published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

Osteocytes are widely regarded as mechanosensors, capable of detecting changes in the mechanical environment of the bone tissue and modifying cellular responses accordingly. Indeed, an intact osteocyte network is required for bone changes in response to unloading, and studies have shown that loading/unloading influences osteocyte expression of proteins that modulate bone turnover, such as sclerostin and receptor activator of nuclear factor kappa B ligand (RANKL). However, mechanisms underlying osteocyte mechanotransduction remain unclear. For instance, one of the earliest responses of bone cells to mechanical stimuli is a rise in intracellular, or cytosolic, calcium (Ca2+cyt), but the mechanisms by which osteocytes generate or utilize Ca2+ signals to direct bone adaptation are largely unknown. In this thesis, I explored the mechanisms underlying the sustainment of Ca2+cyt oscillations in osteocytes as well as downstream consequences of these patterns. I discovered that Ca2+cyt oscillations are generated in osteocytes by Ca2+ release from the endoplasmic reticulum and that the predominant expression of T-Type voltage sensitive Ca2+ channels in these cells facilitates this behavior. I also explored the role of the actin cytoskeleton – another prominent feature in osteocytes – and found that actin dynamics are important for the generation of Ca2+cyt signals. Furthermore, I confirmed that Ca2+cyt transients subsequently activate actomyosin contractions in osteocytes by monitoring interactions of osteocytes exposed to Ca2+ agonists on micropillar substrates. With this information, I sought to relate Ca2+cyt signaling and actomyosin contractility in osteocytes to their roles as coordinators of bone adaptation. Ca2+-dependent contractions have been shown to facilitate the release of extracellular vesicles, small membrane-enclosed packages of proteins that cells use for communication, in other cell types. I found that mechanical stimulation increased the production and release of extracellular vesicles in osteocytes, and this was dependent on Ca2+ signaling. These extracellular vesicles contained key bone regulatory proteins and were small enough to plausibly transport through the lacunocanalicular system. Thus, I uncovered a novel mechanotransduction pathway by which osteocytes may coordinate tissue-level adaptation. As an extension of this work, I also characterized these behaviors in new osteocyte cell lines which may better reflect native cell physiology. The work in this thesis anchors Ca2+ signaling as a critical osteocyte response to mechanical loading and adds to the body of work exploring how and why these signals are generated. The results of these studies add new information to the still limited knowledge of this important bone cell and extend Ca2+ signaling research by connecting early mechanosensation events to subsequent protein responses to mechanical loading. Understanding the mechanisms behind the robust Ca2+cyt oscillations in osteocytes and how they relate to their roles as coordinators of bone adaptation may improve our ability to prevent or treat bone degeneration in diseases like osteoporosis where mechanosensitivity is impaired.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on January 30, 2008) Vita. Includes bibliographical references.

Thesis (M. Sc.)--Memorial University of Newfoundland, 1998. / Bibliography: leaves 68-75.

Thesis (Ph. D.)--University of Hong Kong, 200 . / Title proper from title frame. Also available in printed format.

Effect of added calcium ions on the relative milk clotting activities of porcine pepsin, Mucor miehei protease, Endothia parasitica protease, and Mucor pusillus protease was compared with that of rennin. Skimmilk, maintained at pH 6. 3 with 0.000% to 0.030% added calcium chloride, was used as a substrate. The coagulation activity of Mucor pusillus protease appeared to be most sensitive to calcium ions, followed by Mucor rniehei protease, porcine pepsin, rennin, and Endothia parasitica protease. The clotting activities of Mucor pusillus protease were also more sensitive than rennin to added calcium ions in milk samples maintained at pH values of 6.4, 6.5, and 6.6. Mucor pusillus protease and rennin were standardized to equal activities in skimmilk maintained at pH 6.3 and 6.6 containing 0.020% added CaCl2. For skimmilk maintained at pH 6.3, Mucor pusillus protease concentrations ix that gave the same clotting times as rennin in skimmilk containing 0.000% and 0.010% added CaCl2, were 9.6% and 4.5% higher than its standardized concentration. While at pH 6.6 Mucor pusillus protease concentrations were 13.2% and 5.8% higher.

Li+ ion batteries have been the mainstay of high energy storage devices that have revolutionized the operating life time of consumer electronic devices for the past two decades. However, there is a steady increase in demand for energy storage devices with the ability to store more energy and deliver them at high power at low cost, without comprising safety and lifetime. Li-ion batteries have had significant challenges in increasing the amount of stored energy without affecting the overall lifetime and the ability to deliver stored energy. In order to store and deliver more energy, more lithium ions need to be inserted and extracted from a given electrode (cathode or anode). Upon inserting a large number of Li ions, the crystal lattice of the materials undergo severe mechanical distortions, leading to un-desirable structural changes. This results in underutilization of theoretical energy storage capacities of the electrodes and early failure of the batteries owing to instabilities in the electrode materials. Unlike monovalent Li+ ions, multivalent rechargeable batteries offer a potential solution to the above problems. Multivalent cations, such as Ca2+, are doubly-ionized as opposed to Li+ which is a monovalent cation. The advantages of using Ca2+ ions instead of Li+ ions are multifold. Due to the doubly-ionized nature, only half the number of Ca2+ ions need to be inserted and extracted from a given electrode to store and deliver energy from a high capacity cathode as compared to Li+ ions. This reduces the probability of lattice distortion and un-desirable structural changes, further leading to increased utilization of high theoretical energy storage capacities of the electrodes (cathode and anode). The use of Ca2+ ions also helps in delivering twice the amount of current density as compared to Li+ ions due to its doubly ionized nature. In this work, a set of eight metal hexacyanoferrate compounds were synthesized using the following metal ions: Ba2+, Mn2+, Zn2+, Co2+, Fe3+, Al3+, Sn4+, Mo5+. The resulting metal hexacyanoferrate compounds were subjected to physical characterization using scanning electron microscope (SEM) and powder x-ray diffraction (XRD), to determine physical properties such as size, morphology, unit cell symmetry and unit cell parameters. This was followed by electrochemical characterization utilizing cyclic voltammetry and galvanic cycling, to determine the specific capacity and kinetics involved in the transport of Ca2+ ions to store charge. Optical characterization of the metal hexacyanoferrates using Fourier transform infrared (FTIR) spectroscopy, allowed for the identification of metal-nitrogen stretching frequency, which was used as a measure of the strength of the metal-nitrogen bond to understand the role of the above mentioned metal ions in electron density distribution across the unit cell of the metal hexacyanoferrates. The specific capacity utilization of the metal hexacyanoferrates, when compared to the electronegativity values (Xi) of the above mentioned metal ions, the σ- parameter, and the metal-nitrogen stretching frequency (v), revealed an empirical trend suggesting that the materials (FeHCF, CaCoHCF and CaZnHCF) that possessed intermediates values for the above mentioned parameters demonstrated high capacity utilization (≥50%). Based on these empirical trends, it is hypothesized that a uniform distribution of electron density around a unit cell, as reflected by intermediate values of the electronegativity (Xi) of the above mentioned metal ions, the σ-parameter and the metal-nitrogen stretching frequency (v), results in minimal electrostatic interactions between the intercalating cation and the host unit cell lattice. This results in relatively easy diffusion of the cations, leading to high specific capacity utilization for metal hexacyanoferrate cathodes. These parameters may be used to select high efficiency cathode materials for multivalent batteries.

Molecular dynamics simulations are an important tool for the study of biological systems, such as biomembranes. The missing electronic polarization in classical non- polarizable force fields, however, produces significant inaccuracies in the interactions of membranes with charged particles, such as ions. In this work, we implement the missing electronic polarization effects into CHARMM36 force field for phospatidylcholine lipids. This implementation is done in the mean field way by using electronic continuum correction (ECC) model. We will validate the strength of ion-membrane interactions using the electrometer concept. This concept connects the response of choline order parameters of lipid molecules with the amount of charge present in the surface of the membrane. Keywords: phosphatydylcholine, calcium ions, sodium ions, electronic continuum cor- rection, electrometer concept 1

A procedure was developed for dealing with two problems that have impeded the use of quantal parameters in studies of transmitter release. The first, involving temporal and spatial biasing in the estimates for the number of functional release sites (n̄) and probability of release (p̄), was addressed by reducing temporal variance experimentally and calculating the bias produced by spatial variance in p (var(s)p). The second, involving inaccuracies in the use of nerve-evoked endplate potentials (EPPs), was circumvented by using only miniature EPPs (MEPPs). Intracellular recordings were made from isolated frog cutaneous pectoris, after decapitation and pithing of the animals, and the concentration of K+ ([K+]) was raised to 10 mM to increase the level of transmitter release. The number of quanta released (m̄) by the EPP was replaced by the number of MEPPs in a fixed time interval (bin), and 500 sequential bins used for each quantal estimate. With the use of 50-ms bins, estimates for var(s)p were consistently negative. This was due to too large a bin (and introduction of undetected temporal variance) because the use of smaller bins (5 ms) produced positive estimates of var(s)p. Increases in m, n, and p but not var(s)p were found in response to increases in [K+] or [Ca2+]/[Co2+]. La3+ (20 μM) produced increases in m and n, which peaked after 20 min and declined toward zero. There were also large increases in p and var(s)p, which peaked and declined only to initial control values. The increase in var(s)p was presumed to reflect La3+-induced release of Ca2+ from intracellular organelles. The results suggest that this approach may be used to obtain unbiased estimates of n̄ and p̄ and that the estimates of var(s)p may be useful for studying Ca2+ release from intraterminal organelles.

1 Abstract Adenylate cyclase toxin (CyaA) is one of the major virulence factors of bacterium Bordetella pertussis, which is a causative agent of whooping cough. CyaA belongs to the family of RTX toxin-hemolysins. The toxin targets primarily cells expressing integrin receptor CD11b/CD18 but it can also penetrate cells lacking this receptor. CyaA acts on host cells by two independent activities. One is formation of small cation-selective channels, which can lead to colloid osmotic lysis of target cells. The second is disruption of cell signaling through the translocation of the adenylate cyclase (AC) domain to host cell cytosol, which leads to the conversion of ATP into cyclic AMP. It was recently shown that cholesterol affects endocytosis of CyaA. CyaA translocates it's AC domain after relocation of CyaA molecule to the cholesterol-rich lipid raft (Bumba et al. 2010). In this work I examined the effect of cholesterol on channel- forming activity and selectivity of ion channels created by CyaA. For measurements I used artificial membranes enriched with cholesterol. CyaA channels are voltage-dependent. The positive membrane potential on the side of toxin is rquired for incorporation of CyaA molecule into cell membrane. I tried to find out whether the value of voltage has effect on channels opening time....