Unraveling the role of Arabidopsis ALIX in the trafficking and turnover of abscisic acid receptors

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 22-11-2019Esta tesis tiene embargado el acceso al texto completo hasta el 22-05-2021The plant endosomal trafficking pathway controls the abundance of membrane-associated soluble proteins, as shown for abscisic acid (ABA) receptors of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) family. ABA receptor targeting for vacuolar degradation occurs through the late endosome route and depends on FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FYVE1) and VACUOLAR PROTEIN SORTING 23A (VPS23A), components of the endosomal sorting complex required for transport (ESCRT)-I complexes. FYVE1 and VPS23A interact with ALG-2 INTERACTING PROTEIN-X (ALIX), an ESCRT-III-associated protein, although the functional relevance of such interactions and their consequences in cargo sorting are unknown. Here we show that Arabidopsis thaliana ALIX directly binds to ABA receptors in late endosomes, promoting their degradation. Impaired ALIX function leads to altered endosomal localization and increased accumulation of ABA receptors. In line with this, partial loss-of-function alix-1 mutants display ABA hypersensitivity during growth and stomatal closure, unveiling a role for the ESCRT machinery in the control of water loss through stomata. ABA hypersensitive responses are suppressed in alix-1 plants impaired in PYR/PYL/RCAR activity, in accordance with ALIX affecting ABA responses primarily by controlling ABA receptor stability. ALIX-1 mutant protein displays reduced interaction with VPS23A and ABA receptors, providing a molecular basis for ABA hypersensitivity in alix-1 mutants. Our findings unveil a negative feedback mechanism triggered by ABA that acts via ALIX to control the accumulation of specific PYR/PYL/RCAR receptors.El tráfico endosomal permite a las plantas controlar la abundancia de proteínas transmembrana y proteínas solubles asociadas transitoriamente a ella como ocurre con los receptores de ácido abscísico de la familia PYR/PYL/RCAR (PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS). La degradación vacuolar de estos receptores ocurre a través de la ruta endosomal y está mediada por FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FYVE1) y VACUOLAR PROTEIN SORTING 23A (VPS23A), dos proteínas pertenecientes al complejo ESCRT-I (endosomal sorting complex required for transport). Tanto FYVE1 como VPS23A son capaces de interaccionar con ALIX (ALG-2 INTERACTING PROTEIN-X), una proteína asociada al complejo ESCRT-III. Sin embargo, la relevancia funcional de dichas interacciones y su implicación en la selección y tráfico de proteínas cargo se desconoce. En este trabajo mostramos cómo la proteína ALIX de Arabidopsis thaliana es capaz de interaccionar directamente con los receptors de ABA en endosomas tardíos, provomiendo su degradación. De hecho, alteraciones en la función de ALIX conducen a una acumulación de los receptores de ABA y a una localización subcelular alterada de los mismos. En línea con esto, los mutantes de pérdida de función alix-1, muestran hipersensibilidad al ABA durante el desarrollo y en el cierre estomático, revelando un nuevo papel de la maquinaria ESCRT en el control de la pérdida de agua a través de los estomas. Estas respuestas de hipersensibilidad que presentan los mutantes alix-1 son suprimidas cuando se elimina parcialmente la actividad de los receptores de ABA, lo cuál apoya la idea de que ALIX afecta las respuestas al ABA controlando principalmente la estabilidad de sus receptores. La proteína mutante ALIX-1 muestra una menor interacción con VPS23A y con los receptores de ABA, proporcionando una base molecular que explica la hipersensibilidad al ABA observada en los mutantes alix-1. Nuestro estudio desvela por tanto un nuevo mecanismo de regulación negativa de la ruta del ABA, que actuando a través de la proteína ALIX para controlar la acumulación de PYR/PYL/RCAR específicos

Rheological and hydration characterization of calcium sulfoaluminate cement pastes

Calcium sulfoaluminate (CSA) cements are currently receiving a lot of attention because their manufacture produces less CO2 than ordinary Portland cement (OPC). However, it is essential to understand all parameters which may affect the hydration processes. This work deals with the study of the effect of several parameters, such as superplasticizer (SP), gypsum contents (10, 20 and 30 wt%) and w/c ratio (0.4 and 0.5), on the properties of CSA pastes during early hydration. This characterization has been performed through rheological studies, Rietveld quantitative phase analysis of measured x-ray diffraction patterns, thermal analysis and mercury porosimetry for pastes, and by compressive strength measurements for mortars. The effect of the used SP on the rheological properties has been established. Its addition makes little difference to the amount of ettringite formed but strongly decreases the large pore fraction in the pastes. Furthermore, the SP role on compressive strength is variable, as it increases the values for mortars containing 30 wt% gypsum but decreases the strengths for mortars containing 10 wt% gypsum.This work has been supported by Spanish Ministry of Science and Innovation through MAT2010- 16213 research grant, which is co-funded by FEDER, and Ramón y Cajal Fellowship (RYC-2008- 03523)

Hydration studies of calcium sulfoaluminate cements blended with fly ash

The main objective of this work is to study the hydration and properties of calciumsulfoaluminate cement pastes blended with fly ash (FA) and the corresponding mortars at different hydration ages. Laboratory X-ray powder diffraction, rheological studies, thermal analysis, porosimetry and compressive strength measurements were performed. The analysis of the diffraction data by Rietveld method allowed quantifying crystalline phases and overall amorphous contents. The studied parameters were: i) FA content, 0, 15 and 30 wt.%; and ii) water addition, water-to-CSA mass ratio (w/CSA = 0.50 and 0.65), and water-to-binder mass ratio (w/b = 0.50). Finally, compressive strengths after 6 months of 0 and 15 wt.% FA [w/CSA = 0.50] mortars were similar: 73 ± 2 and 72 ± 3 MPa, respectively. This is justified by the filler effect of the FA as no strong evidences of reactivity of FA with CSA were observed. These results support the partial substitution of CSA cements with FA with the economic and environmental benefitsThis work has been supported by Spanish MINECO through MAT2010-16213 research grant, which is co-funded by FEDER. I. Santacruz thanks a Ramón y Cajal fellowship, RYC-2008-03523

Effect of calcium sulfate source on the hydration of calcium sulfoaluminate eco-cement

The availability of cements, including eco-cements, with tailored mechanical properties is very important for special applications in the building industry. Here we report a full study of the hydration of calcium sulfoaluminate eco-cements with different sulfate sources (gypsum, bassanite and anhydrite) and two water/cement ratios (0.50 and 0.65). These parameters have been chosen because they are known to strongly modify the mechanical properties of the resulting mortars and concretes. The applied multitechnique characterization includes: phase assemblage by Rietveld method, evolved heat, conductivity, rheology, compressive strength and expansion/retraction measurements. The dissolution rate of the sulfate sources is key to control the hydration reactions. Bassanite dissolves very fast and hence the initial setting time of the pastes and mortars is too short (20 min) to produce homogeneous samples. Anhydrite dissolves slowly so, at 1 hydration-day, the amount of ettringite formed (20 wt%) is lower than that in gypsum pastes (26 wt%) (w/c = 0.50), producing mortars with lower compressive strengths. After 3 hydration-days, anhydrite pastes showed slightly larger ettringite contents and hence, mortars with slightly higher compressive strengths. Ettringite content is the chief parameter to explain the strength development in these eco-cements.Universidad de Málaga. Campus de Excelencia Interncaional Andalucía Tech

How to improve the national science curriculum of Spanish primary education. Suggestions from a comparative analysis of goals and content with England and the USA

Se compara el currículo de ciencias español en educación primaria (enseñanzas mínimas nacionales) con los de Inglaterra y EE. UU., y se hacen propuestas de mejora. En las finalidades proponemos la inclusión de nociones de naturaleza de la ciencia y énfasis en la ciencia recreativa. Para los contenidos, sugerimos una revisión general por un panel de expertos en cada una de las materias de ciencias y su didáctica, una mayor sistematización de cada contenido a lo largo de los tres ciclos y una mejor articulación de los contenidos con el resto de diferentes elementos curricularesScience curriculum of Spanish primary education is compared with both the England one and the United States National Science Education Standards, in order to provide improving suggestions for the first one. Concerning the aims, inclusion of nature of science notions; and amusing science learning, are suggested. Regarding to contents, a revision of the whole of them by an expert team on each of the science subjects and science education is suggested; likewise a better organization of contents along the primary stage and a better coordination between contents and the other curricular elements is recommended

Quantitative phase Analisis: A comparative study of Mo and Cu strictly monochromatic radiations

A comparison of the Rietveld quantitative phase analyses (RQPA) obtained using Cu-Kα1, Mo-Kα1, and synchrotron strictly monochromatic radiations is presented. The main aim is to test a simple hypothesis: high energy Mo-radiation, combined with high resolution laboratory X-ray powder diffraction optics, could yield more accurate RQPA, for challenging samples, than well-established Cu-radiation procedure(s). In order to do so, three set of mixtures with increasing amounts of a given phase (spiking-method) were prepared and the corresponding RQPA results have been evaluated. Firstly, a series of crystalline inorganic phase mixtures with increasing amounts of an analyte was studied in order to determine if Mo-Kα1 methodology is as robust as the well-established Cu-Kα1 one. Secondly, a series of crystalline organic phase mixtures with increasing amounts of an organic compound was analyzed. This type of mixture can result in transparency problems in reflection and inhomogeneous loading in narrow capillaries for transmission studies. Finally, a third series with variable amorphous content was studied. Limit of detection in Cu-patterns, ~0.2 wt%, are slightly lower than those derived from Mo-patterns, ~0.3 wt%, for similar recording times and limit of quantification for a well crystallized inorganic phase using laboratory powder diffraction was established ~0.10 wt%. However, the accuracy was comprised as relative errors were ~100%. Contents higher than 1.0 wt% yielded analyses with relative errors lower than 20%. From the obtained results it is inferred that RQPA from Mo-Kα1 radiation have slightly better accuracies than those obtained from Cu-Kα1. This behavior has been established with the calibration graphics obtained through the spiking method and also from Kullback-Leibler distance statistic studies. We explain this outcome, in spite of the lower diffraction power for Mo-radiation (compared to Cu-radiation), due to the larger volume tested with Mo, also because higher energy minimize pattern systematic errors and the microabsorption effect.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

In-situ early age hydration of cement-based materials by synchrotron X-ray powder diffraction

Cement based binders are building materials of worldwide importance. Since these samples are very complex, the knowledge/control of their mineralogical composition are essential to design and predict materials with specific/improved performance. Rietveld quantitative phase analysis (RQPA) allows the quantification of crystalline phases and, when combined with specific methodologies, as the addition of an internal standard or the external standard approach (G-factor), amorphous and non-crystalline phases can also be quantified. However, to carry out a proper RQPA in hydrated cementitious-materials, a good powder diffraction pattern is necessary. In this work, synchrotron X-ray powder diffraction (SXRPD) has been used, allowing in-situ measurements during the early-age hydration process. This work deals with the early hydration study of cement-based materials. The studied samples were: a laboratory-prepared belite calcium sulphoaluminate (BCSAF) clinker (non-active) mixed with 10wt% gypsum, labelled G10B0; two active laboratory-prepared BCSAF clinkers (activated with 2wt% borax), one mixed with 10wt% gypsum and the other one with 10wt% monoclinic-bassanite, hereafter named G10B2 and B10B2, respectively; and an environmentally-friendly cement sample from Henkel, composed of bassanite mixed with 15wt% Portland cement and 10wt% Metakaolin, labelled H1. Anhydrous G10B0 contains beta-belite and orthorhombic-ye'elimite as main phases, while alpha'H-belite and pseudo-cubic-ye'elimite are stabilized in G10B2 and B10B2, with the corresponding sulphate source. Anhydrous H1 contains monoclinic and hexagonal bassanite and alite as main phases. Ye'elimite, in the non-active BCSAF cement pastes, dissolves at a higher pace than in the active one (degree of reaction is α~25% and α~10% at 1 h, respectively) (both prepared with gypsum), with the corresponding differences in ettringite crystallisation (degree of precipitation is α~30% and α~5%, respectively). Moreover, the type of sulphate source has important consequences on the hydration of the active BCSAF cement pastes. Bassanite is quickly dissolved and it precipitates as gypsum within the first hour of hydration (in B10B2). At that time, ettringite starts to crystallize, and after 12 hours is almost fully crystallized, similar to G10B2. In H1, bassanite transforms into gypsum within the first hour, being the principal hydration product; ettringite starts to be formed just after few hydration minutes. These results are crucial in the understanding and development of improved cement materials.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The use of mo and cu monochromatic radiations for quantitative phase analysis: study of the accuracy

Cement hydration is a very complex process in which crystalline phases are dissolving in water and after supersaturation hydrated crystalline and amorphous phases precipitate. Great efforts are being made to develop analytical tools to accurately quantify these processes and X-ray Powder Diffraction (XRPD) combined with Rietveld methodology is a suitable tool to quantify these complex mixtures and their time evolutions. However, some problems/drawbacks should be overcome to fully apply it to cement pastes characterization in order to get accurate phase analyses. In order to tackle this issue, a comparison of the Rietveld quantitative phase analyses (RQPA) obtained using Cu-Kα1, Mo-Kα1, and synchrotron strictly monochromatic radiations of three set of mixtures with increasing amounts of a given phase (spiking-method) is presented. The main aim is to test a simple hypothesis: high energy Mo-radiation, combined with high resolution laboratory X-ray powder diffraction optics, could yield more accurate RQPA, for challenging samples, than well-established Cu-radiation procedure(s). Firstly, a series of crystalline inorganic phase mixtures with increasing amounts of an analyte was studied in order to determine if Mo-Kα1 methodology is as robust as the well-established Cu-Kα1 one. Secondly, a series of crystalline organic phase mixtures with increasing amounts of an organic compound was analyzed. This type of mixture can result in transparency problems in reflection and inhomogeneous loading in narrow capillaries for transmission studies. Finally, a third series with variable amorphous content was studied. Limit of detection in Cu-patterns, ~0.2 wt%, are slightly lower than those derived from Mo-patterns, ~0.3 wt%, for similar recording times and limit of quantification for a well crystallized inorganic phase using laboratory powder diffraction was established ~0.10 wt%. From the obtained results it is inferred that RQPA from Mo-Kα1 radiation have slightly better accuracies than those obtained from Cu-Kα1. The results obtained in the previous comparison have been taken into account to obtain accurate RQPA, including the amorphous component with internal standard methodology, of hydrating cement pastes. The final goal of this second study was understanding the early-stage hydration mechanisms of a variety of cementing systems (Ordinary Portland Cement or Belite Alite Ye’elimite cement) as a function of water content, superplasticizer additives and type and content of sulfate source. In order to do so, X-ray powder diffraction data were taken in-situ with the humidity chamber coupled to the Mo-Kα1 powder diffractometer. Some results of this ongoing investigation will be reported and discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Community and health staff perceptions on non-communicable disease management in El Salvador’s health system: A qualitative study

From Springer Nature via Jisc Publications RouterHistory: received 2019-10-01, accepted 2020-04-23, registration 2020-04-23, online 2020-05-27, epub 2020-05-27, collection 2020-12Funder: Public Health Research Programme; doi: http://dx.doi.org/10.13039/501100001921; Grant(s): Programme 16/136/100Dataset available in eData: https://eresearch.qmu.ac.uk/handle/20.500.12289/9699Background: Non-communicable Diseases (NCDs) are the leading cause of global mortality and disability with a rising burden in low- and middle-income countries. Their multifactorial aetiology, and their requirement of long-term care, implies the need for comprehensive approaches. From 2009, the Ministry of Health (MoH) in El Salvador has developed a national public health system based on comprehensive primary health care. This study aims to describe the different stakeholders’ perceptions about the management of NCDs along the pathways of care in this health system. Methods: During three fieldwork periods in 2018, three complementary qualitative data collection methods were deployed and conducted in settings with high prevalence of NCDs within El Salvador. First, illness narrative methodology was used to document the life histories of people living with a chronic disease and being treated in second and third level health facilities. Second, through social mapping, support resources that NCD patients used throughout the process of their illness within the same settings were analysed. Third, semi-structured interviews were conducted in the same locations, with both chronic patients and health personnel working at different levels of the primary health care setting. Participants were recruited through purposive and snowball sampling, and a deductive approach was implemented for coding during the analysis phase. After grouping codes into potential themes, a thematic framework was developed using a reflexive approach and following triangulation of the data. Results: This innovative approach of combining three well-defined qualitative methods identified key implications for the implementation of a comprehensive approach to NCD management in resource-poor settings. The following elements are identified: 1) social risk factors and barriers to care; 2) patient pathways to NCD care; 3) available resources identified through social connections mapping; 4) trust in social connections; and 5) community health promotion and NCD prevention management. Conclusions: The Salvadoran public health system has been able to strengthen its comprehensive approach to NCDs, combining a clinical approach – including long-term follow-up – with a preventive community-based strategy. The structural collaboration between the health system and the (self-) organised community has been essential for identifying failings, discuss tensions and work out adapted solutions.This study was supported by the NIHR Research Unit on Health in Situations of Fragility (RUHF), through the NIHR Global Health Research Programme 16/136/10020pubpu

Comprehensive primary health care and non-communicable diseases management: A case study of El Salvador

From PubMed via Jisc Publications RouterOne of today's greatest challenges in public health worldwide - and especially its key management from Primary Health Care (PHC) - is the growing burden of non-communicable diseases (NCDs). In El Salvador, since 2009 the Minister of Health (MoH) has scaled up a national public health system based on a comprehensive PHC approach. A national multi-sectorial strategic plan for a comprehensive approach to NCDs has also been developed. This analysis explores stakeholders' perceptions related to the management of NCDs in PHC and, in particular, the role of social participation. A case-study was developed consisting of semi structured interviews and official document reviews. Semi-structured interviews were developed with chronic patients (14) and PHC professionals working in different levels within PHC (12). Purposive sampling was used to recruit participants. A non-pure, deductive approach was implemented for coding. After grouping codes into potential themes, a thematic framework was elaborated through a reflexive approach and the triangulation of the data. The research was conducted between March and August of 2018 in three different departments of El Salvador. The structure and the functioning of the Salvadoran PHC system and its intersectoral approach is firstly described. The interdisciplinary PHC-team brings holistic health care closer to the communities in which health promoters play a key role. The findings reflect the generally positive perception of the PHC system in terms of accessibility, quality and continuity of care by chronic patients. Community engagement and the National Health Forum are ensuring accountability through social controllership mechanisms. However, certain challenges were also noted during the interviews related to the shortage of medication and workforce; coordination between the levels of care and the importance of prevention and health promotion programmes for NCDs. The Salvadoran PHC and its comprehensive approach to NCDs with an emphasis on intersectoral participation has been positively perceived by the range of stakeholders interviewed. Social engagement and the NHF works as a driving force to ensure accountability as well as in the promotion of a preventive culture. The challenges identified provide keys to amplify knowledge for addressing inequalities in health by strengthening PHC and its NCDs management.19pubpu