Thesis (Ph. D.)--University of Nevada, Reno, 2007. / "May, 2007." Includes bibliographical references (leaves 94-101). Online version available on the World Wide Web.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 192-206). Also available in electronic version. Access restricted to campus users.

No description available.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [202]-225).

Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 115 - 121). Also available for download via the World Wide Web; free to University of Oregon users.

The roles of Ca2 + and cAMP in olfactory transduction were explored using agents which affect calcium channels and second messenger systems. These agents were applied at certain calculated final concentrations onto olfactory epithelia of urethane-anesthetized frogs (Sana PiPlens) by two-sec aerosol spray. During extracellular recording, saturated vapors of isoamyl acetate were delivered every 100 sec in 0.3 sec pulses to produce an electroolfactogram (EOG). Inorganic cations that block inward calcium currents inhibit EOG responses with the following rank order: (La3+) > (Zn2+, Cd2+) > (Al3+, Ca2+, Sr2+) > (Co2+). Application of 7.5 mM La3+ eradicates £0G's, while Ba2+ (which can carry more current that Ca2+) initially produces significant enhancement (F=43.04, p<0.001, df=19). Magnesium ion has no effect on EOG's at 7.5 mM, while 1.5 X 10"4M Ca2+ is significantly inhibitory (F=5.74; p=0.0355; df=12). Control aerosol sprays of distilled water depress EOG's by an average of 5%. The organic calcium channel antagonists diltiazem and verapamil inhibit EOG's by 17% and 36X, respectively, at a concentration of 1.5 X 10~*M. Verapamil produces significant inhibition (F=17.17; p=0.002; df=ll) at 1.5 X 10" 5 M, while the 1,4-dihydropyridine calcium channel antagonists, nicardipine and nifedipine, do not inhibit beyond 1% DMSO controls. Several calmodulin antagonists decrease EOG's, but without correlation to their anti-calmodulin potency. Application of 1.5 X 10"*M chlorpromazine and N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide inhibit EOG's by 31% and 27%, respectively, while trifluoperazine inhibits by 23%. Dibutyryl cAMP, a lipophilic mimic of cAMP, produces 54% inhibition at 1.5 X 10" *M. Dibutyryl cGMP, cGMP, cAMP, and adenosine all decrease EOG's by less than 15% compared to distilled water controls. Forskolin, a reversible activator of adenylate cyclase, inhibits EOG's by 57% at 1.5 X 10"5M, which is significant beyond the 1% DMSO controls (F=17.17; p=0.002; df=ll). These data support the hypothesis that Ca2+ participates in olfactory transduction. Calcium ions could serve as charge carriers, second messengers, or both. Cyclic AMP could be involved with the primary excitatory process or sensory adaptation, or both.

Calcium ions (Ca2+) are extremely important intracellular messengers, activating a plethora of cellular processes. Growing evidence now points to a major role for the local Ca2+ signal in driving specific cellular responses. The simplest and most fundamental local Ca2+ signal is the Ca2+ microdomain, which rapidly forms when Ca2+ permeable ion channels open. In non-excitable cells the dominant Ca2+ entry channels are store-operated Ca2+ channels (SOCCs). The best characterised is the Ca2+ release activated Ca2+ (CRAC) channel. How local Ca2+ entry through CRAC channels impacts on channel function however is unclear. I have investigated the interaction between the Ca2+ binding protein calmodulin and CRAC channel activity and subsequent agonist-induced Ca2+ signals. Furthermore, I have investigated a role for mitofusin 2 (a protein that is known to tether the ER and mitochondria) on these Ca2+ signals. Using three different calmodulin mutant constructs with alterations to their Ca2+ binding sensitivities, I have shown that calmodulin facilitates CRAC channel dependent Ca2+ entry and maintains agonist-induced cytosolic Ca2+ oscillations in a lobe-specific manner. Calmodulin has four Ca2+ binding sites, two on the N-lobe and two on the C-lobe. I found a dominant negative calmodulin mutant (CAM4M, where all four binding sites had been mutated), or one where the C-lobe could not bind Ca2+ (CAM2C), impaired both Ca2+ influx through CRAC channels and maintenance of cytosolic Ca2+ oscillations. In contrast, a Ca2+-insensitive N-lobe mutant had little effect, (CAM2N). Knockdown of the mitochondrial Ca2+ uniporter regulator (MICU1) or mitochondrial membrane depolarization had similar effects to those seen with CAM4M or CAM2C, suggesting that at least in part, the action of calmodulin was through regulation of mitochondrial Ca2+ dynamics. This was confirmed by directly measuring the mitochondrial matrix Ca2+ concentration in intact RBL-1 cells using the mitochondrial targeted, fluorescent protein, pericam. Both CAM4M and disruption of mitochondrial Ca2+ buffering impaired agonist-induced mitochondrial Ca2+ uptake, suggesting that the modulation of CRAC channels occurred through Ca2+-calmodulin facilitation of mitochondrial Ca2+ uptake. Using a mutant Orai1 (A73E) that cannot bind calmodulin, I have shown that calmodulin tethered to the CRAC channel provides a major source of calmodulin for effective mitochondrial Ca2+ uptake. Physiological relevance of my proposed pathway was provided from experiments where I showed knockdown of MICU1 impaired agonist-induced CRAC channel dependent NFAT-1-driven gene expression. In addition, I establish a crucial role for mitochondrial MFN2 and presumably its ability to properly link the mitochondria and ER in the control of CRAC channels and agonist-induced Ca2+ signals.

Abstract not available

Abstract not available

The work presented here outlines experiments done using a novel RF exposure chamber. This device allows biological cells to be exposed to microwave radiation similar to those emitted by mobile telephones, whilst imaging them using a laserscanning confocal microscope. Jurkat E6.1 T lymphocytes in the exposure chamber were kept within �0.5�C of 37�C, allowing for the investigation of possible athermal effects of microwave energies. These cells were loaded with the fluorescent probe Fluo-3 AM, which is specific for calcium ions, and were monitored over two 10minute periods. The first period being a control period, the second being a period where the cells were either exposed to RF energy or sham exposed. Another 5min imaging period was for the positive control,where maximal fluorescence can be achieved by the addition of the ionophore A23187. 5 different conditions for cell exposure were investigated. Both continuous wave 900MHz and continuous wave 900MHz pulse modulated at 217Hz exposures were carried out on cells that were either unactivated, or those that were activated by the mitogen phytohaemaglutinin (PHA). For these 4 conditions the average Specific Absorption Rate (SAR) was calculated to be 1.5W/kg. A group of unactivated cells were also exposed to continuous wave 900MHz energy with an average SAR calculation of 7.5W/kg. Results showed that no significant changes in calcium ion levels occurred when averaged fluorescence slopes were compared between RF exposed cells and the control period. The mean change in slopes exposed/sham period � control period)between cells that were exposed and those sham exposed also showed no significant difference. Following an inference made in the work of Galvanovskis et al. (1999)1 who showed there is a change in the calcium ion oscillation spectrum as a result of 50Hz magnetic fields, a measure of the mean frequencies of all cells was determined using a Fast Fourier Transform (FFT) analysis. The change in the average mean frequencies in cells was then measured for all conditions. Of statistical significance was the change in average mean frequency between the control period and the sham/exposed period between cells that were exposed and those sham exposed, when cells were activated by PHA. The results also showed that there was an overall drop in average mean frequency as a result of RF exposure. Assuming there is a biological significance to the findings of this thesis, careful analysis of the calcium dynamics of tissue samples and cell types associated with RF exposure from mobile phones would need to be carried out to determine what they are. This was unfortunately beyond the scope of the present study.