Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi anemia (FA) is a genetic disorder characterized by bone marrow (BM) failure. Previous studies suggest that FA patients exhibit alterations in immunologic function. However, it is unclear whether the immune defects are immune cell autonomous or secondary to leucopenia from evolving BM failure. The aim of the current study was to determine whether FA type C deficient (Fancc-/-) macrophages exhibit impaired function and contribute to an altered inflammatory response. In this study, primary peritoneal macrophage function and the inflammatory response of Fancc-/- immune cells after in vivo intraperitoneal (IP) administration of lipopolysaccharide (LPS) were assessed. Fancc-/- peritoneum exhibit normal macrophage distribution at baseline. However, Fancc-/- macrophages exhibit reduced adhesion both on fibronectin and endothelial cells, impaired migration toward monocyte chemotactic protein-1 (MCP-1) and macrophages-colony stimulating factor (M-CSF), and altered phagocytosis of E.coli and ImmunoglobulinG (IgG)-labeled latex beads compared to WT. An altered F-actin reorganization and impaired activation of RhoA were observed in Fancc-/- macrophages. After single LPS injection IP, Fancc-/- mice exhibited decreased macrophage recruitment, reduced peripheral inflammatory monocytes and impaired myeloid colony formation in presence of M-CSF. Upon M-CSF stimulation, Fancc-/- BM derived macrophages (BMDM) showed a decreased phosphorylation of AKT and ERK compared to WT, leading to reduced proliferation. Collectively, these data suggest that Fancc-/- macrophages and subsequent defects in adhesion, migration, phagocytosis, and recruitment in vivo. These data also support a Fancc-/- macrophage cells autonomous defect predisposing to an altered inflammatory response.

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This research examines the role of actin cytoskeleton filaments in chondroinduction by cyclic hydrostatic pressurization. A chondroinductive hydrostatic pressurization system was developed and characterized. A pressure of 5 MPa at 1 Hz frequency, applied for 7200 cycles (4 hours intermittent) per day, induced chondrogenic differentiation in C3H10T1/2 cells while 1800 cycles (1 hour intermittent) did not induce chondrogenesis. Quantitative analysis of chondrogenesis was determined as sulfated glycosaminoglycan synthesis and rate of collagen synthesis while qualitative analysis was obtained as Alcian Blue staining and collagen type II immunostaining. Actin disruption using 2 uM Cytochalasin D inhibited the enhanced sGAG synthesis in the chondroinductive hydrostatic pressurization environment and significantly inhibited rate of collagen synthesis to the mean level lower than that of the non-pressurized group. These results suggest an involvement of actin cytoskeleton filaments in mechanotransduction of cyclic hydrostatic pressure.

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c-Cbl functions as a multifunctional adaptor and an E3 ubiquitin protein ligase. Several studies have shown that c-Cbl is involved in cytoskeleton-mediated events, but the molecular mechanisms linking c-Cbl to cytoskeletal rearrangements remain to be elucidated. Our previous results indicated that c-Cbl facilitates spreading and migration of v-Abl-transformed NIH 3T3 fibroblasts and suggested that small GTPases play important roles in the cytoskeletal effects of c-Cbl in this system. To elucidate the individual contributions of small GTPases to these effects, we assessed the roles of endogenous Rac1, RhoA and Rap1 in the c-Cbl-dependent spreading and migration of v-Abl-transformed fibroblasts overexpressing c-Cbl, using RNAi. Furthermore, since it has been shown that Rap1 can act as an upstream regulator of Rac1 in inducing cell spreading, we analyzed the interplay between Rap1 and Rac1 in the signaling pathways connecting c-Cbl to the cytoskeletal events. Our results indicate that Rac1 is essential for cell migration and spreading, whereas activation of RhoA exerts a negative effect. We have also shown that Rap1 is essential for cell spreading, although not for migration in our experimental system. Furthermore, we provide evidence that Rap1 is located upstream of Rac1 in one of the signaling pathways that regulate c-Cbl-facilitated cell spreading. Overall, our findings are consistent with the model describing the connection of c-Cbl to the cytoskeletal rearrangements via two pathways, one of which is mediated by PI3K and Rac1, and the other, by CrkL/C3G, Rap1 and Rac1. A major biological feature of glioma is the ability to invade normal brain tissue. The molecular mechanisms of glioma invasion are involved in multiple biological processes which are primarily associated with cytoskeleton-mediated events including adhesion, migration, degradation of extra cellular matrix (ECM). Biological functions of c-Cbl in glioma have not been elucidated. In this study, we examined biological roles of c-Cbl using RNAi-mediated depletion of endogenous c-Cbl and stably c-Cbl expressing glioma cells generated by lentiviral transduction and showed that c-Cbl increases invasion through degradation of ECM by upregulation of MMP2 but not through migration, adhesion, or growth of SNB19, a grade IV glioblastoma cell line. / Microbiology and Immunology / Accompanied by two .avi videos

The neuronal cytoskeleton is vital for normal growth and differentiation of the nervous system, as well as for maintenance of the normal intracellular environment. Without it, major functional deficits occur due to interference with cellular transport of membrane components, proteins and neurotransmitter substances and as a result, inadequate maintenance of the distal axon occurs. Through the study of both nervous tissue and primary neuronal culture, specific cytoskeletal markers have been found to predominate in axonal or dendritic processes, as well as in different stages of neuronal development. In vitro study of neuroblastoma cell lines has also been utilized to develop hypotheses of neuronal development. These hypotheses attempt to explain the appearance of certain cytoskeletal elements, such as phosphorylated neurofilament proteins, in relationship to functional maturity of the neuron. We used SY5Y human neuroblastoma cells as an in vitro model of neurotoxicity to investigate cytoskeletal changes that may occur from the exposure of the nervous system to a known neurotoxicant. Cells were differentiated with either retinoic acid (RA) or dibutyryl cyclic adenosine monophosphate (dbcAMP) and 3-isobutyl-1- methyl-xanthine (IBMX). Differentiation was based morphologically on the appearance of neuritic processes in a majority (>50%) of the cells. Using the peroxidase-antiperoxidase technique, cells were labeled with monoclonal antibodies to cytoskeletal proteins (phosphorylated neurofilament, microtubule associated protein 2, vimentin and low molecular weight neurofilament protein) either specific for axonal markers or linked to stages in neuronal development. Staining patterns were compared to undifferentiated cells using the same protocol. There were no differences in staining patterns found between methods of differentiation or between differentiated cells and undifferentiated controls. Axonal markers of differentiation, defined as phosphorylated neurofilament immunopositivity, were only detected in cells exposed to retinoic acid for 9 days. Once these studies were completed, both differentiated and undifferentiated cultures were exposed to acrylamide as an example of a neurotoxicant with known cytoskeletal effects. Cells were fixed and stained after the observation of cellular swelling 24 hours post acrylamide treatment. In spite of obvious alterations in morphology in unstained cells in culture, the cytoskeletal staining pattern was unchanged after acrylamide treatment. We conclude that there is no difference in the cytoskeletal immunoreactivity of SY5Y neuroblastoma cells when differentiated with RA or dbcAMP/IBMX after three days. Retinoic acid differentiated cells, however, do develop immunoreactivity to axonal markers of differentiation after nine days of treatment. We also conclude that acrylamide does not affect the cytoskeletal structure of SY5Y neuroblastoma cells in undifferentiated or in RA or dbc AMP differentiated cells at the time and concentration tested. / Master of Science

Cell-level and tissue-level mechanical properties are key to healthy biological functions, and many diseases and disorder arise or progress due to altered cell and tissue mechanics. Pulse electric field (PEFs), which employ intense external electric fields to cause electroporation, a phenomenon characterized by increased cell membrane permeability, also can cause significant changes to cell and tissue mechanics. Here, we investigate the mechanics of brain and brain cancer cells, specifically focusing on how PEFs impact cell mechanics and PEF-induced blood-brain barrier disruption. In our first study, we investigate single-cell mechanical disruption of glioblastoma cells after reversible electroporation using Nanonet Force Microscopy (NFM). A precise network of extracellular-matrix mimicking nanofibers enabled cell attachment and contraction, resulting in measurable fiber deflections. Cell contractile forces were shown to be temporarily disrupted after reversible electroporation, in an orientation and field-dependent manner. Furthermore, we found that cell response is often a multi-stage process involving a cell-rounding stage, biphasic stage, and a cell re-spreading stage. Additionally, cell viability post-PEFs was orientation-dependent. In another study, we investigated the mechanical properties of brain cancer for various-grade glioma cells (healthy astrocytes, grade II, grade III, and grade IV (glioblastoma) cells). A microfluidic constriction channel caused cell deformation as cells, driven by hydrostatic pressure, entered a narrow constriction. Finite element models of cell deformation and a neural network were used to convert experimental results (cell entry time and cell elongation within the channel) into elastic modulus values (kPa). We found that the that low-grade glioma cells showed higher stiffnesses compared to healthy and grade IV glioma cells, which both showed similar values. These results warrant future studies to investigate these trends further. PEFs can induce Blood-brain barrier (BBB) disruption, an effect we studied using a multiplexed, PDMS microdevice. A monolayer of human cerebral endothelial cells on a semi-permeable membrane was used to model the BBB, and permeability was assessed by the diffusion of a fluorescent dye from an upper to lower channel. A custom tapered channel and branching channel design created a linear gradient in the electric field within the device that enabled six electric field strengths to be tested at once against two unexposed (control) channels. Normalization of permeability by the control channels significantly removed experimental noise. We found that after high-frequency bipolar irreversible electroporation (HFIRE) electric pulses, permeability transiently increased within the first hour after electroporation, in a voltage- and pulse-number dependent manner. However, we found significant electrofusion events after pulsing at high voltages, which reduced monolayer permeability below baseline values. This device enables efficient exploration of a wide range of electroporation parameters to identify the optimal conditions for blood-brain barrier disruption. In another blood-brain barrier study, we incorporate dense, polystyrene nanofiber networks to create ultra-thin, ultra-porous basement-membrane-mimics for In vitro blood-brain barrier models. Fiber networks are fabricated using the non-electrospinning Spinneret-based Tunable Engineered Parameters (STEP) technique. Endothelial cells cultured on one side of the fiber network are in close contact with supporting cell types (pericytes) cultured on the backside of the fibers. Contact-orientation co-cultures have been shown to increase blood-brain barrier integrity, and our nanofiber networks increase the physiological realism of basement-membrane mimics for improve modeling. Finally, we investigate how cell viability post-electroporation is impacted by cell morphology. The impact of cell morphology (shape and cytoskeletal structure) on cell survival after electroporation is not well understood. Linking specific morphological characteristics with cell susceptibility to electroporation will enhance fundamental knowledge and will be widely useful for improving electroporation techniques where cell viability is desirable (gene transfection, electrofusion, electrochemotherapy) or where cell viability is undesirable (tumor ablation, cardiac ablation). Precise control of cell shape and orientation enabled by nanofiber scaffolds provides a convenient and expedient platform for investigating a wide variety of factors (morphological and experimental) on cell viability. Altogether, these investigations shed new light on cell mechanical changes due to disease and pulsed electric fields, and suggest opportunities for improving brain cancer therapies. / Doctor of Philosophy / In biology, structure and function are interrelated. Cells and tissue have structures that enable them to perform their proper function. In the case of disease, cell and tissue properties are altered, leading to dysfunction. Alternatively, healthy structures sometime hinder effective treatments, and therefore can be therapeutically disrupted to improve treatments. In this study, we investigate single-cell and multi-cellular mechanical change due to disease or after pulsed electric fields (PEFs), with a specific focus on the brain. Pulsed electric fields (PEFs) use electrodes to deliver short, intense pulses of electrical energy to disrupt cell membranes and change cell mechanics. We studied as single-cell contractility, cancer cell stiffness, and blood-brain barrier (BBB) disruption by PEFs. We found that PEFs cause significant change to cell shape and mechanics, and can disrupt the BBB. By studying several grades of brain cancers, we found that low-grade brain cancer (gliomas) showed increased stiffness compared to healthy and highly diseased (grade IV) cells. To mimic the BBB, we used microfluidic devices to grow specialized brain cells (endothelial cells) on permeable membranes and nanofibers networks and showed that these devices can mimic structures found in animals/humans. Finally, we studied how cell properties (such as shape) determine whether cells will survive PEFs. Taken together, our investigations improve the understanding of brain mechanics during disease and after PEFs, and suggest the usefulness of PEFs for improved brain cancer therapies.