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

Throughout their lives, T cells must navigate divergent hemodynamic environments to successfully respond to infection, injury, and stress. From the complex array of fluid dynamics found throughout the bloodstream, to the relatively static, matrix-defined parenchyma of tissues, T cells must effectively perceive and process their extracellular environment to make decisions about motility, growth, differentiation, and effector function. Understanding their environment is accomplished through an array of signaling cascades mediated by specialized proteins that are mechanosensing, including structural components of the cytoskeleton, adhesion molecules, and stretch-activated ion channels. To mount an appropriate immune response, naive T cells must exit from the bloodstream and enter secondary lymphoid organs for antigen surveillance, recognize cognate antigen presented by antigen presenting cells, and effectively proliferate, differentiate, and fine tune its effector function. Effector T cells must then navigate back through the bloodstream to the site of inflammation. Notably, studies of the molecular mechanism of these processes have thus far largely neglected the important contribution of intrinsic mechanical forces. Because each of these processes is regulated, in part, by mechanosensation, the focus of the present work is to assess the role in T cell biology of a stretch-activated ion channel known to be highly expressed in T cells, Piezo1. We first generated and characterized a mouse model with a T cell specific deletion of Piezo1. This model afforded us the opportunity to assess the role of Piezo1 in vivo in all aspects of T cell biology leading us to demonstrate three insights. First, although Piezo1 decreased intrinsic T cell motility, Piezo1 is dispensable in naive T cell homing at steady state and during inflammation. Second, Piezo1 restrains T cell overresponse and upregulation of inhibitory molecules upon TCR activation but is not necessary for succes (open full item for complete abstract)

Master of Science (MS), Ohio University, 2017, Biomedical Engineering (Engineering and Technology)

The role mechanical properties play in the interconnected network of cellular control mechanisms is becoming better understood. Specifically, mechanical stiffness has been shown to be a marker capable of distinguishing between malignant and benign cancer phenotypes. Traditional techniques to measure cell stiffness share the commonality of low throughput. Microfluidic technology has been used to attain stiffness related data at a high throughput, however data collection and analysis is almost exclusively reliant on video spectroscopy. Through the use of a serial multi-constriction microfluidic device, cell ease of transit, i.e., agility, can be measured by the transit through the serial network developed herein. This measure of agility has the capability to differentiate cells based on phenotype, specifically phenotypes characteristic of the epithelial-to-mesenchymal transition, EMT, which occurs in cancer cells upon initiation of metastasis. By developing a compatible microfluidic sensor, the post processing of cell agility data has the potential to be automated and moved toward a non-video spectroscopy dependent system. These improvements push the technology of cellular mechanical property data analysis toward a faster, more convenient platform, thus allowing a better understanding of how mechanical properties correspond with biological behavior of mammalian cells.

Doctor of Philosophy, Case Western Reserve University, 2015, Biomedical Engineering

Existing imaging modalities for tracking cells in vivo have many limitations such as limited resolution or limited field of view. Cryo-imaging is the only imaging technology that enables cell tracking with single cell sensitivity throughout entire animals in small rodents. It provides cell detection anywhere in mice and determines cell densities far below that which can be observed with any other imaging technologies such as MRI, CT, PET, SPECT and BLI. Using the novel imaging and detection technologies, we explored therapeutic mechanisms of mesenchymal stem cell (MSC) in murine models of graft-versus-host disease (GVHD). Many investigators have found that intravascular infusion of exogenous MSCs improves outcomes of GVHD-induced animals. Although, at least 40 clinical trials are in progress, it is still unclear what the therapeutic mechanism is? In this study, the proposed working hypothesis is that the MSCs specifically go to secondary lymphoid organs (SLOs) in order to suppress alloreactive T-cells proliferation which eventually leads to the attenuation of GVHD. In this study, we show that the hypothesis is indeed the mechanism behind the scene and is testable using our proposed imaging technology and experiments. Firstly, we extended/optimized cryo-imaging technology to detect/analyze/visualize of fluorescently labeled cells (MSCs and T-cells). Secondly, we identified bio-distribution of MSCs in a GVHD mouse model. We found evidence showing that MSCs preferentially co-localize with the effector T-cells in the SLOs after intravenous infusion. Lastly, we observed that MSCs could suppress T-cells proliferation in vivo. Novel T-cell proliferation assays were established to study the effectiveness of the MSC therapy. The assays were developed based on SLO volume enlargement approach and CFSE dilution approach. This project is significant as we have developed a new, important technique for the study of stem cell bio-distribution with single cell sensitivity o (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2013, Pathology

Here, we demonstrate that when C3aR and C5aR signals are not transduced into CD4+ T cells, PI-3K¿-AKT-mTOR signaling ceases, PKA activation increases, auto-inductive TGF-ß1 signaling initiates, and CD4+ T cells become Foxp3+ T regulatory cells (iTregs). Endogenous TGF-ß1 suppresses C3aR and C5aR signaling by preventing C3a and C5a production and upregulating C5L2, an alternate C5a receptor. Absent C3aR and C5aR signaling decreases costimulatory molecule and IL-6 production and augments IL-10 production. The resulting iTregs exert robust suppression, possess enhanced stability, and suppress ongoing autoimmune disease. It has been difficult to generate stable human Tregs with potent suppressor activity in vitro with TGF-ß1. We used the insights from this work to generate human iTregs with potent suppressor activity by antagonizing C3aR and C5aR signal transduction during induction. To maintain tolerogenic responses against benign foreign and self antigens, they must be processed and their peptides presented in lymph nodes (LNs) to cognate T cells by immature dendritic cells (DCs). Here we show that maintenance of DCs and CD4+ T cells in an immature state by decay accelerating factor (DAF or CD55) is essential for peripheral Foxp3+ T regulatory cell (iTreg) induction. In the absence of its restraint of C3a/C5a receptor (C3aR/C5aR) signaling, DCs in the anterior chamber (a.c.) of the eye and in Peyer's patch of the gut express costimulatory B7-1,2/CD40 rather than co-inhibitory ICOS-L/PD-L1 and, following their transit to their LNs, evoke a T effector instead of a tolerogenic response. Consistent with this, a.c. pre-injection or oral pre-feeding of ovalbumin (ova) in WT mice and in WT chimeras possessing Daf1–/–C3aR–/–C5aR–/– bone marrow (BM) but not Daf1–/– BM increased ocular TGF-ß1 production, induced iTregs, and suppressed subsequent ova-specific DTH, CD4+, CD8+, and IgG2ab responses. Moreover, DCs from Daf1+/+C3aR–/–C5aR–/– mice expressed higher co-inhibi (open full item for complete abstract)

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)

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.

Doctor of Philosophy, Case Western Reserve University, 2020, EMC - Mechanical Engineering

Cell-cell and cell-protein interactions strongly regulate critical cellular processes such as cell differentiation, cell proliferation, and cell division. To have a better understanding of these interactions, micro-engineered biomimetic platforms providing physiologically relevant environments are needed. Last decades have witnessed remarkable advances in micro/nanotechnologies, through which novel micro-platforms have been developed to mimic physiological microenvironments, providing a means to better understand cellular biomechanics, such as adhesive interaction between different cell types, in vitro. These assays have remarkably enhanced our understanding of many different pathophysiologies that stem from altered cellular mechanical properties as in cancer and sickle cell disease (SCD). The adhesive and deforming characteristics of red blood cells (RBCs) play a critical role in vascular occlusions that lead to life-threatening crises in SCD. The altered properties of sickle hemoglobin (HbS) containing RBCs reduce cellular deformability and increase cellular adhesion onto extracellular matrix (ECM) proteins as well as endothelial cells. This sophisticated dynamic process takes place within a wide range of shear rates indigenous to different types of microvasculature. However, most microfluidic in vitro adhesion assays either do not mimic the shear rate transitioning or they do so through discreet shear rate alterations rather than a continuous transition, which fails to fully recapitulate the flow dynamics in the microvasculature. In Chapter 2, a shear-gradient microfluidic device that creates a variable shear gradient along the flow direction to investigate the adhesion of RBCs under continuously transitioning shear rates is developed and functionalized with endothelium associated biomolecules. With this method, shear gradient dependent adhesion of RBCs from healthy and SCD subjects are quantified, and level of this adhesion is categorized by new parameters such (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2018, Genetics

The ability to culture stem cells in vitro has provided access to a wide variety of cellular states that are inaccessible or too rare to study otherwise. Here we demonstrate the use of stem cells to model two distinct developmental transitions, and examine the role that epigenetic regulation of transcription plays in controlling stem cell fate and function.First, we examine the transition from mouse embryonic stem cells (mESCs), representative of the pre-implantation epiblast, to mouse epiblast stem cells (mEpiSCs), representative of the post-implantation epiblast. These two states, while maintained by distinct signaling pathways, both retain the ability to re-integrate into their respective tissue of origin and contribute to the development of the entire organism. While the transcriptome of the two cell types is largely similar, they rely on dramatically different sets of enhancers to regulate that expression. 97% of the genes that are shared between the two states undergo a change in enhancer usage in the transition. The enhancers that arise in the mEpiSC state are present, but inactive, in the preceding mESC state and become enhancer clusters downstream in development.Second, we examine the development of oligodendrocytes from oligodendrocyte progenitors (OPCs). Oligodendrocytes wrap neural axons in a lipid-rich sheath known as myelin. This myelin is required for proper signal conduction, but is subject to immune attack in multiple sclerosis (MS). OPCs replace damaged oligodendrocytes early in the course of disease, but eventually fail. A chemical genetics screen reveals that BET bromodomain proteins are required for oligodendrocyte development. These proteins are epigenetic readers that integrate chromatin state and other signals to allow transcription elongation at many genes. Other inhibitors of elongation show similar effects on development, and treatment with BET bromodomain inhibitor (S)-JQ1 blocks activation of oligodendrocyte genes. Elongation genes are e (open full item for complete abstract)

Master of Science in Engineering, University of Akron, 2014, Electrical Engineering

An investigation into battery management for lithium-based battery packs was performed. Out of the investigation of the various management/balancing methodologies came a proposed management methodology that is integrated with a charging system and utilizes cost-effective, lossy, bypass resistors for cell balancing. This integration allows the management system to cater the charging current to the needs of the battery pack and overcome the limitations of the lossy bypass on its own. To first investigate this concept, a LiFePO4 cell model was obtained. This was done using a cell discharging procedure and characterization process that provides a mathematical first-principles cell model. The obtained model was then used to simulate various pack configurations, battery management configurations, including the proposed management method. The results from these simulations demonstrated that the proposed management methodology balanced cell voltages within a battery pack in as little as a single charge cycle. To confirm this concept a manually hand-controlled experiment, consisting of voltmeter monitoring cell voltages, manual activation of lossy bypass resistors, and manual adjustments of charging current, was performed. The results from this experiment confirmed the ability to balance the cell voltages within a single cycle. Hardware and software was developed to automate the proposed management methodology. Data collected from the automated implementation was in agreement with the performed simulations and successfully demonstrated a functional automated version of the proposed integrated battery management system and charger.

Doctor of Philosophy, The Ohio State University, 2012, Chemical and Biomolecular Engineering

Magnetic cell separation and related analysis technology continues its maturation with practioners demanding higher system performance using cells with either magnetically labeled or based on the intrinsic magnetic properties of cells. Typical performance metrics include a very high purity and recovery of the targeted cells or a high level of removal of undesired cells while still recovering the majority of desired cells. While this technology is widely used in biological or clinical research laboratories for diagnostic or therapeutic applications, there still exist many engineering challenges. These challenges include unique system designs or cells with various magnetic susceptibilities, and a more fundamental understanding of the magnetic cell separation process. In this dissertation, an instrument referred to as cell tracking velocimetry was used and further perfected as a powerful analytical tool to assist in this continued improvement of magnetic cell separation technology and approaches. For the first time, side by side comparison of two versions of the CTV magnets: permanent verses electromagnet version, on the same targets were compared. The accuracy and sensitivity of the two versions of CTV system was evaluated, and suggestions were made for choice of version for experimental targets with different magnetic susceptibilities. Also, single particle magnetization measurement of micron sized magnetic particles was made possible by the CTV. Three types of commercially available magnetic particles were studied as examples in this study. The average magnetization values from cell tracking velocimetry were found to have good agreements with the reported values from commercially used instruments which usually output only a bulk value. With respect to magnetic cell separation systems, immuno-labeled magnetic cell separation was carried out for the application of T cell depletion for the purpose of a mismatched, bone marrow transplantation. Flow simulations using FL (open full item for complete abstract)

Master of Sciences, Case Western Reserve University, 2008, Genetic Counseling Training Program

This descriptive study assessed 154 pregnant African American women's knowledge of and 11 health care providers' protocols for sickle cell disease carrier screening. The patient survey consisted of questions about sickle cell disease (SCD), incidence, inheritance and knowledge of trait status. Overall, participants were deficient in their knowledge of SCD as evidenced by scoring only approximately 33% on 11 knowledge questions. Factors found to be associated with increased knowledge about SCD included knowing someone with SCD, knowing one's trait status, and completing at least some college. Health care providers' protocols were to report carrier status for sickle cell trait (SCT) to patients who tested positive, but usually not to inform individuals who tested negative for trait. Based on these findings, the role of genetic counselors in educating patients on SCD should be pursued in order to better inform those at highest risk, to decrease unnecessary testing based on patient unawareness of trait status and to assist professionals providing care to this population.

Doctor of Philosophy (PhD), Ohio University, 2013, Chemical Engineering (Engineering and Technology)

During hematogenous metastasis, tumor cells dissociate from a primary tumor, migrate through the tissue space, and enter the circulatory system. The circulating tumor cells (CTCs), once bloodborne, travel to distant sites, where they adhere to the endothelial cells lining the vessel wall, potentially extravasate, and form secondary tumors if directed by niche factors. Determining the mechanisms of each of these steps can provide insights into novel diagnostics and therapeutics for cancer. Of particular interest is elucidating the molecular mechanisms by which metastatic cells adhere to the endothelium while resisting the disruptive shear exerted by the blood flow. We hypothesized that breast cancer cell adhesion is mediated by interaction of endothelial E-selectin with its counter-receptor(s) expressed on breast cancer cells. This hypothesis was tested by using a variety of specialized biochemical techniques and tumor cell/endothelial cell adhesion assays. It was found that breast cancer cells express gangliosides (sialylated lipids), a novel glycoprotein ligand known as Mac-2BP, and CD44 molecules that are functional E-selectin ligands under physiological flow conditions. Further efforts were made to find whether E-selectin ligand activity is related with breast cancer stem-like cells (BCSCs), which are the subset of tumor cells thought to possess properties necessary to maintain and grow tumor mass. For this purpose, breast cancer cell lines which were BCSCs and non-BCSCs were analyzed for E-selectin ligand activity. Interestingly, the non-BCSC cells expressed higher levels of E-selectin ligand activities than that of BCSCs. Epithelial to mesenchymal transition (EMT) is a process by which tumor cells are believed to gain metastatic potential and BCSC properties. The results indicated that E-selectin ligand activity of breast cancer cells may be regulated by EMT. These data suggesting close association of E-selectin ligands with breast cancer metastasis motivated u (open full item for complete abstract)

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.

Master of Science (MS), Wright State University, 2023, Biochemistry and Molecular Biology

Extracellular signal related kinase 3 (ERK3) is one of the atypical mitogen activated protein kinases (MAPK). It is expressed ubiquitously and plays a role in a variety of cellular processes, including cell growth and differentiation. ERK3's role in promoting migration and invasion in various cancers has been well established. ERK3 is upregulated in non-small cell lung cancers (NSCLCs) and has been shown to promote NSCLC tumor growth and progression. However, the regulation of ERK3 in lung cancers remains largely unclear. A recent study indicates that ERK3 phosphorylation at S189, an indicator of ERK3 activity, is upregulated by KRAS in NSCLCs. KRAS is one of the most commonly mutated oncogenes in lung cancers. To study the KRAS dependent regulation of ERK3, knockdown of KRAS was performed and it resulted in a remarkable reduction in ERK3 phosphorylation as well as total ERK3 protein level confirming the regulation of ERK3 by KRAS. Upon knockdown of KRAS a significant reduction of ERK3 mRNA level was observed indicating that KRAS regulates ERK3 at transcriptional level. Further, we found that the regulation of ERK3 by KRAS may be through the transcription factor c-Jun, that is well-known to be activated by KRAS signalling. Our data indicates that c-Jun positively regulates ERK3 transcription in LUAD cell lines. Further, we have found that KRAS upregulates c-Jun activating phosphorylations in LUAD cells, suggesting that KRAS regulates ERK3 through c-Jun. Given the discrepancy regarding the role of ERK3 in NSCLC cell growth reported in previous studies, we have thoroughly investigated the role of ERK3 in cell growth by stable knockdown of ERK3 using shRNA targeting different regions of ERK3 mRNA, as well as by using ERK3 inhibitors in a variety of NSCLS cell lines. While knockdown of ERK3 via targeting the coding region did not affect cell proliferation, targeting the 3'UTR of ERK3 or treatment with ERK3 inhibitors reduced the proliferation of LUAD cells.

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

Advancements in therapy have improved remission and prolonged survival for patients with B cell malignancies. Unfortunately, for many challenging cancer subtypes such as mantle cell lymphoma, most patients will still relapse and die from the cancer. Chimeric antigen receptor (CAR)-T cell therapy uses genetically modified T cells expressing CAR protein to recognize and kill cancers expressing a specific antigen, such as CD19. Although CD19 CAR-T therapy has been very effective against relapsed/refractory B cell cancers, antigen escape and relapse still occur in up to 40% of patients treated with CD19 CAR-T. This work describes the creation and validation of a novel, BAFF ligand-based CAR that aims to prevent antigen escape by being able to bind multiple BAFF receptors expressed by the cancer cell, rather than one antigen alone. Using cytotoxicity assays, we demonstrate the functionality and specificity of BAFF CAR-T cells in killing several types of B cell malignancies, and we further confirm their efficacy in several mouse models of cancer. These results have helped bring BAFF CAR-T therapy to Phase I clinical trials, and we hope they prove efficacious for patients with relapsed/refractory B cell malignancies. Meanwhile, interest has rapidly increased in natural killer (NK) cell-based immunotherapies. NK cells have important roles in cancer immunosurveillance and offer advantages over T cells in clinical safety and time-to-treatment. However, NK therapies face challenges in efficacy and persistence, often due to the presence of TGF-β, a powerful immunosuppressive cytokine that is frequently elevated in cancer patients and a primary cause of NK cell dysfunction. In this work, we characterize an undiscovered role of cyclin dependent kinase 5 (Cdk5) and its coactivator p35 in NK cell cytotoxicity. Using genetic tools to modulate Cdk5/p35 kinase activity in human NK cells, as well as using NK cells from p35 knockout mice, we show that Cdk5/p35 negatively regulates NK (open full item for complete abstract)

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

T cells are a subset of the adaptive immune system that have the capacity to eliminate transforming cells by recognition of minute differences from healthy tissue. Recently, T cell therapies have become an incredible example of scientific progress as human ingenuity is applied to our growing understanding of immunology. In particular, T cells engineered with chimeric antigen receptors (CAR-T cells) have been successful in the treatment of hematological malignancies including B cell leukemias, B cell lymphomas, and multiple myeloma. CARs allow T cells to utilize their anti-tumor functions while bypassing typical T cell constraints such as activation by the innate immune system and human leukocyte antigen dependence. While CAR-T cells are highly efficacious in the treatment of several malignancies, the field is rapidly expanding in their use and application with innumerable strategies remaining to be tested. Here we describe two strategic methods to improve the efficacy and accessibility of CAR-T cell therapy. First, an automated manufacturing method is described that allows in-house generation of clinical grade CAR-T cells from non-Hodgkin's lymphoma (NHL) patients outside a cleanroom environment with a shortened manufacturing time from 12 to 8 days. Second, single-cell RNA sequencing and high dimensional flow cytometry profiling of CAR-T cells derived from the autologous products or blood of patients treated for NHL were used to identify the T cell immunoreceptor with Ig and ITIM domains (TIGIT) as a marker of CAR-T cell dysfunction, particularly in patients with stable or progressive disease. TIGIT antibody blockade was then demonstrated to improve CAR-T cell function with in vitro and in vivo models of NHL. While these strategies were studied in the context of NHL, they may be translated to a variety of disease contexts including solid tumors, as is discussed in this work.

PhD, University of Cincinnati, 2021, Medicine: Molecular and Developmental Biology

Acute kidney injury (AKI) is a rapid renal function decline, with an intensive care unit incidence of up to 67% and merely supportive treatments. This functional deterioration is due to several underlying pathologic processes including pro-fibrotic and pro- inflammatory phenotypic changes and death of the renal tubular cells which are responsible for crucial kidney functions. Furthermore, repetitive insults and maladaptive injury response could lead to the replacement of functional renal parenchyma with fibrotic tissue, which constitutes the pathogenesis of chronic kidney disease (CKD). CKD is a debilitating condition requiring life-time therapeutic maintenance and dialysis support or renal replacement therapy. Thus, there is a need for deeper understanding of the molecular genetics and mechanisms of early AKI since identification of the molecular factors responsible for kidney recovery versus maladaptive injury response could lead to improved preventative treatment. Moreover, examining the long-term renal injury could reveal the molecular and cellular mechanisms of renal fibrotic remodeling, particularly the molecular identity of renal fibroblasts, which would allow for targeted CKD therapy. In recent years, significant progress had been made in kidney injury research. Particularly, single cell RNA sequencing (scRNA-seq) substantially improved our understanding of the genetics and cellular composition of the developing, adult and diseased kidney. However, a thorough atlas of progressive gene expression changes induced by AKI is paramount for understanding of the molecular nature of kidney injury response. In Chapter 2, we present the comprehensive renal cell type specific transcriptional profiles of multiple murine AKI stages created using scRNA-seq and validated with single molecule in situ hybridizations, protein expression and human tissue analyses. We revealed that the injured renal tubules exhibit profound pro-inflammatory and pro-fibrotic phenotype and sig (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2021, Genetics

The pancreatic islet contains multiple endocrine (hormone-producing) cells that play critical roles in glucose homeostasis. Failure of the islet cells, especially the insulin-secreting β cells, is central to the development of diabetes. However, the molecular landscapes of the human pancreatic islet in normal and disease states remain unclear. Here we performed large-scale single-cell RNA-Seq for both adult human islets and stem-cell-derived β-like cells in differentiation. We first analyzed the transcriptome of 39,905 single islet cells from 9 human donors and observed distinct β cell heterogeneity trajectories associated with obesity or type II diabetes (T2D). We therefore developed RePACT, a sensitive single-cell analysis algorithm to identify both common and specific signature genes for obesity and T2D. We mapped both β cell-specific genes and disease signature genes to the insulin regulatory network identified from a genome-wide CRISPR screen. Our integrative analysis discovered the previously unrecognized roles of the cohesin loading complex and the NuA4/Tip60 histone acetyltransferase complex in regulating insulin transcription and release. Second, we generated 95,308 single-cell transcriptomes for the entire differentiation process from hESCs to β-like cells and reconstructed a lineage tree to study temporally 10 regulated genes. We identified so-called `switch genes' at the branch points of endocrine/non-endocrine cell fate choices, revealing insights into the mechanisms of differentiation-promoting reagents, such as NOTCH and ROCKII inhibitors, thus providing the framework for improved differentiation protocols. Over 20% of all detectable genes are activated multiple times during differentiation, even though their enhancer activation is usually unimodal, indicating extensive gene reuse driven by different enhancers. We also identified a stage-specific enhancer in the TCF7L2 diabetes GWAS locus that drives a transient wave of gene expression in pancreatic (open full item for complete abstract)

Doctor of Philosophy (PhD), Ohio University, 2020, Mechanical and Systems Engineering (Engineering and Technology)

In the world of manufacturing, different strategies could be followed to handle the rapidly changing consumer needs and desires in order to remain competitive, and enable their manufacturing systems to respond quickly to new demand and handle the fluctuation in demand. Since the cellular manufacturing system is an important part of the manufacturing system, a new design method, multi-stage cellular manufacturing system design, is proposed in this dissertation. Three performance measures, total number of machines, total machine cost, and %actual risk level, are utilized to evaluate the performance of the proposed design. Considering the uncertainty in the product demand and processing times, two types of the multi-stage cellular manufacturing system are studied. The first type is a deterministic multi-stage cellular manufacturing system. This type of system is propounded to improve the flexibility of the system where the possibility of adding new machines, mini-cells, and stages is existent. Based on the similarity coefficient type used to group the operations into a stage, two design methods are introduced. The first design method is the multi-stage cellular manufacturing system based on the similarity among machines. A new mathematical model is developed to group the machines into stages by maximizing the similarity coefficient among machines. The second design method is the multi-stage cellular manufacturing system based on the similarity among products. A novel heuristic algorithm and mathematical model are proposed to assign machines to stages based on the newly similarity coefficient “cumulative similarity coefficient among products”. In the two design methods, two mini-cell types, regular and flexible flowshop mini-cells, are used in a stage considering the type of products and the possibility to duplicate the machine type. Additionally, the number of stages and product families is un-predetermined and predetermined to minimize the total number of machines. Th (open full item for complete abstract)

Doctor of Philosophy (PhD), Wright State University, 2020, Biomedical Sciences PhD

ΔNp63α is a p53 transcription factor family member that promotes cellular proliferation and survival. In squamous cell carcinoma (SCC), ΔNp63α overexpression is associated with poor prognosis and survival, implicating it as a proto-oncogene. Despite its importance in SCC, the mechanisms regulating ΔNp63α expression are poorly understood. We have identified the acetyltransferase TIP60 as a novel upstream regulator of ΔNp63α levels. We discovered that TIP60 regulates ΔNp63α via two mechanisms. First, TIP60 upregulates ΔNp63α mRNA levels. Moreover, pharmacological inhibition of TIP60 activity reduces ΔNp63α transcript levels, indicating that TIP60-cataltyic activity is vital to amplified transcription of ΔNp63α. Second, we have demonstrated that TIP60 promotes ΔNp63α protein stability by preventing its ubiquitination and proteasomal degradation. This study further shows that TIP60 co-localizes with, interacts with, and directly acetylates ΔNp63α. Utilizing mass spectrometry and site directed mutagenesis, we found that TIP60 acetylates ΔNp63α residues K138, K139, and K494, respectively. We additionally revealed that preventing acetylation of these sites inhibit TIP60-mediated stabilization of ΔNp63α, providing evidence that acetylation by TIP60 enhances ΔNp63α protein stability. We further investigated the functions of the TIP60-ΔNp63α axis in the regulation of SCC proliferation. We discovered that regulation of ΔNp63α by TIP60 increases cellular proliferation. In accordance with the functional role of ΔNp63α, pharmacological inhibition of TIP60 reduced SCC cell proliferation suggesting TIP60 may have therapeutic potential in cancers exhibiting ΔNp63α overexpression. Furthermore, we investigated the mechanisms by which TIP60 regulation of ΔNp63α enhances cancer cell proliferation. We discovered that TIP60 upregulation of ΔNp63α represses p21Cip1/Waf1 levels, resulting in increased G2/M progression. In conclusion, this study uncovers a novel mechanism promoting ΔNp (open full item for complete abstract)