Dissertations / Theses on the topic 'Calcium ions'

Cette thèse s'inscrit dans un projet visant à réaliser un étalon de fréquence optique à ion calcium unique confiné dans un piège de Paul-Straubel. La première partie du manuscrit est consacrée au piégeage et au refroidissement laser des ions. Les trois sources lasers (397 nm, 866 nm et 729 nm) utilisées dans l'expérience sont étudiées en détail. Pour atteindre le régime de Lamb-Dicke et ainsi éliminer l'élargissement par effet Doppler du premier ordre, il est indispensable de réduire le mouvement de l'ion. Différentes techniques de réduction du micromouvement sont exposées, en particulier une nouvelle méthode basée sur l'observation de résonances noires a été mise en place. Des simulations numériques utilisant la matrice densité permettent d'expliquer les observations expérimentales. Une deuxième partie décrit les expériences réalisées pour déterminer les durées de vie des niveaux D3/2 et D5/2 de l'ion calcium 40. La mesure de la durée de vie du niveau D5/2 est une étape importante puisqu'elle permet de contrôler et de réduire les effets qui pourraient élargir la transition d'horloge. La dernière partie de ce mémoire est dédiée à l'évaluation théorique des effets systématiques pouvant affecter la fréquence du futur étalon. Un étalon de fréquence optique basé sur la transition S1/2,F=4,m_F=0 ---> D5/2,F=6,m_F=0 de l'ion 43Ca+ pourra atteindre une exactitude de 4*10^(-16).

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.

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.

In copper amine oxidases the role of the buried active site copper has been extensively studied, while the functions of the peripheral metal ions, assigned as calcium according to crystallographic data, have-largely been ignored. One peripheral metal site slightly buried and called for clarity the 'buried site', is structurally coupled to the copper and two of its ligands are strictly conserved. In Escherichia coli amine oxidase (ECAO), as in Pichia pastoris Iysyl oxidase, Bovine Serum amine oxidase and Vascular adhesion protein, another peripheral metal site, the 'surface site', which is rather poorly conserved, is found near the surface of each subunit. Solution studies have been performed showing that ECAO activity is reduced upon EDTA treatment but can be recovered by addition of calcium and other metal ions (S~+, Mn2+, Mg2+). These studies together with crystallographic data of various ECAO derivatives (EDTA-treated , Strontium soaked and Xenonexposed- ECAO) showed that (i) the EDTA treatment displaces both calcium ions, the buried one being responsible for the reduction of activity upon EDTA treatment (ii) both peripheral sites are normally occupied by calcium ions but can be replaced by other metal ions, with the surface site displaying a lower affinity for calcium than the buried site, (iii) there are some preferred routes for O2 substrate entry, one is also the route for the amine substrate entry, the other one is structurally connected with the peripheral sites. Site-directed mutagenesis studies have also been conducted involving the ligands of both peripheral metals. While all surface site mutants expressed well, none of the buried mutants expressed a full-size ECAO unless the growth medium was supplemented with calcium. Amine oxidase activity is mediated by the redox active cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), which is the result of the post-translational modification of a tyrosine residue. In all surface site mutants studied so far by crystallographic and spectroscopic means, TPQ synthesis was prevented or reduced. The possible mechanisms behind these effects are discussed. Overall this work proposes a role in enhancing activity for the buried site and a surprising but critical role for the surface site in supporting TPQ biogenesis. Both sites could facilitate the folding process.

The interaction of high-Mg calcite and aragonite with a number of different trace metals was studied, in particular with Cu2+, Ni2+ and Co2+. Laboratory experiments into the initial sorption of these three metal ions onto the surfaces of artificial aragonite and high-Mg calcite were performed for a pH range of 6.8-8.2, in buffered solutions saturated with respect to calcite. Below a pH of 8-8.2, substantial dissolution of the unstable polymorph occurred, with little uptake of Co2+ or Ni2+. At a pH of 8.2, uptake was rapid, reaching a plateau in less than 5 min for all three metals. A higher proportion of the available Cu2+ was taken up than of the available Co2+ or Ni2+. Two environmental situations were also investigated. Firstly, samples of water, rock and aragonitic speleothems from Bohemia Cave, Mt. Owen, central Nelson, were examined for major and trace element concentrations. Water samples from the area were found to have very low trace metal ion concentrations, and little consistent relationship between the level of interaction with the environment and the trace metal concentration was seen. Concentration factors of trace metals into the speleothems were much larger than concentration factors of minor or major elements. Metal concentrations in the speleothems were lower than in the surrounding rocks, due to the slow deposition rate of the aragonite crystals, and the exclusion of small cations from the lattice of aragonite. Speleothem samples were analysed by electron microscopy, and structures are described. Secondly, uptake of sub-lethal concentrations of Cu2+, Ni2+ and Co2+ by Corallina officinalis (Corallinales, Rhodophyta) was studied, from a chemically-defined artificial seawater medium. Cu2+ was found to be toxic at even low levels of contamination. All three metal ions were taken up by the live seaweed, in proportion to the metal concentration of the medium. Partitioning of the metal ions was examined, between the inorganic phase of high-Mg calcite, deposited in the plant's cell walls, and the organic material of the plant. Killed seaweed were also "cultured", and found to contain considerably less metal ion than live seaweed. The presence of an increasing excess of Ni2+ had a negative effect on the uptake of Cu2+, but no interaction was seen between Co2+ and the other metal ions. Concentration factors were greatest for Co2+, followed by Cu2+ and Ni2+. C. officinalis has potential as a bio-monitor of pollution levels in seawater, but its usefulness requires further assessment.

The paper presents an analysis of the luminescent properties of Ce3+-doped calcium hydroxyapatite (Ca10(PO4)6(OH)2, HAp) nanopowders prepared by the chemical precipitation and sol-gel method. It was shown that in nanoscale HAp Ce3+ ions are strongly perturbed by surface defects and their local symmetry differs from that of Ce3+ ions in the bulk crystals. Possible origin of defects in nanosized HAp is also discussed. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/34896

Cette thèse s'inscrit dans un projet visant à réaliser un étalon de fréquence optique à ion unique de calcium confiné dans un piège de Paul. Après avoir passé en revue les différents types d'étalons de fréquence dans le domaine optique actuellement développés (atomes neutres en piège magnéto-optique, atomes neutres dans un réseau optique et ions piégés), l'accent est mis sur le concept d'horloge à ion unique confiné dans un piège radiofréquence. Les notions de piégeage des ions, de refroidissement laser, d'effet systématique et les techniques d'asservissement laser y sont étudiées en détail. Les aspects expérimentaux sont ensuite présentés, en insistant particulièrement sur les deux pièges utilisés au cours des expériences ainsi que sur le protocole expérimental qui détermine le cahier des charges du laser d'horloge. Afin de réaliser un étalon de fréquence optique il faut posséder une source laser dont les propriétés spectrales soient meilleures ou identiques à celle de la transition atomique afin de tirer pleinement profit du facteur de qualité de la transition atomique. L'ensemble des systèmes d'asservissement du laser, lui permettant d'atteindre ces performances, sont décrits. Dans une première partie, l'asservissement du laser sur une cavité Invar de pré-stabilisation est présenté. On peut ainsi atteindre une largeur de raie du laser de l'ordre de 1 kHz. Dans une seconde partie la réalisation et le montage de l'asservissement sur une cavité ULE de haute finesse est détaillé. En utilisant ce dernier étage nous avons estimé la largeur de raie du laser à 120 mHz. Enfin le dernier chapitre est consacré à l'utilisation d'une technique d'optique adaptative afin d'améliorer la qualité des faisceaux laser et ainsi réduire le bruit de fond du système de détection des ions, qui a pour conséquence de dégrader la stabilité du système.

NMR studies of water oxygen-17 relaxation in aqueous sucrose and lysozyme solutions have been carried out to investigate the interactions of water with sucrose and lysozyme. The effect of sucrose and lysozyme concentration on water oxygen-17 relaxation has been studied in detail. The dependence of relaxation on frequency and pH has also been analysed. The existing model, describing the relaxation of water oxygen-17 in aqueous protein solutions suggested by Halle in 1981, is tested to see whether it gives a true representation. It is found that at low concentrations of sucrose and lysozyme, the experimental data give good agreement with the model. However, at saturated sucrose concentration the agreement is not so good. An extra contribution to the transverse relaxation rate is seen. A possible explanation for the extra contribution to the transverse relaxation rate at high sucrose content is discussed. The effect of ionic charge on oxygen-17 relaxation in lysozyme solutions is also investigated. It is observed that both the ionic charge of lysozyme as well as lysozyme aggregation strongly affect the relaxation of water oxygen-17. A method for analysing the experimental data for water oxygen-17 relaxation in aqueous sucrose and lysozyme solutions using Halle's model is presented and employed to calculate the various parameters of the model. The relaxation and chemical shift of calcium-43 in simple calcium salts, calcium acetate and calcium ascorbate have been studied as a function of concentration and pH. The complexation of calcium to sucrose and to lysozyme has also been investigated. In almost all cases, a significant calcium-43 chemical shift has been detected. The direct measurement of complexation and binding of calcium by relaxation time and chemical shift measurements has been of particular interest.

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.

Deux types de neurotransmission se produisent dans les neurones du cerveau : la transmission évoquée et la transmission spontanée. Contrairement à la transmission évoquée, le rôle de la transmission spontanée sur la plasticité synaptique - un mécanisme utilisé pour doter le cerveau de capacités d'apprentissage et de mémorisation - reste incertain. Les neurotransmissions spontanées sont localisées et se produisent aléatoirement dans les synapses des neurones. Lorsqu'un tel événement spontané se produit, ce que l'on appelle un influx synaptique miniature d'ions calcium (miniature Synaptic Ca²⁺ Transient, mSCT), des ions calcium messagers secondaires pénètrent dans la synapse, activant les voies de signalisation en aval de la plasticité synaptique. L'utilisation de l'imagerie calcique du neurone in vitro permet la visualisation spatiotemporelle de l'entrée des ions calcium. Les vidéos calciques qui en résultent permettent une étude quantitative de l'impact du mSCT sur la plasticité synaptique. Cependant, la localisation des mSCTs dans l'imagerie du calcium est difficile en raison de leur petite taille, de leur faible intensité par rapport au bruit de l'imagerie et de leur caractère aléatoire inhérent. Dans ce mémoire, nous présentons une méthode d'analyse quantitative à grande échelle des vidéos d'imagerie calcique limitant la variabilité induite par les interventions humaines pour obtenir des données probantes, dans le but de caractériser l'impact des mSCTs sur la plasticité synaptique. En nous basant sur un outil semi-automatique de détection à seuil d'intensité (Intensity Thresholded Detection, ITD), nous sommes capables de générer des données pour entraîner un réseau pleinement convolutionnel (Fully Convolutional Network, FCN) afin de détecter rapidement et automatiquement les mSCTs à partir de vidéos calciques. En utilisant les segmentations bruitées de l'ITD comme données d'entraînement, combinées à un schéma d'entraînement positif (P) et non étiqueté (Unlabeled, U), les performances du FCN surpassent ITD. Le FCN détecte des mSCTs de faible intensité non détectés auparavant par ITD et offre une segmentation supérieure à ITD. Nous avons ensuite caractérisé l'impact des paramètres PU tels que le nombre de P et le ratio P:U. Le FCN entraîné est intégré dans une routine tout-en-un pour permettre une analyse à grande échelle des mSCTs. La routine offre la détection, la segmentation, la caractérisation et la visualisation des mSCTs ainsi qu'une solution logicielle pour gérer plusieurs vidéos avec différentes métadonnées.
Two types of neurotransmission occur in brain’s neurons: evoked transmission and spontaneous transmission. Unlike the former, the role of spontaneous transmission on synaptic plasticity –a mechanism used to endow the brain learning and memory abilities – remain unclear. Spontaneous neurotransmissions are localized and randomly happening in neuron’s synapses. When such spontaneous events happen, so-called miniature synaptic Ca²⁺ transients(mSCT), second messenger calcium ions entered the spine, activating downstream signaling pathways of synaptic plasticity. Using calcium imaging of in vitro neuron enable spatiotemporal visual-ization of the entry of calcium ions. Resulting calcium videos enable quantitative study of mSCT’s impact on synaptic plasticity. However, mSCT localization in calcium imaging can be challenging due to their small size, their low intensity compared with the imaging noise and their inherent randomness. In this master’s thesis, we present a method for quantitative high-through put analysis of calcium imaging videos that limits the variability induced by human interventions to obtain evidence for characterizing the impact of mSCTs on synaptic plasticity. Based on a semi-automatic intensity thresholded detection (ITD) tool, we are able to generate data to train a fully convolutional neural network (FCN) to rapidly and automaticaly detect mSCT from calcium videos. Using ITD noisy segmentations as training data combine with a positive and unlabeled (PU) training schema, we leveraged FCN performances and could even detect previously undetected low instensity mSCTs missed by ITD. The FCN also provide better segmentation than ITD. We then characterized the impact of PU parameters such as the number of P and the ratio P:U. The trained FCN is bundled in a all-in-one pipeline to permit a high-thoughtput analysis of mSCT. The pipeline offers detection, segmentation,characterization and visualization of mSCTs as well as a software solution to manage multiple videos with different metadatas.

The purpose of this study was to synthesize calcium phosphates doped with titanium and fluoride ions in different combinations. Pure and doped calcium phosphates were synthesized by a precipitation method. The synthesized materials were sintered at 1100º
C and 1300º
C for 1h. The ceramics were characterized by density measurements to determine the effect of sintering temperature. Presence of phases and bonds were characterized by XRD diffraction and FTIR spectroscopy. Grain sizes of the samples were obtained by SEM. Microhardness test was applied on the samples to determine the mechanical properties of the samples. It was observed that Ti4+ addition decreased the density of samples significantly at 1100°
C, whereas increasing the sintering temperature to 1300°
C caused an increase. Increasing the F- ion amount increased the densification at 1100°
C when molar ratios were 1.0, 1.25, 1.50 and decreased the density at 1300°
C when Ca /P molar ratios were 1.0, 1.25, 1.67 and 2.0. Ti4+ and F- co-doped samples showed variety in their density behaviour after the sintering at 1100º
C and 1300º
C. The XRD analyses demonstrated that for Ca to P ratio 1 at 1100°
C, &beta
-CPP phase, when sintering temperature was raised to 1300°
C, as a second phase of &beta
-CPP and &alpha
-TCP observed. Increasing the molar ratio to 1.25 and 1.50 demonstrated &beta
-TCP and/or &beta
-CPP and &beta
-TCP/ HA at 1100°
C and &beta
-TCP and/or &beta
-CPP, &alpha
-TCP, TiO2 and HA, &alpha
-TCP, TiO2 phases at 1300°
C, respectively. In higher Ca/P molar ratios of 1.67 and 2.0, HA, &beta
-TCP, &alpha
-TCP, CaO, TiO2, CaTiO3 and HA, CaO, &alpha
-TCP, CaTiO3 phases were determined. Increasing the sintering temperature to 1300°
C resulted in transformation to &alpha
-TCP. In FTIR spectroscopy analysis, when the samples with molar ratio of 1, 1.25, 1.50, 1.67 and 2.0, sintered at 1100°
C, the characteristic bands of &beta
-CPP, OCP/&beta
-TCP, &beta
-TCP/HA, HA and HA were observed, respectively. With increasing the sintering temperature to 1300°
C, samples with molar ratio of 1.0 and 1.25 revealed additional secondary characteristic peaks of HA and &beta
-TCP. SEM images revealed that sintering temperature and ion amounts of dopants had significant effect on grain sizes of the samples. The grain sizes were generally increased when sintering temperature rose from 1100°
C to 1300°
C. The &mu
-hardness test demonstrated that Ti4+ and F- ions in large amounts had positive effect on the mechanical properties at the sintering temperatures of 1100°
C and 1300°
C

The role of metal ions as catalysts for numerous biochemical reactions has been the subject of many investigations. One of the most important classes of ligands are phosphate esters. In this thesis I describe the investigation of some phosphate ester-metal ion equilibria. Formation constants for the complexation of p-nitrophenyl phosphate, phenyl phosphate, 1-naphthyl phosphate, α-D-glucose-1'-phosphate, glycerol-2-phosphate, methyl phosphate, 8-quinolyl phosphate, 8-quinolyl methyl phosphate, triphosphate and fluorotriphosphate with protons, copper, zinc and calcium ions were determined by potentiometry. In addition, the complexation of 1-naphthyl phosphate, 8-quinolyl phosphate and 8-quinolyl methyl phosphate with nickel and cobalt ions was also studied. Protonation enthalpies and copper complexation enthalpies of p-nitrophenyl phosphate, phenyl phosphate, 1-naphthyl phosphate, α-D-glucose-1'-phosphate, glycerol-2-phosphate and methyl phosphate were determined by calorimetry. A correlation between the nucleophilicity of the ester group and the magnitude of the stability constants of the proton, copper and zinc complexes of p-nitrophenyl phosphate, phenyl phosphate, 1-naphthyl phosphate, α-D-glucose-1'-phosphate, glycerol-2-phosphate and methyl phosphate is found and explained in terms of electronic induction effects, i.e. by polarisation of the phosphate oxygens by the ester group. The calorimetric results show that the desolvation of ligand and metal ion during the complexation plays an important role. The possibility of similar correlations for complexes of triphosphates is also discussed.

Nous avons etudie les mecanismes de transport et de regulation du calcium dans des cultures primaires de tubules contournes distaux brillants (dctb) de rein de lapin selon trois approches experimentales: (1) l'identification et la caracterisation des canaux ioniques de la membrane apicale des cellules par la technique de patch-clamp. (2) la mesure des variations de ca#2#+#i au cours du temps par videomicroscopie de fluorescence sur des cellules de dctb poussant sur des boites de petri et par cytometrie a balayage laser lorsque le support de culture est sur un filtre permeable. Nous avons montre dans la membrane apicale des cellules du dctb l'existence d'un canal calcium active par l'hyperpolarisation, sensible au lanthane, au verapamil et aux dihydropyridines. Ce canal interviendrait dans la reabsorption transepitheliale de calcium. Au niveau basolateral, un echangeur na#+-ca#2#+ sensible a l'amp cyclique permettrait l'extrusion du calcium de la cellule. Par ailleurs, nous avons observe que la calcitonine augmente la ca#2#+#i par une voie independante de l'amp cyclique. Sur la base de certaines donnees de la litterature suggerant l'intervention d'une conductance basolaterale au chlore dans la reabsorption transepitheliale de calcium par la calcitonine, nous avons entrepris l'etude des mouvements de chlore dans les cellules du dctb avec les techniques de cytometrie a balayage laser et de patch-clamp. Nous avons montre qu'il existe dans la membrane apicale des cellules du dctb un canal chlore sensible a l'amp cyclique et dans la membrane basolaterale un canal chlore sensible au calcium intracellulaire. D'apres nos resultats, nous proposons le mecanisme suivant: la calcitonine stimulerait la sortie de chlore par les canaux sensibles a l'amp cyclique et/ou au calcium intracellulaire. La diminution de la concentration intracellulaire de chlore resultante entrainerait une hyperpolarisation membranaire favorisant l'entree de calcium via les canaux presents au niveau de la membrane apicale

Ca2+, a signal for death and life, is closely involved in the regulation of numerous important cellular events. Ca2+ carries out its function through its binding to Ca2+-receptors or Ca2+-binding proteins. The EF-hand protein, with a helix-loop-helix Ca2+-binding motif, constitutes one of the largest protein families. To facilitate our understanding of the role of Ca2+ in biological systems (denoted as calciomics) using genomic information, an improved pattern search method (http://www.chemistry.gsu.edu/faculty/Yang/Calciomics.htm) for the identification of EF-hand and EF-like Ca2+-binding proteins was developed. This fast and robust method allows us to analyze putative EF-hand proteins at the genome-wide level and further visualize the evolutionary scenario of the EF-hand protein family. This prediction method further enables us to locate a putative viral EF-hand Ca2+-binding motif within the rubella virus nonstructural protease that cleaves the nonstructural protein precursor into two active replicase components. A novel grafting approach has been used to probe the metal-binding properties of this motif by engineering the predicted 12-residue Ca2+-coordinating loop into a non-Ca2+-binding scaffold protein, CD2 domain 1. Structural and conformational studies were further performed on a purified, bacterially-expressed NS protease minimal metal-binding domain spanning the Zn2+- and EF-hand Ca2+-binding motif. It was revealed that Ca2+ binding induced local conformational changes and increased thermal stability. Furthermore, functional studies were carried out using RUB infectious cDNA clone and replicon constructs. Our studies have shown that the Ca2+ binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions. In addition, we have predicted and characterized a calmodulin-binding domain in the gap junction proteins connexin43 and connexin44. Peptides encompassing the CaM binding motifs were synthesized and their ability to bind CaM was determined using various biophysical approaches. Transient expression in HeLa cells of two mutant Cx43-EYFP constructs without the putative CaM-binding site eliminated the Ca2+-dependent inhibition of gap junction permeability. These results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 and Cx44 in a Ca2+-dependent manner, providing a molecular basis for the well-characterized Ca2+-dependent inhibition of Cx43-containing gap junctions.

Phosphoproteins interact with calcium minerals in numerous biological systems but despite the importance of this phenomenon it is little understood. The interaction of phosphoproteins with calcium minerals is observed in milk where caseins interact with calcium phosphate to form micelles. Further understanding of this interaction was sought through two approaches. First, the adsorption isotherms for binding of the casein β-CN A and its N-terminal phosphopeptide (β-CN (f1-25)) to crystalline dicalcium phosphate dihydrate (DCPD) were measured. Secondly, attempts were made to grow co-crystals of calcium ions with phosphopeptides or with phosphoserine. A method of measuring phosphoprotein and phosphopeptide adsorption to DCPD was developed. From the adsorption isotherms the affinity and capacity of adsorption could be determined using a model of the adsorption process. Adsorption isotherms for β-CN A were all described using a simple Langmuir model of adsorption, adsorption occurring to identical and independent sites on the DCPD crystals. β-CN (f1-25) appears to adsorb to the same adsorption sites as β-CN A and also to a second family of adsorption sites which have a lower intrinsic affinity but which are more abundant. Adsorption isotherms for β-CN (f1-25) were described using a modified version of the Langmuir model in which there are two sets of adsorption sites. A term was included to allow for cooperative adsorption to the second set of adsorption sites. The effects of methylation of lysyl residues, buffer pH, incubation temperature, the presence of 6.0 M urea in the buffer and of crystal size were all examined. The adsorption of β-CN (f1-25) to DCPD crystals appears to be athermal since the measured isotherm does not change significantly with temperature (over the measured range), requiring that adsorption is due to an entropy gain. Comparison of the results of the β-CN A and β-CN (f1-25) adsorption experiments suggests that adsorption probably occurs via the N-terminal region of β-CN A, consistent with the hypothesis that the binding of phosphoproteins to calcium minerals occurs primarily through their phosphoseryl residues.

Sodium linear alkylbenzene sulphonate (NaLAS) is the usual surfactant present in high foam laundry detergents. The foam behaviour of NaLAS is significantly dependent upon the foam generation methodology, water hardness and the antifoam action of deterged sebum soils. Here a study of the foam behaviour of NaLAS (and C12 4-phenyl SO3Na) solutions at different Ca2+ concentrations and pHs in the absence and presence of antifoam is presented. Two foam generation methodologies were used - tumbling tube rotation and cylinder shaking. It has been found that these two methodologies correlate well with a coefficient of ≥ 0.95 when comparing foamabilities. The correlation coefficient however declines to ~0.82 when comparing foam stabilities. The reason of this deterioration has been attributed to the differences of antifoam effect in foam films after foam generation due to differences in bubble size distribution formed by these two methodologies. In the absence of antifoam, the foam behaviour is independent of pH and is dominated by the formation of Ca(LAS)2 (or Ca(C12 4-phenyl SO3)2 lamellar phase liquid crystals. Dynamic surface tension measurements confirm that low foamability after the micellar-precipitate boundary of the Ca2+-LAS- (or Ca2+- C12 4-phenyl SO3-) precipitation phase diagram is due to low rates of transport of surfactant to the rapidly expanded air-water surfaces. Mixtures of triolein/stearic acid and triolein/tristearin are used as models for sebum soil antifoam, as they show similar antifoam effects regardless of pH and calcium concentration. In these two systems, crystalline particles are always present provided, in the case of triolein/stearic acid, formation of soaps is suppressed at low pH. Both stearic acid and tristearin particles adopt an oil-water contact angle >OW > 90° measured through the aqueous phase. They invert the O/W emulsion behaviour shown by triolein alone to W/O by rupturing the oil-water-oil emulsion films. They will also rupture the air-water-oil pseudoemulsion films provided the conditions of θAW > 2.6° for stearic acid and θAW > 0o for tristearin are satisfied. This behaviour of particles will facilitate the emergence of triolein droplets into air-water surfaces. Foam film rupture however only occurs under dynamic conditions, where bridging coefficients for triolein are expected to be positive. However under the near-equilibrium conditions prevailing during foam stability, bridging coefficients for triolein are negative. Little or no antifoam activity is therefore observed under those conditions with these triolein-based mixtures. Oil/particle mixed antifoams probably deactivate through a splitting and coalescence process. Triolein/stearic acid antifoam deactivates more rapidly than sebum soil and triolein/tristearin. This is mainly caused by formation of large inactive agglomerates which occurs both after antifoam dispersion and after continuous foam generation.

Tableau d'honneur de la FÉSP
Tableau d'honneur de la FÉSP
La signalisation calcique dendritique joue un rôle important dans la régulation de mécanismes neuronaux, tels que la plasticité synaptique et l’intégration de l’information transmise. Bien compris chez les neurones principaux, ce processus de régulation est moins étudié chez les divers types d’interneurones GABAergiques qui modulent l’acquisition et l’envoi de signaux neuronaux. Chez les interneurones à décharge rapide, un type d’interneurone commun dans les circuits corticaux, il a été démontré qu’il y a absence de rétropropagation des potentiels d’action dans les dendrites distales (Hu et al., 2010). Cette découverte a des implications fonctionnelles, car la rétropropagation des potentiels d’action est un signal important pour l’induction des formes de plasticité synaptique hebbiennes. Par contre, il a été suggéré que l’activité dendritique locale pourrait compenser pour l’absence de rétropropagation des potentiels d’action. En conséquence, ce travail porte sur l’étude des évènements calciques dans les dendrites distales des interneurones à décharge rapide. Nous avons cherché à déterminer s’il est possible de générer ces signaux calciques par stimulation dendritique locale, à étudier les mécanismes responsables de ces signaux et à déterminer si ces signaux jouent un rôle dans la régulation de la plasticité synaptique à ces synapses. Pour atteindre ces objectifs, nous avons utilisé une combinaison de méthodes électrophysiologiqes (patch-clamp en mode cellule entière), d’imagerie calcique deux-photons et de modélisation computationnelle. Nous avons pu établir qu’il est possible de générer des évènements calciques postsynaptiques supralinéaires dans les synapses excitatrices étudiées par stimulation électrique locale. Ces signaux sont médiés par l’influx calcique provenant de l’activation des récepteurs AMPA perméables au Ca2+, qui déclenche à son tour le relâchement de Ca2+ par les récepteurs ryanodine présents sur réserves calciques intracellulaires. Ces signaux comprennent aussi une contribution calcique mineure des récepteurs NMDA, et ils restent locaux (pas de propagation dans l’arbre dendritique). De plus, nous avons déterminé que ces évènements calciques supralinéaires produisent un revirement de la plasticité synaptique, car ils induisent la dépression à long-terme dans les synapses étudiées, alors que les signaux calciques de basse amplitude induisent la potentiation à long-terme. Nous avons aussi examiné si ces évènements calciques supralinéaires étaient générés de façon équivalente dans les dendrites apicales et basales, qui reçoivent des synapses de différentes sources. Nous avons observé que les signaux des dendrites apicales avaient une plus grande amplitude et étaient associés à un plus haut niveau de dépolarisation. À partir de la modélisation, nous avons pu prédire le nombre de synapses nécessaires à la génération de ces signaux et la contribution potentielle des mécanismes d’extrusion du Ca2+. Finalement, nous avons étudié la spécificité cellulaire des mécanismes d’intégration dendritique en combinant l’imagerie calcique et la modélisation dans un type différent d’interneurone, les interneurones spécifiques aux interneurones type III. En conclusion, nous avons prouvé qu’il existe dans certains interneurones des mécanismes alternatifs, médiés par des hausses de Ca2+ locales, permettant la régulation de la plasticité aux synapses excitatrices.
La signalisation calcique dendritique joue un rôle important dans la régulation de mécanismes neuronaux, tels que la plasticité synaptique et l’intégration de l’information transmise. Bien compris chez les neurones principaux, ce processus de régulation est moins étudié chez les divers types d’interneurones GABAergiques qui modulent l’acquisition et l’envoi de signaux neuronaux. Chez les interneurones à décharge rapide, un type d’interneurone commun dans les circuits corticaux, il a été démontré qu’il y a absence de rétropropagation des potentiels d’action dans les dendrites distales (Hu et al., 2010). Cette découverte a des implications fonctionnelles, car la rétropropagation des potentiels d’action est un signal important pour l’induction des formes de plasticité synaptique hebbiennes. Par contre, il a été suggéré que l’activité dendritique locale pourrait compenser pour l’absence de rétropropagation des potentiels d’action. En conséquence, ce travail porte sur l’étude des évènements calciques dans les dendrites distales des interneurones à décharge rapide. Nous avons cherché à déterminer s’il est possible de générer ces signaux calciques par stimulation dendritique locale, à étudier les mécanismes responsables de ces signaux et à déterminer si ces signaux jouent un rôle dans la régulation de la plasticité synaptique à ces synapses. Pour atteindre ces objectifs, nous avons utilisé une combinaison de méthodes électrophysiologiqes (patch-clamp en mode cellule entière), d’imagerie calcique deux-photons et de modélisation computationnelle. Nous avons pu établir qu’il est possible de générer des évènements calciques postsynaptiques supralinéaires dans les synapses excitatrices étudiées par stimulation électrique locale. Ces signaux sont médiés par l’influx calcique provenant de l’activation des récepteurs AMPA perméables au Ca2+, qui déclenche à son tour le relâchement de Ca2+ par les récepteurs ryanodine présents sur réserves calciques intracellulaires. Ces signaux comprennent aussi une contribution calcique mineure des récepteurs NMDA, et ils restent locaux (pas de propagation dans l’arbre dendritique). De plus, nous avons déterminé que ces évènements calciques supralinéaires produisent un revirement de la plasticité synaptique, car ils induisent la dépression à long-terme dans les synapses étudiées, alors que les signaux calciques de basse amplitude induisent la potentiation à long-terme. Nous avons aussi examiné si ces évènements calciques supralinéaires étaient générés de façon équivalente dans les dendrites apicales et basales, qui reçoivent des synapses de différentes sources. Nous avons observé que les signaux des dendrites apicales avaient une plus grande amplitude et étaient associés à un plus haut niveau de dépolarisation. À partir de la modélisation, nous avons pu prédire le nombre de synapses nécessaires à la génération de ces signaux et la contribution potentielle des mécanismes d’extrusion du Ca2+. Finalement, nous avons étudié la spécificité cellulaire des mécanismes d’intégration dendritique en combinant l’imagerie calcique et la modélisation dans un type différent d’interneurone, les interneurones spécifiques aux interneurones type III. En conclusion, nous avons prouvé qu’il existe dans certains interneurones des mécanismes alternatifs, médiés par des hausses de Ca2+ locales, permettant la régulation de la plasticité aux synapses excitatrices.
Dendritic Ca2+ signaling plays an important role in the regulation of neuronal processes, such as synaptic plasticity and input integration. Well-studied in principal neurons, this form of regulation is not well understood in the various types of GABAergic interneurons that modulate activity in neuronal networks. In fastspiking (FS) interneurons, a common interneuron type in cortical circuits, it has been shown that there is a lack of action potential (AP) backpropagation in distal dendrites (Hu et al., 2010). This discovery has functional implications, AP backpropagation is an important signal for the induction of Hebbian forms of synaptic plasticity. However, it has been suggested that local dendritic activity could compensate for the absence of AP backpropagation. Consequently, this work focuses on the study of Ca2+ transients in distal dendrites of FS interneurons. We sought to determine whether it is possible to generate supralinear Ca2+ transients through local dendritic stimulation, to study the mechanisms responsible for those transients and to determine whether those signals play a role in the regulation of synaptic plasticity at those synapses. To reach those objectives, we used a combination of electrophysiological methods (whole-cell patch-clamp recordings), two-photon Ca2+ imaging and of computational modeling. We were able to establish that supralinear postsynaptic Ca2+ transients can be generated through local electrical stimulation of excitatory synapses in distal dendrites. These Ca2+ transients were mediated by Ca2+ influx from the activation of Ca2+-permeable AMPA receptors, which triggers Ca2+ release through ryanodine receptors present on intracellular Ca2+ stores (Ca2+-induced Ca2+ release). These Ca2+ signals also contain a minor contribution from NMDA receptors, and stay localized (no significant propagation in the dendritic arbor). In addition, we determined that these supralinear Ca2+ signals constitute a switch in the expression of synaptic plasticity, as they induce long-term depression in local synapses, while low-amplitude Ca2+ signals induced synaptic long-term potentiation. We also examined whether these supralinear Ca2+ transients were generated in both apical and basal dendrites, which receive synaptic contacts from different sources (Schaffer collaterals vs local collaterals). We observed that Ca2+ transients in apical dendrites had a higher amplitude and were associated with a higher level of somatic depolarization. We were also able to predict, through computational modeling, the number of synapses necessary to the generation of those signals and the potential contribution of Ca2+ extrusion mechanisms. Finally, we studied the cell-specificity of dendritic integration mechanisms by combining Ca2+ imaging and modeling in a different interneuron type, interneuron-specific interneurons type III. In conclusion, we were able to prove that certain interneurons possess alternative mechanisms, mediated through local Ca2+ transients, that allow for the regulation of plasticity at excitatory synapses.
Dendritic Ca2+ signaling plays an important role in the regulation of neuronal processes, such as synaptic plasticity and input integration. Well-studied in principal neurons, this form of regulation is not well understood in the various types of GABAergic interneurons that modulate activity in neuronal networks. In fastspiking (FS) interneurons, a common interneuron type in cortical circuits, it has been shown that there is a lack of action potential (AP) backpropagation in distal dendrites (Hu et al., 2010). This discovery has functional implications, AP backpropagation is an important signal for the induction of Hebbian forms of synaptic plasticity. However, it has been suggested that local dendritic activity could compensate for the absence of AP backpropagation. Consequently, this work focuses on the study of Ca2+ transients in distal dendrites of FS interneurons. We sought to determine whether it is possible to generate supralinear Ca2+ transients through local dendritic stimulation, to study the mechanisms responsible for those transients and to determine whether those signals play a role in the regulation of synaptic plasticity at those synapses. To reach those objectives, we used a combination of electrophysiological methods (whole-cell patch-clamp recordings), two-photon Ca2+ imaging and of computational modeling. We were able to establish that supralinear postsynaptic Ca2+ transients can be generated through local electrical stimulation of excitatory synapses in distal dendrites. These Ca2+ transients were mediated by Ca2+ influx from the activation of Ca2+-permeable AMPA receptors, which triggers Ca2+ release through ryanodine receptors present on intracellular Ca2+ stores (Ca2+-induced Ca2+ release). These Ca2+ signals also contain a minor contribution from NMDA receptors, and stay localized (no significant propagation in the dendritic arbor). In addition, we determined that these supralinear Ca2+ signals constitute a switch in the expression of synaptic plasticity, as they induce long-term depression in local synapses, while low-amplitude Ca2+ signals induced synaptic long-term potentiation. We also examined whether these supralinear Ca2+ transients were generated in both apical and basal dendrites, which receive synaptic contacts from different sources (Schaffer collaterals vs local collaterals). We observed that Ca2+ transients in apical dendrites had a higher amplitude and were associated with a higher level of somatic depolarization. We were also able to predict, through computational modeling, the number of synapses necessary to the generation of those signals and the potential contribution of Ca2+ extrusion mechanisms. Finally, we studied the cell-specificity of dendritic integration mechanisms by combining Ca2+ imaging and modeling in a different interneuron type, interneuron-specific interneurons type III. In conclusion, we were able to prove that certain interneurons possess alternative mechanisms, mediated through local Ca2+ transients, that allow for the regulation of plasticity at excitatory synapses.

Effects of potassium supplementation were evaluated in four studies in endurance horses during races and treadmill exercise. In the first and second studies a potassium-free experimental formula was compared to potassium rich commercial formulas. The first study showed that supplementation increased plasma [K+], and that the extra sodium in the potassium-free experimental formulas helped to attenuate acidosis at the end of the ride. In the second study supplementation also increased plasma [K+], however speeds were lower and no increases were observed in plasma concentrations during the race. Supplementation of potassium during recovery helped to restore plasma [K+]. Higher plasma [Ca++] was found in horses supplied with experimental feeds, due to a lower dietary cation anion balance (DCAB). Three eliminated horses had heart rate arrhythmias and labile heart rates accompanied with higher plasma [K+] and lower [Ca++] than finishers. Also horses supplied with the experimental sodium-rich formula were less dehydrated than the ones receiving commercial formulas. The third study involved an 80 km endurance exercise test on the treadmill, and plasma [K+] was affected by potassium supplementation during exercise and recovery. The supply of potassium caused higher plasma [K+] helping to restore body stores. Also chloride supply in the electrolyte formulas maintained plasma [Cl-] levels during exercise and affected plasma concentrations during recovery. The fourth study showed that potassium supply affects plasma concentration, but also increases lactate production and glucose during sub-maximal exercise. A potassium-free electrolyte supply caused higher plasma [Ca++] during exercise. Higher sodium supply in the potassium-free electrolytes improved hydration during exercise. These studies show that potassium should supplemented after exercise and but not be done during exercise because of the risk of increased neuromuscular excitability.
Ph. D.

Ce projet vise a determiner le lien entre l'absence de dystrophine et la surcharge calcique observee sur les cellules musculaires humaines dystrophiques (dmd) durant la myogenese in vitro. L'etude porte principalement sur les mecanismes sarcolemmiens qui interviennent dans le maintien de l'homeostasie calcique. Dans ce but, des cellules musculaires normales et dystrophiques sont cocultivees afin d'atteindre un degre de maturation suffisant, permettant a ces cellules de se contracter. Cette contraction produit un stress mecanique sur la membrane plasmique et les proteines qui y sont ancrees. L'absence de la dystrophine sur les cellules dmd pourrait moduler l'activite de certaines de ces proteines et en, particulier, les canaux sensibles au stress. Le but de cette etude est donc d'identifier grace a la technique de patchclamp en configuration cellule-attachee, des canaux mecanosensibles laissant transiter du calcium dans les myotubes humains dmd. Les myotubes humains normaux et dmd cocultives presentent des canaux cationiques qui sont spontanement actifs au potentiel de membrane de repos en absence d'un stress. Cette activite est plus importante sur les cellules dmd ce qui est a rapprocher du manque de stabilisation de la membrane de ces cellules. La seconde observation est que ces cellules possedent des canaux permeables aux ions calcium qui sont actives lors d'un stress (depression ou milieu hypotonique via la pipette). Ces canaux permettent donc une entree significative de calcium lors d'un stress comme cela est le cas lors de la contraction musculaire. Nous pouvons supposer que le taux eleve de calcium de repos des myotubes dmd se contractant peut etre du en partie a une entree de calcium plus importante de calcium par ces canaux sensibles a un stress. Ces resultats nous montrent le role important que detienne la dystrophine et les consequences de son absence dans les cellules dystrophiques.

Thesis (Ph. D.)--University of Nevada, Reno, 2005.
"December, 2005." Includes bibliographical references (leaves 194-212). Online version available on the World Wide Web. Library also has microfilm. Ann Arbor, Mich. : ProQuest Information and Learning Company, [2005]. 1 microfilm reel ; 35 mm.

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.

From a chemical and structural point of view, hydroxyapatite (HA) is a strong candidate in biomedical applications owing to its similarity to the inorganic components of bones and teeth. HA nanoparticles (NPs) as colloidal suspensions are becoming a popular tool in biomedical applications such as gene/drug delivery, bio-imaging etc. Although it is widely acknowledged that ionic substitutions on bulk HA substrates have a strong impact on their biological performance, little is known of their effect on NPs with potential use in gene transfection or drug delivery. In the third chapter carbonate (CO3) and magnesium (Mg) ions, which are the major substitutions in biological apatite, have been explored in the synthesis of ion-doped HA NPs under similar reaction conditions to allow comparison of results. CO3 and Mg ion were incorporated in the crystal lattice of HA and caused various changes mainly in the morphology and solubility of the different nanoparticles. In addition, the impact of ion doping on the interaction of HA NPs with cells was also evaluated under various cell culture conditions: 1) performing the cell culture study on citrate-dispersed NPs and on agglomerated NPs, 2) adding/excluding 10 % of foetal bovine serum (FBS) in the cell culture media and 3) using different types of cells, i.e. osteosarcoma MG-63 cells versus rat mesenchymal stem cells (rMSCs). The in vitro results indicated that Mg-doped HA NPs induced a profound impact on MG63 cells and, in the absence of citrate and FBS these nanoparticles were clearly cytotoxic. However, Mg-doped HA NPs did not alter cell viability in rMSCs under the same conditions. In the fourth chapter, Sr, Zn, Si and Fe(III) ions, which are minor ionic substitutions in biological apatite, were introduced to synthesize additional ion-doped HA NPs. Physicochemical characterization demonstrated that as-synthesized NPs were phase pure and doped ions had little influence on the morphology of NPs as in all cases they kept needle-like structure. Cytotoxicity studies performed using MG63 and rMSCs cells under the conditions of serum-containing and serum-free indicated that all NPs were non-cytotoxic if FBS was present. Interestingly, Zn-doped and Fe-doped HA NPs clearly stimulated MG63 cell proliferation in the absence of FBS. In addition to exploring the effect of ion-doped HA NPs on cell behaviour, it was also the interest of this thesis to investigate calcium phosphate (CaP) mineralization in the presence of organic molecules and also doping ions. In the fifth chapter of this thesis the effect of various organic molecules on CaP precipitation is provided. As shown by transmission electron microscopic studies (TEM) neuron-like CaP structures could be created using organic molecules of diverse nature such as non-ionic surfactant (Tween 80), anionic polymers (sodium polyacrylate) and cationic polymers (polydiallyldimethylammonium chloride). TEM studies through EELS, EFTEM and SAED proved that the neuron-like structures consisting of a dense core and thin filaments surrounding it had calcium, phosphorous and oxygen evenly distributed throughout the dense core as well as the filaments, and were amorphous in nature. Additionally, the co-effect of inorganic additives (i.e. Mg and Sr) together with organic molecules on CaP was also evaluated. It was proved that the addition of small amounts of ions had diverse impact on the stability of the neuron-like structures Mg clearly disrupting them but not Sr. All the findings with organic molecules provide much inspiration not only for the synthesis of more advanced CaP materials with novel structures and useful properties, but also for a better understanding of biomineralization process in nature.
Desde el punto de vista químico y estructural la hidroxiapatita (HA) se considera un gran candidato para aplicaciones biomédicas por su similitud con la fase mineral del hueso y los dientes. Aunque el efecto del dopaje se ha investigado con detalle en la fabricación de implantes viéndose que su presencia tiene un gran impacto en el comportamiento celular, poco se sabe de su efecto en nanopartículas para su uso en terapia génica y liberación de fármacos. En concreto, el tercer capítulo se centra en el dopaje de la apatita con iones carbonato (CO3) y magnesio (Mg) por ser éstas las sustituciones más importantes en la apatita biológica. Para ello todas las reacciones de síntesis se realizan bajo las mismas condiciones con la finalidad de poder comparar resultados. Los resultados muestran que ambas sustituciones acaban incorporando los iones dentro de la estructura del cristal causando diferentes impactos principalmente a nivel de morfología y solubilidad. Con respecto al impacto del dopaje en la caracterización celular, se llevaron a cabo diferentes ensayos: 1) utilizando suspensiones dispersadas con citrato o sin él, 2) en medio de cultivo con o sin 10 % v/v de suero fetal bovino y 3) utilizando dos tipos de células diferentes, células de osteosarcoma (MG63) y células de rata mesenquimales (rMSCs). Los resultados in vitro mostraron que las nanopartículas dopadas con Mg eran claramente citotóxicas a las células MG63 en ausencia de FBS. Sin embargo, las mismas NPs no alteraron la viabilidad celular en rMSCs bajo las mismas condiciones. El capítulo cuarto se centra en la síntesis y caracterización de NPs dopadas con iones Sr, Zn, Si y Fe(III), que representan sustituciones minoritarias en la apatita biológica. Su caracterización fisicoquímica mostró que todas las NPs eran puras con morfología acicular. Los estudios de citotoxicidad con células MG63 y rMSCs, con y sin FBS, mostraron viabilidad en presencia de FBS para todas las NPs. Además, para las NPs dopadas con Zn y Fe se observó un aumento notable en la proliferación celular para las MG63 cultivadas sin FBS. Además de explorar el efecto del dopaje de iones en NP de HA, otro campo de interés en esta tesis ha sido investigar la mineralización de fosfatos de calcio (CaP) en presencia de moléculas orgánicas y de diferentes iones. A través de estudios recientes en el grupo de investigación se ha visto que es posible formar fosfatos de calcio con estructura neuronal con la ayuda de simples moléculas orgánicas. En el quinto capítulo de esta tesis se estudia el efecto de varias moléculas orgánicas en la precipitación de CaP. Estudios por microscopia electrónica de transmisión (TEM) revelaron la presencia de estas estructuras neuronales formadas con la ayuda de moléculas orgánicas de diferente naturaleza: surfactante no iónicos (Tween 80), polímeros aniónicos (poliacrilato sódico) y políremos catiónicos (cloruro de polidialildimetilamonio). Varios estudios por TEM como son EELS, EFTEM y SAED permitieron establecer que las estructuras neuronales consistían de un núcleo denso del cual se extendía una red de filamentos, que en estas estructuras el calcio, fósforo y oxígeno estaba homogéneamente distribuido y que eran de naturaleza amorfa. Además se investigó el efecto simultáneo de añadir aditivos inorgánicos (iones Mg i Sr) junto con moléculas orgánicas. Se observó que la adición de pequeñas cantidades de iones afectaba la estabilidad de las estructuras neuronales. Con Mg las estructuras no eran estables pero sí con Sr. Estos estudios pueden ser fuente no sólo de inspiración en la síntesis de estructuras más avanzadas y con propiedades notablemente diferentes, sino que además proporcionan un mejor conocimiento de los procesos de biomineralización.

L'homeostasie calcique est regulee, en partie par un recepteur couple aux proteines g de la membrane des cellules parathyroidiennes, appele casr pour calcium-sensing receptor, et qui detecte de faibles variations du taux de ca 2 + extracellulaire. Le casr est exprime dans des tissus peripheriques (parathyroide, rein) et dans le systeme nerveux central. Chez l'homme, des mutations du gene codant pour casr sont responsables de maladies genetiques liees a des perturbations de l'homeostasie calcique. Il existe cependant peu de donnees pharmacologiques concernant casr. Nous avons identifie casr sur les cellules tumorales ante-hypophysaires secretrices d'acth, att-20. L'addition de ca 2 + extracellulaire conduit a une stimulation de la formation d'inositol phosphates dans ces cellules. La courbe d'activation par le ca 2 + presente une cooperative elevee. Le ba 2 + et le mg 2 + sont des agonistes partiels du casr. Le compose organique ()nps 568, propose comme un calcimimetique, stimule l'hydrolyse des phosphatidylinositols dans des fibroblastes transfectes avec le gene codant pour casr de rat, ainsi que dans les cellules att-20. Par ailleurs, ce compose, ainsi que le ca 2 + et le ba 2 + stimulent la secretion d'acth par les cellules att-20 par l'intermediaire du casr. Nous avons localise les cellules exprimant les transcrits de casr dans le systeme nerveux de rat par des experiences d'hybridation in situ. Chez l'adulte, les cellules positives sont disseminees dans l'ensemble du cerveau. Elles sont tres concentrees, chez l'adulte et au cours du developpement postnatal, dans divers noyaux neuronaux comme les noyaux olfactifs anterieurs et quelques noyaux du thalamus. En revanche, nous observons un faible marquage de la region ca2 de l'hippocampe chez l'adulte alors qu'il est fort au cours du developpement. Chez l'adulte, une forte densite de cellules positives est presente dans les passages de fibres. Ce marquage evolue de maniere caudo-rostrale, parallelement a l'apparition des transcrits de la mbp, une proteine specifique des oligodendrocytes matures, au cours du developpement postnatal. De plus, une augmentation de ca 2 + extracellulaire stimule l'accumulation d'inositol phosphates et la mobilisation de ca 2 + intracellulaire dans des cultures primaires d'oligodendrocytes.

La culture primaire de cellules ventriculaires de coeur de rat adulte a permis d'etudier la regulation des jonctions communicantes par les ions hydrogene et calcium intracellulaires. Les cellules peuvent etre cultivees jusqu'a 5 jours in vitro sans modification morphologique dans un milieu contenant ou non une faible quantite de serum de veau foetal. La conductance jonctionnelle g et la permeabilite des canaux des paires de cellules sont mesurees par double patch clamp et gap-frap respectivement. Les proprietes electriques et la nature ohmique de ces canaux jonctionnels sont preserves. Les fluorochromes de masse moleculaire variant de 376 a 913 da traversent les canaux avec des temps de passage croissants. Ces canaux principalement constitues de connexine 43 apparaissent peu selectifs. Les effets des ions calcium intracellulaire sur g sont fonction de leur concentration ca#i. Lorsque ca#i < 45 nm, une augmentation transitoire de g suivie d'un retour a l'etat initial est observee. G est stable en presence de ca#i proche de celle des cellules au repos (45 nm). G augmente definitivement lorsque ca#i croit de 100 a 200 nm. G est inhibee lorsque ca#i > 1 m. En presence de l'inhibiteur de calmoduline w7, g augmente encore plus. En presence de l'inhibiteur de proteines kinases h7, les augmentations de g par les fortes ca#i sont partiellement inhibees. L'elevation de ca#i augmenterait g par phosphorylation des connexines ; le complexe cacalmoduline exercerait un effet antagoniste. La diminution du ph intracellulaire renforce souvent les effets stimulants du calcium. Par contre, l'inhibition de la communication jonctionnelle peut se developper meme si la ca#i est voisine du taux basal des cellules au repos et si le ph < 6. 9. Ce decouplage ne fait pas intervenir la calmoduline. En conclusion, ces resultats semblent compatibles avec la fonction physiologique des cellules cardiaques. Lors d'un effort physique, le taux de calcium cytosolique augmente. La communication jonctionnelle serait alors stimulee, un meilleur synchronisme electrique et mecanique des cellules en resulterait permettant un meilleur rendement de l'organe. Les effets des ions calcium sur la communication jonctionnelle seraient renforces par la faible acidose cytosolique, consequence du metabolisme oxydatif.

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.