Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1980. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING. / Vita. / Bibliography: leaves 181-187. / by Ronald Stanton LeFever. / Ph.D.

Mechanical forces regulate biological processes in unique and unexpected ways, but many biochemical methods are unable to reproduce the vectorial stretching experienced by proteins in cells. Force spectroscopy techniques remedy these shortcomings by utilizing microscopic force probes to stretch and relax single protein, DNA, and RNA molecules. The central focus of this thesis is the development and implementation of a custom-built protein magnetic tweezers for unfolding and refolding Ig domains from titin, a critical filament of the sarcomere, and the longest continuous peptide in the human body. Suspended from the Z-disc to the tip of the thick filament, titin sustains the brunt of intracellular forces during muscle elongation. Since the discovery of titin, it has been widely debated whether Ig domain unfolding contributes to muscle mechanics. A combination of single quantum dot tracking in myofibrils extracted from rabbit muscle and single molecule magnetic tweezers experiments on recombinant titin fragments confirms, for the first time, the presence of titin Ig domain unfolding and refolding at physiological sarcomere lengths and stretching forces. The magnetic tweezers experiments show the surprising ability of titin Ig domains to generate piconewton level forces during folding, and we advance the hypothesis that titin folding is an important source of energy during muscle contraction. Muscle elongation recruits Ig domains to the unfolded state, whereby folding is initiated through reduction of force on titin upon actomyosin crossbridges formation. Magnetic tweezers measurements demonstrate that titin Ig folding generates peak work, velocity, and power output of 64 zeptojoules, 1.9 microns per second, and 6,000 zeptowatts, matching or exceeding the equivalent single molecule measurements from single molecule myosin II powerstrokes. The forces generated by protein folding are therefore likely to be an integral part of the contractile process of animal muscle.

by Choi Man. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 146-153). / Abstract --- p.1 / Chapter 1 --- Introduction --- p.3 / Chapter 1.1 --- Anatomy --- p.4 / Chapter 1.2 --- Kinematics of the overhead sports --- p.6 / Chapter 1.3 --- Isokinetics assessment of the shoulder rotators --- p.8 / Chapter 1.4 --- Objectives of the study --- p.10 / Chapter 1.5 --- Method of investigation --- p.11 / Chapter 1.5.1 --- Subject --- p.12 / Chapter 1.5.2 --- Equipment --- p.13 / Chapter 1.5.3 --- Protocol --- p.14 / Chapter 1.6 --- Data Management --- p.17 / Chapter 1.7 --- Clinical application --- p.18 / Chapter 1.8 --- Definition of terms --- p.18 / Chapter 2 --- Literture Review --- p.20 / Chapter 2.1 --- Anatomy --- p.20 / Chapter 2.1.1 --- Ligament --- p.21 / Chapter 2.1.2 --- Muscles --- p.24 / Chapter 2.2 --- Biomechanics of the overhead movement --- p.30 / Chapter 2.2.1 --- Wind up Phase --- p.32 / Chapter 2.2.2 --- Cocking Phase --- p.33 / Chapter 2.2.3 --- Acceleration Phase --- p.36 / Chapter 2.2.4 --- Deceleration Phase --- p.38 / Chapter 2.3 --- Eccentric contraction --- p.40 / Chapter 2.3.1 --- Physiology of eccentric contraction --- p.40 / Chapter 2.3.2 --- Mechanical trauma in eccentric contraction & DOMS --- p.41 / Chapter 2.3.3 --- Eccentric contraction in Plyometric --- p.43 / Chapter 2.3.4 --- Role of eccentric contraction in overhead sports --- p.44 / Chapter 2.3.5 --- Comparison with concentric contraction --- p.45 / Chapter 2.4 --- Isokinetics --- p.46 / Chapter 2.4.1 --- Introduction to isokinetics --- p.46 / Chapter 2.4.2 --- Reliability of isokinetic assessment on Shoulder rotator --- p.48 / Chapter 2.4.3 --- Agonist / Antagonist Ratio --- p.51 / Chapter 2.4.4 --- Variation of torques with testing protocols --- p.52 / Chapter 2.4.5 --- Comparison of muscle torques in different groups --- p.56 / Chapter 2.4.6 --- Isokinetic eccentric contraction of shoulder Rotators --- p.62 / Chapter 3 --- Method of Investigation --- p.63 / Chapter 3.1 --- Subject --- p.64 / Chapter 3.1.1 --- Part One --- p.64 / Chapter 3.1.2 --- Part Two --- p.65 / Chapter 3.1.3 --- Criteria of subject recruitment --- p.65 / Chapter 3.2 --- Equipment --- p.66 / Chapter 3.2.1 --- Collection of medical history and general informations --- p.66 / Chapter 3.2.2 --- Test for general laxity --- p.66 / Chapter 3.2.3 --- Test for shoulder impingement --- p.72 / Chapter 3.2.4 --- Test for shoulder instability --- p.73 / Chapter 3.2.5 --- Joint range measurement --- p.76 / Chapter 3.2.6 --- Isokinetic strength of shoulder rotators --- p.78 / Chapter 3.3 --- Calibration --- p.79 / Chapter 3.4 --- Testing procedure --- p.79 / Chapter 3.4.1 --- "Explanation,warning & consent" --- p.79 / Chapter 3.4.2 --- Warming up --- p.80 / Chapter 3.4.3 --- Screening --- p.81 / Chapter 3.4.4 --- Isokinetic testing of the rotational strength of both shoulders --- p.81 / Chapter 3.5 --- Operator --- p.87 / Chapter 3.6 --- Data Management --- p.88 / Chapter 3.6.1 --- Part One --- p.88 / Chapter 3.6.2 --- Part Two --- p.89 / Chapter 4 --- Result --- p.91 / Chapter 4.1 --- Part One --- p.89 / Chapter 4.1.1 --- Intra-class correlation coefficient --- p.90 / Chapter 4.1.2 --- Correlation between the PTR & the ASMSTR --- p.92 / Chapter 4.2 --- Part Two --- p.94 / Chapter 4.2.1 --- Comparison between the Members in the Hong Kong Badminton Team with the Non-athlete Subjects --- p.94 / Chapter 4.2.2 --- Comparison between the Badminton Players in the Hong Kong Team and the Hong Kong National Junior Team --- p.105 / Chapter 5 --- Discussion --- p.111 / Chapter 5.1 --- General discussion of the design of the study --- p.111 / Chapter 5.1.1 --- Subject --- p.111 / Chapter 5.1.2 --- Specific test --- p.112 / Chapter 5.1.3 --- Warming up --- p.112 / Chapter 5.1.4 --- Testing protocol --- p.113 / Chapter 5.2 --- Part One --- p.116 / Chapter 5.2.1 --- Test-retest reliability --- p.117 / Chapter 5.2.2 --- Correlation between the PTR and the ASMSTR --- p.120 / Chapter 5.3 --- Part two --- p.122 / Chapter 5.3.1 --- Comparison between the HKT and the non- athletes --- p.123 / Chapter 5.3.2 --- Presentation of torque ratio in the HKJ --- p.133 / Chapter 5.3.3 --- Performance in those with history of shoulder problem --- p.134 / Chapter 5.4 --- Clinical application and suggestion for further study --- p.139 / Chapter 6 --- Conclusion --- p.144 / Chapter 7 --- Reference --- p.146 / Chapter 8 --- Appendix

Fatigue is a difficult phenomenon to study because the response can vary based upon task-specific (i.e. contraction type, intensity, position– vs. load-matching and muscle group/joint region) and subject-specific (i.e. sex and age) variables. Although numerous investigations have provided insight into muscle fatigue, further efforts were needed to better characterize the influence of age, sex, joint/muscle group, contraction type, and task complexity have upon fatigue. The primary purpose of this series of three studies was to identify and characterize the influences of potential moderating variables (i.e., sex, joint, age, contraction type, and task complexity) upon fatigue resistance during voluntary muscle contraction fatigue tasks through both empirical (systematic review and meta-analysis) and experimental methods. In general, women demonstrated either the same or better fatigue resistance than men (men never better), the sex advantage was joint specific not systematic, old men were more fatigue resistant than young men, task complexity was not an influential factor and fatigue differences were more readily apparent under isometric conditions. The inclusion of empirical and experimental methods helped clarify the driving factors of localized muscle fatigue. This in turn will better direct future study design and power for mechanistic, training and performance response studies.

One advantage of higher life-forms over less developed organisms is their ability to respond to signals from their environment with motion. This requires highly specialized contractile cells and a whole locomotion apparatus. In vertebrates, the cells responsible for movement are the skeletal muscle cells. They receive signals from the autonomic nervous system in the form of an action potential, and they contract in an appropriate manner. Calcium is a vital intracellular passenger whose role in muscular function is to initiate contraction. It is released via specific channel proteins from an internal Ca++ store, the sarcoplasmic reticulum, and triggers muscular contraction, the actual interplay of actin and myosin filaments. The step that is still not fully understood is the coupling process between arrival of an action potential and the subsequent contraction, called excitation-contraction coupling. Several theories have been proposed to explain this process. Some years ago, our laboratory introduced the hypothesis that an oxidation-reduction reaction of critical sulfhydryls associated with the Ca+t channel protein are involved in the regulation of channel gating. In an effort to understand more about the Ca++ channel gating mechanism at the molecular level, this thesis focuses on the interaction between o-phthalaldehyde, a reagent which specifically forms an isoindole derivative with the amino acids cysteine and lysine, and the Ca++ release channel complex. In this thesis, the planar lipid bilayer technique was used to study the Ca++ release channel protein from skeletal muscle sarcoplasmic reticulum at the single channel level. Utilizing this experimental technique, the direct interaction between OP A and the channel was investigated. In this study, it was shown that the interaction of o-phthalaldehyde with the channel increases the channel's open probability as well as its mean open time. Furthermore, the covalent nature of o-phthalaldehyde binding to the calcium release channel complex is shown and its inhibiting effects on chloride channels are demonstrated.

Muscle contraction is a phenomena which fascinated already the ancient Greeks. People have long sought to understand the mechanism of muscle contraction. Today we know that in order for muscle to contract, an action potential propagates from the nerve cell to the muscle cell. Upon arriving at the muscle cell, via a mechanism called Excitation- Contraction (E-C) coupling, Ca2 + is released from an intracellular membrane system, the sarcoplasmic reticulum (SR), into the intracellular fluid. The increase of intracellular Ca2 + initiates the interaction between the contractile units which results in force development and tension. The least well understood step in the contractile process is mechanism of E-C coupling. During the last 15-20 years various theories have been proposed to describe this process. Our laboratory came up with a theory several years ago, that critical sultbydryl groups on a protein, the ryanodine receptor(RyR)/Ca2 + release channel, are oxidized and subsequently reduced during the process of contraction and relaxation. In this thesis a reagent, o-Phthalaldehyde (OPA), was used to better understand the gating mechanism of the RyR/Ca2 + release channel. This reagent has the ability to form an isoindole derivative with the amino acids cysteine and lysine, if they are separated by not more than 3 A .In this study, it was shown that OP A interacts directly with the Ca2 + release channel by forming a covalent derivative with a critical thiol and a nearby lysine. High affinity [3H]Ryanodine binding to the RyR\Ca2 + release channel is activated by < 130μM OP A, but is inhibited by OPA at concentrations ranging from 200-300 μM OPA. This biphasic behavior indicates that at least two sets of cysteine-lysine pairs regulate Ca2 + channel activity. Moreover, the binding of OP A results in increasing the affinity of the receptor for the binding of ryanodine, in a Ca2 + independent manner, which may indicate that there are two different sets of RyR\Ca2+ release channels present in the SR.

Aging is associated with attenuated blood flow and vasodilator responses during rhythmic exercise. Older adults also demonstrate attenuated blood flow and vasodilator responses following single skeletal muscle contractions (contraction-induced rapid onset vasodilation, ROV) within the forearm. These age-associated attenuations within the forearm have been demonstrated to be a result of endothelial and neural mechanisms. The objective of this research was to examine: 1) whether age-associated attenuations within the forearm are from mechanical factors; 2) whether age-associated attentions in ROV are present within the leg, as well as explore potential mechanisms for these age-associated attenuations in ROV; 3) examine whether aging is associated with a slower rate of adjustment in vasodilation (vasodilator kinetics) during rhythmic exercise preceding steady-state exercise; and 4) examine approaches to ameliorate age-related attenuations in blood flow and vasodilation within the leg across the entire exercise transient (onset to steady-state). The novel findings of this research are that 1) age-associated attenuations in ROV within the forearm are independent of mechanical factors; 2) older adults demonstrate attenuated ROV responses within the leg; 3) age-related attenuations in ROV within the leg are not explained by enhanced sympathetic adrenergic vasoconstriction; 4) older adults exhibit prolonged vasodilator kinetics preceding steady-state exercise; and 5) when examined in a cross-sectional design chronic exercise training improves ROV, vasodilator kinetics, as well as steady-state blood flow and vasodilator responses in older adults; 6) acute supplementation with dietary nitrate fails to exert any effect on blood flow and vasodilator responses during any domain of exercise. Collectively, this work establishes that aging is associated with reductions in blood flow and vasodilation across the entire exercise transient (onset to steady-state) within the leg, which is offset by chronic exercise training. Mechanistically, the current data suggests that mechanical and sympathetic factors do not explain age-related reductions in ROV in the arm and leg, respectively. Furthermore, acute supplementation of dietary nitrate does not impact leg blood flow and vasodilator responses in older adults during any domain of the exercise transient.

The aim of the thesis was to evaluate the effectiveness of static hand splints for the prevention of muscle contracture during early rehabilitation following acquired brain impairment. Three studies were undertaken and are reported in the research. The aim of the first study was to appraise the existing research on the effects of hand splinting for adults with hemiplegia following acquired brain impairment. The aim of the second study was to evaluate the effectiveness of static hand splints which position the wrists and fingers in the common ‘functional position’ when provided in conjunction with a rehabilitation program which included daily motor training and prolonged stretches. The aim of the final study was to evaluate the effectiveness of two hand splinting positions, the ‘functional position’ and a position of wrist and finger extension, in comparison to a control group that did not receive prolonged stretches. Findings indicate that splinting the hand in the ‘functional’ position or in a position of greater wrist extension did not prevent contracture following acquired brain impairment over the course of the study periods (4 weeks with follow up at 4 and 6 weeks in studies 2 and 3 respectively). / Doctor of Philosophy (PhD)

This thesis reports an investigation from the “bedside” back to the “bench”. That is, from the economic evaluation of a medical intervention to basic research and development of a contractility assay. The underlying theme of this thesis is cellular contractility, which was stimulated from our laboratory’s work in the microvascular complications of Diabetic Retinopathy (DR). The health economic perspective of this thesis evaluates the cost effectiveness and cost utility of DR prognosis using the prog-DR test. This novel prognostic test developed in our laboratory relies on the contractile response of blood vessels to detect subjects with high risk of developing DR. Markov modeling based on information in the literature was used to estimate the outcomes of a hypothetical population. The costs, health and utility outcomes of DR were compared to the potential outcomes if the prog-DR test was used. The model show that the prog-DR test can improve the health of the hypothetical population as measured in the number of life years (LY), sight years (SY) and quality-adjusted life years (QALY). The prog-DR test was more cost effective than the benchmark of annual or bi-annual screening and the incremental cost effectiveness ratio (ICER) appears to be at an acceptable level. Scenario and sensitivity analysis also show that the cost effectiveness of the prog-DR test can be improved by (i) better blood glucose management post prog-DR test, (ii) targeted screening (as opposed to population-wide screening) and (iii) reduced costs of both screening and management of DM and DR. The physiological perspective of the thesis aimed to develop a contractility assay for DR that was based on a 3D scaffold, which was affordable, easy to make and mimicked the three dimensional physiological environment of blood vessels. The contractility assay was developed using a 3D, hollow scaffold (PE-PAH capsule) and involved (i) the selection of the optimal core material, (ii) optimisation of the manufacturing process, (iii) characterisation of the scaffold and (iv) ensuring that cells can be grown on it. The cyto-biocompatibility of the candidate polyelectrolyte Poly(Sodium 4-Styrene Sulfonate) (PSS) and Poly(Allylamine Hydrochloride) (PAH) in the thin films format were investigated using three different cell lines and the effects of these thin films were also compared to titanium and titanium nitride thin films. In essence, PSS and PAH are not cytotoxic and was used to develop the contractile scaffold, PE-PAH capsule. This scaffold is relative elastic and the contractile force exerted by the 3T3-L1 cells was calculated based on the deformation of the PE-PAH capsule. The contractility assay was sufficiently sensitive to detect the nano-Newton magnitude of force developed by individual cells and discriminated the change in force due to disruption of the F-actin cytoskeleton by forskolin and cytochalasin D.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: John E . Novotny, Dept. of Mechanical Engineering. Includes bibliographical references.