The effect of thimerosal on cytosolic free Ca2+ concentrations ([Ca2+]i) in human oral cancer cells (OC2) is unclear. This study explored whether thimerosal changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2. Thimerosal at concentrations between 1-50£gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Thimerosal-induced Ca2+ influx was not blocked by L-type Ca2+ entry inhibitors and protein kinase C modulators [phorbol 12-myristate 13-acetate (PMA) and GF109203X]. In Ca2+-free medium, 50£gM thimerosal failed to induce a [Ca2+]i rise after pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Inhibition of phospholipase C with U73122 did not change thimerosal-induced [Ca2+]i rises. At concentrations between 5 and 10£gM thimerosal killed cells in a concentration-dependent manner. The cytotoxic effect of 8£gM thimerosal was potentiated by prechelating cytosolic Ca2+ with the Ca2+ chelator BAPTA/AM. Flow cytometry data suggested that 1-7£gM thimerosal induced apoptosis in a concentration-dependent manner. Collectively, in OC2 cells, thimerosal induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via non-L-type Ca2+ channels. Thimerosal killed cells in a concentration-dependent manner via apoptosis.

Microglia, immune cells of the central nervous system, activate in response to pathophysiological stimuli. One of their reactive phenotypes is to migrate to site of injury where they could have either beneficial or detrimental effects. However, little is known regarding the mechanisms underlying microglial migration and how they traverse the unique extracellular environment in brain tissue to reach their destination. Our laboratory first discovered that microglia express structures called podosomes, which can adhere to as well as degrade extracellular matrix. In this study, I further characterize microglial podosomes, and show that they associate with Iba1, Orai1 and calmodulin, proteins not yet observed in podosomes of other cell types. I also present evidence that podosome formation depends on Ca2+ and its entry through store-operated Ca2+ channels. The findings in this thesis contribute to a better understanding of podosome dynamics and their probable roles in microglia migration.

Microglia, immune cells of the central nervous system, activate in response to pathophysiological stimuli. One of their reactive phenotypes is to migrate to site of injury where they could have either beneficial or detrimental effects. However, little is known regarding the mechanisms underlying microglial migration and how they traverse the unique extracellular environment in brain tissue to reach their destination. Our laboratory first discovered that microglia express structures called podosomes, which can adhere to as well as degrade extracellular matrix. In this study, I further characterize microglial podosomes, and show that they associate with Iba1, Orai1 and calmodulin, proteins not yet observed in podosomes of other cell types. I also present evidence that podosome formation depends on Ca2+ and its entry through store-operated Ca2+ channels. The findings in this thesis contribute to a better understanding of podosome dynamics and their probable roles in microglia migration.

The purpose of this study was to investigate whether skeletal muscle sarcoplasmic reticulum (SR) Ca2+ transport efficiency and expression levels of major SR Ca2+ regulatory proteins are associated with resting metabolic rate (RMR) in humans. Twenty five healthy and weight stable participants with mean age, height and weight of 22±3.6 years, 174.6±8.0 cm and 72.8±21 kg respectively, were recruited for the study. RMR was calculated using the Weir equation based upon measures of VO2 and VCO2, which were collected using the Vmax breath by breath indirect calorimetry system. Ca2+-ATPase activity, Ca2+ uptake and Ca2+ leak analyses were performed in vitro on homogenates that were prepared from vastus lateralis muscle biopsies. Ionophore (IONO) ratio was assessed by measuring Ca2+-ATPase activity in the presence and absence of Ca2+ Ionophore. The coupling ratio, a measure of SR Ca2+ transport efficiency, was calculated by taking the ratio of Ca2+ uptake to Ca2+-ATPase activity. Expression levels of the major SR Ca2+ regulatory proteins, including SERCA1a, SERCA2a, phospholamban (PLN), and calsequestrin (CSQ) were assessed using Western blotting techniques. Pearson correlation coefficient analysis demonstrated a weak but significant negative correlation between coupling ratio and RMR (r2= 0.2108, p =0.0240). Content of the SR Ca2+ regulatory proteins, IONO ratio and Ca2+ leak were not found to be significantly related to either RMR or coupling ratio, with the exception of the ratio of SERCA1a to SERCA2a, which showed a weak but significant positive relationship with RMR (r2=0.1781, p=0.0400). Thus, the relationship between coupling ratio and RMR is not influenced by Ca2+ leak, SERCA pump efficiency or the SR Ca2+ regulatory proteins. Overall, these results suggest that the efficiency of SR Ca2+ transport is weakly related to whole body RMR. Further analysis is needed to assess this relationship, and to determine which SR Ca2+ handling properties are influencing the relationship between coupling ratio and RMR.

Thèse (M.Sc.)--Université Laval, 2007. / Bibliogr.: f. 60-63. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Cytosolic free calcium (Ca2+cyt) is a ubiquitous second messenger in eukaryotes. In Arabidopsis thaliana, diurnal or circadian (diel) rhythms in [Ca2+]cyt have been widely documented. There is evidence to suggest that these diel [Ca2+]cyt oscillations modulate different signalling pathways, including photoperiodic signal transduction, gating responses to endogenous and environmental stimuli and feed-back entrainment of the core circadian clock itself. However, direct evidence for the role of Ca2+ in clock entrainment or as an output from the clock is lacking, and the question of the functional role of diel [Ca2+]cyt oscillations remains open. The role of diel [Ca2+]cyt rhythms in A. thaliana and their relationship relative to the central molecular oscillator was investigated. While it was found that diel [Ca2+]cyt oscillations persist throughout the life cycle of A. thaliana, I found no indication that diel [Ca2+]cyt rhythms are involved in photoperiodic signalling. Furthermore, I demonstrated that normal diel [Ca2+]cyt oscillations persist even in the absence of a functioning core circadian clock, indicating that, contrary to the accepted view, diel [Ca2+]cyt oscillations are not directly controlled by the core circadian clock, but are more probably generated by a non-transcriptional oscillator. In silico analysis of the amino-acid sequences of the 12 core clock proteins revealed that TOC1 contains a putative EF-hand and may therefore provide a route into the molecular oscillator for diel [Ca2+]cyt signals. The TOC1 sequence was altered to eliminate the Ca2+ coordinating residues but attempts to express this protein in E. coli, N. benthamiana and Baculovirus were unsuccessful. Complementation of the A. thaliana toc1-1 mutant with transgenes containing the endogenous TOC1 promoter sequence upstream of the wild type or the altered TOC1 sequences were also unsuccessful. A series of experiments were conducted to provide empirical data for Boolean Logic models of circadian rhythmicity that would enable further characterisation of the potential link between diel [Ca2+]cyt oscillations and TOC1.

Calcium aluminate phases are important parts of Ordinary Portland cement and Alumina cement. Various calcium aluminate hydrates originate during the hydration of calcium alumina phases. Their origin depends on the conditions of hydration. In the diploma thesis was investigated hydration of four calcium aluminate phases – CA, CA2, C12A7, C3A under the conditions of four pH – 6, 9, 11, 12,65. Calcium aluminate phases were prepared from CaCO3 and Al2O3 by clinkering in solid phase in laboratory oven. The phases were hydrated for 48 hours. Kinetics of the hydration was investigated by calorimetry. Calcium aluminate hydrates were identified by XRD and DTA.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20310号 / 薬科博第79号 / 新制||薬科||9(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 金子 周司, 教授 竹島 浩, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Ionized calcium (Ca2+) is a signaling messenger that controls numerous cellular processes essential for life. The fidelity of Ca2+ signaling depends on the mechanisms that dynamically regulate its cytosolic concentration and maintain it at a low level in a resting cell. Plasma Membrane Ca2+ ATPase (PMCA) is a high affinity Ca2+ extrusion pathway involved in Ca2+ homeostasis and signal transduction. PMCA are encoded by 4 genes (PMCA1-4), which are expressed in a tissue dependent manner. The diversity of PMCA isoforms is further increased by alternative splicing. Changes in PMCA activity occur in heart failure and hypertension. Specific inhibitors of other ion transporters such as thapsigargin and digoxin, have made their mark in cell biology, but the currently used inhibitors of PMCA (vanadate and eosin) are non-specific. Thus, selective inhibitors of PMCA are needed to discern its role in Ca2+ signaling in physiology and pathophysiology. We introduced the concept of caloxins - peptides that specifically inhibit the activity of PMCA by binding to one of its five extracellular domains (exdoms) 1 to 5. The earlier caloxins including 2a1 and 3a1 were obtained by screening a phage display random 12-amino acid peptide (Ph.D-12) library for binding to synthetic peptides based on the exdom sequences. However, they all had low affinity. The objective of this research was to develop caloxins with high affinity and PMCA4 isoform selectivity. A two-step screening method was developed to screen the Ph.D-12 library to first bind to the synthetic exdom of PMCA4, followed by affinity chromatography using PMCA protein purified from human erythrocyte ghosts (mainly PMCA4). This method was used to obtain caloxins 1b1 and 1b2 to bind to the N and C-terminal halves of the exdom 1 of PMCA4, respectively. Both caloxins 1b1 and 1b2 had a 10-fold higher affinity than the prototype caloxin 2a1 and showed slight PMCA4 isoform preference. To engineer inhibitors with greater affinity and PMCA4 isoform selectivity, Ph.D caloxin 1b1 like peptide library was constructed. Most of the peptides expressed in this library differed from caloxin 1b1 in 0, 1, 2 or 3 amino acid residues at random. The library was screened to obtain several peptides one of which was caloxin 1c2. Caloxin 1c2 had 200-fold higher affinity than caloxin 2a1 and was isoform selective, with greater than 10-fold affinity for PMCA4 than for PMCA isoforms 1, 2 or 3. Thus, caloxin 1c2 is the first high affinity PMCA inhibitor that also is selective for an individual PMCA isoform. The second aim of this research was to establish that caloxin 1c2 binds to PMCA protein in erythrocyte ghosts. Two photoreactive caloxin 1c2-derivatives containing the photoactivable residue benzoylphenylalanine (Bpa) and a C-terminal biotin tag were used. Bpa substituted tryptophan at position 3 (3Bpa1c2-biotin) and serine at position 16 (16Bpa1c2-biotin) in caloxin 1c2. Both the derivatives inhibited PMCA activity in the erythrocyte ghosts. The intensity of the biotin label in the photolabeled erythrocyte ghosts was much stronger with 3Bpa1c2-biotin, which was then used in the subsequent experiments. The photolabeled proteins in erythrocyte ghosts were detected as a 250-270 kDa doublet in Western blots using streptavidin and the PMCA specific antibody. The degree of photolabeling depended on the UV-crosslinking time, and on the concentrations of 3Bpa1c2-biotin and the ghost protein. The selectivity of the photolabeling site was confirmed by decreased photolabel incorporation at 250-270 kDa doublet in the presence of excess caloxin 1c2 and the synthetic exdom 1X peptide of PMCA4. The photolabeled erythrocyte ghosts were solubilized and analyzed by immunoprecipitation with the PMCA specific antibody. The immunoprecipitate showed a 250-270 kDa doublet in Western blots using streptavidin. This confirmed that PMCA protein was photolabeled by the photoreactive derivatives of caloxin 1c2. Thus, caloxin 1c2 inhibits PMCA activity by binding to the exdom 1X of PMCA4. My work in M.Sc. initiated the concept of caloxins in the literature. This research has taken it to the stage where we can obtain caloxins selective for individual PMCA isoforms. This contrasts with the relative paucity of inhibitors specific for individual isoforms of other ion pumps. The high affinity isoform selective caloxin 1c2 and previous caloxins are being used to study PMCA physiology in our lab and by other researchers. Since caloxins act when added extracellularly and it is possible to obtain PMCA isoform selective caloxins, it is anticipated that they will aid in understanding the role of PMCA in signal transduction and homeostasis in health and disease. / Thesis / Doctor of Philosophy (PhD)

La protéine kinase Ca2+ /calmoduline-dépendante II (CaMKII) est une enzyme impliquée dans le remodelage synaptique dépendant de l'activation des récepteurs NMDA. Une activation importante des récepteurs NMDA provoque, dans l'épine dendritique, une augmentation du calcium libre ainsi que le recrutement de la CaMKII. Une fois dans l'épine, la CaMKII peut interagir avec plusieurs protéines, affectant ainsi sa mobilité et son accessibilité à certains substrats. Mes travaux démontrent que l'interaction de la CaMKII avec la sous-unité NR2B du récepteur NMDA, ainsi que l'auto-association de plusieurs holoenzymes de CaMKII peuvent modifier la localisation de la CaMKII dans des cellules non-neuronales suite à l'activation de la kinase. À l'aide de la technique de fluorescence recovery after photobleaching (FRAP), j'ai montré que l'activation des récepteurs NMDA mène à la rétention de la CaMKII dans l'épine dendritique. Mes résultats suggèrent que le recrutement et la rétention de la CaMKII à la synapse pourraient jouer un rôle dans la plasticité synaptique.