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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.

The sarcoplasmic reticulum is the intramuscular organelle responsible for the regulation of cytoplasmic calcium levels in muscle. This thesis investigates the effects of the cardioprotective drug, dexrazoxane, and its metabolite ADR-925 on doxorubicin induced calcium release from skeletal sarcoplasmic reticulum. Doxorubicin is a widely used antineoplastic agent. One of the major side effects of doxorubicin usage is chronic cardiotoxicity. Doxorubicin is a potent activator of the calcium release mechanism from the SR. The interaction between doxorubicin and the calcium release channel has been proposed as the possible underlying mechanism behind cardiotoxicity. A short overview of different hypotheses describing doxorubicin induced cardiotoxicity and proposed mechanisms of cardioprotection by dexrazoxane are presented. While dexrazoxane did not appear to affect the calcium permeability of the SR, its metabolite, ADR-925, modulates the ryanodine receptor complex. ADR-925 inhibits high affinity ryanodine binding to the ryanodine receptor/calcium release channel complex by decreasing the sensitivity of the receptor for stimulation by calcium. ADR-925's ability to inhibit doxorubicin stimulated ryanodine binding is independent of the doxorubicin concentration. These results demonstrate that ADR-925 directly affects the ryanodine receptor complex of the SR by desensitizing the receptor to activation by calcium. Furthermore, ADR-925 reduces the inhibitory effect of hydrogen peroxide on the ryanodine receptor/ calcium release channel. This suggests that ADR-925 may protect the SR from oxidative effects of free radicals. It has been somewhat controversial whether doxorubicin induced cardiotoxicity is due to a specific interaction with the calcium release mechanism of SR. The findings presented in this thesis which demonstrate that the cardioprotectant ADR-925 interacts directly with the ryanodine receptor from SR, further support the hypothesis that the ryanodine receptor is a primary target of doxorubicin's action.

The unfolded protein response (UPR) is a cellular mechanism whose primary functions are to sense perturbations in the protein-folding capacity of the endoplasmic reticulum and to take corrective steps to restore homeostasis. Although the UPR is conserved across all eukaryotic cells, it is considerably more complex in mammalian cells, due to the presence of three interconnected pathways triggered by separate sensor proteins, a translation attenuation mechanism, and a negative feedback loop. The mechanisms of these interacting biochemical pathways in the mammalian UPR allow for a better fine-tuning of the response than in the case of lower eukaryotes, such as yeasts. The present thesis develops a quantitative mathematical model for the dynamics of the UPR in mammalian cells, which incorporates all the proteins and interactions between them that are known to play a role in this response. This model can be used to provide quantitative information about the levels of its components throughout the response, and to analyze the ramifications of perturbations of the UPR. The model uses a system of ordinary nonlinear differential equations based on biochemical rate equations to describe the dynamics of the UPR as a network of interacting proteins and mRNAs. An early model is presented as a first attempt to investigate the UPR network and construct an inclusive wiring diagram, as well as suggesting a framework to model the differential equations. Then, a refined, quantitative model is designed based on experimental data collected on Mouse Embryonic Fibroblasts treated with Thapsigargin to induce stress and trigger the UPR. The model defines the differential equations and determines the unknown kinetic parameters by optimizing the fit of the system's solution to the experimental data. It includes the UPR's intrinsic feedback loops and allows for the integration of various forms of external stress signals. To the best of our knowledge, it is the first, data-validated, quantitative model in the literature for the UPR in mammalian cells. The last chapters of the thesis address, from a modeling point of view, two important questions for the UPR: (1) cell survival versus apoptosis; and (2) incompleteness of the biological wiring diagram. Recent experimental results show that the UPR is capable of producing qualitatively different results leading to cell survival or death depending on the nature, strength, and persistence of the inducing stress. This thesis proposes several approaches by which the equations can be modified to model the transition from adaptation to apoptosis as a dynamic switch, while taking into account the various hypotheses of cell death mechanisms. Finally, we use recently-developed computational algebra techniques to infer an optimal structure of the UPR network, based solely on the experimental data; the resulting wiring diagram provides insights on elements of the structure of the model that may have been overlooked during the classical (mechanistic) approach to our original data-based model.

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Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

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Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 130-155). Also available on the Internet.