Understanding the temporal and spatial changes in calcium concentration has been a difficult endeavor for many years due to the relatively small changes in calcium concentration during messenging events, the rapid changes upon physiological messenging, and the unavailability of fast, efficient, and sensitive sensors to detect calcium changes. In addition, the key factors in calcium binding have yet to be determined due to the metal-metal interactions, cooperativity, and conformational change involved in calcium binding to natural calcium-binding proteins. To overcome these obstacles and to engineer calcium sensors for in vivo studies of calcium signaling events, calcium binding sites have been engineered into Green Fluorescent Protein. The engineered binding sites demonstrate terbium binding affinity from 2-30 ƒÝM and calcium binding affinity from 50-100 ƒÝM. Site 177 demonstrates green fluorescence when expressed in mammalian cells and produces a response to calcium concentration changes when expressed in the cytosol. Addition of the cycle 3 mutations (M153T, V163A, F99S) to Site 177 allowed for increased brightness in the emission of the chromophore but still exhibited calcium response. The second generation Site 1 demonstrates fluorescence response to calcium concentration changes when expressed both in the cytosol and in the endoplasmic reticulum. Addition of M153T and V163A to Site 1 allowed for expression of fluorescent protein at 37 ¢XC in HeLa cells and at 30 ¢XC in bacteria. Site 1-M153T/V163A exhibits chromophore fluorescence response to calcium with a Kd of 100 ƒÝM and competition with Rhodamine-5N produced a calcium Kd of 107 ƒÝM. This designed sensor, Site 1-M153T/V163A is the first demonstration of a designed calcium binding GFP with calcium response measured both in vivo and in vitro.

In recent years, dietary calcium intake among children and adolescents has fallen sharply. This trend is alarming because childhood and adolescence are the primary ages for building bone mass. Therefore, an interactive website, Clueless in the Mall (http://calcium.tamu.edu) was developed targeting youth to increase knowledge, improve attitudes, and equip them to incorporate calcium-rich foods into their diets. This website was created with the help of adolescents at every stage of development and has been pilot tested with adolescents at home and in school, showing improvements in knowledge and attitudes. However, the website has not been tested for effectiveness in changing behaviors, specifically for increasing calcium intake. The present study was conducted to measure the effectiveness of the website for changing knowledge, attitudes, and behaviors. The hypothesis of this investigation was that the website intervention would significantly improve attitudes and knowledge about calcium and increase dietary calcium intake among adolescents and pre-adolescents who took part in the study. After the group-administered Food Frequency Questionnaire (FFQ) was validated among a local church youth group, a total of 126 middle school and high school students were recruited from 12 local public school classes to participate. Each student took three questionnaires during class time to measure baseline knowledge, attitudes, and behaviors (including calcium intake using the FFQ). Then each student took one class period to view the calcium website. Four to six weeks later, the investigators returned to administer the same three tests to each student. Demographic information was collected, and data were analyzed using paired samples t-tests and analyses of variance (ANOVA). Results demonstrated that the website alone was sufficient to improve knowledge scores; however, it was not enough to change attitudes and behaviors significantly. In conclusion, the website should be used to promote and reinforce health behaviors, but should not be expected to stand alone as an intervention.

Thèse doctorat : Biologie Cellulaire : Nancy 1 : 2007. / Titre provenant de l'écran-titre.

In this study the mechanisms of Ca²⁺-induced-Ca²⁺-release, effects of membrane depolarizations and the actions of pharmacological intracellular Ca²⁺-modulators were examined in cultured rat dorsal root ganglion (DRG) neurones. The whole cell configuration of the patch clamp technique was used to record action potentials, action potential after-potentials and voltage-activated calcium currents, (I_Ca), calcium-activated chloride currents, (I_CI(Ca)), and non-selective cation currents, (I_CAN), under current and voltage clamp recording conditions, respectively. A sub population of DRG neurones expressed action potential after-depolarizations and I_CI(Ca)tail currents which were due to activation of Ca²⁺-activated Cl^- channels as a result of Ca²⁺ entry. I_CANwas dominantly activated due to Ca²⁺ release from intracellular stores evoked by pharmacological Ca²⁺-releasing agents such as caffeine, ryanodine and dihydrosphingosine. Calcium-activated conductances were identified by estimating reversal potentials of the activated currents, using selective pharmacological blockers and extracellular ionic replacement studies. Calcium-dependence of activated currents was also examined by using high concentration of intracellular Ca²⁺ buffer, EGTA, to prevent elevation of intracellular Ca²⁺-levels and by rapidly buffering raised intracellular Ca²⁺ using intracellular 'caged Ca²⁺ chelator', diazo-2. The involvement of intracellular Ca²⁺- stores was examined by performing experiments in Ca²⁺-free extracellular recording medium and pharmacologically inhibiting release of Ca²⁺ from intracellular stores, using dantrolene. Ryanodine had complex actions on DRG neurones, which reflected its ability to mobilize Ca²⁺, deplete Ca²⁺ stores, and inhibit Ca²⁺ release channels. Ryanodine inhibited action potential after-depolarizations and I_CI(Ca)tail currents by interacting with intracellular stores and preventing amplification of Ca²⁺ signalling by CICR. It was found that CICR observed under physiological conditions in rat DRG neurones involves intracellular Ca²⁺stores which were sensitive to ryanodine. In addition to ryanodine sensitivity these intracellular Ca²⁺ stores could be mobilized by caffeine and dihydrosphingosine.

Thesis (MScAgric)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

Thèses (Ph.D.)--Université de Sherbrooke (Canada), 1998. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Thesis (M.S.)--University of Michigan, 1998. / Includes bibliographical references.

Thesis (Ph. D. in materials science)--Washington State University. / Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1934. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2002. / Bibliography: leaves 104-111.