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El metabolisme embrionari s’ha relacionat amb la viabilitat embrionària, fet que suggereix el seu us per a ajudar en la selecció d’embrions d’alta qualitat en tecnologies de reproducció assistida (TRA). Malgrat els nous avenços per avaluar el metabolisme dels oòcits i embrions per determinar-ne la qualitat, encara falten mètodes per mesurar de manera no invasiva i robusta l’estat metabòlic. Fluorescence lifetime imaging microscopy (FLIM) és una tècnica d’imatge no invasiva capaç de mesurar l’estat metabòlic de les cèl·lules. FLIM mesura l’autofluorescència del dinucleòtid de nicotinamida adenina (fosfat) (NAD(P)H) i el dinucleòtid de flavina adenina (FAD+), coenzims intracel·lulars essencials per a diversos processos metabòlics. FLIM permet mesurar no només la intensitat de fluorescència d’aquestes molècules, que es correlaciona amb la seva concentració, sinó també la vida mitja dels coenzims, que proporciona informació sobre la unió enzimàtica. Una imatge de FLIM produeix 9 paràmetres quantitatius que permeten la caracterització l’estat metabòlic cel·lular. Juntament amb FLIM, també es pot obtenir second harmònic generation (SHG), una tècnica que permet l’obtenció imatges d’estructures polaritzades com el fus mitòtic. El primer estudi d’aquesta tesi te com a objectiu explorar la viabilitat, sensibilitat i seguretat de les tècniques FLIM i SHG per ser utilitzades per a l’avaluació d’oòcits i embrions de ratolí. Aquest estudi ha demostrat que FLIM en combinació amb SHG permet detectar canvis metabòlics significatius durant el desenvolupament embrionari de ratolí o canvis en l’estat metabòlic causats per pertorbacions metabòliques, sense afectar el desenvolupament embrionari. La cooperació metabòlica entre els oòcits i les cèl·lules del cúmul que l’envolten és essencial per al correcte desenvolupament d’oòcits i embrions. L’objectiu del segon estudi era determinar si FLIM podia detectar diferències en l’estat metabòlic de cèl·lules del cúmul humanes. FLIM permet mesures quantitatives de l’estat metabòlic de les cèl·lules del cúmul i detecta variacions entre diferents grups de cèl·lules del cúmul, amb una major variació entre els grups de cèl·lules de cada pacient que entre pacients. A més, hem pogut observar associacions entre els paràmetres metabòlics de les cèl·lules del cúmul i l’edat del pacient i els nivells d’hormones anti Mulleriana, però no amb l’IMC. També s’han trobat variacions entre cèl·lules del cúmul que embolcallen oòcits madurs o immadurs, indicant la possible utilització d’aquesta tècnica com a mètode d’avaluació oocitària. Els embrions de mamífer experimenten variacions en el metabolisme al llarg del seu desenvolupament preimplantacional. Aquest metabolisme s’ha relacionat amb la viabilitat embrionària, fet que suggereix el seu possible ús per a seleccionar embrions d’alta qualitat en TRA. No obstant, el metabolisme dels embrions humans encara no està completament caracteritzat, probablement a causa de la manca de mètodes no invasius per mesurar el seu estat metabòlic. El tercer estudi s’ha enfocat a determinar si la tècnica no invasiva FLIM detecta variacions en els perfils metabòlics de blastocists humans donats per recerca. S’han observat grans canvis en l’estat metabòlic en el desenvolupament en 36 hores, associats al dia des de la fecundació de l’embrió o al seu estat de desenvolupament del blastocist. Hem pogut observar una significativa heterogeneïtat metabòlica dins dels mateixos blastocists, concretament entre la massa cel·lular interna i el trofectoderm, i entre les parts de blastocists en eclosió dins i fora de la zona pel·lúcida. Aquests resultats demostren encara més que el metabolisme embrionari pre-implantacional es altament dinàmic i plàstic. En conjunt, els resultats d’aquesta tesi presenten que FLIM en combinació amb SHG pot proporcionar informació detallada i biològicament rellevant d’oòcits, embrions i cèl·lules del cúmul. Aquests nous enfocaments son prometedors per ajudar en l’avaluació de la qualitat oocitària i embrionària mitjançant mesures quantitatives no invasives del seu metabolisme i morfologia del fus mitòtic o meiòtic. El metabolismo embrionario se ha relacionado con la viabilidad embrionaria, lo que sugiere su utilización para ayudar en la selección de embriones de alta calidad en tecnologías de reproducción asistida (TRA). A pesar de nuevos avances en técnicas de evaluación el metabolismo de ovocitos y embriones para determinar su calidad, todavía faltan métodos para medir de forma no invasiva y robusta el estado metabólico. La fluorescencia lifetime imaging microscopy (FLIM) es una técnica de imagen no invasiva capaz de medir el estado metabólico celular. FLIM mide la autofluorescencia del dinucleótido de nicotinamida adenina (fosfato) (NAD(P)H) y el dinucleótido de flavina adenina (FAD+), coenzimas intracelulares esenciales para varios procesos metabólicos. FLIM permite medir la intensidad de fluorescencia de estas moléculas, que se correlaciona con su concentración, y también la vida media de las coenzimas, proporcionando información sobre la unión con enzimas. Una imagen de FLIM produce 9 parámetros cuantitativos que permiten la caracterización del estado metabólico celular. Junto con FLIM, se puede obtener second harmonic generation (SHG), una técnica que permite la obtención imágenes de estructuras polarizadas como el huso mitótico. El primer estudio de esta tesis tiene como objetivo explorar la viabilidad, sensibilidad y seguridad de las técnicas FLIM y SHG para ser utilizadas en la evaluación de ovocitos y embriones. Este estudio ha demostrado que FLIM en combinación con SHG puede detectar cambios significativos en el metabolismo durante el desarrollo embrionario de ratón o cambios causados por perturbaciones metabólicas, sin perjudicar el desarrollo embrionario. La cooperación metabólica entre los ovocitos y las células del cúmulo (CC) que le rodean es esencial para el desarrollo correcto de ovocitos y embriones. El objetivo del segundo estudio era determinar si FLIM podía detectar diferencias en el estado metabólico entre grupos de CC humanas. FLIM permite medidas cuantitativas del estado metabólico de las CC y detecta variaciones entre distintos grupos de CC, con una mayor variación entre grupos de células de cada paciente que entre pacientes. Además, hemos observado asociaciones entre los parámetros metabólicos de las CC y la edad del paciente y los niveles de hormonas anti Mulleriana, pero no con el IMC. También se han encontrado variaciones entre CC que rodean ovocitos maduros o inmaduros, indicando la posible utilización de esta técnica como método de evaluación ovocitária. Los embriones de mamífero experimentan variaciones en el metabolismo a lo largo de su desarrollo preimplantacional. Este metabolismo se ha relacionado con la viabilidad embrionaria, lo que sugiere su posible uso para seleccionar embriones de alta calidad en TRA. Sin embargo, el metabolismo de los embriones humanos todavía no está completamente caracterizado. El tercer estudio se ha enfocado a determinar si la técnica no invasiva FLIM puede detectar variaciones en los perfiles metabólicos de los blastocitos humanos donados para investigación. Hemos observado grandes cambios en el estado metabólico de los blastocitos humanos durante su desarrollo en 36 horas, cambios en el estado metabólico asociados al día desde la fecundación del embrión y a su estado de desarrollo del blastocito. Hemos podido observar una significativa heterogeneidad metabólica dentro de los propios blastocitos, concretamente entre la masa celular interna y el trofectodermo, y entre las partes de blastocitos en eclosión dentro y fuera de la zona pelúcida. Estos resultados demuestran aún más que el metabolismo embrionario preimplantacional es altamente dinámico y plástico. En conjunto, los resultados de esta tesis presentan que FLIM en combinación con SHG pueden proporcionar información detallada y biológicamente relevante de ovocitos, embriones y CC. Estos nuevos enfoques son prometedores para ayudar en la evaluación de la calidad oocitária y embrionaria mediante medidas cuantitativas no invasivas de su metabolismo y morfología del huso mitótico y meiótico. Embryo metabolism has long been linked to embryo viability, suggesting its potential utility to aid in selecting high quality embryos in assisted reproductive technologies (ART). Despite new advances to assess oocyte and embryo metabolism to determine their quality, non-invasive methods to robustly measure metabolic state are still lacking. Fluorescence lifetime imaging microscopy (FLIM) is a non-invasive method for measuring the metabolic state of cells. FLIM measures the autofluorescence of nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD+), intracellular coenzymes essential for cellular respiration and glycolysis. FLIM enables measures of not only the fluorescence intensity of these molecules, which correlates with their concentration, but also the fluorescence lifetimes, that provides information on enzyme engagement. A metabolic measurement produces 9 quantitative parameters for characterizing the metabolic state of cells. Along with FLIM, second harmonic generation (SHG), which allows imaging of polarized structures such as the spindle, can also be obtained. The first study of this thesis aimed to explore the feasibility, sensitivity, and safety of FLIM and SHG to be used for oocyte and embryo assessment. This study showed that FLIM in combination with SHG can detect sensitively meaningful changes in metabolism through mouse embryo development. FLIM detected changes in the metabolic state due to metabolic perturbations and does not significantly impair mouse embryo development. Metabolic cooperativity between the oocytes and its surrounding cumulus cells is essential for correct oocyte and embryo development. The aim of the second study was to determine whether FLIM could detect differences in the metabolic state among human cumulus cell clusters. FLIM enabled quantitative measurements of the metabolic state of cumulus cells and detected variations between clusters, with greater variance among clusters from each patient than between patients. Additionally, cumulus cell metabolic parameters were associated with patient age and anti-Mullerian hormone levels, but not with BMI. Variations between cumulus cells associated with mature and immature oocytes were also found, suggesting the potential utility of this technique in ART assessments. Mammalian embryos undergo variations in metabolism over the course of preimplantation development. Embryo metabolism has long been linked to embryo viability, suggesting its potential use in selecting high quality embryos in ART. Nonetheless, the metabolism of human embryos is not fully characterized yet, probably due to a lack of non-invasive methods to measure their metabolic state. The third study sought to determine whether non-invasive FLIM could detect variations in metabolic profiles of human discarded blastocysts. FLIM revealed extensive changes in the metabolic state of discarded human blastocysts associated with blastocyst development over 36h, the day after fertilization and blastocyst developmental stage. Significant metabolic heterogeneity was observed within individual blastocysts, between the inner cell mass and the trophectoderm, and between the portions of hatching blastocysts within and without the zona pellucida. These results further demonstrate the highly dynamic and plastic metabolism of preimplantation embryos and the potential utility of measures of metabolic state to aid in embryo selection in ART. This thesis has sought to deepen the knowledge of non-invasive assessments of oocyte, cumulus cell and embryo metabolism. Taken together, the results of the present thesis present that FLIM in combination with SHG can provide detailed biologically relevant information and could be promising approaches to assess oocyte and embryo developmental competency through non-invasive quantitative measures of their metabolism and spindle morphology. Universitat Autònoma de Barcelona. Programa de Doctorat en Biologia Cel·lular

Background: Dry-cured ham is one of the most valued food products by Mediterranean consumers. In this sense, the appropriate development of its different production stages is essential to ensure the quality requirements. For this reason, non-invasive technologies have gained popularity and have been reported as useful not only to ensure the food safety of different products, but also to monitor fundamental stages in the production process, such as the salting stage, to analyze the content of different compounds without sample losses, and to correct possible defects in the final product. Scope and approach: This work has been focused on summarizing the studies that describe and have successfully applied these techniques, as well as on mentioning other technologies with potential use in dry-cured ham manufacture which have not been studied enough. Finally, the potential next steps to improve and optimize the process, as well as the suitability of creating new products with added value based on the new quality standards, have also been evaluated. Key findings and conclusions: Innovative non-invasive technologies such as high pressure (HP), ultrasound (US), pulsed electric fields (PEF), microwaves, irradiation, etc. can be used as promising tools to effectively control salting and curing stages as well as for checking defects of the final product and/or ensuring food safety. HP and US are useful tools for the determination of salt and fat content, and for monitoring the salting process. Moreover, HP enhances salty taste perception, which makes it a useful tool to reduce salt addition. Both, HP and US, can correct texture defects. In addition, NIRS allows predicting the state of the meat to remove those pieces that could result in defective products. Moreover, RAMAN or MRI are able to detect anomalous textures at the end of the process. Microwaves could be useful for the online estimation of salt, water and fat contents easily with portable equipment. Finally, data mining, that allows to make predictions based on an immense data file, is the most promising discovery in recent years for detecting defects or classifying products according to sensory attributes., This research was supported by Grant RTA 2013-00030-CO3-03 from INIA (Spain). Acknowledgements to INIA for granting Cristina Pérez Santaescolástica with a predoctoral scholarship (grant number CPD2015-0212). José M. Lorenzo is member of the MARCARNE network, funded by CYTED (ref. 116RT0503).

Background: Dry-cured ham is one of the most valued food products by Mediterranean consumers. In this sense, the appropriate development of its different production stages is essential to ensure the quality requirements. For this reason, non-invasive technologies have gained popularity and have been reported as useful not only to ensure the food safety of different products, but also to monitor fundamental stages in the production process, such as the salting stage, to analyze the content of different compounds without sample losses, and to correct possible defects in the final product. Scope and approach: This work has been focused on summarizing the studies that describe and have successfully applied these techniques, as well as on mentioning other technologies with potential use in dry-cured ham manufacture which have not been studied enough. Finally, the potential next steps to improve and optimize the process, as well as the suitability of creating new products with added value based on the new quality standards, have also been evaluated. Key findings and conclusions: Innovative non-invasive technologies such as high pressure (HP), ultrasound (US), pulsed electric fields (PEF), microwaves, irradiation, etc. can be used as promising tools to effectively control salting and curing stages as well as for checking defects of the final product and/or ensuring food safety. HP and US are useful tools for the determination of salt and fat content, and for monitoring the salting process. Moreover, HP enhances salty taste perception, which makes it a useful tool to reduce salt addition. Both, HP and US, can correct texture defects. In addition, NIRS allows predicting the state of the meat to remove those pieces that could result in defective products. Moreover, RAMAN or MRI are able to detect anomalous textures at the end of the process. Microwaves could be useful for the online estimation of salt, water and fat contents easily with portable equipment. Finally, data mining, that allows to make predictions based on an immense data file, is the most promising discovery in recent years for detecting defects or classifying products according to sensory attributes. This research was supported by Grant RTA 2013-00030-CO3-03 from INIA (Spain). Acknowledgements to INIA for granting Cristina Pérez Santaescolástica with a predoctoral scholarship (grant number CPD2015-0212). José M. Lorenzo is member of the MARCARNE network, funded by CYTED (ref. 116RT0503).

Póster presentado en el 11th International Terroir Congress, celebrado en Willamette Valley, Oregon (Estados Unidos) del 10 al 14 de julio de 2016., Soil malfunctioning, caused by an improper land preparation before vine plantation and/or management, is a common problem in European vineyards. Soil malfunctioning can include: reduced contribution of the soil fauna, poor organic matter content, imbalance nutritional status, altered pH, water deficiency, soil compaction and/or scarce oxygenation. To address these problems, ReSolVe, a transnational European research project, aimed at testing the effects of selected agronomic strategies for restoring optimal soil functionality in degraded areas within organic vineyard. The project involves 8 research groups in 6 different EU countries (Italy, Spain, France, Sweden, Slovenia, and Turkey) with experts from several disciplines including soil science, ecology, microbiology, grapevine physiology, viticulture, and biometry. The experimental vineyards are situated in Italy (Chianti hills and Maremma plain, Tuscany), Spain (La Rioja), France (Bordeaux and Languedoc), and Slovenia (Primorska) for winegrapes, and in Turkey (Adana and Mersin) for tablegrapes. Three different restoring strategies have been implemented: (i) compost, (ii) green manure with winter legumes, and (iii) dry mulching with cover crops. These strategies have being tested according to their efficiency to improve i) plant and root growth; and ii) grape yield and quality; optimize iii) the quality of soil ecosystem services; and iv) the terroir effect. The first activity of the project was characterizing and mapping the degraded areas within experimental vineyards. In the work we used non-invasive technologies to characterize soil and plant status. In Spanish and Italian vineyards, the delineation of degraded areas was performed by gamma-ray spectroscopy for topsoil, RGB machine vision for canopy status and thermography for plant water status. Gamma-ray spectroscopy measured continuously the natural gamma-ray emitted from the first 30-40 cm of soil, calculating the contribution of the main radionuclides (40K, 232Th, and 238U). The spectra of gamma-ray were able to provide information about mineralogy, texture, surficial stoniness and carbonates. RGB and thermal cameras were used to assess canopy porosity, leaf area exposure and vine water status of both degraded and non-degraded areas. All soil, canopy and water status parameters were mapped.

Tesis por compendio de publicaciones, [EN]Since the late twentieth century, Geotechnologies are being applied in different research lines in Agroforestry Engineering aimed at advancing in the modeling of biophysical parameters in order to improve the productivity. In this study, low-cost and close range photogrammetry has been used in different agroforestry scenarios to solve identified gaps in the results and improve procedures and technology hitherto practiced in this field. Photogrammetry offers the advantage of being a non-destructive and non-invasive technique, never changing physical properties of the studied element, providing rigor and completeness to the captured information. In this PhD dissertation, the following contributions are presented divided into three research papers: • A methodological proposal to acquire georeferenced multispectral data of high spatial resolution using a low-cost manned aerial platform, to monitor and sustainably manage extensive áreas of crops. The vicarious calibration is exposed as radiometric calibration method of the multispectral sensor embarked on a paraglider. Low-cost surfaces are performed as control coverages. • The development of a method able to determine crop productivity under field conditions, from the combination of close range photogrammetry and computer vision, providing a constant operational improvement and a proactive management in the crop monitoring. An innovate methodology in the sector is proposed, ensuring flexibility and simplicity in the data collection by non-invasive technologies, automation in processing and quality results with low associated cost. • A low cost, efficient and accurate methodology to obtain Digital Height Models of vegatal cover intended for forestry inventories by integrating public data from LiDAR into photogrammetric point clouds coming from low cost flights. This methodology includes the potentiality of LiDAR to register ground points in areas with high density of vegetation and the better spatial, radiometric and temporal resolution from photogrammetry for the top of vegetal covers., [ES]Desde finales del siglo XX se están aplicando Geotecnologías en diferentes líneas de investigación en Ingeniería Agroforestal orientadas a avanzar en la modelización de parámetros biofísicos con el propósito de mejorar la productividad. En este estudio se ha empleado fotogrametría de bajo coste y rango cercano en distintos escenarios agroforestales para solventar carencias detectadas en los resultados obtenidos y mejorar los procedimientos y la tecnología hasta ahora usados en este campo. La fotogrametría ofrece como ventaja el ser una técnica no invasiva y no destructiva, por lo que no altera en ningún momento las propiedades físicas del elemento estudiado, dotando de rigor y exhaustividad a la información capturada. En esta Tesis Doctoral se presentan las siguientes contribuciones, divididas en tres artículos de investigación: • Una propuesta metodológica de adquisición de datos multiespectrales georreferenciados de alta resolución espacial mediante una plataforma aérea tripulada de bajo coste, para monitorizar y gestionar sosteniblemente amplias extensiones de cultivos. Se expone la calibración vicaria como método de calibración radiométrico del sensor multiespectral embarcado en un paramotor empleando como coberturas de control superficies de bajo coste. • El desarrollo de un método capaz de determinar la productividad del cultivo en condiciones de campo, a partir de la combinación de fotogrametría de rango cercano y visión computacional, facilitando una mejora operativa constante así como una gestión proactiva en la monitorización del cultivo. Se propone una metodología totalmente novedosa en el sector, garantizando flexibilidad y sencillez en la toma de datos mediante tecnologías no invasivas, automatismo en el procesado, calidad en los resultados y un bajo coste asociado. • Una metodología de bajo coste, eficiente y precisa para la obtención de Modelos Digitales de Altura de Cubierta Vegetal destinados al inventario forestal mediante la integración de datos públicos procedentes del LiDAR en las nubes de puntos fotogramétricas obtenidas con un vuelo de bajo coste. Esta metodología engloba la potencialidad del LiDAR para registrar el terreno en zonas con alta densidad de vegetación y una mejor resolución espacial, radiométrica y temporal procedente de la fotogrametría para la parte superior de las cubiertas vegetales.

© 2014 Elsevier Ltd. Wine quality depends mostly on the features of the grapes it is made from. Cluster and berry morphology are key factors in determining grape and wine quality. However, current practices for grapevine quality estimation require time-consuming destructive analysis or largely subjective judgment by experts.The purpose of this paper is to propose a three-dimensional computer vision approach to assessing grape yield components based on new 3D descriptors. To achieve this, firstly a partial three-dimensional model of the grapevine cluster is extracted using stereo vision. After that a number of grapevine quality components are predicted using SVM models based on new 3D descriptors. Experiments confirm that this approach is capable of predicting the main cluster yield components, which are related to quality, such as cluster compactness and berry size (R2 > 0.80, p < 0.05). In addition, other yield components: cluster volume, total berry weight and number of berries, were also estimated using SVM models, obtaining prediction R2 of 0.82, 0.83 and 0.71, respectively., This work has been partially funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria de España (INIA – Spanish National Institute for Agriculture and Food Research and Technology) through research project RTA2012-00062-C04-02, support of European FEDER funds, UPV-SP20120276 and AGL2011-23673 project.

[Background] Flowers, flowering and fruit set are key determinants of grapevine yield. Currently, practical methods to assess the flower number per inflorescence, necessary for fruit set estimation, are time and labour demanding. This work aims at developing a simple, cheap, fast, accurate and robust machine vision methodology to be applied to RGB images taken under field conditions, to estimate the number of flowers per inflorescence automatically., [Results] Ninety images of individual inflorescences of Vitis vinifera L. cultivars Tempranillo, Graciano and Carignan were acquired in the vineyard with a pocket RGB camera prior to flowering, and used to develop and test the 'flower counting' algorithm. Strong and significant relationships, with R2 above 80% for the three cultivars were observed between actual and automated estimation of inflorescence flower numbers, with a precision exceeding 90% for all cultivars., [Conclusion] The developed algorithm proved that the analysis of digital images captured by pocket cameras under uncontrolled outdoors conditions was able to automatically provide a useful estimation of the number of flowers per inflorescence of grapevines at early stages of flowering. © 2013 Society of Chemical Industry.