Browsing by Author "Forsten-Williams, Kimberly"

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    Characterization of Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) interaction with the Bovine Aortic Endothelial (BAE) cell surface: Examination of the Role of Heparan Sulfate Proteoglycans (HSPG)
    Parghi, Nirav (Virginia Tech, 1998-07-15)
    Insulin-like growth factor binding proteins (IGFBPs) are known to be important modulators of the insulin-like growth factor (IGF-I). However, their precise role is as yet unclear. Further, recent studies have indicated that IGFBP-3 has a receptor mediated growth inhibitory response of its own. In the present study, we quantified the binding characteristics of IGFBP-3 to bovine aortic endothelial (BAE) cells. Binding studies at 4 oC were conducted and a specific binding curve for IGFBP-3 was obtained. IGFBP-3 was found to bind with an equilibrium dissociation constant (KD) value of 3.1 x 10-10 M. The role of heparan sulfate proteoglycans (HSPG) in the IGFBP-3 binding mechanism was also examined. It was seen that inactivation of the cell surface HSPGs with 75 mM sodium chlorate did not affect IGFBP-3 binding. Further, there have been reports of inhibition of IGFBP-3 binding by heparin in the media. Hence, the most probable interaction of HSPG with IGFBP-3 occurs in the extracellular region, with soluble HSPGs acting as receptors for IGFBP-3 and decreasing the net cell associated ligand receptor interaction. This is likely, since IGFBP-3 is known to possess a heparin binding domain. Simultaneous introduction of IGF-I and IGFBP-3 into the extracellular media decreased IGFBP-3 binding to the cell surface, which might imply that IGF-I and IGFBP-3 regulate each other's action.
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    Characterization of proteins and tissue remodeling components in porcine aqueous humor
    Chandran, Jayanth Sankrit (Virginia Tech, 2000-08-22)
    Connective tissue remodeling is an important area of study in biomedical engineering with respect to cancer and wound healing. Tissue remodeling components may be involved in the pathogenesis of open-angle glaucoma. Risk factors for open angle glaucoma include increased intraocular pressure (IOP), male gender, and advanced age. In a 1963 study, the hormone relaxin decreased IOP in the human eye through a mechanism that may involve the up-regulation of tissue remodeling matrix metalloproteinases (MMPs). The effects of age and gender on MMP and protein activity in porcine aqueous humor were determined in this study to identify correlations existing between MMP activity and glaucoma risk factors. Gelatin zymography identified MMPs at 66 kD and approximately 105 kD. The concentration of the 66 kD band compared to human MMP-2 standard was 0.22 ± 0.06 ng/ml for the adult female (AF) samples and 0.28 ± 0.04 ng/ml for the juvenile samples. This difference in concentration was statistically significant (p < 0.05). The concentration of the protease migrating to 66 kD was statistically independent of gender. Casein zymograms identified two non-MMP proteinases at 51 kD and 80 kD. The average total protein concentration for all aqueous humor samples was 2.54 ± 0.89 mg/ml. The mean IgG, transferrin, and albumin concentrations for all aqueous humor samples was 11.4 ± 4.2 mg/ml, 17.11 ± 6.8 mg/ml, and 78.0 ± 26.3 mg/ml respectively. Results from these experiments establish baseline levels of MMP and protein activity, allowing for identification of potential changes caused by relaxin in tissue culture studies.
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    Characterization of the Recombinant Human Factor VIII Expressed in the Milk of Transgenic Swine
    Hodges, William Anderson (Virginia Tech, 2001-02-19)
    Factor VIII is a protein which has therapeutic applications for the treatment of Hemophilia A. Its deficiency, either qualitative or quantitative, results in Hemophilia A, a disorder affecting approximately 1 in 10,000 males. Currently, FVIII replacement therapy uses FVIII derived from plasma or cell culture. The current cost of this therapy is in excess of $150,000 per patient per year. Thus, alternative sources that are more economical are attractive. The present work focuses upon the characterization of recombinant FVIII (rFVIII) made in the milk of transgenic pigs. Two dimensional western analysis of rFVIII obtained from pig whey showed a range of FVIII species having different isoelectric points (pI) consistent with diverse glycosylation patterns. The pI of these diverse FVIII populations were accurately predicted using theoretical calculations based upon primary protein structure as variable biantennary glycosylation patterns having 0, 1, or 2 sialic acid groups present. Kinetic limitations in the adsorption of rFVIII to anion exchange media due to the nature of the complex milk environment were observed. rFVIII was purified quantitatively using batch equilibration of whey with DEAE Sepharose. This material showed proteolytic processing that was very similar to FVIII obtained from human plasma. Based upon these results, it was postulated that a dissociation of the light (A3C1C2) and heavy (A1A2B) chain due to a lack of vWF may be responsible for the low FVIII activity.
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    Chronic Shear Stress Effects on Endothelial Cell Response
    Elhadj, Selim (Virginia Tech, 2001-12-10)
    The overall focus of this dissertation is on how chronic shear stress alters the synthesis and secretion of important regulatory molecules by endothelial cells. Our hypothesis was that inclusion of chronic pulsatile shear stress in our model would lead to changes in endothelial cell release of regulatory molecules. We distinguished between high arterial shear stresses and low venous shear stresses and used static cell cultures as reference. The first part of this research thus entailed the complete characterization of the flow dynamics in our experimental biomechanical model. Cell stretching can have a physiological effect on endothelial cells; hence we implemented a laser based optical technique for real time strain measurement of the growth fibers used in our culture system, and found that no significant strains were occurring during shear treatment. After characterization of the mechanical environment of the cells, we focused the scope of our research on metabolism of proteoglycans and insulin-like growth factor-I (IGF-I) and related IGF binding proteins (IGFBPs) in bovine aortic endothelial cells cultured under chronic pulsatile shear. We found that shear stress increased the release of proteoglycans and significantly altered proteoglycans distribution. We also found that there was an inverse relationship between the shear level treatment used to obtain the purified proteoglycans from endothelial cells and their potency in inhibiting coagulation. IGF-I release and message (IGF-I mRNA) was decreased at high shear stress compared to low shear stress. Further, the levels found under shear were significantly greater than those observed in the static cell culture model. IGFBPs released were also significantly increased by shear. This research thus establishes a link between chronic pulsatile shear stress and the metabolism of both primary (IGF-I) and secondary (IGFBPs, proteoglycans) regulators of vascular cell activity. The improved realism of our experimental biomechanical model has proved to be a valuable tool in improving the relevance of this study to vascular research. Ultimately, this research calls for further investigation in the molecular mechanisms underlying the phenomenological effects documented, which may help in understanding fundamental aspects in cardiovascular disease and its link to hemodynamics but our work is an important first step.
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    Complex receptor-ligand dynamics control the response of the VEGF system to protease injury
    Forsten-Williams, Kimberly; Kurtagic, Elma; Nugent, Matthew A. (2011-10-21)
    Background Vascular homeostasis and response to injury are dependent on the coordinated activity of growth factors such as vascular endothelial growth factor-A (VEGF). VEGF signaling is mediated by VEGF receptors 1 (VEGFR1) and 2 (VEGFR2). VEGF also binds to extracellular matrix (ECM) and neuropilin (NP), a cell surface glycoprotein that enhances VEGF binding to VEGFR2 while inhibiting VEGF-VEGFR1 interactions. Proteases such as neutrophil elastase release VEGF bound to ECM; however, this results in proteolytic processing of VEGF to a smaller species termed VEGF fragment (VEGFf). We hypothesized that the generation and presence of VEGFf would have significant effects on the binding distribution of VEGF. Results We show that VEGFf, unlike VEGF, does not bind ECM, fibronectin, or NP-1. Using computational simulations, we find that excess VEGFf can lead to increased binding of VEGF to VEGFR2 through VEGFf binding to VEGFR1 and subsequent liberation of NP-1. We show experimentally that VEGF-induced migration has a biphasic response to conversion of VEGF to VEGFf. Simulations suggest that a simple change in VEGFR1 or VEGFR2 complexes are unlikely to be responsible and that a more complex integration of signals is more likely involved. Conclusions These findings suggest that proteolytic damage at sites of tissue injury and inflammation has the potential to modulate the VEGF system through a complex process and highlight the need for quantitative analysis to reveal mechanisms of growth factor control.
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    Composite Films for Modifying Evanescent Wave Characteristics in Long-Period Grating Biosensors
    Martin, Jennifer E. (Virginia Tech, 2001-02-09)
    Biosensors are detection devices that couple biological recognition elements to physiochemical transducers to generate quantifiable signals. Immunosensors are biosensors that use antibodies as the recognition element. The highly specific nature of antibody-antigen binding is exploited to create immunosensors that are sensitive to analytes in complex mixtures and demonstrate a rapid response. Fiber optical immunosensors based on long-period gratings have limited sensitivity at the refractive index of ordinary aqueous solutions (~1.33). A composite film was designed to raise the local refractive index of the sensor, thus increasing sensitivity. Titanium dioxide deposition raised the refractive index of the sensor to ~1.42. Bovine serum albumin was immobilized onto a dextran hydrogel and attached to the LPG element via reductive amination. The thickness of the hydrogel was estimated to be 500 nm using Environmental Scanning Electron Microscopy. The affinity film was probed by an evanescent wave to detect changes in refractive index due to the binding of anti-BSA IgG. Under these conditions, the sensor yielded a signal ratio of approximately 10-4 refractive index units per nm signal. Reproducible binding was shown over multiple exposures, with no cross reactivity for non-specific antibodies and other proteins. Anti-BSA IgG (20 µg/mL) in whole serum was recycled through the fiber holder with an accompanying peak wavelength shift that averaged 2 nm on an Optical Spectrum Analyzer with a noise level of 0.1 nm. The BSA affinity film was regenerated 50 times and showed a baseline shift of -1.3 nm.
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    Effects of receptor clustering on ligand dissociation kinetics: Theory and simulations
    Gopalakrishnan, Mahima; Forsten-Williams, Kimberly; Nugent, Matthew A.; Täuber, Uwe C. (Cell Press, 2005-12-01)
    Receptor-ligand binding is a critical first step in signal transduction and the duration of the interaction can impact signal generation. In mammalian cells, clustering of receptors may be facilitated by heterogeneous zones of lipids, known as lipid rafts. In vitro experiments show that disruption of rafts significantly alters the dissociation of fibrbroblast growth factor-2 (FGF2) from heparan sulfate proteoglycans (HSPGs), co-receptors for FGF-2. In this article, we develop a continuum stochastic formalism to address how receptor clustering might influence ligand rebinding. We find that clusters reduce the effective dissociation rate dramatically when the clusters are dense and the overall surface density of receptors is low. The effect is much less pronounced in the case of high receptor density and shows nonmonotonic behavior with time. These predictions are verified via lattice Monte Carlo simulations. Comparison with FGF-2-HSPG experimental results is made and suggests that the theory could be used to analyze similar biological systems. We further present an analysis of an additional cooperative internal-diffusion model that might be used by other systems to increase ligand retention when simple rebinding is insufficient.
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    Effects of receptor clustering on ligand dissociation kinetics: Theory and simulations
    Gopalakrishnan, Mahima; Forsten-Williams, Kimberly; Nugent, M. A.; Täuber, Uwe C. (Cell Press, 2005-12-01)
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    Endothelial Cell Capture of Heparin-Binding Growth Factors under Flow
    Zhao, Bing; Zhang, Changjiang; Forsten-Williams, Kimberly; Zhang, Jun; Fannon, Michael (PLOS, 2010-10)
    Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule's fate, are difficult to interpret using traditional approaches. In this work, we analyzed and predicted growth factor capture under flow using computer modeling and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process was desired. The experimental module consisted of a bioreactor with synthetic endothelial-lined hollow fibers under flow. The physical design of the system was incorporated into the model parameters. The heparin-binding growth factor fibroblast growth factor-2 (FGF-2) was used for both the experiments and simulations. Our computational model was composed of three parts: (1) media flow equations, (2) mass transport equations and (3) cell surface reaction equations. The model is based on the flow and reactions within a single hollow fiber and was scaled linearly by the total number of fibers for comparison with experimental results. Our model predicted, and experiments confirmed, that removal of heparan sulfate (HS) from the system would result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicted a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that should prove advantageous for biologicals or drug delivery investigations.
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    Investigation of Cellular Responses Activated by Mechanical Compression in Equine Chondrocytes: Device Design, Construction and Testing
    Cassino, Theresa R. (Virginia Tech, 2006-01-17)
    The metabolic activity of cartilage cells (chondrocytes) is regulated by mechanical forces which act on them. Chondrocytes can respond to these forces through synthesis or degradation of extracellular matrix and changes in gene expression. The overall objective of this study was to investigate the effects of mechanical compression on gene regulation, proteoglycan (PG) synthesis and activation of signaling pathways. To achieve this goal a simple oscillatory displacement controlled device was designed to provide uniaxial unconfined strain to cell constructs. Static compression and dynamic compression with various waveforms are utilized with a stroke range of 0.25 mm to 4 mm and a frequency range of 0.1 Hz to 3 Hz. Poly-L-lactic acid (PLLA/)alginate disks and alginate disks with equine chondrocytes embedded in them were developed and showed unchanged viability for 24 hr under static and dynamic compression. Testing to relate the strains applied to forces experienced in cell constructs was completed and the simple procedure outlined for companion use with our device. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) revealed changes in expression of collagen II and matrix metalloproteinase-3 under dynamic compression for 24 hr. Equine chondrocytes compressed for 48 hr showed lower PG synthesis for both static and dynamic compression when compared to uncompressed samples in replicate experiments. Repeatability of this experiment was problematic possibly due to decreased viability and inefficient extraction. Different patterns of extracellular signal regulated kinase (ERK) activation with time were found for uncompressed and compressed samples (static at 15% strain and dynamic at 15% strain, 1 Hz) and protein kinase B (also called Akt) was not regulated by compression. Results from experiments involving frequency and strain for dynamic compression were inconclusive. These studies show that regulation of gene expression, PG synthesis and intracellular signaling can be studied with our device but optimization of the experimental procedure is still needed. To our knowledge these studies are the first to show these types of studies utilizing equine chondrocytes. Despite issues encountered, our studies provide valuable insights into the effects of compression on equine chondrocytes and detail a simple device for use in a wide variety of compression studies.
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    Investigations of Insulin-Like Growth Factor I Cell Surface Binding: Regulation by Insulin-Like Growth Factor Binding Protein-3 and Heparan Sulfate Proteoglycan
    Balderson, Stephanie D. (Virginia Tech, 1997-05-22)
    The primary aim of this text is to gain insight on how cellular activation by a insulin-like growth factor (IGF-I), in the presence of insulin-like growth factor binding protein-3 (IGFBP-3), is influenced by heparan sulfate proteoglycans (HSPG). Initial research will be presented, assumptions and hypotheses that were included in the development of mathematical models will be discussed, and the future enhancements of the models will be explored. There are many potential scenarios for how each component might influence the others. Mathematical modeling techniques will highlight the contributions made by numerous extracellular parameters on IGF-I cell surface binding. Tentative assumptions can be applied to modeling techniques and predictions may aid in the direction of future experiments. Experimentally, it was found that IGFBP-3 inhibited IGF-I Bovine Aortic Endothelial (BAE) cell surface binding while p9 HS slightly increased IGF-I BAE cell surface binding. IGFBP-3 has a higher binding affinity for IGF-I (3 x 10-9 M) than p9 HS has for IGF-I (1.5 x 10-8 M) as determined with cell-free binding assays. The presence of p9 HS countered the inhibiting effect of IGFBP-3 on IGF-I BAE cell surface binding. Although preliminary experiments with labeled p9 HS and IGFBP-3 indicated little to no cell surface binding, later experiments indicated that both IGFBP-3 and p9 HS do bind to the BAE cell surface. Pre-incubation of BAE cells with either IGFBP-3 or p9 HS resulted in an increase of IGF-I BAE cell surface binding . There was a more substantial increase of IGF-I surface binding when cells were pre-incubated with IGFBP- 3 than p9 HS. There was a larger increase of IGF-I BAE cell surface binding when cells were pre-incubated with p9 HS than when p9 HS and IGF-I were added simultaneously. This suggests that IGFBP-3 and p9 HS surface binding plays key role in IGF-I surface binding, however, p9 HS surface binding does not alter IGF-I surface binding as much as IGFBP-3 surface binding seems to. Experimental work helps further the understanding of IGF-I cellular activation as regulated by IGFBP-3 and p9 HS. Developing mathematical models allows the researcher to focus on individual elements in a complex systems and gain insight on how the real system will respond to individual changes. Discrepancies between the model results and the experimental data presented indicate that soluble receptor inhibition is not sufficient to account for experimental results. The alliance of engineering analysis and molecular biology helps to clarify significant principles relevant to the conveyance of growth factors into tissue. Awareness of the effects of individual parameters in the delivery system, made possible with mathematical models, will provide guidance and save time in the design of future therapeutics involving growth factors.
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    Ligand rebinding: self-consistent mean-field theory and numerical simulations applied to surface plasmon resonance studies
    Gopalakrishnan, Mahima; Forsten-Williams, Kimberly; Cassino, T. R.; Padro, L.; Ryan, T. E.; Täuber, Uwe C. (Springer, 2005-10-01)
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    Ligand-induced coupling versus receptor pre-association: cellular automaton simulations of FGF-2 binding
    Gopalakrishnan, Mahima; Forsten-Williams, Kimberly; Täuber, Uwe C. (Academic Press – Elsevier, 2004-03-21)
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    Ligand-receptor binding kinetics in surface plasmon resonance cells: A Monte Carlo analysis
    Carroll, J.; Raum, M.; Forsten-Williams, Kimberly; Täuber, Uwe C. (2016-09-14)
    Surface plasmon resonance (SPR) chips are widely used to measure association and dissociation rates for the binding kinetics between two species of chemicals, e.g., cell receptors and ligands. It is commonly assumed that ligands are spatially well mixed in the SPR region, and hence a mean-field rate equation description is appropriate. This approximation however ignores the spatial fluctuations as well as temporal correlations induced by multiple local rebinding events, which become prominent for slow diffusion rates and high binding affinities. We report detailed Monte Carlo simulations of ligand binding kinetics in an SPR cell subject to laminar flow. We extract the binding and dissociation rates by means of the techniques frequently employed in experimental analysis that are motivated by the mean-field approximation. We find major discrepancies in a wide parameter regime between the thus extracted rates and the known input simulation values. These results underscore the crucial quantitative importance of spatio-temporal correlations in binary reaction kinetics in SPR cell geometries, and demonstrate the failure of a mean-field analysis of SPR cells in the regime of high Damk\"ohler number Da > 0.1, where the spatio-temporal correlations due to diffusive transport and ligand-receptor rebinding events dominate the dynamics of SPR systems.
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    Microphysiometry Studies of Rapid Binding of Insulin-Like Growth Factor I by Parental and Transfected Mammary Epithelial Cell Lines
    Robinson, Rose Marie (Virginia Tech, 1998-10-16)
    Breast cancer is a leading cause of cancer death of women in the U.S. today. Members of the family of insulin-like growth factors (IGFs) are proposed to play a major role in the development and subsequent uncontrolled proliferation of breast cancer cells. Insulin-like growth factor-I (IGF-I) is known to be a potent mitogen for mammary epithelial cells. IGF-I acts by binding to cell surface receptors, thereby stimulating a cascade of events leading to cell division. In the interest of interrupting the effect of IGF-I on cancerous mammary epithelial cells, an understanding of how IGF-I behaves in the presence of other extracellular components is needed. This study examines the IGF-I response of SV40-IGF-I, an immortalized bovine mammary epithelial cell line which secretes IGF-I constitutively. The microphysiometer allows real-time sampling of cellular activity by measuring the excretion of protons from a sample of cells stimulated by IGF-I binding. The contributions of other factors in enhancing or suppressing stimulation can be compared by examining the pH response of cells exposed to IGF-I in the presence of these factors. We present data showing the stimulatory effect of IGF-I in a dose dependent manner on the SV40-IGF-I cell line. In addition, we compare IGF-I stimulation with stimulation by long R3IGF-I, a substituted analogue of IGF-I having a reduced binding affinity for the IGF binding proteins. We examine the effect of insulin-like binding protein-3 (IGFBP-3) both in the presence and absence of IGF-I, finding no IGF-I independent effect in the rapid binding experiment and no effect on stimulation of IGFBP-3 pre-incubated cells by subsequent IGF-I challenge. This is of particular interest due to recent work demonstrating an IGF-independent IGFBP-3 response in a number of cell lines. Binding studies to correlate with the rapid binding stimulation show binding of the IGFBP-3 molecule with high affinity to a small number of surface receptors on the SV40-IGF-I cell. Analysis of the extracellular environment and the components contributing to the binding of IGF-I to the cell membrane receptor will provide information for the development of interventions to slow or interrupt the process of IGF-I binding and therefore cancer growth. Optimization of the Cytosensor(r) Microphysiometer System for the (transfected) SV40-IGF-I and the (parental) MAC-T cell lines was achieved to continue comparison studies of autocrine and paracrine stimulation of bovine mammary epithelial cells by IGF-I. This work was supported by the Whitaker Foundation Biomedical Engineering Grant.
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    New Catalysts for Hydroprocessing: Molybdenum and Tungsten Phosphide
    Clark, Paul Alexander (Virginia Tech, 1998-10-14)
    This dissertation describes the preparation and application of a novel class of hydroprocessing catalysts, transition metal phosphides. Concentration was placed on molybdenum and tungsten monophosphides because of the importance of these elements in standard sulfidic hydrotreating catalysts. Transition metal phosphides exist over a wide range of stoichiometry, and their properties have a great deal of variation, ranging from phosphorus poor compounds with metallic electrical properties to phosphorus rich compounds with semiconducting or insulating properties. The x-ray diffraction patterns of the phosphides studied here were unchanged under the conditions of catalytic hydroprocessing, demonstrating their stability toward the hydroprocessing conditions and allowing study of their intrinsic catalytic properties. Materials were prepared in bulk form, supported on alumina, and supported on silica. The mechanism of hydrodenitrogenation on MoP/SiO2 and WP/SiO2 catalysts was investigated by comparison of hydrodenitrogenation reactions of pyridine, piperidine, n-pentylamine, tert-pentylamine, and neo-pentylamine.
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    Preparation of Heparin Surface for Quantification of Fibroblast Growth Factor-2 (FGF-2) Binding Using Surface Plasmon Resonance (SPR)
    Kirtland, David Rand (Virginia Tech, 2005-05-04)
    A mixed self assembling monolayer (mSAM) chip with attached heparin was developed to analyze heparin-protein interactions using a Reichert Inc, SR7000, surface plasmon resonance (SPR) instrument. The heparin was attached via streptavidin-biotin linkage where the streptavidin was covalently coupled to the mSAM and biotinylated heparin bound to it. These chips were then used to quantify the interactions of fibroblast growth factor-2 (FGF-2) with the surface bound heparin. Kinetic rate constants of association and disassociation were calculated. The association data of FGF-2 with heparin was fit to a single compartment, well-mixed model as the data did not exhibit mass transfer limitations. The results suggested that rebinding was prevalent and observed disassociation rates differed significantly in the presence of competing soluble heparin during disassociation. Our results indicate that the Reichert instrument and mSAM chips can be used to analyze heparin-protein interactions but that a careful protocol, outlined in this thesis, should be followed to obtain optimal data.
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    Purification and Characterization of Proteoglycan from Bovine Aortic Endothelial Cells Conditioned Media, and its Interaction with Basic Fibroblast Growth Factor (bFGF)
    Wang, Ningling III (Virginia Tech, 1997-08-27)
    Cultured bovine aortic endothelial (BAE) cells were found to synthesize and secrete heparan sulfate proteoglycans (HSPG), which bound basic fibrobalst growth factor (bFGF). bFGF is a known mitogen for vascular smooth muscle cells, and is indicated to have a role in some proliferative vascular disorders. In the present study, we have purified proteoglycans from BAE cells conditioned media (BAE PG), and further separated the PG into two fractions, PG-I and PG-II, by ion exchange chromatography on a Q-Sepharose column using a linear salt gradient (0.15 M to 1.2 M). PG-I and PG-II elute at 0.85M salt and 0.1M salt respectively. BAE PG is primarily composed of heparan sulfate, which is accessible to the digestion of Heparinase I/III and nitrous acid treatment; and a small amount of chondroitin sulfate, which can be digested by Chondroitinase ABC. Gel filtration chromatography (Sepharose CL-2B and CL-4B columns) showed that BAE PG consisted of two different sized peaks, and had an average molecular weight of approximately 5 x 10⁵ Da. SDS-PAGE with silver staining indicated that BAE PG had two core proteins with estimated sizes of 300kDa and 320kDa, which corresponded to the core protein of PG-I and PG-II respectively. Western blotting with anti-perlecan primary antibody recognized the core proteins of BAE PG. Size exclusion chromatography (Sepharose CL-6B column) following β-elimination showed that BAE PG had GAG chains with an estimated size less than 2 x 10⁵ Da. A protocol to investigate the cell free binding of bFGF with purified BAE PG was established using the BioRad Bio-Dot apparatus - the cationic filtration assay (CAFAS). Using a simple monovalent binding model, we obtained values for the equilibrium dissociation constant, KD, of (1.6 ± 0.8) x 10⁻¹⁰ M; the dissociation rate constant, kr, of 0.01 min⁻¹; the association rate constant, kf, of 6.2 x 10⁷ M⁻¹min⁻¹ and the total binding sites of the proteoglycan, RT, of 0.1~0.2 (# of site)/(molecule of PG). The comparison of experimental data with model predictions indicates that when the number of binding sites provided by the PG is similar or greater than that of bFGF, the monovalent binding model is valid. When the number of binding sites is less than that of bFGF, one possibility is that the binding might not be the described simple monovalent reaction, and bFGF might bind to the PG as dimers or oligomers. In addition, a model is proposed for BAE PG, in which 5 ~ 10 BAE PG molecules form a high affinity binding site for bFGF. Experimentally we find that exogenous heparan sulfate competes with BAE PG for binding with bFGF, while chondroitin sulfate seems to facilitate the binding. This result may be a useful consideration when we want to design possible pharmaceutical compounds.
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    Recombinant Equine Interleukin-1 Induced Models of Equine Joint Disease
    Takafuji, Vivian Ann (Virginia Tech, 2003-11-13)
    Osteoarthritis (OA) is a debilitating disease of joints that afflicts horses of all ages and breeds and can result in lameness, suboptimal performance, and decreased quality of life. The pro-inflammatory cytokine interleukin-1 (IL-1) has been associated with the initiation and pathogenesis of joint disease. In part, this occurs by induction of proteases and oxidative pathways that contribute to the degradation of structural components of the articular cartilage extracellular matrix. Elucidating the complex macromolecular and molecular effects of IL-1 on articular tissues may further our understanding of the roles of IL-1 and inflammation in OA pathobiology. Full-length gene sequences encoding three recombinant equine interleukin-1 proteins (EqIL-1a, EqIL-1b, and EqIL-1 receptor antagonist), were previously cloned and expressed in-vitro. The objectives of this dissertation were to 1) establish EqIL-1 induced experimental models of equine OA, and 2) to investigate specific IL-1-induced immuno-inflammatory responses. Effects of EqIL-1 on articular cartilage explant proteoglycan metabolism and synthesis of a downstream inflammatory product, prostaglandin E2, established culturing conditions and furthered the rationale to use EqIL-1 in the in-vitro modeling of early joint disease. A customized cDNA array was used to profile changes in mRNA levels resulting from EqIL-1 treatments of cultured articular cartilage chondrocytes. EqIL-1a induced elevated mRNA levels corresponding to six genes after 1 hour relative to media control chondrocytes (p<0.05). EqIL-1b increased transcript levels of seven genes after 6 hours (p<0.0004); 102 additional transcripts were elevated > 2-fold over controls. A subset of the array-generated data was verified using optimized reverse transcriptase-PCR amplification. Results of principal component analysis indicate co-regulation of EqIL-1 induced transcript levels to relate to chondrocyte differentiation and cell-cycle processes. Subtractive hybridization-PCR identified 148 differentially expressed cDNAs in synovium resulting from a 6-hour intra-articular EqIL-1b injection. Combined results demonstrate the potent bioactivity of our equine IL-1 proteins and support the argument for crucial roles of IL-1 in pro-inflammatory processes and cytokine imbalances underlying early OA pathogenesis. These results add to the current knowledge of IL-1 modulated transcription that may precede ECM catabolic processes characteristic of OA. The culture systems, assays, and techniques for gene expression analysis may be useful for future studies attempting to elucidate macromolecular and transcriptional events underlying inflammatory-associated joint disease processes in horses. Reported information may further efforts toward improved diagnostic and preventive strategies and development of anti-IL-1 directed therapies.
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    Thermal Degradation Studies of Polycarbonate
    Robertson, Jennifer E. (Virginia Tech, 2001-05-04)
    Polymeric materials are increasingly being used in diverse, very demanding applications. Either pre- or post- application environments may require exposures to conditions hostile to the polymer's integrity. Frequently, these demanding conditions result in degradation of the polymer and subsequent decreases in desirable properties. Clearly then, a methodology to predict important properties, such as Tg, molecular weight, and tensile strength, from knowledge of the environmental history of a polymeric-based specimen is beneficial. The current study focuses on bisphenol A polycarbonate and tracks changes in the properties of this material as a function of the degree of degradation, t. For the purposes of the present research, the environmental effects have been limited to those associated with elevated temperature, although the methodology is general. This t parameter is a product of the kinetic rate constant, k, found from isothermal kinetics, and the time of degradation, t. Elucidation of t has been linked to measurement of the molecular weight distribution which in turn can be related to various properties to yield predictive relationships for these properties. Only the thermal history of the polymer and its initial properties are required for the model. This technique is not limited to a specific polymer or even to thermal degradation. As long as the kinetics of the process can be mathematically modeled, this approach should apply to a host of other situations, providing property prediction simply from knowledge of the material history. The research seeks to better understand the thermal degradation of polycarbonate. Kinetics of the process was explored, and the chemical mechanisms were examined. A key part of the project was the determination of the molecular weights and molecular weight distributions at each level of degradation. Furthermore, mechanical stress-strain properties, glass transition temperatures, and melt viscosities were also measured. This information, together with the kinetic expressions, facilitated prediction of these types of material properties for a known thermal history.