Master of Science (MS), Wright State University, 2021, Pharmacology and Toxicology

Endothelial progenitor cells (EPCs) have been shown to provide beneficial effects on oxidative stress. Exosomes (EXs) released from these stem cells could be one of the major contributors, as they are known to convey the benefit of one cell to another cell via microRNAs (miRNA). At first, we determined that EPCs release more EXs when they are serum-starved for 48 hours., and by determining the microRNA-210 (miR-210) levels in the EXs, we found that miRNA is being transferred from cells to EXs. Meanwhile, miR-210 is gaining popularity in reducing elevated oxidative stress levels. In this study, we investigated the role of endothelial progenitor cells-EXs (EPC-EXs) and the beneficial effects of loading miR-210 into EPC-EXs (miR-210-EPC-EXs) on hypoxia and reoxygenation (H/R) induced reactive oxygen species (ROS) overproduction, apoptosis, and reduced viability of neuronal cells. It was found that EXs were uptaken by neurons and elevated the miR-210 levels in the neurons. In comparison with the vehicle, the EPC-EXs and EXs from scramble transfected EPCs (Scramble-EPC-EXs), were efficient in attenuating neuronal apoptosis, elevated oxidative stress, and restoring cell viability. Whereas the miR-210-EPC-EXs were more efficient in attenuating these inimical effects induced by H/R injury in neurons

Committee: Ji Chen Bihl M.D., Ph.D. (Advisor); Yanfang Chen M.D., Ph.D. (Committee Member); Ravi Sahu Ph.D. (Committee Member) Subjects: Biomedical Research; Medicine; Pharmaceuticals; Pharmacology; Science Education; Toxicology

PhD, University of Cincinnati, 2023, Medicine: Pathobiology and Molecular Medicine

Advances in neonatal medicine have made it possible to improve the survival of extremely preterm infants who are born at the biological edge of viability. Preterm birth and exposure to oxygen, in addition to other compounding factors such as inflammation and infection, result in vascular injury and an arrest in lung development, increasing the risk of developing chronic lung diseases such as bronchopulmonary dysplasia (BPD). Despite gentle assisted ventilation, corticosteroid administration, and exogenous pulmonary surfactant therapy, BPD remains the most frequent complication of extremely preterm birth. The pulmonary microvascular network is formed by two distinct biological processes, vasculogenesis and angiogenesis. Vasculogenesis is defined as the de novo formation of blood vessels via the differentiation of endothelial progenitor cells (EPCs), while angiogenesis describes vascular formation via sprouting from preexisting vessels. Since their first description in 1997, great strides have been made to identify and characterize new pulmonary EPC populations, in an effort to better understand the role that these cells play in lung development, injury, and repair. Recent studies have demonstrated that c-KIT+FOXF1+ EPCs are a subset of CAP1 cells which reside in the pulmonary microvasculature. Single-cell RNA sequencing identified a unique gene signature of this EPC population in both human and mouse newborn lungs which is highly enriched in expression of FOXF1 downstream target genes. Importantly, c-KIT+FOXF1+ EPCs are highly sensitive to elevated levels of oxygen exposure (hyperoxia), as seen in both human and mouse BPD lungs. The negative effects of c-KIT+FOXF1+ EPC loss have been previously described and adoptive transfer of endogenous (lung-derived) c-KIT+FOXF1+ EPCs into the neonatal circulation of hyperoxia-injured mice prevented alveolar simplification and increased angiogenesis. However, limited work has been done to derive c-KIT+FOXF1+ EPCs in vitr (open full item for complete abstract)

Committee: Vladimir Kalinichenko M.D. Ph.D. (Committee Chair); Richard Lang Ph.D. (Committee Member); Kathryn Wikenheiser-Brokamp M.D. (Committee Member); James Wells Ph.D. (Committee Member); Francis McCormack M.D. (Committee Member) Subjects: Biology

MS, University of Cincinnati, 2023, Medicine: Biomedical Research Technology

It has been established that the early presence of Glial cell line-derived neurotrophic factor (GDNF) in the metanephric mesenchyme is essential for the outgrowth of the ureteric bud from the nephric duct. Also, GDNF in the cap mesenchyme promotes branching of the collecting duct throughout kidney development. We have shown that GNDF is expressed by self-renewing nephron progenitor cells (NPCs) at mouse embryonic day 10.5 (E10.5); however, single-cell RNAseq by Magella et al., demonstrated that Gdnf is strongly expressed by stromal progenitor cells at E14.5. To reconcile these seemingly opposed results, we used a transgenic GDNF-CreERT2 and R26R-tdTomato mouse model to conduct lineage tracing experiments activated through tamoxifen injections at different time points during development. We hypothesized that the expression of Gdnf was dynamic, with early expression in NPCs transitioning into stromal progenitors at later stages. Our lineage studies demonstrate that Gdnf is expressed primarily by NPCs at early stages (from E9.5 to E12.5) and primarily by stromal cells during the later stages of development (E13.5 and later). Unexpectedly, we revealed that Gdnf is also expressed by nephric duct (ND) progenitors at even earlier stages. Expression of Gdnf was transient in the ND, and quantification of recombination shows that expression in the ND progenitors starts as early as E7.5 and peaks around E8.5. Altogether, our results indicate that Gdnf is expressed by all kidney progenitor cells (ND progenitors, NPCs, and stromal progenitors) but at different time points during kidney development. The physiological relevance of these transitions remains to be elucidated.

Committee: Cristina Cebrian-Ligero Ph.D. (Committee Chair); Kyle McCracken M.D. Ph.D. (Committee Member); Samantha Brugmann Ph.D. (Committee Member) Subjects: Developmental Biology

PhD, University of Cincinnati, 2023, Medicine: Molecular and Developmental Biology

Respiratory pathologies are central causes of human morbidity and mortality. While few solutions have been developed to address acute lung injury, much less is known about how to prevent chronic disease/dysfunction or promote functional regeneration. Severe acute lung injuries (caused by influenza, bacterial pneumonia, COVID-19, etc.) lead to Acute Respiratory Distress Syndrome (ARDS), which biologically represents a combination of cell injury/death, barrier dysfunction, and airspace edema, resulting in a dysfunctional gas exchange and hypoxia. Patients with similar medical histories and presentations can have disparate outcomes following ARDS, and very little is known about determinants of this heterogeneity. Here, I will describe our efforts to explore this complex question, first looking at molecular mechanisms of progenitor populations of the alveolar epithelium using in vitro models and genetic knockouts, and second, using a perinatal non-human primate injury model to evaluate longitudinal regenerative impacts after early life injury. In Chapter 1, I discuss the history of alveolar epithelial regeneration driven by Alveolar Epithelial Progenitor (AEP) cells, framing the challenges facing the field at the outset of this work. In Chapters 2 and 3, I characterize our refined AEP-derived mouse clonal organoid model, wherein a single AEP expands into organoids that recapitulate in vivo epithelial cell type differentiation. Transcriptional regulatory network (TRN) analysis of time-series scRNAseq and scATACseq data from these organoids identified novel transcriptional regulators of each alveolar epithelial cell state. Using a newly optimized genetic perturbation model of progenitors using AAV6.2FF-Cre, we show novel cell-specific functions of the transcription factor Nkx2-1, a known master regulator in lung development. Loss of Nkx2-1 drives irreversible acquisition of a stressed cell state, a hypothesized initiator of many chronic lung diseases. Finally, we c (open full item for complete abstract)

Committee: William Zacharias M.D. Ph.D. (Committee Chair); Claire Chougnet Ph.D. (Committee Member); Leah Claire Kottyan Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member); Jeffrey Whitsett M.D. (Committee Member); Kathryn Wikenheiser-Brokamp M.D. Ph.D. (Committee Member); Emily Miraldi Ph.D. (Committee Member) Subjects: Developmental Biology

PhD, University of Cincinnati, 2022, Medicine: Molecular and Developmental Biology

Lifelong renal function in mammals relies on the number of nephrons, the filtration units of the kidney, that form during development. Importantly, low nephron endowment is associated with increased risk and earlier onset of chronic kidney disease (CKD) and end stage kidney disease (ESKD). Two salient, incompletely understood features of mammalian kidney development that motivated the experimental work presented herein are 1) the synchronous nature of cessation of nephrogenesis, occurring with the exhaustion of nephron progenitor cells (NPCs) and 2) a striking 10-fold variation in the number of nephrons in kidneys from different individuals. To investigate the first feature, we examined a mouse model exhibiting delayed nephrogenesis cessation timing, from which one allele of the gene Tsc1 (encoding hamartin, a negative regulator of mTOR complex 1 (mTORC1) activity), was removed from Six2-expressing NPCs during development. It is known that nephron progenitor cell self-renewal and differentiation are both reliant on signaling induced by the canonical ligand WNT9b secreted by adjacent ureteric bud tip cells. RNA-sequencing and subsequent genetic validation solidified a differential abundance (reduction) of the Wnt agonist Rspo3 among transcripts poised for translation in older (postnatal day 0) Tsc1+/- NPCs as a mechanistic basis for the nephron gain. To address the question of variation in nephron endowment, we utilized a variety of mouse models from different genetic backgrounds (strains) and mutants which exhibited highly consistent nephron numbers within a given strain, but importantly, approximately 2-fold differences in magnitude between strains. We quantified nephron number in the Collaborative Cross cohort (a set of stable recombinant inbred strains derived from eight distinct parental lines), identifying a region of genome-wide significance on chromosome 13 which will inform future investigations of the genetic contributions to nephron endowment. Furth (open full item for complete abstract)

Committee: Raphael Kopan Ph.D. (Committee Member); Vladimir Kalinichenko M.D. Ph.D. (Committee Member); Matthew Weirauch Ph.D. (Committee Member); Rolf Stottmann Ph.D. (Committee Member); Juan Sanchez-Gurmaches Ph.D. (Committee Member) Subjects: Developmental Biology

Doctor of Philosophy, The Ohio State University, 2020, Neuroscience Graduate Studies Program

Microglia, the innate immune cell of brain, have many important functions during brain development. They engulf and digest living and dying cells, synaptic elements and debris via a process called phagocytosis. They also release a host of diffusible factors that support cell genesis and synapse formation. However, there is very little data on the long-term impact of directly disrupting microglia function on brain development and resulting behavioral outcomes. Developing a better understanding of microglia function(s) during development could lead to new therapies for psychiatric disorders that are targeted at specific microglia functions. This dissertation examines the role of microglia in supporting cell genesis, the long-term impact of disrupting cell genesis, and what behaviors microglia might be programming early in development. Chapter two will focus on whether microglia are important for normal behavioral development. We temporarily depleted microglia in the neonatal period using an agent that is specifically toxic to macrophages, called liposomal clodronate. We found that microglia depletion decreased some social and anxiety behaviors, and increased locomotor activity in juvenile rats. We found that microglia depletion decreased passive social interactions, decreased anxiety, decreased behavioral despair and increased locomotor activity in adult rats. Additionally, we found that acute stress induced glucocorticoid release was blunted in females in adulthood after neonatal microglia loss. Collectively these studies show that microglia are important for normal behavioral development Chapter three will focus on sex differences in microglia morphology and phagocytosis in the hippocampus during development. We show that while there are no sex differences in microglia morphology or number during development, females have more microglia phagocytosing cells in the hippocampus compared to males. The sex difference in phagocytic microglia is not present before the (open full item for complete abstract)

Committee: Kathryn Lenz PhD (Advisor); Jonathan Godbout PhD (Committee Member); Laurence Coutellier PhD (Committee Member); John Sheridan PhD (Committee Member); Tamar Gur PhD (Committee Member) Subjects: Neurosciences

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

Pluripotent stem cells have been widely used to gain insights about embryonic development and study the mechanisms underlying complex diseases, as these cells can be differentiated into a broad range of cells types. In these studies, we describe two instances where the use of pluripotent stem cells enabled us to model early developmental processes. In our first study, we generated mouse embryos and embryonic stem cells (ESCs) to investigate the regulatory mechanisms of Dishevelled (Dvl)-mediated Wnt signaling on early developmental processes, such as axis specification and gastrulation. To overcome the functional redundancy of the Wnt signaling pathways, we deleted the three Dvl genes to completely disrupt canonical Wnt/β-catenin and non-canonical Wnt/planar cell polarity (PCP) signaling in vivo. We discovered that Dvl triple knockout embryos manifest severe defects in mesoderm formation and anterior-posterior axis patterning. We further model the process of gastrulation and germ lineage specification in vitro using Dvl triple knockout blastocyst-derived mouse ESCs and identified major transcriptional dysregulation in axis specification, gastrulation, and mesodermal genes. Furthermore, we assessed the effects of modulating other key developmental pathways in the absence of Dvl-mediated Wnt signaling to determine the mechanisms of germ lineage specification. In our second study, we developed and optimized three methods that are highly efficient in generating a stable and homogenous population of dorsal telencephalic neural progenitor cells (NPCs), which are representative of a subset of neural progenitors in the cerebral cortex. We improve upon currently available protocols for differentiating human induced pluripotent stem cells, human ESCs, and mouse ESCs into dorsal NPCs. These NPCs represent important tools that allow modeling of neurological and neurodegenerative disorders in both mouse and human. Altogether, both studies describe genetic tools and meth (open full item for complete abstract)

Committee: Anthony Wynshaw-Boris (Advisor); Paul Tesar (Committee Chair); Radhika Atit (Committee Member); Peter Scacheri (Committee Member) Subjects: Developmental Biology; Genetics; Neurosciences

Doctor of Philosophy in Engineering, Cleveland State University, 2019, Washkewicz College of Engineering

Development of nervous system has been greatly explored in the framework of genetics, biochemistry and molecular biology. With the growing evidence that mechanobiology plays a crucial role in morphogenesis, current studies are geared towards understanding the role of mechanical cues in nervous system development and progression of neurological disorders. Formation, maturation and differentiation of various development related cells are sensitive to extrinsic and intrinsic perturbations. Based on this hypothesis, the objective of this study was to investigate the effects of environmental toxicants, mutations in molecular clutch proteins, and matrix stiffness cues on the biophysical, biomechanical, and phenotypic changes in brain-derived neural progenitor cells (NPCs) and microglia. In the first aim, we established the utility of biophysical and biomechanical properties of NPCs as indicators of developmental neurotoxicity. Significant compromise (p < 0.001) in NPC mechanical properties was observed with increase in concentration (p < 0.001) and exposure duration (p < 0.001) of four distinct classes of toxic compounds. We propose the utility of mechanical characteristics as a crucial maker of developmental neurotoxicity (mechanotoxicology). In the second aim, we elucidated the critical role of molecular clutch proteins, specifically that of kindlin-3 (K3) in murine brain-derived microglia, on the cell membrane mechanics and physical characteristics. Using genetic knockouts of K3 and AFM analysis, we established the role of K3 in regulating microglia membrane mechanics.Mutation at the K3-β1 integrin binding site revealed that the connection serves as the major contributor of membrane to cortex attachment (MCA). Finally, in aim 3, we identified the molecular mechanisms (non-muscle myosin II) by which NPCs transduce mechanical input from external substrate into fate decisions such as differentiation and phenotype. We established cell mechanics as a label-free marker of d (open full item for complete abstract)

Committee: Chandra Kothapalli (Advisor); Moo-Yeal Lee (Committee Member); Nolan Holland (Committee Member); Xue-Long Sun (Committee Member); Parthasarathy Srinivasan (Committee Member) Subjects: Biomechanics; Biomedical Engineering; Biomedical Research; Biophysics; Engineering; Materials Science; Mechanics; Neurosciences

Doctor of Philosophy, Miami University, 2019, Cell, Molecular and Structural Biology (CMSB)

Destruction of the human neural retina (NR), by either disease or injury, results in irreversible loss of vision. None of the current treatments restore lost retinal tissue. Under specific exogenous stimuli during a specific developmental window, both the embryonic chick and mouse can induce NR reprogramming from the retinal pigmented epithelium (RPE), whereas adult mammals lack the ability to undergo NR regeneration following NR injury. In contrast, following NR detachment, adult newts regenerate a functional NR from the RPE without any exogenous stimuli. Subsequent to NR injury, both the newt and adult human RPE dedifferentiate, and proliferate. However, the hRPE undergoes an epithelial to mesenchymal transition (EMT) that leads to scar formation. The common initial reaction of the RPE upon NR injury in the newt and human RPE suggests that a proper stimulus could reprogram hRPE-NR. Identifying the exogenous stimuli (small molecules/growth factors) to induce the hRPE-NR reprogramming represents this dissertation's goal. Here, we exploited genetic engineering of human induced pluripotent stem cells (hiPSCs) to design fluorescence-based reporters to identify compounds that induce RPE-NR progenitor (RPE-NRP) reprogramming. Specifically, we generated a single hiPSC transgenic line to report real-time expression of VSX2, a gene expressed by NRPs, via cyan fluorescent protein expression. We used RPE differentiated from our transgenic hiPSC line in a pilot experiment, where we identified a combination of 4 factors that induced hRPE-NRP reprogramming in this line and in three other hiPSC-derived RPE lines. Furthermore, we created an hiPSC (PGP1) that reports real-time expression of VSX2, BRN3b (a ganglion marker), and RCVRN (a photoreceptor marker) via Cerulean, eGFP, and mCherry expression, respectively. PGP1-derived RPE (PGP1.RPE) was used in high-throughput screening of two small molecule libraries. In the developmental library, we identified three compounds tha (open full item for complete abstract)

Committee: Michael Robinson L (Advisor); Katia Del Rio-Tsonis (Committee Chair); Chun Liang (Committee Member); Lori Isaacson (Committee Member); Justin Saul (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2019, Biological Sciences

Reports have described cells with stem-like protein expression in the hypothalamic suprachiasmatic nucleus (SCN), which contains the principal circadian pacemaker of the body. Additionally, there are oligodendrocyte progenitor cells (OPCs) scattered throughout the SCN and other brain areas with reported abilities to differentiate into neurons and glia. The SCN is a particularly good structure for studying adult neurogenesis because its cellular manipulation has known quantifiable effects on specific parameters of circadian rhythms. The objectives of this study were to characterize stem and progenitor cells in the SCN and to study neurogenesis from SCN OPCs in vitro. We first performed a meta-analysis to identify the expression of stem cell-related genes in the SCN and then used defined serum-free media for inducing stem and progenitor cell proliferation in SCN explant cultures, identified by immunocytochemistry and confocal microscopy. In the meta-analysis, we analyzed 25 genes associated with stem cell maintenance and increased motility, out of which over 90% were expressed at higher levels in the SCN than in other brain areas. In explant cultures maintained in stem and progenitor cell medium (SPM), cells expressed stem cell proteins: SOX2, nestin, MSI2 and OCT4. Explant cultures had ongoing mitotic activity and extensive cell loss. Despite neuronal loss, tissue remained viable for over 7 weeks in culture, as shown by bioluminescence imaging. The circadian rhythm in SCN gene expression persisted in brain slice cultures in SPM. SCN explants maintained in NeuralX medium supporting OPC proliferation, formed a cell monolayer and a suspended cell culture that included 87% OPCs. These cells were then induced to differentiate into neurons, which were identified by immunocytochemistry and electrical impulses recorded with microelectrode arrays. In differentiating cultures, a subset of OPCs formed oligodendrocytes that myelinated nascent neurons. These results provide evid (open full item for complete abstract)

Committee: Michael Geusz PhD (Advisor); George Bullerjahn PhD (Committee Member); Howard Cromwell PhD (Committee Member); Paul Morris PhD (Committee Member); Pascal Bizarro PhD (Other) Subjects: Biology; Biomedical Research; Cellular Biology; Neurosciences

Master of Science (MS), Wright State University, 2019, Pharmacology and Toxicology

In this study, we tested the protective effects of EPC-EXs (endothelial progenitor cell derived exosomes) and miR-126 EPC-EXs (microRNA-126 EPC-EXs) on the astrocytes injured by HG (high glucose) plus H/R (hypoxia/reoxygenation) model. At first, we determined the concentration and time dependent uptake of EPC-EXs by astrocytes. It was also found that the EPC-EXs were uptaken via macropinocytosis, caveolin-dependent and clathrin-mediated pathways in astrocytes. Furthermore, the astrocyte cell line was injured through the HG + H/R model. EXs, isolated by ultracentrifugation from the EPC culture supernatant were co-incubated with the injured cells. It was found that EPC-EXs and miR- 126 EPC-EXs decrease apoptosis, lipid peroxidation, oxidative stress and cytotoxicity in the injured cells, hence protecting them.

Committee: Ji C. Bihl M.D., Ph.D. (Advisor); Adrian M. Corbett Ph.D. (Committee Member); Yanfang Chen M.D., Ph.D. (Committee Member) Subjects: Pharmacology

Master of Arts, The Ohio State University, 2019, Psychology

In the adult mammalian brain, there are two regions where neural stem and progenitor cells reside and proliferate throughout life: the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. While much of the current research focuses on these cells' ability to create new neurons, a process known as neurogenesis, new findings indicate that neural stem and progenitor cells (NSPCs) may influence their niches through the secretion of growth factors. Our previous work indicates that NSPCs express 1/3 of the vascular endothelial growth factor (VEGF) in the DG. While global VEGF has been shown to support the proliferation and maturation of adult-born DG neurons, the role of NSPC-derived VEGF is not entirely understood. Our data suggest that VEGF plays a role in regulating NSPC stemness in the DG. Currently, we aim to investigate the role of a VEGF/VEGFR2 intracellular autocrine (i.e. intracrine) signaling pathway in regulating NSPC stemness and maintenance. This thesis contributes to our ongoing work by investigating the immediate effects of VEGF knockdown on NSPC stemness in vitro and modeling VEGF knockdown to determine the NSPC-derived VEGF signaling pathway in vivo. My results suggest NSPC-derived VEGF knockdown increases NSPC proliferation, which is indicative of impaired stemness, in vitro. To investigate VEGF intracrine signaling in vivo we utilized a transgenic mouse line of inducible NSPC-derived VEGF knockdown was accompanied by EYFP reporter expression. I investigated the possible limitations of this commonly used genetic model and discovered recombination induced expression of one fluorescent reported does not accurately predict recombination of another gene at a single cell level. These data indicate that we may not accurately identify NSPC-derived VEGF intracrine signaling using our mouse model.

Committee: Elizabeth Kirby Ph.D. (Advisor); Kathryn Lenz Ph.D. (Committee Member); Jonathan Godbout Ph.D. (Committee Member) Subjects: Psychology

PhD, University of Cincinnati, 2018, Medicine: Cancer and Cell Biology

Background: While c-Kit+ adult progenitor cells were initially reported to produce new cardiomyocytes in the heart, recent genetic evidence suggests that such events are exceedingly rare. However, to determine if these rare events represent true de novo cardiomyocyte formation we deleted the necessary cardiogenic transcription factors Gata4 and Gata6 from c-Kit-expressing cardiac progenitor cells (CPCs). Methods: Kit allele-dependent lineage tracing and fusion analysis was performed in mice following simultaneous Gata4 and Gata6 cell-type specific deletion to examine rates of putative de novo cardiomyocyte formation from c-Kit+ cells. Bone marrow transplantation experiments were used to define the contribution of Kit allele-derived hematopoietic cells versus Kit lineage-dependent cells endogenous to the heart in contributing to apparent de novo lineage-traced cardiomyocytes. A Tie2-CreERT2 transgene was also used to examine the global impact of Gata4 deletion on the mature cardiac endothelial cell network, which was further evaluated with select angiogenesis assays. Results: Deletion of Gata4 in Kit lineage-derived endothelial cells or in total endothelial cells using the Tie2-CreERT2 transgene, but not from bone morrow cells, resulted in profound endothelial cell expansion, defective endothelial cell differentiation, leukocyte infiltration into the heart and a dramatic increase in Kit allele-dependent lineage-traced cardiomyocytes. However, this increase in labeled cardiomyocytes was an artifact of greater leukocyte-cardiomyocyte cellular fusion due to defective endothelial cell differentiation in the absence of Gata4. Conclusions: Past identification of presumed de novo cardiomyocyte formation in the heart from c-Kit+ cells using Kit allele lineage tracing appears to be an artifact of labeled leukocyte fusion with cardiomyocytes. Deletion of Gata4 from c-Kit+ endothelial progenitor cells or adult endothelial cells negatively impacted angiogenesis and ca (open full item for complete abstract)

Committee: Jeff Molkentin Ph.D. (Committee Chair); Burns Blaxall Ph.D. (Committee Member); Rafeeq Habeebahmed Ph.D. (Committee Member); Susan Waltz Ph.D. (Committee Member); Kathryn Wikenheiser-Brokamp M.D. Ph.D. (Committee Member) Subjects: Molecular Biology

Master of Sciences (Engineering), Case Western Reserve University, 2018, Biomedical Engineering

Peripheral nerve injuries have traditionally been treated with a variety of different surgical procedures, but the use of allografts for these injuries remain to be a largely unexplored concept. As a result, there has yet to be a successful protocol created for imaging cells on the surface of nerve allografts. In this thesis, I developed a z-stack projection imaging protocol for nerve allografts and demonstrated the ability to take clear and focused images of cell retention and proliferation on the surface of the allograft. I also optimized the preparation of the nerve graft to improve cell and connective progenitor cell (CTP) retention rates.

Committee: George Muschler MD (Committee Chair); Eben Alsberg PhD (Advisor); Robert Kirsch PhD (Committee Member); Cynthia Boehm (Committee Member) Subjects: Biomedical Engineering; Cellular Biology; Optics

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)

Committee: Paul Tesar PhD (Advisor); Peter Scacheri PhD (Committee Chair); Helen Salz PhD (Committee Member); Ahmad Khalil PhD (Committee Member) Subjects: Bioinformatics; Biology; Developmental Biology; Genetics

PHD, Kent State University, 2016, College of Arts and Sciences / School of Biomedical Sciences

Stem and progenitor cells are a key component of regenerative medicine with the potential to fully heal damaged tissues and organs, offering ultimate solutions for people who live with long-suffering conditions; such as diabetes, heart failure, and degenerative nerve, bone, and joint pathologies, that today are beyond repair. All tissue formation, regeneration or repair requires a population of cells (i.e. stem cells or progenitor cells) that can be activated to proliferate and/or differentiate, generating progeny that will contribute to the formation of new tissue. Hierarchical populations of stem and progenitor cells have been defined, with varying degrees of precision, in many tissues. Understanding the kinetics of tissue remodeling and responses to disease is increasingly important for the development of therapies in settings where the stem/progenitor population has become deficient or dysfunctional. Understanding of these systems is also essential for the rational optimization of cell sourcing and processing strategies for cell therapy applications. Regeneration and repair of tissues depend on stem and progenitor cell populations that are resident in tissues. The choice of the source of cells for tissue engineering or cell therapy applications depends mainly on factors such as the ease of harvest, low morbidity, consistency (with respect to the yield and biological potential). Several cell populations from various tissues have been isolated and characterized. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) have been considered as a source of multipotent cells and can be derived from various tissue sources. However, controversial findings, with respect to the best tissue source for isolation and utilization in regenerative applications, have been presented 9. Therefore, an alternative source of cell populations with better proliferation potential and defined marker profile represents a promising tool in clinical applications. Connective t (open full item for complete abstract)

Committee: George Muschler M.D. (Advisor); Gary Koski Ph.D. (Committee Member); Christopher Malcuit Ph.D. (Committee Member); Ronald Midura Ph.D. (Committee Member); Vincent Hascall Ph.D. (Committee Member); Douglas Kline Ph.D. (Other) Subjects: Biomedical Research; Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2016, Neuroscience Graduate Studies Program

Myelin accelerates action potential conduction velocity and provides essential metabolic support for axons. Unfortunately, myelin and myelinating cells are often vulnerable to injury or disease, resulting in myelin damage, which in turn can lead to axon dysfunction, overt pathology and neurological impairment. Inflammation is a common component of CNS trauma and disease, and therefore an active inflammatory response is often considered deleterious to myelin health. While inflammation can certainly damage myelin, inflammatory processes also benefit oligodendrocyte (OL) lineage progression and myelin repair. Consistent with the divergent nature of inflammation, intraspinal toll-like receptor 4 (TLR4) activation, an innate immune pathway, kills OL lineage cells, but also initiates oligodendrogenesis. Soluble factors produced by TLR4-activated microglia can reproduce these effects in vitro, however the exact factors are unknown. To determine what microglial factors might contribute to TLR4-induced OL loss and oligodendrogenesis, mRNA of factors known to affect OL lineage cells was quantified in TLR4-activated microglia and spinal cords (chapter 2). Results indicate that TLR4-activated microglia transcribe numerous factors that induce OL loss, OL progenitor cell (OPC) proliferation and OPC differentiation. However, some factors upregulated after intraspinal TLR4 activation were not upregulated by microglia, suggesting that other cell types contribute to transcriptional changes in vivo. Many factors produced by TLR4-activated microglia have no known effects on OL lineage cells. In chapter 3, colony stimulating factor 3 (CSF3) and interleukin 7 (IL-7) were identified as factors produced by TLR4-activated microglia that might contribute to OPC proliferation or differentiation. Indeed, CSF3 injection into the uninjured spinal cord promoted OPC proliferation. Conversely, IL-7 injection into the uninjured spinal cord promoted OPC differentiation. Although it is unclear if t (open full item for complete abstract)

Committee: Dana McTigue (Advisor); Phillip Popovich (Committee Member); DeVries Courtney (Committee Member); Godbout Jonathan (Committee Member) Subjects: Biology; Biomedical Research; Immunology; Medicine; Molecular Biology; Neurobiology; Neurosciences

Doctor of Philosophy, The Ohio State University, 2016, Neuroscience Graduate Studies Program

Traumatic spinal cord injury (SCI) disrupts not only the central nervous system, but also peripheral homeostatic processes. Macrophages are immune cells that accumulate in the SCI lesion and peripheral organs after injury and respond to a variety of endogenous ligands, including damage-associated molecular patterns (DAMPs) generated by trauma to the spinal cord. Ligands for the innate immune receptor toll-like receptor 4 (TLR4) expressed on macrophages are present in the intraspinal lesion and in peripheral organs after injury. DAMPs such as HMGB1 and heat-shock proteins can bind TLR4, while bacteria originating in the gut can translocate and activate peripheral TLR4. TLR4 signaling regulates key macrophage functions that influence outcome after SCI including iron regulation, phagocytosis, and expression of cytokines. Thus it is likely that manipulating this single receptor will have a profound effect on intraspinal and peripheral responses after SCI. After SCI, DAMPs initiate secondary injury processes including apoptosis and inflammation. Acutely, oligodendrocytes (OLs) and their progenitor cells (OPCs) are lost, followed by robust OPC proliferation, formation of new OLs, and remyelination of bare axons. Previously, our group showed that macrophage TLR4 signaling promoted beneficial OL lineage cell responses in the intact spinal cord and mice deficient for TLR4 signaling (TLR4d) displayed reduced white matter sparing after SCI. Here, we determined that reduced white matter sparing in TLR4d mice was due to reduced OL lineage survival and replacement (Chapter 2). These differences in OL survival and replacement were accompanied by alterations in macrophage iron regulation and myelin debris phagocytosis, cytokine and growth factor expression, and axon survival, processes which all directly impact OL and OPC responses. We next asked whether exogenous intraspinal TLR4 activation would enhance oligogenesis and remyelination in a lysolecithin demyelination model (Chap (open full item for complete abstract)

Committee: Dana McTigue PhD (Advisor); Phillip Popovich PhD (Committee Member); Jonathan Godbout PhD (Committee Member); Jessica Lerch PhD (Committee Member) Subjects: Immunology; Neurobiology; Neurosciences

Doctor of Philosophy, The Ohio State University, 2015, Molecular, Cellular and Developmental Biology

Sight-threatening diseases of the eye are prevalent across the world and result in a progressive loss of visual acuity that often culminates in blindness. These ocular diseases are caused by the degeneration and death of neurons in the retina, the neurosensory tissue of the eye. A promising possible treatment for retinal diseases is to stimulate neuronal regeneration from the Muller glia. Muller glia regenerate the retina in cold blooded vertebrates, but fail to do so mammalian retinas. To achieve retinal regeneration, Muller glia must de-differentiate into Muller glia-derived progenitor cells (MGPCs), proliferate, differentiate into neurons, and functionally integrate into neuronal circuits. Identification of the signaling pathways that influence the reprogramming of Muller glia into MGPCs is key to harnessing the potential of these cells to regenerate the retina. This dissertation focuses on understanding the role Glucocorticoid Receptor (GCR)- and Wnt-signaling pathways, in regulating proliferative, regenerative and neuroprotective properties of Muller glia in the avian retina in vivo. In the first part of this dissertation, we analyze the impact that the GCR- signaling pathway has on the MGPCs in the presence and absence of damage. The primary amino acid sequence of GCR and its expression by Muller glia is highly conserved across vertebrate species, including chickens, mice, guinea pigs, dogs and humans. We find that, in damaged retinas, activation of GCR-signaling suppresses the formation MGPCs via inhibition of MAPK-signaling, and inhibition of GCR-signaling stimulates the formation of proliferating MGPCs. In undamaged retina, FGF2/MAPK-signaling stimulates the formation of MGPCs, and activation of GCR-signaling reduces the number of proliferating MGPCs in FGF2 treated retinas. We also find that inhibition of the GCR-signaling enhances the neuronal differentiation of MGPC-derived cells in damaged retina. The second part of this dissertation describes th (open full item for complete abstract)

Committee: Andy Fischer (Advisor) Subjects: Biology; Cellular Biology; Developmental Biology; Molecular Biology; Neurosciences

PhD, University of Cincinnati, 2014, Engineering and Applied Science: Biomedical Engineering

Tendon and ligament injuries often interfere with normal activities of daily living. As current surgical techniques are not uniformly successful, tissue engineers seek to develop tissue engineered constructs to accelerate and improve musculoskeletal soft tissue healing. Our Functional Tissue Engineering Lab has decades of experience in using adult progenitor cell-based therapies to improve the mechanics of repair tissue. However, even the best repairs to date do not achieve minimum mechanical design limits of matching tendon tangent stiffness beyond peak in vivo loads. As tendons are living tissues, composed of cells and extracellular matrix, we propose that advances in the design of tissue engineered constructs require establishing biological design criteria. In an effort to identify possible biological design parameters, our lab collaborates with developmental biologists to understand normal tendon formation.

Committee: David Butler Ph.D. (Committee Chair); Balakrishna Haridas Ph.D. (Committee Member); Marepalli Rao Ph.D. (Committee Member); Jason Shearn Ph.D. (Committee Member) Subjects: Biomedical Research