Caracterización e identificación de 449 accesiones de Vitis vinifera L. procedentes de dos colecciones ampelográficas

Vitis vinifera L. és una de les moltes espècies que està sotmesa a una important erosió genètica. Un primer pas per a la preservació d’aquesta espècie és conèixer bé la seva biodiversitat, és per això que el present treball aborda l’estudi de la caracterització molecular de 449 accessions de Vitis vinifera L. procedents de 23 països mitjançant la tècnica dels microsatèl•lits. Prèviament a la caracterització, es fonamental fer una bona extracció d’ADN, ja que sinó, es pot veure compromès el procés d’amplificació. En aquesta tesi s’han dissenyat dues metodologies d’extracció d’ADN. La més polivalent es caracteritza per poder extraure ADN pur de diferents tipus de mostres (tan de fulla jove com adulta, sarment (fusta) i de llavor) en unes 2 h 30 minuts (sent ràpida com un “kit” comercial, però aquest només pot extreure a partir de teixit foliar). La segona metodologia és més ràpida, donat que en només 90 minuts es realitza l’extracció d’ADN, però queda restringida per a mostres de fulla, tan jove com adulta. Posteriorment s’aborda l’estudi de caracterització varietal on s’han identificat 340 varietats diferents, quedant palesa la problemàtica dels errors comesos en denominació de les accessions. Aquests resultats han servit per establir els fonaments per a la creació d’una Base de Dades de Microsatèl•lits d’identificació varietal de Vitis vinifera L. de la Universitat Rovira i Virgili. Finalment, els resultats obtinguts en l’estudi de l’estructura genètica suggereixen l’existència de 4 nous centres de domesticació: 2 localitzats al nord-est i al centre sud-oest de la Península Ibèrica, i 2 més localitzats al sud-est i nord-est de França.La Vitis vinifera L. es una de las muchas especie que se encuentra sometida a una importante erosión genética. Un primer paso para la preservación de esta especie es conocer bien su biodiversidad, es por ello que el presente trabajo aborda el estudio de la caracterización molecular de 449 accesiones de Vitis vinifera L. procedentes de 23 países mediante la técnica de los microsatélites. Previamente a la caracterización, es fundamental realizar una buena extracción de ADN, ya que si no, se puede ver afectado el proceso de amplificación. En esta tesis se han diseñado dos metodologías de extracción de ADN. La más polivalente se caracteriza por poder extraer ADN puro de distintos tipos de muestra (tanto de hoja joven como adulta, sarmiento (madera) y de semilla) en unas 2 h y 30 minutos (siendo rápida como un “kit” comercial, pero éste sólo puede extraer a partir de tejido foliar). La segunda metodología es más rápida, dado que en sólo 90 minutos se realiza la extracción de ADN, pero queda restringida para muestra de hoja, tanto joven como adulta. Posteriormente se aborda el estudio de caracterización varietal donde se han identificado 340 variedades distintas, siendo clara la problemática de los errores en la denominación de las accesiones. Estos resultados han servido para sentar las bases para la creación de un Base de Datos de Microsatélites de identificación varietal de Vitis vinifera L. de la Universidad Rovira i Virgili. Finalmente, los resultados obtenidos en el estudio de la estructura genética sugieren la existencia de 4 nuevos centros de domesticación: 2 localizados al noreste y al centro suroeste de la Península Ibérica, y 2 más localizados al sureste y noreste de Francia.Vitis vinifera L. is one of the species which is subject to important genetic erosion. A first step for the preservation of this species is to know its biodiversity, for this reason this paper deals with the study of the molecular characterization of 449 accessions of Vitis vinifera L. from 23 countries by the microsatellites analysis. Prior the characterization, it is essential to carry out a good DNA extraction. In this thesis two methods of DNA extraction have been designed. The most versatile extracts pure DNA from different types of samples (both young and adult leaf, stem (wood) and seed) in about 2 hours and 30 minutes (fast like a commercial kit, but this only useful to extract DNA from leaf. The second method is faster, because in just 90 minutes the DNA extraction is performed, but is restricted to leaves, both young and adult. Subsequently, it is tackled the study of varietal characterization, 340 different varieties are identified; being present the problems of mislabeling. These results are the groundwork for the creation of a Vitis vinifera Microsatellite Database of the Universitat Rovira i Virgili. Finally, the results obtained in the study of the genetic structure suggest the existence of 4 new Centers of Domestication: 2 are located in the northeast and centre-southwest of the Iberian Peninsula, and 2 more are located in the southeast and northeast of France

ATP crossing the cell plasma membrane generates an ionic current in Xenopus oocytes

The presence of ATP within cells is well established. However, ATP also operates as an intercellular signal via specific purinoceptors. Furthermore, nonsecretory cells can release ATP under certain experimental conditions. To measure ATP release and membrane currents from a single cell simultaneously, we used Xenopus oocytes. We simultaneously recorded membrane currents and luminescence. Here, we show that ATP release can be triggered in Xenopus oocytes by hyperpolarizing pulses. ATP release (3.2 +/- 0.3 pmol/oocyte) generated a slow inward current (2.3 +/- 0.1 microA). During hyperpolarizing pulses, the permeability for ATP(4-) was more than 4000 times higher than that for Cl(-). The sensitivity to GdCl(3) (0. 2 mm) of hyperpolarization-induced ionic current, ATP release and E-ATPase activity suggests their dependence on stretch-activated ion channels. The pharmacological profile of the current inhibition coincides with the inhibition of ecto-ATPase activity. This enzyme is highly conserved among species, and in humans, it has been cloned and characterized as CD39. The translation, in Xenopus oocytes, of human CD39 mRNA encoding enhances the ATP-supported current, indicating that CD39 is directly or indirectly responsible for the electrodiffusion of ATP

Resultats de la tècnica dels microsatèl·lits o SSR aplicada al trepat

L?objectiu d?aquest treball és fer la caracterització de la varietat trepat mitjançant la tècnica dels microsatèl·lits (SSR), i a partir del germoplasma de Bodegues Sumarroca, SL. Es tracta, doncs, de fer una exhaustiva revisió de les bases de dades ampelogràfiques, de la bibliografia i de tota la informació en línia, i comparar-la amb els resultats obtinguts a partir de l?estudi del genoma (microsatèl·lits en aquest cas).El objetivo de este trabajo es hacer la caracterización de la variedad trepat mediante la técnica de microsatélites (S. S. R.) y a partir del germoplasma de Bodegas Sumarroca, S. L. Se trata, pues, de hacer una exhaustiva revisión de las bases de datos ampelográficas, de la bibliografía y de toda la información en línea y compararla con los resultados obtenidos a partir del estudio del genoma (microsatélites en este caso)

Resultats de la tècnica dels microsatèl·lits o SSR aplicada al trepat

L?objectiu d?aquest treball és fer la caracterització de la varietat trepat mitjançant la tècnica dels microsatèl·lits (SSR), i a partir del germoplasma de Bodegues Sumarroca, SL. Es tracta, doncs, de fer una exhaustiva revisió de les bases de dades ampelogràfiques, de la bibliografia i de tota la informació en línia, i comparar-la amb els resultats obtinguts a partir de l?estudi del genoma (microsatèl·lits en aquest cas).El objetivo de este trabajo es hacer la caracterización de la variedad trepat mediante la técnica de microsatélites (S. S. R.) y a partir del germoplasma de Bodegas Sumarroca, S. L. Se trata, pues, de hacer una exhaustiva revisión de las bases de datos ampelográficas, de la bibliografía y de toda la información en línea y compararla con los resultados obtenidos a partir del estudio del genoma (microsatélites en este caso)

Estudi de l'evolució de la climatologia a la Conca de Barberà en els darrers cinquanta anys i les possibles repercussions en la viticultura del segle XXI

L'estudi s'ha dut a terme a Montblanc (capital de la Conca de Barberà). S'han analitzat tendències de temperatures i pluviometries per a cada estadi fenològic de la vinya, amb la finalitat de poder determinar els impactes sobre la viticultura de la zona. La base de dades correspon a l'observatori de Montblanc (coordenades UTM: X: 341.175; Y: 4.584.350; Z: 441 m s. n. m.). La sèrie climàtica de temperatures va des de l'any 1950 fins al 2007 i la pluviometria va des de l'any 1914 fins al 2007. Es poden observar tendències creixents de temperatures màximes i una lleugera pujada de les temperatures mínimes. Les precipitacions disminueixen progressivament cada any i es concentren en determinades èpoques de l'any, per tant, es desestacionalitzen els períodes de pluges, i s'incrementa el dèficit hídric entre la brotada i el verol (és important recalcar que en aquest període l'estrès hídric ha de ser nul).El estudio se ha llevado a cabo en Montblanc (capital de la Conca de Barberà). Se ha analizado tendencias de temperaturas y pluviometrías para cada estado fenológico de la vid, con la finalidad de poder determinar los impactos sobre la viticultura de la zona. La base de datos corresponde al observatorio de Montblanc (coordenadas UTM: X: 341.175; Y: 4.584.350; Z: 441 m s. n. m.) La serie climática de temperaturas va desde el año 1950 hasta el 2007 y la pluviometría va desde el año 1914 hasta el 2007. Se pueden observar tendencias crecientes de temperaturas máximas y una ligera subida de las temperaturas mínimas. Las precipitaciones disminuyen progresivamente cada año y se concentran en determinadas épocas del año, por lo tanto se desestacionalizan los periodos de lluvia y se incrementa el déficit hídrico entre el brote y el envero (es importante recalcar que en este periodo el estrés hídrico ha de ser nulo)

G9a Inhibition Promotes Neuroprotection through GMFB Regulation in Alzheimer’s Disease

Epigenetic alterations are a fundamental pathological hallmark of Alzheimer’s disease (AD). Herein, we show the upregulation of G9a and H3K9me2 in the brains of AD patients. Interestingly, treatment with a G9a inhibitor (G9ai) in SAMP8 mice reversed the high levels of H3K9me2 and rescued cognitive decline. A transcriptional profile analysis after G9ai treatment revealed increased gene expression of glia maturation factor β (GMFB) in SAMP8 mice. Besides, a H3K9me2 ChIP-seq analysis after G9a inhibition treatment showed the enrichment of gene promoters associated with neural functions. We observed the induction of neuronal plasticity and a reduction of neuroinflammation after G9ai treatment, and more strikingly, these neuroprotective effects were reverted by the pharmacological inhibition of GMFB in mice and cell cultures; this was also validated by the RNAi approach generating the knockdown of GMFB/Y507A.10 in Caenorhabditis elegans. Importantly, we present evidence that GMFB activity is controlled by G9a-mediated lysine methylation as well as we identified that G9a directly bound GMFB and catalyzed the methylation at lysine (K) 20 and K25 in vitro. Furthermore, we found that the neurodegenerative role of G9a as a GMFB suppressor would mainly rely on methylation of the K25 position of GMFB, and thus G9a pharmacological inhibition removes this methylation promoting neuroprotective effects. Then, our findings confirm an undescribed mechanism by which G9a inhibition acts at two levels, increasing GMFB and regulating its function to promote neuroprotective effects in age-related cognitive decline</p

Protective effects of the succinate/SUCNR1 axis on damaged hepatocytes in NAFLD

Objective: Succinate and succinate receptor 1 (SUCNR1) are linked to fibrotic remodeling in models of non-alcoholic fatty liver disease (NAFLD), but whether they have roles beyond the activation of hepatic stellate cells remains unexplored. We investigated the succinate/SUCNR1 axis in the context of NAFLD specifically in hepatocytes. Methods: We studied the phenotype of wild-type and Sucnr1-/- mice fed a choline-deficient high-fat diet to induce non-alcoholic steatohepatitis (NASH), and explored the function of SUCNR1 in murine primary hepatocytes and human HepG2 cells treated with palmitic acid. Lastly, plasma succinate and hepatic SUCNR1 expression were analyzed in four independent cohorts of patients in different NAFLD stages. Results: Sucnr1 was upregulated in murine liver and primary hepatocytes in response to diet-induced NASH. Sucnr1 deficiency provoked both beneficial (reduced fibrosis and endoplasmic reticulum stress) and detrimental (exacerbated steatosis and inflammation and reduced glycogen content) effects in the liver, and disrupted glucose homeostasis. Studies in vitro revealed that hepatocyte injury increased Sucnr1 expression, which when activated improved lipid and glycogen homeostasis in damaged hepatocytes. In humans, SUCNR1 expression was a good determinant of NAFLD progression to advanced stages. In a population at risk of NAFLD, circulating succinate was elevated in patients with a fatty liver index (FLI) ≥60. Indeed, succinate had good predictive value for steatosis diagnosed by FLI, and improved the prediction of moderate/severe steatosis through biopsy when added to an FLI algorithm. Conclusions: We identify hepatocytes as target cells of extracellular succinate during NAFLD progression and uncover a hitherto unknown function for SUCNR1 as a regulator of hepatocyte glucose and lipid metabolism. Our clinical data highlight the potential of succinate and hepatic SUCNR1 expression as markers to diagnose fatty liver and NASH, respectively.This study was supported by grants from MCIN/AEI/10.13039/501100011033 (SAF2015-65019-R, RTI2018-093919-B-100 and PID2021-122480OB-100 to S.F.-V.; PID2021-122766OB-100 to A.M.V; PID2021-124425OB-I00 to P.A.) co-financed by the European Regional Development Fund (ERDF) and Grupos consolidados Gobierno Vasco IT1476-22 to P.A. This research was funded by the Institute of Health “Carlos III” (ISCIII) and co-financed by ERDF (PI20/00095 to V.C.-M.; PI20/00338 to J.V. and PI20/00505 to B.R.-M.). This study was also supported by a grant from ISCIII and CIBERDEM, DEM19PI01/2019 to V.C.-M. and P.R. The project that gave rise to these results received funding from “La Caixa” Foundation (ID 100010434), under the grant agreement LCF/PR/HR20/52400013 (to S.F.-V.). This study was also supported by Rovira i Virgili University and Tarragona Provincial Council with the Talent Salut fellowship to A.R.-C. A.M.-B. is a recipient of an FPU grant (FPU20/05633) from MCIN/AEI/10.13039/501100011033. B.A. acknowledges support from the PERIS program 2016–2020 (LT017/20/000033), from Departament de Salut de la Generalitat de Catalunya. V.C.-M. acknowledges support from the Ramón y Cajal program (RYC2019-02649-I), from MCIN/AEI/10.13039/501100011033/ and the European Social Fund (ESF), “Investing in your future”. B.R.-M. acknowledges support from the Miguel Servet Type I program (CP19/00098) from the Fondo de Investigación Sanitaria, co-financed by the ERDF. SFV and JVO acknowledge support from the Agency for Management of University Research Grants of the Generalitat de Catalunya (2021 SGR 01409, 2021 SGR 0089). The study was also supported by CIBER–Consorcio Centro de Investigación Biomédica en Red (CB07708/0012), ISCIII, Ministerio de Ciencia e Innovación

Comparison of the efficiency of some of the most usual DNA extraction methods for woody plants in different tissues of <em>Vitis vinifera</em> L.

Aim: To compare different methods for extracting DNA from non-recalcitrant and recalcitrant tissues of Vitis vinifera woody plants and propose a modification of a previously published method to reduce the time and cost of extraction. Methods and results: DNA was extracted from young and mature leaves as well as from stems and seeds using some of the most common methods of DNA isolation and two commercial kits. Another commercial kit, which does not require DNA extraction prior to PCR, was also used. Only two methods provided adequate results in all tissues. Other methods were only applicable to some tissues and some did not yield any functional DNA in any tissue. A modification of the method reported by Marsal et al. (2011) is proposed to reduce handling time and cost. Conclusion: All of the methods studied here use a surfactant to improve the extractions. For DNA extraction from recalcitrant tissues to be optimal, it is best to use a combination of dodecyltrimethylammonium bromide (DTAB) and cetyltrimethylammonium bromide (CTAB). The changes made to the protocol reported by Marsal et al. (2011) enable functional DNA to be obtained from leaves in only 90 minutes and at very low cost (17 €/8 samples). However, this method cannot adequately isolate DNA from recalcitrant tissues (stems and seeds) and so, for this type of sample, we would recommend using the original method. Significance and impact of the study: Nowadays, handling time and cost are key factors in selecting the most suitable DNA extraction method. This study compares not only the effectiveness of the various methods but also the handling time and cost. It also proposes a modification of the fastest and most economic DNA extraction method for leaves so that handling time and processing cost will be reduced even further