Self-organizing cultured neural networks: Image analysis techniques for longitudinal tracking and modeling of the underlying network structure

Esta tesis estudia la evolución estructural de conjuntos de neuronas como la capacidad de auto-organización desde conjuntos de neuronas separadas hasta que forman una red (clusterizada) compleja. Esta tesis contribuye con el diseño e implementación de un algoritmo no supervisado de segmentación basado en grafos con un coste computacional muy bajo. Este algoritmo proporciona de forma automática la estructura completa de la red a partir de imágenes de cultivos neuronales tomadas con microscopios de fase con una resolución muy alta. La estructura de la red es representada mediante un objeto matemático (matriz) cuyos nodos representan a las neuronas o grupos de neuronas y los enlaces son las conexiones reconstruidas entre ellos. Este algoritmo extrae también otras medidas morfológicas importantes que caracterizan a las neuronas y a las neuritas. A diferencia de otros algoritmos hasta el momento, que necesitan de fluorescencia y técnicas inmunocitoquímicas, el algoritmo propuesto permite el estudio longitudinal de forma no invasiva posibilitando el estudio durante la formación de un cultivo. Además, esta tesis, estudia de forma sistemática un grupo de variables topológicas que garantizan la posibilidad de cuantificar e investigar la progresión de las características principales durante el proceso de auto-organización del cultivo. Nuestros resultados muestran la existencia de un estado concreto correspondiente a redes con configuracin small-world y la emergencia de propiedades a micro- y meso-escala de la estructura de la red. Finalmente, identificamos los procesos físicos principales que guían las transformaciones morfológicas de los cultivos y proponemos un modelo de crecimiento de red que reproduce el comportamiento cuantitativamente de las observaciones experimentales. ABSTRACT The thesis analyzes the morphological evolution of assemblies of living neurons, as they self-organize from collections of separated cells into elaborated, clustered, networks. In particular, it contributes with the design and implementation of a graph-based unsupervised segmentation algorithm, having an associated very low computational cost. The processing automatically retrieves the whole network structure from large scale phase-contrast images taken at high resolution throughout the entire life of a cultured neuronal network. The network structure is represented by a mathematical object (a matrix) in which nodes are identified neurons or neurons clusters, and links are the reconstructed connections between them. The algorithm is also able to extract any other relevant morphological information characterizing neurons and neurites. More importantly, and at variance with other segmentation methods that require fluorescence imaging from immunocyto- chemistry techniques, our measures are non invasive and entitle us to carry out a fully longitudinal analysis during the maturation of a single culture. In turn, a systematic statistical analysis of a group of topological observables grants us the possibility of quantifying and tracking the progression of the main networks characteristics during the self-organization process of the culture. Our results point to the existence of a particular state corresponding to a small-world network configuration, in which several relevant graphs micro- and meso-scale properties emerge. Finally, we identify the main physical processes taking place during the cultures morphological transformations, and embed them into a simplified growth model that quantitatively reproduces the overall set of experimental observations.

Self-organizing cultured neural networks: Image analysis techniques for longitudinal tracking and modeling of the underlying network structure (versión actualizada)

Esta tesis estudia la evolución estructural de conjuntos de neuronas como la capacidad de auto-organización desde conjuntos de neuronas separadas hasta que forman una red (clusterizada) compleja. Esta tesis contribuye con el diseño e implementación de un algoritmo no supervisado de segmentación basado en grafos con un coste computacional muy bajo. Este algoritmo proporciona de forma automática la estructura completa de la red a partir de imágenes de cultivos neuronales tomadas con microscopios de fase con una resolución muy alta. La estructura de la red es representada mediante un objeto matemático (matriz) cuyos nodos representan a las neuronas o grupos de neuronas y los enlaces son las conexiones reconstruidas entre ellos. Este algoritmo extrae también otras medidas morfológicas importantes que caracterizan a las neuronas y a las neuritas. A diferencia de otros algoritmos hasta el momento, que necesitan de fluorescencia y técnicas inmunocitoquímicas, el algoritmo propuesto permite el estudio longitudinal de forma no invasiva posibilitando el estudio durante la formación de un cultivo. Además, esta tesis, estudia de forma sistemática un grupo de variables topológicas que garantizan la posibilidad de cuantificar e investigar la progresión de las características principales durante el proceso de auto-organización del cultivo. Nuestros resultados muestran la existencia de un estado concreto correspondiente a redes con configuracin small-world y la emergencia de propiedades a micro- y meso-escala de la estructura de la red. Finalmente, identificamos los procesos físicos principales que guían las transformaciones morfológicas de los cultivos y proponemos un modelo de crecimiento de red que reproduce el comportamiento cuantitativamente de las observaciones experimentales. ABSTRACT The thesis analyzes the morphological evolution of assemblies of living neurons, as they self-organize from collections of separated cells into elaborated, clustered, networks. In particular, it contributes with the design and implementation of a graph-based unsupervised segmentation algorithm, having an associated very low computational cost. The processing automatically retrieves the whole network structure from large scale phase-contrast images taken at high resolution throughout the entire life of a cultured neuronal network. The network structure is represented by a mathematical object (a matrix) in which nodes are identified neurons or neurons clusters, and links are the reconstructed connections between them. The algorithm is also able to extract any other relevant morphological information characterizing neurons and neurites. More importantly, and at variance with other segmentation methods that require fluorescence imaging from immunocyto- chemistry techniques, our measures are non invasive and entitle us to carry out a fully longitudinal analysis during the maturation of a single culture. In turn, a systematic statistical analysis of a group of topological observables grants us the possibility of quantifying and tracking the progression of the main networks characteristics during the self-organization process of the culture. Our results point to the existence of a particular state corresponding to a small-world network configuration, in which several relevant graphs micro- and meso-scale properties emerge. Finally, we identify the main physical processes taking place during the cultures morphological transformations, and embed them into a simplified growth model that quantitatively reproduces the overall set of experimental observations.

Use of the energy-release rate in a 2 scale damage analysis of a bonded joint

Stress singularities appear at the extremities of an adhesive bond. They can produce a damage mechanism that we assimilate in this Note to a crack. The energy release rate permits to characterize its evolution. But a very refined mesh would be necessary for a real structure. Using an asymptotic method based on the small thickness of the bond a limit model with a different local behaviour is suggested. It leads to an approximation of the energy release rate

Rapid prototyping of multi-scale biomedical microdevices by combining additive manufacturing technologies

The possibility of designing and manufacturing biomedical microdevices with multiple length-scale geometries can help to promote special interactions both with their environment and with surrounding biological systems. These interactions aim to enhance biocompatibility and overall performance by using biomimetic approaches. In this paper, we present a design and manufacturing procedure for obtaining multi-scale biomedical microsystems based on the combination of two additive manufacturing processes: a conventional laser writer to manufacture the overall device structure, and a direct-laser writer based on two-photon polymerization to yield finer details. The process excels for its versatility, accuracy and manufacturing speed and allows for the manufacture of microsystems and implants with overall sizes up to several millimeters and with details down to sub-micrometric structures. As an application example we have focused on manufacturing a biomedical microsystem to analyze the impact of microtextured surfaces on cell motility. This process yielded a relevant increase in precision and manufacturing speed when compared with more conventional rapid prototyping procedures.

Liquid crystal temperature sensor based on a micrometric structure and a metallic nanometric layer

This letter presents a novel temperature sensor, which consists of an interdigitated comb electrode structure with a micrometric-scale size, nanometric metallic layer, and nematic liquid crystal (NLC) film. This sensor exploits the permittivity dependence of the NLC with temperature and principle of electrical conductivity above the percolation threshold in thin film metallic layers. The latter has been demonstrated to increase the temperature sensitivity considerably. The high impedance input reduces the power dissipation, and the high enough voltage output makes it easy to measure the output signal with high precision. The operation principle and fabrication process as well as the characterization of the temperature sensor are presented. Experimental results show that the device offers a sensitivity of 9 mV/°C and is dependent on the applied voltage. This is six times greater than the same structure without the use of a nanometric layer.

Guidelines for the design of an environmental flow regime program at the regional scale: Application of the ELOHA framework in the Ebro River basin

La gestión de los recursos hídricos se convierte en un reto del presente y del futuro frente a un panorama de continuo incremento de la demanda de agua debido al crecimiento de la población, el crecimiento del desarrollo económico y los posibles efectos del calentamiento global. La política hidráulica desde los años 60 en España se ha centrado en la construcción de infraestructuras que han producido graves alteraciones en el régimen natural de los ríos. Estas alteraciones han provocado y acrecentado los impactos sobre los ecosistemas fluviales y ribereños. Desde los años 90, sin embargo, ha aumentado el interés de la sociedad para conservar estos ecosistemas. El concepto de caudales ambientales consiste en un régimen de caudales que simula las características principales del régimen natural. Los caudales ambientales están diseñados para conservar la estructura y funcionalidad de los ecosistemas asociados al régimen fluvial, bajo la hipótesis de que los elementos que conforman estos ecosistemas están profundamente adaptados al régimen natural de caudales, y que cualquier alteración del régimen natural puede provocar graves daños a todo el sistema. El método ELOHA (Ecological Limits of Hydrological Alteration) tiene como finalidad identificar las componentes del régimen natural de caudales que son clave para mantener el equilibrio de los ecosistemas asociados, y estimar los límites máximos de alteración de estas componentes para garantizar su buen estado. Esta tesis presenta la aplicación del método ELOHA en la cuenca del Ebro. La cuenca del Ebro está profundamente regulada e intervenida por el hombre, y sólo las cabeceras de los principales afluentes del Ebro gozan todavía de un régimen total o cuasi natural. La tesis se estructura en seis capítulos que desarrollan las diferentes partes del método. El primer capítulo explica cómo se originó el concepto “caudales ambientales” y en qué consiste el método ELOHA. El segundo capítulo describe el área de estudio. El tercer capítulo realiza una clasificación de los regímenes naturales de la cuenca (RNC) del Ebro, basada en series de datos de caudal mínimamente alterado y usando exclusivamente parámetros hidrológicos. Se identificaron seis tipos diferentes de régimen natural: pluvial mediterráneo, nivo-pluvial, pluvial mediterréaneo con una fuerte componente del caudal base, pluvial oceánico, pluvio-nival oceánico y Mediterráneo. En el cuarto capítulo se realiza una regionalización a toda la cuenca del Ebro de los seis RNC encontrados en la cueca. Mediante parámetros climáticos y fisiográficos se extrapola la información del tipo de RNC a puntos donde no existen datos de caudal inalterado. El patrón geográfico de los tipos de régimen fluvial obtenido con la regionalización resultó ser coincidente con el patrón obtenido a través de la clasificación hidrológica. El quinto capítulo presenta la validación biológica de los procesos de clasificación anteriores: clasificación hidrológica y regionalización. La validación biológica de los tipos de regímenes fluviales es imprescindible, puesto que los diferentes tipos de régimen fluvial van a servir de unidades de gestión para favorecer el mantenimiento de los ecosistemas fluviales. Se encontraron diferencias significativas entre comunidades biológicas en cinco de los seis tipos de RNC encontrados en la cuenca. Finalmente, en el sexto capítulo se estudian las relaciones hidro-ecológicas existentes en tres de los seis tipos de régimen fluvial encontrados en la cuenca del Ebro. Mediante la construcción de curvas hidro-ecológicas a lo largo de un gradiente de alteración hidrológica, se pueden sugerir los límites de alteración hidrológica (ELOHAs) para garantizar el buen estado ecológico en cada uno de los tipos fluviales estudiados. Se establecieron ELOHAs en tres de los seis tipos de RNC de la cuenca del Ebro Esta tesis, además, pone en evidencia la falta de datos biológicos asociados a registros de caudal. Para llevar a cabo la implantación de un régimen de caudales ambientales en la cuenca, la ubicación de los puntos de muestreo biológico cercanos a estaciones de aforo es imprescindible para poder extraer relaciones causa-efecto de la gestión hidrológica sobre los ecosistemas dependientes. ABSTRACT In view of a growing freshwater demand because of population raising, improvement of economies and the potential effects of climate change, water resources management has become a challenge for present and future societies. Water policies in Spain have been focused from the 60’s on constructing hydraulic infrastructures, in order to dampen flow variability and granting water availability along the year. Consequently, natural flow regimes have been deeply altered and so the depending habitats and its ecosystems. However, an increasing acknowledgment of societies for preserving healthy freshwater ecosystems started in the 90’s and agreed that to maintain healthy freshwater ecosystems, it was necessary to set environmental flow regimes based on the natural flow variability. The Natural Flow Regime paradigm (Richter et al. 1996, Poff et al. 1997) bases on the hypothesis that freshwater ecosystems are made up by elements adapted to natural flow conditions, and any change on these conditions can provoke deep impacts on the whole system. Environmental flow regime concept consists in designing a flow regime that emulates natural flow characteristics, so that ecosystem structure, functions and services are maintained. ELOHA framework (Ecological Limits of Hydrological Alteration) aims to identify key features of the natural flow regime (NFR) that are needed to maintain and preserve healthy freshwater and riparian ecosystems. Moreover, ELOHA framework aims to quantify thresholds of alteration of these flow features according to ecological impacts. This thesis describes the application of the ELOHA framework in the Ebro River Basin. The Ebro River basin is the second largest basin in Spain and it is highly regulated for human demands. Only the Ebro headwaters tributaries still have completely unimpaired flow regime. The thesis has six chapters and the process is described step by step. The first chapter makes an introduction to the origin of the environmental flow concept and the necessity to come up. The second chapter shows a description of the study area. The third chapter develops a classification of NFRs in the basin based on natural flow data and using exclusively hydrological parameters. Six NFRs were found in the basin: continental Mediterranean-pluvial, nivo-pluvial, continental Mediterranean pluvial (with groundwater-dominated flow pattern), pluvio-oceanic, pluvio-nival-oceanic and Mediterranean. The fourth chapter develops a regionalization of the six NFR types across the basin by using climatic and physiographic variables. The geographical pattern obtained from the regionalization process was consistent with the pattern obtained with the hydrologic classification. The fifth chapter performs a biological validation of both classifications, obtained from the hydrologic classification and the posterior extrapolation. When the aim of flow classification is managing water resources according to ecosystem requirements, a validation based on biological data is compulsory. We found significant differences in reference macroinvertebrate communities between five over the six NFR types identified in the Ebro River basin. Finally, in the sixth chapter we explored the existence of significant and explicative flow alteration-ecological response relationships (FA-E curves) within NFR types in the Ebro River basin. The aim of these curves is to find out thresholds of hydrological alteration (ELOHAs), in order to preserve healthy freshwater ecosystem. We set ELOHA values in three NFR types identified in the Ebro River basin. During the development of this thesis, an inadequate biological monitoring in the Ebro River basin was identified. The design and establishment of appropriate monitoring arrangements is a critical final step in the assessment and implementation of environmental flows. Cause-effect relationships between hydrology and macroinvertebrate community condition are the principal data that sustain FA-E curves. Therefore, both data sites must be closely located, so that the effects of external factors are minimized. The scarce hydro-biological pairs of data available in the basin prevented us to apply the ELOHA method at all NFR types.

Mycorrhizal preferences and fine spatial structure of the epiphytic orchid Epidendrum rhopalostele

• Premise of the study: The presence of compatible fungi is necessary for epiphytic orchid recruitment. Thus, identifying associated mycorrhizal fungi at the population level is essential for orchid conservation. Recruitment patterns may also be conditioned by factors such as seed dispersal range and specific environmental characteristics. • Methods: In a forest plot, all trees with a diameter at breast height >1 cm and all individuals of the epiphytic orchid Epidendrum rhopalostele were identified and mapped. Additionally, one flowering individual of E. rhopalostele per each host tree was randomly selected for root sampling and DNA extraction. • Key results: A total of 239 E. rhopalostele individuals were located in 25 of the 714 potential host trees. Light microscopy of sampled roots showed mycorrhizal fungi in 22 of the 25 sampled orchids. Phylogenetic analysis of ITS1-5.8S-ITS2 sequences yielded two Tulasnella clades. In four cases, plants were found to be associated with both clades. The difference between univariate and bivariate K functions was consistent with the random labeling null model at all spatial scales, indicating that trees hosting clades A and B of Tulasnella are not spatially segregated. The analysis of the inhomogenous K function showed that host trees are not clustered, suggesting no limitations to population-scale dispersal. χ2 analysis of contingency tables showed that E. rhopalostele is more frequent on dead trees than expected. • Conclusions: Epidendrum rhopalostele establishes mycorrhizal associations with at least two different Tulasnella species. The analysis of the distribution patterns of this orchid suggests a microsite preference for dead trees and no seed dispersal limitation.

Non prismatic folded roof structure

The design of a prestressed concrete roof is described. A special linear elastic analysis of non-prismatic folded structures has been applied. The obtained results have been compared to the results deducted from a small scale model test. Conclusions about the efficiency of these type of structures are shown.

Unearthing the roots of degradation of Quercus pyrenaica coppices: an integrative perspective from clonal structure to carbon budgets

Quercus pyrenaica es una especie rebrotadora de raíz intensa e históricamente aprovechada en monte bajo para la obtención de leñas, carbón y pastos. Debido al éxodo rural y a la aparición de nuevas fuentes energéticas, este aprovechamiento fue abandonado en la década de 1970. Desde entonces, las bajas producciones de madera y bellota y el puntisecado de los pies evidencian el generalizado estancamiento de estas masas. Uno de los mayores retos actuales de la selvicultura en el ámbito mediterráneo es encontrar usos alternativos para estos montes abandonados, siendo la conversión a monte alto una de las alternativas preferidas. Se han realizado resalveos de conversión, sin embrago, éstos se aplican sin un conocimiento integral de las causas de la degradación. En esta tesis doctoral, estudiamos un hipotético desequilibrio entre la parte radical y la parte aérea (R:S) de las cepas de rebollo como causa subyacente de su decaimiento. En una parcela experimental, aprovechada al menos desde el siglo XII, se realizaron análisis genéticos a priori para elucidar la estructura genética del rodal, y así estudiar la influencia del tamaño clonal en el funcionamiento de las cepas. Las cepas de mayor tamaño presentaron un menor crecimiento diametral de sus pies, así como mayores tasas de respiración radical, estimadas a partir de flujos internos de CO2 a través del xilema (FT) y de los flujos de CO2 del suelo. Estos resultados sugieren que el desequilibrio R:S aumenta con el tamaño clonal, dado que la eliminación periódica de órganos aéreos, al mismo tiempo que las raíces permanecen intactas, da lugar a un gran desarrollo del sistema radical que consume gran parte de los carbohidratos no estructurales (NSC) en respiración de mantenimiento, comprometiendo así el desarrollo de órganos aéreos. Se excavaron y pesaron dos cepas compuestas por cuatro y ocho pies, las cuales mostraron ratios R:S (0.5 y 1, respectivamente) superiores a los registrados en pies de origen sexual. Al igual que en otras especies rebrotadoras de raíz, se observaron altas concentraciones de NSC en las raíces (> 20% en primavera) y una gran proporción de albura en el sistema radical (52%) que alberga una notable reserva de NSC (87 kg en la cepa de mayor tamaño). En el sistema radical de dicha cepa, estimada mediante dataciones radiocarbónicas en 550 años de edad, se contaron 248 uniones radicales. La persistencia de sistemas radicales grandes, viejos, y altamente interconectados sugiere que la gran cantidad de recursos almacenados y consumidos en las raíces compensan un pobre desarrollo aéreo con una alta resiliencia vegetativa. Para un mejor entendimiento de los balances de carbono y del agotamiento de NSC en las cepas de rebollo, se midieron los flujos internos y externos de CO2 en troncos y los flujos de CO2 del suelo, y se estimó la respiración de órganos aéreos (RS) y subterráneos (RR). Estacionalmente, RS y RR reflejaron las dinámicas de flujo de savia y de crecimiento del tronco, y estuvieron determinadas principalmente por los flujos externos de CO2, dada la escasa contribución de FT a RS y RR (< 10% y < 2%, respectivamente). En una escala circadiana, la contribución de FT a RS aumentó hasta un 25% en momentos de alta transpiración. Las bajas concentraciones de CO2 en el xilema ([CO2] hasta un 0.11%) determinaron comparativamente unos bajos FT, probablemente causados por una limitada respiración del xilema y una baja resistencia a la difusión radial del CO2 impuestos por la sequía estival. Los pulsos de [CO2] observados tras las primeras lluvias de otoño apoyan esta idea. A lo largo del periodo vegetativo, el flujo medio de CO2 procedente del suelo (39 mol CO2 day-1) fue el mayor flujo respiratorio, tres y cuatro veces superior a RS (12 mol CO2 day-1) y RR (8-9 mol CO2 day-1), respectivamente. Ratios RR/RS menores que la unidad evidencian un importante peso de la respiración aérea como sumidero de carbono adicional. Finalmente, se ensayó el zanjado de raíces y el anillamiento de troncos como tratamientos selvícolas alternativos con el objetivo de aumentar las reservas de NSC en los troncos de las cepas. Los resultados preliminares desaconsejan el zanjado de raíces por el alto coste derivado posiblemente de la cicatrización de las heridas. El anillado de troncos imposibilitó el transporte de NSC a las raíces y aumentó la concentración de almidón por encima de la zona anillada, mientras que sistema radical se mantiene por los pies no anillados de la cepa. Son necesarias más mediciones y datos adicionales para comprobar el mantenimiento de esta respuesta positiva a largo plazo. Para concluir, destacamos la necesidad de estudios multidisciplinares que permitan una comprensión integral de la degradación de los rebollares ibéricos para poder aplicar a posteriori una gestión adecuada en estos montes bajos abandonados. ABSTRACT Quercus pyrenaica is a vigorous root-resprouting species intensively and historically coppiced for firewood, charcoal and woody pastures. Due to the rural exodus and the appearance of new energy sources, coppicing was abandoned towards 1970. Since then, tree overaging has resulted in stand stagnation displayed by slow stem growth, branch dieback, and scarce acorn production. The urgent need to find new alternative uses for abandoned coppices is recognized as one of the biggest challenges which currently faces Mediterranean silviculture; conversion into high forest by thinning is one of the preferred alternatives. For this aim, thinning has been broadly applied and seldom tested, although without a comprehensive understanding of the causes of stand stagnation. In this PhD study, we test the hypothesis of an imbalance between above- and below-ground organs, result of long term coppicing, as the underlying cause of Q. pyrenaica decay. In an experimental plot coppiced since at least the 12th century, genetic analyses were performed a priori to elucidate inconspicuous clonal structure of Q. pyrenaica to evaluate how clonal size affects the functioning of these multi-stemmed trees. Clonal size negatively affected diametric stem growth, whereas root respiration rates, measured by internal fluxes of CO2 through xylem (FT) and soil CO2 efflux, increased with clonal size. These results suggest root-to-shoot (R:S) imbalance intensifying with clonal size: periodic removal of aboveground organs whilst belowground organs remain undisturbed may have led to massive root systems which consume a great proportion of non-structural carbohydrates (NSC) for maintenance respiration, thus constraining aboveground performance. Furthermore, excavation of two multi-stemmed trees, composed by four and eight stems, revealed R:S ratios (0.5 and 1, respectively) greater than those reported for sexually regenerated trees. Moreover, as similarly observed in several root-resprouting species, NSC allocation to roots was favored ([NSC] > 20% in spring): a large proportion of sapwood maintained throughout the root system (52%) stored a remarkable NSC pool of 87 kg in the case of the largest clone. In this root system of the eight-stemmed tree, 248 root connections were counted and, by radiocarbon dating, its age was estimated to be 550-years-old. Persistence of massive, old and highly interconnected root systems suggests that enhanced belowground NSC storage and consumption reflects a trade-off between vegetative resilience and aboveground development. For a better understanding of tree carbon budget and the potential role of carbon starvation in Q. pyrenaica decay, internal and external stem CO2 fluxes and soil CO2 effluxes were monitored to evaluate respiratory costs above- and below-ground. On a seasonal scale, stem and root respiration (RS and RR) mirrored sap flow and stem growth dynamics. Respiration was determined to the greatest extent by external fluxes of CO2 to the atmosphere or soil, since FT accounted for a low proportion of RS and RR (< 10% and < 2%, respectively). On a diel scale, the contribution of FT to RS increased up to 25% at high transpiration rates. Comparatively low FT was determined by the low concentration of xylem CO2 registered ([CO2] as low as 0.11%), likely as a consequence of constrained xylem respiration and reduced resistance to CO2 radial diffusion imposed by summer drought. Xylem [CO2] pulses following first autumn rains support this idea. Averaged over the growing season, soil CO2 efflux was the greatest respiratory flux (39 mol CO2 day-1), three and four times greater than RS (12 mol CO2 day-1) and RR (8-9 mol CO2 day-1), respectively. Ratios of RR/RS below one evidence an additional and important weight of aboveground respiration as a tree carbon sink. Finally, root trenching and stem girdling were tested as complimentary treatments to thinning as a means to improve carbon reserves in stems of clonal trees. Preliminary results discouraged root trenching due to the high cost likely incurred for wound closure. Stem girdling successfully blocked NSC translocation downward, increasing starch concentrations above the girdled zone whilst the root system is fed by non-girdled stems within the clone. Further measurements and ancillary data are necessary to verify that this positive effect hold over time. To conclude, the need of multidisciplinary approaches for an integrative understanding on the functioning of abandoned Q pyrenaica coppices is highlighted for an appropriate management of these stands.

A multi-scale analysis of transforming agricultural markets in the context of globalization: implications for natural resources, food prices and rural poverty in Latin America

Vivimos una época en la que el mundo se transforma aceleradamente. La globalización está siguiendo un curso imparable, la población mundial así como la población urbana siguen creciendo, y en los países emergentes los ingresos promedios aumentan, resultando en un cambio también acelerado de las dietas y hábitos alimentarios. En conjunto esos factores están causando un aumento fundamental de la demanda de alimentos. Junto con la apertura de los mercados agrícolas, estos procesos han provocado un crecimiento del comercio internacional de alimentos durante la última década. Dado que muchos países de América Latina están dotados de abundancia de recursos naturales, estas tendencias han producido un crecimiento rápido de las exportaciones de bienes primarios desde América Latina al resto del mundo. En sólo 30 años la participación en el mercado agrícola de América Latina casi se ha duplicado, desde 10% en 1980 a 18% en 2010. Este aumento del comercio agrícola ha dado lugar a un debate sobre una serie de cuestiones cruciales relacionadas con los impactos del comercio en la seguridad alimentaria mundial, en el medio ambiente o en la reducción de la pobreza rural en países en desarrollo. Esta tesis aplica un marco integrado para analizar varios impactos relacionados con la transformación de los mercados agrícolas y los mercados rurales debidos a la globalización y, en particular, al progresivo aumento del comercio internacional. En concreto, la tesis aborda los siguientes temas: En primer lugar, la producción mundial de alimentos tendrá que aumentar considerablemente para poder satisfacer la demanda de una población mundial de 9000 millones personas en 2050, lo cual plantea grandes desafíos sobre los sistemas de la producción de alimentos. Alcanzar este logro, sin comprometer la integridad del medio ambiente en regiones exportadoras, es un reto aún mayor. En este contexto, la tesis analiza los efectos de la liberalización del comercio mundial, considerando distintas tecnologías de producción agraria, sobre unos indicadores de seguridad alimentaria en diferentes regiones del mundo y sobre distintos indicadores ambientales, teniendo en cuenta escalas diferentes en América Latina y el Caribe. La tesis utiliza el modelo “International Model for Policy Analysis of Agricultural Commodities and Trade (IMPACT)” – un modelo dinámico de equilibrio parcial del sector agrícola a escala global – para modelar la apertura de los mercados agrícolas así como diferentes escenarios de la producción hasta el año 2050. Los resultados del modelo están vinculados a modelos biofísicos para poder evaluar los cambios en la huella hídrica y la calidad del agua, así como para cuantificar los impactos del cambio en el uso del suelo sobre la biodiversidad y los stocks de carbono en 2050. Los resultados indican que la apertura de los mercados agrícolas es muy importante para mejorar la seguridad alimentaria a nivel mundial, sin embargo, produce también presiones ambientales indeseables en algunas regiones de América Latina. Contrastando dos escenarios que consideran distintas modos de producción, la expansión de la tierra agrícola frente a un escenario de la producción más intensiva, se demuestra que las mejoras de productividad son generalmente superiores a la expansión de las tierras agrícolas, desde un punto de vista económico e ambiental. En cambio, los escenarios de intensificación sostenible no sólo hacen posible una mayor producción de alimentos, sino que también generan menos impactos medioambientales que los otros escenarios futuros en todas sus dimensiones: biodiversidad, carbono, emisiones de nitratos y uso del agua. El análisis muestra que hay un “trade-off” entre el objetivo de alcanzar la sostenibilidad ambiental y el objetivo de la seguridad alimentaria, independiente del manejo agrícola en el futuro. En segundo lugar, a la luz de la reciente crisis de los precios de alimentos en los años 2007/08, la tesis analiza los impactos de la apertura de los mercados agrícolas en la transmisión de precios de los alimentos en seis países de América Latina: Argentina, Brasil, Chile, Colombia, México y el Perú. Para identificar las posibles relaciones de cointegración entre los índices de precios al consumidor de alimentos y los índices de precios de agrarios internacionales, sujetos a diferentes grados de apertura de mercados agrícolas en los seis países de América Latina, se utiliza un modelo simple de corrección de error (single equation error correction). Los resultados indican que la integración global de los mercados agrícolas ha dado lugar a diferentes tasas de transmisión de precios en los países investigados. Sobre todo en el corto plazo, las tasas de transmisión dependen del grado de apertura comercial, mientras que en el largo plazo las tasas de transmisión son elevadas, pero en gran medida independientes del régimen de comercio. Por lo tanto, durante un período de shocks de precios mundiales una mayor apertura del comercio trae consigo más inestabilidad de los precios domésticos a corto plazo y la resultante persistencia en el largo plazo. Sin embargo, estos resultados no verifican necesariamente la utilidad de las políticas comerciales, aplicadas frecuentemente por los gobiernos para amortiguar los shocks de precios. Primero, porque existe un riesgo considerable de volatilidad de los precios debido a cambios bruscos de la oferta nacional si se promueve la autosuficiencia en el país; y segundo, la política de proteccionismo asume el riesgo de excluir el país de participar en las cadenas de suministro de alto valor del sector agrícola, y por lo tanto esa política podría obstaculizar el desarrollo económico. Sin embargo, es indispensable establecer políticas efectivas para reducir la vulnerabilidad de los hogares a los aumentos repentinos de precios de alimentos, lo cual requiere una planificación gubernamental precisa con el presupuesto requerido disponible. En tercer lugar, la globalización afecta a la estructura de una economía y, por medios distintos, la distribución de los ingreso en un país. Perú sirve como ejemplo para investigar más profundamente las cuestiones relacionadas con los cambios en la distribución de los ingresos en zonas rurales. Perú, que es un país que está cada vez más integrado en los mercados mundiales, consiguió importantes descensos en la pobreza extrema en sus zonas rurales, pero a la vez adolece de alta incidencia de pobreza moderada y de desigualdad de los ingresos en zonas rural al menos durante el periodo comprendido entre 2004 y 2012. Esta parte de la tesis tiene como objetivo identificar las fuerzas impulsoras detrás de estas dinámicas en el Perú mediante el uso de un modelo de microsimulación basado en modelos de generación de ingresos aplicado a nivel los hogares rurales. Los resultados indican que la fuerza principal detrás de la reducción de la pobreza ha sido el crecimiento económico general de la economía, debido a las condiciones macroeconómicas favorables durante el periodo de estudio. Estos efectos de crecimiento beneficiaron a casi todos los sectores rurales, y dieron lugar a la disminución de la pobreza rural extrema, especialmente entre los agricultores de papas y de maíz. En parte, estos agricultores probablemente se beneficiaron de la apertura de los mercados agrícolas, que es lo que podría haber provocado un aumento de los precios al productor en tiempos de altos precios mundiales de los alimentos. Sin embargo, los resultados también sugieren que para una gran parte de la población más pobre existían barreras de entrada a la hora de poder participar en el empleo asalariado fuera de la agricultura o en la producción de cultivos de alto valor. Esto podría explicarse por la falta de acceso a unos activos importantes: por ejemplo, el nivel de educación de los pobres era apenas mejor en 2012 que en 2004; y también las dotaciones de tierra y de mano de obra, sobre todo de los productores pobres de maíz y patata, disminuyeron entre 2004 y 2012. Esto lleva a la conclusión de que aún hay margen para aplicar políticas para facilitar el acceso a estos activos, que podría contribuir a la erradicación de la pobreza rural. La tesis concluye que el comercio agrícola puede ser un importante medio para abastecer una población mundial creciente y más rica con una cantidad suficiente de calorías. Para evitar adversos efectos ambientales e impactos negativos para los consumidores y de los productores pobres, el enfoque debe centrarse en las mejoras de la productividad agrícola, teniendo en cuenta los límites ambientales y ser socialmente inclusivo. En este sentido, será indispensable seguir desarrollando soluciones tecnológicas que garanticen prácticas de producción agrícola minimizando el uso de recursos naturales. Además, para los pequeños pobres agricultores será fundamental eliminar las barreras de entrada a los mercados de exportación que podría tener efectos indirectos favorables a través de la adopción de nuevas tecnologías alcanzables a través de mercados internacionales. ABSTRACT The world is in a state of rapid transition. Ongoing globalization, population growth, rising living standards and increasing urbanization, accompanied by changing dietary patterns throughout the world, are increasing the demand for food. Together with more open trade regimes, this has triggered growing international agricultural trade during the last decade. For many Latin American countries, which are gifted with relative natural resource abundance, these trends have fueled rapid export growth of primary goods. In just 30 years, the Latin American agricultural market share has almost doubled from 10% in 1980 to 18% in 2010. These market developments have given rise to a debate around a number of crucial issues related to the role of agricultural trade for global food security, for the environment or for poverty reduction in developing countries. This thesis uses an integrated framework to analyze a broad array of possible impacts related to transforming agricultural and rural markets in light of globalization, and in particular of increasing trade activity. Specifically, the following issues are approached: First, global food production will have to rise substantially by the year 2050 to meet effective demand of a nine billion people world population which poses major challenges to food production systems. Doing so without compromising environmental integrity in exporting regions is an even greater challenge. In this context, the thesis explores the effects of future global trade liberalization on food security indicators in different world regions and on a variety of environmental indicators at different scales in Latin America and the Caribbean, in due consideration of different future agricultural production practices. The International Model for Policy Analysis of Agricultural Commodities and Trade (IMPACT) –a global dynamic partial equilibrium model of the agricultural sector developed by the International Food Policy Research Institute (IFPRI)– is applied to run different future production scenarios, and agricultural trade regimes out to 2050. Model results are linked to biophysical models, used to assess changes in water footprints and water quality, as well as impacts on biodiversity and carbon stocks from land use change by 2050. Results indicate that further trade liberalization is crucial for improving food security globally, but that it would also lead to more environmental pressures in some regions across Latin America. Contrasting land expansion versus more intensified agriculture shows that productivity improvements are generally superior to agricultural land expansion, from an economic and environmental point of view. Most promising for achieving food security and environmental goals, in equal measure, is the sustainable intensification scenario. However, the analysis shows that there are trade-offs between environmental and food security goals for all agricultural development paths. Second, in light of the recent food price crisis of 2007/08, the thesis looks at the impacts of increasing agricultural market integration on food price transmission from global to domestic markets in six Latin American countries, namely Argentina, Brazil, Chile, Colombia, Mexico and Peru. To identify possible cointegrating relationships between the domestic food consumer price indices and world food price levels, subject to different degrees of agricultural market integration in the six Latin American countries, a single equation error correction model is used. Results suggest that global agricultural market integration has led to different levels of price path-through in the studied countries. Especially in the short-run, transmission rates depend on the degree of trade openness, while in the long-run transmission rates are high, but largely independent of the country-specific trade regime. Hence, under world price shocks more trade openness brings with it more price instability in the short-term and the resulting persistence in the long-term. However, these findings do not necessarily verify the usefulness of trade policies, often applied by governments to buffer such price shocks. First, because there is a considerable risk of price volatility due to domestic supply shocks if self-sufficiency is promoted. Second, protectionism bears the risk of excluding a country from participating in beneficial high-value agricultural supply chains, thereby hampering economic development. Nevertheless, to reduce households’ vulnerability to sudden and large increases of food prices, effective policies to buffer food price shocks should be put in place, but must be carefully planned with the required budget readily available. Third, globalization affects the structure of an economy and, by different means, the distribution of income in a country. Peru serves as an example to dive deeper into questions related to changes in the income distribution in rural areas. Peru, a country being increasingly integrated into global food markets, experienced large drops in extreme rural poverty, but persistently high rates of moderate rural poverty and rural income inequality between 2004 and 2012. The thesis aims at disentangling the driving forces behind these dynamics by using a microsimulation model based on rural household income generation models. Results provide evidence that the main force behind poverty reduction was overall economic growth of the economy due to generally favorable macroeconomic market conditions. These growth effects benefited almost all rural sectors, and led to declines in extreme rural poverty, especially among potato and maize farmers. In part, these farmers probably benefited from policy changes towards more open trade regimes and the resulting higher producer prices in times of elevated global food price levels. However, the results also suggest that entry barriers existed for the poorer part of the population to participate in well-paid wage-employment outside of agriculture or in high-value crop production. This could be explained by a lack of sufficient access to important rural assets. For example, poor people’s educational attainment was hardly better in 2012 than in 2004. Also land and labor endowments, especially of (poor) maize and potato growers, rather decreased than increased over time. This leads to the conclusion that there is still scope for policy action to facilitate access to these assets, which could contribute to the eradication of rural poverty. The thesis concludes that agricultural trade can be one important means to provide a growing and richer world population with sufficient amounts of calories. To avoid adverse environmental effects and negative impacts for poor food consumers and producers, the focus should lie on agricultural productivity improvements, considering environmental limits and be socially inclusive. In this sense, it will be crucial to further develop technological solutions that guarantee resource-sparing agricultural production practices, and to remove entry barriers for small poor farmers to export markets which might allow for technological spill-over effects from high-value global agricultural supply chains.

Quantifying the relationship between drainage networks at hillslope scale and particle size distribution at pedon scale

Nowadays, translating information about hydrologic and soil properties and processes across scales has emerged as a major theme in soil science and hydrology, and suitable theories for upscaling or downscaling hydrologic and soil information are being looked forward. The recognition of low-order catchments as self-organized systems suggests the existence of a great amount of links at different scales between their elements. The objective of this work was to research in areas of homogeneous bedrock material, the relationship between the hierarchical structure of the drainage networks at hillslope scale and the heterogeneity of the particle-size distribution at pedon scale. One of the most innovative elements in this work is the choice of the parameters to quantify the organization level of the studied features. The fractal dimension has been selected to measure the hierarchical structure of the drainage networks, while the Balanced Entropy Index (BEI) has been the chosen parameter to quantify the heterogeneity of the particle-size distribution from textural data. These parameters have made it possible to establish quantifiable relationships between two features attached to different steps in the scale range. Results suggest that the bedrock lithology of the landscape constrains the architecture of the drainage networks developed on it and the particle soil distribution resulting in the fragmentation processes.

Quantifying the relationship between drainage networks at hillslope scale and particle size distribution at pedon scale

Nowadays, translating information about hydrologic and soil properties and processes across scales has emerged as a major theme in soil science and hydrology, and suitable theories for upscaling or downscaling hydrologic and soil information are being looked forward. The recognition of low-order catchments as self-organized systems suggests the existence of a great amount of links at different scales between their elements. The objective of this work was to research in areas of homogeneous bedrock material, the relationship between the hierarchical structure of the drainage networks at hillslope scale and the heterogeneity of the particle-size distribution at pedon scale. One of the most innovative elements in this work is the choice of the parameters to quantify the organization level of the studied features. The fractal dimension has been selected to measure the hierarchical structure of the drainage networks, while the Balanced Entropy Index (BEI) has been the chosen parameter to quantify the heterogeneity of the particle-size distribution from textural data. These parameters have made it possible to establish quantifiable relationships between two features attached to different steps in the scale range. Results suggest that the bedrock lithology of the landscape constrains the architecture of the drainage networks developed on it and the particle soil distribution resulting in the fragmentation processes.

A regularised continuum damage model based on the mesoscopic scale for soft tissue

Material properties of soft fibrous tissues are highly conditioned by the hierarchical structure of this kind of composites. Collagen based tissues present, at decreasing length scales, a complex framework of fibres, fibrils, tropocollagen molecules and amino-acids. Understanding the mechanical behaviour at nano-scale level is critical to accurately incorporate this structural information in phenomenological damage models. In this work we derive a relationship between the mechanical and geometrical properties of the fibril constituents and the soft tissue material parameters at macroscopic scale. A Hodge–Petruska two-dimensional model has been used to describe the fibrils as staggered arrays of tropocollagen molecules. After a mechanical characterisation of each of the fibril components, two fibril failures modes have been defined related with two planes of weakness. A phenomenological continuous damage model with regularised softening was presented along with meso-structurally based definitions for its material parameters. Finally, numerical analysis at fibril, fibre and tissue levels are presented to show the capabilities of the model

Brownian dynamics simulations of protein folding: Access to milliseconds time scale and beyond

Protein folding occurs on a time scale ranging from milliseconds to minutes for a majority of proteins. Computer simulation of protein folding, from a random configuration to the native structure, is nontrivial owing to the large disparity between the simulation and folding time scales. As an effort to overcome this limitation, simple models with idealized protein subdomains, e.g., the diffusion–collision model of Karplus and Weaver, have gained some popularity. We present here new results for the folding of a four-helix bundle within the framework of the diffusion–collision model. Even with such simplifying assumptions, a direct application of standard Brownian dynamics methods would consume 10,000 processor-years on current supercomputers. We circumvent this difficulty by invoking a special Brownian dynamics simulation. The method features the calculation of the mean passage time of an event from the flux overpopulation method and the sampling of events that lead to productive collisions even if their probability is extremely small (because of large free-energy barriers that separate them from the higher probability events). Using these developments, we demonstrate that a coarse-grained model of the four-helix bundle can be simulated in several days on current supercomputers. Furthermore, such simulations yield folding times that are in the range of time scales observed in experiments.

Structure-based conformational preferences of amino acids

Proteins can be very tolerant to amino acid substitution, even within their core. Understanding the factors responsible for this behavior is of critical importance for protein engineering and design. Mutations in proteins have been quantified in terms of the changes in stability they induce. For example, guest residues in specific secondary structures have been used as probes of conformational preferences of amino acids, yielding propensity scales. Predicting these amino acid propensities would be a good test of any new potential energy functions used to mimic protein stability. We have recently developed a protein design procedure that optimizes whole sequences for a given target conformation based on the knowledge of the template backbone and on a semiempirical potential energy function. This energy function is purely physical, including steric interactions based on a Lennard-Jones potential, electrostatics based on a Coulomb potential, and hydrophobicity in the form of an environment free energy based on accessible surface area and interatomic contact areas. Sequences designed by this procedure for 10 different proteins were analyzed to extract conformational preferences for amino acids. The resulting structure-based propensity scales show significant agreements with experimental propensity scale values, both for α-helices and β-sheets. These results indicate that amino acid conformational preferences are a natural consequence of the potential energy we use. This confirms the accuracy of our potential and indicates that such preferences should not be added as a design criterion.