8,041 research outputs found
The effect of polystyrene sodium sulfonate grafting on polyethylene terephthalate artificial ligaments on in vitro mineralisation and in vivo bone tissue integration
No full textThis study investigates the impact of polystyrene sodium sulfonate (PolyNaSS) grafting onto the osseointegration of a polyethylene terephthalate artificial ligament (Ligament Advanced Reinforcement System, LARS ) used for Anterior Cruciate Ligament (ACL). The performance of grafted and non-grafted ligaments was assessed in vitro by culturing human osteoblasts under osteogenic induction and this demonstrated that the surface modification was capable of up-regulating the secretion of ALP and induced higher level of mineralisation as measured 6 weeks post-seeding by Micro-Computed Tomography. Grafted and non-grafted LARS were subsequently implanted in an ovine model for ACL reconstruction and the ligament-to-bone interface was evaluated by histology and biomechanical testings 3 and 12 months post-implantation. The grafted ligaments exhibited more frequent direct ligament-tobone contact and bone formation in the core of the ligament at the later time point than the nongrafted specimens, the grafting also significantly reduced thefibrous encapsulation of the ligament 12 months post-implantation. However, this improved osseo-integration was not translated into a significant increase in the biomechanical pull-out loads. These results provide evidences that PolyNaSS grafting improved the osseo-integration of the artificial ligament within the bone tunnels. This might positively influence the outcome of the surgical reconstructions, as higher ligament stability is believed to limit micro-movement and therefore permits earlier and enhanced healing.Programme ANR LIGAR
Quantitative Computational and Experimental Characterization of Functionalized Nanoparticles
No full textThesis (Ph.D.)--University of Washington, 2017Nanoparticles are widely used in many fields of science and can often be found in everyday commercial products. This widespread use of nanoparticles in our daily lives and the industry have raised several concerns regarding the safety and environmental impact of these nanoparticles. In the biomedical field, understanding how nanoparticles interact with the biological environment is crucial for public safety and advancing the development of nanomedicine. In this work, both computational and experimental methods were developed to aid the surface chemical characterization of functionalized nanoparticles. The major experimental project focuses on controlling and probing the orientation of immobilized proteins on gold nanoparticles. Protein G B1, a protein that will selectively bind to the Fc region of IgG, was immobilized onto gold NPs (AuNPs) functionalized with oligo(ethylene glycol)-Maleimide (OEG-MEG) self-assembled monolayers (SAMs). The orientation of the protein can be controlled via a site-specific maleimide-sulfhydryl reaction between the OEG-MEG SAMs and the cysteine amino acid in the protein. Utilizing site-specific chemistry and surface sensitive analysis techniques of X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), it was possible to both control and determine the orientation of immobilized Protein G B1on gold nanoparticles. In addition to experimental analysis, robust computer simulations using the Simulation of Electron Spectra for Surface Analysis (SESSA) program were incorporated to aid the characterization of a wide variety of nanoparticles. It was demonstrated that SESSA can accurately simulate XPS spectra and peak intensities of nanoparticles and verify existing methods of calculating overlayer thickness of core-shell nanoparticles. Further, SESSA can be applied to assess the structure and thickness of various SAMs on both flat and nanoparticle surfaces by incorporating experimentally collected XPS and sum-frequency generation (SFG) results. In another study, SESSA was applied to model citrate stabilized Au/Ag-core/shell nanoparticles with complex geometrical properties. The Au/Ag-core/shell NPs were polydispersed in size, non-spherical, and contained off-centered Au-cores. The NPs were characterized using XPS and scanning transmission electron microscopy (STEM) to determine the composition and morphology of the NPs. Simulating NPs with average dimensions and not accounting for the geometrical properties of the NPs resulted in significant underestimation of the gold intensity. Simulations based on the combined effect of NP non-sphericity and off-centered Au-core resulted in reduced effective Ag-shell thickness and provided simulated elemental compositions that matched the experimental XPS results
Using Surface Analysis to Investigate how Adsorbed Protein Structure Changes with Controlled Changes in Surface Chemistry: A Case Study Involving Bare & Sodium Styrene Sulfonate-Grafted Gold Surfaces
No full textThesis (Ph.D.)--University of Washington, 2015Protein adsorption on synthetic surfaces is an important and widely studied phenomenon in biomaterials research. Despite its importance, it is still poorly understood. Therefore, the ultimate goal of this thesis is to contribute toward a better understanding of the protein-biomaterial interface, with application toward the next generation of biomaterial implants. Narrowing this broad objective down to something more specific, this thesis represents a body of work focused on the interaction of blood plasma proteins with sodium styrene sulfonate (NaSS) grafted surfaces. The approach to achieving this objective is straightforward. First, proof-of-concept must be established that NaSS can be successfully grafted to a given surface. A thorough characterization of NaSS films grafted from titanium and silicon oxide surfaces using atom transfer radical polymerization (ATRP) is presented herein. Next, the grafting procedure must be optimized and scaled in order to reliably produce sufficient quantities of NaSS-grafted samples to meet the demands of protein adsorption studies. Using a 24 factorial experimental design and “activators are continuously regenerated by electron transfer” (ARGET) ATRP chemistry, an optimized NaSS grafting procedure was developed. The findings here may be generally applied as a starting point for grafting other polymers to a variety of surfaces. The last step is application—to use the NaSS films for their intended purpose. Surface analysis techniques were applied to characterize changes in adsorbed bovine serum albumin (BSA), bovine fibrinogen (Fgn), bovine immunoglobulin G (IgG), and bovine plasma films between bare and NaSS-grafted gold surfaces. All three proteins and plasma adsorb more readily to, and have a higher affinity for gold than NaSS surfaces. However, at higher concentrations NaSS adsorbs similar amounts (for plasma) or more (for BSA and Fgn) total protein than gold. The only protein that NaSS surfaces adsorb less of than gold is IgG, because IgG adopts a highly denatured conformation on NaSS. Each adsorbed IgG molecule takes up more space on NaSS compared to gold surfaces, resulting in less total protein adsorbed at all concentrations. Still, with the exception of BSA and plasma on gold surfaces, neither surface appeared to have saturated at the highest protein solution concentration studied. Using principal component analysis (PCA) of just the amino acid ToF-SIMS mass fragments, it was determined that all three proteins and plasma adsorb differently on NaSS and gold surfaces, and that the structure of the xii adsorbed protein films change with surface concentration. One difference between adsorbed films on both surfaces, determined using peak ratios for buried/surface amino acids for each protein, is that adsorbed proteins denature more on NaSS than gold. Also, using peak ratios of non-uniformly distributed amino acids, small differences in average orientations were found between the two surfaces for BSA and IgG films. Finally, principal component (PC) modeling, was used to track changes in adsorbed plasma films with time. On NaSS surfaces the plasma films appear to be more BSA-like at short adsorption times, and more Fgn-like at longer adsorption times. Similarly, on gold surfaces the plasma films on appear to start out more IgG-like and become more Fgn-like with increasing adsorption time. However, the PC model included only the three proteins studied here, where plasma is a complex mixture of hundreds of proteins. Therefore, while both gold and NaSS appear to adsorb more Fgn with time, further study is required to confirm that this is truly representative of the final state of the adsorbed plasma films
Rabbit menace in New South Wales : an abridgement of the report / by David G. Stead ... commissioned on 30th April 1925 to inquire into matters connected with the rabbit menace in New South Wales.
No full textAt head of title: Department of Agriculture, New South Wales.; Electronic reproduction. Canberra, A.C.T. : National Library of Australia, 2012.; Library's copy signed by the author
Combining Surface Analytical and Computational Techniques to Investigate Orientation Effects of Immobilized Proteins
No full textThesis (Ph.D.)--University of Washington, 2017-06Controlling how proteins are immobilized (e.g. controlling their orientation and conformation) is essential for developing and optimizing the performance of in vitro protein-binding devices, such as enzyme-linked immunosorbent assays. The objective of this work is to develop new methodologies to study proteins and complex mixtures of proteins immobilized onto surfaces. The focus of this study was to control and characterize the orientation of protein G B1, an IgG antibody-binding domain of protein G, on well-defined surfaces as well as measure the effect of protein G B1 orientation on IgG antibody binding using a variety of surface analytical and computational techniques. The surface sensitivity of time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to distinguish between different proteins and their orientation by monitoring the changes in intensity of characteristic amino acid mass fragments. Amino acids distributed asymmetrically were used to calculate peak intensity ratios from ToF-SIMS data to determine the orientation of five different cysteine mutants of protein G B1 covalently attached to a maleimide surface. To study the effect of protein orientation on antibody binding, we formed multilayer protein films by binding IgG to protein G B1 films. Quartz crystal microbalance with dissipation monitoring (QCM-D) detected protein coverages of 69 – 130 ng/cm^2 (theoretical mass of a monolayer of protein G B1 is 110 – 160 ng/cm^2). QCM-D and X-ray photoelectron spectroscopy analysis revealed that packing density along with orientation affected the antibody binding process. Spectra from ToF-SIMS using large Ar gas cluster ion sources distinguished between different proteins in multilayer protein systems. A Monte Carlo algorithm was developed to predict protein orientation on surfaces. Two distinct orientations of protein G B1 adsorbed onto a hydrophobic surface were found and characterized as two mutually exclusive sets of amino acids on the outermost β-sheets contacting the surface. This prediction was consistent with sum frequency generation (SFG) vibrational spectroscopy results. In fact, theoretical SFG spectra calculated from an equal combination of the two predicted orientations exhibited reasonable agreement with measured spectra of protein G B1 on polystyrene surfaces. These results show that computational methods to study proteins on surfaces can complement surface analytical data
Cult: A Composite Novel
No full textCult (redacted)
The first component of the thesis is a composite novel called Cult which falls into two parts with seven narratives in each. Part 1 tracks the protagonist, Ellen, from her first involvement with the cult through to her eventually leaving it. Although fiction, the first half of the book answers the kinds of questions the author is asked when people discover that she was once a sannyasin (a follower of the guru Bhagwan Shree Rajneesh). While the experiences of meditation, group therapy and communal living are all faithfully rendered within the stories, the need for strong characters, narrative drive and a lightness of touch takes precedence.
Part 2 picks up Ellen’s story some twenty or so years later and explores what becomes of her in middle age. It also looks at other groups in society, such as academia, the law and the internet dating community which each have their own jargon, hierarchies, rituals and rules but are not considered to be cults.
The book examines the question raised in the Epigraph, ‘how do we be together when we feel so alone’ with a focus on relationships other than the familial and the romantic.
Collisions, Chasms and Connections: a Performative Exploration of the Composite Novel Form
The second part of the thesis is both a critical and creative response to three contemporary American books: Olive Kitteridge by Elizabeth Strout; A Visit from the Goon Squad by Jennifer Egan; and Legend of a Suicide by David Vann. The critical element comprises a close reading of the three books; a chronological reconstruction of their overarching storylines; and a consideration of what their authors have said about writing the books. It concludes that, in the composite novel, the simultaneous presentation of multiple views and storylines operate much like a 3D image to give the impression of depth to the characters and situations rendered. The creative element of the essay is a playful and personal response to the texts
Shadowing the Legendary Dizzy Gillespie
No full textShadowing the Legencary Dizzy Gillespie with author and jazz aficionado David G. Brown
Surface Analysis of Adsorbed Proteins: A Multi-Technique Approach to Characterize Surface Structure
No full textThesis (Ph.D.)--University of Washington, 2012Adsorbed proteins on surfaces are important in many applications, including medical implants, sensors, marine materials, and in vitro substrates for cell culture and other uses. Understanding the protein structure on the surface would allow better control of the interaction of the material with the surrounding environment and a more reproducible system response. Protein adsorption is a complex process, and characterization requires the combination of multiple analysis techniques. In this thesis, adsorbed protein amounts were measured using x-ray photoelectron spectroscopy (XPS) and radiolabeled proteins. Protein conformation and orientation were measured using time-of-flight secondary ion mass spectrometry (ToF-SIMS), near edge x-ray absorption fine structure (NEXAFS), sum frequency generation (SFG), and enzyme-linked immunosorbent assays (ELISA). Surface type influenced the adsorbed surface concentration of albumin and fibronectin on surfaces of glass, polystyrene, titanium, and sulfonated polystyrene as measured by XPS and radiolabeled protein adsorption. More albumin adsorbed onto polystyrene than glass. More albumin and fibronectin adsorbed onto sulfonated polystyrene than titanium. ToF-SIMS also showed differences in structure of the proteins adsorbed onto the different surfaces. The A1 domain of von Willebrand Factor adsorbed in similar amounts onto glass, tissue culture polystyrene, and polystyrene surfaces, as measured by XPS. However, ToF-SIMS showed differences in solution exposure of A1 domain amino acids when adsorbed onto the three surfaces and NEXAFS showed the most ordered beta-sheet structure when A1 was adsorbed onto polystyrene. ELISA showed lowest binding of antibodies recognizing a nonlinear epitopes within A1 when A1 was adsorbed onto polystyrene. Functional studies using a parallel plate flow chamber measured platelet binding to A1 adsorbed onto the three surfaces. At high shear (20dyne/cm2), platelets showed most detachment from A1 adsorbed onto glass. At low shear (0.2dyne/cm2), platelets showed most detachment from A1 adsorbed onto polystyrene. Surface analysis was also useful in characterizing collagen substrates created under different experimental conditions, including material source and pH. Collagen obtained from different sources exhibited altered adsorption behavior, both in amount adsorbed as measured by XPS and interaction with the A1 domain of von Willebrand Factor as measured by ToF-SIMS. SFG was used to identify differences in ordering of collagen adsorbed from solutions at different pH values. Collagen adsorbed at pH 8.0 showed higher SFG amide signal than collagen adsorbed at pH 6.5, suggesting greater ordering of the peptide backbone at pH 8.0. The differences in SFG signal were not due to the amount adsorbed protein, as XPS showed more collagen adsorbed onto tissue culture polystyrene at pH 6.5 than pH 8.0. These studies demonstrated that the surface type can have a large impact on adsorbed proteins, both in amount adsorbed and surface structure. These studies also showed that surface analysis is very useful in creating defined in vitro protein substrates. In all cases, it was crucial to use multiple analysis techniques to understand these systems
Chemical Analysis of Cells and Tissues with Time-of-Flight Secondary Ion Mass Spectrometry
No full textThesis (Ph.D.)--University of Washington, 2013In this work the chemical analysis of biological cells and tissues with time-of-flight secondary ion mass spectrometry (ToF-SIMS) was explored. ToF-SIMS has the ability to obtain a mass spectrum with submicron spatial resolution for imaging and is extremely surface sensitive. ToF-SIMS for biological sample analysis is still an emerging field, so the development and characterization of novel sample preparation and analysis methods is key to acquiring useable information. In this work, three different methods to prepare NIH/3T3 fibroblasts were investigated: chemically fixed, freeze-dried and frozen-hydrated. Chemical fixation followed by rinsing removed a majority of intracellular Cl-, improving the secondary ion yields of all organic positively charged secondary ions an average of 2.6x. Damage cross sections were reduced during frozen-hydrated analysis, improving the secondary ion yields of higher mass organic fragments. In a separate experiment, accurate 3D reconstructions of NIH/3T3 fibroblasts were produced. A simple z-correction was applied to the data cube, and the biggest assumption for that correction was validated. An intracellular lipid-rich region surrounding the nucleus was visualized. ToF-SIMS applied to two different breast cancer systems. In the first, eight human breast cancer cell lines were distinguished form one another using mass spectra and principal component analysis (PCA). Not only was PCA to distinguish the cell lines form one another, it also highlighted the largest sources of variance between the cells. Phosphocholine, fatty acids, cholesterol and diacylglycerols (DAGs) were identified as key peaks. The identification of these species indicate that differences in lipid metabolism play an important role in separating the cell types from one another. Breast cancer tumor tissues were also investigated. Data from four tumors was collected. PCA applied to the spectra distinguished the four tissues from one another. Imaging PCA determined the largest sources of variance within an analysis area. Structures were identified by PCA that matched structures observed in serial-sectioned, conventionally-stained slices, and other domains that were not visible in the conventionally-stained slices. As with the breast cancer cell lines, phosphocholine, fatty acids, DAGs, cholesterol and vitamin were found to be large sources of variance, indicating lipid metabolism plays in important role in tumor differentiation
Investigations of Protein Fiber Structures and the Interactions at Their Interfaces Using Nonlinear Optical Spectroscopy
No full textThesis (Ph.D.)--University of Washington, 2018Protein fibers are ubiquitous in nature, either as functional components of cells and tissues, or as hallmarks of severe diseases. Examples include amyloid fibers in the brain of patients with Alzheimer’s disease, or collagen fibers in the extracellular matrix of tissues - central design targets for scaffolds in tissue engineering. Characterizing the structure and surface chemistry of protein fibers is therefore vital, but notoriously challenging due to their complexity. In this work, fundamental nonlinear optical properties of type I collagen fibers and amyloid structures from insulin and beta-lactoglobulin have been investigated and used for structural characterization. The collagen fibers were characterized with vibrational sum-frequency scattering spectroscopy, in a first demonstration of this technique for protein fiber investigations. Spectral features were shown to be dependent on the scattering angle and the signals away from the phase-matched direction were highly reproducible for samples with random fiber orientations. Furthermore, a scattering angle of 22˚ exhibits surface specificity for the fibers when probed in the amide I region, which was utilized to detect the initial effects of treatments with sodium dodecyl sulfate surfactants. Information from such studies of the fiber surface structure may complement the information gained from techniques with sensitivity for the overall structure, such as second-harmonic generation (SHG). For the amyloid structures, the intrinsic linear and nonlinear optical properties were investigated. In the amyloid state, beta-lactoglobulin develops a weak fluorescence in the visible regime, which could be identified by tracking the red-edge excitation shift (REES). The intrinsic optical properties allowed imaging of amyloid spherulites from both proteins, with high contrast especially for the nonlinear techniques, which included SHG and two-photon excitation fluorescence (TPEF). By monitoring the total signal from insulin amyloid spherulites in two separate detection channels while shifting the excitation wavelength, indications of the REES in TPEF were observed for the first time. The nonlinear optical techniques and phenomena explored in this work opens up for label-free detection and detailed structural investigations of protein fibers in aqueous and biological 3D environments
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