SlyD is a protein involved in [NiFe]-hydrogenase enzyme maturation and, together with HypB and HypA proteins, contributes to the nickel delivery step. To understand the molecular details of this in vivo function, the nickel-binding activity of SlyD was investigated in vitro. SlyD is a monomeric protein that can chelate up to 7 nickel ions with an affinity in the sub-nanomolar range. By truncation and mutagenesis studies we show that the unique C-terminal metal-binding domain of this protein is required for Ni(II) binding and that the protein coordinates this metal non-cooperatively. This activity of SlyD supports the proposed in vivo role of SlyD in nickel homeostasis. In addition to nickel, SlyD can bind a variety of other types of transition metals. Therefore it was feasible that the protein contributes to homeostasis of metals other than nickel. To test this hypothesis, the metal selectivity of the protein was examined. The preference of SlyD for the metals examined could be ordered as follows, Mn(II), Fe(II) < Co(II) < Ni(II) ~ Zn(II) << Cu(I) indicating that the affinity of SlyD for the different metals follows the Irving-Williams series of metal-complex stabilities. Although the protein is unable to overcome the large thermodynamic preference in vitro for Cu(I) and exclude Zn(II) chelation, in vivo studies suggest a Ni(II)-specific function for the protein. To understand the function of SlyD as a metallochaperone, its interaction with HypB was investigated. This investigation revealed that SlyD plays a role in Ni(II) storage in E. coli and can function as a Ni(II)-donor to HypB. This study also revealed that SlyD can modulate the metal-binding as well as the GTPase activities of HypB. Based on the experimental data, a role for the HypB-SlyD complex in [NiFe]-hydrogenase biosynthesis is presented.

The underlying mechanism for the origin of ultradian rhythms is not clearly understood at present. Based on a recent study from our laboratory, we have conceptualized a model for the origin of quasiperiodic ultradian rhythms in sleep-wake state. This model hypothesizes that the ultradian rhythms of sleep-wake state may be generated by a mechanism that includes the sleep-wake homeostat. The main purpose of the present study was to test the hypothesis that sleep homeostasis and sleep-wake ultradian rhythms share a common underlying mechanism. The present study has refuted that hypothesis. I conclude that: (1) the proposed model for the generation of quasiperiodic ultradian rhythms in sleep-wake state in mammals is overly simplistic in its present form, (2) the generation of ultradian rhythms in sleep-wake state probably arise from a more complex systemic interactions between the sleep-wake oscillatory network and other internal/external inputs, rather than the simple expression of sleep homeostat.

The underlying mechanism for the origin of ultradian rhythms is not clearly understood at present. Based on a recent study from our laboratory, we have conceptualized a model for the origin of quasiperiodic ultradian rhythms in sleep-wake state. This model hypothesizes that the ultradian rhythms of sleep-wake state may be generated by a mechanism that includes the sleep-wake homeostat. The main purpose of the present study was to test the hypothesis that sleep homeostasis and sleep-wake ultradian rhythms share a common underlying mechanism. The present study has refuted that hypothesis. I conclude that: (1) the proposed model for the generation of quasiperiodic ultradian rhythms in sleep-wake state in mammals is overly simplistic in its present form, (2) the generation of ultradian rhythms in sleep-wake state probably arise from a more complex systemic interactions between the sleep-wake oscillatory network and other internal/external inputs, rather than the simple expression of sleep homeostat.

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 (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Thesis (Ph.D.). / Written for the Dept. of Medicine, Division of Experimental Medicine. Title from title page of PDF (viewed 2008/05/08). Includes bibliographical references.

Thesis (Ph.D.)--University of Missouri-Columbia, 2006. / 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 April 27, 2009) Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Bibliography: leaves 157-175. Also available online.