Use of Saccharomyces cerevisiae immobilized in agarose gel as a binding agent for diffusive gradients in thin films

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

Trocas gasosas, índices fisiológicos e acúmulo iônico em plantas jovens de Annona emarginata (Schltdl.) H. Rainer em solução nutritiva

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Desenvolvimento de plantas jovens de Annona emarginata (SHLTDL.) H. Rainer (Araticum-de-terra-fria) cultivadas e solução nutritiva

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Water–montmorillonite systems: Neutron scattering and tracer throughdiffusion studies

Designs for deep geological respositories of nuclear waste include bentonite as a hydraulic and chemisorption buffer material to protect the biosphere from leakage of radionuclides. Bentonite is chosen because it is a cheap, naturally occurring material with the required properties. It consists essentially of montmorillonite, a swelling clay mineral. Upon contact with groundwater such clays can seal the repository by incorporating water in the interlayers of their crystalline structure. The intercalated water exhibits significantly different properties to bulk water in the surrounding interparticle pores, such as lower diffusion coefficients (González Sánchez et. al. 2008). This doctoral thesis presents water distribution and diffusion behavior on various time and space scales in montmorillonite. Experimental results are presented for Na- and Cs-montmorillonite samples with a range of bulk dry densities (0.8 to 1.7 g/cm3). The experimental methods employed were neutron scattering (backscattering, diffraction, time-of-flight), adsorption measurements (water, nitrogen) and tracer-through diffusion. For the tracer experiments the samples were fully saturated via the liquid phase under volume-constrained conditions. In contrast, for the neutron scattering experiments, the samples were hydrated via the vapor phase and subsequently compacted, leaving a significant fraction of interparticle pores unfilled with water. Owing to these differences in saturation, the water contents of the samples for neutron scattering were characterized by gravimetry whereas those for the tracer experiments were obtained from the bulk dry density. The amount of surface water in interlayer pores could be successfully discriminated from the amount of bulk-like water in interparticle pores in Na- and Csmontmorillonite using neutron spectroscopy. For the first time in the literature, the distribution of water between these two pore environments was deciphered as a function of gravimetric water content. The amount was compared to a geometrical estimation of the amount of interlayer and interparticle water determined by neutron diffraction and adsorption measurements. The relative abundances of the 1 to 4 molecular water layers in the interlayer were determined from the area ratios of the (001)-diffraction peaks. Depending on the characterization method, different fractions of surface water and interlayer water were obtained. Only surface and interlayer water exists in amontmorillonite with water contents up to 0.18 g/g according to spectroscopic measurements and up to 0.32 g/g according to geometrical estimations, respectively. At higher water contents, bulk-like and interparticle water also exists. The amounts increase monotonically, but not linearly, from zero to 0.33 g/g for bulk-like water and to 0.43 g/g for interparticle water. It was found that water most likely redistributes between the surface and interlayer sites during the spectroscopic measurements and therefore the reported fraction is relevant only below about -10 ºC (Anderson, 1967). The redistribution effect can explain the discrepancy in fractions between the methods. In a novel approach the fractions of water in different pore environments were treated as a fixed parameter to derive local diffusion coefficients for water from quasielastic neutron scattering data, in particular for samples with high water contents. Local diffusion coefficients were obtained for the 1 to 4 molecular water layers in the interlayer of 0.5·10–9, 0.9·10–9, 1.5·10–9 and 1.4·10–9 m²/s, respectively, taking account of the different water fractions (molecular water layer, bulk-like water). The diffusive transport of 22Na and HTO through Na-montmorillonite was measured on the laboratory experimental scale (i.e. cm, days) by tracer through-diffusion experiments. We confirmed that diffusion of HTO is independent of the ionic strength of the external solution in contact with the clay sample but dependent on the bulk dry density. In contrast, the diffusion of 22Na was found to depend on both the ionic strength of the pore solution and on the bulk dry density. The ratio of the pore and surface diffusion could be experimentally determined for 22Na from the dependence of the diffusion coefficient on the ionic strength. Activation energies were derived from the temperaturedependent diffusion coefficients via the Arrhenius relation. In samples with high bulk dry density the activation energies are slightly higher than those of bulk water whereas in low density samples they are lower. The activation energies as a function of ionic strengths of the pore solutions are similar for 22Na and HTO. The facts that (i) the slope of the logarithmic effective diffusion coefficients as a function of the logarithmic ionic strength is less than unity for low bulk dry densities and (ii) two water populations can be observed for high gravimetric water contents (low bulk dry densities) support the interlayer and interparticle porosity model proposed by Glaus et al. (2007), Bourg et al. (2006, 2007) and Gimmi and Kosakowski (2011).

Experimental Characterization and Quantification of Cement-Bentonite Interaction using Core Infiltration Techniques Coupled with X-ray Tomography

Deep geological storage of radioactive waste foresees cementitious materials as reinforcement of tunnels and as backfill. Bentonite is proposed to enclose spent fuel canisters and as drift seals. Sand/bentonite (s/b) is foreseen as backfill material of access galleries or as drift seals. The emplacement of cementitious material next to clay material generates an enormous chemical gradient in pore-water composition that drives diffusive solute transport. Laboratory studies and reactive transport modeling predicted significant mineral alteration at and near interfaces, mainly resulting in a decrease of porosity in bentonite. The goal of this thesis was to characterize and quantify the cement/bentonite interactions both spatially and temporally in laboratory experiments. A newly developed mobile X-ray transparent core infiltration device was used to perform X-ray computed tomography (CT) scans without interruption of running experiments. CT scans allowed tracking the evolution of the reaction plume and changes in core volume/diameter/density during the experiments. In total 4 core infiltration experiments were carried out for this study with the compacted and saturated cores consisting of MX-80 bentonite and sand/MX-80 bentonite mixture (s/b; 65/35%). Two different high-pH cementitious pore-fluids were infiltrated: a young (early) ordinary Portland cement pore-fluid (APWOPC; K+–Na+–OH-; pH 13.4; ionic strength 0.28 mol/kg) and a young ‘low-pH’ ESDRED shotcrete pore-fluid (APWESDRED; Ca2+–Na+–K+–formate; pH 11.4; ionic strength 0.11 mol/kg). The experiments lasted between 1 and 2 years. In both bentonite experiments, the hydraulic conductivity was strongly reduced after switching to high-pH fluids, changing eventually from an advective to a diffusion-dominated transport regime. The reduction was mainly induced by mineral precipitation and possibly partly also by high ionic strength pore-fluids. Both bentonite cores showed a volume reduction and a resulting transient flow in which pore-water was squeezed out during high-pH infiltration. The outflow chemistry was characterized by a high ionic strength, while chloride in the initial pore water got replaced as main anionic charge carrier by sulfate, originating from gypsum dissolution. The chemistry of the high-pH fluids got strongly buffered by the bentonite, consuming hydroxide and in case of APWESDRED also formate. Hydroxide got consumed by mineral reactions (saponite and possibly talc and brucite precipitation), while formate being affected by bacterial degradation. Post-mortem analysis showed reaction zones near the inlet of the bentonite core, characterized by calcium and magnesium enrichment, consisting predominately of calcite and saponite, respectively. Silica got enriched in the outflow, indicating dissolution of silicate-minerals, identified as preferentially cristobalite. In s/b, infiltration of APWOPC reduced the hydraulic conductivity strongly, while APWESDRED infiltration had no effect. The reduction was mainly induced by mineral precipitation and probably partly also by high ionic strength pore-fluids. Not clear is why the observed mineral precipitates in the APWESDRED experiment had no effect on the fluid flow. Both s/b cores showed a volume expansion along with decreasing ionic strengths of the outflow, due to mineral reactions or in case of APWESDRED infiltration also mediated by microbiological activity, consuming hydroxide and formate, respectively. The chemistry of the high-pH fluids got strongly buffered by the s/b. In the case of APWESDRED infiltration, formate reached the outflow only for a short time, followed by enrichment in acetate, indicating most likely biological activity. This was in agreement to post-mortem analysis of the core, observing black spots on the inflow surface, while the sample had a rotten-egg smell indicative of some sulfate reduction. Post-mortem analysis showed further in both cores a Ca-enrichment in the first 10 mm of the core due to calcite precipitation. Mg-enrichment was only observed in the APWOPC experiment, originating from newly formed saponite. Silica got enriched in the outflow of both experiments, indicating dissolution of silicate-minerals, identified in the OPC experiment as cristobalite. The experiments attested an effective buffering capacity for bentonite and s/b, a progressing coupled hydraulic-chemical sealing process and also the preservation of the physical integrity of the interface region in this setup with a total pressure boundary condition on the core sample. No complete pore-clogging was observed but the hydraulic conductivity got rather strongly reduced in 3 experiments, explained by clogging of the intergranular porosity (macroporosity). Such a drop in hydraulic conductivity may impact the saturation time of the buffer in a nuclear waste repository, although the processes and geometry will be more complex in repository situation.

Brownian flocculation of polymer colloids in the presence of a secondary minimum

Most analyses of Brownian flocculation apply to conditions where London–van der Waals attractive forces cause particles to be strongly bound in a deep interparticle potential well. In this paper, results are reported that show the interaction between primary- and secondary-minimum flocculation when the interparticle potential curve reflects both attractive and electrostatic repulsive forces. The process is highly time-dependent because of transfer of particles from secondary- to primary-minimum flocculation. Essential features of the analysis are corroborated by experiments with 0.80-μm polystyrene spheres suspended in aqueous solutions of NaCl over a range of ionic strengths. In all cases, experiments were restricted to the initial stage of coagulation, where singlets and doublets predominate.

Chromatin structure mapping in Saccharomyces cerevisiae in vivo with DNase I

Most methods for assessment of chromatin structure involve chemical or nuclease damage to DNA followed by analysis of distribution and susceptibility of cutting sites. The agents used generally do not permeate cells, making nuclear isolation mandatory. In vivo mapping strategies might allow detection of labile constituents and/or structures that are lost when chromatin is swollen in isolated nuclei at low ionic strengths. DNase I has been the most widely used enzyme to detect chromatin sites where DNA is active in transcription, replication or recombination. We have introduced the bovine DNase I gene into yeast under control of a galactose-responsive promoter. Expression of the nuclease leads to DNA degradation and cell death. Shorter exposure to the active enzyme allows mapping of chromatin structure in whole cells without isolation of nuclei. The validity and efficacy of the strategy are demonstrated by footprinting a labile repressor bound to its operator. Investigation of the inter-nucleosome linker regions in several types of repressed domains has revealed different degrees of protection in cells, relative to isolated nuclei.

Rapid preparation of giant unilamellar vesicles.

We report here a rapid evaporation method that produces in high yield giant unilamellar vesicles up to 50 microns in diameter. The vesicles are obtained after only 2 min and can be prepared from different phospholipids, including L-alpha-phosphatidylcholine (lecithin), dipalmitoleoyl L-alpha-phosphatidylcholine, and beta-arachidonoyl gamma-palmitoyl L-alpha-phosphatidylcholine. Vesicles can be produced in distilled water and in Hepes, phosphate, and borate buffers in the pH range of 7.0 to 11.5 with ionic strengths up to 50 mM. The short preparation time allows encapsulation of labile molecular targets or enzymes with high catalytic activities. Cell-sized proteoliposomes have been prepared in which gamma-glutamyltransferase (EC 2.3.2.2) was functionally incorporated into the membrane wall.

Binding studies of cationic thymidyl deoxyribonucleic guanidine to RNA homopolynucleotides.

Deoxyribonucleic guanidine is a potential antisense agent that is generated via the replacement of the negative phosphodiester linkages of DNA [--O--(PO2-)--O--] with positively-charged guanidinium (g) linkages [--NH--C(==NH2+)--NH--]. A pentameric thymidyl deoxyribonucleic guanidine molecule [d(Tg)4T-azido] has been shown to base pair specifically to poly(rA) with an unprecedented affinity. Both double and triple strands consisting of one and two equivalents of d(Tg)4T-azido paired with one equivalent of poly(rA) are indicated by thermal denaturation experiments. At an ionic strength of 0.22, the five bases of d(Tg)4T-azido are estimated to dissociate from a double helix with poly(rA) at > 100 degrees C! The effect of ionic strength on thermal denaturation is very pronounced, with stability greatest at low ionic strengths. The method of continuous variation indicates that there is an equilibrium complex with a molar ratio of d(Tg) to r(Ap) or d(Ap) of 2:1. Based on this evidence, models of the structures of d(Tg)9T-azido bound to r(Ap)9A are proposed.

Measurement of exchangeable cations in saline soils

The measurement of exchangeable cations in saline soils is limited by the difficulty in accurately separating soluble cations from exchangeable cations. A method is examined for saline soils in which exchangeable cations are calculated as the total extractable cations minus the concentration of soil solution (soluble) cations. In addition, a further two standard methods were investigated, one which assumes the total soil extractable cations are exchangeable, the other utilises a pretreatment to remove soluble salts prior to measurement of the remaining (exchangeable) cations. After equilibration with a range of sodium adsorption ratio (SAR) solutions at various ionic strengths, the exchangeable cation concentrations of two soils (Dermosol and Vertosol) were determined by these methods and compared to known values. The assumption that exchangeable cations can be estimated as the total soil extractable cations, although valid at low ionic strength, resulted in an overestimation of exchangeable Na and Ca concentrations at higher ionic strengths due to the presence of soluble salts. Pretreatment with ethanol and glycerol was found to effectively remove soluble salts thus allowing the accurate measurement of the effective cation exchange capacity (ECEC), however, dilution associated with the pretreatment process decreased concentrations of exchangeable Ca while simultaneously increasing exchangeable Na. Using the proposed method, good correlations were found between known and measured concentrations of exchangeable Na (Dermosol: y=0.873x and Vertosol: y=0.960x) and Ca (Dermosol: y=0.906x, and Vertosol: y=1.05x). Therefore, for soils with an ionic strength of approximately 50 mM (ECse 4 dS m-1) or greater (in which exchangeable cation concentrations are overestimated by assuming the total soil cations are exchangeable), concentrations can be calculated as difference between total extractable cations and soluble cations.

Extruded colour-based plastic film for the measurement of dissolved CO2

Dissolved CO2 measurements are usually made using a Severinghaus electrode, which is bulky and can suffer from electrical interference. In contrast, optical sensors for gaseous CO2, whilst not suffering these problems, are mainly used for making gaseous (not dissolved) CO2 measurements, due to dye leaching and protonation, especially at high ionic strengths (>0.01 M) and acidity (<pH 4). This is usually prevented by coating the sensor with a gas-permeable, but ion-impermeable, membrane (GPM). Herein, we introduce a highly sensitive, colourimetric-based, plastic film sensor for the measurement of both gaseous and dissolved CO2, in which a pH-sensitive dye, thymol blue (TB) is coated onto particles of hydrophilic silica to create a CO2-sensitive, TB-based pigment, which is then extruded into low density polyethylene (LDPE) to create a GPM-free, i.e. naked, TB plastic sensor film for gaseous and dissolved CO2 measurements. When used for making dissolved CO2 measurements, the hydrophobic nature of the LDPE renders the film: (i) indifferent to ionic strength, (ii) highly resistant to acid attack and (iii) stable when stored under ambient (dark) conditions for >8 months, with no loss of colour or function. Here, the performance of the TB plastic film is primarily assessed as a dissolved CO2 sensor in highly saline (3.5 wt%) water. The TB film is blue in the absence of CO2 and yellow in its presence, exhibiting 50% transition in its colour at ca. 0.18% CO2. This new type of CO2 sensor has great potential in the monitoring of CO2 levels in the hydrosphere, as well as elsewhere, e.g. food packaging and possibly patient monitoring.

Dynamics and Heterogeneity of Pb(II) Binding by SiO2 Nanoparticles in an Aqueous Dispersion

Pb(II) binding by SiO2 nanoparticles in an aqueous dispersion was investigated under conditions where the concentrations of Pb2+ ions and nanoparticles are of similar magnitude. Conditional stability constants (log K) obtained at different values of pH and ionic strength varied from 4.4 at pH 5.5 and I = 0.1 M to 6.4 at pH 6.5 and I = 0.0015 M. In the range of metal to nanoparticle ratios from 1.6 to 0.3, log K strongly increases, which is shown to be due to heterogeneity in Pb(II) binding. For an ionic strength of 0.1 M the Pb2+/SiO2 nanoparticle system is labile, whereas for lower ionic strengths there is loss of lability with increasing pH and decreasing ionic strength. Theoretical calculations on the basis of Eigen-type complex formation kinetics seem to support the loss of lability. This is related to the nanoparticulate nature of the system, where complexation rate constants become increasingly diffusion controlled. The ion binding heterogeneity and chemodynamics of oxidic nanoparticles clearly need further detailed research.

Chemical Immobilization Of Crosslinked Polymeric Ionic Liquids On Nitinol Wires Produces Highly Robust Sorbent Coatings For Solid-phase Microextraction.

Super elastic nitinol (NiTi) wires were exploited as highly robust supports for three distinct crosslinked polymeric ionic liquid (PIL)-based coatings in solid-phase microextraction (SPME). The oxidation of NiTi wires in a boiling (30%w/w) H2O2 solution and subsequent derivatization in vinyltrimethoxysilane (VTMS) allowed for vinyl moieties to be appended to the surface of the support. UV-initiated on-fiber copolymerization of the vinyl-substituted NiTi support with monocationic ionic liquid (IL) monomers and dicationic IL crosslinkers produced a crosslinked PIL-based network that was covalently attached to the NiTi wire. This alteration alleviated receding of the coating from the support, which was observed for an analogous crosslinked PIL applied on unmodified NiTi wires. A series of demanding extraction conditions, including extreme pH, pre-exposure to pure organic solvents, and high temperatures, were applied to investigate the versatility and robustness of the fibers. Acceptable precision of the model analytes was obtained for all fibers under these conditions. Method validation by examining the relative recovery of a homologous group of phthalate esters (PAEs) was performed in drip-brewed coffee (maintained at 60 °C) by direct immersion SPME. Acceptable recoveries were obtained for most PAEs in the part-per-billion level, even in this exceedingly harsh and complex matrix.

Development Of Egg Pc/cholesterol/α-tocopherol Liposomes With Ionic Gradients To Deliver Ropivacaine.

Ropivacaine (RVC) is an aminoamide local anesthetic widely used in surgical procedures. Studies with RVC encapsulated in liposomes and complexed in cyclodextrins have shown good results, but in order to use RVC for lengthy procedures and during the postoperative period, a still more prolonged anesthetic effect is required. This study therefore aimed to provide extended RVC release and increased upload using modified liposomes. Three types of vesicles were studied: (i) large multilamellar vesicle (LMV), (ii) large multivesicular vesicle (LMVV) and (iii) large unilamellar vesicle (LUV), prepared with egg phosphatidylcholine/cholesterol/α-tocopherol (4:3:0.07 mol%) at pH 7.4. Ionic gradient liposomes (inside: pH 5.5, pH 5.5 + (NH4)2SO4 and pH 7.4 + (NH4)2SO4) were prepared and showed improved RVC loading, compared to conventional liposomes (inside: pH 7.4). An high-performance liquid chromatography analytical method was validated for RVC quantification. The liposomes were characterized in terms of their size, zeta potential, polydispersion, morphology, RVC encapsulation efficiency (EE(%)) and in vitro RVC release. LMVV liposomes provided better performance than LMV or LUV. The best formulations were prepared using pH 5.5 (LMVV 5.5in) or pH 7.4 with 250 mM (NH4)2SO4 in the inner aqueous core (LMVV 7.4in + ammonium sulfate), enabling encapsulation of as much as 2% RVC, with high uptake (EE(%) ∼70%) and sustained release (∼25 h). The encapsulation of RVC in ionic gradient liposomes significantly extended the duration of release of the anesthetic, showing that this strategy could be a viable means of promoting longer-term anesthesia during surgical procedures and during the postoperative period.