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

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, 2013, Biomedical Engineering

Current stem cell and tissue engineering R&D aim to initiate and guide de novo tissue generation for the repair or replacement of native tissue compromised due to injury, disease, or other disorders. Biophysical cues present a safe and controllable means to modulate cell behavior and emergent cell fate. Scaling up from cellular to systems length scales, there is great future potential for prescriptive physical therapies that synergistically promote healing in conjunction with approaches including stem cell based regenerative tissue engineering and next generation implants cum delivery devices. This dissertation addresses key obstacles for biophysically-enhanced de novo tissue generation by (i) examining the potential of the femoral neck periosteum in discarded tissue from arthritic patients undergoing hip replacement surgery as a feasible source of adult stem cells for tissue engineering, (ii) unraveling the emergent spatial and temporal mechanoadaptive structure – function relationships of stem cells after exposure to volume and shape changing stresses, (iii) and implementing a coupled computational fluid dynamics and finite element method model to define future experiments to test the ability of mechanical cues to guide stem cell fate during healing. Addressing the first aim, an IRB approved study highlighted the potential of femoral neck periosteum as a new source for stem cell acquisition and banking of autologous multipotent cells for middle aged to aging individuals. The second aim, to unravel emergent anisotropy in model embryonic mesenchymal stem cell structure and nascent lineage commitment, showed that the actin and tubulin cytoskeleton exhibit emergent anisotropy in the apical-basal direction and that these changes correlate to regulation of transcription factors indicative of fate. Finally, the third aim addresses a parametric approach using a CFD-FEM model to predict the local normal and shear stresses at cell-fluid interfaces in a tissue template with (open full item for complete abstract)

Committee: Melissa Knothe Tate (Advisor); Edward Greenfield (Committee Member); Daniela Calvetti (Committee Member); Eben Alsberg (Committee Member) Subjects: Biomechanics; Biomedical Engineering; Biomedical Research; Cellular Biology; Developmental Biology; Engineering; Mechanical Engineering; Medicine; Molecular Biology; Scientific Imaging

Doctor of Philosophy, Case Western Reserve University, 2023, Molecular Medicine

Autism Spectrum Disorders (ASDs) are a group of neurodevelopmental disorders for which there is no direct treatment. Understanding the disease mechanisms may lead to future therapeutic targets. In this thesis, we performed bi-directional CRISPR-Cas9 genome editing on induced pluripotent stem cells (iPSCs) derived from individuals with ASD with macrocephaly as well as controls, and uncovered that in addition to PTEN p.Ile135Leu variant, ASD genetic backgrounds also contributed to dysregulating cortical neurogenesis in both 2D neural progenitor cells and 3D cortical organoid models. Surprisingly, ASD specifc PTEN p.Ile135Leu variant dysregulates cortical neurogenesis in an ASD genetic background dependent fashion, as we found that this variant led to overproduction of neural progenitor cell (NPC) subtypes including intermediate progenitor cells (IPCs) and outer radial glia cells (oRGs) as well as neuronal subtypes such as deep and upper layer neurons in the ASD genetic background but not in the control genetic background in the cortical organoids. This study provides strong evidence that both an ASD-specifc PTEN p.Ile135Leu variant and autism genetic background are contributing to the cortical neurogenesis dysregulation. We also developed a lineage tracing system to track neurogenesis in human cortical organoids and applied this system to a control iPSC line as well as autistic isogenic CTNNB1 iPSC lines, we uncovered that the majority of the cortical neurons in the cortical organoids were indirectly generated through IPCs, and neurons derived from diferent lineages were transcriptionally distinct. An ASD-linked CTNNB1 p.Gln76* variant altered the lineage specific production of deep and upper layer neurons as well as the landscape of gene expression profiles among and within different neural progenitor lineages of both deep and upper layer neuron production. Overall, this thesis provides direct evidence that variants in the PTEN and WNT pathways as well as ASD (open full item for complete abstract)

Committee: Anthony Wynshaw-Boris M.D., Ph.D. (Advisor); Justin Lathia Ph.D. (Committee Chair); Tara DeSilva Ph.D. (Committee Member); Fulai Jin Ph.D. (Committee Member); Charis Eng M.D., Ph.D. (Committee Member) Subjects: Genetics; Neurosciences

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

PHD, Kent State University, 2012, College of Arts and Sciences / School of Biomedical Sciences

This dissertation is divided into two Chapters: Chapter I focuses on the requirement of maternal Smad4 in mouse preimplantation development; Chapter II focuses on the function of zygotic Smad4 in trophoblast lineage development. Chapter I: Maternally contributed transcripts and proteins are crucial for shaping the earliest developmental programs. Smad4 is a central mediator of the Transforming Growth Factor beta (TGF-β) superfamily, molecules of which are important regulators of development and tissue homeostasis. We found that Smad4 gene products are abundant in unfertilized eggs as well as cleaving blastomeres of mouse preimplantation embryos. To investigate the role of maternal Smad4 during mouse preimplantation development, we have conditionally inactivated Smad4 in mouse female germline. Here we report that Smad4 is a new member of mouse maternal effect genes that lacking of maternal Smad4 results in peri-implantation lethality severe preimplantation development defects in mitosis, polarization, and first lineage segregation between trophectoderm (TE) and inner cell mass (ICM). Not only have our studies uncovered novel functions of Smad4-dependent signaling in preimplantation development, but also have provided important insights into how maternal effect genes influence the earliest development programs in mammals. Chapter II: Trophoblast lineage gives rise to embryonic portion of the placenta, the function of which is essential for the establishment and maintenance of pregnancy. Mouse trophoblast stem (TS) cells established from the outgrowth of the polar trophectoderm of blastocysts or the extraembryonic ectoderm of post-implantation embryos are the precursors of trophoblasts and can contribute to all trophoblast lineage derivatives in vivo, providing a powerful in vitro system for the study of trophoblast stem cell self-renewal and differentiation. Although it is known that TGF-β/Nodal-related signaling together with FGF4 signaling is critical for TS self- (open full item for complete abstract)

Committee: Yijing Chen (Advisor) Subjects: Developmental Biology

Doctor of Philosophy, Case Western Reserve University, 2009, Biology

Our concept of adipose tissue has evolved a great deal over the past century. Initially thought only to provide insulation and mechanical support, the definition of adipose tissue was later revised to include a role in systemic metabolism. Genetic approaches in recent years have revealed its endocrine function, as well as the inter-depot, molecular-level diversity within what is now known as the adipose organ. Because much of this research was driven by the global spread of obesity, medullary adipose tissue, the ostensibly isolated depot within bone marrow, has received comparatively little attention. It is widely hypothesized and commonly accepted that medullary adipocytes derive from multipotent progenitor cells of the marrow, such as human mesenchymal stem cells (hMSCs); however, molecular-level tracking of this development is lacking. In order to conduct a comparative study between the two cell populations, methods were adapted both for hMSC adipogenesis and for enrichment of medullary adipocytes from whole marrow aspirate. Using PCR, a high degree of similarity was determined to exist between hMSC-derived adipocytes and medullary adipocytes, as shown through transcription factor upregulation and the expression of lipid metabolism-related genes, adipokines, and other bioactive macromolecules. Furthermore, there was no detection in either population of UCP-1, demonstrating that neither population developed characteristics of, nor a substantial subpopulation of, brown adipocytes. Having supported the hypothesis that medullary adipocytes can derive from hMSCs, this culture system was then used as an in vitro model for the development of mature adipocytes from stem cells. With this tool, we investigated temporal protein expression by monitoring the occurrence and localization of PPARγ and C/EBPα, the major players in transcription, as well as two well-characterized lipid droplet-associated proteins, adipophilin and perilipin. We finally approached the subject of fun (open full item for complete abstract)

Committee: Stephen Haynesworth PhD (Advisor); Arnold Caplan PhD (Committee Member); Radhika Atit PhD (Committee Member); Mary Laughlin MD (Committee Member); Jennifer Liang PhD (Committee Member) Subjects: Biology; Cellular Biology

Doctor of Philosophy, Case Western Reserve University, 1990, Biomedical Engineering

Studies of the origin, commitment, and differentiation of osteogenic cells can be facilitated by cell-specific markers. To this end, monoclonal antibodies against surface determinants on osteogenic cells were generated by immunizing naive mice with a heterogeneous population of chick embryonic bone cells. Four monoclonal cell lines, SB-1, SB-2, SB-3, and SB-5, which each secrete a different antibody against the surface of osteogenic cells, were immortalized and subsequently used to study the developmental progression of osteogenic cells in a variety of developmental systems. Detailed morphologic analyses during the process of bone formation in embryonic chick tibiae, organ culture of embryonic chick periosteum, and in vivo diffusion chamber culture of avian marrow stem cells reveal that a precise sequence of cell surface alterations occur during osteogenic differentiation. Of particular interest is the observation that this sequence of cellular events is conserved in all the developmental systems studied. Results of these analyses indicate the Pre-Osteoblastic cells, which are recognized by antibody SB-1, proceed through their lineage to become Transitory 1 Osteoblasts, which are immunostained by SB-1 and SB-3. These cells continue differentiating to become Transitory 2 Osteoblasts, which are reactive wit h antibodies SB-1, SB-3, and SB-2. These non-secretory cells then elaborate a type I collagen-rich osteoid matrix as SB-1, SB-3, and SB-2-positive Secretory Osteoblasts. Those Secretory Osteoblasts which are incorporated into the bone matrix then loose surface antigens SB-1 and SB-3, while they begin expressing the SB-5 antigen on their surface. The resulting Osteocytic Osteoblasts, which are reactive with both the SB-2 and SB-5 antibodies, finally undergo a terminal differentiation step to become Osteocytes. This final lineage step is characterized by the loss of the SB-2 antigen and the retention of SB-5 immunoreactivity. Additional studies have focused on charac (open full item for complete abstract)

Committee: Arnold Caplan (Advisor) Subjects: