szczesniak Ph.D. (Committee Member); Surya Prasath Ph.D. (Committee Member); ChangHui Pak PhD (Committee Member) Subjects: Bioinformatics

Doctor of Philosophy, Case Western Reserve University, 2023, Genetics

Type II Diabetes (T2D) is a metabolic disorder characterized by dysfunction of insulin-producing β cells in the pancreatic islet. The pancreas is a very heterogeneous tissue with both endocrine and exocrine cell-types. As such, single-cell sequencing technologies are a powerful tool to allow dissection of cell-type differences as well as heterogeneity within the same cell type. We utilized a single-cell multiomics approach to identify T2D-relevant disease genes in pancreatic islets. First, we developed a framework to integrate single-cell transcriptome, single-nuclei chromatin accessibility, and cell-type specific 3D genome profiles from human islets. We identified T2D-associated β cell heterogeneity driven by a transcription factor, HNF1A, at both the transcriptome and epigenome levels. Second, we identified key differences between prediabetic and diabetic β cell states. We further characterized obesity and aging profiles in human islets including a sex dimorphic response in obese states. Finally, we identified a T2D-associated disease signature in pancreatic stellate cells (PSCs) and propose a potential mechanism for PSC-β crosstalk. Overall, our study demonstrates the powerful applications of single-cell genomics technologies to identify novel biological and disease pathways.

Committee: Yan Li (Advisor); Betul Hatipoglu (Committee Member); Anthony Wynshaw-Boris (Committee Member); Drew Adams (Committee Chair) Subjects: Biomedical Research; Genetics

PhD, University of Cincinnati, 2020, Medicine: Molecular and Developmental Biology

Vertebrate skeletal muscle is formed by the fusion of mononucleated progenitor cells into multinucleated myofibers, which comprise the functional units of the tissue and perform both contractile and metabolic functions. Muscle cell fusion is driven by the skeletal muscle-specific membrane protein Myomaker, which is expressed during development and is reactivated following injury to drive regenerative fusion. Myonuclei within the resulting syncytial myofibers must coordinate activity in order to accomplish the distinct tasks necessary for cellular function, but understanding of myonuclear diversity has been lacking due to the technical difficulties of working with multinucleated cell types. We performed single-nucleus RNA-sequencing of skeletal muscle across the murine lifespan in order to achieve nuclear-level resolution of transcriptional dynamics during development, homeostasis, and aging. We uncovered the transient manifestation of distinct myonuclear transcriptional states in postnatal development, enriched for genes involved in myofibrillogenesis and sarcomere assembly, as well as their reemergence in aging muscle. In addition, our datasets comprise a nuclear atlas of skeletal muscle that serve as a platform for interrogation of rare myonuclear subpopulations such as the neuromuscular and myotendinous junctions, for which we identified numerous previously unknown enriched genes. Functional testing of novel postsynaptic genes using an siRNA knockdown screen in C2C12 myoblasts generated validated hits as well as proof-of-concept for our datasets as a resource for gene discovery and elucidation of cellular mechanisms. We also sought to investigate the consequences of ongoing cell fusion in chronic muscle pathology. Genetic muscle diseases such as Duchenne muscular dystrophy (DMD) are characterized by ongoing fusion of activated satellite cells (SCs). Using the mdx mouse model of DMD, we assessed the role of cell fusion by inducible knockout of Myomaker in eithe (open full item for complete abstract)

Committee: Douglas Millay Ph.D. (Committee Chair); Steven Crone Ph.D. (Committee Member); Jeffery Molkentin Ph.D. (Committee Member); Sakthivel Sadayappan Ph.D. (Committee Member); Kathryn Wikenheiser-Brokamp M.D. (Committee Member) Subjects: Molecular Biology

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

AML is the most common acute leukemia in adults with an overall poor prognosis and high relapse rate. Multiple factors including genetic abnormalities, differentiation defects and altered cellular metabolism contribute to AML development and progression. Though the roles of oxidative phosphorylation and glycolysis are defined in AML, the role of the HBP, which regulates the O-GlcNAcylation of cytoplasmic and nuclear proteins, remains poorly defined. We studied the expression of the key enzymes involved in the HBP in AML blasts and stem cells at the single-cell and bulk level. We found higher expression levels of the key enzymes in the HBP in AML as compared to healthy donors in whole blood. We also observed elevated OGT and OGA expression in AML stem and bulk cells as compared to normal HSPCs. Gene set analysis showed substantial enrichment of the NF-κB pathway in AML cells expressing high OGT levels. We found AML bulk cells and stem cells show enhanced OGT protein expression and global O-GlcNAcylation compared to normal HSPCs, validating our in-silico findings. Our study suggests the HBP may prove a potential target, alone or in combination with other therapeutic approaches, to impact both AML blasts and stem cells. Moreover, as insufficient targeting of AML stem cells by traditional chemotherapy is thought to lead to relapse, blocking HBP and O-GlcNAcylation in AML stem cells may represent a novel promising target to control relapse. Additionally, prognostic biomarker discovery approaches based upon bulk analysis are unable to capture key attributes of rare subsets of cells that play a critical role in patient outcomes. Single-cell RNA sequencing is a powerful technique that enables the assessment of rare subsets of cells, but this technique is not amenable to clinical diagnostics. One area where improved prognostic biomarkers are important is for the management of pediatric AML patients with a FLT3-ITD genetic abnormality. We utilized single-cell data from the ra (open full item for complete abstract)

Committee: Brian Cobb (Committee Chair); David Wald (Advisor); Tae Hyun Hwang (Advisor); Stanley Huang (Committee Member); Li Lily Wang (Committee Member); Clive Hamlin (Committee Member) Subjects: Biostatistics; Immunology; Oncology

Master of Sciences, Case Western Reserve University, 2024, Systems Biology and Bioinformatics

Alzheimer's disease (AD) is the most common neurodegenerative disease and the leading cause of dementia. Cerebrospinal fluid (CSF) is a neuroprotector fluid that carries brain metabolites away from the blood-brain barrier. It is an optimal sample for studying neuroinflammation in central nervous system diseases. However, the role of cells carried in CSF in remains underexplored. In this thesis, we investigated the single-cell RNA sequencing data of leukocytes in CSF. The ratio of CD11B+ cells versus T cells increased in amyloid-healthy individuals and gradually decreased with AD progression. Differential expression analysis of the same leukocyte subtype in different AD stages showed that CCL3 and its variants are up-regulated in monocytes from MCI to AD. IL1B is down-regulated in IM and NCM in MCI patients vs healthy individuals. Pathways enrichment analysis shows that interferon-gamma response, interferon-alpha response, and allograft rejection pathways are up-regulated through AD progress in most cell types.

Committee: Gurkan Bebek (Committee Chair); Cheryl Cameron (Committee Member); Jagan Pillai (Committee Member) Subjects: Bioinformatics; Biomedical Research; Immunology; Neurobiology

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

Understanding the viral infection and lifecycle of Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) and Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is important in improving disease outcomes and reducing viral prevalence. In our SFTSV study, we discovered specific cytokine profiles associated with the severity of clinical symptoms. We used single cell RNA sequencing (scRNAseq) on patient blood samples to identify a unique expansion of the B cell population in SFTSV-induced fatal cases which indicated that plasma B cells are a primary reservoir of SFSTV replication. These findings present a potential method of reducing the severity of SFTSV infection, especially in aged patients who are more susceptible to adverse outcomes. In our KSHV study, we developed a novel oral 3D infection model and demonstrated that KSHV can only infect exposed basal epithelial cells in the oral epithelia. We used scRNAseq to show that keratinocyte differentiation and cell death pathways were affected by KSHV infection, suggesting that epithelial differentiation could contribute to KSHV reactivation through changes in epigenetic regulation. In addition, we found a unique population of infected cells with limited early lytic gene expression and a unique gene expression profile, which we termed latent-2 cells. These findings demonstrate that our in vitro 3D epithelial ALI culture model should be a valuable tool to further understand oral KSHV infection for the development of future anti-viral therapeutics to block KSHV transmission.

Committee: Jae Jung (Advisor); Feixiong Cheng (Committee Chair); Weiqiang Chen (Committee Member); Christine O'Connor (Committee Member); Frank Esper (Committee Member) Subjects: Bioinformatics; Biology; Molecular Biology; Virology

Doctor of Philosophy, The Ohio State University, 2023, Biomedical Engineering

A trachea defect from malformation or obstruction can result in significant morbidities and mortalities. Long segment tracheal defects, where the length of the defective tracheal segment is >50% for adults and >30% for the pediatric population, are often fatal due to the lack of autologous tissue suitable for reconstruction. Tissue engineered tracheal grafts (TETG) have the potential to be used to replace diseased tracheal tissue. However, TETG success is often limited by delayed epithelial formation and biocompatibility. The Chiang lab developed a partially decellularized tracheal graft (PDTG) that removes the immunogenic epithelium and lamina propria while preserving donor cartilage that is immune-protected. We have previously shown that PDTG can support host-derived neotissue formation but the composition, long-term outcome, and drivers of PDTG neotissue formation have been largely uncharacterized. The characterization of PDTG neotissue will facilitate the translation of PDTG into large animals as we better understand the regenerated tissue and possible drivers behind neotissue formation. This dissertation aims to examine PDTG neotissue using a combination of immunofluorescent and transcriptomic approaches to characterize PDTG neotissue before uncovering drivers of tracheal graft epithelialization. Functional vasculature in PDTG was established by 2 weeks post-implant. Graft epithelialization was reestablished by 1 month. PDTG neo-epithelialization is driven by basal progenitor cells that were able to proliferate and differentiate into secretory and multiciliated cells. This neo-epithelialization process mirrors the epithelial repair processes seen in epithelial injury models. There was also a macrophage-dominant immune response post-implantation for both PDTG and syngeneic transplants. I then examine the immune response associated with graft implantation to study the impact of macrophages on graft epithelialization. To do that, macrophage phenotypes in the (open full item for complete abstract)

Committee: Tendy Chiang (Advisor); Susan Reynolds (Committee Member); Kaitlin Swindle-Reilly (Committee Member); Christopher Breuer (Committee Co-Chair) Subjects: Biomedical Engineering; Medicine

Doctor of Philosophy, The Ohio State University, 2021, Neuroscience Graduate Studies Program

Degenerative retinal diseases result in neuronal cell death, which causes permanent vision loss in humans. The response to the loss of retinal neuron across species is diverse, with some species capable of complete and functional retinal regeneration. One source of these regenerated neurons is Muller glia (MG), the primary glial cell of the retina. In zebrafish, MG respond to damage by dedifferentiating into progenitor cells (MGPCs) which proliferate before they undergo neurogenesis. While mice completely lack a regenerative response by MG, chick MG form progenitors with limited neurogenesis, proving to be an effective phenotype for understanding molecular pathways that influence reprogramming and cell fate. The focus of this dissertation was on understanding various cellular factors that regulate MG de-differentiation and proliferation or reactive gliosis. The work outlined in these chapters utilize new advancements in single cell RNA sequencing technology (scRNA-seq) to better understand the glial biology of cellular reprogramming. In recent years, we have been able to quantitatively capture the transcriptome of tens of thousands of individual cells in an individual library. Generating these libraries has allowed us probe for transcriptional changes in MG in response to damage or growth factor treatment. These large libraries have enabled us to model the transition from MG to MGPC, probe for novel factors, and guide hypotheses about the functional implications of these target genes. Chapter one focuses on Matrix metalloproteinases (MMP) in damaged retinas, and their regulation in response to retinal damage, and how these changes feed back onto Muller glia to influence their regenerative potential in the chick. Gelatinases in the retina are temporally regulated in retinal development, cellular migration, and axonal guidance. We found that oligodendrocytes were the primary expressors of gelatinase MMP-2, and that expression did not fluctuate with damage. We did ob (open full item for complete abstract)

Committee: Andy Fischer (Advisor); Dana McTigue (Committee Member); Colleen Cebulla (Committee Member); Karl Obrietan (Committee Member) Subjects: Bioinformatics; Biology; Cellular Biology; Neurobiology; Neurosciences

PhD, University of Cincinnati, 2019, Medicine: Molecular and Developmental Biology

Lifelong hematopoiesis requires balanced hematopoietic stem cell (HSC) self-renewal and differentiation into mature blood populations. Such cell fate decisions are often initiated in response to extracellular cues (cytokines) to induce distinct transcriptional programs through coordinated teams of transcription factors that vary between cell states. Inherited or acquired genetic mutations in transcription factors or signaling molecules disrupts this balance to initiate any number of hematologic diseases that each arise from a single or a concise set of cell states. More specifically, cytopenias are life-threatening conditions characterized by low levels of mature hematopoietic cells that are associated with a diverse number of hematopoietic disorders in turn caused by distinct genetic alterations. For example, severe congenital neutropenia (SCN) presents early in life and is characterized by the relative absence of neutrophils, which predisposes these patients to life threatening infections. In contrast to SCN, myelodysplastic syndrome (MDS) typically affects the elderly, is usually accompanied by anemia (can include neutropenia) and presents with abnormal/dysplastic cells. The proper understanding of these conditions is hampered by a lack of accurate models and techniques to enable proper analyses of heterogeneous populations. Furthermore, elucidating the sequence of normal cell state transitions that occur during steady-state hematopoietic development at the single cell level is prerequisite to understanding the corresponding disease states. In this dissertation, I explore the cell states and signaling pathways of murine hematopoietic stem cells (HSC) and granulocytic progenitors and precursors that are disrupted by genetic mutations found in humans with neutropenia or MDS. This was achieved by utilizing novel murine models of disease, many single cell analytical procedures, novel bioinformatics algorithms and techniques, and global proteomics. My findings from t (open full item for complete abstract)

Committee: H. Grimes Ph.D. (Committee Chair); Jose Cancelas-Perez M.D. (Committee Member); Marie-Dominique Filippi Ph.D. (Committee Member); Nathan Salomonis M.D. (Committee Member); Daniel Starczynowski Ph.D. (Committee Member) Subjects: Molecular Biology