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

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

Committee: Monica M. Burdick (Advisor) Subjects: Biochemistry; Biology; Biomedical Engineering; Biomedical Research; Biophysics; Chemical Engineering

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 (PhD), Ohio University, 2011, Physics and Astronomy (Arts and Sciences)

White blood cells (WBC) protect the body from infectious pathogens. They are recruited to the site of infection, and respond quickly and potently. There have been many studies of WBC, but the mechanism of leukocyte arrest in pulmonary capillary has not been brought to a consensus even though the pulmonary capillary bed is a key site of neutrophil recruitment and sequestration in the lung and is important to prevent damage to healthy tissue and fight infection [1]. It has been shown that the selectins are responsible for early adhesion steps in the recruitment of leukocyte into the sites of inflammation in venules, but the role of adhesion molecules in leukocyte arrest in the pulmonary capillaries is still controversial. In this study, we aimed to determine the mechanical and biochemical properties of neutrophils for understanding of neutrophil arrest, using micropipette aspiration with various sizes of micropipettes coated with soluble P-selectins and heat-treated BSA used as a control. Our observations demonstrate that very small concentrations of sP-selectin have an effect on neutrophils motion and arrest in non-tapered micropipette. The larger contact area formed by the aspirated cell leads to greater adhesion with higher disrupting forces in capillaries than in venules. The gap width between the neutrophil surface and the micropipette wall varies from 3 nm to 228 nm and this gap thickness decreases with increasing concentration of sP-selectin. Higher concentrations of sP-selectin produce more bonds, which leads to closer contact and smaller gap width than in lower concentrations or BSA. Additionally, it was shown that high concentrations of anti-P-selectin F(ab΄)2 fragments are effective to inhibit the P-selectin mediated adhesion. There is no mechanical arrest observed in tapering vessels with taper angles of up to 9°. However, there is a transit point where cells slow down, which does not depend on the endothelial cell adhesion molecule concentration, tap (open full item for complete abstract)

Committee: David Tees PhD (Advisor); Douglas Goetz PhD (Committee Chair); Monica Burdick PhD (Committee Member); Alexander Neiman PhD (Committee Member); Eric Stinaff PhD (Committee Member) Subjects: Biophysics

Doctor of Philosophy (PhD), Ohio University, 2010, Chemical Engineering (Engineering and Technology)

Atherosclerosis is an inflammatory disease that affects major arteries of the vasculature. Extensive research on atherosclerosis has indicated that among other inflammatory markers, vascular cell adhesion molecule-1 (VCAM-1) is a major player in atherosclerosis development and progression. VCAM-1 has been shown to be elevated at vascular sites prone to atherosclerosis. The elevated expression of VCAM-1 takes part in the adhesion and transmigration of leukocytes across the endothelium. This leads to the formation of atherosclerotic plaques which are the most common cause of adverse cardiovascular events. Hence, this doctoral work was motivated by the need to improve current diagnostics and therapeutics for atherosclerosis wherein the elevated expression of VCAM-1 was exploited for the development of novel therapeutic and diagnostic approaches for atherosclerosis.In the first study, a VCAM-1 ligand (α-VCAM-1, an antibody to VCAM-1) was conjugated to polymeric particles and their adhesion to the vasculature was probed using a mouse model of atherosclerosis. Specifically, their adhesion to the aorta and the femoral artery was studied. Results from this study demonstrated that the ligand conjugated polymeric particles exhibit an avid and focal adhesion to sites of atherosclerosis. It was also observed that the particle adhesion is a function of the location within a plaque, position along the length of the aorta and the type of the vascular bed. Particles conjugated with α-VCAM-1 exhibited significantly higher adhesion (as high as 5-fold higher) to the aorta of atherosclerotic mice compared to their adhesion to the aorta of control mice. Additionally, a 2.5-fold higher adhesion was observed for α-VCAM-1 particles to the plaque regions of the aorta compared to the non-plaque regions of the aorta. These results demonstrate that the ligand conjugated polymeric particles can selectively target sites of atherosclerosis and may potentially serve as the vehicles for delivering (open full item for complete abstract)

Committee: Douglas J. Goetz PhD (Advisor); David F. J. Tees PhD (Committee Member); Darin Ridgway PhD (Committee Member); Monica Burdick PhD (Committee Member); Ramiro Malgor MD (Committee Member) Subjects: Biomedical Research; Chemical Engineering

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

The goal of this work is to investigate magnetic materials (MM) and the influence of magnetic field on different shapes and types of MM with innovative magnetic devices. Using magneto-optical detection devices, we examined the physical science of two MM systems: malarial hemozoin (a paramagnetic crystal) in rod-like form, and synthetic magnetic particles with iron oxide cores in spherical form. The second study focuses on capturing and imaging targeted cells using a newly developed CAPGLO device that combines magnetic and fluorescent mechanisms. Due to their magnetic properties, the crystalline hemozoin shows unique alternation in the presence of a magnetic field. Using a light beam, we were able to detect and verify the changes in the direction of the crystal by measuring the polarization-dependent optical absorption when the magnetic field is alternated between on and off. Our results show a factor of two increase in absorption as it grows with light polarization along the easy-axis crystallographic direction compared to polarization along the hard-axis (ax/az = σx/σz = 2), which shows a correlation between the basic physics and the chemical structure of the crystal. The characteristics of magnetophoresis – that is, motion under a gradient magnetic field of magnetic particles (MP) suspended in fluid – are analyzed. As the MP migrate from the center of the sample toward the magnets, we measure the changes in the photodiode detector voltage for the light beam over time. Our results show the effect of different concentrations and MP sizes. In addition, we notice a universal pattern that displays multiple phases that are correlated with the motion of fluid, the chain formation of MPs, and the complete separation of the MPs. Lastly, CAPGLO is an original magnetic detector designed to capture the composite made up of an MP binding to a cell. The cancer cell example has concentrations corresponding to biopsies. The composites are imaged using a fluorescence technique. (open full item for complete abstract)

Committee: Robert Brown (Committee Chair); Michael Martens (Committee Member); Robert Deissler (Committee Member); Susann Brady-Kalnay (Committee Member); Gary Chottiner (Committee Member) Subjects: Experiments; Physics

Doctor of Philosophy, The Ohio State University, 2023, Biophysics

Cadherins are a family of large transmembrane glycoproteins instrumental in facilitating organ formation during morphogenesis in vertebrates and invertebrates. At the cellular level, they are involved in adhesion, signaling, recognition, mechanotransduction, and motility. In the modern classification of the cadherin superfamily, classical cadherins with five extracellular cadherin (EC) repeats as well as clustered and non-clustered -protocadherins with six or seven EC repeats have been well-studied and their homophilic/heterophilic interactions with molecules on the same (cis) cell or opposite (trans) cells have been characterized. Complexity arises when the number of EC repeats increases with diverse Ca2+ coordination at linker regions between two consecutive EC repeats. In larger cadherins, such as cadherin-23 (CDH23), protocadherin-15 (PCDH15) and cadherin epithelial growth factor (EGF) Laminin-G (LAG or LamG) seven pass G-type receptor-1 (CELSR1), the structural flexibility afforded by different Ca2+ coordination plays determinant roles in their adhesion capacity during inner-ear mechanotransduction and planar cell polarity (PCP). CDH23 and PCDH15, each with 27 and 11 EC repeats, connect two adjacent hair- like protrusions known as stereocilia together atop of a hair cell, the primary mechanosensory cell in the inner ear. Through heterophilic interactions between their first two N-terminal EC repeats, CDH23 and PCDH15 form a filament known as the tip link. In response to sound, stereocilia undergo displacement and the tip link experiences tension, which opens the ion-conducting mechanotransduction channels on the tip-link's lower end to send signals to the brain. The heterophilic trans tetrameric complex formed by CDH23 and PCDH15, and the cis interactions along the length of PCDH15 have been well-studied in the past but full-length ectodomain structures and high-resolution structural models of complete CDH23 and PCHD15 ectodomain have not been resolved. H (open full item for complete abstract)

Committee: Marcos Sotomayor (Advisor) Subjects: Biochemistry; Biophysics

Doctor of Philosophy, Case Western Reserve University, 2022, Physiology and Biophysics

Gasdermins (GSDMs) are a family of structurally-related proteins, with Gasdermin D (GSDMD) being the best characterized known for its role in pore formation within the plasma membrane during pyroptosis, providing preliminary evidence towards the function of other GSDMs. Gasdermin B (GSDMB) is the least studied member of the group due to a lack of a mouse ortholog, rendering functional studies examining biological relevance as challenging. However, GWAS have revealed an association between SNPs within the gene encoding GSDMB towards an increased susceptibility of acquiring chronic, mucosal inflammatory disorders, including inflammatory bowel disease (IBD), and alterations to the structural confirmation of the protein. Yet, the characterization of GSDMB expression in IBD and functional consequences of structural changes of the protein, remain understudied. Herein, we report increased GSDMB in IBD, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in epithelial cells and intestinal epithelial organoids after stimulation with methotrexate, a novel inducer of GSDMB expression. Instead, we find functions 9 of full-length GSDMB towards increasing proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display a hyper- adhesive phenotype, due in part to the formation of vinculin-based actomyosin stress fibers. Mechanistically, we uncover novel associations of GSDMB modulation of PDGFA which in turn controls phosphorylation of FAK, underscoring the process of epithelial wound closure. Importantly, biological significance in carriage of disease-associated GSDMB SNPs are demonstrated, conferring defects disrupting epithelial restitution and repair, which is further supported by trends observed in intestinal epithelial organoids. Together, we establish inher (open full item for complete abstract)

Committee: George Dubyak (Committee Chair); Theresa Pizarro (Advisor) Subjects: Immunology; Medicine; Pathology; Physiology

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

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

Committee: Umut Gurkan (Committee Chair); Ozan Akkus (Committee Member); Jaikrishnan Kadambi (Committee Member); Jane Little (Committee Member) Subjects: Biomechanics; Mechanical Engineering

Master of Science (MS), Ohio University, 2018, Mechanical Engineering (Engineering and Technology)

Cell rolling onto vascular endothelium under hydrodynamic blood flow is an important mechanism in many physiological and pathological processes, such as inflammatory response and tumor metastasis. The blood-borne cells are in direct contact with the most inner layer of endothelium formed by a highly compliant layer of endothelial cells. The effect of substrate stiffness on the adhesive dynamics of rolling cells is poorly understood. The goal of this thesis is to develop a modeling framework to understand the physics underlying the specific adhesion of a rolling cell to a soft bio-adhesive substrate subjected to a viscous shear flow. Of particular importance is to predict the effect of substrate stiffness on stable adhesion and rolling velocity of the cell. The developed models show a direct correlation between the state of adhesion and the substrate compliance. The results of this Thesis are anticipated to enhance our understanding of cellular arrest by endothelium and provide a quantitative basis to rational design of the nano-particles for vascular drug delivery.

Committee: Sarvestani Alireza (Advisor); Farnoud Amir (Committee Member); Tees David (Committee Member); Cotton John (Committee Member) Subjects: Biomechanics; Biomedical Engineering; Biomedical Research; Biophysics; Chemical Engineering; Materials Science; Mathematics; Mechanical Engineering; Mechanics; Molecular Biology; Molecular Physics; Physics

Doctor of Philosophy (PhD), Ohio University, 2016, Chemistry and Biochemistry (Arts and Sciences)

Cell adhesion is an important early step of cancer metastasis, yet the roles of sialylation in regulating integrin-mediated breast cancer cell adhesion in comparison to migration and invasion are not well-understood. The role of sialylation on alpha5 beta1 and alpha2 beta1 integrins in the regulation of adhesion between breast cancer cells and extracellular matrix (ECM) was studied. Our data showed that alpha2, alpha5 and beta1 integrins had considerable alpha 2,6 sialylation on MDA-MB-231 cells, whereas signals from MCF-7 cells were undetectable. Cleavage of alpha 2,6 sialylation increased adhesion of MDA-MB-231 cells to ECM, while adhesion of MCF-7 cells was unaffected, consistent with the latter's lack of endogenous alpha 2,6 sialylated surface integrins. Neither surface expression of alpha5 beta1 and alpha2 beta1 integrins, nor activated beta 1 integrin, changed in MDA-MB-231 cells after sialidase treatment. However, sialidase treatment did not have significant impact on migration or invasion of MDA-MB-231 cells. Integrins not only play an important role in adhesion of cancer cells, but also have a direct connection with ionizing radiation-induced atherosclerosis, which is an adverse effect observed after radiotherapy. However, minimal attention has been given to monocytes/macrophages, which are exposed to the radiation at the same time. Under flow conditions using a parallel plate flow chamber to mimic physiological shear stress, we demonstrate here that the avidity between very late antigen-4 (VLA-4) of RAW264.7 cells and its ligand vascular cell adhesion molecule-1 (VCAM-1), was increased after low dose (0.5 Gy), but was reduced after higher dose (5 Gy) treatment of ionizing radiation. Treating the cells with free radical scavenger N-acetyl-L-cysteine reduced the avidity between RAW264.7 cells and VCAM-1 to a similar level. These results suggest that ionizing radiation regulates adhesive interactions between VLA-4 and VCAM-1, and that reactive oxygen spe (open full item for complete abstract)

Committee: Shiyong Wu (Advisor) Subjects: Biochemistry

Doctor of Philosophy, University of Toledo, 2016, Biology (Cell-Molecular Biology)

Despite the effort to discover molecular pathways regulating breast cancer, there is little understood about the role of the hormone prolactin (PRL) in breast cancer cell motility, invasion, and epithelial-to-mesenchymal transition (EMT). PRL, which is typically secreted from the anterior pituitary and regulates lactation, is also synthesized and secreted by breast cancer cells and promotes cell proliferation, motility, and invasion. Additionally, increased expression and activity of the serine-threonine kinase p21-activated kinase 1 (PAK1) has been observed in human breast tumors and enhances breast tumor growth, cell motility, and epithelial-to-mesenchymal transition (EMT). We have previously demonstrated that PAK1 is a novel target of PRL-activated Janus kinase 2 (JAK2). Active JAK2 can phosphorylate PAK1 on three tyrosines; 153, 201, and 285. Here we implicate tyrosyl phosphorylated PAK1 (pTyr-PAK1) in PRL-induced breast cancer cell motility, adhesion, and EMT. We also describe a novel JAK2/PAK1-independent pathway coordinating PRL-induced breast cancer cell invasion. To start uncovering a role of PRL-activated PAK1 in the regulation of the actin cytoskeleton, we analyzed the effect of PRL on the actin-dependent processes of membrane ruffling and cell migration. We show that prolactin treatment promoted T47D and MCF7 breast cancer cell membrane ruffling. Membrane ruffling was enhanced upon overexpression of PAK1 WT, but abrogated in cells overexpressing a phospho-tyrosyl mutant PAK1, PAK1 Y3F, where the three tyrosines phosphorylated by JAK2 have been mutated to phenylalanine. Using PAK1-specific siRNA, we demonstrate that PAK1 is required for PRL-induced cell migration. T47D cell migration was augmented in response to PRL and dependent on tyrosyl phosphorylation of PAK1, as overexpression of PAK1 Y3F significantly diminished PRL-induced cell migration. Knockdown of the actin crosslinking protein Filamin A (FLNa) eliminated PRL-mediated cell motilit (open full item for complete abstract)

Committee: Maria Diakonova (Committee Chair); Douglas Leaman (Committee Member); Rafael Garcia-Mata (Committee Member); Song-Tao Liu (Committee Member); Kathryn Eisenmann (Committee Member); Randall Ruch (Committee Member) Subjects: Biology; Cellular Biology

Doctor of Philosophy, The Ohio State University, 2009, Integrated Biomedical Sciences

Osteoclasts are large, multinucleated cells that are derived from differentiated macrophages and are mainly responsible for breakdown of bone (bone resorption). They are highly motile cells and adopt a polarized phenotype briefly during bone resorption. Because of the fluidity in cell shape associated with these processes, osteoclasts possess a dynamic cytoskeleton that contain specialized actin structures involved in motility (podosomes) and attachment during bone resorption (an F-actin ring). In our initial studies, we showed that various non-muscle tropomyosin isoforms are distributed to different intracellular locations where they regulate specific actin pools. Results presented here describe the role that alpha gene tropomyosins, TM-2/3 and TM-5a/5b play in the regulation of mature osteoclast activity. TM-2 and -3 are closely related high molecular weight tropomyosins that differ from the low molecular weight TM-5a and -5b only by alternately spliced exons that encode the amino termini. However, this difference of 80 amino acid residues mediates markedly different patterns of expression, subcellular distribution, and function for these TMs. The high molecular weight TM-2/3 were not expressed in the monocytic and macrophage precursors of osteoclasts, but were strongly upregulated late in osteoclastogenesis. In contrast, low molecular weight TM-5a/5b were present in macrophage precursors and were modestly upregulated early during osteoclast formation. Further, TM-5a/b were strongly enriched in adhesion complexes (podosomes and actin ring) of osteoclasts, while TM-2/3 were distributed internally throughout the cells, and were not strongly associated with these actin-rich adhesion structures. RNAi-mediated suppression of TM-2/3 caused increased spreading and flattening of the cells, accompanied by diminished motility and altered resorptive capacity, while overexpression of TM-2 caused decreased spreading and induction of actin patches or stress-fiber like actin fi (open full item for complete abstract)

Committee: Beth Lee (Advisor); Michael Ostrowski (Committee Member); Richard Burry (Committee Member); Susheela Tridandapani (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology

Doctor of Philosophy (PhD), Ohio University, 2007, Chemical Engineering (Engineering)

Leukocyte sequestration in pulmonary capillaries is a key step in the inflammatory response to lung infection. P-selectin and Inter Cellular Adhesion Molecule-1 (ICAM-1) have well defined roles in leukocyte (White Blood Cell) adhesion in the systemic venules but their role in pulmonary capillaries is still unclear. Studies using transgenic animals and monoclonal antibodies (mAbs) suggest no role for P-selectin in leukocyte adhesion in pulmonary capillaries while subsequent retention and migration following initial mechanical arrest may be mediated by ICAM-1/β 2-integrins. In this study, a novel in vitroassay Micropipette Cell Adhesion Assay(MCAA) was developed which used P-selectin, ICAM-1 or BSA coated pulmonary capillary-sized glass microvessels as an in vitromodel for a pulmonary capillary. Leukocytes were aspirated into these in vitrocapillaries and the pause times and velocities were determined under pressures representative of pulmonary capillaries. The average pause time and percent arrest for HL-60 cells in the MCAA was significantly larger on P-selectin than BSA. The HL-60 adhesion to similar densities of P-selectin was greater in MCAA than in a Parallel Plate Flow Assay and increased with increasing contact area of the cell. However, this adhesion to P-selectin was hard to block with mAb and anti-adhesion treatments. Neutrophil velocities in MCAA were significantly lower on P-selectin than BSA and decreased with increasing P-selectin concentration. Pre-treating P-selectin coated microvessels with an anti-P-selectin mAb resulted in a small but significant increase in velocity. Interestingly, presenting the anti-P-selectin mAb in the fluid phase during the assay caused many neutrophils to arrest. Neutrophil velocities in MCAA were not significantly different on ICAM-1 than BSA. Neutrophil velocities in MCAA were not significantly different on P-selectin and ICAM-1 than P-selectin alone. These results demonstrate that there is no mechanical force to mediate l (open full item for complete abstract)

Committee: David Tees (Advisor) Subjects:

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

Master of Science, Miami University, 2023, Mechanical and Manufacturing Engineering

Owing to their superior properties like excellent mechanical strength, thermal and oxidation resistance, chemical stability, biocompatibility, chemical resistance, etc. zirconia-based ceramics find their applications in industries, including aerospace, automotive, biomedical, energy, etc. But manufacturing parts of zirconia-based ceramic by traditional manufacturing processes like injection molding, hot pressing, cold pressing, etc., provides difficulties in fabricating high-quality parts with complex geometrical shapes. Additive manufacturing (AM) can be a solution to this problem. Various AM techniques, including binder jetting, selective laser sintering, material extrusion, etc., have been utilized to manufacture complex shapes using zirconia-based ceramic. It remains a challenge to fabricate good-quality parts using AM techniques. From the published report, it is also evident that zirconia-based ceramics show inferior mechanical properties. In this research, inkjet-based AM, which is a material extrusion-based AM technique, is used to fabricate high-quality zirconia-based ceramic. Moreover, zirconia-based ceramic is reinforced with graphene to improve mechanical, thermal, and microstructural properties, and the effects of graphene on these properties as well as on cell adhesion have been analyzed.

Committee: Dr. Yingbin Hu (Advisor); Dr. Muhammad P. Jahan (Committee Member); Dr. Jinjuan She (Committee Member) Subjects: Mechanical Engineering

Doctor of Philosophy, Case Western Reserve University, 2022, EMC - Mechanical Engineering

Cellular processes strongly regulate the biophysical and biomechanical properties of each blood cell including density, adhesion, and motility, all of which can rapidly change during various healthy and pathological states. To have a better understanding of the cellular processes, an assessment of the biophysical and biomechanical signatures of single cells is needed. Microfluidics has the remarkable ability to mimic the physiological environment of microvasculature, providing a means to better characterize biomechanical and biophysical signatures of blood cells, such as adhesive interactions between different blood cells. As a result, microfluidics has found applications in studies of many different pathophysiologies that stem from altered biomechanical and biophysical properties of blood cells. Despite the advances in microfluidics for the characterization of the biophysical and biomechanical properties of blood cells, the use of microfluidics for mechanistic and mechanobiology studies remains limited due to a lack of standardization and single-cell level imaging. Microfluidics can harness image analysis for standardized characterization of fundamental properties of single cells and reveal subpopulations in heterogeneous cell populations to unfold the mechanobiology and mechanisms of physiology reliably. This dissertation presents the design and engineering of standardized microfluidic systems that utilize image analysis at various technical complexity levels (i.e., manual pixel-to-pixel distance measurements, and automated computer vision with and without machine learning). These microfluidic single-cell level imaging systems demonstrate effective multi-variate measurement of blood cells' biophysical and biomechanical properties and inform the investigation of microcirculatory cellular action mechanisms. The specific aims of this dissertation were: 1) To develop a microfluidic magnetic levitation platform that can perform size and density measurements of single r (open full item for complete abstract)

Committee: Umut Gurkan (Advisor); Bryan Schmidt (Committee Chair); Michael Hinczewski (Committee Member); Ozan Akkus (Committee Member) Subjects: Biomechanics; Biomedical Engineering; Biophysics

Doctor of Philosophy, The Ohio State University, 2021, Biochemistry Program, Ohio State

Cell adhesion is an incredibly important process for multicellular life and is essential for development and maintenance of tissues, immunological response, cancer suppression and mechanosensation. The cadherin superfamily of calcium-dependent cell-adhesion proteins is a large group of diverse glycoproteins which play roles in the processes listed above. The superfamily can be broken down in roughly three subfamilies: classical cadherins, clustered protocadherins, and non-clustered protocadherins. Of those subfamilies, the cadherins in the non-clustered protocadherins have the most diverse range of adhesive functions. To mediate adhesion, cadherins make use of their extracellular domain, which is composed of tandem extracellular cadherin (EC) repeats. Within the repeat sequence, conserved motifs are present encoding for residues which bind three calcium ions at the linker region between repeats, thus imparting rigidity to the extracellular domain. Calcium binding is essential for adhesive function to occur. Here, I focus on several unique non-clustered protocadherins which use their ectodomains to help support the development and tissue morphogenesis of various systems. My work on the 7D-cadherins has focused primarily on cadherin 17 (CDH17) and its homophilic adhesion relevant for the function of the intestinal epithelia. In addition, its sibling, cadherin 16 (CDH16), is expected to mediate similar adhesion in the kidney epithelia. Structures of CDH17 EC1-2 reveal that the 7D-cadherins are unique and distinct from their relatives, the classical cadherins, in that they lack the tryptophan necessary for the conserved strand-swap interaction observed in classical cadherins. Bead aggregation assays using the full ectodomain of CDH17 as well as N- and C-terminal truncation series reveal that CDH17 relies on its EC7, but not its EC1 repeat, to carry out trans adhesion. A mutation in EC1 interferes with aggregation of the full-length ectodomain, indicating the entire e (open full item for complete abstract)

Committee: Marcos Sotomayor (Advisor); Richard Swenson (Committee Member); Ross Dalbey (Committee Member); Kotaro Nakanishi (Committee Member) Subjects: Biochemistry; Biology; Biophysics

Doctor of Philosophy (PhD), Ohio University, 2020, Molecular and Cellular Biology (Arts and Sciences)

Breast cancer cells (BCCs) potentiate hematogenous metastasis by expressing specialized glycosylated proteins and lipids that act as ligands to E-selectin, a cell adhesion molecule expressed on cytokine-activated endothelial cells that line blood vessel walls, under hemodynamic shear stresses. Recently, the functional E-selectin ligand activities of BCCs were shown to be modulated by Snail and Twist transcription factors (TFs) that regulate the epithelial-to-mesenchymal transition (EMT) and the mesenchymal-to-epithelial transition (MET), key processes in metastasis. However, the influence on the functional E-selectin ligand activities of BCCs by physiologically important molecules reported to initiate EMT or MET signaling cascades has yet to be determined. Consequently, the potential for microRNA-200c (miR-200c), an miR overexpressed in the blood of metastatic BC patients, to modify the functional E-selectin ligand activities of BCCs via the MET was determined. Transient overexpression of miR-200c in MDA-MB-231 BCCs induced the MET and flow cytometry analysis revealed that the expression of binding epitopes recognized by E-selectin was significantly higher in these cells compared to cells treated with a negative control miR. Furthermore, cells overexpressing miR-200c had higher functional E-selectin ligand activities, as these cells had significantly lower rolling velocities on E-selectin, significantly higher levels of firm adhesion to E-selectin, and significantly lower detachment from E-selectin in shear flow adhesion assays. Consistent with these findings, gene expression of fucosyltransferase 3 (FUT3) and FUT6, the primary enzymes responsible for the synthesis of functional E-selectin ligands on epithelial BCCs, was significantly higher in cells following miR-200c-induced MET. MiR-200c-induced MET is caused by Zeb1 and Zeb2 TFs, suggesting a novel pathway by which the MET regulates the functional E-selectin ligand activities of BCCs. Over-activation of Notch si (open full item for complete abstract)

Committee: Monica Burdick (Advisor); Douglas Goetz (Committee Member); Fabian Benencia (Committee Member); Amir Farnoud (Committee Member) Subjects: Bioinformatics; Cellular Biology; Molecular Biology

Master of Science (MS), Ohio University, 2020, Biomedical Engineering (Engineering and Technology)

The overexpression of sialofucosylated glycans on cancer cells, which can function as L-selectin ligands, may be involved in promoting cancer metastasis. However, the exact role of sialylated L-selectin ligands of cancer cells and cancer-derived extracellular vesicles (EVs) in promoting metastasis is not fully understood. Evidence suggests circulating tumor cells (CTCs) can interact with white blood cells (WBCs) promoting metastasis under certain circumstances. Breast and colon cancer cells and giant plasma membrane vesicles (GPMVs) isolated from these cancer cells express α2,3 sialylated L-selectin ligands that interact with L-selectin/hFc under physiological flow conditions. In addition, breast and colon cancer cells and GPMVs isolated from these cancer cells interact with WBCs in an L-selectin/ligand mediated manner. Understanding the role of α2,3 sialylated L-selectin ligands of cancer cells and EVs released from cancer cells may help elucidate a mechanism underlying cancer metastasis.

Committee: Monica Burdick (Advisor) Subjects: Biomedical Engineering; Engineering

Doctor of Philosophy, University of Toledo, 2019, Biology (Cell-Molecular Biology)

Cell migration is a critical physiological process that requires the careful cooperation of all cytoskeletal elements within the cell. A multitude of biological events such as embryogenesis, wound healing, tissue maintenance, and cancer metastasis rely upon the ability of the cell to effectively and efficiently migrate. Migration is comprised of four distinct steps: polarization (or reorientation of the cell in the intended direction of migration), protrusion, adhesion, and retraction or contractility. All of these steps are dictated by both internal and external cues, many of which are mechanical in nature. Here, we will primarily focus on the regulation of adhesion and contractility through a structure known as focal adhesions (FA). FA are a complex of proteins formed to allow the cell physical contact between the cytoskeleton and the extracellular matrix (ECM). FA are dynamic structures involved in force transduction and the indirect regulation of the cytoskeleton, including actin and myosin II activity. FA form at the leading edge of cells to stabilize protrusions and disassemble at a later time to allow the cell to retract and progress forward. Many proteins have been identified in the regulation of FA formation; however, the underlying mechanisms that regulate adhesion turnover remain poorly understood. The small family of Rho GTPases are known to play a role in cell migration, including FA dynamics. Several Rho GTPases have been extensively studied in the context of cell migration; however, here we present data showing that the lesser studied RhoG, a Rho GTPase related to Rac, modulates FA dynamics and contractility. Using cell imaging techniques and automated quantification, we have demonstrated that when RhoG expression is silenced (KD), there is a distinct phenotype of increased FA within the cell and a greater number located centrally. Through live imaging, we have shown that this phenotype is the result of increased stability, and therefore longer FA (open full item for complete abstract)

Committee: Rafael Garcia-Mata (Committee Chair); Tomer Avidor-Reiss (Committee Member); Kathryn Eisenmann (Committee Member); Guofa Liu (Committee Member); Kam Yeung (Committee Member) Subjects: Biology; Cellular Biology