A novel HS-SBSE system coupled with gas chromatography and mass spectrometry for the analysis of organochlorine pesticides in water samples

A methodology to analyze organochlorine pesticides (OCPs) in water samples has been accomplished by using headspace stir bar sorptive extraction (HS-SBSE). The bars were in house coated with a thick film of PDMS in order to properly work in the headspace mode. Sampling was done by a novel HS-SBSE system whereas the analysis was performed by capillary GC coupled mass spectrometric detection (HS-SBSE-GC-MS). The extraction optimization, using different experimental parameters has been established by a standard equilibrium time of 120 min at 85 degrees C. A mixture of ACN/toluene as back extraction solvent promoted a good performance to remove the OCPs sorbed in the bar. Reproducibility between 2.1 and 14.8% and linearity between 0.96 and 1.0 were obtained for pesticides spiked in a linear range between 5 and 17 ng/g in water samples during the bar evaluation.

Plasmid DNA partitioning and separation using poly(ethylene glycol)/poly(acrylate)/salt aqueous two-phase systems

Phase diagrams of poly(ethylene glycol)/polyacrylate/Na2SO4 systems have been investigated with respect to polymer size and pH. Plasmid DNA from Escherichia coil can depending on pH and polymer molecular weight be directed to a poly(ethylene glycol) or to a polyacrylate-rich phase in an aqueous two-phase system formed by these polymers. Bovine serum albumin (BSA) and E. coil homogenate proteins can be directed opposite to the plasmid partitioning in these systems. Two bioseparation processes have been developed where in the final step the pDNA is partitioned to a salt-rich phase giving a total process yield of 60-70%. In one of them the pDNA is partitioned between the polyacrylate and PEG-phases in order to remove proteins. In a more simplified process the plasmid is partitioned to a PEG-phase and back-extracted into a Na2SO4-rich phase. The novel polyacrylate/PEG system allows a strong change of the partitioning between the phases with relatively small changes in composition or pH. (C) 2012 Elsevier B.V. All rights reserved.

Photo-Fenton oxidation of phenol and organochlorides (2,4-DCP and 2,4-D) in aqueous alkaline medium with high chloride concentration

A highly concentrated aqueous saline-containing solution of phenol, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2.4-DCP) was treated by the photo-Fenton process in a system composed of an annular reactor with a quartz immersion well and a medium-pressure mercury lamp (450 W). The study was conducted under special conditions to minimize the costs of acidification and neutralization, which are usual steps in this type of process. Photochemical reactions were carried out to investigate the influence of some process variables such as the initial concentration of Fe2+ ([Fe2+](0)) from 1.0 up to 2.5 mM, the rate in mmol of H2O2 fed into the system (F-H2O2,F-in) from 3.67 up to 7.33 mmol of H2O2/min during 120 min of reaction time, and the initial pH (pH(0)) from 3.0 up to 9.0 in the presence and absence of NaCl (60.0 g/L). Although the optimum pH for the photo-Fenton process is about 3.0, this particular system performed well in experimental conditions starting at alkaline and neutral pH. The results obtained here are promising for industrial applications, particularly in view of the high concentration of chloride, a known hydroxyl radical scavenger and the main oxidant present in photo-Fenton processes. (C) 2012 Elsevier Ltd. All rights reserved.

Impact of Aqueous Plant Extracts on Trigona spinipes (Hymenoptera: Apidae)

The stingless bees are an important component of the insect biomass in many tropical areas, due to their collection of nectar and pollen. Trigona spinipes is a widely distributed species in South America, and described as a pollinator of many crops that can be used in a commercial pollinating system. The effects of plant extracts on insects are studied because of the demand for organic food and their selectivity to natural enemies. Plant insecticides are reported as a potential agent for the control of insect pests, however little is known about their impact on beneficial insects. This study investigated the survival of Trigona spinipes (Hymenoptera: Apidae, Meliponini) Fabricius, after exposure to the leaf extracts of Azadiracha indica (Meliaceae), Lippia sidoides (Verbenaceae), Sapindus saponaria (Sapindaceae), Anonna squamosa (Anonnaceae) Cymbopogon winterianum (Poaceae), Corimbia citriodora (Myrtaceae), Jatropha curcas (Euphorbiaceae) and Ricinus communis (Euphorbiaceae) and of seeds of Azadiracha indica, Ricinus communis Nordestina and AL Guarany varieties and Jatropha curcas. The extracts that had the greatest influence on the survival of the bees were A. indica at 3% and 7% of concentration, A. squamosa at a concentration of 10% with 68.89% survival and green leaf of R. communis at a concentration of 7%. The results show that although the extracts were effective in controlling pests, they may also affect the pollinator Trigona spinipes.

Toward the Optimization of an e-Tongue System Using Information Visualization: A Case Study with Perylene Tetracarboxylic Derivative Films in the Sensing Units

The wide variety of molecular architectures used in sensors and biosensors and the large amount of data generated with some principles of detection have motivated the use of computational methods, such as information visualization techniques, not only to handle the data but also to optimize sensing performance. In this study, we combine projection techniques with micro-Raman scattering and atomic force microscopy (AFM) to address critical issues related to practical applications of electronic tongues (e-tongues) based on impedance spectroscopy. Experimentally, we used sensing units made with thin films of a perylene derivative (AzoPTCD acronym), coating Pt interdigitated electrodes, to detect CuCl(2) (Cu(2+)), methylene blue (MB), and saccharose in aqueous solutions, which were selected due to their distinct molecular sizes and ionic character in solution. The AzoPTCD films were deposited from monolayers to 120 nm via Langmuir-Blodgett (LB) and physical vapor deposition (PVD) techniques. Because the main aspects investigated were how the interdigitated electrodes are coated by thin films (architecture on e-tongue) and the film thickness, we decided to employ the same material for all sensing units. The capacitance data were projected into a 2D plot using the force scheme method, from which we could infer that at low analyte concentrations the electrical response of the units was determined by the film thickness. Concentrations at 10 mu M or higher could be distinguished with thinner films tens of nanometers at most-which could withstand the impedance measurements, and without causing significant changes in the Raman signal for the AzoPTCD film-forming molecules. The sensitivity to the analytes appears to be related to adsorption on the film surface, as inferred from Raman spectroscopy data using MB as analyte and from the multidimensional projections. The analysis of the results presented may serve as a new route to select materials and molecular architectures for novel sensors and biosensors, in addition to suggesting ways to unravel the mechanisms behind the high sensitivity obtained in various sensors.

Aqueous dispersions of DMPG in low salt contain leaky vesicles

Aqueous dispersions of dimyristoyl phosphatidylglycerol (DMPG), at low ionic strength, display uncommon thermal behavior. Models for such behavior need to assign a form to the lipid aggregate. Although most studies accept the presence of lipid vesicles in the lipid gel and fluid phases, this is still controversial. With electron spin resonance (ESR) spectra of spin labels incorporated into DMPG aggregates, quantification of [C-14]sucrose entrapped by the aggregates, and viscosity measurements, we demonstrate the existence of leaky vesicles in dispersions of DMPG at low ionic strength, in both gel and fluid phases of the lipid. As a control system, the ubiquitous lipid dimyristoyl phosphatidylcholine (DMPC) was used. For DMPG in the gel phase, spin labeling only indicated the presence of lipid bilayers, strongly suggesting that DMPG molecules are organized as vesicles and not micelles or bilayer fragments (bicelles), as the latter has a non-bilayer structure at the edges. Quantification of [C-14]sucrose entrapping by DMPG aggregates revealed the presence of highly leaky vesicles. Due to the short hydrocarbon chains (C-14 atoms), DMPC vesicles were also found to be partially permeable to sucrose, but not as much as DMPG vesicles. Viscosity measurements, with the calculation of the intrinsic viscosiiy of the lipid aggregate, showed that DMPG vesicles are rather similar in the gel and fluid phases, and quite different from aggregates observed along the gel-fluid transition. Taken together, our data strongly supports that DMPG forms leaky vesicles at both gel and fluid phases. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Photophysical properties and interactions of xanthene dyes in aqueous micelles

Photosensitizers (PS) photodynamic activities are regulated by their location in the biological target, which modulates their photophysical and photochemical features. In this work the PS partition for the Xanthene Dyes Fluorescein (FSC), Eosin Y(EOS), Erythrosin B (ERY) and Rose Bengal B (RBB) in biomimetic models (SDS, CTAB and Pluronic P-123 micelles) and the effects on their photophysical characteristics are evaluated. The hydrophobic and electrostatic forces that govern the PS-micelle interaction are analyzed. At physiological pH (7.25), the ability of the dianionic protolytic form of the dyes to be positioned into the micelle palisade and its micelle interaction depends not only on the hydrophobicity of the dye but also on the micellar surface charge. The Binding Constants obey exactly the same order of the Partition Coefficients for the dyes in P-123 and CTAB micelles. The Stern-Volmer treatment pointed out that dyes are located inside the micelle, especially ERY and RBB. The magnitude of the dye-micelle interaction increased from SDS, P-123 and finally CTAB micelles due to the charges between dye and micelle, and among the xanthenes, their hydrophobic characteristics. Within the micelle pseudo phase, ERY and RBB are still very efficient photosensitizers exhibiting high quantum yield of singlet oxygen, which turns them very attractive especially with P-123 polymeric system as drug delivery systems in photodynamic therapy. (C) 2012 Elsevier B.V. All rights reserved.

Thermodynamics of aqueous biopolymer solutions and fractionation of Dextran

Flory-Huggins interaction parameters and thermal diffusion coefficients were measured for aqueous biopolymer solutions. Dextran (a water soluble polysaccharide) and bovine serum albumin (BSA, a water soluble protein) were used for this study. The former polymer is representative for chain macromolecules and the latter is for globular macromolecules. The interaction parameters for the systems water/dextran and water/BSA were determined as a function of composition by means of vapor pressure measurements, using a combination of headspace sampling and gas chromatography (HS-GC). A new theoretical approach, accounting for chain connectivity and conformational variability, describes the observed dependencies quantitatively for the system water/dextran and qualitatively for the system water/BSA. The phase diagrams of the ternary systems water/methanol/dextran and water/dextran/BSA were determined via cloud point measurements and modeled by means of the direct minimization of the Gibbs energy using the information on the binary subsystems as input parameters. The thermal diffusion of dextran was studied for aqueous solutions in the temperature range 15 < T < 55 oC. The effects of the addition of urea were also studied. In the absence of urea, the Soret coefficient ST changes its sign as T is varied; it is positive for T > 45.0 oC, but negative for T < 45.0 oC. The positive sign of ST means that the dextran molecules migrate towards the cold side of the fluid; this behavior is typical for polymer solutions. While a negative sign indicates the macromolecules move toward the hot side; this behavior has so far not been observed with any other binary aqueous polymer solutions. The addition of urea to the aqueous solution of dextran increases ST and reduces the inversion temperature. For 2 M urea, the change in the sign of ST is observed at T = 29.7 oC. At higher temperature ST is always positive in the studied temperature range. To rationalize these observations it is assumed that the addition of urea opens hydrogen bonds, similar to that induced by an increase in temperature. For a future extension of the thermodynamic studies to the effects of poly-dispersity, dextran was fractionated by means of a recently developed technique called Continuous Spin Fractionation (CSF). The solvent/precipitant/polymer system used for the thermodynamic studies served as the basis for the fractionation of dextran The starting polymer had a weight average molar mass Mw = 11.1 kg/mol and a molecular non-uniformity U= Mw / Mn -1= 1.0. Seventy grams of dextran were fractionated using water as the solvent and methanol as the precipitant. Five fractionation steps yielded four samples with Mw values between 4.36 and 18.2 kg/mol and U values ranging from 0.28 to 0.48.

Oxidative properties of the Iron - Thymine-1-acetic acid - Hydrogen peroxide catalytic system

The oxidation of alcohols and olefins is a pivotal reaction in organic synthesis. However, traditional oxidants are toxic and they often release a considerable amounts of by-products. Here, two IronIII-based systems are shown as oxidative catalyst, working in mild conditions with hydrogen peroxide as primary oxidant. An efficient catalytic system for the selective oxidation of several alcohols to their corresponding aldehydes and ketones was developed and characterized, [Fe(phen)2Cl2]NO3 (phen=1,10-Phenantroline). It was demonstrated that the adoption of a buffered aqueous solution is of crucial importance to ensure both considerable activity and selectivity.The Iron - Thymine-1-acetic acid in-situ complex was studied as catalyst in alcohol oxidations and C-H oxidative functionalization, involving hydrogen peroxide as primary oxidant in mild reaction conditions. The catalytic ability in alcohol oxidations was investigated by Density Functional Theory calculations, however the catalyst still has uncertain structure. The system shows satisfactory activity in alcohol oxidation and aliphatic rings functionalization. The Fe-THA system was studied in cyclohexene oxidation and oxidative halogenations. Halide salts such as NBu4X and NH4X were introduced in the catalytic system as halogens source to obtain cyclohexene derivatives such as halohydrins, important synthetic intermediates.The purpose of this dissertation is to contribute in testing new catalytic systems for alcohol oxidations and C-H functionalization. In particular, most of the efforts in this work focus on studying the Iron - Thymine-1-acetic acid (THA) systems as non-heme oxidative model, which present: •an iron metal centre(s) as a coordinative active site, •hydrogen peroxide as a primary oxidant, •THA as an eco-friendly, biocompatible, low cost coordinating ligand.

Cannabinoid CB1 receptor in the regulation of sociability, stress coping, and its interaction with the serotonergic system

Cannabinerge Substanzen können das Verhalten in einer dosisabhängigen, aber biphasischen Weise beeinflussen. Eine Erklärung für diese Art der Effekte könnte die Verteilung des CB1 Rezeptors auf verschiedenen Neuronentypen sein. CB1 Rezeptoren in glutamatergen und GABAergen Neuronen sind hier besonders wichtig, da die entsprechenden Neurotransmitter als Gegenspieler die neuronale Erregung kontrollieren. Spezifische Deletion des CB1 Rezeptor-Gens von einer der beiden Populationen führte zu gegensätzlichen Phenotypen, genauer gesagt, einem erniedrigten, bzw. einem gesteigerten Interaktiondrang. Tiere, bei denen der CB1 Rezeptor ausschließlich in striatalen, GABAergen „Medium Spiny“ Neuronen deletiert wurde, zeigten keinen veränderten Phänotyp. Dies legt nahe, dass der CB1 Rezeptor in kortikalen glutamatergen und GABAergen Neuronen für einen ausgeglichenen Interaktionsdrang entscheidend ist (siehe Kapitel 3).rnDiese dosisabhängigen, biphasischen Effekte auf das Verhalten können auch im „Forced Swim Test“ (FST) beobachtet werden. Ein möglicher Mechanismus, durch den Cannabinoide das Stressverhalten beeinflussen können, wäre die Regulierung der Monoaminausschüttung. Um die Abhängigkeit der Cannabinoideffekte von der Serotonintransmission zu untersuchen, wurden Dosen von CB1 Rezeptoragonisten und –antagonisten mit antidepressiv-induzierenden Eigenschaften bei gleichzeitiger Inhibition der Serotonintransmission im FST getestet. Die Ergebnisse zeigten, dass lediglich der Agonisteffekt durch die Inhibition der Serotoninauschüttung beeinflusst wird. Zusätzlich konnte die Abhängigkeit des Antagonisteneffekts von funktionsfähigen GABAergen CB1 Rezeptoren nachweisen werden. Interessanter Weise konnte der durch die Deletion von glutamatergen CB1 Rezeptoren induzierte Phänotyp durch Inhibition der Serotoninausschüttung blockiert werden (siehe Kapitel 4).rnEin indirekter Einfluss auf Serotoninausschüttung scheint also wahrscheinlich zu sein. Bis jetzt blieb jedoch unklar, inwieweit cannabinerge Substanzen direkt auf serotonerge Neuronen wirken können. Im Jahr 2007 konnte unsere Gruppe die Expression des CB1 Rezeptors in serotonergen Neuronen auf mRNA- und Proteinebene nachweisen. Die Züchtung und Analyse einer mutanten Mauslinie, in welcher der CB1-Rezeptor spezifisch in serotonergen Neuronen ausgeschaltet wurde, zeigte bei männlichen Tieren eine schwache, aber signifikante Verhaltensänderungen, die durch soziale Stimuli und lebensbedrohlichen Situationen ausgelöst wurde. So ist es erstmals gelungen nachzuweisen, dass serotonerge CB1-Rezeptoren eine physiologische Relevanz besitzen (siehe Kapitel 5).rn

Non-aqueous dispersions of particles and their applications

In the current work, three studies about non-aqueous dispersions of particles were carried out by using an amphiphilic block copolymer poly(isoprene)-block-poly(methyl methacrylate) (PI-b-PMMA) as stabilizer:rn1. Dispersions of polyurethane and polyurea porous particles for polymer compositesrn2. Dispersions of PMMA and PU particles with PDI dye for study of Single Molecule Spectroscopy Detectionrn3. Dispersions of graphene nanosheets for polymer compositesrnrnIn the first study, highly porous polyurethane and polyurea particles were prepared in a non-aqueous emulsion. The preparation of porous particles consisted of two parts: At first, a system was developed where the emulsion had high stability for the polymerization among diisocyanate, diol and water. In the second part, porous particles were prepared by using two methods fission/fusion and combination by which highly porous particles were obtained. In this study, the applications of porous particles were also investigated where polyurethane particles were tested as filling material for polymer composites and as catalyst carrier for polyethylene polymerization. rnrnIn the second study, PMMA and PU particles from one non-aqueous emulsion were investigated via single molecule fluorescence detection. At first the particles were loaded with PDI dye, which were detected by fluorescence microscopy. The distribution and orientation of the PDI molecules in the particles were successfully observed by Single Molecule Fluorescence Detection. The molecules were homogenously distributed inside of the particles. In addition they had random orientation, meaning that no aggregations of dye molecules were formed. With the results, it could be supposed that the polymer chains were also homogenously distributed in the particles, and that the conformation was relatively flexible. rnrnIn the third part of the study, graphene nanosheets with high surface area were dispersed in an organic solvent with low boiling point and low toxicity, THF, stabilized with a block copolymer PI-b-PMMA. The dispersion was used to prepare polymer composites. It was shown that the modified graphene nanosheets had good compatibility with the PS and PMMA matrices. rn

Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system

PURPOSE: To evaluate the expression and presence of surfactant protein (SP) A and SP-D in the lacrimal apparatus, at the ocular surface, and in tears in healthy and pathologic states. METHODS: Expression of mRNA for SP-A and SP-D was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts as well as in a spontaneously immortalized conjunctival epithelial cell line (HCjE; IOBA-NHC) and a SV40-transfected cornea epithelial cell line (HCE). Deposition of SP-A and SP-D was determined by Western blot, dot blot, and immunohistochemistry in healthy tissues, in tears, aqueous humor, and in sections of different corneal abnormalities (keratoconus, herpetic keratitis, and Staphylococcus aureus-based ulceration). Cell lines were stimulated with different cytokines and bacterial components and were analyzed for the production of SP-A and SP-D by immunohistochemistry. RESULTS: The presence of SP-A and SP-D on mRNA and protein levels was evidenced in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal duct samples. Moreover, both proteins were present in tears but were absent in aqueous humor. Immunohistochemistry revealed the production of both peptides by acinar epithelial cells of the lacrimal gland and epithelial cells of the conjunctiva and nasolacrimal ducts, whereas goblet cells revealed no reactivity. Healthy cornea revealed weak reactivity on epithelial surface cells only. In contrast, SP-A and SP-D revealed strong reactivity in patients with herpetic keratitis and corneal ulceration surrounding lesions and in several immigrated defense cells. Reactivity in corneal epithelium and endothelium was also seen in patients with keratoconus. Cell culture experiments revealed that SP-A and SP-D are produced by both epithelial cell lines without and after stimulation with cytokines and bacterial components. CONCLUSIONS: These results show that SP-A, in addition to SP-D, is a peptide of the tear film. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D seem to be involved in several ocular surface diseases.

Quantification of anandamide and 2-arachidonoylglycerol plasma levels to examine potential influences of tetrahydrocannabinol application on the endocannabinoid system in humans

The effects of tetrahydrocannabinol (THC) and endogenous cannabinoids (endocannabinoids, ECs) are both mediated by activation of the cannabinoid receptors CB1 and CB2. Exogenous activation of these receptors by THC could therefore alter EC levels. We tested this hypothesis in healthy volunteers (n = 25) who received a large intravenous dose of THC (0.10 mg/kg). Effects on the EC system were quantified by serial measurements of plasma ECs after THC administration. Eleven blood samples were drawn during the first 5 h after THC administration and two more samples after 24 and 48 h. THC, its metabolites THC-OH (biologically active) and THC-COOH (non-active), and the ECs anandamide and 2-arachidonoylglycerol (2-AG) were quantified by liquid chromatography-mass spectrometry. EC-plasma levels showed a biphasic response after THC injection reaching maximal values at 30 min. Anandamide increased slightly from 0.58 ± 0.21 ng/ml at baseline to 0.64 ± 0.24 ng/ml (p < 0.05) and 2-AG from 7.60 ± 4.30 ng/ml to 9.50 ± 5.90 ng/ml (p < 0.05). After reaching maximal concentrations, EC plasma levels decreased markedly to a nadir of 300 min after THC administration (to 0.32 ± 0.15 ng/ml for anandamide and to 5.50 ± 3.01 ng/ml for 2-AG, p < 0.05). EC plasma concentrations returned to near baseline levels until 48 h after the experiment. THC (0.76 ± 0.16 ng/ml) and THC-OH (0.36 ± 0.17 ng/ml) were still measurable at 24 h and remained detectible until 48 h after THC administration. Although the underlying mechanism is not clear, high doses of intravenous THC appear to influence endogenous cannabinoid concentrations and presumably EC-signalling.

Diffusible, nonfibrillar ligands derived from Aβ1–42 are potent central nervous system neurotoxins

Aβ1–42 is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer’s pathogenesis. Neurotoxicity of amyloid β protein (Aβ) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible Aβ oligomers (referred to as ADDLs, for Aβ-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer’s disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer’s disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of Aβ-derived diffusible ligands acting upon particular neural signal transduction pathways.

Direct measurement of salt-bridge solvation energies using a peptide model system: implications for protein stability.

The solvation energies of salt bridges formed between the terminal carboxyl of the host pentapeptide AcWL- X-LL and the side chains of Arg or Lys in the guest (X) position have been measured. The energies were derived from octanol-to-buffer transfer free energies determined between pH 1 and pH 9. 13C NMR measurements show that the salt bridges form in the octanol phase, but not in the buffer phase, when the side chains and the terminal carboxyl group are charged. The free energy of salt-bridge formation in octanol is approximately -4 kcal/mol (1 cal = 4.184 J), which is equal to or slightly larger than the sum of the solvation energies of noninteracting pairs of charged side chains. This is about one-half the free energy that would result from replacing a charge pair in octanol with a pair of hydrophobic residues of moderate size. Therefore, salt bridging in octanol can change the favorable aqueous solvation energy of a pair of oppositely charged residues to neutral or slightly unfavorable but cannot provide the same free energy decrease as hydrophobic residues. This is consistent with recent computational and experimental studies of protein stability.