1 Abstract Adenylate cyclase toxin (CyaA) is one of the major virulence factors of bacterium Bordetella pertussis, which is a causative agent of whooping cough. CyaA belongs to the family of RTX toxin-hemolysins. The toxin targets primarily cells expressing integrin receptor CD11b/CD18 but it can also penetrate cells lacking this receptor. CyaA acts on host cells by two independent activities. One is formation of small cation-selective channels, which can lead to colloid osmotic lysis of target cells. The second is disruption of cell signaling through the translocation of the adenylate cyclase (AC) domain to host cell cytosol, which leads to the conversion of ATP into cyclic AMP. It was recently shown that cholesterol affects endocytosis of CyaA. CyaA translocates it's AC domain after relocation of CyaA molecule to the cholesterol-rich lipid raft (Bumba et al. 2010). In this work I examined the effect of cholesterol on channel- forming activity and selectivity of ion channels created by CyaA. For measurements I used artificial membranes enriched with cholesterol. CyaA channels are voltage-dependent. The positive membrane potential on the side of toxin is rquired for incorporation of CyaA molecule into cell membrane. I tried to find out whether the value of voltage has effect on channels opening time....

O objetivo do presente trabalho foi avaliar as propriedades físico-químicas de um cimento endodôntico biocerâmico, Endosequence BC Sealer (Brasseler USA, Savannah, GA, EUA). Radiopacidade, escoamento, pH e liberação de íons cálcio (Ca2+) foram analisadas e os resultados foram comparados aos do cimento AH Plus (Dentsply De Trey Gmbh, Konstanz, Alemanha). A radiopacidade e o escoamento foram avaliados de acordo com as normas ISO 6876/2001. Para o teste de radiopacidade, anéis metálicos com 10 mm de diâmetro e 1 mm de altura foram preenchidos com os cimentos. O valor da radiopacidade foi determinado de acordo com a densidade radiográfica, representada em mm Al. A análise do escoamento foi feita com 0,05 mL de cada cimento colocados sobre uma placa de vidro. Outra placa de vidro, com 20 g, foi cuidadosamente colocada sobre o cimento e sobre esta um peso de 100 g, totalizando 120 g, que permaneceram por 7 minutos. Em seguida, foram medidos os diâmetros maiores e menores dos discos formados, sendo esta feita com um paquímetro digital. A liberação de íons cálcio (Ca2+) e o pH foram medidos nos períodos de 3, 24, 72, 168 e 240 horas, com o auxílio de um espectrofotômetro e pHmetro, respectivamente. Os resultados foram analisados estatisticamente pelos testes ANOVA e Tukey, sendo considerado significante quando p<0,05. O cimento biocerâmico analisado apresentou radiopacidade (3,84 mm Al) significantemente menor do que o AH Plus (6,90 mm Al). Foi observado que o cimentos Endosequence BC Sealer e o AH Plus apresentaram escoamentos de 26,96 mm e 21,17 mm, respectivamente (p<0,05). O cimento biocerâmico apresentou maior pH e maior liberação de íons cálcio (Ca2+) do que o cimento AH Plus (p<0,05). Concluiu-se que o cimento Endosequence BC Sealer apresentou valores de radiopacidade e de escoamento em concordância com as norma ISO 6876/2001. Os valores de pH e de liberação de íons cálcio (Ca2+) foram favoráveis para um cimento endodôntico. / The aim of the present study was to evaluate the physicochemical properties of a bioceramic root canal sealer, Endosequence BC Sealer. Radiopacity, pH, release of calcium ions (Ca2+) and flow were analyzed and the results were compared with AH Plus cement. Radiopacity and flow were evaluated according to ISO 6876/2001 standards. For the radiopacity analysis, metallic rings with 10 mm in diameter and 1 mm of thickness were filled with cements. The radiopacity value was determined according to radiographic density (mm Al). The flow test was performed with 0.05 mL of cement placed on a glass plate. A 120-g weight was carefully placed over the cement. The largest and smallest diameters of the disks formed were measured using a digital caliper. The release of Ca2+ and pH were measured at periods of 3, 24, 72, 168 and 240 hours, with spectrophotometer and pHmeter, respectively. Data were analyzed by ANOVA and Tukeys test (p<0.05). The bioceramic endodontic cement showed radiopacity (3.84 mm Al) significantly lower than that of AH Plus (6.90 mm Al). The pH analysis showed that Endosequence BC Sealer showed pH and release of Ca2+ greater than those of AH Plus (p<0.05), during the experimental periods. The flow test revealed that the BC Sealer and AH Plus presented flow 26.96 mm and 21.17 mm, respectively (p<0.05). Endosequence BC Sealer showed radiopacity and flow according to ISO 6876/2001 recommendations. The others physicochemical properties analyzed demonstrated favorable values for a root canal sealer.

This thesis reports on the setup of a new ion trap apparatus designed for experiments with single ⁴⁰Ca⁺ ions to perform molecular spectroscopy. The calcium ion is laser cooled, allowing for sympathetic cooling of the nonfluorescing molecular ion. The aim of these experiments is to explore loading and identifying molecular ions in RF-Paul traps, as well as developing new spectroscopic tools to measure transitions of molecular ions via the fluorescence of co-trapped ⁴⁰Ca⁺ ions. Ground state cooling of a mixed ion pair is implemented as a first step towards increasing the sensitivity of our technique to the level necessary to measure transitions with low scattering rates (like those present in molecular ions). Doppler cooling on the S(1/2)->P(1/2) transition of the calcium ion results in the formation of a Coulomb crystal, the behavior of which may be used to infer properties of the molecular ion. Following cooling, sideband spectroscopy on the narrow S(1/2)->D(5/2) quadrupole transition of calcium may be used to identify the mass of single molecular ions. This method is verified via a non-destructive measurement on ⁴⁰CaH⁺ and ⁴⁰Ca¹⁶O⁺. The normal modes of the Coulomb crystal can also be used to extract information from the target ion to the control ion. By driving the blue side of a transition, laser induced heating can be put into the two ion system, which leads to changes in fluorescence of the ⁴⁰Ca⁺ ion, first demonstrated with two Ca⁺ isotopes. Increasing the sensitivity of this technique requires ground state cooling of both the ⁴⁰Ca⁺ ion and the ion of interest, enabling the transfer of the ion's motional state into the ground state with high probability. This thesis demonstrates ground state cooling of the atomic ion and sympathetic cooling of a second ion (⁴⁴Ca⁺). Once in the ground state, heating of the Coulomb crystal by scattering photons off of the spectroscopy ion can be measured by monitoring the resolved motional sidebands of the S(1/2)->D(5/2) transition of ⁴⁰Ca⁺, allowing for spectral lines to be inferred. Future experiments will investigate this technique with molecular ions.

Calcium ions (Ca2+) are involved in almost all neuronal functions, providing the link between electrical signals and cellular activity. This work examines the mechanisms by which a neuron can regulate the movement and sequestration of Ca2+ through specific channels such that this ubiquitous ion can encode specific functions. My initial focus was using intracellular calcium ([Ca2+]i) imaging techniques to study the influence of the inhibition of specific voltage gated calcium channels (VGCC) by ethanol on a depolarization induced rise in [Ca2+]i in neurohypophysial nerve terminals. This research took an unexpected turn when I observed an elevation of [Ca2+]i during perfusion with ethanol containing solutions. Control experiments showed this to be an artifactual result not directly attributable to ethanol. It was necessary to track down the source of this artifact in order to proceed with future ethanol experiments. The source of the artifact turned out to be a contaminant leaching from I.V. drip chambers. Due to potential health implications stemming from the use of these drip chambers in a clinical setting as well as potential artifactual results in the ethanol field where these chambers are commonly used, I choose to investigate this phenomenon more rigorously. The agent responsible for this effect was shown to be di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer that has been shown to be carcinogenic in rats and mice. The extraction of this contaminant from the I.V. drip chamber, as measured by spectrophotometry, was time-dependent, and was markedly accelerated by the presence of ethanol in the solution. DEHP added to saline solution caused a rise in [Ca2+]i similar to that elicited by the contaminant containing solution. The rise in calcium required transmembrane flux through membrane channels. Blood levels of DEHP in clinical settings have been shown to exceed the levels which we found to alter [Ca2+]i. This suggests that acute alterations in intracellular calcium should be considered in addition to long-term effects when determining the safety of phthalate-containing plastics. As part of a collaboration between Steven Treistman and Robert Messing's laboratory at UCSF, I participated in a study of how ethanol regulates N-type calcium channels which are known to be inhibited acutely, and upregulated in the chronic presence of ethanol. Specific mRNA splice variants encoding N-type channels were investigated using ribonuclease protection assays and real-time PCR. Three pairs of N-type specific α-subunit Cav2.2 splice variants were examined, with exposure to ethanol observed to increase expression of one alternative splice form in a linker that lacks six bases encoding the amino acids glutamate and threonine (ΔET). Whole cell electrophysiological recordings that I carried out demonstrated a faster rate of channel activation and a shift in the voltage dependence of activation to more negative potentials after chronic alcohol exposure, consistent with increased expression of ΔET variants. These results demonstrate that chronic ethanol exposure not only increases the abundance of N-type calcium channels, but also increases the expression of a Cav2.2 splice variant with kinetics predicted to support a larger and faster rising intracellular calcium signal. This is the first demonstration that ethanol can up-regulate ion channel function through expression of a specific mRNA splice variant, defining a new mechanism underlying the development of drug addiction. Depolarizing a neuron opens voltage gated Ca2+ channels (VGCC), leading to an influx of Ca2+ ions into the cytoplasm, where Ca2+ sensitive signaling cascades are stimulated. How does the ubiquitous calcium ion selectively modulate a large array of neuronal functions? Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca2+ current and the resulting rise and decay of [Ca2+]i, showing that equal amounts of Ca2+ entering through N-type and L-type voltage gated Ca2+ channels result in significantly different [Ca2+]i temporal profiles. When the contribution of N-type channels was reduced, a faster [Ca2+]i decay was observed. Conversely, when the contribution of L-type channels was reduced, [Ca2+]i decay was slower. Potentiating L-type current or inactivating N-type channels both resulted in a more rapid decay of [Ca2+]i. Channel-specific differences in [Ca2+]i decay rates were abolished by depleting intracellular Ca2+ stores suggesting the involvement of Ca2+-induced Ca2+ release (CICR). I was able to conclude that Ca2+ entering through N-type, but not L-type channels, is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR generates a unique intracellular Ca2+ signal depending on the route of entry, potentially encoding the selective activation of a subset of Ca2+ -sensitive processes within the neuron.

O objetivo do presente trabalho foi avaliar as propriedades físico-químicas de um cimento endodôntico biocerâmico, Endosequence BC Sealer (Brasseler USA, Savannah, GA, EUA). Radiopacidade, escoamento, pH e liberação de íons cálcio (Ca2+) foram analisadas e os resultados foram comparados aos do cimento AH Plus (Dentsply De Trey Gmbh, Konstanz, Alemanha). A radiopacidade e o escoamento foram avaliados de acordo com as normas ISO 6876/2001. Para o teste de radiopacidade, anéis metálicos com 10 mm de diâmetro e 1 mm de altura foram preenchidos com os cimentos. O valor da radiopacidade foi determinado de acordo com a densidade radiográfica, representada em mm Al. A análise do escoamento foi feita com 0,05 mL de cada cimento colocados sobre uma placa de vidro. Outra placa de vidro, com 20 g, foi cuidadosamente colocada sobre o cimento e sobre esta um peso de 100 g, totalizando 120 g, que permaneceram por 7 minutos. Em seguida, foram medidos os diâmetros maiores e menores dos discos formados, sendo esta feita com um paquímetro digital. A liberação de íons cálcio (Ca2+) e o pH foram medidos nos períodos de 3, 24, 72, 168 e 240 horas, com o auxílio de um espectrofotômetro e pHmetro, respectivamente. Os resultados foram analisados estatisticamente pelos testes ANOVA e Tukey, sendo considerado significante quando p<0,05. O cimento biocerâmico analisado apresentou radiopacidade (3,84 mm Al) significantemente menor do que o AH Plus (6,90 mm Al). Foi observado que o cimentos Endosequence BC Sealer e o AH Plus apresentaram escoamentos de 26,96 mm e 21,17 mm, respectivamente (p<0,05). O cimento biocerâmico apresentou maior pH e maior liberação de íons cálcio (Ca2+) do que o cimento AH Plus (p<0,05). Concluiu-se que o cimento Endosequence BC Sealer apresentou valores de radiopacidade e de escoamento em concordância com as norma ISO 6876/2001. Os valores de pH e de liberação de íons cálcio (Ca2+) foram favoráveis para um cimento endodôntico. / The aim of the present study was to evaluate the physicochemical properties of a bioceramic root canal sealer, Endosequence BC Sealer. Radiopacity, pH, release of calcium ions (Ca2+) and flow were analyzed and the results were compared with AH Plus cement. Radiopacity and flow were evaluated according to ISO 6876/2001 standards. For the radiopacity analysis, metallic rings with 10 mm in diameter and 1 mm of thickness were filled with cements. The radiopacity value was determined according to radiographic density (mm Al). The flow test was performed with 0.05 mL of cement placed on a glass plate. A 120-g weight was carefully placed over the cement. The largest and smallest diameters of the disks formed were measured using a digital caliper. The release of Ca2+ and pH were measured at periods of 3, 24, 72, 168 and 240 hours, with spectrophotometer and pHmeter, respectively. Data were analyzed by ANOVA and Tukeys test (p<0.05). The bioceramic endodontic cement showed radiopacity (3.84 mm Al) significantly lower than that of AH Plus (6.90 mm Al). The pH analysis showed that Endosequence BC Sealer showed pH and release of Ca2+ greater than those of AH Plus (p<0.05), during the experimental periods. The flow test revealed that the BC Sealer and AH Plus presented flow 26.96 mm and 21.17 mm, respectively (p<0.05). Endosequence BC Sealer showed radiopacity and flow according to ISO 6876/2001 recommendations. The others physicochemical properties analyzed demonstrated favorable values for a root canal sealer.

The primary focus of this dissertation is the development of novel fluorescent near-infrared molecules for various applications. In Chapter 1, a compound dU-BZ synthesized via Sonogashira coupling reaction methodology is described. A deoxyuridine building block was introduced to enhance hydrophilic properties and reduce toxicity, while an alkynylated benzothiazolium dye was incorporated for near-IR emission and reduce photodamage and phototoxicity that is characteristic of common fluorphores that are excited by UV or visible light. A 30-fold enhancement of fluorescence intensity of dU-BZ was achieved in a viscous environment. Values of fluorescence quantum yields in 99% glycerol/1% methanol (v/v) of varying temperature from 293 K to 343 K, together with fluorescence quantum yields, radiative and nonradiative rate constants and fluorescence lifetimes in glycerol/methanol solutions of varying viscosities from 4.8 to 950 cP were determined. It was found that both fluorescence quantum yields and fluorescence lifetimes increased with increasing viscosity, which is consistent with results predicted by theory. This suggests that the newly designed compound dU-BZ is capable of functioning as a probe of local microviscosity, and was later confirmed by in vitro bioimaging experiments. In Chapter 2, a new BAPTA (O,O*-bis(2-aminophenyl)ethyleneglycol-N,N,N*,N*-tetra acetic acid) and BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-based calcium indicator, BAPBO-3, is reported. A new synthetic route was employed to simplify both synthesis and purification, which tend to be low yielding and cumbersome for BAPTA derivatives. Upon excitation, a 1.5-fold increase in fluorescence intensity in buffer containing 39 ?? Ca2+ and a 3-fold increase in fluorescence intensity in buffer containing 1 M Ca2+ was observed; modest but promising fluorescence turn-on enhancements. In Chapter 3, a newly-designed unsymmetrical squaraine dye, SQ3, was synthesized. A one-pot synthesis was employed resulting in a 10% yield, a result that is generally quite favorable for the creation of unsymmetrical squaraines Photophysical and photochemical characterization was conducted in various solvents, and a 678 nm absorption maximum and a 692 nm emission maximum were recorded in DMSO solution with a fluorescence quantum yield of 0.32. In vitro cell studies demonstrated that SQ3 can be used as a near-IR probe for bioimaging.

No / Patients with acute vitiligo have low epidermal catalase expression/activities and accumulate 10 -3 M H 2O 2. One consequence of this severe oxidative stress is an altered calcium homeostasis in epidermal keratinocytes and melanocytes. Here, we show decreased epidermal calmodulin expression in acute vitiligo. Since 10 -3M H 2O 2 oxidises methionine and tryptophan residues in proteins, we examined calcium binding to calmodulin in the presence and absence of H 2O 2 utilising 45calcium. The results showed that all four calcium atoms exchanged per molecule of calmodulin. Since oxidised calmodulin looses its ability to activate calcium ATPase, enzyme activities were followed in full skin biopsies from lesional skin of patients with acute vitiligo (n = 6) and healthy controls (n = 6). The results yielded a 4-fold decrease of ATPase activities in the patients. Computer simulation of native and oxidised calmodulin confirmed the loss of all four calcium ions from their specific EF-hand domains. Taken together H 2O 2-mediated oxidation affects calcium binding in calmodulin leading to perturbed calcium homeostasis and perturbed L-phenylalanine-uptake in the epidermis of acute vitiligo.

Paclitaxel is one of the most successful drugs for the treatment of cancer because of its ability to target tubulin, block cell cycle progression at mitosis, and induce apoptosis. Despite the success of Paclitaxel, the development of drug resistance hampers its clinical applicability. Paclitaxel is used in malignant tumors in the present research, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), head-neck scale epitheliomatous, urinary bladder cancer, tumor of the reproduction organ and so on. Clinical treatment of paclitaxel be injected 250mg/m2 to previously non-treated patients of small lung cancer its effect about 34% and previously non-treated patients of non-small lung cancer its effect about 21% to 24% . On the other hand we had established a taxol-resistant human lung carcinoma subline A549R by paclitaxel to compare the different proteins with A549 wild type and treat the different concention of paclitaxel with MTT assay so as to observe the tolerance dosage of subline growth.We obtained the patient¡¦s specimens of lung cancer to treat with paclitaxel some of resistant and some of non-resistant to compare differentially expressed proteins between normal and tumor. When we cultured taxol-resistant human lung carcinoma subline in which paclitaxel and calcium regulate growth, owing to the proteins of changes result to resistance. The extraction cell subline and specimens were analyzed by 2-D electrophoresis patterns and we found that interact paclitaxel with calcium it is the important factor of drug resistant. Verified from clinical treatment might have hypercalcemia in malignant tumors and calcium ion may increase paclitaxel drug resistance and hypercalcemia patient will be more insensitive to paclitaxel treatment.

[Truncated abstract] Self-filtration is here defined as particle detachment and re-deposition causing re-arrangement of the particles and therefore pore space which affects water flow in soil by decreasing hydraulic conductivity. This is of particular important in soils which are susceptible to structural breakdown. The objective of this thesis was to examine the dynamics of the self-filtration process in sodic sandy soils as affected by ionic strength and soil solution composition. The temporal changes of hydraulic conductivity and the elution of fine particles from soil columns were used as the main criteria to assess selffiltration. Two porous media exhibiting significantly different structural cohesion were examined, one a loamy sand (Balkuling soil) from agricultural land use and the second a mining residue from mineral sands operations . . . The effects of the composition of mixed calcium (Ca) and sodium (Na) ions in solution (sodium adsorption ratio (SAR)) on the exchange behaviour and saturated hydraulic conductivity were examined by carrying out batch binary exchange and saturated column transport experiments. A strong preference for Ca2+ ions in the exchange complex was observed for both soils. Generally K/Ko was found to decrease with increasing sodium adsorption ratio with the more structured Balkuling soil maintaining K/Ko for SARs 3 and 5 at an electrolyte concentration of 100 mmol/L. However measurements at the critical threshold and turbidity concentrations at a SAR of 15 revealed structural breakdown of the pore matrix system attributed to various extents of slaking, swelling, dispersion and decreases of pore radii as a result of selffiltration during leaching. These experiments illustrate the wide range of complex interactions involving clay mineralogy, solution composition and structural factors which can influence the extent of mobilization, transport and re-deposition of colloidal particles during the leaching process in soil profiles.

Species of the Bordetella genus cause the highly contagious whooping cough disease in humans (B. pertussis, B. parapertussis) and related respiratory diseases in other mammals (B. bronchiseptica, B. parapertussis). One of the virulence systems of Bordetellae is the type III secretion system (T3SS) employed for translocation of effector proteins directly from bacterial cytosol into the cytosol of host cells. The T3SS protein BopN protein has been categorized as a Bordetella effector protein. Nevertheless, the homologous proteins in other gram-negative bacteria function in establishing the secretion hierarchy through T3SS and some of them block T3SS secretion in high calcium environments before bacteria-host cell contact has been established. In this thesis I examined the function of the BopN protein and the role of calcium ions in T3SS activity of B. bronchiseptica. Two independent methods have been used for determination of T3SS secretion activity. Addition of 2 mM calcium ions into bacterial media decreased secretion of the T3SS reporter, while no such effect was observed in a B. bronchiseptica strain lacking the bopN gene. Mass spectrometry data confirmed the inhibition of T3SS activity in the presence of calcium ions. Enhanced calcium levels resulted in decreased mobilization and secretion of...