Influence of enzymes and technology on the composition of Cobrançosa and Galega vulgar virgin olive oils

Doutoramento em Engenharia Alimentar - Instituto Superior de Agronomia - UL

Influence of Pseudomonas putida AF7 inoculation on soil enzymes.

There has been some concern about the environmental impact of microbial agents. Pseudomonas may be used as bioremediator and as biopesticide. In this study, we report the use of soil enzyme assays as biological indicator of possible negative effects in soil functioning after the P. putida AF7 inoculation. For that, P. putida AF7 was originally isolated from the rizosphere of rice and was inoculated on three soil types: Rhodic Hapludox (RH), Typic Hapludox (TH); and Arenic Hapludult (AH). The acid phosphatase, b-glucosidase and protease enzymes activities were measured for three period of evaluation (7, 14 and 21 days). In general, the enzymatic activities pre- sented variation among the tested soils. The highest activities of b-glucosidase and acid phosphatase were observed in the RH and AH soils, while the protease activity was higher in the TH soil. Also, the soil charac- teristics were measured for each plot. The activity of enzymes from the carbon cycle was positively correlated with the N and the P and the enzyme from the nitrogen cycle was negatively correlated with N and C.org. The presented data indicate that soil biochemical properties can be an useful tool for use as an indicator of soil perturba- tions by microbial inoculation in a risk assessment.

Sostenibilidad de los servicios de salud en el área No. 6, Centro de Salud Hospital de Baba, Los Ríos, año 2001

El proceso de investigación buscará determinar si los servicios de salud brindados a la población que pertenecen al área n. 6, Centro de Salud Hospital Baba, de la provincia de Los Ríos, tiene una sustentación económica y pueden seguir siendo entregados a la población geográfica de la zona, determinando además los problemas que sobre acceso a los servicios d salud tenga la comunidad del área.-au

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (ionic exchangers, silicates). We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailor-made heterofunctional supports as a tool to immobilize–stabilize–purify some proteins will be discussed in deep, using low concentration of adsorbent groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be the last part of the review.

Importance of the Support Properties for Immobilization or Purification of Enzymes

Immobilization and purification of enzymes are usual requirements for their industrial use. Both purification and immobilization have a common factor: they use a solid activated support. Using a support for enzyme purification means having mild conditions for enzyme release and a selective enzyme–support interaction is interesting. When using a support for immobilization, however, enzyme desorption is a problem. The improvement of enzyme features through immobilization is a usual objective (e.g., stability, selectivity). Thus, a support designed for enzyme purification and a support designed for enzyme immobilization may differ significantly. In this review, we will focus our attention on the requirements of a support surface to produce the desired objectives. The ideal physical properties of the matrix, the properties of the introduced reactive groups, the best surface activation degree to reach the desired objective, and the properties of the reactive groups will be discussed.

Análise da expressão de APE1 após estresse oxidativo induzido em células proficientes e deficientes na via de reparo por excisão de nucleotídeos.

Riboflavin is a vitamin very important in aerobic organisms, as a precursor of many coenzymes involved in the electron transporter chain. However, after photosensitization of riboflavin with UV or visible light, it generates reactive oxygen species (ROS), which can oxidize the DNA. The repair of oxidative lesions on DNA occurs through the base excision repair pathway (BER), where APE1 endonuclease plays a central role. On the other hand, the nucleotide excision repair pathway (NER) repairs helix-distorting lesions. Recently, it was described the participation of NERproteins in the repair of oxidative damage and in stimulation of repair function fromAPE1. The aim of this research was to evaluate the cytotoxic effects of photosensitized riboflavin (RF*) in cells proficient and deficient in NER, correlating with APE1 expression. For this propose, the cells were treated with RF* and it was performed the cell viability assay, extraction of whole proteins, cells fractionation, immunoblotting, indirect immunofluorescence and analysis of polymorphisms of BER gens. The results evidenced that cells deficient in XPA and CSB proteins were more sensitive to RF*. However, XPC-deficient cells presented similar resistance to MRC5- SV cells, which is proficient in NER. These results indicate that XPA and CSB proteins have an important role on repair of oxidative lesions induced by RF*. Additionally, it was evidenced that single nucleotide polymorphisms (SNPs) in BER enzymes may influence in sensitivity of NER-deficient cell lines. Concerning the APE1 expression, the results showed that expression of this protein after treatment with RF* only changed in XPC-deficient cells. Though, it was observed that APE1 is recruited and is bound to chromatin in MRC5-SV and XPA cells after treatment with RF*. The results also showed the induction of DNA damage after treatment with RF*, through the analysis of-H2AX, since the treatment promoted an increase of endogenous levels of this phosphorylated protein, which acts signaling double strand-break on DNA. On the other hand, in XPC-deficient cells, regardless of resistance of RF*, the endogenous levels of APE1 are extremely reduced when compared with other cell lines and APE1 is not bound to chromatin after treatment with RF*. These results conclude that RF* was able to induce cell death in NERdeficient cells, where XPA and CSB cells were more sensitive when compared with MRC5-SV and XPC-deficient cells. This last result is potentially very interesting, since XPC-deficient cell line presents low levels of APE1. Additionally, the results evidenced that APE1 protein can be involved in the repair of oxidative damage induced by RF*, because APE1 is recruited and bound strongly to chromatin after treatment.

El agua en la tradición lírica manierista: ríos y mares en las "Flores" de Pedro Espinosa (1605)

El presente trabajo aborda el recorrido del agua como imagen poética en sus dos principales manifestaciones —aguas dulces y saladas— en las "Flores de poetas ilustres de España" (1605), antología lírica ordenada por el antequerano Pedro Espinosa paradigmática del manierismo literario español. Desde la cita erudita hasta el símbolo, pasando por el particular tratamiento de la tópica clásica y petrarquista que le sirve de sustancia en un contexto contrarreformista, así como por la característica personificación de ríos y mares que distingue a la obra, se hace patente el protagonismo otorgado a este elemento tanto en la selección de textos como en su disposición y en la particular elocución de la retórica del movimiento. Como sucede con los juegos de agua del jardín manierista, integrando el agua de modo sustancial en su estética intelectualizante y preciosista y en sus diseños geométricos, el libro ofrece la oportunidad como ninguna otra de nuestra literatura áurea de apreciar la innovadora significación y versatilidad de este elemento como motivo poético.

The decrease of NAD(P)H:quinone oxidoreductase 1 activity and increase of ROS production by NADPH oxidases are early biomarkers in doxorubicin cardiotoxicity

info:eu-repo/semantics/publishedVersion

Cellular ROS imaging with hydro-Cy3 dye is strongly influenced by mitochondrial membrane potential

Background: Hydrocyanines are widely used as fluorogenic probes to monitor reactive oxygen species (ROS) generation in cells. Their brightness, stability to autoxidation and photobleaching, large signal change upon oxidation, pH independence and red/near infrared emission are particularly attractive for imaging ROS in live tissue. Methods: Using confocal fluorescence microscopy we have examined an interference of mitochondrial membrane potential (ΔΨm) with fluorescence intensity and localisation of a commercial hydro-Cy3 probe in respiring and non-respiring colon carcinoma HCT116 cells. Results: We found that the oxidised (fluorescent) form of hydro-Cy3 is highly homologous to the common ΔΨm-sensitive probe JC-1, which accumulates and aggregates only in ‘energised’ negatively charged mitochondrial matrix. Therefore, hydro-Cy3 oxidised by hydroxyl and superoxide radicals tends to accumulate in mitochondrial matrix, but dissipates and loses brightness as soon as ΔΨm is compromised. Experiments with mitochondrial inhibitor oligomycin and uncoupler FCCP, as well as a common ROS producer paraquat demonstrated that signals of the oxidised hydro-Cy3 probe rapidly and strongly decrease upon mitochondrial depolarisation, regardless of the rate of cellular ROS production. Conclusions: While analysing ROS-derived fluorescence of commercial hydrocyanine probes, an accurate control of ΔΨm is required. General significance: If not accounted for, non-specific effect of mitochondrial polarisation state on the behaviour of oxidised hydrocyanines can cause artefacts and data misinterpretation in ROS studies.

Kinetic modelling of in vitro data of PI3K, mTOR1, PTEN enzymes and on-target inhibitors Rapamycin, BEZ235, and LY294002

The phosphatidylinositide 3-kinases (PI3K) and mammalian target of rapamycin-1 (mTOR1) are two key targets for anti-cancer therapy. Predicting the response of the PI3K/AKT/mTOR1 signalling pathway to targeted therapy is made difficult because of network complexities. Systems biology models can help explore those complexities but the value of such models is dependent on accurate parameterisation. Motivated by a need to increase accuracy in kinetic parameter estimation, and therefore the predictive power of the model, we present a framework to integrate kinetic data from enzyme assays into a unified enzyme kinetic model. We present exemplar kinetic models of PI3K and mTOR1, calibrated on in vitro enzyme data and founded on Michaelis-Menten (MM) approximation. We describe the effects of an allosteric mTOR1 inhibitor (Rapamycin) and ATP-competitive inhibitors (BEZ2235 and LY294002) that show dual inhibition of mTOR1 and PI3K. We also model the kinetics of phosphatase and tensin homolog (PTEN), which modulates sensitivity of the PI3K/AKT/mTOR1 pathway to these drugs. Model validation with independent data sets allows investigation of enzyme function and drug dose dependencies in a wide range of experimental conditions. Modelling of the mTOR1 kinetics showed that Rapamycin has an IC50 independent of ATP concentration and that it is a selective inhibitor of mTOR1 substrates S6K1 and 4EBP1: it retains 40% of mTOR1 activity relative to 4EBP1 phosphorylation and inhibits completely S6K1 activity. For the dual ATP-competitive inhibitors of mTOR1 and PI3K, LY294002 and BEZ235, we derived the dependence of the IC50 on ATP concentration that allows prediction of the IC50 at different ATP concentrations in enzyme and cellular assays. Comparison of the drug effectiveness in enzyme and cellular assays showed that some features of these drugs arise from signalling modulation beyond the on-target action and MM approximation and require a systems-level consideration of the whole PI3K/PTEN/AKT/mTOR1 network in order to understand mechanisms of drug sensitivity and resistance in different cancer cell lines. We suggest that using these models in systems biology investigation of the PI3K/AKT/mTOR1 signalling in cancer cells can bridge the gap between direct drug target action and the therapeutic response to these drugs and their combinations.

Rapamycin reverses age-related increases in mitochondrial ROS production at complex I, oxidative stress, accumulation of mtDNA fragments inside nuclear DNA, and lipofuscin level, and increases autophagy, in the liver of middle-aged mice

Rapamycin consistently increases longevity in mice although the mechanism of action of this drug is unknown. In the present investigation we studied the effect of rapamycin on mitochondrial oxidative stress at the same dose that is known to increase longevity in mice (14 mg of rapamycin/kg of diet). Middle aged mice (16 months old) showed significant age-related increases in mitochondrial ROS production at complex I, accumulation of mtDNA fragments inside nuclear DNA, mitochondrial protein lipoxidation, and lipofuscin accumulation compared to young animals (4 months old) in the liver. After 7 weeks of dietary treatment all those increases were totally or partially (lipofuscin) abolished by rapamycin, middle aged rapamycin-treated animals showing similar levels in those parameters to young animals. The decrease in mitochondrial ROS production was due to qualitative instead of quantitative changes in complex I. The decrease in mitochondrial protein lipoxidation was not due to decreases in the amount of highly oxidizable unsaturated fatty acids. Rapamycin also decreased the amount of RAPTOR (of mTOR complex) and increased the amounts of the PGC1-α and ATG13 proteins. The results are consistent with the possibility that rapamycin increases longevity in mice at least in part by lowering mitochondrial ROS production and increasing autophagy, decreasing the derived final forms of damage accumulated with age which are responsible for increased longevity. The decrease in lipofuscin accumulation induced by rapamycin adds to previous information suggesting that the increase in longevity induced by this drug can be due to a decrease in the rate of aging. © 2016 Elsevier Inc.

Influence of Pseudomonas putida AF7 inoculation on soil enzymes.

Pseudomonas may use as bioremediator and as biopesticide. The use of soil enzymatic assays as biological indicator of possible negative effects in soil functioning was evaluated after P.putida AF7 inoculation. For that, AF7 was originally isolated from the rizosphere of rice and was inoculated on three soils: Rhodic Hapludox (RH), Typic Hapludox (TH); and Arenic Hapludult (AH). Soil characteristics were measured in each plot. Acid phosphatase, ?-glucosidase and protease activities were measured at 7, 14 and 21 days. The enzyme activity waved during the experimental period but there is a significant reduction of ?-glucosidase activity in RH soil on day 14. Corg was positively correlated to the activities of ?-glucosidase and protease. The presented data indicate that soil biochemical properties may be useful as indicator of soil perturbations.

Influence of pseudomonas putida af7 inoculation on soil enzymes activities.

2008

Influence of Pseudomonas putida AF7 inoculation on soil enzymes activities.

2008

VALORACIÓN ECONÓMICA DE LOS RECURSOS HÍDRICOS EN LAS CUENCAS DE LOS RÍOS BANANO Y BANANITO

Entre los meses de marzo y setiembre del 2004, el Centro Internacional en Política Económica para el Desarrollo Sostenible de la Universidad Nacional (CINPE-UNA) elaboró el estudio Valoración económica de los recursos hI’dricos en las cuencas de los rfos Banano y Bananito3 . El objetivo general de éste responde a “estimar el valor del recurso hídrico en las cuencas de los ríos Banano y Bananito, en función de los usos prioritarios, para la recomendación de mecanismos efectivos para la sostenibilidad de un sistema de compensación por los servicios hidrológicos”. Para cumplir con este objetivo se plantean los siguientes objetivos específicos: (1) identificar el método de valoración económica para el recurso hídrico más apropiado dadas las condiciones de la zona, (2) cuantificar los servicios hidrológicos en las cuencas de los ríos Banano y Bananito en función de los principales usos, y (3) recomendar un mecanismo de pago voluntario por el servicio ambiental hídrico, basado en los resultados del estudio, que provea al Área de Conservación La Amistad Caribe (ACLA-C) los elementos para el desarrollo de una experiencia concreta.Producto de una exhaustiva revisión bibliográfica y dados los objetivos del estudio se seleccionó el Análisis de Múltiples Criterios (AMC), como la metodología marco para la valoración económica. Esto implica la aplicación de un método no tradicional, que permite combinar variables cuantitativas y cualitativas. A este respecto, el uso de una metodología participativa y multisectorial hizo posible la participación de los principales grupos de actores usuarios del agua. que se clasificaron en cinco grupos: (1) Asociaciones Administradoras de Acueductos Rurales (ASADAS) y acueductos rurales, (2) ONG’s y organizaciones comunales, (3) sociedad civil, (4) empresa privada y (5) sector público.Luego de sistematizar la información recabada durante las 23 sesiones de consulta participativa. se procede al análisis de los datos, que sirven de insumos para realizar la valoración económica y, finalmente, el diseño de la propuesta de mecanismos de cobro por grupo de usuarios para el pago por servicios ambientales. Los resultados finales se presentan mediante el uso de matrices y gráficos de telaraña.Este informe presenta los avances correspondientes al primer objetivo específico. Asimismo, incluye los comentarios y las sugerencias realizadas durante y posterior al taller de discusión con representantes de PROARCA-APM, TNC y ACLA-C. El documento contiene la caracterización económica y social del área en estudio, una revisión de estudios de valoración, y métodos, así como el método de valoración seleccionado y su forma de aplicación.A bstract:Between March and September 2004, the International Center of Economic Policy for Sustainable Development of the National University (CINPE.IJNA) elaborated the study Economic Valuation of ihe Water Resources in ihe Basins of ihe rivers Banano and Bananito4. The general objcctive of it answers to “estimate the value of the water resource in the basin of the rivers Banano and Bananito, depending on the prior uses, for the recommendation of effective mechanisms for the sustainability of a system of compensation for the hydrological serv ices”.The following specific objectives appear to reach the general objective: (1) To identify the most appropriate method of economic valuation for water resource given (he conditions of the zone. (2) To quantify the hydrological services in the basins of the rivers Banano and Bananito depending on (he principal uses. And (3) to recommend a mechanism of voluntary payment for the environmental water service based on the results of the study, which provides to La Amistad Caribe Conservation Area (ACLA-C), the elements for the development of a concrete experience.Product and given of an exhaustive bibliographical review, the Analysis of Multiple Criteria (AMC) is selected as (he methodology for the economic valuation. It implies (he application of a not traditional method, which allows combining quantitative and qualitative variables. In this regard, the use of a participatory methodoiogy there made possible the participation of the principal groups of actors users of the water, who quatified ¡n five groups to knowing, (1) administrative associations of rural aqueducts (ASADAS), (2) ONG and communal organizations, (3) civil society, (4) private sector and (5) public sector.After systematizing the information obtained during 23 meetings of participatory consultation. one proceeds to the analysis of the information, which use as ¡nputs to realize the economic valuation. Finally, the design of the offer of collection mechanisms for user group for the payment for environmental services is presented. The final results appear by means of the use of counterfoils and spider web graphs.This repon includes the commentaries and suggestions realized during and later to ihe workshop of discussion with representatives of PROARCA-APM, TNC and ACLA-C. The document also contains the economic and social characterization of the area under study, a review of studies of valuation, and methods. as well as the method of valuation selected and its form of application.