Master of Sciences, Case Western Reserve University, 2021, Biology

ACF7 (actin crosslinking family protein-7) is a large actin-bundling and microtubule/actin crosslinking protein that is primarily responsible for cytoskeletal organization and integrity. Hair cells contain specialized subcellular structures to enable hearing, including the actin-rich cuticular plate and the circumferential band. ACF7 localizes to the cuticular plate and circumferential band of both mice and zebrafish. Due to known roles of ACF7 in the cytoskeleton of cell types throughout metazoans, it is plausible that ACF7 plays a vital role in the subcellular architecture of hair cell. To determine the function of ACF7 in hair cells, we used a Pax2-Cre/loxP system that removes three exons of the Macf1 actin binding domain in all descending cells of Pax2-expressing otic progenitors. Surprisingly, our data in four-week old conditional knockout mice suggests that hair cells have normal survivability, morphology, polarity, and hearing capabilities, demonstrating that the loss of ACF7 does not impact hearing in adult mice.

Committee: Brian McDermott Jr. (Advisor); Martín Basch (Committee Member); Ruben Stepanyan (Committee Member); Yolanda Fortenberry (Committee Chair) Subjects: Audiology; Biology; Biomedical Research; Genetics; Molecular Biology; Physiology

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

CD4+ T “helper” populations comprise a key subset of adaptive immune cells that are critical for orchestrating antigen-specific immune responses for both the clearance of pathogens and elimination of cancers. This population responds to insult-specific environmental signals, including those from cytokines, by differentiating into a number of functionally distinct subsets, which produce cytokines and interact with additional immune cells to effect their diverse functions. Of these, T follicular helper (TFH) cells are established coordinators of humoral immune responses, as they engage in bi-directional signaling with B cells, via both cell surface receptors and cytokine signals. Ultimately, this interaction is critical for the germinal center reaction, during which B cells are activated, proliferate, and are ultimately selected to support the generation of high-affinity B cell clones, and thus, high-affinity antibodies. This process is also required for the formation of long-lived plasma cell populations, which are a key part of both natural and vaccine-induced immunological memory. In contrast to this important role, TFH cells have also been implicated in autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, and others, for which the production of autoantibodies is a key aspect of pathogenesis. To date, the full scope of mechanisms underlying TFH cell differentiation are incompletely understood. Complicating this process, TFH cells are not comprised of a single, monolithic population, and numerous studies support the existence and function of ‘polyfunctional' TFH populations which exhibit characteristics of other CD4+ T cell subsets (recently reviewed in (1)). Thus, it will be important for TFH-focused work to identify not only shared, but also TFH-subset-specific, regulatory mechanisms. Here, I present findings regarding both cytokine- and transcription-mediated factor mechanisms by which TFH populations are regulated. First, we ide (open full item for complete abstract)

Committee: Kenneth Oestreich Ph.D. (Advisor); Eugene Oltz Ph.D. (Committee Chair); Hazem Ghoneim Ph.D. (Committee Member); Amy Lovett-Racke Ph.D. (Committee Member); Purnima Dubey Ph.D. (Committee Member) Subjects: Immunology; Molecular Biology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2019, College of Sciences and Health Professions

Differentiation and apoptosis are coordinately regulated biological endpoints in many cell types, including skeletal myoblasts. In adult skeletal muscle, the muscle transcription factor MyoD is necessary for the regeneration of damaged tissue. When myoblasts encounter differentiation cues, mimicked in vitro by removing serum from culture media (differentiation media; DM), ~70% of cells undergo differentiation, while ~30% undergo apoptosis. We have previously reported that the expression of apoptotic versus differentiation-associated genes is temporally distinct, with PUMA expression (apoptosis) and myogenin expression (differentiation) detected after 3 or 18 hours of culture in DM, respectively. The underlying goal of the current study was to identify distinctions influencing MyoD induced transcription of PUMA from MyoD induced transcription of myogenin. We hypothesized that epigenetic modifications of histones at MyoD-responsive promoter regions and post-translational modifications of MyoD may influence MyoD's ability to promote differentiation vs. apoptosis. We first discovered higher levels of acetylated histones surrounding the MyoD-responsive element in the PUMA promoter. Chromatin immunoprecipitation showed MyoD binding at the PUMA and myogenin promoter regions at 3 and 18 hours in DM, respectively. However, MyoD was not detected at the PUMA promoter after 18 hours in DM despite the fact that the PUMA promoter remained highly acetylated. We next assessed the effect of the cell cycle position. By synchronizing myoblasts, we determined that cells in early S-phase have a greater propensity to undergo apoptosis in response to DM, than cells in any other cell cycle phase, while cells in G1 or G2 undergo differentiation. Since MyoD becomes phosphorylated at serine200 near the G1/S border, we hypothesized that MyoDPser200 may be responsible for driving PUMA expression in response to differentiation cues. Herein, we report that a MyoD mutant (MyoDs200a) is unable to i (open full item for complete abstract)

Committee: Crystal Weyman Dr. (Advisor); Anton Komar Dr. (Committee Member); Aaron Severson Dr. (Committee Member); Roman Kondratov Dr. (Committee Member); Aimin Zhou Dr. (Committee Member); Michelle Longworth Dr. (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology

Doctor of Philosophy (PhD), Wright State University, 2022, Biomedical Sciences PhD

Gene expression in mammalian cells requires complex nuclear choreography, and there is increasing evidence that spatiotemporal organization of chromatin and nuclear compartments plays an important role in gene expression. In this dissertation, I examined the function of SON, a splicing factor with a known role in nuclear organization, in chromatin-mediated gene expression. SON association with a transcriptionally inactive U2OS 2-6-3 reporter gene array provided a useful model to study SON's chromatin dynamics. I demonstrated that SON associates with the inactive but not the activated array, and that SON's RNA binding domains are not necessary for that association. Second, I discovered a new role for SON in maintaining chromatin condensation. Whereas chromatin decondensation is typically correlated with transcription activation, I have demonstrated that reporter transcripts are not produced at decondensed SON-depleted U2OS 2-6-3 reporter gene loci, and that SON-depleted loci contain histone H3 that is trimethylated on lysine 9, a marker for transcriptionally silent chromatin. We found that SON depleted reporter loci are still transcriptionally activatable, and that inhibiting transcription elongation is not sufficient to condense the enlarged SON depleted reporter locus. These findings suggest that higher order chromatin structure and transcription activation are functionally distinct mechanisms of gene regulation that can be uncoupled. Finally, I investigated SON's role in genome-wide chromatin organization. SON-depleted cells are more susceptible to DNase digestion, implicating SON in the maintenance of chromatin stability globally. In conclusion, this study demonstrates a new function for the splicing factor SON in maintaining chromatin organization.

Committee: Paula Bubulya Ph.D. (Advisor); Quan Zhong Ph.D. (Committee Member); Labib Rouhana Ph.D. (Committee Member); Weiwen Long Ph.D. (Committee Member); Michael Leffak Ph.D. (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2021, Biomedical Sciences

Few therapeutic options exist for the 15-25% of melanoma patients whose disease is driven by oncogenic NRAS. NRAS is a member of the RAS family of proto-oncogenic GTPase proteins which trigger signal transduction pathways involved in cellular motility, survival, proliferation, and metabolism. Therapeutic targeting of NRAS is a decades-old challenge, hindered by the inability to develop small molecule inhibitors specific for the mutant protein. Furthermore, oncogenic NRAS can circumvent treatments targeting post-translational RAS modifications, interacting partners, and downstream signaling pathways. Current first-line therapies for NRAS-driven melanoma are immune-based. While such drugs are effective in 40-50% of individuals, many patients suffer from high-grade adverse events and only a subset of responders experience durable remissions. With NRAS-driven melanomas being the most aggressive subtype of this disease, new and effective therapeutic options are needed. Oncogenic NRAS mutations primarily affect codons 12, 13, and 61, resulting in constitutive GTP-binding, activation, and downstream signal transduction. However, each NRAS-driven malignancy shows selection bias for a different subset of NRAS mutants. For example, NRAS-driven melanomas are enriched for genetic mutations in codon 61 (>80%) while mutations in acute myeloid leukemia primarily occur in codons 12 and 13. This mutational bias remains poorly understood, especially in melanoma where the codon 61 alterations are not directly attributed to ultraviolet light. I developed a suite of conditional, Nras knock-in mouse models (LSL-Nras Q61R, -K, -L, -H, -P, -Q; G12D and G13D, -R) to test the hypothesis that NRAS mutants commonly observed in melanoma possess functional properties required for efficient melanocyte transformation. Expression of these alleles in melanocytes revealed that the melanomagenic potential of each NRAS variant parallels the frequency of that mutation in human melanoma. Specifical (open full item for complete abstract)

Committee: Christin Burd E (Advisor); Joanna Groden (Committee Chair); Michael Freitas A (Committee Member); Terence Williams M (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology; Oncology

Doctor of Philosophy, The Ohio State University, 2018, Mathematics

Understanding the effects of unknown parameters and estimating their values has become a major task in all areas of systems biology. This task is especially challenging in models that are expensive to evaluate or that contain a large number of parameters. This dissertation consists of two major parts, each one addressing the issue of parameter analysis in a different biological context. In the first chapter, we present a methodology for parameter sensitivity analysis and parameter estimation and apply this methodology to a large spatial model for yeast mating polarization. The model consists of 11 partial differential equations with 35 unknown parameters, and we seek to understand the effects of these parameters and estimate their values from experimental data. In models with such a large number of parameters, traditional methods for parameter estimation can become computationally intractable. Our methodology provides a dramatic improvement in computational efficiency from the replacement of model simulation by evaluation of a polynomial surrogate model. This allows us to perform derivative-based parameter sensitivity analysis to reduce the parameter count, followed by rapid Bayesian parameter estimation that would otherwise be prohibitively expensive to perform. We first tested our methodology on a smaller ordinary differential equation (ODE) model of the heterotrimeric G-protein cycle, which shows results consistent with published single-point parameter estimates. Then, applying our methodology to the full spatial model, we are able to reduce the parameter count via sensitivity analysis and obtain probability distributions of the 15 most sensitive parameters. We show that a wide range of parameter values permit polarization in the model. In the second chapter, we consider a compartmental ODE stem cell lineage model for tissue growth. We compare three variants of hierarchical stem cell lineage tissue models with different combinations of negative feedbacks. (open full item for complete abstract)

Committee: Ching-Shan Chou (Advisor); Adriana Dawes (Committee Member); Dongbin Xiu (Committee Member) Subjects: Mathematics

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

Epilepsy is a neurological disorder characterized by repeated seizures. It affects 1% of the US population. Despite the recent progress in antiepileptic drug discovery and development, approximately 30% of the patients do not respond to current therapies. A major pitfall of the current antiepileptic drug discovery pipeline is current animal seizure models. The most widely used animal models are mechanically or chemically induced models, and they fail to recapitulate drug-resistant seizures, which are largely genetic epilepsies. Induced pluripotent stem cells (iPSCs) are promising alternative preclinical epilepsy models. iPSCs preserve the genetic information of epilepsy patients and can be an unlimited source of neuronal cell types of interest. Many studies have shown that iPSC disease models successfully reproduce epilepsy phenotypes. Herein, we successfully leveraged stem cell reprogramming technologies and CRISPR/Cas9 gene editing tools to generate iPSCs models of three drug-resistant epilepsies with monogenic cause: Early infantile epileptic encephalopathy, subtype 76 (EIEE-76), Microcephaly, Epilepsy, Diabetes Syndrome (MEDS), and GRIN2A-related epilepsy. Using these models, we uncovered pathophysiological mechanisms underlying the epilepsy phenotype of these rare genetic epilepsies that were previously unknown. We demonstrated EIEE-76 patient variant of ACTL6B dysregulates genes essential for neuronal development and that EIEE-76 patient-derived neurons demonstrated elevated electrophysiological activity than control, consistent with the patient phenotype. Also, we elucidated MEDS patient-derived neural progenitor cells (NPCs) have defects in secretory protein trafficking. Impaired protein trafficking during neurogenesis resulted in abnormal neuronal differentiation and electrophysiologically less active neuronal population. Next, we took a step further and developed a disease-relevant, high-throughput, phenotypic screen platform to identify potential an (open full item for complete abstract)

Committee: Ashleigh Schaffer (Advisor); Yan Li (Committee Member); Drew Adams (Committee Chair); Anthony Wynshaw-Boris (Committee Member); Andrew Pieper (Committee Member) Subjects: Genetics

PhD, University of Cincinnati, 2023, Medicine: Immunology

Hematopoiesis is a complex and tightly regulated biological system in which the stem cells and progenitors balance between mature lineage outputs and self-renewal. Environmental stimuli, genetic predispositions, and host responses together control and alter underlying transcriptional network. Various animal models and single-cell multi-omics approaches have been developed to unravel the network and mechanisms associated with it. This dissertation characterizes relationship between gut microbiome (environmental stimuli) and host; describes human and murine hematopoietic stem cells and progenitors; and demonstrates methods to incorporate with biological models to derive insightful biological readouts. Microbiome models have informed our understanding of microbiome-dependent metabolites, their role in homeostatic hematopoiesis, and nominated some metabolites as potential therapeutics. However, conclusions about gut microbiota-induced biological functions have been called into question after recent results from metabolite clinical trials and conflicting findings across studies. To systematically evaluate the impact of durably different gut microbiome on homeostatic hematopoiesis, we analyzed three C57BL/6 mouse microbiome models with defined flora, and contrasted with germ-free and standard-pathogen-free C57BL/6 mice. Metagenomics and metabolomics confirmed distinct microbial species present, their relative diversity, and associated changes in physiology and serum metabolites. We find that a minimal microbiome composition restores the majority of metabolites absent in germ-free mice, but community complexity controls metabolite abundance and perturbs metabolite pathways and transcriptional signatures. However, immunophenotyping reveals comparable innate and adaptive immune populations across models. Bone marrow transplant reveals normal homeostatic HSC function, and in vitro assays reveal similar neutrophil innate immune function across models. Thus, defined flo (open full item for complete abstract)

Committee: H. Leighton Grimes Ph.D. (Committee Chair); Daniel Lucas Ph.D. (Committee Member); Jose Cancelas-Perez M.D. Ph.D. (Committee Member); Nathan Salomonis Ph.D. (Committee Chair) Subjects: Immunology

Master of Science (MS), Bowling Green State University, 2021, Forensic Science

An increase of DNA testing sensitivity has led to an elevated variety of sample testing, including complex DNA mixtures, making the interpretation of DNA profiling results more complex. Currently there is no proscribed method used in laboratories to separate complex DNA mixtures by their contributors, therefore a method is needed that could reduce complex mixtures into their component parts. In this study methods of obtaining whole, intact cells from desiccated forensic samples for later cytometric sorting and downstream DNA analysis were examined. This study observed the effects of three different elution buffers (AutoMACS®, Phosphate Buffered Saline, and water) on recovery of whole, intact cells from standard cotton swabs, and then the effect of three different swab types (cotton, flocked, and dissolvable), of different composition, on recovery of whole, intact cells. This was accomplished by washing the swab containing the sample with an elution buffer that maintains the integrity of the cell membrane, resulting in a solution of intact cells. The results of this study demonstrated that the combination of AutoMACS® buffer and flocked swabs provided the highest yield of intact cells post elution. In the future, a solution of whole cells could then be grouped into categories by their cell surface proteins and sorted through cytometric sorting techniques. Once the mixtures have been separated into their component parts by the cell's surface proteins, then DNA analysis could proceed as normal, potentially resulting in single-source samples.

Committee: Crystal Oechsle Ph.D. (Advisor); Travis Worst Ph.D. (Committee Member); Lewis Maddox Ph.D. (Committee Member) Subjects: Biology

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

Master of Science in Biological Sciences, Youngstown State University, 2020, Department of Biological Sciences and Chemistry

C2C12 cells are an immortalized mouse myogenic stem cell line, a model for muscle development. Expression of MyoD, Cdk1, and Titin are good genetic markers for cell cycle, myogenesis and sarcomerogenesis, respectively. This study was conducted to evaluate the effects of different growth media on myogenesis and gene expression during myogenesis. The various serum-based media used are Dulbeccos's Modified media base media supplemented with, either Fetal Bovine Serum (FBS) (10% or 1%) or Horse Serum Media (10% or 1%). In addition to these undefined media (serum) we also looked at defined media, PC-1 defined media (transferrin, insulin like growth factor). The effects of the different medias on C2C12 cell morphology and the gene expression of MyoD and the titin isoforms was determined. Cells were grown over a 12-day time course study with imaging, media change, and pelleting cells for mRNA isolation. Quantitative Real- Time Polymerase Chain Reaction (QRT-PCR) analysis was carried out to measure expression of the specific myogenic genes mentioned above and correlated to changes in morphology. In addition to the time course study a switch-back experiment was done on days 2 and 8 where the cells were changed from their experimental media back into the control 10% FBS. Images and pelleting of cells was done 2 days post-switch-back. The results show changing culture conditions leads to an alteration in cell morphology and genetic expression in C2C12 cells. Cells will proliferate and grow into normal myoblasts however; differentiation does not normally proceed. Myotubes are observed to differentiate but do not do so in the normal fashion or density. In normal differentiation myotubes are observed in long dense sheets of muscle fibers. In the switch-back experiments cells in the cultures did not differentiate normally and were at low densities. When switched back into 10% FBS the density increased and myotube development begins to resemble the controls. To confirm that the cel (open full item for complete abstract)

Committee: Gary Walker PhD (Advisor); David Asch PhD (Committee Member); Xiangjia "Jack" Min PhD (Committee Member) Subjects: Biology

Doctor of Philosophy, Case Western Reserve University, 2020, Pharmacology

Aberrant signal transduction resulting from dysregulated phosphorylation is a hallmark of human cancer. Altered phosphorylation has broad implications on cancer biology. Much work has been done characterizing the effects of individual kinases with their cancer phenotypes. However, the structural complexity of their counterparts, phosphatases, has limited our knowledge of these signaling events. Protein Phosphatase 2A (PP2A), one such negative regulator of multiple oncogenic kinases, has been well characterized as a tumor suppressor protein that when inhibited can lead to cellular transformation. PP2A is a heterotrimeric complex whose substrate specificity is dependent on one of 23 different regulatory subunits that can bind to form over 60 distinct holoenzyme complexes. Although PP2A's function as a general tumor suppressor is well studied, the role of PP2A on specific tumor suppressive signaling pathways and the specific holoenzymes mediating this signaling are not completely understood. Through chemical and genetic approaches, this work characterizes a new role for PP2A in the regulation of DNA replication, and links PP2A effects on replication with its ability to induce apoptosis. 2 Utilizing both a gain of function chemical biology approach and loss of function genetic approaches to modulate PP2A activity, we demonstrate that increasing PP2A activity can interrupt ongoing DNA replication resulting in a collapse of replication forks, the induction of double-stranded DNA (dsDNA) breaks, and a replication stress response that is PP2A dependent. Additionally, we show that increasing PP2A activity during replication causes a dissociation of the replisome, a common mechanism of inhibiting ongoing replication. Furthermore, patients harboring mutations in PP2A are shown to have a higher fraction of their genome altered, suggesting that PP2A regulates ongoing replication as a mechanism for maintaining genomic integrity. Moreover, knockdown of the (open full item for complete abstract)

Committee: Goutham Narla M.D./Ph.D. (Advisor); Derek Taylor Ph.D. (Committee Chair); John Mieyal Ph.D. (Committee Member); Youwei Zhang Ph.D. (Committee Member); Amar Desai Ph.D. (Committee Member) Subjects: Biomedical Research; Cellular Biology; Pharmacology

Doctor of Philosophy, The Ohio State University, 2017, Molecular Genetics

Eukaryotic cells have established elaborate mechanisms to ensure genomic fidelity between generations. Many of these pathways are conserved throughout Eukarya. One such mechanism is the utilization of a bipolar mitosis to separate chromosomes to the two daughter cells. The regulation of the duplication of the single spindle pole body, or centrosome in higher eukaryotes, inherited by each daughter cell after mitosis has been an intriguing question for cell biologists for decades. Additionally, scientific studies aimed at elucidating the mechanism of how the maternal centriole, a component of centrosomes, transitions into a basal body to form a primary cilium used for signaling purposes, has seen a dramatic increase. Methods in microscopy and biochemistry have enabled investigators to increase the knowledge of the molecular players involved in both the duplication of animal centrosomes and primary cilium formation. The present study illustrates centrosomal regulation of one such protein, Mps1 (monopolar spindle 1), that was originally identified as a key regulator of spindle pole body duplication in yeast. The role of Mps1 at mammalian centrosomes has been controversial due to some studies having conflicting data and the potential for mitotic functions at kinetochores confounding centrosomal findings. Using a series of truncation mutants in a combination of fluorescent microscopy and biochemical assays including interaction assays and immunoblotting, this study provides the foundation for a true separation of function for this complexly regulated kinase. Non-overlapping localization motifs target Mps1 to either the centrosome or kinetochore. Localization to the centrosome is critical for Mps1 function in centriole biogenesis, but not ciliogenesis. Mps1 and its targeting partner VDAC3 both play integral roles in regulating the localization and activation of the centrosomal kinase Aurora A to facilitate cilia disassembly. Mps1 and VDAC3 paly similar but separate role (open full item for complete abstract)

Committee: Harold Fisk PhD (Advisor); Stephen Osmani PhD (Committee Member); Kirk Mykytyn PhD (Committee Member); Jian-Qiu Wu PhD (Committee Member) Subjects: Biology; Cellular Biology; Genetics; Molecular Biology

Doctor of Philosophy (PhD), Ohio University, 2014, Mathematics (Arts and Sciences)

Mathematical models are used to study two biological systems: pulmonary innate immunity and autonomous oscillation in yeast. In order to better understand the dynamics of an early infection of the lungs, we construct a predator-prey ODE model of pulmonary innate immunity which describes several observed properties of the pulmonary innate immune system. Under reasonable biological assumptions, the model predicts a single nontrivial equilibrium point with a stable and unstable manifold. Trajectories to one side of the stable manifold are asymptotic to the disease-free equilibrium and on the other side are unbounded in the size of the infection. The model also reproduces a phenomenon observed by Ben-David et al whereby the innate response to an infectious challenge reduces the ability of further infections to take hold. The model may be useful in analyzing and understanding time series data obtained by new methods in pathogen detection in ventilated patients. We also examine several models of autonomous oscillation in yeast (YAO), called the Immediate, Gap, and Mediated models. These models are based on a new concept of Response / Signaling (RS) coupled oscillator models, where feedback signaling and response are phase-dependent. In all three models, clustering of the type seen in YAO is a robust and generic phenomenon. The Gap and Mediated models add a dynamical delay, the latter by modeling a signaling agent present in the culture. For dense populations the Mediated model approximates the Immediate model, but the Mediated model includes dynamical quorum sensing where clustered solutions become stable through density-dependent bifurcations. A partial differential equations model is also examined, and we demonstrate existence and uniqueness of solutions for most parameter values.

Committee: Todd Young (Advisor); Winfried Just (Committee Member); Alexander Neiman (Committee Member); Tatiana Savin (Committee Member) Subjects: Applied Mathematics; Cellular Biology; Immunology

Master of Science (MS), Bowling Green State University, 2024, Biological Sciences

Student success, defined broadly by a student's academic achievement and retention, can be impacted by a number of internal and external factors. One factor is a student's sense of community in the classroom. At BGSU, the sense of community within the classroom was positively impacted by the implementation of a learning assistant (LA) program. Sloan (2020) found that students in LA classrooms had an increased sense of community based on classroom community subscale scores. My research continued to follow student performance and success within LA classes and their correlation with identifiers and scores of classroom community, with a focus on the impact of the corona virus pandemic on the student classroom community. My study considered 4,066 student surveys from introductory STEM courses taught with undergraduate LAs at Bowling Green State University before, during, and after the onset of the corona virus pandemic. Student survey results were paired with sociodemographic data provided by the Bowling Green State University academic affairs office. These data tracked students by race, sex, retention status, and housing in their respective courses The complexity of this data set required a flexible and robust analysis strategy. To this end, I applied principles of disturbance ecology, where in changes in variance over time are considered. This strategy considered multiple factors; here, applied to understand the impact of the coronavirus pandemic on cohort resistance, resilience, and recovery to the pandemic. Changes in the distribution of student scores of classroom community for both subscales between pre-pandemic and post- corona virus pandemic periods were evident. The data indicated an increase in the percentage of failing students in these courses. Sociodemographic differences iii were noted, with specific groups having experienced higher fail rates and more changes in their classroom community scale questionnaires. Specifically, students that reported as whit (open full item for complete abstract)

Committee: Karen Sirum Ph.D (Committee Chair); Julie Matuga Ph.D (Committee Member); Christopher Ward Ph.D (Committee Member) Subjects: Biology; Ecology; Education; Higher Education

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, Pathology

Steroid resistance is a major challenge in the management of autoimmune diseases, including multiple sclerosis (MS). While TH17 cells are widely implicated in steroid-resistant pathology, the underlying mechanism is currently unknown. In this study we found that IL-1R-blockade renders experimental autoimmune encephalomyelitis (EAE) mice highly sensitive to dexamethasone (Dex) therapy. Mechanistically, IL-1b induces a STAT5-mediated steroid-resistant gene program in TH17 cells, promoting inflammatory cytokine production in a steroid-resistant manner and suppressing Dex-induced anti-inflammatory genes. Consistently, TH17-specific deletion of STAT5 ablates the IL-1b-induced steroid-resistant gene program, rendering EAE mice sensitive to Dex treatment. Furthermore, IL-1b synergizes with Dex to promote a STAT5-dependent expression of the tissue residence receptor CD69 in TH17 cells, giving rise to CD69+ TH17 cells that reside in the central nervous system (CNS). Combined treatment with IL-1R blockade and Dex ablates these CNS-resident TH17 cells, reduces endpoint disease severity and prevents disease relapse in a relapsing-remitting EAE model. Importantly, CD69+ tissue-resident TH17 cells are also detected in the brain lesions of MS patients. Collectively, the data identify a novel TH17-intrinsic IL-1b-STAT5 axis that mediates steroid-resistant CNS inflammation, revealing a novel therapeutic strategy for reversing steroid resistance and preventing disease relapse in MS patients.

Committee: George Dubyak (Committee Chair); Xiaoxia Li (Advisor); Clive Hamlin (Committee Member); Stanley Adoro (Committee Member); Serpil Erzurum (Committee Member); Thaddeus Stappenbeck (Advisor) Subjects: Immunology; Pathology

PhD, University of Cincinnati, 2024, Medicine: Pathobiology and Molecular Medicine

Heart disease continues to be the number one killer in the United States accounting for 1 in every 9 deaths. Furthermore, over 6 million people are currently living with heart failure in the US, costing 30 billion dollars each year. In essence, heart disease develops because the cardiac tissue, unlike skeletal muscle or skin, lacks a robust regenerative capacity. Thus, an ischemic injury to the heart almost always results in a permanent loss of cardiac myocytes, inducing a cascade of pathological responses that result in interstitial fibrosis, loss of ventricular compliance, and chamber remodeling. For years, development of new therapies to cure heart failure has primarily focused on the repair and replacement of cardiomyocytes since they constitute most of the heart by volume and are the fundamental contractile cells that generate the force to pump blood to the body. However, more and more studies are now showing the importance of noncardiomyocytes and their interactions with cardiomyocytes in maintaining cardiac homeostasis during health and disease. In fact, recent calculations show that cardiomyocytes account for only 30% of the total cell count in the adult mouse with the majority represented by supportive cell populations such as endothelial cells, hematopoietic-derived cells, fibroblasts, and smooth muscle cells. This new avenue of non-cardiomyocyte biology is at the forefront of the efforts in developing new therapies to treat heart disease which requires extensive research to decipher the relevance and the role of these non-myocyte populations in homeostasis, disease, and injury to the heart.

Committee: Onur Kanisicak PhD (Committee Chair); David Askew Ph.D. (Committee Member); Yi-Gang Wang M.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member); Michael Tranter Ph.D. (Committee Member) Subjects: Cellular Biology

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

The formation of morphological structures of multicellular organisms relies on appropriate cell fate decisions being made. These decisions depend on highly coordinated, tightly regulated signaling network dynamics. Many cell fate decisions rely on the highly conserved EGF signaling pathway. In particular, the EGF pathway is important for the cell fate decisions of the vulval precursor cells (VPCs) in Caenorhabditis nematodes. These cells go on to form the egg-laying structure called the vulva. Reducing EGF signaling pathway activity via small molecules, like the U0126 MEK inhibitor, and mutants, like null mutants of the sur-2 Mediator complex subunit, cause species-specific fates in C. elegans and C. briggsae. In C. elegans, the P12 also uses EGF signaling activity to induce cell fates; this cell becomes part of the hindgut in later development. I hypothesized that knockdown of the EGF pathway results in the loss of cell fates in both C. elegans and C. briggsae P12 with a stronger response in C. elegans, as we see in the VPCs. I found that both U0126 treatment and sur-2 mutation resulted in fewer induced cells in both organs, with a stronger effect in C. elegans lab strains than in C. briggsae lab strains. This effect was compounded in response to combination treatment. Across multiple wild-type strains, C. elegans isolates generally had more loss of cell fates in both the VPCs and P12 compared to the C. briggsae isolates. Finally, I identified several perturbations that were insufficient to induce species-specific differences, either by causing no phenotype or by resulting in organism death. Loss of EGF pathway activity reduces the cell fates taken in both the C. elegans and C. briggsae P12 with a stronger response in C. elegans, mirroring the VPCs. In summary, we can conclude that loss of EGF pathway activity results in species-specific phenotypes of both the VPCs and P12 in C. elegans and C. briggsae; these species-specific cell fate phenotypes are similar across (open full item for complete abstract)

Committee: Adriana Dawes (Advisor); Harald Vaessin (Committee Member); Sharon Amacher (Committee Member); Helen Chamberlin (Committee Member) Subjects: Developmental Biology; Genetics

Doctor of Philosophy, The Ohio State University, 2024, Biomedical Sciences

My interdisciplinary research integrated bioinformatics and immuno-oncology, focusing on leveraging high-dimensional biological data to understand immune cell characteristics in tumor microenvironment (TME). From the biological point of view, my focus has been on the diverse CD8+ T cell landscape of TME, especially the phenomenon of T cell exhaustion, which impairs anti-tumor immunity. My work aims to understand underlying exhaustion mechanisms and to enhance the effectiveness of immunotherapies by elucidating the heterogeneity and regulatory mechanismss of exhausted T cells using single-cell RNA-seq and spatially resolved omics data. Specifically, I validated biological hypotheses by leveraging both in-house and publicly available scRNA-seq and spatially resolved omics data. In the sex bias study, I confirmed the regulatory role of the androgen receptor in T cell exhaustion in bladder cancer. Furthermore, I also corroborated the function of the GARP-TGFβ axis in immune evasion using TCGA's bulk RNA-seq data, which indirectly promoted T cell exhaustion and undermined immunotherapy. Moving from biology to computation, I leveraged general graph representation models to learn and represent patterns of regulatory mechanisms in specific cell types and functional tissue units (FTUs) from scRNA-seq and spatially resolved omics data. I established an R package and web server, IRIS-FGM and IRIS3, to discover the regulatory pattern using scRNA-seq. Regarding spatially resolved omics data, I formulated RESEPT, a deep learning framework, to effectively characterize and visualize histological patterns and gene expression coherence in spatial domains. Furthermore, I developed SpaGFT to provide an unbiased representation method of FTU patterns. After establishing those general methods, I also fine-tuned these computational tools using specific cases and provided novel hypotheses. Refinement of IRIS3 with sex-specific scRNA-seq data led to the identification of unrecognized (open full item for complete abstract)

Committee: Qin Ma (Advisor); Zihai Li (Advisor); Gang Xin (Committee Member); Dongjun Chung (Committee Member) Subjects: Bioinformatics; Biomedical Research; Immunology