Impaired cortical energy metabolism but not major antioxidant defenses in experimental bacterial meningitis.

The loss of soluble brain antioxidants and protective effects of radical scavengers implicate reactive oxygen species in cortical neuronal injury caused by bacterial meningitis. However, the lack of significant oxidative damage in cortex [J. Neuropathol. Exp. Neurol. 61 (2002) 605-613] suggests that cortical neuronal injury may not be due to excessive parenchymal oxidant production. To see whether this tissue region exhibits a prooxidant state in bacterial meningitis, we examined the state of the major cortical antioxidant defenses in infant rats infected with Streptococcus pneumoniae. Adenine nucleotides were co-determined to assess possible changes in energy metabolism. Arguing against heightened parenchymal oxidant production, the high NADPH/NADP(+) ratio ( approximately 3:1) and activities of the major antioxidant defense and pentose phosphate pathway enzymes remained unchanged at the time of fulminant meningitis. In contrast, cortical ATP, ADP and total adenine nucleotides were on average decreased by approximately 25%. However, energy depletion did not lead to a significant decrease in adenylate energy charge (AEC). ATP depletion was likely a consequence of metabolic degradation, since it correlated with both the loss of total adenine nucleotides and accumulation of purine degradation products. Furthermore, the loss of ATP and decrease in AEC correlated significantly with the extent of neuronal injury. These results strongly suggest that energy depletion rather than parenchymal oxidative damage is involved in the observed cortical neuronal injury.

The regulation of protein kinase C by thiol oxidation

The Ser/Thr protein kinase C (PKC) isozyme family plays an important role in cell growth and differentiation and also contributes to key events in the development and progression of cancer. PKC isozymes are activated by phospholipid-dependent mechanisms, and they are also subject to oxidative activation and inactivation. Oxidative regulatory mechanisms are important in the governance of PKC isozyme action. While oxidative PKC activation involves phospho-tyrosine (P-Y) stabilization, the molecular mechanism(s) for oxidative PKC inactivation have not been defined. We previously reported that Thr → Cys peptide-substrate analogs inactivate several PKC isozymes including PKC-α via S-thiolation, i.e., by forming disulfides with PKC thiols. This inactivation mechanism is chemically analogous to protein S-glutathiolation, a post-translational modification that has been shown to oxidatively regulate several enzymes. To determine if PKC-α could be inactivated by S-glutathiolation, we employed the thiol-specific oxidant diamide (0.01–10mM) and 100μM glutathione (GSH). Diamide alone (0.1–5.0 mM) weakly inactivated PKC-α (<20%), and GSH alone had no effect on the isozyme activity. Marked potentiation of diamide-induced PKC-α inactivation (>90%) was achieved by 100μM GSH, resulting in full inactivation of the isozyme. Inactivation was reversed by DTT, consistent with a mechanism involving PKC-α S-glutathiolation. S-glutathiolation was demonstrated as DTT-reversible incorporation of [35S] GSH into PKC-α isozyme structure. These results indicate that a mild oxidative stimulus can inactivate purified PKC-α via S-glutathiolation. In addition, diamide treatment of metabolically labeled NIH3T3 cells induced potent PKC-α inactivation via isozyme [35S] S-thiolation. These results indicate that cellular PKC-α can be regulated via S-glutathiolation. ^

Molecular mechanism of jet fuel induced immune suppression

Dermal exposure to jet fuel suppresses the immune response. Immune regulatory cytokines, and biological modifiers, including platelet activating factor, prostaglandin E2, and interleukin-10 have all been implicated in the pathway leading to immunosuppression. It is estimated that approximately 260 different hydrocarbons are found in JP-8 (jet propulsion-8) jet fuel, and the identity of the immunotoxic compound is not known. The recent availability of synthetic jet fuel (S-8), which is devoid of aromatic hydrocarbons, made it feasible to design experiments to test the hypothesis that the aromatic hydrocarbons are responsible for jet fuel induced immune suppression. Applying S-8 to the skin of mice does not up-regulate the expression of epidermal cyclooxygenase-2 nor does it induce immune suppression. Adding back a cocktail of 7 of the most prevalent aromatic hydrocarbons found in jet fuel to S-8 up-regulated cyclooxygenase-2 expression and induced immune suppression. Cyclooxygenase-2 induction can be initiated by reactive oxygen species (ROS). JP-8 treated keratinocytes increased ROS production, S-8 did not. Antioxidant pre-treatment blocked jet fuel induced immune suppression and cyclooxygenase-2 up-regulation. Accumulation of reactive oxygen species induces oxidant stress and affects activity of ROS sensitive transcription factors. JP-8 induced activation of NFκB while S-8 did not. Pre-treatment with antioxidants blocked activation of NFκB and parthenolide, an NFκB inhibitor, blocked jet fuel induced immune suppression and cyclooxygenase-2 expression in skin of treated mice. p65 siRNA transfected keratinocytes demonstrated NFκB is critically involved in jet fuel induced COX-2 expression. These findings clearly implicate the aromatic hydrocarbons found in jet fuel as the agents responsible for inducing immune suppression, in part by the production of reaction oxygen species, NFκB dependent up-regulation of cyclooxygenase-2, and the production of immune regulatory factors and cytokines. ^

Caspase 8: Mediating the effects of a novel proteasome inhibitor, NPI-0052

Targeting the proteasome with the sole FDA approved proteasome inhibitor (PI), bortezomib, has been fruitful in specific cancers. Its success has generated an interest in next-generation PIs that might have a therapeutic advantage in cancers, such as leukemia, where bortezomib monotherapy was less effective. This study focuses on a novel, clinically relevant PI, NPI-0052. Experiments show that NPI-0052 targets chymotrypsin- and caspase-like activities more potently than the trypsin-like activity in leukemia cells. NPI-0052 induced apoptosis, as determined by caspase-3 activation and DNA fragmentation. Using caspase inhibitors and caspase-8 (I9.2) or FADD (I2.1) deficient cells revealed that caspase-8 was essential for NPI-0052-induced apoptosis. NPI-0052 killed cells via a caspase-8-tBid-mitochondrial pathway, relying on caspase-8, whereas bortezomib relies on several caspases. NPI-0052 increased reactive oxygen species (ROS) levels, which contributed towards cytotoxicity since an antioxidant conferred protection. To improve the clinical efficacy of PIs, NPI-0052 was combined with epigenetic anti-cancer agents, histone deacetylase inhibitors (HDACi). NPI-0052 with MS-275 or vorinostat (FDA approved HDACi), synergistically induced apoptosis more effectively than an HDACi/bortezomib regimen in Jurkat cells. Caspase-8 and ROS contributed towards NPI-0052/HDACi cytotoxicity and caspase-8 mediated superoxide production by NPI-0052 or NPI-0052/HDACi. The proximal targets of these agents: proteasome activity and histone acetylation were examined to determine if they contributed towards synergistic effects. HDACi targeted proteasomal β subunits and corresponding catalytic activities responsible for degrading proteins. Immunoblotting showed increases in histone-H3 expression and its acetylation with NPI-0052 or NPI-0052/HDACi in Jurkat and primary cells. Importantly, the hyper-acetylation by NPI-0052 was not detected with bortezomib, suggesting that this effect may be unique to NPI-0052. An antioxidant attenuated histone-H3 expression and acetylation induced by NPI-0052 alone or with HDACi. Furthermore, the hyper-acetylation by NPI-0052 relied on caspase-8. These novel results show that a PI is eliciting classical epigenetic alterations, demonstrated by hyper-acetylation of histone-H3. This alteration was oxidant and caspase-8 dependent. Overall, results reveal that caspase-8 mediates many effects induced by NPI-0052. Data show overlapping activities by NPI-0052 and HDACi which are contributing, along with caspase-8 activation and oxidative stress, to cytotoxic interactions in leukemia cells, reinforcing the potential clinical utility of combining these two compounds. ^

Iron and manganese in bottom sediments and interstitial waters sampled in the White Sea on August 21-30, 2003

Iron and manganese in bottom sediments studied along the sublatitudinal transect from Kandalaksha to Arkhangelsk are characterized by various contents and speciations depending on sedimentation environment, grain size of sediments, and diagenetic processes. The latter include redistribution of reactive forms leading to enrichment in Fe and Mn of surface sediments, formation of films, incrustations, and ferromanganese nodules. Variations in total Fe content (2-8%) are accompanied by changes in concentration of its reactive forms (acid extraction) and concentration of dissolved Fe in interstitial waters (1-14 µM). Variations in Mn content in bottom sediments (0.03-3.7%) and interstitial waters (up to 500 µM) correspond to high diagenetic mobility of this element. Changes in oxidation degree of chemical elements result in redox stratification of sediment strata with maximum concentrations of Fe, Mn, and sulfides. Organic matter of bottom sediments with considerable terrestrial constituent is oxidized by bottom water oxygen mainly at the sediment surface or in anaerobic conditions within the sediment strata. The role of inorganic components in organic matter oxidation changes from surface layer bottom sediments (where manganese oxyhydroxide dominates among oxidants) to deeper layers (where sulfate of interstitial water serves as the main oxidant). Differences in river runoff and hydrodynamics are responsible for geochemical asymmetry of the transect. The deep Kandalaksha Bay serves as a sediment trap for manganese (Mn content in sediments varies within 0.5-0.7%), whereas the sedimentary environment in the Dvina Bay promotes its removal from bottom sediments (Mn 0.05%).

Pore water and sediment geochemistry of the Bothian Sea

Methane is a powerful greenhouse gas and its biological conversion in marine sediments, largely controlled by anaerobic oxidation of methane (AOM), is a crucial part of the global carbon cycle. However, little is known about the role of iron oxides as an oxidant for AOM. Here we provide the first field evidence for iron-dependent AOM in brackish coastal surface sediments and show that methane produced in Bothnian Sea sediments is oxidized in distinct zones of iron- and sulfate-dependent AOM. At our study site, anthropogenic eutrophication over recent decades has led to an upward migration of the sulfate/methane transition zone in the sediment. Abundant iron oxides and high dissolved ferrous iron indicate iron reduction in the methanogenic sediments below the newly established sulfate/methane transition. Laboratory incubation studies of these sediments strongly suggest that the in situ microbial community is capable of linking methane oxidation to iron oxide reduction. Eutrophication of coastal environments may therefore create geochemical conditions favorable for iron-mediated AOM and thus increase the relevance of iron-dependent methane oxidation in the future. Besides its role in mitigating methane emissions, iron-dependent AOM strongly impacts sedimentary iron cycling and related biogeochemical processes through the reduction of large quantities of iron oxides.

Investigación técnica y económica sobre desinfección de aguas residuales por sistemas de oxidación

Un incremento de la demanda del agua, junto con el aumento de la contaminación, ha provocado que hoy en día la reutilización de las aguas depuradas sea necesaria, pero la reutilización de aguas debe garantizar y minimizar los posibles riesgos sanitarios y medioambientales que su práctica pueda provocar. En España estos parámetros se encuentran regulados por el RD 1620/2007 relativo al régimen jurídico de la reutilización de las aguas depuradas. Las aguas regeneradas son aguas que ya han sido sometidas a un tratamiento de depuración, y a las cuales se aplica un posterior tratamiento adicional o complementario que permita adecuar su calidad al uso al que vaya a destinarse. Siendo requeridos para los distintos reúsos procesos de desinfección, uno de los principales sistemas utilizados es el cloro, debido a su sencilla aplicación y costos bajos, sin tomar en cuenta la posible formación de compuestos organohalogenados potencialmente cancerígenos. Es por esto que surge la necesidad de aplicar distintos sistemas de oxidación objeto de estudio en esta tesis, como el dióxido de cloro estabilizado, ozono y los procesos avanzados de oxidación (Advanced Oxidation Processes, AOP), ozono/peróxido y uv/peróxido. En esta tesis se investiga los rendimientos que pueden alcanzar estos sistemas en la eliminación de los ácidos húmicos y los fenoles, siendo las principales sustancias formadoras de subproductos de la desinfección, así mismo, se considera necesario garantizar la desinfección del agua a través del estudio de tres grupos de microrganismos, los coliformes totales, e. coli y enterococos, siendo un punto importante el posible recrecimiento microbiológico debido a una desinfección escasa, por la permanencia en el agua de los compuestos antes mencionados, o por alguna fuente de alimento que pudieran encontrar en el sistema de distribución. Lo más importante será la calidad que se pueda alcanzar con estos desinfectantes, con el fin de obtener agua para los distintos reúsos que existen en la actualidad. Y así no limitar los alcances que puede tener la reutilización de las aguas residuales. Basándose en lo antes mencionado se procedió a realizar la caracterización del agua del rio Manzanares, con el fin de determinar la cantidad de ácidos húmicos disueltos y fenoles, obteniendo valores bajos, se decidió incorporar a las muestras de rio 5 mg/L de estos compuestos, con el fin de observar de que manera podrían interferir en la desinfección de esta agua. De esta forma se logran obtener resultados óptimos de los sistemas de desinfección estudiados, siendo el Ozono un oxidante eficiente en la desinfección de los microrganismos y en la eliminación de ácidos húmicos y fenoles con tiempos de contacto cortos, mostrando deficiencias al permitir el recrecimiento de los coliformes totales. Del sistema de oxidación avanzada UV/Peróxido se determino como un eficiente desinfectante para garantizar la inexistencia de rebrotes, al paso del tiempo. Así mismo se concluye que tiene buenos rendimientos en la eliminación del ácido húmico y los fenoles. An increase in water demand, coupled with increasing pollution, has caused today reuse of treated water is necessary, but must ensure water reuse and minimize potential health and environmental risks that their practice is cause. In Spain these parameters are regulated by Royal Decree 1620/2007 on the legal regime of the reuse of treated water. The reclaimed water is water that has already been subjected to a depuration treatment, which is applied as a subsequent further treatment that will bring quality to the use to which is to be delivered. As required for various reuses disinfection processes, one of the main systems used is chlorine, due to its simple implementation and low costs, without taking into account the possible formation of potentially carcinogenic halogenated organic compounds. That is why there is a need to apply different oxidation systems studied in this thesis, as stabilized chlorine dioxide, ozone and advanced oxidation processes (AOP), ozone/peroxide and UV/peroxide. This thesis investigates the rates can reach these systems in removing humic acids and phenols, the main substances forming disinfection byproducts, likewise, it is considered necessary to ensure water disinfection through the study of three groups of microorganisms, total coliform, e. coli and enterococci, the important point being a possible regrowth due to microbiological disinfection scarce, the water remaining on the aforementioned compounds, or a food source which may be found in the distribution system. The most important quality is that achievable with these disinfectants, with the water to obtain various reuses that exist today. And thus not limit the scope that can be reuse of wastewater. Based on the above we proceeded to perform characterization Manzanares river water, in order to determine the quantity of dissolved humic acids and phenols, obtaining low values, it was decided to incorporate river samples 5 mg / L of these compounds, in order to observe how they might interfere with the disinfection of the water. Thus optimum results are achieved for disinfection systems studied, being efficient ozone oxidant in the disinfection of microorganisms and the removal of humic acids and phenols with short contact times, showing gaps to allow regrowth total coliforms. Advanced oxidation system UV / peroxide were determined as an efficient disinfectant to ensure the absence of volunteers, the passage of time. Also it is concluded that has good yields in removing humic acid and phenols.

Mechanical Characterization of Tungsten-Titanium-Lanthana alloy: Influence of Temperature and Atmosphere

The target is to evaluate the mechanical behavior of Ti and La2O3 dispersed W alloy, processed by HIP and compare it with a reference pure-W. Tests were performed in both oxidant (air) and inert (vacuum) atmosphere in a temperature range from -196 to 1200 °C.

Characterization of spontaneous combustuion tendency of dried sewage sludge

The general purpose of this study was the determination of the safety conditions to avoid the presence of explosive atmospheres in the wastewater industry. Eight Spanish plants located in Madrid, Barcelona and Málaga were considered and several sludge samples were taken in different seasons. The base for the assessment of the spontaneous ignition behaviour of dust accumulations is the experimental determination of the self-ignition temperature under isothermal conditions. Self-ignition temperatures at four volumes were obtained for one sample of sewage sludge, allowing their extrapolation to large storage facilities. A simple test method, based also on an isothermal study of samples, is the UN classification of substances liable to spontaneous combustion. Two different samples were so tested, obtaining unlike results if transported in packages of different volumes. By means of thermogravimetric techniques it is possible to analyse the thermal susceptibility of dried sewage sludge. Apparent activation energy can be obtained from the rate of weight loss. It is also applied to the study of self-ignition susceptibility by modifying test conditions when oxygen stream is introduced. As a consequence of this oxidant contribution, sample behaviour can be very different during testing and a step drop or sudden loss of weight is observed at a characteristic temperature for every substance, associated to a rapid combustion. Plotting both the activation energy and the characteristic temperature, a map of self-ignition risk was obtained for 10 samples, showing different risk levels for samples taken in different locations and at different seasons. A prediction of the self-ignition risk level can be also determined.

Tratamientos avanzados de agua potable para eliminación de materia orgánica disuelta : aplicación del BAC

Los procesos de biofiltración por carbón activo biológico se han utilizado desde hace décadas, primeramente en Europa y después en Norte América, sin embargo no hay parámetros de diseño y operación específicos que se puedan utilizar de guía para la biofiltración. Además, el factor coste a la hora de elegir el carbón activo como medio de filtración impacta en el presupuesto, debido al elevado coste de inversión y de regeneración. A la hora de diseñar y operar filtros de carbón activo los requisitos que comúnmente se buscan son eliminar materia orgánica, olor, y sabor de agua. Dentro de la eliminación de materia orgánica se precisa la eliminación necesaria para evitar subproductos en la desinfección no deseados, y reducir los niveles de carbono orgánico disuelto biodegradable y asimilable a valores que consigan la bioestabilidad del agua producto, a fin de evitar recrecimiento de biofilm en las redes de distribución. El ozono se ha utilizado durante años como un oxidante previo a la biofiltración para reducir el olor, sabor, y color del agua, oxidando la materia orgánica convirtiendo los compuestos no biodegradables y lentamente biodegradables en biodegradables, consiguiendo que puedan ser posteriormente eliminados biológicamente en los filtros de carbón activo. Sin embargo la inestabilidad del ozono en el agua hace que se produzcan ácidos carboxilos, alcoholes y aldehídos, conocidos como subproductos de la desinfección. Con esta tesis se pretende dar respuesta principalmente a los siguientes objetivos: análisis de parámetros requeridos para el diseño de los filtros de carbón activo biológicos, necesidades de ozonización previa a la filtración, y comportamiento de la biofiltración en un sistema compuesto de coagulación sobre un filtro de carbón activo biológico. Los resultados obtenidos muestran que la biofiltración es un proceso que encaja perfectamente con los parámetros de diseño de plantas con filtración convencional. Aunque la capacidad de eliminación de materia orgánica se reduce a medida que el filtro se satura y entra en la fase biológica, la biodegradación en esta fase se mantienen estable y perdura a lo lago de los meses sin preocupaciones por la regeneración del carbón. Los valores de carbono orgánico disuelto biodegradable se mantienen por debajo de los marcados en la literatura existente para agua bioestable, lo que hace innecesaria la dosificación de ozono previa a la biofiltración. La adición de la coagulación con la corrección de pH sobre el carbón activo consigue una mejora en la reducción de la materia orgánica, sin afectar a la biodegradación del carbón activo, cumpliendo también con los requerimientos de turbidez a la salida de filtración. Lo que plantea importantes ventajas para el proceso. Granular activated carbon filters have been used for many years to treat and produce drinking water using the adsorption capacity of carbon, replacing it once the carbon lost its adsorption capacity and became saturated. On the other hand, biological activated carbon filters have been studied for decades, firstly in Europe and subsequently in North America, nevertheless are no generally accepted design and operational parameters documented to be used as design guidance for biofiltration. Perhaps this is because of the cost factor; to choose activated carbon as a filtration media requires a significant investment due to the high capital and regeneration costs. When activated carbon filters are typically required it is for the reduction of an organic load, removal of colour, taste and / or odour. In terms of organic matter reduction, the primary aim is to achieve as much removal as possible to reduce or avoid the introduction of disinfection by products, the required removal in biodegradable dissolved organic carbon and assimilable organic carbon to produce a biologically stable potable water which prohibits the regrowth of biofilm in the distribution systems. The ozone has historically been used as an oxidant to reduce colour, taste and odour by oxidizing the organic matter and increasing the biodegradability of the organic matter, enhancing the effectiveness of organic removal in downstream biological activated carbon filters. Unfortunately, ozone is unstable in water and reacts with organic matter producing carboxylic acids, alcohols, and aldehydes, known as disinfection by products. This thesis has the following objectives: determination of the required parameters for the design of the biological activated filters, the requirement of ozonization as a pre-treatment for the biological activated filters, and a performance assessment of biofiltration when coagulation is applied as a pretreatment for biological activated carbon filters. The results show that the process design parameters of biofiltration are compatible with those of conventional filtration. The organic matter removal reduces its effectiveness as soon as the filter is saturated and the biological stage starts, but the biodegradation continues steadily and lasts for a long period of time without the need of carbon regeneration. The removal of the biodegradable dissolved organic carbon is enough to produce a biostable water according to the values shown on the existing literature; therefore ozone is not required prior to the filtration. Furthermore, the addition of coagulant and pH control before the biological activated carbon filter achieves a additional removal of organic matter, without affecting the biodegradation that occurs in the activated carbon whilst also complying with the required turbidity removal.

Diseño y síntesis de materiales nanoestructurados basados en aluminatos de estroncio con propiedades fotoluminiscentes

Durante la última década, se han llevado acabo numeroso estudios sobre la síntesis de materiales fotoluminiscentes sub-micrónicos, en gran medida, al amplio número de aplicaciones que demandan este tipo de materiales. En concreto dentro de los materiales fosforescentes o también denominados materiales con una prolongada persistencia de la luminiscencia, los estudios se han enfocado en la matriz de SrAl2O4 dopada con Europio (Eu2+) y Disprosio (Dy3+) dado que tiene mayor estabilidad y persistencia de la fosforescencia con respecto a otras matrices. Estos materiales se emplean mayoritariamente en pinturas luminiscentes, tintas, señalización de seguridad pública, cerámicas, relojes, textiles y juguetes fosforescentes. Dado al amplio campo de aplicación de los SrAl2O4:Eu, Dy, se han investigado múltiples rutas de síntesis como la ruta sol-gel, la síntesis hidrotermal, la síntesis por combustión, la síntesis láser y la síntesis en estado sólido con el fin de desarrollar un método eficiente y que sea fácilmente escalable. Sin embargo, en la actualidad el método que se emplea para el procesamiento a nivel industrial de los materiales basados en aluminato de estroncio es la síntesis por estado sólido, que requiere de temperaturas de entre 1300 a 1900oC y largos tiempos de procesamiento. Además el material obtenido tiene un tamaño de partícula de 20 a 100 μm; siendo este tamaño restrictivo para el empleo de este tipo de material en determinadas aplicaciones. Por tanto, el objetivo de este trabajo es el desarrollo de nuevas estrategias que solventen las actuales limitaciones. Dentro de este marco se plantean una serie de objetivos específicos: Estudio de los parámetros que gobiernan los procesos de reducción del tamaño de partícula mediante molienda y su relación en la respuesta fotoluminiscente. Estudio de la síntesis por combustión de SrAl2O4:Eu, Dy, evaluando el efecto de la temperatura y la cantidad de combustible (urea) en el proceso para la obtención de partículas cristalinas minimizando la presencia de fases secundarias. Desarrollo de nuevas rutas de síntesis de SrAl2O4:Eu, Dy empleando el método de sales fundidas. Determinación de los mecanismos de reacción en presencia de la sal fundida en función de los parámetros de proceso que comprende la relación de sales y reactivos, la naturaleza de la alúmina y su tamaño, la temperatura y atmósfera de tratamiento. Mejora de la eficiencia de los procesos de síntesis para obtener productos con propiedades finales óptimas en procesos factibles industrialmente para su transferencia tecnológica. Es este trabajo han sido evaluados los efectos de diferentes procesos de molienda para la reducción del tamaño de partícula del material de SrAl2O4:Eu, Dy comercial. En el proceso de molienda en medio húmedo por atrición se observa la alteración de la estructura cristalina del material debido a la reacción de hidrólisis generada incluso empleando como medio líquido etanol absoluto. Con el fin de solventar las desventajas de la molienda en medio húmedo se llevo a cabo un estudio de la molturación en seco del material. La molturación en seco de alta energía reduce significativamente el tamaño medio de partícula. Sin embargo, procesos de molienda superiores a una duración de 10 minutos ocasionan un aumento del estado de aglomeración de las partículas y disminuyen drásticamente la respuesta fotoluminiscente del material. Por tanto, se lleva a cabo un proceso de molienda en seco de baja energía. Mediante este método se consigue reducir el tamaño medio de partícula, d50=2.8 μm, y se mejora la homogeneidad de la distribución del tamaño de partícula evitando la amorfización del material. A partir de los resultados obtenidos mediante difracción de rayos X y microscopia electrónica de barrido se infiere que la disminución de la intensidad de la fotoluminiscencia después de la molienda en seco de alta energía con respecto al material inicial se debe principalmente a la reducción del tamaño de cristalito. Se observan menores variaciones en la intensidad de la fotoluminiscencia cuando se emplea un método de molienda de baja de energía ya que en estos procesos se preserva el dominio cristalino y se reduce la amorfización significativamente. Estos resultados corroboran que la intensidad de la fotoluminiscencia y la persistencia de la luminiscencia de los materiales de SrAl2O4:Eu2+, Dy3+ dependen extrínsecamente de la morfología de las partículas, del tamaño de partícula, el tamaño de grano, los defectos superficiales e intrínsecamente del tamaño de cristalito. Siendo las características intrínsecas las que dominan con respecto a las extrínsecas y por tanto tienen mayor relevancia en la respuesta fotoluminiscente. Mediante síntesis por combustión se obtuvieron láminas nanoestructuradas de SrAl2O4:Eu, Dy de ≤1 μm de espesor. La cantidad de combustible, urea, en la reacción influye significativamente en la formación de determinadas fases cristalinas. Para la síntesis del material de SrAl2O4:Eu, Dy es necesario incluir un contenido de urea mayor que el estequiométrico (siendo m=1 la relación estequiométrica). La incorporación de un exceso de urea (m>1) requiere de la presencia de un agente oxidante interno, HNO3, para que la reacción tenga lugar. El empleo de un mayor contenido de urea como combustible permite una quelación efectiva de los cationes en el sistema y la creación de las condiciones reductoras para obtener un material de mayor cristalinidad y con mejores propiedades fotoluminiscentes. El material de SrAl2O4:Eu, Dy sintetizado a una temperatura de ignición de 600oC tiene un tamaño medio 5-25 μm con un espesor de ≤1 μm. Mediante procesos de molturación en seco de baja energía es posible disminuir el tamaño medio de partícula ≈2 μm y homogenizar la distribución del tamaño de partícula pero hay un deterioro asociado de la respuesta luminiscente. Sin embargo, se puede mejorar la respuesta fotoluminiscente empleando un tratamiento térmico posterior a 900oC N2-H2 durante 1 hora que no supone un aumento del tamaño de partícula pero si permite aumentar el tamaño de cristalito y la reducción del Eu3+ a Eu2+. Con respecto a la respuesta fotoluminiscente, se obtiene valores de la intensidad de la fotoluminiscencia entre un 35%-21% con respecto a la intensidad de un material comercial de referencia. Además la intensidad inicial del decaimiento de la fosforescencia es un 20% de la intensidad del material de referencia. Por tanto, teniendo en cuenta estos resultados, es necesario explorar otros métodos de síntesis para la obtención de los materiales bajo estudio. Por esta razón, en este trabajo se desarrollo una ruta de síntesis novedosa para sintetizar SrAl2O4:Eu, Dy mediante el método de sales fundidas para la obtención de materiales de gran cristalinidad con tamaños de cristalito del orden nanométrico. Se empleo como sal fundente la mezcla eutéctica de NaCl y KCl, denominada (NaCl-KCl)e. La principal ventaja de la incorporación de la mezcla es el incremento la reactividad del sistema, reduciendo la temperatura de formación del SrAl2O4 y la duración del tratamiento térmico en comparación con la síntesis en estado sólido. La formación del SrAl2O4 es favorecida ya que se aumenta la difusión de los cationes de Sr2+ en el medio líquido. Se emplearon diferentes tipos de Al2O3 para evaluar el papel del tamaño de partícula y su naturaleza en la reacción asistida por sales fundidas y por tanto en la morfología y propiedades del producto final. Se obtuvieron partículas de morfología pseudo-esférica de tamaño ≤0.5 μm al emplear como alúmina precursora partículas sub-micrónicas ( 0.5 μm Al2O3, 0.1 μm Al2 O3 y γ-Al2O3). El mecanismo de reacción que tiene lugar se asocia a procesos de disolución-precipitación que dominan al emplear partículas de alúmina pequeñas y reactivas. Mientras al emplear una alúmina de 6 μm Al2O3 prevalecen los procesos de crecimiento cristalino siguiendo un patrón o plantilla debido a la menor reactividad del sistema. La nucleación y crecimiento de nanocristales de SrAl2O4:Eu, Dy se genera sobre la superficie de la alúmina que actúa como soporte. De esta forma se desarrolla una estructura del tipo coraza-núcleo («core-shell» en inglés) donde la superficie externa está formada por los cristales fosforescentes de SrAl2O4 y el núcleo está formado por alúmina. Las partículas obtenidas tienen una respuesta fotoluminiscente diferente en función de la morfología final obtenida. La optimización de la relación Al2O3/SrO del material de SrAl2O4:Eu, Dy sintetizado a partir de la alúmina de 6 μm permite reducir las fases secundarias y la concentración de dopantes manteniendo la respuesta fotoluminiscente. Comparativamente con un material comercial de SrAl2O4:Eu, Dy de referencia, se han alcanzado valores de la intensidad de la emisión de hasta el 90% y de la intensidad inicial de las curvas de decaimiento de la luminiscencia de un 60% para el material sintetizado por sales fundidas que tiene un tamaño medio ≤ 10μm. Por otra parte, es necesario tener en cuenta que el SrAl2O4 tiene dos polimorfos, la fase monoclínica que es estable a temperaturas inferiores a 650oC y la fase hexagonal, fase de alta temperatura, estable a temperaturas superiores de 650oC. Se ha determinado que fase monoclínica presenta propiedades luminiscentes, sin embargo existen discordancias a cerca de las propiedades luminiscentes de la fase hexagonal. Mediante la síntesis por sales fundidas es posible estabilizar la fase hexagonal empleando como alúmina precursora γ-Al2O3 y un exceso de Al2O3 (Al2O3/SrO:2). La estabilización de la fase hexagonal a temperatura ambiente se produce cuando el tamaño de los cristales de SrAl2O4 es ≤20 nm. Además se observó que la fase hexagonal presenta respuesta fotoluminiscente. El diseño de materiales de SrAl2O4:Eu,Dy nanoestructurados permite modular la morfología del material y por tanto la intensidad de la de la fotoluminiscencia y la persistencia de la luminiscencia. La disminución de los materiales precursores, la temperatura y el tiempo de tratamiento significa la reducción de los costes económicos del material. De ahí la viabilidad de los materiales de SrAl2O4:Eu,Dy obtenidos mediante los procesos de síntesis propuestos en esta memoria de tesis para su posterior escalado industrial. ABSTRACT The synthesis of sub-micron photoluminescent particles has been widely studied during the past decade because of the promising industrial applications of these materials. A large number of matrices has been developed, being SrAl2O4 host doped with europium (Eu2+) and dysprosium (Dy3+) the most extensively studied, because of its better stability and long-lasting luminescence. These functional inorganic materials have a wide field of application in persistent luminous paints, inks and ceramics. Large attention has been paid to the development of an efficient method of preparation of SrAl2O4 powders, including solgel method, hydrothermal synthesis, laser synthesis, combustion synthesis and solid state reaction. Many of these techniques are not compatible with large-scale production and with the principles of sustainability. Moreover, industrial processing of highly crystalline powders usually requires high synthesis temperatures, typically between 1300 a 1900oC, with long processing times, especially for solid state reaction. As a result, the average particle size is typically within the 20-100 μm range. This large particle size is limiting for current applications that demand sub-micron particles. Therefore, the objective of this work is to develop new approaches to overcome these limitations. Within this frame, it is necessary to undertake the following purposes: To study the parameters that govern the particle size reduction by milling and their relation with the photoluminescence properties. To obtain SrAl2O4:Eu, Dy by combustion synthesis, assessing the effect of the temperature and the amount of fuel (urea) to synthesize highly crystalline particles minimizing the presence of secondary phases. To develop new synthesis methods to obtain SrAl2O4:Eu, Dy powders. The molten salt synthesis has been proposed. As the method is a novel route, the reaction mechanism should be determine as a function of the salt mixture, the ratio of the salt, the kind of Al2O3 and their particle size and the temperature and the atmosphere of the thermal treatment. To improve the efficiency of the synthesis process to obtain SrAl2O4:Eu, Dy powders with optimal final properties and easily scalable. On the basis of decreasing the particle size by using commercial product SrAl2O4:Eu2+, Dy3+ as raw material, the effects of different milling methods have been evaluated. Wet milling can significantly alter the structure of the material through hydrolysis reaction even in ethanol media. For overcoming the drawbacks of wet milling, a dry milling-based processes are studied. High energy dry milling process allows a great reduction of the particle size, however milling times above 10 min produce agglomeration and accelerates the decrease of the photoluminescence feature. To solve these issues the low energy dry milling process proposed effectively reduces the particle size to d50=2.8 μm, and improves the homogeneity avoiding the amorphization in comparison with previous methods. The X-ray diffraction and scanning electron microscope characterization allow to infer that the large variations in PL (Photoluminescence) values by high energy milling process are a consequence mainly of the crystallite size reduction. The lesser variation in PL values by low energy milling proces is related to the coherent crystalline domain preservation and the unnoticeable amorphization. These results corroborate that the photoluminescence intensity and the persistent luminescence of the SrAl2O4:Eu2+, Dy3+ powders depend extrinsically on the morphology of the particles such as particle size, grain size, surface damage and intrinsically on the crystallinity (crystallite size); being the intrinsically effects the ones that have a significant influence on the photoluminescent response. By combustion method, nanostructured SrAl2O4:Eu2+, Dy3+ sheets with a thickness ≤1 μm have been obtained. The amount of fuel (urea) in the reaction has an important influence on the phase composition; urea contents larger than the stoichiometric one require the presence of an oxidant agent such as HNO3 to complete the reaction. A higher amount of urea (excess of urea: denoted m>1, being m=1 the stoichiometric composition) including an oxidizing agent produces SrAl2O4:Eu2+,Dy3+ particles with persistent luminescence due to the effective chelation of the cations and the creation of suitable atmospheric conditions to reduce the Eu3+ to Eu2+. Therefore, optimizing the synthesis parameters in combustion synthesis by using a higher amount of urea and an internal oxidizing agent allows to complete the reaction. The amount of secondary phases can be significantly reduced and the photoluminescence response can be enhanced. This situation is attributed to a higher energy that improves the crystallinity of the powders. The powders obtained have a particle size c.a. 5-25 μm with a thickness ≤1 μm and require relatively low ignition temperatures (600oC). It is possible to reduce the particle size by a low energy dry milling but this process implies the decrease of the photoluminescent response. However, a post-thermal treatment in a reducing atmosphere allows the improvement of the properties due to the increment of crystallinity and the reduction of Eu3+ to Eu2+. Compared with the powder resulted from solid state method (commercial reference: average particle size, 20 μm and heterogeneous particle size distribution) the emission intensity of the powder prepared by combustion method achieve the values between 35% to 21% of the reference powder intensity. Moreover, the initial intensity of the decay curve is 20% of the intensity of the reference powder. Taking in account these results, it is necessary to explore other methods to synthesize the powders For that reason, an original synthetic route has been developed in this study: the molten salt assisted process to obtain highly crystalline SrAl2O4 powders with nanometric sized crystallites. The molten salt was composed of a mixture of NaCl and KCl using a 0.5:0.5 molar ratio (eutectic mixture hereafter abbreviated as (NaCl-KCl)e). The main advantages of salt addition is the increase of the reaction rate, the significant reduction of the synthesis temperature and the duration of the thermal treatment in comparison with classic solid state method. The SrAl2O4 formation is promoted due to the high mobility of the Sr2+ cations in the liquid medium. Different kinds of Al2O3 have been employed to evaluate the role of the size and the nature of this precursor on the kinetics of reaction, on the morphology and the final properties of the product. The SrAl2O4:Eu2+, Dy3+ powders have pseudo-spherical morphology and particle size ≤0.5 μm when a sub-micron Al2O3 ( 0.5 μm Al2O3, 0.1 μm Al2O3 and γ-Al2O3) has been used. This can be attributed to a higher reactivity in the system and the dominance of dissolution-precipitation mechanism. However, the use of larger alumina (6 μm Al2O3) modifies the reaction pathway leading to a different reaction evolution. More specifically, the growth of SrAl2O4 sub-micron particles on the surface of hexagonal platelets of 6μm Al2O3 is promoted. The particles retain the shape of the original Al2O3 and this formation process can be attributed to a «core-shell» mechanism. The particles obtained exhibit different photoluminescent response as a function of the final morphology of the powder. Therefore, through this study, it has been elucidated the reaction mechanisms of SrAl2O4 formation assisted by (NaCl-KCl)e that are governed by the diffusion of SrCO3 and the reactivity of the alumina particles. Optimizing the Al2O3/SrO ratio of the SrAl2O4:Eu, Dy powders synthesized with 6 μm Al2O3 as a precursor, the secondary phases and the concentration of dopant needed can be reduced keeping the photoluminescent response of the synthesized powder. Compared with the commercial reference powder, up to 90% of the emission intensity of the reference powder has been achieved for the powder prepared by molten salt method using 6μm Al2O3 as alumina precursor. Concerning the initial intensity of the decay curve, 60% of the initial intensity of the reference powder has been obtained. Additionally, it is necessary to take into account that SrAl2O4 has two polymorphs: monoclinic symmetry that is stable at temperatures below 650oC and hexagonal symmetry that is stable above this temperature. Monoclinic phase shows luminescent properties. However, there is no clear agreement on the emission of the hexagonal structure. By molten salt, it is possible to stabilize the hexagonal phase of SrAl2O4 employing an excess of Al2O3 (Al2O3/SrO: 2) and γ-Al2O3 as a precursor. The existence of nanometric crystalline domains with lower size (≤20 nm) allows the stabilization of the hexagonal phase. Moreover, it has been evidenced that the hexagonal polymorph exhibits photoluminescent response. To sum up, the design of nanostructured SrAl2O4:Eu2+, Dy3+ materials allows to obtain different morphologies and as consequence different photoluminescent responses. The reduction of temperature, duration of the thermal treatment and the precursors materials needed imply the decrease of the economic cost of the material. Therefore, the viability, suitability and scalability of the synthesis strategy developed in this work to process SrAl2O4:Eu2+, Dy3+ are demonstrated.

Xanthine oxidase activity associated with arterial blood pressure in spontaneously hypertensive rats

Recent evidence in vivo indicates that spontaneously hypertensive rats (SHR) exhibit an increase in oxyradical production in and around microvascular endothelium. This study is aimed to examine whether xanthine oxidase plays a role in overproduction of oxidants and thereby may contribute to hypertensive states as a consequence of the increasing microvascular tone. The xanthine oxidase activity in SHR was inhibited by dietary supplement of tungsten (0.7 g/kg) that depletes molybdenum as a cofactor for the enzyme activity as well as by administration of (−)BOF4272 [(−)-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo(1,5-α)-1,3,5-triazine-4-monohydrate], a synthetic inhibitor of the enzyme. The characteristic elevation of mean arterial pressure in SHR was normalized by the tungsten diet, whereas Wistar Koto (WKY) rats displayed no significant alteration in the pressure. Multifunctional intravital videomicroscopy in mesentery microvessels with hydroethidine, an oxidant-sensitive fluoroprobe, showed that SHR endothelium exhibited overproduction of oxyradicals that coincided with the elevated arteriolar tone as compared with WKY rats. The tungsten diet significantly repressed these changes toward the levels observed in WKY rats. The activity of oxyradical-producing form of xanthine oxidase in the mesenteric tissue of SHR was ≈3-fold greater than that of WKY rats, and pretreatment with the tungsten diet eliminated detectable levels of the enzyme activity. The inhibitory effects of the tungsten diet on the increasing blood pressure and arteriolar tone in SHR were also reproducible by administration of (−)BOF4272. These results suggest that xanthine oxidase accounts for a putative source of oxyradical generation that is associated with an increasing arteriolar tone in this form of hypertension.

Identification of the prooxidant site of human ceruloplasmin: A model for oxidative damage by copper bound to protein surfaces

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.

Functional consequences of NR2 subunit composition in single recombinant N-methyl-d-aspartate receptors

Single-channel recordings were obtained from Chinese hamster ovary cells transfected with the N-methyl-d-aspartate (NMDA) receptor subunit NR1 in combination with NR2A, NR2B, NR2C, or NR2A/NR2B. NMDA-activated currents were recorded under control conditions and in the presence of a thiol reductant (DTT), an oxidant (5,5′-dithio-bis[2-nitrobenzoic acid], DTNB), or the noncompetitive antagonist CP101,606 (CP). For all subunit combinations, DTT increased the frequency of channel opening when compared with DTNB. In addition, channels obtained from NR1/NR2A-transfected cells also exhibited a pronounced difference in mean open dwell-time between redox conditions. CP dramatically reduced both the open dwell-time and frequency of channel opening of NR1/NR2B-containing receptors, but only modestly inhibited NR1/NR2A and NR1/NR2C channel activity. A small number of patches obtained from cells transfected with NR1/NR2A/NR2B had channels with properties intermediate to NR1/NR2A and NR1/NR2B receptors, including insensitivity to CP block but redox properties similar to NR1/NR2B, consistent with the coassembly of NR2A with NR2B. Hence, NMDA receptors containing multiple types of NR2 subunits can have functionally distinguishable attributes.

Nitration of γ-tocopherol and oxidation of α-tocopherol by copper-zinc superoxide dismutase/H2O2/NO2−: Role of nitrogen dioxide free radical

Copper-zinc superoxide dismutase (Cu,ZnSOD) is the antioxidant enzyme that catalyzes the dismutation of superoxide (O2•−) to O2 and H2O2. In addition, Cu,ZnSOD also exhibits peroxidase activity in the presence of H2O2, leading to self-inactivation and formation of a potent enzyme-bound oxidant. We report in this study that lipid peroxidation of l-α-lecithin liposomes was enhanced greatly during the SOD/H2O2 reaction in the presence of nitrite anion (NO2−) with or without the metal ion chelator, diethylenetriaminepentacetic acid. The presence of NO2− also greatly enhanced α-tocopherol (α-TH) oxidation by SOD/H2O2 in saturated 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine liposomes. The major product identified by HPLC and UV-studies was α-tocopheryl quinone. When 1,2-diauroyl-sn-glycero-3-phosphatidylcholine liposomes containing γ-tocopherol (γ-TH) were incubated with SOD/H2O2/NO2−, the major product identified was 5-NO2-γ-TH. Nitrone spin traps significantly inhibited the formation of α-tocopheryl quinone and 5-NO2-γ-TH. NO2− inhibited H2O2-dependent inactivation of SOD. A proposed mechanism of this protection involves the oxidation of NO2− by an SOD-bound oxidant to the nitrogen dioxide radical (•NO2). In this study, we have shown a new mechanism of nitration catalyzed by the peroxidase activity of SOD. We conclude that NO2− is a suitable probe for investigating the peroxidase activity of familial Amyotrophic Lateral Sclerosis-linked SOD mutants.