Identificación de la gelsolina como posible biomarcador de las enfermedades mitocondriales del sistema OXPHOS

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina. Departamento de Bioquímica. Fecha de lectura: 3-02-2017.Esta tesis tiene embargado el acceso al texto completo hasta el 03-08-2018Las mitocondrias son orgánulos intracelulares cuya principal función es generar energía celular en forma de adenosín trifosfato (ATP), a partir de un proceso conocido como fosforilación oxidativa (OXPHOS). El sistema OXPHOS tiene un origen bi-genómico y está formado por cinco complejos enzimáticos multiproteicos, que catalizan la síntesis de ATP acoplada a un potencial electroquímico de protones en la membrana interna mitocondrial. Las enfermedades que afectan al sistema OXPHOS constituyen la primera causa de errores congénitos del metabolismo energético y están asociados a un grupo heterogéneo de enfermedades neuromusculares o multisistémicas de severidad variable, que pueden manifestarse tanto en la infancia como en adultos. Su variabilidad genética, clínica y fenotípica, junto con la inexistencia de biomarcadores suficientemente específicos, dificulta enormemente el diagnóstico de las enfermedades mitocondriales OXPHOS. Este trabajo se ha centrado en la identificación, validación y caracterización funcional de nuevos biomarcadores proteicos de enfermedades mitocondriales del sistema OXPHOS. Estudios previos revelaron que en células con un déficit enzimático del complejo III, se producía un aumento significativo de una proteína implicada en el mantenimiento del citoesqueleto: la Gelsolina (GSN) citosólica (Marín-Buera et al., 2015). Los resultados obtenidos en este trabajo demuestran que la GSN se acumula específicamente en las mitocondrias de células humanas como respuesta a las alteraciones enzimáticas de los complejos de la cadena respiratoria. La GSN se localiza en la membrana externa mitocondrial, donde forma parte de un complejo de 150 kDa e interactúa con la proteína del poro de permeabilidad de membrana VDAC1. La disminución de los niveles de GSN conduce a un aumento específico de la dimerización de VDAC1 y de la liberación del Citocromo c mitocondrial al citosol, sin alterar las estructuras supramoleculares de VDAC1 ni la estabilidad de los complejos OXPHOS. Además, el tratamiento con estaurosporina provoca la proteolización de la GSN específicamente en la mitocondria de células con déficits enzimáticos de cadena respiratoria. Los niveles de la isoforma plasmática de la GSN disminuyen significativamente, en respuesta a déficits enzimáticos de cadena respiratoria, tanto en modelos celulares como en muestras sanguíneas de pacientes con enfermedad mitocondrial. Por ello, se propone que la ratio entre los niveles de GSN mitocondrial y plasmática podría servir como un nuevo indicador de los cambios moleculares asociados a la disfunción de la cadena respiratoria mitocondrialMitochondria are intracellular organelles that generate most of the cellular energy in the term of adenosine triphosphate (ATP) in a process known as oxidative phosphorylation (OXPHOS). The OXPHOS system, of dual genetic origin, is located in the mitochondrial inner membrane. It consists of five multiprotein enzyme complexes, which are responsible for the transfer of electrons coupled to the generation of the proton gradient in the mitochondrial intermembrane space, which is used by the H+-ATP synthase to produce ATP. The OXPHOS system enzyme deficiencies are the leading cause of the inborn errors of energy metabolism. They comprise a heterogeneous group of neuromuscular and multisystemic disorders of variable severity that are present in both childhood and adulthood. Currently, there are very limited means for objectively monitoring disease progression, and non invasive ways to measure respiratory chain disorders based on the use of specific biomarkers are not currently available. Consequently, the diagnosis of these diseases can be challenging. This work has focused on the identification, validation and functional characterization of novel protein biomarkers for mitochondrial diseases. Previous studies showed that the citosolic isoform of Gelsolin (GSN), a cytoskeleton maintenance protein, was significantly overexpressed in complex III-deficient cells (Marín-Buera et al., 2015). In this work, we show that the increase in GSN levels occurs specifically in the mitochondria from human cells with respiratory chain enzyme deficiencies. Gelsolin is located in the mitochondrial outer membrane, where it forms a ~150 kDa complex and it interacts with the voltage-dependent annion channel protein 1 (VDAC1). GSN downregulation led to the specific accumulation of VDAC1 dimers and to the mitochondrial Cytochrome c release into the cytosol, without a major effect on the levels of larger VDAC1- containing complexes or on the stability of the OXPHOS system. Moreover, citosolic GSN is specifically proteolyzed in the mitochondrial fraction from OXPHOS-deficient cells upon staurosporine treatment. In addition, the levels of the plasma isoform of GSN were significantly decreased in the extracellular media from cells with OXPHOS deficiencies and in blood samples from patients biochemically and/or genetically diagnosed with mitochondrial disease. Therefore, the ratio between mitochondrial and plasma GSN is proposed as a novel indicator of the molecular alterations associated with mitochondrial respiratory chain dysfunction.El presente trabajo ha sido financiado por los proyecto de investigación del Instituto de Salud Carlos III (números FIS 11-00182 y FIS 14-00209), así como por el proyecto del National Institute of General Medical Sciences (NIGMS-NIH, número 1R01GM105781-01) concedidos a la Dra. Cristina Ugalde Bilbao

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Features of the annual evolution of CO2 and CH4 in the atmosphere of a Mediterranean climate site studied using a nonparametric and a harmonic function

Concentrations of CO2 and CH4 measured over 3 years at a rural site in the Spanish northern plateau were investigated together with vegetation and meteorological variables. Two procedures were implemented to study the annual evolution. Kernel estimation provided a detailed time description, and the harmonic model may be fitted easily. The site was characterised by grass from autumn to spring. However, vigorous growth was observed during the latter season due to the biological cycle of plants under favourable meteorological conditions. A CO2 peak was observed a fortnight before the time of maximum NDVI, and was attributed to the prevalence of respiration over photosynthesis. A pronounced trough was apparent in summer and was explained by the death of vegetation and active dispersion in a highly developed boundary layer. CH4 evolution was characterised by a deficit period from May to October, indicating that meteorological evolution played a key role. The harmonic model showed that annual and half-annual cycles evidenced a similar contribution for CO2, whereas said weight for the half-annual cycle was considerably smaller for CH4.Ministry of Economy and Competitiveness and ERDF funds (grant numbers CGL2009-11979 and CGL2014-53948-P

Trend analysis of CO2 and CH4 recorded at a semi-natural site in the northern plateau of the Iberian Peninsula

CO2 and CH4 were recorded from October 2010 to February 2016 with a Picarro G1301 analyser at the centre of the upper plateau of the Iberian Peninsula. Large CO2 values were observed during the vegetation growing season, and were reinforced by the stable boundary layer during the night. Annual CH4 evolution may be explained by ecosystem activity and by the dispersion linked with the evolution of the boundary layer. Their trends were studied using an equation that considers one polynomial and one harmonic part. The polynomial part revealed an increasing trend from 0.8 to 2.3 ppm year 1 for CO2 and from 0.004 to 0.011 ppm year 1 for CH4. The harmonic part considered four harmonics whose amplitudes were noticeable for the first and second harmonics for CO2 and for the first harmonic for CH4. Long-term evolution was similar with alternative equations. Finally, seasonal study indicated summer minima for both gases, which may be explained by the lack of vegetation in this season. Harmonic analysis showed two maxima for CO2, one in spring linked with vegetation growth, which decreased with time, and another in autumn related with the onset of plant activity after the summer, which increased with time. CH4 presented only one maximum in winter and a short time with steady concentration in spring where the evolution of the boundary layer may play a noticeable role. The harmonic equation, which takes into account all the observations, revealed opposite behaviour between CO2, whose minima decreased, and CH4, whose maxima increased.Ministry of Economy and Competitiveness and ERDF funds (projects CGL2009-11979 and CGL2014-53948-P)

Boundaries of air mass trajectory clustering: key points and applications

Calculating air mass trajectories is common in atmospheric analyses. However, if explainable results are to be achieved, several procedures are needed to process the vast amount of information handled. Clustering methods are statistical tools usually considered for such a purpose. Although they are based on rigorous algorithms, certain questions still remain when these methods are applied. The current review is organised in sections according to the sequence followed by such procedures. First, the types of clustering methods are described, with their core being the distance used. One key point is the stopping rule, which determines the final number of clusters. A simple classification based on this number is then suggested. Finally, the graphical presentation of the results is examined and the main drawbacks are commented on. A range of applications and results are considered to illustrate each section, and certain caveats and recommendations are also presented.Ministry of Economy and Competitiveness, Spain and ERDF funds (grant numbers CGL2009-11979 and CGL2014-53948-P

Trend analysis and outlier distribution of CO2 and CH4: A case study at a rural site in northern Spain

Producción CientíficaCO2 and CH4 outliers may have a noticeable impact on the trend of both gases. Nine years of measurements since 2010 recorded at a rural site in northern Spain were used to investigate these outliers. Their influence on the trend was presented and two limits were established. No more than 23.5% of outliers should be excluded from the measurement series in order to obtain representative trends, which were 2.349 ± 0.012 ppm year−1 for CO2 and 0.00879 ± 0.00004 ppm year−1 for CH4. Two types of outliers were distinguished. Those above the trend line and the rest below the trend line. Outliers were described by skewed distributions where the Weibull distribution figures prominently in most cases. A qualitative procedure was presented to exclude the worst fits, although five statistics were considered to select the best fit. In this case, the modified Nash-Sutcliffe efficiency is prominent. Finally, three symmetrical distributions were added to fit the observations when outliers are excluded, with the Gaussian and beta distributions providing the best fits. As a result, certain skewed functions, such as the lognormal distribution, whose use is frequent for air pollutants, could be questioned in certain applications.Ministerio de Economía y Competitividad y fondos FEDER, (project numbers CGL-2009-11979 and CGL2014-53948-P

Influence of wind speed on CO2 and CH4 concentrations at a rural site

Producción CientíficaMeteorological variables have a noticeable impact on pollutant concentrations. Among these variables, wind speed is typically measured, although research into how pollutants respond to it can be improved. This study considers nine years of hourly CO2 and CH4 measurements at a rural site, where wind speed values were calculated by the METEX model. Nine wind speed intervals are proposed where concentrations, distribution functions, and daily as well as annual cycles are calculated. Contrasts between local and transported concentrations are around 5 and 0.03 ppm for CO2 and CH4, respectively. Seven skewed distributions are applied, and five efficiency criteria are considered to test the goodness of fit, with the modified Nash–Sutcliffe efficiency proving to be the most sensitive statistic. The Gumbel distribution is seen to be the most suitable for CO2, whereas the Weibull distribution is chosen for CH4, with the exponential function being the worst. Finally, daily and annual cycles are analysed, where a gradual decrease in amplitude is observed, particularly for the daily cycle. Parametric and nonparametric procedures are used to fit both cycles. The latter gave the best fits, with the agreement being higher for the daily cycle, where evolution is smoother than for the annual cycle.Ministerio de Economía, Industria y Competitividad y Fondo Europeo de Desarrollo Regional (FEDER) - (Projects CGL-2009-11979 and CGL2014-53948-P)Junta de Castilla y León - (Project VA027G19

Snail1 induces IL-17 expression to inhibit adipogenesis

44 p.-7 fig.-1 tab. Peláez-García, Alberto et al.Adipogenesis requires a differentiation program driven by multiple transcription factors, where PPARγ and C/EBPα play a central role. Recent findings indicate that Snail inhibits adipocyte differentiation in 3T3-L1 and murine mesenchymal stem cells (mMSC). An in-depth quantitative SILAC analysis of the nuclear fraction of Snail-induced alterations of 3T3-L1 cells was carried out. In total, 2251 overlapping proteins were simultaneously quantified in forward and reverse experiments. We observed 574 proteins deregulated by Snail1 using a fold-change ≥1.5, with 111 up- and 463 down-regulated proteins, respectively. Among other proteins, multiple transcription factors such as Trip4, OsmR, Nr2f6, Cbx6, and Prrx1 were down-regulated. Results were validated in 3T3-L1 cells and mMSC cells by Western blot and quantitative PCR. Knock-down experiments in 3T3-L1 cells demonstrated that only Nr2f6 (and Trip4 at minor extent) was required for adipocyte differentiation. Ectopic expression of Nr2f6 reversed the effects of Snail1 and promoted adipogenesis. Because Nr2f6 inhibits the expression of IL-17, we tested the effect of Snail on IL-17 expression. IL-17 and TNFα were among the most up-regulated pro-inflammatory cytokines in Snail-transfected 3T3-L1 and mMSC cells. Furthermore, the blocking of IL-17 activity in Snail-transfected cells promoted adipocyte differentiation, reverting Snail inhibition. In summary, Snail inhibits adipogenesis through a down-regulation of Nr2f6, which in turn facilitates the expression of IL-17, an anti-adipogenic cytokine. These results would support a novel and important role for Snail and Nr2f6 in obesity control.This research was supported by a grant to established research groups (AECC), grant BIO2012-31023 from the Ministry of Economy and Competitiveness, grant S2011/BMD-2344/ (Colomics2) from Comunidad de Madrid and ProteoRed-ISCIII support. Work at AGH’s lab was supported by a grant from the Ministry of Economy and Competitiveness (SAF2010-16089)Peer reviewe

SCOPE model applied for rapeseed in Spain

The integrated SCOPE (Soil, Canopy Observation, Photochemistry and Energy balance) model, coupling radiative transfer theory and biochemistry, was applied to a biodiesel crop grown in a Spanish agricultural area. Energy fluxes and CO2 exchange were simulated with this model for the period spanning January 2008 to October 2008. Resultswere compared to experimentalmeasurements performed using eddy covariance and meteorological instrumentation. The reliability of the model was proven by simulating latent (LE) and sensible (H) heat fluxes, soil heat flux (G), and CO2 exchanges (NEE and GPP). LAI data used as input in the model were retrieved from the MODIS and MERIS sensors. SCOPE was able to reproduce similar seasonal trends to those measured for NEE, GPP and LE. When considering H, the modelled values were underestimated for the period covering July 2008 to mid-September 2008. The modelled fluxes reproduced the observed seasonal evolution with determination coefficients of over 0.77 when LE and H were evaluated. The modelled results offered good agreement with observed data for NEE and GPP, regardless of whether LAI data belonged to MODIS or MERIS, showing slopes of 0.87 and 0.91 for NEE-MODIS and NEE-MERIS, and 0.91 and 0.94 for GPP-MODIS and GPP-MERIS, respectively. Moreover, SCOPE was able to reproduce similar seasonal behaviou s to those observed for the experimental carbón fluxes, clearly showing the CO2 sink/source behaviour for the whole period studied