Protein content of Calanus finmarchicus from the North Atlantic collected during Maria S. Merian cruise MSM26 in spring 2013

For the determination of water-soluble protein content of C. finmarchicus of the different stations the Qubit® Protein Assay Kit (Invitrogen) was used. Analysis was performed with extracts of 10 copepods. Working solution was prepared with Qubit® protein reagent and Qubit® protein buffer (1:200). 190 µL working solution was pipetted into each well of a micro plate and 10 µL of sample or Qubit® protein standard (0, 200 and 400 ng/µL) was added. Solutions were mixed and incubated for 15 min at room temperature. Measurements were conducted with a micro plate reader (TriStar LB 941, Berthold Technologies) at 485 nm excitation and 590 nm emission, using the software MikroWin2000 (Berthold Technologies).

Residual effect of synthetic Zn chelates on the availability of this metal in different soils under waterlogged conditions.

Adding Zn improves crop growth, increases seed yield and also positively affects nutritional quality. After Zn fertilization, there is normally a period of several years in which residual effects provide an adequate supply of Zn to successive crops. Immediately after the application of Zn sources water-soluble Zn slowly but continually decreases. Various factors, including time and moisture conditions, affect the aging process and modify the solubility of the metal in soil and therefore its availability. In previous experiments, we studied the residual effect of synthetic chelates, obtained that the amounts of potentially available Zn decreased in the second cropping year due to aging processes. The present study was undertaken to verify variations in the residual effects of applying four different synthetic Zn sources

LHPP, the light-harvesting NADPH:protochlorophyllide (Pchlide) oxido¬reductase:Pchlide complex of etiolated plants, is developmentally expressed across the barley leaf gradient

NADPH:protochlorophyllide oxidoreductase is a key enzyme for the light-induced greening of etiolated angiosperm plants. In barley, two POR proteins exist termed PORA and PORB that have previously been proposed to structurally and functionally cooperate in terms of a higher molecular mass light-harvesting complex named LHPP, in the prolamellar body of etioplasts [Nature 397 (1999) 80]. In this study we examined the expression pattern of LHPP during seedling etiolation and de-etiolation under different experimental conditions. Our results show that LHPP is developmentally expressed across the barley leaf gradient. We further provide evidence that LHPP operates both in plants that etiolate completely before being exposed to white light and in plants that etiolate only partially and begin light-harvesting as soon as traces of light become available in the uppermost parts of the soil. As a result of light absorption, in either case LHPP converts Pchlide a to chlorophyllide (Chlide) a and in turn disintegrates. The released Chlide a, as well as Chlide b produced upon LHPP’s light-dependent dissociation, which leads to the activation of the PORA as a Pchlide b-reducing enzyme, then bind to homologs of water-soluble chlorophyll proteins of Brassicaceae. We propose that these proteins transfer Chlide a and Chlide b to the thylakoids, where their esterification with phytol and assembly into the photosynthetic membrane complexes ultimately takes place. Presumably due to the tight coupling of LHPP synthesis and degradation, as well as WSCP formation and photosynthetic membrane assembly, efficient photo-protection is conferred onto the plant.

Influencia de los fármacos presentes en el agua residual sobre la resistencia de la bacteria Escherichia coli y su eliminación por oxidación avanzada

Los fármacos en el agua han sido considerados en los últimos años un problema medioambiental grave, y se ha incrementado el interés por los efectos que pueden producirse en el medio acuático. Aunado a este problema se encuentra el consumo excesivo de medicamentos no controlados, los cuales pueden ser desechados sin tener el tratamiento adecuado; por lo que se ingresan a los cursos de agua. Estos contaminantes emergentes son compuestos cuyo vertido supone un problema sanitario y ambiental. Se trata de contaminantes solubles en agua por lo que son capaces de estar presentes en todas las etapas del ciclo del agua. Han sido numerosos estudios los que se han realizado en diferentes países, ya que su presencia se ha convertido en un tema emergente en la química del medio ambiente, debido a que en las investigaciones realizadas muestran que no hay una eliminación completa a pesar de los distintos procesos que se aplican en las plantas de tratamiento de aguas residuales. Esta contaminación, incrementa la necesidad de conocer cuál es el efecto toxicológico sobre los organismos acuáticos y, en consecuencia, en las personas. La bacteria Escherichia Coli, es un organismo muy estudiado, debido a que se encuentra en los intestinos de los animales y humanos y por lo consiguiente en las aguas negras. Teniendo en cuenta la crítica situación, se planteó estudiar el efecto sobre la bacteria E. coli de 4 fármacos: Atenolol, Azitromicina, Estradiol e Ibuprofeno, para conocer cual era su comportamiento y el efecto que podían producir la presencia de los fármacos en la eliminación por procesos de oxidación. Así también, los efectos producidos sobre E. Coli, después de estar en contacto con los fármacos 1, 3 y 7 días. Se observó que los fármacos tienen efectos en el aumento o eliminación de los microrganismos dependiendo de los tiempos de exposición y la concentración del fármaco. Así mismo se observó que los microorganismos asimilan mejor las concentraciones menores de fármacos, a tiempos de contacto mayores de 24 horas. Con todos los desinfectantes de estudio se observaron ligeras resistencias de la bacteria ante la presencia de los fármacos. Drugs in water have been considered in recent years a serious environmental problem, and has increased interest in the effects that may occur in the aquatic environment. Added to this problem is the excessive consumption of non-controlled drugs, which can be disposed of without proper treatment, so they enter waterways. These are compounds emerging contaminants being discharged is a health and environmental problem. It is water soluble contaminants and are therefore able to be present in all stages of the water cycle. There have been numerous studies conducted in different countries, since their presence has become an emerging issue in environmental chemistry, because in the research shows that there isn’t a removal despite the different processes used in wastewater treatment plants. This contamination, increases the need to know what is the toxicological effects on aquatic organisms and, consequently, in people. The bacterium Escherichia coli, is a well-studied organism because it is found in the intestines of animals and humans and is therefore in the wastewater. Given the critical situation, was proposed to study the effect on the bacterium E. coli of 4 drugs: Atenolol, Azithromycin, Estradiol and Ibuprofen, to know what his behavior and the effect it could produce the presence of drugs in the removal by oxidation processes. Also, the effects on E. Coli, after being in contact with the drug 1, 3 and 7 days. It was noted that the drugs have effects on the growth or elimination of microorganisms depending on exposure time and the drug concentration. Also it was observed that the microorganisms assimilate lower concentrations of drug better over 24 hours. With all disinfectants study were observed resistances of the bacteria in the presence of the drugs.

Estudios de especiación de arsénico y acumulación de metales en muestras de interés medioambiental : Arsenic speciation and metal accumulation studies in environmental samples

Se ha estudiado la determinación de especies de arsénico y de contenidos totales de arsénico y metales pesados, específicamente cadmio, cromo, cobre, níquel, plomo y cinc, en muestras de interés medioambiental por su elevada capacidad acumuladora de metales, concretamente algas marinas comestibles y plantas terrestres procedentes de suelos contaminados por la actividad minera. La determinación de contenidos totales se ha llevado a cabo mediante espectrometría de emisión atómica con plasma de acoplamiento inductivo (ICP‐AES), así como por espectrometría de fluorescencia atómica con generación de hidruros (HG‐AFS), para bajos contenidos de arsénico. Las muestras fueron mineralizadas en medio ácido y calentamiento en horno de microondas. Los métodos fueron validados a través de su aplicación a materiales de referencia de matriz similar a la de las muestras, certificados en contenidos totales de los elementos seleccionados. Los resultados obtenidos mostraron su elevada capacidad de bioabsorción, especialmente en relación a los elevados contenidos de arsénico encontrados en algunas especies de algas pardas (Phaeophytas). En las plantas, se calcularon los factores de translocación, acumulación y biodisponibilidad de los elementos estudiados, permitiendo identificar a la especie Corrigiola telephiifolia como posible acumuladora de plomo e hiperacumuladora de arsénico. La determinación de especies de arsénico hidrosolubles en las muestras objeto de estudio, se llevó a cabo por cromatografía líquida de alta eficacia (HPLC) acoplado a ICP‐AES, HG‐ICP‐AES y HG‐AFS, incluyendo una etapa previa de foto‐oxidación. Los métodos desarrollados, mediante intercambio aniónico y catiónico, permitieron la diferenciación de hasta once especies de arsénico. Para el análisis de las muestras, fue necesaria la optimización de métodos de extracción, seleccionándose la extracción asistida por microondas (MAE) con agua desionizada. Asimismo, se realizaron estudios de estabilidad de arsénico total y de las especies hidrosolubles presentes en las algas, tanto sobre la muestra sólida como en sus extractos acuosos, evaluando las condiciones de almacenamiento adecuadas. En el caso de las plantas, la aplicación del diseño factorial de experimentos permitió optimizar el método de extracción y diferenciar entre las especies de arsénico presentes en forma de iones sencillos de mayor movilidad y el arsénico más fuertemente enlazado a componentes estructurales. Los resultados obtenidos permitieron identificar la presencia de arseniato (As(V)) y arsenito (As(III)) en las plantas, así como de ácido monometilarsónico (MMA) y óxido de trimetilarsina (TMAO) en algunas especies. En la mayoría de las algas se encontraron especies tóxicas, tanto mayoritarias (arseniato) como minoritarias (ácido dimetilarsínico (DMA)), así como hasta cuatro arsenoazúcares. Los resultados obtenidos y su estudio a través de la legislación vigente, mostraron la necesidad de desarrollar una reglamentación específica para el control de este tipo de alimentos. La determinación de especies de arsénico liposolubles en las muestras de algas se llevó a cabo mediante HPLC, en modo fase inversa, acoplado a espectrometría de masas con plasma de acoplamiento inductivo (ICP‐MS) y con ionización por electrospray (ESI‐MS), permitiendo la elucidación estructural de estos compuestos a través de la determinación de sus masas moleculares. Para ello, fue necesaria la puesta a punto de métodos extracción y purificación de los extractos. La metodología desarrollada permitió identificar hasta catorce especies de arsénico liposolubles en las algas, tres de ellas correspondientes a hidrocarburos que contienen arsénico, y once a arsenofosfolípidos, además de dos especies desconocidas. Las masas moleculares de las especies identificadas fueron confirmadas mediante cromatografía de gases acoplada a espectrometría de masas (GC‐MS) y espectrometría de masas de alta resolución (HR‐MS). ABSTRACT The determination of arsenic species and total arsenic and heavy metal contents (cadmium, chromium, cooper, nickel, lead and zinc) in environmental samples, with high metal accumulator capacity, has been studied. The samples studied were edible marine algae and terrestrial plants from soils polluted by mining activities. The determination of total element contents was performed by inductively coupled plasma atomic emission spectrometry (ICP‐AES), as well as by hydride generation atomic fluorescence spectrometry (HG‐AFS) for low arsenic contents. The samples studied were digested in an acidic medium by heating in a microwave oven. The digestion methods were validated against reference materials, with matrix similar to sample matrix and certified in total contents of the elements studied. The results showed the high biosorption capacity of the samples studied, especially regarding the high arsenic contents in some species of brown algae (Phaeophyta division). In terrestrial plants, the translocation, accumulation and bioavailability factors of the elements studied were calculated. Thus, the plant species Corrigiola telephiifolia was identified as possible lead accumulator and arsenic hyperaccumulator. The determination of water‐soluble arsenic species in the samples studied was carried out by high performance liquid chromatography (HPLC) coupled to ICP‐AES, HG‐ICP‐AES and HG‐AFS, including a prior photo‐oxidation step. The chromatographic methods developed, by anion and cation exchange, allowed us to differentiate up to eleven arsenic species. The sample analysis required the optimization of extraction methods, choosing the microwave assisted extraction (MAE) with deionized water. On the other hand, the stability of total arsenic and water‐soluble arsenic species in algae, both in the solid samples and in the water extracts, was studied, assessing the suitable storage conditions. In the case of plant samples, the application of a multivariate experimental design allowed us to optimize the extraction method and differentiate between the arsenic species present as simple ions of higher mobility and the arsenic more strongly bound to structural components. The presence of arsenite (As(III)) and arsenate (As(V)) was identified in plant samples, as well as monomethylarsonic acid (MMA) and trimethylarsine oxide (TMAO) in some cases. Regarding algae, toxic arsenic species were found in most of them, both As(V) and dimethylarsinic acid (DMA), as well as up to four arsenosugars. These results were discussed according to the current legislation, showing the need to develop specific regulations to control this kind of food products. The determination of lipid‐soluble arsenic species in alga samples was performed by reversed‐phase HPLC coupled to inductively coupled plasma and electrospray mass spectrometry (ICP‐MS and ESI‐MS), in order to establish the structure of these compounds by determining the corresponding molecular masses. For this purpose, it was necessary to develop an extraction method, as well as a clean‐up method of the extracts. The method developed permitted the identification of fourteen lipid‐soluble arsenic compounds in algae, corresponding to three arsenic‐hydrocarbons and eleven arsenosugarphospholipids, as well as two unknown compounds. Accurate mass measurements of the identified compounds were performed by gas chromatography coupled to mass spectrometry (GC‐MS) and high resolution mass spectrometry (HR‐MS).

Bioavailability and extraction of heavy metals from contaminated soil by Atriplex halimus

Pot experiments were performed to evaluate the phytoremediation capacity of plants of Atriplex halimus grown in contaminated mine soils and to investigate the effects of organic amendments on the metal bioavailability and uptake of these metals by plants. Soil samples collected from abandoned mine sites north of Madrid (Spain) were mixed with 0, 30 and 60 Mg ha?1 of two organic amendments, with different pH and nutrients content: pine-bark compost and horse- and sheep-manure compost. The increasing soil organic matter content and pH by the application of manure amendment reduced metal bioavailability in soil stabilising them. The proportion of Cu in the most bioavailable fractions (sum of the water-soluble, exchangeable, acid-soluble and Fe?Mn oxides fractions) decreased with the addition of 60 Mg ha?1 of manure from 62% to 52% in one of the soils studied and from 50% to 30% in the other. This amendment also reduced Zn proportion in water-soluble and exchangeable fractions from 17% to 13% in one of the soils. Manure decreased metal concentrations in shoots of A. halimus, from 97 to 35 mg kg?1 of Cu, from 211 to 98 mg kg?1 of Zn and from 1.4 to 0.6 mg kg?1 of Cd. In these treatments there was a higher plant growth due to the lower metal toxicity and the improvement of nutrients content in soil. This higher growth resulted in a higher total metal accumulation in plant biomass and therefore in a greater amount of metals removed from soil, so manure could be useful for phytoextraction purposes. This amendment increased metal accumulation in shoots from 37 to 138 mg pot?1 of Cu, from 299 to 445 mg pot?1 of Zn and from 1.8 to 3.7 mg pot?1 of Cd. Pine bark amendment did not significantly alter metal availability and its uptake by plants. Plants of A. halimus managed to reduce total Zn concentration in one of the soils from 146 to 130 mg kg?1, but its phytoextraction capacity was insufficient to remediate contaminated soils in the short-to-medium term. However, A. halimus could be, in combination with manure amendment, appropriate for the phytostabilization of metals in mine soils.

Stability and mechanical evaluation of bovine pericardium cross-linked with polyurethane prepolymer in aqueous medium

The present study investigates the potential use of non-catalyzed water-soluble blocked polyurethane prepolymer (PUP) as a bifunctional cross-linker for collagenous scaffolds. The effect of concentration (5, 10, 15 and 20%), time (4, 6, 12 and 24 h), medium volume (50, 100, 200 and 300%) and pH (7.4, 8.2, 9 and 10) over stability, microstructure and tensile mechanical behavior of acellular pericardial matrix was studied. The cross-linking index increased up to 81% while the denaturation temperature increased up to 12 °C after PUP crosslinking. PUP-treated scaffold resisted the collagenase degradation (0.167 ± 0.14 mmol/g of liberated amine groups vs. 598 ± 60 mmol/g for non-cross-linked matrix). The collagen fiber network was coated with PUP while viscoelastic properties were altered after cross-linking. The treatment of the pericardial scaffold with PUP allows (i) different densities of cross-linking depending of the process parameters and (ii) tensile properties similar to glutaraldehyde method.

Residuos orgánicos como fuentes de fósforo

En este trabajo, se han llevado a cabo distintos experimentos en laboratorio, con el objetivo de estudiar el efecto de la aplicación de residuos orgánicos como fuentes de P en las pérdidas de este elemento, que se producen en suelo, tanto por escorrentía superficial como por lixiviación. El interés por evaluar las pérdidas de P se debe a la necesidad de conocer mejor los factores que influyen en los procesos de pérdidas de este elemento y así, poder reducir los problemas de eutrofización de aguas, tanto superficiales como subterráneas, provocadas por un exceso de este nutriente, junto con otros como el nitrógeno. Los trabajos experimentales que se han llevado a cabo se detallan a continuación: Se ha realizado el estudio de las formas de P contenidas en una serie de 14 residuos orgánicos, de distinto origen y tratamiento (compost, lodos, purines y digestato), comparando la información aportada por tres protocolos de fraccionamientos de P diferentes, seleccionados entre los principales métodos utilizados: protocolo de fraccionamiento de Ruttemberg (1992), protocolo de Normas, medidas y ensayos (Ruban et al., 2001a) y protocolo de Huang et al. (2008). Todos los métodos de fraccionamiento empleados aportaron información útil para conocer las formas de P de los residuos, a pesar de que alguno de ellos fue descrito para sedimentos o suelos. Sin embargo, resulta difícil comparar los resultados entre unos y otros, ya que cada uno emplea extractantes y tiempos de ensayos diferentes. Las cantidades de P total determinadas por cada método mantienen una relación lineal, aunque el método SMT, por ser más directo, obtiene las cantidades más elevadas para todos los residuos. Los métodos secuenciales (métodos de Huang y Ruttemberg), a pesar de ser más largos y tediosos, aportan información más detallada de la disponibilidad de las formas de P, y con ello, permiten obtener una mejor estimación de las pérdidas potenciales de este elemento tras su aplicación en suelo. Se han encontrado relaciones positivas entre las fracciones determinadas por estos dos métodos. Así mismo, se encuentra una correlación entre las formas solubles de P y la concentración de los iones [Ca + Fe + Al] de los residuos, útiles como indicadores de la disponibilidad de este elemento. Sin embargo, el protocolo SMT, no obtiene información de la solubilidad o disponibilidad de las formas de P contenidas, ni las formas determinadas mantienen relaciones directas con las de los otros métodos, con excepción del P total. Para el estudio del comportamiento de los residuos aplicados en suelos, se pusieron a punto sistemas de lluvia simulada, con el objetivo de caracterizar las pérdidas de P en la escorrentía superficial generada. Por otra parte, se emplearon columnas de suelos enmendados con residuos orgánicos, para el estudio de las pérdidas de P por lixiviación. Los ensayos de simulación de lluvia se llevaron a cabo de acuerdo al “National Phosphorus Research proyect“ (2001), que consigue simular eventos sucesivos de lluvia en unas condiciones semejantes a la realidad, empleando cajas llenas de suelo del horizonte superficial, con residuos aplicados tanto superficialmente como mediante mezcla con el propio suelo. Los ensayos se realizaron con seis residuos de diferente naturaleza y sometidos a distintos tratamientos. Se encontraron diferencias significativas en las pérdidas de las formas de P analizadas, tanto disueltas como particuladas, en las aguas de escorrentía generadas. En general, las pérdidas en el primer evento de lluvia tras la aplicación de los residuos fueron mayores a las generadas en el segundo evento, predominando las formas de P particuladas respecto a las disueltas en ambos. Se encontró una relación positiva entre las pérdidas de P en las aguas de escorrentía generadas en cada ensayo, con los contenidos de P soluble en agua y fácilmente disponible de los residuos empleados, determinados por los protocolos de fraccionamientos secuenciales. Además, se emplearon los modelos matemáticos desarrollados por Vadas et al. (2005, 2007), de evaluación de las pérdidas de P por escorrentía para fertilizantes y estiércoles. La predicción de estos modelos no se cumple en el caso de todos los residuos. Las distintas propiedades físicas de los residuos pueden afectar a las diferencias entre las pérdidas experimentales y las esperadas. Los ensayos de simulación del proceso de lixiviación se llevaron a cabo en columnas de percolación, con suelos enmendados con residuos orgánicos, de acuerdo a la norma “CEN/TS 14405–2004: Caracterización de los residuos – Test de comportamiento de lixiviación – Test de flujo ascendente”. Las pérdidas de P por procesos de lixiviación de agua, han sido despreciadas durante mucho tiempo respecto a las pérdidas por escorrentía. Sin embargo, se ha demostrado que deben tenerse en consideración, principalmente en algunos tipos de suelos o zonas cercanas a acuíferos. Se utilizaron tres suelos de distinta procedencia para los ensayos, de manera que se pudo estudiar la influencia del tipo de suelo en las pérdidas de P para cada tipo de residuo (purín, compost, digestato y lodo de EDAR). Los índices de adsorción de P determinados para cada suelo permiten evaluar aquellos que presentarán más riesgo de producir pérdidas de este elemento al aplicarse fuentes externas de P, encontrando una relación positiva entre ambos. Las pérdidas de P en los lixiviados varían en función tanto del residuo como del suelo empleado. Para el compost, el purín y el lodo, se encontró una relación entre las pérdidas generadas en el agua lixiviada de las columnas y las formas de P soluble contenidas en los residuos. Sin embargo, en el caso del digestato, no existía esta correlación. Las pérdidas para este residuo fueron en todos los casos menores a las estimadas, considerando las formas de P contenido. El estudio de la mojabilidad, propiedad física del residuo que evalúa la capacidad de interacción residuo-agua, permitió explicar el comportamiento anómalo de este residuo, con una mayor resistencia a que el agua entrara en su estructura y por tanto, una mayor dificultad de solubilizar el P contenido en el propio residuo, que en el caso de otros residuos. En general, podemos considerar que el estudio de las formas de P más disponibles o solubles en agua, aporta información útil de las pérdidas potenciales de P. Sin embargo, es necesario estudiar las propiedades físicas de los residuos orgánicos aplicados y la capacidad de adsorción de P de los suelos, para estimar las pérdidas de P y con ello, colaborar a controlar los procesos de eutrofización en aguas. ABSTRACT This dissertation explores the effect of organic wastes application as sources of P in losses of this element that occur by both surface runoff and leaching in soil. To do so, diverse laboratory experiments are conducted and presented here. Evaluating P losses is necessary to better understand the factors that influence the processes behind the loss of this element. Reducing P losses reduces eutrophication problems of both surface water and groundwater caused by an excess of this nutrient, along with other as nitrogen. Details of the experiments are presented below: The first experiment studies the forms of P contained in a series of 14 organic wastes of different origin and treatment (compost, sludge, slurry and digestate), comparing the information provided by three methods of P fractionation. The methods selected were: Ruttemberg protocol (1992); Standards, Measurements and Testing protocol (Ruban et al., 2001a); and Huang protocol (Huang et al., 2008). All fractionation methods employed successfully contribute to our knowledge of P forms in wastes, even though one of them was originally described for sediments or soils information. However, it is difficult to compare results among each other, as each protocol employs different extractants and time in the trials. Total amounts of P obtained by each method show a linear relationship, although the SMT method, which is more direct, obtains the highest amounts for all residues. Sequential methods (Huang and Ruttemberg’s protocols), despite being longer and more tedious, provide more detailed information on the availability of the forms of P. Therefore, allow the estimation of the potential losses of P after application in soil. Furthermore, positive relationships have been found among fractions obtained by these methods. Positive relationship has been found also among soluble forms of P and the concentration of ions Fe + Ca + Al, which is useful as an indicator of the availability of this element. However, the SMT protocol does not collect information about solubility or availability of forms of P contained; neither do certain forms maintain direct relations with the forms from other methods, with the exception of total P methods. To study the behavior of wastes applied to soils two experiments were conducted. Simulated rain systems were prepared to characterize P losses in the surface runoff generated. In addition, columns of soils amended with organic waste were developed for the study of P leaching losses. Simulated rain systems were carried out according to the ’National Phosphorus Research Project’ (2001), which manages to simulate successive rainfall events in conditions resembling reality. The experiment uses boxes filled with soil from the surface horizon amended with residues, both superficially and by mixing with the soil. Tests were conducted with six residues of different type and subjected to diverse treatments. Findings show significant differences in losses of the P forms analyzed in the generated runoff water, in both solution and particulate forms. In general, losses in the first rainfall event after application of waste were higher than the losses generated in the second event, predominating particulate forms of P over dissolved forms in both events. In all trials, a positive relationship was found between various P forms determined by sequential fractionation protocols (water soluble P and readily available P forms) and P losses in runoff. Furthermore, results from Vadas´s mathematical models (Vadas et al., 2005; 2007) to assess P losses by runoff fertilizers and manures indicate that the prediction of this model is not fulfilled in the case of all residues. The diverse physical properties of wastes may affect the differences between experimental and expected losses. Finally, leaching simulation processes were carried out in percolation columns, filled with soils amended with organic wastes, following the ‘CEN/TS 14405-2004 standard: Characterization of waste - Leaching behavior test - Test Flow ascending ’. P losses by leaching have been neglected for a long time with respect to runoff losses. However, findings corroborate previous studies showing that these P losses have to be taken into account, especially in certain types of soils and in zones near aquifers. To study the influence of soil type on P losses, experiments were carried out with three different soils and for each type of waste (manure, compost, digestate and sludge WWTP). Each soil’s P adsorption rates allow assessing which soils imply a higher risk of P losses when external sources of P are applied. P losses in leachate vary according to the type of soil employed and according to the specific residue. In the case of compost, manure and sludge, there is a relationship between leaching losses and residues’ soluble forms of P. The exception being the digestate, where there was no such correlation. Digestate P losses by leaching were lower than expected in all cases considering the forms of P contained. Moreover, examining digestate wettability -- that is, the physical property of the residue that assesses the capacity of waste-water interaction -- allowed explaining the anomalous behavior of this residue. Digestate has a high resistance to water entering its structure and thus higher difficulty to solubilize the P contained. Overall, studying the more available or soluble P forms provides useful information about the potential loss of P. However, this dissertation shows that it is necessary to examine the physical properties of organic residues applied as well as the P adsorption capacity of soils to estimate P losses, and thus to control eutrophication in water.

Risk of Leaching in Soils Amended by Compost and digestate from Municipal Solid Waste

New European directives have proposed the direct application of compost and digestate produced from municipal solid wastes as organic matter sources in agricultural soils. Therefore information about phosphorus leaching from these residues when they are applied to the soil is increasingly mportant. Leaching experiments were conducted to determine the P mobility in compost and digestate mixtures, supplying equivalent amounts to 100 kg P ha?1 to three different types of soils. The tests were performed in accordance with CEN/TS 14405:2004 analyzing the maximum dissolved reactive P and the kinetic rate in the leachate. P biowaste fractionation indicated that digestate has a higher level of available P than compost has. In contrast, P losses in leaching experiments with soil-compost mixtureswere higher than in soil-digestate mixtures. For bothwastes, therewas no correlation between disolved reactive P lost and the water soluble P.The interaction between soil and waste, the long experimentation time, and the volume of leachate obtained caused the waste?s wettability to become an influential parameter in P leaching behavior. The overall conclusion is that kinetic data analysis provides valuable information concerning the sorption mechanism that can be used for predicting the large-scale behavior of soil systems.

Carboxyfullerenes as neuroprotective agents

Two regioisomers with C3 or D3 symmetry of water-soluble carboxylic acid C60 derivatives, containing three malonic acid groups per molecule, were synthesized and found to be equipotent free radical scavengers in solution as assessed by EPR analysis. Both compounds also inhibited the excitotoxic death of cultured cortical neurons induced by exposure to N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or oxygen-glucose deprivation, but the C3 regioisomer was more effective than the D3 regioisomer, possibly reflecting its polar nature and attendant greater ability to enter lipid membranes. At 100 μM, the C3 derivative fully blocked even rapidly triggered, NMDA receptor-mediated toxicity, a form of toxicity with limited sensitivity to all other classes of free radical scavengers we have tested. The C3 derivative also reduced apoptotic neuronal death induced by either serum deprivation or exposure to Aβ1–42 protein. Furthermore, continuous infusion of the C3 derivative in a transgenic mouse carrying the human mutant (G93A) superoxide dismutase gene responsible for a form of familial amyotrophic lateral sclerosis, delayed both death and functional deterioration. These data suggest that polar carboxylic acid C60 derivatives may have attractive therapeutic properties in several acute or chronic neurodegenerative diseases.

Extraction of Cholesterol with Methyl-β-Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-β-cyclodextrin (MβCD) to selectively extract cholesterol from the plasma membrane. MβCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MβCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MβCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MβCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.

The vacuolating toxin from Helicobacter pylori forms hexameric pores in lipid bilayers at low pH

Pathogenic strains of Helicobacter pylori secrete a cytotoxin, VacA, that in the presence of weak bases, causes osmotic swelling of acidic intracellular compartments enriched in markers for late endosomes and lysosomes. The molecular mechanisms by which VacA causes this vacuolation remain largely unknown. At neutral pH, VacA is predominantly a water-soluble dodecamer formed by two apposing hexamers. In this report, we show by using atomic force microscopy that below pH ≈5, VacA associates with anionic lipid bilayers to form hexameric membrane-associated complexes. We propose that water-soluble dodecameric VacA proteins disassemble at low pH and reassemble into membrane-spanning hexamers. The surface contour of the membrane-bound hexamer is strikingly similar to the outer surface of the soluble dodecamer, suggesting that the VacA surface in contact with the membrane is buried within the dodecamer before protonation. In addition, electrophysiological measurements indicate that, under the conditions determined by atomic force microscopy for membrane association, VacA forms pores across planar lipid bilayers. This low pH-triggered pore formation is likely a critical step in VacA activity.