Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2015, College of Sciences and Health Professions

Cell movement is essential for the development and maintenance of complex multicellular organism. Excessive migration contributes to cancer metastasis and autoimmune diseases, whereas reduced migration can cause developmental defects and immunodeficiencies. In many biological process cells migrate in coordinate groups called “collectives”. Moreover, Collective cell migration is a now well established mode of metastasis in certain carcinomas. Despite the broad impact on normal development and disease, the molecular mechanisms that facilitate collective migration in vivo are still not well understood. We use border cell migration during Drosophila oogenesis as a genetically tractable model to study collective cell migration in the native three-dimensional environment of the tissue. During oogenesis, a pair of specialized cells, the polar cells, signal to recruit 6-8 cells follicular epithelial cells to form the border cell cluster. The cluster is composed of migratory border cells and the non-migratory pair of polar cells. The cluster then detaches from the follicular epithelium and migrates as a cohesive cluster from the anterior to posterior end of the egg chamber. The mechanism that controls detachment and migration as cohesive group are still poorly understood. Presence of cell – cell adhesion is a defining characteristic of collectively migrating cells, including border cells. The regulation of E-cadherin, cell adhesion molecule, is complex in migrating border cells, because E-cadherin forms differential but robust adhesions during migration. In this thesis, I investigated role of two proteins PDZ-GEF and its target Rap1 GTPase in collective border cell migration. We discovered that both of these proteins were required for collective cell migration. Loss of Rap1 and PDZ-GEF cause disorganization of E-cadherin protein localization at border cell-border cell and border cell-nurse cell junctions, resulting in migration failure. In contrast, hyperactivation of Rap1 c (open full item for complete abstract)

Committee: Jocelyn McDonald PhD (Advisor); Aaron Severson PhD (Committee Member); Michelle Longworth PhD (Committee Member); Jun Qin PhD (Committee Member) Subjects: Biology; Cellular Biology; Genetics; Molecular Biology

Master of Science, The Ohio State University, 2012, Biomedical Engineering

Cancer affects a large percentage of the population in the United States, and innumerable research efforts are dedicated to studying the attributes of cancer to develop effective therapies. The mortality rate increases significant when the primary tumor begins to metastasize to different parts of the body. For example, women with localized breast cancer have a 93% survival rate while women with metastatic disease have a 15% survival rate. During metastasis, cancer cells acquire a highly mobile phenotype allowing them to invade into the surrounding tissues. One mechanism by which cancer cells acquire this highly invasive and mobile phenotype is known as Epithelial to Mesenchymal Transition (EMT). EMT is a fundamental biological process that plays an important role in organogenesis and wound healing but is also thought to play an important role in cancer progression where adherent epithelial cells acquire a highly invasive mesenchymal phenotype. Although EMT is believed to be a key process in cancer cell metastasis, the role of substrate stiffness on EMT and motility patterns is not completely understood. In addition, EMT can be induced in some cell types transforming growth factor-¿¿(TGF-¿¿)but it is not known how changes in substrate stiffness influence TGF-¿¿ induced EMT and the associated changes in cancer cell migration. The goal of this thesis was to examine whether substrate stiffness affects migration pattern and behavior of cancer cells in vitro. We used a two dimensional invasion assay on substrates with varying stiffness in the physiologically relevant range, and assessed migration parameters including migration velocity, directionality and distance. We also examined how substrate stiffness alters cell migration patterns in the presence of TGF-¿¿. Finally, the migration results were compared with changes in cell morphology and EMT protein marker expression to investigate how substrate stiffness influences correlations between morphology/protein markers and (open full item for complete abstract)

Committee: Samir Ghadiali PhD (Advisor); Douglass Kniss PhD (Committee Member) Subjects: Biomedical Engineering

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)

Committee: Alex Huang (Advisor); Alan Levine (Committee Chair); George Dubyak (Committee Member); Wendy Goodman (Committee Member); Tsan Xiao (Committee Member); Clive Hamlin (Committee Member) Subjects: Cellular Biology; Immunology; Pathology

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.

Committee: Monica Burdick Dr. (Advisor); Robert Williams Dr. (Committee Member); Douglas Goetz Dr. (Committee Member); Allan Showalter Dr. (Committee Member) Subjects: Biomechanics; Biomedical Engineering

Doctor of Philosophy, University of Toledo, 2017, Biology (Cell-Molecular Biology)

Prostate cancer is the second leading cause of cancer-related death in men in the U.S. Hereditary Prostate Cancer (HPC) accounts for 43% of early onset cases and 9% of all cases of cancer. Positional cloning and linkage studies mapped Hereditary Prostate Cancer 1 (HPC1) to an antiviral gene, RNase L. RNase L is a latent endoribonuclease that is activated by a unique ligand, 2-5A, produced from cellular ATP in virally-infected cells. To date there is no correlation of viral infections with prostate cancer, suggesting that RNase L may play additional roles in tumor suppression. In these studies we demonstrate the role of RNase L, which does not require nuclease activity, in regulating transcription of androgen-responsive genes, cell migration and activity of matrix metalloproteinases, suggesting a novel role as a tumor suppressor. Here we show that both RNase L and Filamin A bind to AR, and the interaction is regulated by androgens. Further, RNase L regulates ligand-dependent AR translocation to the nucleus and transcription of androgen-response genes. Cells with reduced levels of RNase L or Filamin A show increased AR translocation to the nucleus and this is accompanied by an increase in expression of androgen-response genes, PSA, ETV1 and SGCa1. Expression of RNase L mutants R462Q and E265X, which are most prevalent in HPC patients, in cells lacking endogenous RNase L resulted in increased AR translocation accompanied by increased transcription of AR-responsive genes. In addition, RNase L negatively regulates cell migration and cell attachment on various extracellular matrices. Cells with reduced RNase L levels promote cell surface expression of integrin ß1 which in turn activates FAK-Src pathway and Rac-GTPase activity to increase cell migration. Activity of MMP-2 and -9 is significantly increased in cells where RNase L levels are ablated. Mutants of RNase L with defects in binding the ligand 2-5A, defective in dimerization or lacking nuclease activity suggest that (open full item for complete abstract)

Committee: Malathi Krishnamurthy (Committee Chair); Lirim Shemshedini (Committee Member); Roger Taylor (Committee Member); Douglas Leaman (Committee Member); Eda Yildirim (Committee Member) Subjects: Biology

Doctor of Philosophy, Case Western Reserve University, 2014, Biomedical Engineering

The key role of smooth muscle cell (SMC) migration and proliferation in vascular physiological and pathological remodeling necessitates the exploration of mechanisms underlying these functions. This work focuses on engineering a poly (ethylene glycol) (PEG) hydrogel as a model system to evaluate SMC migration and proliferation in three dimensions (3D). We hypothesized that 3D SMC migration and proliferation can be regulated by the properties of a cell-instructive scaffold, including cell-matrix adhesion, degradability, and cross-linking density. To accomplish this, bio-inert PEG-based hydrogels were designed as the scaffold substrate. To mimic the properties of the extracellular matrix (ECM), cell-adhesive peptide (GRGDSP) and enzyme-sensitive peptide (VPMSMRGG or GPQGIAGQ) were incorporated into the PEG macromer chain. Copolymerization of the biomimetic macromers by photopolymerization resulted in the formation of bioactive hydrogels with the dual properties of cell adhesion and proteolytic degradation. Studies of mass swelling ratio as a function of gel compositions indicated that this hydrogel can be engineered quantitatively to allow for uncoupled investigation of scaffold properties on cell functions. By utilizing these biomimetic scaffolds, we studied the effect of adhesive ligand concentration, proteolysis, and network cross-linking density on 3D SMC migration and proliferation. Our results indicated that 3D SMC migration and proliferation were critically dependent on cell-matrix adhesiveness, proteolysis, and cross-linking density. The incorporation of cell-adhesive ligand significantly enhanced SMC spreading, migration and proliferation, with cell-adhesive ligand concentration mediating 3D SMC migration and proliferation in a biphasic manner. The faster degrading hydrogels promoted SMC migration and proliferation. In particular, higher cross-linking density could impede 3D SMC migration and proliferation despite the presence of cell-adhesive ligands and pro (open full item for complete abstract)

Committee: Kandice Kottke-Marchant (Committee Chair); Anirban Sen Gupta (Committee Member); Horst von Recum (Committee Member); Stuart Rowan (Committee Member) Subjects: Biomedical Engineering; Polymers

Master of Science in Chemical Engineering, Cleveland State University, 2013, Fenn College of Engineering

Under this research, live cell imaging of osteoblast-like marrow stromal cells has been carried out on polished and nanotextured (NaOH-etched) medical-grade titanium alloy (Ti-6Al-4V) surfaces to examine cellular adhesion and migration. The purpose of this research was to: 1) Build and assemble suitable hardware and software to conduct live cell imaging in a micro-incubator over an extended period of time. 2) Monitor and record live osteoblast-like marrow stromal cells on polished and NaOH-etched titanium alloy surfaces from cell inoculation to about one week of culture. 3) Measure location, area and perimeter of individual cells as a function of time, and examine if, as compared / contrasted with the polished titanium surface, that the NaOH-etched titanium surface promotes adhesion and migration of cells. This was achieved by describing the mobility, morphology and overall behavior of osteoblast-like marrow stromal cells. During the cell growth cycles, data generated from image analysis included the cells' center of mass (X,Y), their area, perimeter and shape as a function of incubation time. From the change in center of mass after each 15-minute interval, the real time speed of the cells was obtained. Major observations to support comparison studies between the surfaces determined that compared with polished titanium, NaOH-etched titanium promotes cellular filopodia growth, thus, promotes attachment. Filopodia provide cellular anchoring support and when prevalent, make cells more angular in shape. The median aspect ratio (length / width) of cells was found to be 1.38 on polished and 2.36 on NaOH-etched titanium. This, in addition to lower mean circularity shape factor values of 0.26 ± 0.03 on polished and 0.11 ± 0.01 on NaOH-etched titanium imply that the nanotextured surface promotes growth of cells more anchored to the substrate. This is also confirmed by increased perimeters of cells found on the NaOH-etched surface (950.92 ± 84.88 μm) compared with perime (open full item for complete abstract)

Committee: Surendra Tewari PhD (Committee Chair); Joanne Belovich PhD (Committee Member); Ronald Midura PhD (Committee Member) Subjects: Biomedical Engineering

MS, University of Cincinnati, 2012, Medicine: Molecular and Developmental Biology

SAM Pointed Domain Containing ETS Transcription Factor (SPDEF) is a new member of ETS transcription factor family that is originally discovered in the epithelium of prostate. Like other ETS family members, SPDEF is involved in multiple biological processes including cell fate determination and specification, cell proliferation, epithelial-to-mesenchymal transformation and migration. Although much work has been done to elucidate the role of SPDEF in normal physiological conditions and in cancers, its expression level in cancer is still controversial. Similarly, there is no conclusive message on the role of SPDEF in cancer despite the fact that several in vitro studies have been done. We first overviewed the discovery and properties of SPDEF, followed by a comprehensive review of the role of SPDEF, with a special focus on its role in the development of cancer. In the second chapter, we utilized a xenograft model to manipulate SPDEF level in mouse prostate cancer. After orthotopic injection, we found that overexpression of SPDEF resulted in smaller tumors in mice. We also showed that several target genes related to cell proliferation and migration are down-regulated by SPDEF overexpression. We confirmed down-regulation of these genes in prostate cancer cell lines. In fact, we found that SPDEF overexpression inhibited cell migration, resulting in reduced aggressiveness of tumor cells. In addition, SPDEF overexpression was shown to suppress tumor growth via alteration of cell cycle profile. Collectively, these data indicate that SPDEF suppresses tumor growth via inhibition of cell proliferation and migration.

Committee: Tanya Kalin PhD (Committee Chair); Vladimir Kalinichenko MD PhD (Committee Member); Jeffrey Whitsett MD (Committee Member) Subjects: Molecular Biology

PhD, University of Cincinnati, 2011, Medicine: Developmental Biology

Primordial germ cells (PGCs) are the embryonic precursors of adult gametes. In the mouse, they arise around E7.5 in the allantois, and migrate through the developing hindgut and midline dorsal body wall mesenchyme to colonize the gonad primordia by E11.5. PGC behavior, including proliferation, survival, and motility, is controlled by cellular signaling during migration. Steel factor is known as an essential survival factor for PGCs. It is the protein product of the Steel locus, and the ligand for the receptor tyrosine kinase c-kit, which is expressed by PGCs throughout migration. Steel factor exists in two forms, membrane-bound and the soluble, generated by alternative splicing. This thesis addresses two general questions: 1) Is PGC behavior controlled by Steel factor from the beginning of their migration? 2) Do the two different forms of Steel factor control different aspects of PGC behavior? Using the mouse reporter line Stella-GFP, in which PGCs express GFP under the control of the promoter of Stella gene, I demonstrate that PGC number is significantly reduced in Steel-/- embryos at E7.5. Similarly, in the absence of Steel factor, either by null mutation or antibody blockade, PGCs aggregate together and show dramatically decreased motility, but their directionality is maintained. These data indicate an essential role for Steel factor in PGC survival and motility. I then show that Steel factor-expressing cells surround PGCs from the time of their initial specification in the allantois, to the time of their colonization of the gonad primordia, providing a “spatio-temporal niche” that travels with PGCs to regulate their survival, proliferation and motility throughout migration. Further, I show that these functions of Steel factor in PGC behavior are distributed between the membrane-bound and soluble forms by analyzing PGC behavior in Steel-dickie mutant embryos, which make only the soluble form. Soluble Steel factor alone is sufficient for PGC survival at E7.5. How (open full item for complete abstract)

Committee: Christopher Wylie PhD (Committee Chair); Aaron Zorn PhD (Committee Member); Kenneth Campbell PhD (Committee Member); Iain Cartwright PhD (Committee Member); James Wells PhD (Committee Member) Subjects: Developmental Biology

PhD, University of Cincinnati, 2008, Engineering : Chemical Engineering

Controlling the spatial organization of cells is a critical step towards engineering tissues with distributed networks of blood vessels or nerve cells. Here we report a new soft-lithography based approach for micropatterning proteins and cells on the surface of biodegradable chitosan substrates that are more applicable to engineering tissues than the gold, silver, silicone, glass, or silicone substrates currently used in cell micropatterning studies. In this approach, we use random copolymers of oligoethyleneglycol methacrylate (OEGMA), which resists protein and cell adsorption, and methacrylic acid (MA), which adheres strongly onto chitosan substrate via acid-base interactions, to form stable protein and cell resistant micropatterns on chitosan surfaces. We have also developed a non-invasive in vitro migration assay that operates through release of confluent groups of cells initially confined within patterns of cell-resistant polyelectrolyte. Cell-resistant patterns of polyelectrolyte, separating groups of confluent cells, are rendered cell-adhesive by adsorption of a second, cell adhesive polyelectrolyte of opposite charge; thereby, resulting in migration of cells into the separating regions. By dynamically controlling cell-surface interactions through self-assembly of cell-adhesive and cell resistant polyelectrolytes, this method eliminates the need to mechanically wound cells, as is done in current cell migration assays. We used this technique in identifying molecules and mechanisms that regulate cell migration is demonstrated by its application as an assay for the effects of platelet derived growth factors, cytoskeleton disrupting agents, and Merlin over-expression, on the migration of NIH 3T3 fibroblasts. We further used our patterning technique to precisely control the directional migration of a cell. Directional control of cell migration is essential for engineering tissues with defined cellular architectures, promoting wound healing, and non-invasively mani (open full item for complete abstract)

Committee: Chia-Chi Ho PhD (Advisor); Carlos Co PhD (Committee Member); Ian Papautsky PhD (Committee Member); Stephen Clarson PhD (Committee Member) Subjects: Biomedical Research; Chemical Engineering

PhD, University of Cincinnati, 2008, Medicine : Pathobiology and Molecular Medicine

Fibrinogen, a plasma protein central to clot formation, has long been considered to play a role in inflammation and immunity. Fibrin(ogen) interactions with various immune cells have been heavily investigated; lacking is an understanding of the protein's influence on dendritic cell activity. Results from Chapter 2 demonstrate that fibrinogen initiates human dendritic cell production of inflammatory cytokines and chemokines. Adsorbed fibrin(ogen) has increased stimulatory capacity over fibrin(ogen) free in solution, indicating the bound protein, acting through the ß2-integrins, is the active species. Adsorbed fibrin(ogen) also stimulates the focal accumulation of dendritic cells. This is likely due to ß2-integrin-mediated chemotaxis of dendritic cells toward released chemokines and fibrin(ogen) degradation fragments. Because studies suggested adsorbed fibrinogen might be exploited to enhance vaccine adjuvanticity, fibrinogen-coated olive oil droplets were investigated as vaccine adjuvant. Results from Chapter 3 demonstrate the importance of surface in adjuvant activity, and the possible use of olive oil droplets as a safe and effective vaccine adjuvant. Having investigated the interactions between fibrinogen-coated particles and human dendritic cells, Chapter 4 describes the existence of a not yet recorded phenomenon: extracellular transport of cell-sized particles by dendritic cells. Results presented in that chapter not only demonstrate particles can be carried extracellularly by dendritic cells, but also that the process can be directed. Adsorbed fibrin(ogen) appears to enhance particle/dendritic cell interactions mediated through the ß2-integrins. The influence of adsorbed fibrin(ogen) on dendritic cells provides new knowledge into the protein's involvement in initiating inflammatory and immune responses, knowledge that may be applied to the development of new therapeutics to treat and prevent disease.

Committee: Gregory Retzinger MD,PhD (Committee Chair); Alison Weiss PhD (Committee Member); George Deepe MD (Committee Member); Judith Rhodes PhD (Committee Member); Philip Howles PhD (Committee Member) Subjects: Immunology; Pathology

PhD, University of Cincinnati, 2005, Medicine : Molecular and Cellular Physiology

Regulation of epithelial cell motility requires coordinated actin rearrangement, yet the signaling pathways that lead to such modulation are not fully described. Previously we showed that protein kinase C (PKC) regulates actin remodeling in T84 intestinal epithelial cells but little is known regarding the role of PKC during cellular spreading. In subconfluent T84 cells, phorbol 12-myristate 13-acetate (PMA), which activates the conventional cPKC isoform PKCα and the novel nPKC isoforms PKCδ and PKCε of T84 cells, dramatically accelerated cell spreading with concurrent increase in tyrosine phosphorylation of the focal adhesion-associated proteins, Src, FAK and paxillin. These effects were abolished by the PKC inhibitor Go6850 and PKCδ-specific inhibitor rottlerin. In contrast, the cPKC-selective inhibitor Go6976 had no effect. Furthermore, siRNA-mediated down-regulation of PKCδ and PKCε, but not PKCα, attenuated cell spreading, implicating PKCδ and PKCε as the critical elements participating in regulation of epithelial cell spreading. Phosphorylation of FAK and paxillin by PMA was mediated by ligand-dependent transactivation of epidermal growth factor receptor (EGFr). Inhibition of EGFr signaling abolished PMA-induced phosphorylation of FAK and paxillin as well as cell spreading, indicating that EGFr transactivation plays a central role in mediating PMA-elicited cellular spreading. PMA caused PKCδ to translocate to the basal compartment, yet pretreatment with Go6850 or siRNA against PKCε redistributed PKCδ to the lateral membrane; Go6976, rottlerin, or siRNAs against PKCα or PKCδ were ineffective in preventing basal translocation of PKCδ. Inhibition of actin remodeling also redirected PKCδ to the lateral membrane, preventing PMA-induced phosphorylation of Src, FAK, and paxillin. These data demonstrate that actin remodeling by PKCε is critical for basal translocation of PKCδ and subsequent phosphorylation of focal adhesion targets, emphasizing the cooperation between (open full item for complete abstract)

Committee: Dr. Jeffrey Matthews (Advisor) Subjects: Biology, Cell

Doctor of Philosophy, Case Western Reserve University, 2010, Genetics

Many species have evolved to propagate through sexual reproduction. This process provides reliable transmission of genetic material to offspring while allowing for the accumulation of mutations that contribute to diversity. Sexual reproduction requires the development of specialized cell types. In mammals, the germ cells are specified early in embryogenesis and undergo an elaborate process of migration before colonizing the nascent gonads. There, they undergo gradual differentiation, culminating in the production of gametes in the adult. Germ cell development relies on communication between germ cells and surrounding somatic cells. These signals control germ cell survival, proliferation, migration, and sexual differentiation, without which, life as we know it would cease. For my thesis, I examined the signals that control primordial germ cell (PGC) migration from the hindgut to the genital ridges. Using tissue culture and conditional gene targeting in mice, I demonstrated that bone morphogenetic proteins (BMPs) play a pivotal role in PGC migration. I discovered that BMPs expressed in the pronephric compartment of the E9.5 genital ridge establish and maintain a PGC nice within the nascent gonads. BMP signaling supports somatic survival in the mesonephric mesenchyme and represses expression of Scarb1, an epithelial marker. In the coelomic epithelium, BMP signaling promotes the expression of the PGC survival factor Kitl and putative chemo-attractant Sdf1a, creating a niche environment for arriving PGCs. Integral to the effectiveness of the genital ridge niche is high expression of Kitl. During PGC migration KITL is present as a gradient with the highest levels in the genital ridges. Disruption of this through KIT receptor inhibition, decreased Kitl expression, or exogenous soluble KITL led to decreased PGC survival and defects in PGC targeting resulting in increased ectopic accumulation of PGCs. I propose a model where PGCs interact with their somatic neighbors through (open full item for complete abstract)

Committee: Molyneaux Kathleen Ph.D. (Advisor); Conlon Ron Ph.D. (Committee Chair); Bai Brian Ph.D. (Committee Member); Watanabe Michiko Ph.D. (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Genetics; Molecular Biology

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

Cell migration is crucial for various physiological and pathologicalprocesses, including embryonic morphogenesis, wound healing, immune responses, cancer progression, and atherosclerosis development. Particularly, endothelial cell (EC) migration is a fundamental process in angiogenesis and vascular immune responses. Cell migration is driven by actin polymerization-mediated lamellipodia protrusion, and proceeds by repeated cycles of protrusion, adhesion, and contraction. Intensive studies have shown that these processes are subjected to spatiotemporal regulation by signalling molecules, e.g. PI(3)K and Rho GTPases, and by actin-binding proteins, e.g. Arp2/3 and cofilin. Here our data show that 1) spatially restricted dissociation of G-actin–Tβ4 complexes at the leading edge liberates actin for filament assembly, and simultaneously facilitates Tβ4 binding to integrin-linked kinase in the lamellipodia, followed by Akt2-mediated matrix metalloproteinase-2 production, providing a coordination mechanism that couples actin polymerization to matrix degradation; 2) Myo1c delivers lamellipodial G-actin to the leading edge to facilitate local actin polymerization, indicating a mechanism for G-actin transport-mediated polarization of actin polymerization; 3) During VEGF-induced endothelial cell chemotaxis migration, profilin phosphorylation at Tyr129 preferentially occurs at the leading edge to increase local actin polymerization, suggesting a mechanism for establishment and maintenance of cell polarity during chemotaxis. Taken together, these findings elucidate novel molecular mechanisms underlying coordination of actin polymerization and matrix degradation, as well as polarization of actin polymerization during EC migration, which contributes to better molecular resolution of cell movement and may lead to the development of therapeutic strategies for EC migration-related diseases.

Committee: Paul L Fox (Advisor); Alan D Levine (Committee Chair); Sanjay W Pimplikar (Committee Member); Tom Egelhoff (Committee Member); Clive Hamlin (Committee Member) Subjects: Cellular Biology; Pathology

Doctor of Philosophy, The Ohio State University, 2024, Oral Biology

Tooth enamel is a highly mineralized tissue of epithelial origin and a product of specialized cells known as ameloblasts. Once completely mineralized, enamel does not possess the ability to regenerate. Therefore, any developmental aberrations during the process of enamel formation can affect the volume and quality of enamel. A Disintegrin and Metalloproteinase-10 (ADAM10) is a catalytically active, membrane bound proteinase, best known for extracellular shedding of its substrates. Mice with Adam10 conditionally deleted from the epithelium, exhibit tooth defects suggesting ADAM10's role in tooth formation. Previous studies in our lab have shown that ADAM10 is expressed in the secretory stage ameloblasts. Here we sought to determine if ADAM10 plays a functional role that supports enamel formation. Our first series of experiments concluded that ADAM10 expression does not rely on the activation of p38 MAPK signaling. Our second and third set of experiments focused on establishing a conditional knockout Adam10 mouse using an amelogenin promoter driven Cre recombinase (Adam10fl/fl /Amelx-iCre+/-), which we verify is only expressed in ameloblasts. Structural and morphological assessment of the Adam10 conditional knockout (cKO) mice reveal an abnormal enamel phenotype. Teeth are discolored, abraded and mechanically weak. Notably, the Adam10 cKO enamel density and volume were significantly reduced in both incisors and molars. Moreover, the incisor enamel rod pattern became progressively more disorganized moving from the DEJ to the outer enamel surface, creating large gaps between and among the enamel rods. Our results show for the first time that ADAM10 is essential for proper enamel formation, likely through its ability to facilitate ameloblast movement and/or its ability to cleave multiple substrates essential for enamel formation.

Committee: John Bartlett Dr. (Advisor) Subjects: Dentistry

PHD, Kent State University, 2024, College of Arts and Sciences / Department of Chemistry and Biochemistry

DNA origami structures have provided versatile tools in a wide variety of applications. Due to higher stability, tunability and mechanical strength compared to single stranded or double stranded DNA, origami-based DNA nanostructures have garnered special interest in the scientific community. Many of these biomedical applications exploit mechanical properties and therefore, need a discrete tool/method to analyze the dynamic structural changes in these nanoassemblies. Herein, we synthesized different origami based nanosprings and mechanically characterized their properties and eventually applied them to halt the cancer cell motions. To that end, the nanosprings were synthesized from 6 helix bundles of DNA with specific bridge elements made up of either i-motifs, or G-quadruplexes or duplex DNAs. The bridge elements served as junctions which pulled/pushed nearby elements to bend the structures; multiple of which exhibited a coiled spring. To characterize the mechanical properties, we employed optical tweezers as a single molecule force manipulation tool that record the extension behaviors under tensile force in real time, assisting to compute spring constant, kinetics, recoil and uncoil distances, and pitch of nanosprings. With the strength 17 times more than previous nanosprings, these nanostructures were exploited to modulate cell migrations. To that end, we modified nanosprings with arginyl-glycyl-aspartate (RGD) domains with a spacing such that when the nanospring is coiled, the RGD ligands trigger the clustering of integrin molecules, which changes cell motions. The coiling or uncoiling of the nanospring is controlled, respectively, by the formation or dissolution of pH responsive i-motifs. Results showed significant inhibition to the migration of HeLa cells in acidic extracellular environment under the influence of nanosprings. Likewise, to divulge the structural-property relationship and the modulation factors behind the long-range, higher order arrangement of s (open full item for complete abstract)

Committee: Hanbin Mao (Committee Chair); Sanjaya Abeysirigunawardena (Committee Member); Yao-Rong Zheng (Committee Member); Thorsten-Lars Schmidt (Committee Member); Manabu Kurokawa (Other) Subjects: Biochemistry; Biology; Biophysics; Cellular Biology; Chemistry

Doctor of Philosophy, The Ohio State University, 2023, Molecular, Cellular and Developmental Biology

Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in the western world, accounting for more than 4000 deaths in the United States every year. It is a malignancy of CD5+CD23+CD19+ B lymphocytes, characterized by activation of B cell receptor (BCR) signaling that in turn activates several downstream cell survival pathways, and migration of these B cells (B-CLL cells) to pro-survival niches like the bone marrow, lymph nodes and spleen. The only curative therapy is allogeneic hematopoietic stem cell transplantation, but this therapy is associated with common risks like graft-versus-host-disease and pulmonary complications. BCR signaling inhibitors are effective agents for CLL treatment, but therapeutic resistance and relapse remain problematic, necessitating the identification of new CLL targets that can bypass current disease resistance. Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of cell surface glycoproteins that demonstrate cell-type specific expression and can modulate receptor signaling. Many studies have shown the importance of Siglecs in tumor immunosurveillance including immunosuppression, thereby making them attractive anti-cancer molecular targets. Our study focuses on evaluating a novel Siglec member, Siglec-6, which was recently found to be upregulated on the surface of B cells from CLL patients but not on B cells from healthy donors. CLL-specific upregulation of Siglec-6 thus makes it a valuable therapeutic target and warrants the development of transgenic mouse models and cell line systems to further explore the biological role of Siglec-6. While Siglec-6 has been extensively studied in trophoblastic cells, mast cells and bladder cancer cells, the physiological role of Siglec-6 in immune cells is still poorly understood. In the first part of this dissertation, we utilized our novel Siglec-6 transgenic mouse models to identify a specific role for Siglec-6 in regulating B cell functions. Siglec-6 transgenic (open full item for complete abstract)

Committee: Natarajan Muthusamy (Advisor); Meixiao Long (Committee Member); Lalit Sehgal (Committee Member); Paul Martin (Committee Member) Subjects: Cellular Biology; Molecular Biology

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

Though the basic building blocks of the cytoskeleton were first identified more than fifty years ago, there remains much to learn about how cytoskeletal dynamics impact cellular function, and ultimately contribute to cellular homeostasis and human disease. In this project, we have sought to determine how a microtubule-associated protein, the dynein arm assembly factor Coiled-coiled domain-containing protein 103 (CCDC103) contributes to the normal maturation and effector function of human and zebrafish neutrophils by secondarily impacting α-tubulin lysine 40 (K40) acetylation, a critical post-translational modification affecting microtubule stability. CCDC103 plays a critical role in the proper assembly of the axonemal dynein complex, and, which mutated, causes a disorder known in humans as Primary Ciliary Dyskinesia or PCD. Interestingly, studies have shown that myeloid cells from PCD patients have multiple functional and cytostructural defects, even though myeloid cells themselves do not possess motile cilia. We have previously shown that ccdc103 mutations are sufficient to disrupt neutrophil and macrophage migration and proliferation in zebrafish, mostly likely through its well-characterized role as a microtubule stabilizing factor. Additionally, we identified multiple novel CCDC103 binding partners that may support its function, including the flagellar central-apparatus protein SPAG6 and the NAD-dependent deacetylase SIRT2, and showed that PCD-associated CCDC103 mutations abrogate interactions with both of these proteins. Zebrafish spag6 mutant embryos display multiple phenotypic hallmarks of motile cilia dysfunction, including low fertility and situs inversus, and recapitulate many of the same proliferation, morphology, and directed migration capacity defects also observed in ccdc103 mutants, suggesting that SPAG6 acts together with CCDC103 to stabilize cytoplasmic microtubules. Conversely, SIRT2 is a key deacetylase of α-tubulin, a pos (open full item for complete abstract)

Committee: Joshua Waxman Ph.D. (Committee Member); Lindsey Barske Ph.D. (Committee Member); Ashish Kumar M.D. (Committee Member); Marie-Dominique Filippi Ph.D. (Committee Member); Tony De Falco Ph.D. (Committee Member) Subjects: Cellular Biology

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

Extracellular-regulated kinase 3 (ERK3) is overexpressed in a variety of cancers, including lung cancer. This atypical mitogen-activated protein kinase (MAPK) has a unique structure which includes a C34 domain and C-terminus and about which relatively little is known. ERK3 has been demonstrated to promote cell migration and metastasis in multiple cancer types. A yeast two-hybrid assay using ERK3 as bait indicated Diacylglycerol kinase ζ (DGKζ) is a binding partner. DGKζ is an isoform in the DGK family, all the members of which phosphorylate the lipid diacylglycerol (DAG) to generate phosphatidic acid (PA). DGKζ was shown to promote migration in mouse embryonic fibroblasts (MEFs) and enhance invasion in some types of cancer cells, but its role in lung cancer has yet to be described. The interaction of these two proteins was confirmed by immunoprecipitation in both endogenous and overexpression conditions in cells as well as by in vitro assays. Further, as observed by immunofluorescence, these proteins colocalize in cells primarily near the cell periphery. Interestingly, DGKζ interacts with ERK3 via the C34 domain of ERK3 and deletion of this domain drastically reduces the ability of these proteins to form a stable complex. ERK3 interacts with DGKζ though the N-terminal and the C1 regions of DGKζ. Lipid mass spectrometry analysis revealed that knockdown of ERK3 reduced phosphatidic acid levels but promoted diacylglycerol and triacylglycerol levels. Changes to triacylglycerol and phosphatidic acid levels following ERK3 knockdown were confirmed in additional lung cancer cell lines. Despite these exciting findings, ERK3 had no effect on DGKζ localization or enzymatic activity, suggesting the effect of ERK3 on lipids may be independent of DGKζ. In lung cancer cells, DGKζ surprisingly decreased cell motility with DGKζ overexpression reducing cell migration and DGKζ knockdown increasing cell migration. With co-overexpression, DGKζ entirely inhibited the ability of ERK3 to p (open full item for complete abstract)

Committee: Weiwen Long Ph.D. (Advisor); Hongmei Ren Ph.D. (Committee Member); Yong-jie Xu M.D., Ph.D. (Committee Member); Paula A. Bubulya Ph.D. (Committee Member); Michael Leffak Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Molecular Biology

PhD, University of Cincinnati, 2019, Medicine: Cancer and Cell Biology

The rate-limiting GTP-biosynthetic enzyme inosine 5'-monophosphate dehydrogenase (IMPDH) has been implicated in the tumorigenesis and progression of multiple cancer types. Overexpression of the IMPDH2 isozyme is correlated with worse prognosis, the presence of metastatic disease, and disease progression and is also readily inhibited by the FDA-approved drug mycophenolic acid (MPA), making IMPDH a potential therapeutic target for cancer therapy. In recent years, the GTP biosynthetic pathway has been increasingly associated with cell migration, the important first step to cancer metastasis. How IMPDH promotes cancer cell migration has not been extensively studied. In this thesis, the role of IMPDH in renal cell carcinoma (RCC) migration is assessed. RCC has a very high rate of metastasis, with nearly 25% of patients presenting with metastatic disease at the time of diagnosis and another 20-30% progressing to metastatic disease following nephrectomy. Understanding the underlying mechanisms of RCC migration may enhance our ability to develop effective therapeutics for preventing and treating RCC metastasis. Chapter 1 and chapter 2 encompass the importance of purine nucleotide biosynthesis in cancer, including an explanation of the enzymes involved in biosynthesis and the literature connecting their activities with tumorigenesis, as well as background information regarding RCC subtypes, metabolism, staging and diagnosis, and an introduction to the current therapeutic options for patients with metastatic disease. In chapter 3 of this thesis, the role of IMPDH in RCC proliferation and migration in vitro is reported. IMPDH knockout or inhibition in RCC cell lines impedes both proliferation and migration ability on a global level. Chapter 4 delves deeper into the subcellular localization of the entire purine biosynthesis pathway and its role in cell migration. In this thesis, we report the novel finding that enzymes involved in the purine de novo pathway, ATP and GTP b (open full item for complete abstract)

Committee: Atsuo Sasaki Ph.D. (Committee Chair); Maria Czyzyk-Krzeska M.D. (Committee Member); Francis McCormack M.D. (Committee Member); Satoshi Namekawa Ph.D. (Committee Member); Susan Waltz Ph.D. (Committee Member) Subjects: Biology