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Browsing by Subject "muscle"

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    3D Orientation of Alpha Helix in Muscle Myosin Regulatory Light Chain Using Bifunctional Electron Paramagnetic Resonance
    (2021-06) Savich, Yahor
    Muscle contraction is a coordinated work of nanometer-sized force generators, myosin molecules. These molecules are out of equilibrium: they use the energy stored in the form of ATP to move collectively along the track protein actin. The myosin molecules transfer their work via lever arms that connect force generators to their cargo. Orientation of these lever arms has been studied thoroughly since 1) their structural dynamics is fundamental for understanding the muscle contraction and 2) their particular orientations are associated with disease states of cardiac and skeletal muscle. Electron microscopy, fluorescence polarization, and X-ray diffraction have provided insight into the structure of muscle, but there is still no high-resolution data of the vertebrate lever arm orientation available at ambient (not vitrified or crystallized) conditions. The present work establishes a method of measuring the orientation of the alpha helices in three dimensions using electron paramagnetic resonance (EPR). Chapter 3 introduces the use of EPR with bifunctional spin labels attached to different helices of the myosin regulatory light chain (RLC) protein with and without ATP. Demembranated skeletal muscle fibers were aligned with the slowly-varying magnetic field; RLC was chemically substituted by labeled RLC; axial orientational dynamics of the probe with respect to the muscle axis was determined. Chapter 4 utilizes 1) directional statistics that replaces the previous use of a Gaussian distribution and provides new insights into the degree of disorder and 2) a new bifunctional probe that adds an azimuthal dimension to the orientational data. Together, these techniques allow determination of the tilt and roll angles of the alpha helix without relying on the myosin structure.
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    Investigation of Sarcopenia in a Murine Model: Symptoms of Age-Related Neuromuscular Decline and Resistance Training Intervention
    (2015-05) Graber, Ted
    The world population demographic is rapidly aging. With advancing age comes the onset of age-related diseases and syndromes such as sarcopenia, the age-associated loss of muscle mass and strength. Sarcopenia leads to a multitude of adverse outcomes including a reduced quality of life, increased mortality, functional disability, and eventual loss of independence. Currently no cure for sarcopenia exists and its etiology is still largely undefined. Thus, there is a need for animal models for preclinical investigation of novel interventions. The overall purpose was to first investigate, characterize, and describe the neuromuscular healthspan of the C57BL/6 mouse, a common animal model of aging, and then to subsequently create a treatment model for sarcopenia by producing and validating a voluntary resistance training protocol for mice. The first research chapters (2 and 3) investigate and define the neuromuscular healthspan and the age-related decline of contractile parameters in the mouse. Chapter 2 outlines the neuromuscular healthspan scoring system, a composite score consisting of two functional measures combined with in vitro maximum isometric force of the extensor digitorum longus (EDL). This composite outcome measurement increased the power to detect change beyond the capacity of the individual component measures alone. Chapter 3 examines unique aspects of contractile velocity and power production in the soleus and EDL, revealing that age has a greater effect on concentric contraction performed at higher percentages of maximum force. Because the only consensus treatment for sarcopenia is resistance exercise, in Chapter 4 a mouse model of voluntary resistance training was designed and validated. The protocol was designed using human principles of weight training and was assessed with a comprehensive battery of outcome measurements. The outcomes were selected to test whether the mice had the same type of adaptations as would be observed in humans undergoing a similar training intervention. In Chapter 5, the resistance training protocol was applied to a cohort of aged mice to test if signs of anabolic resistance would be detected. Overall, the thesis tells the story of age-related neuromuscular dysfunction that can be partially rescued though exercise and creates a novel preclinical animal model of voluntary resistance training.