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Browsing by Subject "Muscle"

Now showing 1 - 8 of 8
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    Bone's functional and geometric properties in dystrophin-deficient mice and the efficacy of low intensity vibration training to improve musculoskeletal function
    (2013-03) Novotny, Susan Anne
    Overall, my dissertation work has shown that bone health is affected in dystrophic mice secondary to the muscle disease (Chapter 3), and both prednisolone and physical inactivity accentuate these declines (Chapter 4). I identified two sets of low intensity, high frequency vibration parameters (45 Hz at 0.6 g and 90 Hz at 0.6 g) that initiated an osteogenic response in mdx mice. Further experiments were performed utilizing the 45 Hz and 0.6 g setting, the results of which indicated that vibration was safe for dystrophic muscle (Chapters 5 and 6). However, long-term training adaptations for musculoskeletal function were not realized (Chapter 6). The lack of adaptations following vibration training in mdx or wildtype mice does not negate the utility of vibration as a potential therapeutic exercise modality for DMD, but further research, utilizing alternative strategies, is needed to determine the full extent of vibration's capacity to improve musculoskeletal health.
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    Copolymer-based membrane stabilizers for Duchenne Muscular Dystrophy
    (2016-04) Houang, Evelyne
    The overarching objective of this work centers on a structure-function approach to investigate the mechanism of action of synthetic copolymer-based membrane stabilization in the context of Duchenne Muscular Dystrophy (DMD). The guiding theme is the investigation of mechanism of interaction of membrane stabilizing copolymers using cellular and whole animal physiology, chemical engineering, and supercomputational approaches. DMD is an X-linked recessive disease of marked striated muscle deterioration affecting 1 in 3500-5000 boys. DMD results from the lack of the cytoskeletal protein dystrophin, which is essential for maintaining the structural integrity of the muscle cell membrane. DMD patients develop severe skeletal muscle degeneration, along with clinically significant cardiomyopathy. There is no cure for DMD patients, or any effective treatment to halt, prevent or reverse DMD striated muscle deterioration. The primary pathophysiological defect in DMD is the marked susceptibility to contraction-induced membrane stress and the subsequent muscle damage and degeneration that occurs due to loss of muscle membrane barrier function. In this context, a unique therapeutic approach is the use of synthetic membrane stabilizers to prevent muscle damage by directly stabilizing the dystrophin-deficient muscle membrane. The triblock copolymer poloxamer 188 (P188) has numerous features that make it an attractive synthetic membrane stabilizer candidate for DMD treatment and has been demonstrated to target and stabilize damaged membranes in various pathophysiological contexts. The efficacy of P188 in protecting the dystrophic myocardium has been well established, but its effect on the dystrophic skeletal muscle has remained unclear. This work for the first time demonstrates that P188 stabilizes the dystrophic skeletal muscle membrane in vivo and protects it against the mechanical stress associated with lengthening contractions. This result validates P188 as a therapeutic strategy to directly target the hallmark of DMD: impaired membrane stability in all striated muscles. Very little is known on how P188 interacts with and stabilizes biological membranes. To fundamentally probe the mechanism of action of synthetic copolymers as membrane stabilizers, a structure-function approach was undertaken. The aim was to gain insight into the essential critical chemical parameters of copolymers in terms of membrane interacting properties. This work shows for the first time that copolymer mass, composition, architecture, and functional end group chemistries significantly define mechanism of action at the membrane. Based on these insights, an “anchor and chain” model is advanced whereby membrane interaction is critically dependent on end group hydrophobicity. Finally, leveraging the power of supercomputational approaches, Molecular Dynamics simulations were developed to further evaluate and understand copolymer-membrane interactions at atom level resolution. Using increases in surface tension applied to the lipid bilayer, an area-per- lipid dependence of adsorption vs. insertion was uncovered, supporting the hypothesis that copolymers insert into areas of decreased lipid density and then are “squeezed-out” once membrane integrity is restored. Collectively, these findings shed new light on block copolymer dynamic interaction with biological membranes.
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    Direct Reprogramming Of Fibroblasts Into Muscle Or Neural Lineages By Using Single Transcription Factor With Or Without Myod Transactivation Domain
    (2014-02) BELUR, NANDKISHORE RAGHAV
    The generation of induced pluripotent stem cells (iPSCs) from somatic cells has opened new doors for regenerative medicine by overcoming the ethical concerns surrounding embryonic stem (ES) cell research. However, iPSC technology presents several safety concerns, such as the potential risk of tumor formation, that have caused apprehension for use of iPSCs in humans. One such approach is "direct reprogramming" which can bypass the iPSC or pluripotent stage and obtain tissue-specific cell types from somatic cells. In this study, we examined whether an important transcription factor involved in myogenesis (Pax3) or neurogenesis (NeuroD1) alone can directly reprogram the mouse embryonic fibroblasts (MEFs) into myogenic or neurogenic lineages, respectively. In addition, we created fusion transcription factors (Pax3 or NeuroD1) with the potent MyoD transactivation domain (MDA) that could facilitate radical acceleration of reprogramming into the desired cell type through chromatin modification compared to wild-type factors. Here, we showed that Pax3 can reprogram MEFs towards a myogenic lineage and that MDA-Pax3 further enhances this myogenic reprogramming event. In addition, ectopic expression of NeuroD1 and MDA-NeuroD1 is able to induce neurogenic genes in MEFs, suggesting the partial neurogenic conversion of MEFs. Furthermore, we also showed that the ectopic expression of NeuroD1 but not MDA-NeuroD1 in myoblasts could suppress myogenic differentiation. These data suggest that single gene transduction such as Pax3 or NeuroD1 will become a feasible therapeutic approach for neuro- and muscle degenerative diseases, respectively.
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    Duchenne muscular dystrophy and extraocular muscle: a potential sparing mechanism with therapeutic implications.
    (2009-10) Kallestad, Kristen Marie
    This project investigates the role of extraocular muscle (EOM) progenitor cells in sparing the muscles from pathology associated with Duchenne Muscular Dystrophy (DMD). Mouse models of muscular dystrophy and wild type mice were analyzed by flow cytometry and cell culture for the size, heterogeneity and functional characteristics of stem and satellite cell populations of EOM and limb muscles. EOM have a 5-fold increase in progenitor cells compared with limb muscles. Additionally, an enriched population of cells expressing the stem cell marker CD34 but no other typical stem or differentiation markers (Sca-1, CD45, CD31, pax-7, m-cadherin) exists in the EOM. We refer to this population as EOMCD34 cells. The EOMCD34 cells are present in developing muscle, but only maintained in adult EOM, surviving in very aged animals. The EOMCD34 cells are also present in EOM of DMD model animals, but not their limb muscles. EOMCD34 cells are resistant to apoptosis and proliferate in vivo. Finally, these cells are capable of forming myotubes in vitro. The EOMCD34 cells may represent a primitive stem cell population, which is capable of maintaining life-long pools of myogenic precursor cells. Since EOM continuously remodel throughout life, unlike other skeletal muscle, it is logical that the proliferative potential of their precursor cells is enhanced. Since one proposed mechanism of DMD muscle destruction is exhaustion of the reparative progenitor cells, the EOMCD34 cells might prove useful for myoblast transplant therapies for DMD.
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    Elucidating the Structural Dynamics of Muscle Myosin Using Novel Methods in Electron Paramagnetic Resonance
    (2016-11) Binder, Benjamin
    Muscle contraction is fundamentally driven by an interaction between two proteins: actin and myosin. Myosin is a molecular motor, and assumes the active role in this relationship, coupling energy from hydrolysis of ATP with conformational changes to generate force on actin. In the context of a muscle fiber, this force causes filaments of myosin and actin to slide past one another in an ordered lattice, drawing the ends of individual contractile units (called sarcomeres) together. Concerted shortening of sarcomeres along the length of a fiber results in large-scale shortening of the entire fiber. Although muscle myosin has been the focus of intense study for many years, crucial details regarding its mechanism remain unknown. In particular, few structures of actin and myosin together have been reported—this is largely due to the inherent difficulties of handling large, filamentous protein complexes in traditional methods for structure determination. Myosin's interactions with actin are absolutely essential for macroscopic function, and this lack of structural information has created a knowledge gap: there is an abundance of functional and kinetic data for myosin in both normal and pathological states, but often no direct insight into the underlying structural causes for the observed behavior. In the present work, I seek to address this knowledge gap by providing high-resolution insight into the structural states of actin-bound myosin. My work is based on the hypothesis that allosteric coupling in myosin's catalytic domain (the domain responsible for actin binding, ATP hydrolysis, and initiation of force-generating conformational change) is accomplished via subtle internal rearrangements of individual structural elements. Furthermore, I hypothesize that these changes can be detected and quantified by innovative applications of site-directed spectroscopy. In Chapter 4, I establish a method using electron paramagnetic resonance (EPR) of a bifunctional spin label to probe nucleotide-dependent changes in the actomyosin complex. In Chapter 5, this method is expanded to include two complementary EPR techniques, ultimately providing sufficient constraints for direct modeling of nucleotide-dependent changes. Following these results, Chapter 6 addresses the ongoing development and further application of these methods within myosin and other protein systems.
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    Oxidative Stress: aging and disuse.
    (2009-05) Chen, Chiao-nan
    Sarcopenia, the age-related decline of muscle mass and strength, is one major risk factor for frailty and mobility disability of the elderly. Muscle disuse due to bed rest or surgery (such as joint replacements) exacerbates the ongoing decline of muscle function in the elderly. The decline of muscle function with disuse is greater in aging muscles. However, the cellular mechanism responsible for the greater functional decline of aging muscles with disuse is unknown. Oxidative stress, a condition where the balance between oxidant production and removal is disrupted, is a shared mechanism of age and disuse related muscle dysfunction. Thus, the overall aim of my dissertation is to understand the role of oxidative stress in the age-related muscle dysfunction with disuse.Using an animal model of muscle disuse (hindlimb unloading), I tested the hypothesis that the ability of aging muscles to cope with the increased oxidative stress associated with muscle disuse is compromised. There are three major findings: (1) the regulation of glutathione (GSH), an essential endogenous antioxidant, is impaired in aging muscles with disuse; (2) the decline of GSH levels in aging muscles with disuse is associated with the decrease of glutamate cysteine ligase (GCL) activity and the reduction of the catalytic subunit of GCL content; (3) using proteomic techniques, I identified two proteins (carbonic anhydrase III and four-and-a-half LIM protein1, FHL1), which show changes in the oxidation levels with disuse and aging. The changes in the oxidation levels of these two proteins with disuse occur in adult rats but not old rats. However, old rats have greater baseline levels of oxidized FHL1.In summary, the series of studies demonstrate that the response of muscles with disuse is age-dependent. The ability to maintain GSH levels with disuse is compromised in aging muscles. In addition, the changes of protein oxidation with muscle disuse occur in specific proteins and that the changes are age-related.
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    The structural dynamics of force generation in muscle, probed by electron paramagnetic resonance of bifunctionally labeled myosin.
    (2009-05) Thompson, Andrew Russell
    Two proteins in muscle, actin and myosin, are the key structural components that interact in order to produce muscle contraction. Myosin is a molecular motor that utilizes the chemical energy of ATP to undergo conformational changes that translate actin linearly, resulting in mechanical work. While previous studies have provided high-resolution measurement of these structural changes, many are unable to do so in intact muscle or in systems where myosin and actin can interact. This project seeks to make high-resolution structural measurements of myosin in actomyosin complexes during the different biochemical states associated with contraction. These measurements are being made using electron paramagnetic resonance (EPR), a spectroscopic technique sensitive to protein dynamics and orientation. In order to study myosin with EPR, a spin label is chemically attached to cysteine within the protein structure. In certain cases, native cysteines are used for spin labeling whereas in others, mutant protein is created with cysteines engineered in desired locations, a process known as site-directed spin labeling.Traditional spin probes attach via a single, flexible bond. This monofunctional attachment limits the sensitivity of EPR to protein orientation and dynamics because the resultant spectra are a mixture of probe and protein states. This project, on the other hand, uses a novel bifunctional spin label that is rigidly coupled to the protein via attachment to two engineered cysteines. Due to this rigid coupling, high-resolution structural measurements can be made with a degree of sensitivity not available to other techniques.
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    Structural transitions of myosin associated with force generation in spin-labeled muscle fibers.
    (2012-06) Mello, Ryan Nicholas
    Muscle contraction is driven by the actin-activated hydrolysis of ATP by myosin, resulting in the relative sliding of actin and myosin filaments. Current models propose that filament sliding is driven by a structural transition of myosin’s catalytic domain (CD) and light chain domain (LCD). The goal of this research is to measure structural transitions of myosin II (muscle and nonmuscle) that are associated for force generation. Structural measurements were made using electron paramagnetic resonance (EPR) spectroscopy. This work is comprised of two separate, but related, projects. In the first project (Chapter 3), thiol crosslinking and EPR were used to resolve structural transitions of myosin’s LCD and CD that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain (RLC) for endogenous RLC, with full retention of function. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), post-hydrolysis intermediate (A.M.D.P), and rigor (A.M.D). To trap myosin in a structural state analogous to the elusive post-hydrolysis ternary complex A.M.D.P, we used pPDM to crosslink SH1 (Cys707) to SH2 (Cys697) on the CD. EPR showed that the LCD of crosslinked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to crosslink SH1 to SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 crosslinking traps a state in which both the LCD and CD are in a structural state intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the crosslinked state is an A.M.D.P analog on the force generation pathway. In the second project, we present a method for obtaining high-resolution structural information of proteins using EPR of a bifunctional spin label (BSL). Two complimentary EPR techniques were employed to measure dynamics and orientation (conventional EPR) and intraprotein distances (dipolar electron-electron resonance). The exploitation of BSL is a key feature of this work. BSL attaches at residue positions i and i+4, which drastically restricts probe motion compared to monofunctional probes. For comparison, measurements were also made with the monofunctional spin label MSL. Subfragment 1 of Dictyostelium myosin II (S1dC) was used to exemplify the increased resolution provided by BSL. Using this approach, we demonstrate with experiments that BSL significantly increases resolution when measuring distance and orientation compared to MSL. And while this work does focus on the methodology, there is significant biological insight into myosin’s nucleotide-dependent structural transitions.