Browsing by Subject "Atomic Force Microscopy"

Now showing 1 - 5 of 5
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    Biophysical and functional consequences of sequence changes on dystrophin and utrophin
    (2017-04) McCourt, Jackie
    Mutations in the DMD gene result in Duchenne (DMD) and Becker (BMD) muscular dystrophies. The DMD gene encodes the protein, dystrophin that is predominantly expressed in skeletal muscle. Dystrophin is part of a larger protein complex known as the dystrophin-glycoprotein complex (DGC) and, as part of the DGC, provides a mechanical link between the sub-sarcolemmal cytoskeleton and the extracellular matrix. BMD is typically caused by mutations that maintain the reading frame and most often produce variable levels of internally truncated, partially functional dystrophin wheras DMD is most frequently characterized by a complete loss of dystrophin protein or disruption of key ligand binding domains. Utrophin has a highly similar overall structure to dystrophin and is part of the homologous utrophin-glycoprotein complex (UGC) present during fetal development and is localized to neuromuscular and myotendinous junctions in adult muscle. Our lab has previously demonstrated that dystrophin protein in vitro thermal stability is sensitive to disease-causing missense mutations and internal deletions. In contrast, utrophin displays uniform stability upon internal deletion or terminal truncation. Several therapeutic strategies to treat DMD utilize internally deleted dystrophins, including the recently FDA approved exon-skipping drug, eteplirsen, as well as adeno-associated virus (AAV) mediated delivery of therapeutic micro-dystrophins. Here, we characterized therapeutically relevant, internally truncated dystrophin constructs that have been proposed by leading scientists in the field. We show that, as a group, gene therapy micro-dystrophins are significantly less stable in vitro than full-length dystrophin whereas exon-skipped dystrophins have stability profiles congruent with full-length dystrophin. To address the consequences of dystrophin instability in vivo, we generated two novel transgenic mouse models expressing missense mutant dystrophins reported in human DMD (L54R) and BMD (L172H) patients. The L54R and L172H missense mutants were previously evaluated in cultured myoblasts and shown to have missense-mutant dystrophin levels that were inversely proportional to in vitro stability and disease severity of the corresponding patients. Analysis of the L54R and L172H mouse lines as well as a homozygous L172H mouse reveal that disease severity inversely correlates with expression levels of dystrophin protein. Based on the increase of mutant dystrophin upon proteasome inhibition in cultured myoblasts, our hypothesis is that missense dystrophin proteins are being targeted to the proteasome for degradation through the ubiquitin-proteasome pathway. To determine the specific ligases involved in targeting missense dystrophins to the proteasome, we screened an siRNA library of over 500 ubiquitin-conjugating enzymes in cultured myoblasts and identified five putative dystrophin-specific E3 ligases. Two of the identified ligases, Amn1 and FBXO33, were observed in our transgenic mouse lines, with Amn1 protein levels showing significant increases correlating with the amount of missense dystrophin present. Our future studies will continue to evaluate the impact of Amn1 and FBXO33 activity on dystrophin protein levels in order to identify potential therapeutic targets. In addition to the characterization of dystrophin and utrophin stability, we have begun to interrogate a long-hypothesized but understudied function of dystrophin and utrophin, namely, their roles as molecular springs to mechanically stabilize the muscle membrane during muscle contraction. Using atomic force microscopy (AFM), we show here the first mechanical characterization of utrophin and functionally relevant utrophin fragments. Our data reveal striking differences in the mechanical properties of N- and C-terminal halves of utrophin despite having nearly identical thermal stabilities and high structural homology. The high unfolding forces observed in utrophin and the evidence of stiffening spring behavior suggest that utrophin may be acting as a stiff elastic element in series with the giant muscle protein, titin, at the myotendinous junction. Future studies will include evaluation of myotendinous defects in utrophin-deficient mice as well as mechanical characterization of full-length dystrophin.
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    The Effect of α-Synuclein on Lipid Membrane Properties Characterized by Molecular Dynamics and Atomic Force Microscopy
    (2018-08) Brummel, Benjamin
    The protein α-synuclein (αSyn), primarily recognized for its link to neurodegenerative disorders, has multiple reported functions. One well-established role of αSyn is its ability to bind and remodel lipid membranes. This ability has been characterized in synthetic lipid bilayers and has been observed both in cellular and in vivo models. The native environment of αSyn—the presynaptic terminal of neurons—contains mitochondria and synaptic vesicles, which have unique membranes that differ from previously studied models. The goal of this dissertation was to characterize how lipids enriched in synaptic vesicles and mitochondria affect how αSyn changes membrane properties. First, molecular dynamics (MD) simulations of synaptic vesicle-mimic bilayers showed how lipids with polyunsaturated fatty acids modify membrane properties and interact with αSyn. Next, tubulation experiments were combined with MD simulations to explore how αSyn remodels bilayers containing cardiolipin and phosphatidylethanolamine, two lipids enriched in mitochondria. Finally, methods were developed to characterize lipid vesicle mechanical properties using pulsed force mode (PFM) atomic force microscopy (AFM). This work provides insight into the specifics of how αSyn affects the properties of synaptic vesicle and mitochondrial membranes and demonstrates how PFM-AFM can identify the mechanical properties of lipid vesicles.
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    Model mismatch paradigm for probe based nanoscale imaging.
    (2010-10) Agarwal, Pranav
    Scanning Probe Microscopes (SPMs) are widely used for investigation of material properties and manipulation of matter at the nanoscale. These instruments are considered critical enablers of nanotechnology by providing the only technique for "direct" observation of dynamics at the nanoscale and affecting it with sub Angstrom resolution. Current SPMs are limited by low throughput and lack of quantitative measurements of material properties. Various applications like the high density data storage, sub-20 nm lithography, fault detection and functional probing of semiconductor circuits, direct observation of dynamical processes involved in biological samples viz. motor proteins and transport phenomena in various materials demand high throughput operation. Researchers involved in material characterization at nanoscale are interested in getting quantitative measurements of stiffness and dissipative properties of various materials in a least invasive manner. In this work, system theoretic concepts are used to address these limitations. The central tenet of the thesis is to model, the known information about the system and then focus on perturbations of these known dynamics or model, to sense the effects due to changes in the environment such as changes in material properties or surface topography. Thus a model mismatch paradigm for probe based nanoscale imaging is developed. The topic is developed by presenting physics based modeling of a particular mode of operation of SPMs called the dynamic mode operation. This mode is modeled as a forced Lure system where a spring mass damper system is in feedback with an unknown static memoryless nonlinearity. Tools from averaging theory are used to tame this rich nonlinear system by approximating it as a linear system with time varying parameters. Material properties are thus transformed from being parameters of unknown nonlinear functions to being unknown coefficients of a linear plant. The first contribution of this thesis deals with real time detection and reduction of spurious areas in the image which are also known as probe-loss areas. These areas become critical during high speed operations. The detection strategy is based on thresholding of a distance measure, which captures the difference between the sensor models in absence and presence of probe-loss. A switching gain control strategy based on the output of a Kalman Filter is used to reduce probe-loss areas in real time. The efficacy of this technique is demonstrated through experimental results showing increased image fidelity at scan rates that are 10 times faster than conventional scan rates. The second contribution of this thesis deals with developing multi-frequency input excitation strategy and deriving a bias compensated adaptive parameter estimation strategy to determine the instantaneous equivalent cantilever model. This is used to address the challenge of quantitative imaging at high bandwidth operation by relating the estimated plant coefficients to conservative and dissipative components of tip-sample interaction. The efficacy of the technique is demonstrated for quantitative material characterization of a polymer sample, resulting in material information not previously obtainable during dynamic mode operation. This information is obtained at speeds which are two orders faster than existing techniques. Quantitative verification strategies for the accuracy of estimated parameters are presented. The third contibution of this thesis deals with developing real time tractable models and characterization methodology for an electrostatically actuated MEMS cantilever with an integrated solid state thermal sensor. Appropriate modeling assumptions are made to delineate various nonlinear forces on the cantilever viz. electrostatic force, tip-sample interaction force and capacitive coupling. Experimental strategy is presented to measure the thermal sensing transfer function from DC-100kHz. A quantitative match between experimental and simulated data is obtained for the large range nonlinearities and small signal dynamics.
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    Reconstructing oxide surfaces.
    (2009-06) Riesterer, Jessica Lori
    The work presented here is concentrated on surfaces and interfaces in alumina (Al2O3), anorthite (CaAl2Si2O8), silica (SiO2) and rutile (TiO2). While each of these materials have different crystal structures and measurable properties, they all exhibit similar mechanisms for fundamental behavior. The topics researched and discussed lead into each other. Faceting describes the movement of atoms to a lower energy configuration. While faceting of the surface is only considered, grain boundaries can be faceted. In cross-section, facets resemble grain boundary grooves. Grooves and ridges form where a grain boundary intersects the surface of a material. The grooves facilitate grain boundary migration and diffusion. The surface tension at the groove is governed by Young's equation, which balances the interfacial forces between the solid and vapor. Glass films can wet or dewet the surface a grain boundaries. Whether the film wets or dewets depends on the surface energy of the surface and the liquid. Capillary forces determine the type of dewet patterns formed on the surface. Again, the surface-vapor-liquid interfaces are governed by Young's equation. Liquid films at grain boundaries facilitate densification and grain boundary migration. Liquid phase sintering (LPS) uses capillary forces and the dissolution/reprecipitation process to sinter green compacts to a high density at lower temperatures. Capillary forces and surface tension can also cause the liquid film to penetrate or exude from the grain boundary. Various forms of microscopy have been used to characterize and relate these phenomena.
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    Signals and Systems Tools for Advanced Nanoscale Investigation with Atomic Force Microscopy
    (2017-03) Ghosal, Sayan
    The atomic force microscope (AFM) is one of the major advancements in recent science that has enabled imaging of samples at the nanometer and sub-nanometer scale. Over the years, different techniques have been developed to improve the speed, resolution and accuracy of imaging using AFM. Further, the application spectrum of AFMs has extended beyond topography imaging, examples of which include material characterization, probe based data storage systems, and also single molecule force spectroscopy. In spite of the remarkable achievements by AFM technologies, many challenges exist. While majority of this thesis aims to address important challenges that exist with state of the art AFM methodologies using tools from signal processing and systems theory, it also reports some surprising new phenomena that are observed from AFM based mechanical characterization of protein molecules. The techniques developed in each chapter are extensively verified with simulation and experimental results. A key issue that remains largely unaddressed in the AFM literature is the assessment of fidelity of the measurement data. The first contribution of this thesis is to develop a quantitative measure for the fidelity of images obtained from a fast dynamic mode AFM technique. The developed paradigm facilitates user specific priority for either detection of sample features with high decision confidence or on not missing detection of true features. The fidelity measures developed in this thesis are suitable for real-time implementation. The second contribution of this thesis is to develop and compare the performance of different methods to characterize mechanical properties of materials utilizing the dynamic mode of AFM operation. The dynamic mode AFM is particularly suitable for investigating soft-matter. Here, an important enabler is the viewpoint of an equivalent cantilever. The parameters of the equivalent cantilever need to be estimated to derive material properties. In this thesis, we develop a new steady-state based estimation of equivalent parameters (SEEP) and compare it with the recursive estimation of equivalent parameters (REEP). We show that the SEEP is considerably simpler to implement, however, SEEP is a low bandwidth method when compared to REEP. Both methods yield material parameters that quantitatively agree in the domain of validity of the methods. This thesis also streamlines the process of material identification and outlines the key pitfalls that need to be avoided for quantitative estimation of material parameters. Extensive design of a system identification module is reported which implements the REEP algorithm on modern field programmable gate arrays (FPGA). The step by step design procedure of the module explained in this thesis is employable to the development of a wide variety of FPGA based signal processing systems. The third contribution of this thesis is a new system model detection technique called the innovations squared mismatch. Such detection of a model from a set of models that best describes the behavior of a system is of primary importance in many applications. Here, two discriminating signals are derived from measurements for a plant that switches between two model behaviors, where the transfer functions from inputs to the two signals are identical when one model is effective while they are negative of one another when the other model is effective. Further, we report sequence based detection approaches to extend the use of the signals for high bandwidth applications. In such applications the plant behavior can switch from one model to another at high rates and the transients from a previous behavior affect the current behavior causing inter-symbol interference (ISI). Methods developed are specialized for probe based data storage where experimental data demonstrates that they offer significant advantages over current methods. The fourth contribution of this thesis is the first ever characterization of mechanical properties of utrophin protein molecule and its different terminal fragments using AFM based force spectroscopy experiments. Utrophin and its homologue dystrophin are proteins which are believed to play vital roles in mechanically stabilizing the muscle cells during stretch and relax cycles. These proteins are also under active research for finding possible cure for the disease muscular dystrophy. In this thesis we report markedly different mechanical characteristics for the utrophin constructs where previous thermodynamic studies measured identical thermal denaturation profiles. Our findings signify the need for force spectroscopy based characterization of molecules that are believed to play important mechanical roles in human body.