Effect of knockdown of Giα proteins using antisense oligodeoxynucleotides encapsulated in cationic liposomes on the development of hypertension in spontaneously hypertensive rats

L'hypertension artérielle est l'une des principales causes de morbidité et de mortalité dans le monde. La compréhension des mécanismes qui sont à la base du développement de l'hypertension offrira de nouvelles perspectives pour un meilleur contrôle de l'hypertension. Nous avons précédemment montré que le niveau des protéines Giα-2 et Giα-3 est augmenté chez les rats spontanément hypertendus (SHR) avant l'apparition de l'hypertension. Le traitement avec les inhibiteurs de l’enzyme de conversion de l’Angiotensine (IEC) est associé à une diminution de l’expression des protéines Gi. De plus, l'injection intrapertoneale de la toxine de la coqueluche inactive les deux protéines Giα et empêche le développement de l'hypertension chez les SHR. Cependant, la contribution spécifique des protéines Giα-2 et Giα-3 dans le développement de l'hypertension n'est pas encore connue. Dans la présente étude, l’Anti-sens oligodésoxynucléotide (AS-ODN) de Giα-2 et Giα-3 (1mg/Kg en poids corporel) encapsulé dans des liposomes cationiques PEG / DOTAP/ DOPE ont été administrés par voie intraveineuse aux SHR pré-hypertendus âgé de trois semaines et aux Wistar Kyoto (WKY) rats de même âge. Les contrôles des WKY et SHR non traités ont été injectés avec du PBS stérile, liposomes vides ou oligomères sens. La pression artérielle (PA) a été suivie chaque semaine en utilisant la technique manchon caudal. Les rats ont été sacrifiés à l'âge de six semaines et neuf semaines. Le coeur et l'aorte ont été utilisés pour étudier l'expression des protéines Gi. Le knockdown des protéines Giα-2 par l’injection de Giα-2-AS a empêché le développement de l'hypertension à l'âge de six semaines. Par la suite, la PA a commencé à augmenter rapidement et a atteint le niveau que l'on retrouve dans les groupes témoins à l'âge de neuf semaines. D'autre part, la PA du groupe traité avec le Giα-3-AS a commencé à augmenter à l'âge de quatre semaines. Dans le groupe des SHR-Giα-3-AS, la PA a augmenté à l’âgé de six semaines, mais moins que celle de SHR-CTL. Le coeur et l'aorte obtenues des SHR Giα-2-AS et Giα-3-AS à partir de l’âgé de six semaines ont eu une diminution significative de l’expression des protéines Giα-2 et Giα-3 respectivement. Dans le groupe des WKY Giα-2-AS et Giα-3-AS l'expression des protéines Giα-2 et Giα-3 respectivement a diminué malgré l'absence de changement dans la PA par rapport aux WKY CTL. À l'âge de neuf semaines, les SHR traités avec du Giα-2-AS et Giα-3-AS avaient la même PA et expression des protéines Gi que le SHR CTL. Ces résultats suggèrent que les deux protéines Giα-2 et Giα-3 sont impliqués dans le développement de l'hypertension chez les SHR, mais le knockdown de Giα-2 et pas de Giα-3 a empêché le développement de l'hypertension.

Biochemical Effects of Anionic, Cationic and Non Ionic Surfactants on a Tropical Teleost Oreochromis Mossambicus(Peters) and a Marine Cyanobacterium Synechocystis Salina Wislouch

The present work is a base line attempt to investigate and assess the toxicities of three surfactants viz. anionic sodium dodecyl sulfate (SDS), non ionic Triton X-1OO (TX-IOO) and cationic cetyl trimethyl ammonium bromide (CTAB). These compounds represent simple members of the often neglected group of aquatic pollutants i.e. the anionic alkyl sulfates, non ionics and the cationics. These compounds are widely used In plastic industry, pesticide/herbicide formulations, detergents, oil spill dispersants, molluscicides etc. The test organisms selected for the present study are the cyanobacterium Synechocystis salina Wislouch representing a primary producer in the marine environment and a fresh water adapted euryhaline teleost Oreochromis mossambicus (peters) at the consumer level of the ecological pyramid. The fish species, though not indigenous to our country, is now found ubiquitously in fresh water systems and estuaries. Also it is highly resistant to pollutants and has been suggested as an indicator of pollution in tropical region .

Electron energy loss spectroscopy assessment of cationic migration in La2/3Ca1/3MnO3 thin films

Màster en Nanociència i Nanotecnologia curs 2006-2007. Directors: Francesca Peiró i Martínez and Jordi Arbiol i Cobos

A "Trojan horse" strategy to reverse drug-resistance in brain tumors

Los gliomas malignos representan una de las formas más agresivas de los tumores del sistema nervioso central (SNC). De acuerdo con la clasificación de los tumores cerebrales de la Organización Mundial de la Salud (OMS), los astrocitomas han sido categorizados en cuatro grados, determinados por la patología subyacente. Es así como los gliomas malignos (o de alto grado) incluyen el glioma anaplásico (grado III) así como el glioblastoma multiforme (GBM, grado IV),estos últimos los más agresivos con el peor pronóstico (1). El manejo terapéutico de los tumores del SNC se basa en la cirugía, la radioterapia y la quimioterapia, dependiendo de las características del tumor, el estadio clínico y la edad (2),(3), sin embargo ninguno de los tratamientos estándar es completamente seguro y compatible con una calidad de vida aceptable (3), (4). En general, la quimioterapia es la primera opción en los tumores diseminados, como el glioblastoma invasivo y el meduloblastoma de alto riesgo o con metástasis múltiple, pero el pronóstico en estos pacientes es muy pobre (2),(3). Solamente nuevas terapias dirigidas (2) como las terapias anti-angiogénicas (4); o terapias génicas muestran un beneficio real en grupos limitados de pacientes con defectos moleculares específicos conocidos (4). De este modo, se hace necesario el desarrollo de nuevas terapias farmacológicas para atacar los tumores cerebrales. Frente a las terapias los gliomas malignos son con frecuencia quimioresistentes, y esta resistencia parece depender de al menos dos mecanismos: en primer lugar, la pobre penetración de muchas drogas anticáncer a través de la barrera hematoencefálica (BBB: Blood Brain Barrier), la barrera del fluido sangre-cerebroespinal (BCSFB: Blood-cerebrospinal fluid barrier) y la barrera sangre-tumor (BTB: blood-tumor barrier). Dicha resistencia se debe a la interacción de la droga con varios transportadores o bombas de eflujo de droga ABC (ABC: ATP-binding cassette) que se sobre expresan en las células endoteliales o epiteliales de estas barreras. En segundo lugar, estos transportadores de eflujo de drogas ABC propios de las células tumorales confieren un fenotipo conocido como resistencia a multidrogas (MDR: multidrug resistance), el cual es característico de varios tumores sólidos. Este fenotipo también está presente en los tumores del SNC y su papel en gliomas es objeto de investigación (5). Por consiguiente el suministro de medicamentos a través de la BBB es uno de los problemas vitales en los tratamientos de terapia dirigida. Estudios recientes han demostrado que algunas moléculas pequeñas utilizadas en estas terapias son sustratos de la glicoproteína P (Pgp: P-gycoprotein), así como también de otras bombas de eflujo como las proteínas relacionadas con la resistencia a multidrogas (MRPs: multidrug resistance-related proteins (MRPs) o la proteína relacionada con cáncer de seno (BCRP: breast-cancer resistance related protein)) que no permiten que las drogas de este tipo alcancen el tumor (1). Un sustrato de Pgp y BCRP es la DOXOrubicina (DOXO), un fármaco utilizado en la terapia anti cáncer, el cual es muy eficaz para atacar las células del tumor cerebral in vitro, pero con un uso clínico limitado por la poca entrega a través de la barrera hematoencefálica (BBB) y por la resistencia propia de los tumores. Por otra parte las células de BBB y las células del tumor cerebral tienen también proteínas superficiales, como el receptor de la lipoproteína de baja densidad (LDLR), que podría utilizarse como blanco terapéutico en BBB y tumores cerebrales. Es asi como la importancia de este estudio se basa en la generación de estrategias terapéuticas que promuevan el paso de las drogas a través de la barrera hematoencefalica y tumoral, y a su vez, se reconozcan mecanismos celulares que induzcan el incremento en la expresión de los transportadores ABC, de manera que puedan ser utilizados como blancos terapéuticos.Este estudio demostró que el uso de una nueva estrategia basada en el “Caballo de Troya”, donde se combina la droga DOXOrubicina, la cual es introducida dentro de un liposoma, salvaguarda la droga de manera que se evita su reconocimiento por parte de los transportadores ABC tanto de la BBB como de las células del tumor. La construcción del liposoma permitió utilizar el receptor LDLR de las células asegurando la entrada a través de la BBB y hacia las células tumorales a través de un proceso de endocitosis. Este mecanismo fue asociado al uso de estatinas o drogas anticolesterol las cuales favorecieron la expresión de LDLR y disminuyeron la actividad de los transportadores ABC por nitración de los mismos, incrementando la eficiencia de nuestro Caballo de Troya. Por consiguiente demostramos que el uso de una nueva estrategia o formulación denominada ApolipoDOXO más el uso de estatinas favorece la administración de fármacos a través de la BBB, venciendo la resistencia del tumor y reduciendo los efectos colaterales dosis dependiente de la DOXOrubicina. Además esta estrategia del "Caballo de Troya", es un nuevo enfoque terapéutico que puede ser considerado como una nueva estrategia para aumentar la eficacia de diferentes fármacos en varios tumores cerebrales y garantiza una alta eficiencia incluso en un medio hipóxico,característico de las células cancerosas, donde la expresión del transportador Pgp se vió aumentada. Teniendo en cuenta la relación entre algunas vías de señalización reconocidas como moduladores de la actividad de Pgp, este estudio presenta no solo la estrategia del Caballo de Troya, sino también otra propuesta terapéutica relacionada con el uso de Temozolomide más DOXOrubicina. Esta estrategia demostró que el temozolomide logra penetrar la BBB por que interviene en la via de señalización de la Wnt/GSK3/β-catenina, la cual modula la expresión del transportador Pgp. Se demostró que el TMZ disminuye la proteína y el mRNA de Wnt3 permitiendo plantear la hipótesis de que la droga al disminuir la transcripción del gen Wnt3 en células de BBB, incrementa la activación de la vía fosforilando la β-catenina y conduciendo a disminuir la β-catenina nuclear y por tanto su unión al promotor del gen mdr1. Con base en los resultados este estudio permitió el reconocimiento de tres mecanismos básicos relacionados con la expresión de los transportadores ABC y asociados a las estrategias empleadas: el primero fue el uso de las estatinas, el cual condujo a la nitración de los transportadores disminuyendo su actividad por la via del factor de transcripción NFκB; el segundo a partir del uso del temozolomide, el cual metila el gen de Wnt3 reduciendo la actividad de la via de señalización de la la β-catenina, disminuyendo la expresión del transportador Pgp. El tercero consistió en la determinación de la relación entre el eje RhoA/RhoA quinasa como un modulador de la via (no canónica) GSK3/β-catenina. Se demostró que la proteína quinasa RhoA promovió la activación de la proteína PTB1, la cual al fosforilar a GSK3 indujo la fosforilación de la β-catenina, lo cual dio lugar a su destrucción por el proteosoma, evitando su unión al promotor del gen mdr1 y por tanto reduciendo su expresión. En conclusión las estrategias propuestas en este trabajo incrementaron la citotoxicidad de las células tumorales al aumentar la permeabilidad no solo de la barrera hematoencefálica, sino también de la propia barrera tumoral. Igualmente, la estrategia del “Caballo de Troya” podría ser útil para la terapia de otras enfermedades asociadas al sistema nervioso central. Por otra parte estos estudios indican que el reconocimiento de mecanismos asociados a la expresión de los transportadores ABC podría constituir una herramienta clave en el desarrollo de nuevas terapias anticáncer.

Synthesis and evaluation of cyclic cationic peptides as antimicrobial agents for use in plant protection

Aquesta tesi doctoral se centra en l'estudi de l'aplicació de pèptids antimicrobians en la lluita contra agents patògens de cultius de plantes d'interès econòmic.L'estratègia sintètica s'ha portat a terme utilitzant metodologies convencionals de síntesi de pèptids en fase sòlida com l'estratègia tridimensional ortogonal Fmoc/tBut/Allyl. Ha calgut fer la recerca de les condicions òptimes per a l'eliminació del grup Allyl i la ciclació. D'entre els pèptids cíclics de 4-10 aminoacids sintetitzats, el decapèptid c(Lys-Leu-Lys-Leu-Lys-Phe-Lys-Lys-Leu-Gln) ha resultat ésser el més efectiu i s'ha pres com a base per al disseny d'una quimioteca de 56 pèptids. Dels resultats obtinguts s'ha sintetitzat una segona quimioteca basada en l'estructura general c(X1-X2-X3-X4-Lys-Phe-Lys-Lys-Leu-Gln) determinada com la que posseix el millor perfil d'activitat. Els pèptids més efectius obtinguts constituixen els primers exemples de pèptids cíclics actius contra E. amylovora i poden ser considerats com a bons candidats pel desenvolupament d'agents antimicrobians efectius en protecció vegetal.

Determination of the total antioxidant activity of fruits and vegetables by a liposome assay

The effects of mixtures of antioxidants on the oxidation of phospholipids have been investigated in large unilamellar liposomes following initiation by 2,2'-azobis(2-aminopropane) dihydrochloride. The lag phase increased linearly with antioxidant concentration. The lag phases of mixtures containing alpha-tocopherol with ascorbic acid showed synergy between the antioxidants, but mixtures of beta-carotene with cc-tocopherol or ascorbic acid were not synergistic. The liposome system was used to investigate the total antioxidant activity of lipid- and water-soluble extracts from 16 samples of fruits, vegetables, and related food products. The water-soluble extracts caused greater increases in lag phase than the lipid-soluble extracts. The lag phase of liposomes containing the water-soluble extracts from fruits and vegetables increased linearly with the total phenolic concentration, with the continental salad extract having the longest lag phase. The lipid-soluble extract from apples caused the largest increase in lag phase of the lipid-soluble extracts. The lag phases of the lipid-soluble and water-soluble extracts of all fruits and vegetables studied were additive, but no synergy was detected. The lag phase of the liposomes containing both the water-soluble and lipid-soluble extracts varied from 611.5 min for the continental salad extracts to 47.5 min for the cauliflower extracts.

Selective separation of beta-lactoglobulin from sweet whey using CGAs generated from the cationic surfactant CTAB

The selective separation of whey proteins was studied using colloidal gas aphrons generated from the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). From the titration curves obtained by zeta potential measurements of individual whey proteins, it was expected to selectively adsorb the major whey proteins, i.e., bovine serum albumin, alpha-lactalbumin, and beta-lactoglobulin to the aphrons and elute the remaining proteins (lactoferrin and lactoperoxidase) in the liquid phase. A number of process parameters including pH, ionic strength, and mass ratio of surfactant to protein (M-CTAB/M-TP) were varied in order to evaluate their effect on protein separation. Under optimum conditions (2 mmol/l CTAB, M-CTAB/M-TP = 0.26-0.35, pH 8, and ionic strength = 0.018 mol/l), 80-90% beta-lactoglobulin was removed from the liquid phase as a precipitate, while about 75% lactoferrin and lactoperoxidase, 80% bovine serum albumin, 95% immunoglobulin, and 65% alpha-lactalbumin were recovered in the liquid fraction. Mechanistic studies using zeta potential measurements and fluorescence spectroscopy proved that electrostatic interactions modulate only partially the selectivity of protein separation, as proteins with similar surface charges do not separate to the same extent between the two phases. The selectivity of recovery of beta-lactoglobulin probably occurs in two steps: the first being the selective interaction of the protein with opposite-charged surfactant molecules by means of electrostatic interactions, which leads to denaturation of the protein and subsequent formation and precipitation of the CTAB-beta-lactoglobulin complex. This is followed by the separation of CTAB-beta-lactoglobulin aggregates from the bulk liquid by flotation in the aphron phase. In this way, CGAs act as carriers which facilitate the removal of protein precipitate. (c) 2005 Wiley Periodicals, Inc.

Synthesis of cationic water-soluble copolymers and hydrogels based on [2-(methacryloyloxy)ethyl]trimethylammonium cloride and 2-hydroxyethylacrylate and their complex formation with poly(acrylic acid)

Water-soluble cationic copolymers and hydrogels were synthesized by radical copolymerization of [2-(methacryloyloxy)ethyl]trimetilylammonium chloride (MADQUAT) and 2-hydroxyethylacrylate (HEA). The kinetics of copolymerization has been studied and the reactivity ratios were determined. It was found that MADQUAT exhibits higher reactivity in copolymerization. The complexation between linear MADQUAT-HEA and linear poly(acrylic acid) (PAA) has been studied in aqueous solutions at different pH. It results in the formation of insoluble polyelectrolyte complexes, whose composition and stability to aggregate depends on MADQUAT content in copolymers and pH. The hydrogels were synthesized by three-dimensional radical copolymerization of MADQUAT and HEA in the presence of a crosslinker. The effects of the feed mixture composition on yield and swelling properties of the hydrogels were studied. The interactions of these hydrogels with linear PAA result in formation of gel-polyelectrolyte complexes and contraction of the samples. It was found that the contraction depends on copolymer composition, PAA molecular weight, and solution pH. (c) 2006 Wiley Periodicals, Inc.

Mucoadhesive interactions of amphiphilic cationic copolymers based on [2-(methacryloyloxy)ethyl]trimethylammonium chloride

Three series of water-soluble cationic copolymers have been synthesised by free-radical copolymerisation of [2-(methacryloyloxy)ethyl]-trimethylammonium chloride (MADQUAT) with methyl acrylate (MA), butyl acrylate (BA) and butyl methacrylate (BMA). The interactions between these copolymers and porcine stomach mucin have been studied in aqueous solutions using dynamic light scattering, zeta-potential measurements, turbidimetric titration and transmission electron microscopy (TEM). It was demonstrated that mixing aqueous dispersions of mucin with solutions of the cationic copolymers results in significant changes in size distribution and zeta-potential of its particles. It was found that an increase in the content of hydrophobic groups in copolymers leads to more efficient adsorption of macromolecules on the surface of mucin particles, which evidences the importance of hydrophobic effects in mucoadhesion. The efficiency of mucoadhesive interactions was found to be significantly dependent on pH, which affects the surface charge and aggregation stability of mucin. (C) 2007 Elsevier B.V. All rights reserved.

Rhodium cationic complexes using dithioethers as chiral ligands. Application in styrene hydroformylation

Addition of the dithioethers (−)-DIOSR2 (R=Me, iPr) (2,3-O-isopropylidene-1,4-dimethyl (and diisopropyl) thioether-L-threitol) to a dichloromethane solution of [Rh(COD)2]ClO4 (COD=1,5-cyclooctadiene) yielded the mononuclear complexes [Rh(COD)(DIOSR2)]ClO4. X-ray diffraction methods showed that the [Rh(COD)(DIOSiPr2)]ClO4 complex had an square-planar coordination geometry at the rhodium atom with the iPr groups in anti position. Cyclooctadiene complexes react with carbon monoxide to form dinuclear tetracarbonylated complexes [(CO)2Rh(μ-DIOSR2)2(CO)2](ClO4)2. [Rh(COD)(DIOSR2)]ClO4 are active catalyst precursors in styrene hydroformylation at 30 atm and 65°C which give conversions of up to 99% with a regioselectivity in 2-phenylpropanal as high as 74%. In all cases enantioselectivities are low.

Recovery of gallic acid with colloidal gas aphrons generated from a cationic surfactant

In a previous study we have demonstrated that gallic acid (GA) in its anionic form can be recovered from aqueous solutions using colloidal gas aphrons (CGA) generated from the cationic surfactant cetyltrimethylammonium bromide (CTAB). The aim of the present work is to get a better understanding of the separation mechanism in order to determine the optimum operating conditions to maximise the recovery of GA while preserving its antioxidant properties. Zeta potential measurements were carried out to characterise the surface charge of GA, CTAB and their mixtures at three different pH conditions (both in buffers and in aqueous solutions). GA interacted strongly with CTAB at pH higher than its pKa 3.14 where it is ionised and negatively charged. However, at pH higher than 7 GA becomes oxidised and loses its antioxidant power. GA recovery was mainly affected by pH, ionic strength, surfactant/GA molar ratio, mixing conditions and contact time. Scale-up of the separation using a flotation column resulted in both higher recovery and reproducibility. Preliminary experiments with grape marc extracts confirmed the potential application of this separation for the recovery of polyphenols from complex feedstocks

Interaction between a cationic surfactant-like peptide and lipid vesicles and its relationship to antimicrobial activity

We investigate the properties of an antimicrobial surfactant-like peptide (Ala)6(Arg), A6R, containing a cationic headgroup. The interaction of this peptide with zwitterionic (DPPC) lipid vesicles is investigated using a range of microscopic, X-ray scattering, spectroscopic, and calorimetric methods. The β-sheet structure adopted by A6R is disrupted in the presence of DPPC. A strong effect on the small-angle X-ray scattering profile is observed: the Bragg peaks from the DPPC bilayers in the vesicle walls are eliminated in the presence of A6R and only bilayer form factor peaks are observed. All of these observations point to the interaction of A6R with DPPC bilayers. These studies provide insight into interactions between a model cationic peptide and vesicles, relevant to understanding the action of antimicrobial peptides on lipid membranes. Notably, peptide A6R exhibits antimicrobial activity without membrane lysis.

Efficient masking of corrosion and fission products such as Ni(II) and Pd(II) in the presence of the minor actinide Am(III) using hydrophilic anionic or cationic bis-triazines

Water soluble anionic and cationic bis-triazine ligands are able to suppress (mask) the extraction of corrosion and fission products such as Ni(II) and Pd(II) that are found in PUREX raffinates. Thus it is possible to separate these elements from the minor actinide Am(III). Although some masking agents have previously been developed that retard the extraction of Pd(II), this is the first time a masking agent has been developed for Ni(II).

Effects of the cationic antimicrobial peptide eumenitin from the venom of solitary wasp Eumenes rubronotatus in planar lipid bilayers: Surface charge and pore formation activity

Eumenitin, a novel cationic antimicrobial peptide from the venom of solitary wasp Eumenes rubronotatus, was characterized by its effects on black lipid membranes of negatively charged (azolectin) and zwitterionic (1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or DPhPC-cholesterol) phospholipids: surface potential changes, single-channel activity, ion selectivity, and pore size were studied. We found that eumenitin binds preferentially to charged lipid membranes as compared with zwitterionic ones. Eumenitin is able to form pores in azolectin (G(1) = 118.00 +/- 3.67 pS or G(2) = 160.00 +/- 7.07 pS) and DPhPC membranes (G = 61.13 +/- 7.57 pS). Moreover, cholesterol addition to zwitterionic DPhPC membranes inhibits pore formation activity but does not interfere with the binding of peptide. Open pores presented higher cation (K (+)) over anion (Cl-) selectivity. The pore diameter was estimated at between 8.5and 9.8 angstrom in azolectin membranes and about 4.3 angstrom in DPhPC membranes. The results are discussed based on the toroidal pore model for membrane pore-forming activity and ion selectivity. (c) 2007 Elsevier Ltd. All rights reserved.

Novel immunoadjuvants based on cationic lipid: Preparation, characterization and activity in vivo

The interactions between three different protein antigens and dioctadecyldimethylammonium bromide (DODAB) dispersed in aqueous solutions from probe sonication or adsorbed its one bilayer onto particles was comparatively investigated. The three model proteins were bovine serum albumin (BSA), purified 18 kDa/14 kDa antigens from Taenia crassiceps (18/14-Tcra) and a recombinant, heat-shock protein hsp-18 kDa from Mycobacterium leprae. Protein-DODAB complexes in water solution were characterized by dynamic light scattering for sizing and zeta-potential analysis. Cationic complexes (80-100 nm of mean hydrodynamic diameter) displayed sizes similar to those of DODAB bilayer fragments (BF) in aqueous solution and good colloid stability over a range of DODAB and protein concentrations. The amount of cationic lipid required for attaining zero of zeta-potential at a given protein amount depended on protein nature being smaller for 18 kDa/14 kDa antigens than for BSA. Mean diameters for DODAB/protein complexes increased, whereas zeta-potentials decreased with NaCl or protein concentration. In mice, weak IgG production but significant cellular immune responses were induced by the complexes in comparison to antigens alone or carried by aluminum hydroxide as shown from IgG in serum determined by ELISA, delayed type hypersensitivity reaction from footpad swelling tests and cytokines analysis. The novel cationic adjuvant/protein complexes revealed good colloid stability and potential for vaccine design at a reduced DODAB concentration. (C) 2009 Elsevier Ltd. All rights reserved.