Caracterización de transportadores de cobre de Arabidopsis thaliana: efectos de la alteración de su expresión en plantas transgénicas.

RESUMEN Con la finalidad de diseñar nuevas estrategias biotecnológicas relacionadas con la contaminación por cobre (Cu), los esfuerzos de esta Tesis se centraron en tres objetivos: 1. Identificación de la respuesta molecular a diferentes niveles de Cu en el medio en Arabidopsis thaliana mediante análisis global de los cambios de expresión génica entre exceso y déficit de Cu. Determinamos que el medio estándar de crecimiento en placa ½ MS es deficitario en Cu para las plántulas (~ 4 g/g P.S.), mientras que el mismo medio suplementado con Cu 10 M provoca una elevada concentración endógena del metal próxima al exceso (~ 30 g/g P.S.). Las respuestas moleculares observadas mediante micromatrices a estos niveles de Cu en el medio se dividen en tres categorías: - Respuesta al estrés y alteración del metabolismo, incluyendo la respuesta al estrés hídrico y salino, la división celular y la modificación de DNA en exceso de Cu, así como la respuesta al estrés por patógenos, expansión celular y metabolismo del azufre en déficit del metal. El ritmo circadiano también se encuentra alterado por los niveles de Cu. En estas respuestas están implicados factores de regulación por déficit de Cu como las SPLs y los miRNAs. - Prioridad en el uso del Cu para proteínas esenciales, como la plastocianina, y sustitución de proteínas con función equivalente que utilizan otros metales en sus centros activos, como la Cu/ZnSOD y la FeSOD cloroplásticas. En este sentido, CSD2 y FSD1 son buenos indicadores de niveles elevados y deficitarios de Cu, respectivamente, dentro de la célula. - Regulación de los niveles intracelulares de Cu mediante alteración de los mecanismos de incorporación del metal en la célula, como los transportadores de Cu de alta afinidad COPT, y de destoxificación del metal, como la familia de ATPasas tipo P transportadoras de Cu. 2. Caracterización de transportadores de Cu de familias génicas que responden a los diferentes niveles de Cu en Arabidopsis thaliana. - HMA5, ATPasa tipo P transportadora de Cu. HMA5 se expresa principalmente en raíces y su expresión se induce en plántula entera específicamente por Cu. Mutantes por pérdida de función hma5 son hipersensibles específicamente al Cu y acumulan más Cu en las raíces que las plantas silvestres en exceso de Cu. - COPT3, transportador de Cu de alta afinidad. COPT3 se expresa principalmente en flores y semillas. COPT3 se localiza intracelularmente, probablemente en algún compartimento de la ruta de secreción. La sobrexpresión de COPT3 en plantas transgénicas provoca retraso en el desarrollo de la planta, cotiledones en forma caída y estambres más cortos que los de plantas silvestres. Estas plantas sobrexpresoras muestran hipersensibilidad al Cu, acumulan más Cu en las semillas y, en exceso de Cu, acumulan más Cu que las plantas silvestres en plántula entera. 3. Obtención de plantas transgénicas de Arabidopsis thaliana con posibles aplicaciones biotecnológicas en procesos de fitorremediación, mediante el estudio del transportador de Cu de alta afinidad COPT1. La expresión de COPT1 en plántula entera se reprime específicamente por Cu. COPT1 se localiza en la membrana plasmática. La sobrexpresión de COPT1 en plantas transgénicas provoca reducción del tamaño global de la planta, cotiledones en forma de remo, curvatura cóncava de las hojas, pétalos y estambres protegidos por los sépalos y escasa fertilidad. Estas plantas sobrexpresoras son hipersensibles específicamente al Cu y acumulan más Cu respecto a las plantas silvestres. La sobrexpresión simultánea de COPT1 y TcMT3 podría ser una estrategia candidata en procesos de fitorremediación de suelos contaminados por Cu. Estas plantas reúnen dos características fundamentales para este tipo de aplicaciones biotecnológicas: acumulación y tolerancia al Cu. __________________________________________________________________________________________________With the aim of designing new biotechnological strategies related to copper (Cu), the objectives of this Thesis were: 1. Identification of the Arabidopsis molecular responses to Cu status in the medium by microarray global analysis of gene expression. The internal Cu content in seedlings grown in the ½ MS medium is below the sufficiency levels, whereas the same medium supplemented with Cu 10 M provoke a high endogenous metal concentration close to excess. The responses observed are divided in three categories: - Metabolic changes and stress responses. Circadian rythm is also alterated. Transcription factors and miRNAs are implicated. - Prioritizing the use of Cu for essential proteins and substitution of counterpart proteins with different metals at their active sites. CSD2 and FSD1 expression is a good marker of Cu levels in Arabidopsis. - Regulation of intracellular Cu levels by modification of expression of metal homeostasis components, such as the COPT and HMA families of transporters. 2. Characterization of Cu transporters from gene families that respond to Cu in Arabidopsis. - HMA5, Cu transporting P-type ATPase. HMA5 is mainly expressed in roots and specifically induced by Cu. The hma5 knockout mutants are specifically Cu hypersensitive and accumulate Cu in roots under Cu excess. - COPT3, high affinity Cu transporter. COPT3 is mainly expressed in flowers and seeds. COPT3 is intracellulary located, probably at some secretory pathway compartment. COPT3 overexpression provokes a delay in plant development, phenotypically characteristic cotyledons and short stamens. These overexpressing plants are Cu hypersensitive, accumulate Cu in seeds and, under Cu excess, accumulate Cu in the whole seedling. 3. Obtaining Arabidopsis transgenic plants with possible biotechnological applications, by overexpressing the high affinity Cu transporter COPT1. COPT1 expression is specifically repressed by Cu. COPT1 is located at the plasma membrane. COPT1 overexpression provokes biomass reduction, phenotypically characteristic cotyledons, concave leaves, petals and stamens protected by sepals and low fertility. These overexpressing plants are specifically Cu hypersensitive and accumulate Cu. Simultaneous overexpression of COPT1 and TcMT3 could be a candidate strategy for phytoremediation of Cu contaminated soils. These plants possess two fundamental characteristics for this biotechological application: Cu accumulation and tolerance

Optimization of non-denaturing protein extraction conditions for plant PPR proteins

[EN] Pentatricopeptide repeat proteins are one of the major protein families in flowering plants, containing around 450 members. They participate in RNA editing and are related to plant growth, development and reproduction, as well as to responses to ABA and abiotic stresses. Their characteristics have been described in silico; however, relatively little is known about their biochemical properties. Different types of PPR proteins, with different tasks in RNA editing, have been suggested to interact in an editosome to complete RNA editing. Other non-PPR editing factors, such as the multiple organellar RNA editing factors and the organelle RNA recognition motif-containing protein family, for example, have also been described in plants. However, while evidence on protein interactions between non-PPR RNA editing proteins is accumulating, very few PPR protein interactions have been reported; possibly due to their high instability. In this manuscript, we aimed to optimize the conditions for non denaturing protein extraction of PPR proteins allowing in vivo protein analyses, such as interaction assays by co-immunoprecipitation. The unusually high protein degradation rate, the aggregation properties and the high pl, as well as the ATP-dependence of some PPR proteins, are key aspects to be considered when extracting PPR proteins in a non-denatured state. During extraction of PPR proteins, the use of proteasome and phosphatase inhibitors is critical. The use of the ATP-cofactor reduces considerably the degradation of PPR proteins. A short centrifugation step to discard cell debris is essential to avoid PPR precipitation; while in some cases, addition of a reductant is needed, probably caused by the pl/pH context. This work provides an easy and rapid optimized non-denaturing total protein extraction protocol from plant tissue, suitable for polypeptides of the PPR family.This work was supported by Research Foundation Flanders (FWO) (project G.0C84.14N). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.r D.V.D.S. gratefully acknowledges Ghent University and the Research Foundation Flanders (FWO) for financial support (project G.0084.14N). N.A.-C. and D.V.D.S. acknowledge Maria Helena S. Goldman for the N. benthamiana seeds, helpful suggestions regarding experimental design and data interpretation, and critical reading of the manuscript.Andrés-Colás, N.; Van Der Straeten, D. (2017). Optimization of non-denaturing protein extraction conditions for plant PPR proteins. PLoS ONE. 12(11):1-15. https://doi.org/10.1371/journal.pone.0187753S1151211Barkan, A., & Small, I. (2014). Pentatricopeptide Repeat Proteins in Plants. 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Evaluación de la aproximación al aprendizaje de estudiantes de ciencias de la vida usando la versión revisada del Study Process Questionnaire (R-SPQ-2F)

[EN] The revised version of Study Process Questionnaire (R-SPQ-2F) analyzes the approach to learning on different scales. In the present study we analyzed the answers to the questionnaire of students of degree and master level of different subjects related to the sciences of the life in the sociocultural context of Spain. The adjustment of the model analyzed by the alpha of Cronbach was adequate to measure the approach to learning, deep or superficial, but not for the subscales of measurement of strategy and motivation. The students mainly expressed a deep learning approach. Although differences were detected among students according to subjects, they were not observed at the global level for the level of studies (degree or master), year or gender[ES] El cuestionario revisado Study Process Questionnaire (R-SPQ-2F) analiza la aproximación al aprendizaje en diferentes escalas. En el presente estudio se analizaron las respuestas al cuestionario de alumnos de grado y máster de distintas asignaturas relacionadas con las ciencias de la vida en el entorno sociocultural de España. El ajuste del modelo analizado mediante el alfa de Cronbach fue adecuado para medir la aproximación al aprendizaje, profunda o superficial, pero no para las subescalas de medición de estrategia y motivación. Los alumnos manifiestaron mayoritariamente una aproximación profunda al aprendizaje. Si bien se detectaron diferencias entre los estudiantes según asignaturas, no se observaron a nivel global para el nivel de estudios (grado o máster), curso o género.La publicación de este trabajo ha sido parcialmente financiada por un proyecto de innovación educativa (PIME/2017/A/016/A) concedido por el Vicerrectorado de Estudios, Calidad y Acreditación de la Universitat Politècncia de València (UPV).Leiva Brondo, M.; Cebolla Cornejo, J.; Andrés Colás, N.; Peiró Barber, RM.; Pérez De Castro, AM. (2019). Evaluación de la aproximación al aprendizaje de estudiantes de ciencias de la vida usando la versión revisada del Study Process Questionnaire (R-SPQ-2F). En IN-RED 2019. V Congreso de Innovación Educativa y Docencia en Red. Editorial Universitat Politècnica de València. 156-170. https://doi.org/10.4995/INRED2019.2019.10406OCS15617

Saccharomyces cerevisiae as a Tool to Investigate Plant Potassium and Sodium Transporters

[EN] Sodium and potassium are two alkali cations abundant in the biosphere. Potassium is essential for plants and its concentration must be maintained at approximately 150 mM in the plant cell cytoplasm including under circumstances where its concentration is much lower in soil. On the other hand, sodium must be extruded from the plant or accumulated either in the vacuole or in specific plant structures. Maintaining a high intracellular K+/Na+ ratio under adverse environmental conditions or in the presence of salt is essential to maintain cellular homeostasis and to avoid toxicity. The baker's yeast, Saccharomyces cerevisiae, has been used to identify and characterize participants in potassium and sodium homeostasis in plants for many years. Its utility resides in the fact that the electric gradient across the membrane and the vacuoles is similar to plants. Most plant proteins can be expressed in yeast and are functional in this unicellular model system, which allows for productive structure-function studies for ion transporting proteins. Moreover, yeast can also be used as a high-throughput platform for the identification of genes that confer stress tolerance and for the study of protein-protein interactions. In this review, we summarize advances regarding potassium and sodium transport that have been discovered using the yeast model system, the state-of-the-art of the available techniques and the future directions and opportunities in this field.This work was supported by the Spanish Ministry of Economy and Competitiveness (BIO201677776-P and BIO2016-81957-REDT) and the Valencian Government (AICO/2018/300)Locascio, AAM.; Andrés-Colás, N.; Mulet, JM.; Yenush, L. (2019). Saccharomyces cerevisiae as a Tool to Investigate Plant Potassium and Sodium Transporters. International Journal of Molecular Sciences. 20(9):1-37. https://doi.org/10.3390/ijms20092133S137209Schroeder, J. I. (2003). Knockout of the guard cell K+out channel and stomatal movements. Proceedings of the National Academy of Sciences, 100(9), 4976-4977. doi:10.1073/pnas.1031801100Hurst, A. C., Meckel, T., Tayefeh, S., Thiel, G., & Homann, U. (2004). Trafficking of the plant potassium inward rectifier KAT1 in guard cell protoplasts of Vicia faba. The Plant Journal, 37(3), 391-397. doi:10.1046/j.1365-313x.2003.01972.xBRAG, H. (1972). The Influence of Potassium on the Transpiration Rate and Stomatal Opening in Triticum aestivum andPisum sativum. Physiologia Plantarum, 26(2), 250-257. doi:10.1111/j.1399-3054.1972.tb08553.xMohd Zain, N. A., & Ismail, M. R. (2016). Effects of potassium rates and types on growth, leaf gas exchange and biochemical changes in rice (Oryza sativa) planted under cyclic water stress. 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Study Approaches of Life Science Students Using the Revised Two-Factor Study Process Questionnaire (R-SPQ-2F)

[EN] Students' approaches to learning can vary between students of different ages, genders, years, degrees, or cultural contexts. The aim of this study was to assess the approaches to learning of different students of life science degrees. The Revised Two-Factor Study Process Questionnaire (R-SPQ-2F) has been used to assess the approaches to learning of 505 students of thirteen different subjects of four different degrees at Universitat Politecnica de Valencia in order to study the factors that influence their approaches. Results show a higher deep approach of the students. Differences were observed between subjects and gender, not related to level (bachelor or master) or year. The item reliability analysis showed a high consistency for the main scales, but not for the secondary scales of the R-SPQ-2F questionnaire. High correlation between the deep and surface scales were observed. These data can provide more information to the teachers, which may help them to develop strategies focused on promoting a deeper approach to learning for the students, more adapted to their subject, level, and year.This research was partially funded by innovation educative projects (PIME/2017/A/016/A and PIME/19-20/168) by Vice-Rectorate for Studies, Quality and Accreditation of Universitat Politecnica de Valencia (UPV, Valencia, Spain).Leiva-Brondo, M.; Cebolla Cornejo, J.; Peiró Barber, RM.; Andrés-Colás, N.; Esteras Gómez, C.; Ferriol Molina, M.; Merle Farinós, HB.... (2020). Study Approaches of Life Science Students Using the Revised Two-Factor Study Process Questionnaire (R-SPQ-2F). Education Sciences. 10(7):1-18. https://doi.org/10.3390/educsci10070173S118107Sinatra, G. M., Heddy, B. C., & Lombardi, D. (2015). The Challenges of Defining and Measuring Student Engagement in Science. Educational Psychologist, 50(1), 1-13. doi:10.1080/00461520.2014.1002924Jeong, J. 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European Journal of Psychology of Education, 23(3), 355-372. doi:10.1007/bf03173004Fryer, L. K., Ginns, P., Walker, R. A., & Nakao, K. (2011). The adaptation and validation of the CEQ and the R-SPQ-2F to the Japanese tertiary environment. British Journal of Educational Psychology, 82(4), 549-563. doi:10.1111/j.2044-8279.2011.02045.xGurpinar, E., Kulac, E., Tetik, C., Akdogan, I., & Mamakli, S. (2013). Do learning approaches of medical students affect their satisfaction with problem-based learning? Advances in Physiology Education, 37(1), 85-88. doi:10.1152/advan.00119.2012Parpala, A., Lindblom-Ylänne, S., Komulainen, E., Litmanen, T., & Hirsto, L. (2010). Students’ approaches to learning and their experiences of the teaching-learning environment in different disciplines. British Journal of Educational Psychology, 80(2), 269-282. doi:10.1348/000709909x476946Gijbels, D., Van de Watering, G., Dochy, F., & Van den Bossche, P. (2005). 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British Journal of Educational Psychology, 71(4), 511-530. doi:10.1348/000709901158659Zeegers, P. (2001). Approaches to learning in science: A longitudinal study. British Journal of Educational Psychology, 71(1), 115-132. doi:10.1348/000709901158424Snelgrove, S., & Slater, J. (2003). Approaches to learning: psychometric testing of a study process questionnaire. Journal of Advanced Nursing, 43(5), 496-505. doi:10.1046/j.1365-2648.2003.02747.xROSSUM, E. J., & SCHENK, S. M. (1984). THE RELATIONSHIP BETWEEN LEARNING CONCEPTION, STUDY STRATEGY AND LEARNING OUTCOME. British Journal of Educational Psychology, 54(1), 73-83. doi:10.1111/j.2044-8279.1984.tb00846.xTrigwell, K., Prosser, M., & Waterhouse, F. (1999). Higher Education, 37(1), 57-70. doi:10.1023/a:1003548313194Crawford, K., Gordon, S., Nicholas, J., & Prosser, M. (1998). Qualitatively different experiences of learning mathematics at university. Learning and Instruction, 8(5), 455-468. doi:10.1016/s0959-4752(98)00005-xApproaches and Study Skills Inventory for Students (ASSIST) (Incorporating the Revised Approaches to Studying Inventory—RASI) https://www.researchgate.net/publication/260291730_Approaches_and_Study_Skills_Inventory_for_Students_ASSIST_incorporating_the_Revised_Approaches_to_Studying_Inventory_-_RASIImmekus, J. C., & Imbrie, P. K. (2009). A Test and Cross-Validation of the Revised Two-Factor Study Process Questionnaire Factor Structure Among Western University Students. Educational and Psychological Measurement, 70(3), 495-510. doi:10.1177/0013164409355685Socha, A., & Sigler, E. A. (2014). Exploring and «reconciling» the factor structure for the Revised Two-factor Study Process Questionnaire. Learning and Individual Differences, 31, 43-50. doi:10.1016/j.lindif.2013.12.010Chamorro-Premuzic, T., Furnham, A., & Lewis, M. (2007). Personality and approaches to learning predict preference for different teaching methods. Learning and Individual Differences, 17(3), 241-250. doi:10.1016/j.lindif.2006.12.001Furnham, A., Monsen, J., & Ahmetoglu, G. (2009). Typical intellectual engagement, Big Five personality traits, approaches to learning and cognitive ability predictors of academic performance. British Journal of Educational Psychology, 79(4), 769-782. doi:10.1348/978185409x412147Phan, H. P. (2017). Multiple regression analysis of epistemological beliefs, learning approaches, and self-regulated learning. Electronic Journal of Research in Education Psychology, 6(14). doi:10.25115/ejrep.v6i14.1262Stes, A., De Maeyer, S., & Van Petegem, P. (2013). Examining the Cross-Cultural Sensitivity of the Revised Two-Factor Study Process Questionnaire (R-SPQ-2F) and Validation of a Dutch Version. PLoS ONE, 8(1), e54099. doi:10.1371/journal.pone.0054099Baeten, M., Kyndt, E., Struyven, K., & Dochy, F. (2010). 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REMYP_07 Evaluación de la adquisición de la Competencia Transversal “Responsabilidad Ética, Medioambiental y Profesional” en los estudios de grado de la Universitat Politècnica de València.

[EN] The text presents the work developed by the EICE (educational innovation and quality team) REMYP_07, related to the acquisition of the generic outcome "Ethical, environmental and professional responsibility" by UPV graduated students. To this end, the paper develops the diagnosis of the current situation in four engineering bachelor programs taught at this university . Based on the diagnosis, the work focuses in actions to improve the inclusion of this competence throughout the curriculum to guarantee its acquisition by students who complete their studies.[ES] En el texto se presenta el trabajo desarrollado por el EICE (equipo de innovación y calidad educativa) REMYP_07, relacionado con la adquisición de la competencia transversal “Responsabilidad ética, medioambiental y profesional” por parte de los alumnos egresados de la UPV. Para ello se indica como se ha realizado el diagnóstico de la situación actual en cuatro grados impartidos en dicha universidad y a partir del diagnóstico se muestran las acciones a llevar a cabo para mejorar la inclusión de dicha competencia a lo largo del plan de estudios de manera que se pueda garantizar su adquisición por parte de los alumnos egresados.Andrés Doménech, I.; Cabedo Fabrés, M.; Martí Campoy, A.; Coll Aliaga, PE.; Fenollosa Forner, EJ.; Giménez Carbó, E.; Gómez Martín, ME.... (2021). REMYP_07 Evaluación de la adquisición de la Competencia Transversal “Responsabilidad Ética, Medioambiental y Profesional” en los estudios de grado de la Universitat Politècnica de València. En IN-RED 2021: VII Congreso de Innovación Edicativa y Docencia en Red. Editorial Universitat Politècnica de València. 1402-1409. https://doi.org/10.4995/INRED2021.2021.13723OCS1402140

Evolving trends in the management of acute appendicitis during COVID-19 waves. The ACIE appy II study

Background: In 2020, ACIE Appy study showed that COVID-19 pandemic heavily affected the management of patients with acute appendicitis (AA) worldwide, with an increased rate of non-operative management (NOM) strategies and a trend toward open surgery due to concern of virus transmission by laparoscopy and controversial recommendations on this issue. The aim of this study was to survey again the same group of surgeons to assess if any difference in management attitudes of AA had occurred in the later stages of the outbreak. Methods: From August 15 to September 30, 2021, an online questionnaire was sent to all 709 participants of the ACIE Appy study. The questionnaire included questions on personal protective equipment (PPE), local policies and screening for SARS-CoV-2 infection, NOM, surgical approach and disease presentations in 2021. The results were compared with the results from the previous study. Results: A total of 476 answers were collected (response rate 67.1%). Screening policies were significatively improved with most patients screened regardless of symptoms (89.5% vs. 37.4%) with PCR and antigenic test as the preferred test (74.1% vs. 26.3%). More patients tested positive before surgery and commercial systems were the preferred ones to filter smoke plumes during laparoscopy. Laparoscopic appendicectomy was the first option in the treatment of AA, with a declined use of NOM. Conclusion: Management of AA has improved in the last waves of pandemic. Increased evidence regarding SARS-COV-2 infection along with a timely healthcare systems response has been translated into tailored attitudes and a better care for patients with AA worldwide

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Electroforesis en gel de poliacrilamida

Para separar las proteínas presentes en una muestra por electroforesis en un gel de poliacrilamida, una vez tengamos el gel preparado y el sistema de electroforesis montado, añadiremos el tampón y cargaremos las muestras para proceder a la electroforesis, tal y como se detalla en este vídeo.https://polimedia.upv.es/visor/?id=ece536a0-b18a-11eb-b7fc-e95eca16729bAndrés Colás, N.; Yenush, LP. (2021). Electroforesis en gel de poliacrilamida. http://hdl.handle.net/10251/167244DE

Montaje para preparar un gel de poliacrilamida

Para separar las proteínas presentes en una muestra por electroforesis en un gel de poliacrilamida, debemos montar el sistema en el que polimerizar el gel, tal y como se detalla en este vídeo.https://media.upv.es/player/?id=74239bc0-8274-11eb-9c0f-ff7aa61993b1Andrés Colás, N.; Yenush, LP. (2021). Montaje para preparar un gel de poliacrilamida. http://hdl.handle.net/10251/164935DE