Responses of human dental pulp cells after application of a low-concentration bleaching gel to enamel

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Dormancy breaking of the fig tree with hydrogen cyanamide and garlic extrate

The supply of cold hours needed to the dormancy breaking of shoots is the limiting factor for the cultivation of temperate climate fruit trees in warmer regions. In subtropical conditions, it is necessary to use chemical products to promote uniform sprouting. This research aimed at evaluating the effect of garlic extract and hydrogen cyanamide in sprouting, growth, production and production cycle of the fig tree. The experiment was conducted during the production cycles of 2011/12 and 2012/13. We used plants from the cultivar Roxo de Valinhos. Production pruning was made in the months of July/2011 and July/2012, and the following treatments were applied immediately after it: 2% hydrogen cyanamide and garlic extract in 4%, 8% and 12% doses, and a control treatment. Split plots were used as the experimental design, with five repetitions in blocks; each plot consisted of five treatments with hydrogen cyanamide, garlic extract and control; the subplots consisted of two production cycles. The use of hydrogen cyanamide promoted an anticipation of sprouting and the use of hydrogen cyanamide and garlic extract promoted a concentration of the productive period, when compared to the control. The estimated garlic extract dose that promoted the highest production per plant was 3%.

Interface tailoring for adhesion enhancement of diamond-like carbon thin films

We have explored the suitability and characteristics of interface tailoring as a tool for enhancing the adhesion of hydrogen-free diamond-like carbon (DLC) thin films to silicon substrates. DLC films were deposited on silicon with and without application of an initial high energy carbon ion bombardment phase that formed a broad Si-C interface of gradually changing Si:C composition. The interface depth profile was calculated using the TRIDYN simulation program, revealing a gradient of carbon concentration including a region with the stoichiometry of silicon carbide. DLC films on silicon, with and without interface tailoring, were characterized using Raman spectroscopy, scanning electron microscopy, atomic force microscopy and scratch tests. The Raman spectroscopy results indicated sp3-type carbon bonding content of up to 80%. Formation of a broadened Si:C interface as formed here significantly enhances the adhesion of DLC films to the underlying silicon substrate. (C) 2012 Elsevier B.V. All rights reserved.

Effect of some surfactants on the chemiluminescent reactions of bis(2,4,6-trichlorophenyl)oxalate and bis(2-nitrophenyl)oxalate with hydrogen peroxide

The chemiluminescent reactions of bis(2,4,6-trichlorophenyl)oxalate (TCPO) and bis(2-nitrophenyl)oxalate (2-NPO) with hydrogen peroxide in acetonitrile/water micellar systems (anionic, cationic, and non-ionic) and gamma-cyclodextrin were studied in the presence of fluoranthene or 9,10-diphenylanthracene, imidazole, and two buffer solutions, HTRIS+/TRIS and H2PO4-/HPO42-. The relative chemiluminenscence (CL) intensity is higher in the presence of the cationic (DDAB, CTAC, DODAC, and OTAC), anionic (SDS), and non-ionic (Tween 80) surfactants. In the presence of some non-ionic surfactants (Brij 35, Brij 76, and Tween 20), the CL intensity was partially quenched compared with the reaction with no surfactant. The sensitivity for hydrogen peroxide determination in the range 0.01 x 10(-4) to 1.0 x 10(-4) mol L-1, considering the slope of the calibration curves (maximum peak height of CL vs. concentration), improved with the introduction of DDAH, CTAB, and SDS in HTRIS+/TRIS buffer.

Hydrogen peroxide monitoring in Fenton reaction by using a ruthenium oxide hexacyanoferrate/multiwalled carbon nanotubes modified electrode

A novel amperometric sensor based on the incorporation of ruthenium oxide hexacyanoferrate (RuOHCF) into multiwalled carbon nanotubes (MWCNTs) immobilized on a glassy carbon electrode is described. Cyclic voltammetry experiments indicated that the cathodic reduction of hydrogen peroxide at the RuOHCF/MWCNTs100/GC modified electrode is facilitated, occurring at 0.0 V vs. Ag/AgCl/KCl(sat). Following the optimization of the experimental conditions, the proposed sensor presented excellent analytical properties for hydrogen peroxide determination, with a low limit of detection (4.7 mu mol L-1), a large dynamic concentration range (0.1-10 mmol L-1) and a sensitivity of 1280 mu A mmol(-1) L cm(-2). The usefulness of the RuOHCF/MWCNTs100/GC electrochemical sensor was confirmed by monitoring the consumption of hydrogen peroxide during the degradation of phenol by the Fenton reaction. (C) 2012 Elsevier B.V. All rights reserved.

Gold nanoparticle formation in diamond-like carbon using two different methods: Gold ion implantation and co-deposition of gold and carbon

We describe work in which gold nanoparticles were formed in diamond-like carbon (DLC), thereby generating a Au-DLC nanocomposite. A high-quality, hydrogen-free DLC thin film was formed by filtered vacuum arc plasma deposition, into which gold nanoparticles were introduced using two different methods. The first method was gold ion implantation into the DLC film at a number of decreasing ion energies, distributing the gold over a controllable depth range within the DLC. The second method was co-deposition of gold and carbon, using two separate vacuum arc plasma guns with suitably interleaved repetitive pulsing. Transmission electron microscope images show that the size of the gold nanoparticles obtained by ion implantation is 3-5 nm. For the Au-DLC composite obtained by co-deposition, there were two different nanoparticle sizes, most about 2 nm with some 6-7 nm. Raman spectroscopy indicates that the implanted sample contains a smaller fraction of sp(3) bonding for the DLC, demonstrating that some sp(3) bonds are destroyed by the gold implantation. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4757029]

Performance and composition of bacterial communities in anaerobic fluidized bed reactors for hydrogen production: Effects of organic loading rate and alkalinity

This study evaluated the effects of the organic loading rate (OLR) and pH buffer addition on hydrogen production in two anaerobic fluidized bed reactors (AFBRs) operated simultaneously. The AFBRs were fed with glucose, and expanded clay was used as support material. The reactors were operated at a temperature of 30 degrees C, without the addition of a buffer (AFBR1) and with the addition of a pH buffer (AFBR2, sodium bicarbonate) for OLRs ranging from 19.0 to 140.6 kg COD m(-3) d(-1) (COD: chemical oxygen demand). The maximum hydrogen yields for AFBR1 and AFBR2 were 2.45 and 1.90 mol H-2 mol(-1) glucose (OLR of 84.3 kg COD m(-3) d(-1)), respectively. The highest hydrogen production rates were 0.95 and 0.76 L h(-1) L-1 for AFBR1 and AFBR2 (OLR of 140.6 kg COD m(-3) d(-1)), respectively. The operating conditions in AFBR1 favored the presence of such bacteria as Clostridium, while the bacteria in AFBR2 included Clostridium, Enterobacter, Klebsiella, Veillonellaceae, Chryseobacterium, Sporolactobacillus, and Burkholderiaceae. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Vanadium and Titanium Mixed Oxide Films: Synthesis, Characterization and Application as Ion Sensor

Vanadium/titanium mixed oxide films were produced using the sol-gel route. The structural investigation revealed that increased TiO2 molar ratio in the mixed oxide disturbs the V2O5 crystalline structure and makes it amorphous. This blocks the TiO2 phase transformation, so TiO2 stabilizes in the anatase phase. In addition the surface of the sample always presents larger amounts of TiO2 than expected, revealing a concentration gradient along the growth direction. For increased TiO2 molar ratios the roughness of the surface is reduced. Ion sensors were fabricated using the extended gate field effect transistor configuration. The obtained sensitivities varied in the range of 58 mV/pH down to 15 mV/pH according to the composition and morphology of the surface of the samples. Low TiO2 amounts presented better sensing properties that might be related to the cracked and inhomogeneous surfaces. Rising the TiO2 quantity in the films produces homogeneous surfaces but diminishes their sensitivities. Thus, the present paper reveals that the compositional and structural aspects change the surface morphology and electrical properties accounting for the final ion sensing properties of the V2O5/TiO2 films. (C) 2012 The Electrochemical Society. [DOI: 10.1149/2.053206jes] All rights reserved.

HYDROGEN SULFIDE AS A CRYOGENIC MEDIATOR OF HYPDXIA-INDUCED ANAPYREXIA

Hypoxia causes a regulated decrease in body temperature (Tb), a response that has been aptly called anapyrexia, but the mechanisms involved are not completely understood. The roles played by nitric oxide (NO) and other neurotransmitters have been documented during hypoxia-induced anapyrexia, but no information exists with respect to hydrogen sulfide (H(2)S), a gaseous molecule endogenously produced by cystathionine beta-synthase (CBS). We tested the hypothesis that HA production is enhanced during hypoxia and that the gas acts in the anteroventral preoptic region (AVPO; the most important thermosensitive and thermointegrative region of the CNS) modulating hypoxia-induced anapyrexia. Thus, we assessed CBS and nitric oxide synthase (NOS) activities [by means of H2S and nitrite/nitrate (NO(x)) production, respectively] as well as cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) levels in the anteroventral third ventricle region (AV3V; where the AVPO is located) during normoxia and hypoxia. Furthermore, we evaluated the effects of pharmacological modifiers of the H2S pathway given i.c.v. or intra-AVPO. I.c.v. or intra-AVPO microinjection of CBS inhibitor caused no change in Tb under normoxia but significantly attenuated hypoxia-induced anapyrexia. During hypoxia there were concurrent increases in H2S production, which could be prevented by CBS inhibitor, indicating the endogenous source of the gas. cAMP concentration, but not cGMP and NOR, correlated with CBS activity. CBS inhibition increased NOS activity, whereas H2S donor decreased NO. production. In conclusion, hypoxia activates H2S endogenous production through the CBS-H(2)S pathway in the AVPO, having a cryogenic effect. Moreover, the present data are consistent with the notion that the two gaseous molecules, H(2)S and NO, play a key role in mediating the drop in Tb caused by hypoxia and that a fine-balanced interplay between NOS-NO and CBS-H(2)S pathways takes place in the AVPO of rats exposed to hypoxia. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Counter-ion effect on surfactant-DNA gel particles as controlled DNA delivery systems

The ability to entrap drugs within vehicles and subsequently release them has led to new treatments for a number of diseases. Based on an associative phase separation and interfacial diffusion approach, we developed a way to prepare DNA gel particles without adding any kind of cross-linker or organic solvent. Among the various agents studied, cationic surfactants offered particularly efficient control for encapsulation and DNA release from these DNA gel particles. The driving force for this strong association is the electrostatic interaction between the two components, as induced by the entropic increase due to the release of the respective counter-ions. However, little is known about the influence of the respective counter-ions on this surfactant-DNA interaction. Here we examined the effect of different counter-ions on the formation and properties of the DNA gel particles by mixing DNA (either single-(ssDNA) or double-stranded (dsDNA)) with the single chain surfactant dodecyltrimethylammonium (DTA). In particular, we used as counter-ions of this surfactant the hydrogen sulfate and trifluoromethane sulfonate anions and the two halides, chloride and bromide. Effects on the morphology of the particles obtained, the encapsulation of DNA and its release, as well as the haemocompatibility of these particles are presented, using counter-ion structure and DNA conformation as controlling parameters. Analysis of the data indicates that the degree of counter-ion dissociation from the surfactant micelles and the polar/hydrophobic character of the counter-ion are important parameters in the final properties of the particles. The stronger interaction with amphiphiles for ssDNA than for dsDNA suggests the important role of hydrophobic interactions in DNA.

Time course of hydrogen peroxide-thioredoxin balance and its influence on the intracellular signalling in myocardial infarction

We investigated the myocardial thioredoxin-1 and hydrogen peroxide concentrations and their association with some prosurvival and pro-apoptotic proteins, during the transition from myocardial infarction (MI) to heart failure in rats. Male Wistar rats were divided into the following six groups: three sham-operated groups and three MI groups, each at at 2, 7 and 28 days postsurgery. Cardiac function was analysed by echocardiography; the concentration of H2O2 and the ratio of reduced to oxidized glutathione were measured spectrophotometrically, while the myocardial immunocontent of thioredoxin-1, angiotensin II, angiotensin II type 1 and type 2 receptors, p-JNK/JNK, p-ERK/ERK, p-Akt/Akt, p-mTOR/mTOR and p-GSK3 beta/GSK3 beta was evaluated by Western blot. Our results show that thioredoxin-1 appears to make an important contribution to the reduced H2O2 concentration. It was associated with lower JNK expression in the early period post-MI (2 days). However, thioredoxin-1 decreased, while reninangiotensin system markers and levels of H2O2 increased, over 28 days post-MI, in parallel with some signalling proteins involved in maladaptative cardiac remodelling and ventricular dysfunction. These findings provide insight into the time course profile of endogenous antioxidant adaptation to ischaemic injury, which may be useful for the design of therapeutical strategies targeting oxidative stress post-MI.

Baccharin Prevents Genotoxic Effects Induced by Methyl Methanesulfonate and Hydrogen Peroxide in V79 Cells

Baccharin is one of the major chemical compounds isolated from the aerial parts of Baccharis dracunculifolia DC (Asteraceae), a native plant of South America and the most important botanical source of the Brazilian green propolis that has been used in alternative medicine to treat inflammation, liver disorders, and stomach ulcers. The present study was carried out in V79 cells to determine the possible genotoxic and antigenotoxic activities of baccharin utilizing comet and micronucleus assays, where 2 known mutagenic agents with different mechanisms of DNA damage were used as positive controls. The V79 cells were treated with concentrations of baccharin (0.25, 0.5, 1.0, and 2.0 mu g/mL) and for to investigate the antigenotoxicity these concentrations were associated with methyl methanesulfonate (MMS; 200 mu M-comet assay and 400 mu M-micronucleus assay) or hydrogen peroxide (H2O2; 50 mu M-comet assay and 100 mu M-micronucleus assay). Statistically significant differences in the rate of DNA damage were observed in cultures treated with the highest concentration of baccharin when compared to the control group, but this difference was not found in the micronucleus assay. The results also showed that the frequencies of DNA damage and micronuclei induced by MMS and H2O2 were significantly reduced after treatment with baccharin. The baccharin showed a chemoprevention effect and can be the chemical compound responsible for the antigenotoxicity also demonstrated by the B. dracunculifolia. The antioxidant potential of baccharin may be related to its chemoprevention activity induced against both genomic and chromosomal damages.

Surface modification by metal ion implantation forming metallic nanoparticles in insulating matrix.

There is special interest in the incorporation of metallic nanoparticles in a surrounding dielectric matrix for obtaining composites with desirable characteristics such as for surface plasmon resonance, which can be used in photonics and sensing, and controlled surface electrical conductivity. We investigated nanocomposites produced through metallic ion implantation in insulating substrate, where the implanted metal self-assembles into nanoparticles. During the implantation, the excess of metal atom concentration above the solubility limit leads to nucleation and growth of metal nanoparticles, driven by the temperature and temperature gradients within the implanted sample including the beam-induced thermal characteristics. The nanoparticles nucleate near the maximum of the implantation depth profile (projected range), that can be estimated by computer simulation using the TRIDYN. This is a Monte Carlo simulation program based on the TRIM (Transport and Range of Ions in Matter) code that takes into account compositional changes in the substrate due to two factors: previously implanted dopant atoms, and sputtering of the substrate surface. Our study suggests that the nanoparticles form a bidimentional array buried few nanometers below the substrate surface. More specifically we have studied Au/PMMA (polymethylmethacrylate), Pt/PMMA, Ti/alumina and Au/alumina systems. Transmission electron microscopy of the implanted samples showed the metallic nanoparticles formed in the insulating matrix. The nanocomposites were characterized by measuring the resistivity of the composite layer as function of the dose implanted. These experimental results were compared with a model based on percolation theory, in which electron transport through the composite is explained by conduction through a random resistor network formed by the metallic nanoparticles. Excellent agreement was found between the experimental results and the predictions of the theory. It was possible to conclude, in all cases, that the conductivity process is due only to percolation (when the conducting elements are in geometric contact) and that the contribution from tunneling conduction is negligible.

Computational analyses on the structure-function relation in ion channels

Ion channels are protein molecules, embedded in the lipid bilayer of the cell membranes. They act as powerful sensing elements switching chemicalphysical stimuli into ion-fluxes. At a glance, ion channels are water-filled pores, which can open and close in response to different stimuli (gating), and one once open select the permeating ion species (selectivity). They play a crucial role in several physiological functions, like nerve transmission, muscular contraction, and secretion. Besides, ion channels can be used in technological applications for different purpose (sensing of organic molecules, DNA sequencing). As a result, there is remarkable interest in understanding the molecular determinants of the channel functioning. Nowadays, both the functional and the structural characteristics of ion channels can be experimentally solved. The purpose of this thesis was to investigate the structure-function relation in ion channels, by computational techniques. Most of the analyses focused on the mechanisms of ion conduction, and the numerical methodologies to compute the channel conductance. The standard techniques for atomistic simulation of complex molecular systems (Molecular Dynamics) cannot be routinely used to calculate ion fluxes in membrane channels, because of the high computational resources needed. The main step forward of the PhD research activity was the development of a computational algorithm for the calculation of ion fluxes in protein channels. The algorithm - based on the electrodiffusion theory - is computational inexpensive, and was used for an extensive analysis on the molecular determinants of the channel conductance. The first record of ion-fluxes through a single protein channel dates back to 1976, and since then measuring the single channel conductance has become a standard experimental procedure. Chapter 1 introduces ion channels, and the experimental techniques used to measure the channel currents. The abundance of functional data (channel currents) does not match with an equal abundance of structural data. The bacterial potassium channel KcsA was the first selective ion channels to be experimentally solved (1998), and after KcsA the structures of four different potassium channels were revealed. These experimental data inspired a new era in ion channel modeling. Once the atomic structures of channels are known, it is possible to define mathematical models based on physical descriptions of the molecular systems. These physically based models can provide an atomic description of ion channel functioning, and predict the effect of structural changes. Chapter 2 introduces the computation methods used throughout the thesis to model ion channels functioning at the atomic level. In Chapter 3 and Chapter 4 the ion conduction through potassium channels is analyzed, by an approach based on the Poisson-Nernst-Planck electrodiffusion theory. In the electrodiffusion theory ion conduction is modeled by the drift-diffusion equations, thus describing the ion distributions by continuum functions. The numerical solver of the Poisson- Nernst-Planck equations was tested in the KcsA potassium channel (Chapter 3), and then used to analyze how the atomic structure of the intracellular vestibule of potassium channels affects the conductance (Chapter 4). As a major result, a correlation between the channel conductance and the potassium concentration in the intracellular vestibule emerged. The atomic structure of the channel modulates the potassium concentration in the vestibule, thus its conductance. This mechanism explains the phenotype of the BK potassium channels, a sub-family of potassium channels with high single channel conductance. The functional role of the intracellular vestibule is also the subject of Chapter 5, where the affinity of the potassium channels hEag1 (involved in tumour-cell proliferation) and hErg (important in the cardiac cycle) for several pharmaceutical drugs was compared. Both experimental measurements and molecular modeling were used in order to identify differences in the blocking mechanism of the two channels, which could be exploited in the synthesis of selective blockers. The experimental data pointed out the different role of residue mutations in the blockage of hEag1 and hErg, and the molecular modeling provided a possible explanation based on different binding sites in the intracellular vestibule. Modeling ion channels at the molecular levels relates the functioning of a channel to its atomic structure (Chapters 3-5), and can also be useful to predict the structure of ion channels (Chapter 6-7). In Chapter 6 the structure of the KcsA potassium channel depleted from potassium ions is analyzed by molecular dynamics simulations. Recently, a surprisingly high osmotic permeability of the KcsA channel was experimentally measured. All the available crystallographic structure of KcsA refers to a channel occupied by potassium ions. To conduct water molecules potassium ions must be expelled from KcsA. The structure of the potassium-depleted KcsA channel and the mechanism of water permeation are still unknown, and have been investigated by numerical simulations. Molecular dynamics of KcsA identified a possible atomic structure of the potassium-depleted KcsA channel, and a mechanism for water permeation. The depletion from potassium ions is an extreme situation for potassium channels, unlikely in physiological conditions. However, the simulation of such an extreme condition could help to identify the structural conformations, so the functional states, accessible to potassium ion channels. The last chapter of the thesis deals with the atomic structure of the !- Hemolysin channel. !-Hemolysin is the major determinant of the Staphylococcus Aureus toxicity, and is also the prototype channel for a possible usage in technological applications. The atomic structure of !- Hemolysin was revealed by X-Ray crystallography, but several experimental evidences suggest the presence of an alternative atomic structure. This alternative structure was predicted, combining experimental measurements of single channel currents and numerical simulations. This thesis is organized in two parts, in the first part an overview on ion channels and on the numerical methods adopted throughout the thesis is provided, while the second part describes the research projects tackled in the course of the PhD programme. The aim of the research activity was to relate the functional characteristics of ion channels to their atomic structure. In presenting the different research projects, the role of numerical simulations to analyze the structure-function relation in ion channels is highlighted.

Analysis of ion channel stochastic signals for biosensing

In biological world, life of cells is guaranteed by their ability to sense and to respond to a large variety of internal and external stimuli. In particular, excitable cells, like muscle or nerve cells, produce quick depolarizations in response to electrical, mechanical or chemical stimuli: this means that they can change their internal potential through a quick exchange of ions between cytoplasm and the external environment. This can be done thanks to the presence of ion channels, proteins that span the lipid bilayer and act like switches, allowing ionic current to flow opening and shutting in a stochastic way. For a particular class of ion channels, ligand-gated ion channels, the gating processes is strongly influenced by binding between receptive sites located on the channel surface and specific target molecules. These channels, inserted in biomimetic membranes and in presence of a proper electronic system for acquiring and elaborating the electrical signal, could give us the possibility of detecting and quantifying concentrations of specific molecules in complex mixtures from ionic currents across the membrane; in this thesis work, this possibility is investigated. In particular, it reports a description of experiments focused on the creation and the characterization of artificial lipid membranes, the reconstitution of ion channels and the analysis of their electrical and statistical properties. Moreover, after a chapter about the basis of the modelling of the kinetic behaviour of ligand gated ion channels, a possible approach for the estimation of the target molecule concentration, based on a statistical analysis of the ion channel open probability, is proposed. The fifth chapter contains a description of the kinetic characterisation of a ligand gated ion channel: the homomeric α2 isoform of the glycine receptor. It involved both experimental acquisitions and signal analysis. The last chapter represents the conclusions of this thesis, with some remark on the effective performance that may be achieved using ligand gated ion channels as sensing elements.