Targeted Deletion Of Functionally Validated Enhancers Defines Their Role in Mouse Limb Development

Transcriptional enhancers are genomic DNA sequences that contain clustered transcription factor (TF) binding sites. When combinations of TFs bind to enhancer sequences they act together with basal transcriptional machinery to regulate the timing, location and quantity of gene transcription. Elucidating the genetic mechanisms responsible for differential gene expression, including the role of enhancers, during embryological and postnatal development is essential to an understanding of evolutionary processes and disease etiology. Numerous methods are in use to identify and characterize enhancers. Several high-throughput methods generate large datasets of enhancer sequences with putative roles in embryonic development. However, few enhancers have been deleted from the genome to determine their roles in the development of specific structures, such as the limb. Manipulation of enhancers at their endogenous loci, such as the deletion of such elements, leads to a better understanding of the regulatory interactions, rules and complexities that contribute to faithful and variant gene transcription – the molecular genetic substrate of evolution and disease. To understand the endogenous roles of two distinct enhancers known to be active in the mouse embryo limb bud we deleted them from the mouse genome. I hypothesized that deletion of these enhancers would lead to aberrant limb development. The enhancers were selected because of their association with p300, a protein associated with active transcription, and because the human enhancer sequences drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. To confirm that the orthologous mouse enhancers, mouse 280 and 1442 (M280 and M1442, respectively), regulate expression in the developing limb we generated stable transgenic lines, and examined lacZ expression. In M280-lacZ mice, expression was detected in E11.5 fore- and hindlimbs in a region that corresponds to digits II-IV. M1442-lacZ mice exhibited lacZ expression in posterior and anterior margins of the fore- and hindlimbs that overlapped with digits I and V and several wrist bones. We generated mice lacking the M280 and M1442 enhancers by gene targeting. Intercrosses between M280 -/+ and M1442 -/+, respectively, generated M280 and M1442 null mice, which are born at expected Mendelian ratios and manifest no gross limb malformations. Quantitative real-time PCR of mutant E11.5 limb buds indicated that significant changes in transcriptional output of enhancer-proximal genes accompanied the deletion of both M280 and M1442. In neonatal null mice we observed that all limb bones are present in their expected positions, an observation also confirmed by histology of E18.5 distal limbs. Fine-scale measurement of E18.5 digit bone lengths found no differences between mutant and control embryos. Furthermore, when the developmental progression of cartilaginous elements was analyzed in M280 and M1442 embryos from E13.5-E15.5, transient development defects were not detected. These results demonstrate that M280 and M1442 are not required for mouse limb development. Though M280 is not required for embryonic limb development it is required for the development and/or maintenance of body size – adult M280 mice are significantly smaller than control littermates. These studies highlight the importance of experiments that manipulate enhancers in situ to understand their contribution to development.

Computational methods to create and analyze a digital gene expression atlas of embryo development from microscopy images

Abstract The creation of atlases, or digital models where information from different subjects can be combined, is a field of increasing interest in biomedical imaging. When a single image does not contain enough information to appropriately describe the organism under study, it is then necessary to acquire images of several individuals, each of them containing complementary data with respect to the rest of the components in the cohort. This approach allows creating digital prototypes, ranging from anatomical atlases of human patients and organs, obtained for instance from Magnetic Resonance Imaging, to gene expression cartographies of embryo development, typically achieved from Light Microscopy. Within such context, in this PhD Thesis we propose, develop and validate new dedicated image processing methodologies that, based on image registration techniques, bring information from multiple individuals into alignment within a single digital atlas model. We also elaborate a dedicated software visualization platform to explore the resulting wealth of multi-dimensional data and novel analysis algo-rithms to automatically mine the generated resource in search of bio¬logical insights. In particular, this work focuses on gene expression data from developing zebrafish embryos imaged at the cellular resolution level with Two-Photon Laser Scanning Microscopy. Disposing of quantitative measurements relating multiple gene expressions to cell position and their evolution in time is a fundamental prerequisite to understand embryogenesis multi-scale processes. However, the number of gene expressions that can be simultaneously stained in one acquisition is limited due to optical and labeling constraints. These limitations motivate the implementation of atlasing strategies that can recreate a virtual gene expression multiplex. The developed computational tools have been tested in two different scenarios. The first one is the early zebrafish embryogenesis where the resulting atlas constitutes a link between the phenotype and the genotype at the cellular level. The second one is the late zebrafish brain where the resulting atlas allows studies relating gene expression to brain regionalization and neurogenesis. The proposed computational frameworks have been adapted to the requirements of both scenarios, such as the integration of partial views of the embryo into a whole embryo model with cellular resolution or the registration of anatom¬ical traits with deformable transformation models non-dependent on any specific labeling. The software implementation of the atlas generation tool (Match-IT) and the visualization platform (Atlas-IT) together with the gene expression atlas resources developed in this Thesis are to be made freely available to the scientific community. Lastly, a novel proof-of-concept experiment integrates for the first time 3D gene expression atlas resources with cell lineages extracted from live embryos, opening up the door to correlate genetic and cellular spatio-temporal dynamics. La creación de atlas, o modelos digitales, donde la información de distintos sujetos puede ser combinada, es un campo de creciente interés en imagen biomédica. Cuando una sola imagen no contiene suficientes datos como para describir apropiadamente el organismo objeto de estudio, se hace necesario adquirir imágenes de varios individuos, cada una de las cuales contiene información complementaria respecto al resto de componentes del grupo. De este modo, es posible crear prototipos digitales, que pueden ir desde atlas anatómicos de órganos y pacientes humanos, adquiridos por ejemplo mediante Resonancia Magnética, hasta cartografías de la expresión genética del desarrollo de embrionario, típicamente adquiridas mediante Microscopía Optica. Dentro de este contexto, en esta Tesis Doctoral se introducen, desarrollan y validan nuevos métodos de procesado de imagen que, basándose en técnicas de registro de imagen, son capaces de alinear imágenes y datos provenientes de múltiples individuos en un solo atlas digital. Además, se ha elaborado una plataforma de visualization específicamente diseñada para explorar la gran cantidad de datos, caracterizados por su multi-dimensionalidad, que resulta de estos métodos. Asimismo, se han propuesto novedosos algoritmos de análisis y minería de datos que permiten inspeccionar automáticamente los atlas generados en busca de conclusiones biológicas significativas. En particular, este trabajo se centra en datos de expresión genética del desarrollo embrionario del pez cebra, adquiridos mediante Microscopía dos fotones con resolución celular. Disponer de medidas cuantitativas que relacionen estas expresiones genéticas con las posiciones celulares y su evolución en el tiempo es un prerrequisito fundamental para comprender los procesos multi-escala característicos de la morfogénesis. Sin embargo, el número de expresiones genéticos que pueden ser simultáneamente etiquetados en una sola adquisición es reducido debido a limitaciones tanto ópticas como del etiquetado. Estas limitaciones requieren la implementación de estrategias de creación de atlas que puedan recrear un multiplexado virtual de expresiones genéticas. Las herramientas computacionales desarrolladas han sido validadas en dos escenarios distintos. El primer escenario es el desarrollo embrionario temprano del pez cebra, donde el atlas resultante permite constituir un vínculo, a nivel celular, entre el fenotipo y el genotipo de este organismo modelo. El segundo escenario corresponde a estadios tardíos del desarrollo del cerebro del pez cebra, donde el atlas resultante permite relacionar expresiones genéticas con la regionalización del cerebro y la formación de neuronas. La plataforma computacional desarrollada ha sido adaptada a los requisitos y retos planteados en ambos escenarios, como la integración, a resolución celular, de vistas parciales dentro de un modelo consistente en un embrión completo, o el alineamiento entre estructuras de referencia anatómica equivalentes, logrado mediante el uso de modelos de transformación deformables que no requieren ningún marcador específico. Está previsto poner a disposición de la comunidad científica tanto la herramienta de generación de atlas (Match-IT), como su plataforma de visualización (Atlas-IT), así como las bases de datos de expresión genética creadas a partir de estas herramientas. Por último, dentro de la presente Tesis Doctoral, se ha incluido una prueba conceptual innovadora que permite integrar los mencionados atlas de expresión genética tridimensionales dentro del linaje celular extraído de una adquisición in vivo de un embrión. Esta prueba conceptual abre la puerta a la posibilidad de correlar, por primera vez, las dinámicas espacio-temporales de genes y células.

The human side of social technology for climate change mitigation and human development: the case of "efficient stoves" in Brazil

Carbon management has gradually gained attention within the overall environmental management and corporate social responsibility agendas. The clean development mechanism, from Kyoto Protocol, was envisioned as connecting carbon market and sustainable development objectives in developing countries. Previous research has shown that this potential is rarely being achieved. The paper explores how the incorporation of the human side into carbon management reinforces its contribution to generate human development in local communities and to improve the company's image. A case study of a Brazilian company is presented, with the results of the application of an analytical model that incorporates the human side and human development. The selected project is an "efficient stoves" programme. "Efficient stoves" are recognised in Brazil as social technologies. Results suggest that the fact that social technologies value the human side of the technology plays a key role when it comes to analysing the co-benefits of the project implementation.

Perspectives on climate change and human development in the Mediterranean agriculture

The Mediterranean region is one of the world's climate change hotspots. Future climate projections envisage dramatic implications for the agricultural and water sectors that will endanger economic development and lead to natural resources degradation and social instability.

Development of a human movement monitoring system based on wearable devices

It is essential to remotely and continuously monitor the movements of individuals in many social areas, for example, taking care of aging people, physical therapy, athletic training etc. Many methods have been used, such as video record, motion analysis or sensor-based methods. Due to the limitations in remote communication, power consumption, portability and so on, most of them are not able to fulfill the requirements. The development of wearable technology and cloud computing provides a new efficient way to achieve this goal. This paper presents an intelligent human movement monitoring system based on a smartwatch, an Android smartphone and a distributed data management engine. This system includes advantages of wide adaptability, remote and long-term monitoring capacity, high portability and flexibility. The structure of the system and its principle are introduced. Four experiments are designed to prove the feasibility of the system. The results of the experiments demonstrate the system is able to detect different actions of individuals with adequate accuracy.

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.

Study and analysis of the Leap Motion sensor and the Software Development Kit (SDK) for the implementation of visual Human Computer Interfaces

En este proyecto, se presenta un informe técnico sobre la cámara Leap Motion y el Software Development Kit correspondiente, el cual es un dispositivo con una cámara de profundidad orientada a interfaces hombre-máquina. Esto es realizado con el propósito de desarrollar una interfaz hombre-máquina basada en un sistema de reconocimiento de gestos de manos. Después de un exhaustivo estudio de la cámara Leap Motion, se han realizado diversos programas de ejemplo con la intención de verificar las capacidades descritas en el informe técnico, poniendo a prueba la Application Programming Interface y evaluando la precisión de las diferentes medidas obtenidas sobre los datos de la cámara. Finalmente, se desarrolla un prototipo de un sistema de reconocimiento de gestos. Los datos sobre la posición y orientación de la punta de los dedos obtenidos de la Leap Motion son usados para describir un gesto mediante un vector descriptor, el cual es enviado a una Máquina Vectores Soporte, utilizada como clasificador multi-clase.

Development and implementation of an air quality integrated assessment modelling system for the Iberian Peninsula

La mejora de la calidad del aire es una tarea eminentemente interdisciplinaria. Dada la gran variedad de ciencias y partes involucradas, dicha mejora requiere de herramientas de evaluación simples y completamente integradas. La modelización para la evaluación integrada (integrated assessment modeling) ha demostrado ser una solución adecuada para la descripción de los sistemas de contaminación atmosférica puesto que considera cada una de las etapas involucradas: emisiones, química y dispersión atmosférica, impactos ambientales asociados y potencial de disminución. Varios modelos de evaluación integrada ya están disponibles a escala continental, cubriendo cada una de las etapas antesmencionadas, siendo el modelo GAINS (Greenhouse Gas and Air Pollution Interactions and Synergies) el más reconocido y usado en el contexto europeo de toma de decisiones medioambientales. Sin embargo, el manejo de la calidad del aire a escala nacional/regional dentro del marco de la evaluación integrada es deseable. Esto sin embargo, no se lleva a cabo de manera satisfactoria con modelos a escala europea debido a la falta de resolución espacial o de detalle en los datos auxiliares, principalmente los inventarios de emisión y los patrones meteorológicos, entre otros. El objetivo de esta tesis es presentar los desarrollos en el diseño y aplicación de un modelo de evaluación integrada especialmente concebido para España y Portugal. El modelo AERIS (Atmospheric Evaluation and Research Integrated system for Spain) es capaz de cuantificar perfiles de concentración para varios contaminantes (NO2, SO2, PM10, PM2,5, NH3 y O3), el depósito atmosférico de especies de azufre y nitrógeno así como sus impactos en cultivos, vegetación, ecosistemas y salud como respuesta a cambios porcentuales en las emisiones de sectores relevantes. La versión actual de AERIS considera 20 sectores de emisión, ya sea equivalentes a sectores individuales SNAP o macrosectores, cuya contribución a los niveles de calidad del aire, depósito e impactos han sido modelados a través de matrices fuentereceptor (SRMs). Estas matrices son constantes de proporcionalidad que relacionan cambios en emisiones con diferentes indicadores de calidad del aire y han sido obtenidas a través de parametrizaciones estadísticas de un modelo de calidad del aire (AQM). Para el caso concreto de AERIS, su modelo de calidad del aire “de origen” consistió en el modelo WRF para la meteorología y en el modelo CMAQ para los procesos químico-atmosféricos. La cuantificación del depósito atmosférico, de los impactos en ecosistemas, cultivos, vegetación y salud humana se ha realizado siguiendo las metodologías estándar establecidas bajo los marcos internacionales de negociación, tales como CLRTAP. La estructura de programación está basada en MATLAB®, permitiendo gran compatibilidad con software típico de escritorio comoMicrosoft Excel® o ArcGIS®. En relación con los niveles de calidad del aire, AERIS es capaz de proveer datos de media anual y media mensual, así como el 19o valor horario más alto paraNO2, el 25o valor horario y el 4o valor diario más altos para SO2, el 36o valor diario más alto para PM10, el 26o valor octohorario más alto, SOMO35 y AOT40 para O3. En relación al depósito atmosférico, el depósito acumulado anual por unidad de area de especies de nitrógeno oxidado y reducido al igual que de azufre pueden ser determinados. Cuando los valores anteriormente mencionados se relacionan con características del dominio modelado tales como uso de suelo, cubiertas vegetales y forestales, censos poblacionales o estudios epidemiológicos, un gran número de impactos puede ser calculado. Centrándose en los impactos a ecosistemas y suelos, AERIS es capaz de estimar las superaciones de cargas críticas y las superaciones medias acumuladas para especies de nitrógeno y azufre. Los daños a bosques se calculan como una superación de los niveles críticos de NO2 y SO2 establecidos. Además, AERIS es capaz de cuantificar daños causados por O3 y SO2 en vid, maíz, patata, arroz, girasol, tabaco, tomate, sandía y trigo. Los impactos en salud humana han sido modelados como consecuencia de la exposición a PM2,5 y O3 y cuantificados como pérdidas en la esperanza de vida estadística e indicadores de mortalidad prematura. La exactitud del modelo de evaluación integrada ha sido contrastada estadísticamente con los resultados obtenidos por el modelo de calidad del aire convencional, exhibiendo en la mayoría de los casos un buen nivel de correspondencia. Debido a que la cuantificación de los impactos no es llevada a cabo directamente por el modelo de calidad del aire, un análisis de credibilidad ha sido realizado mediante la comparación de los resultados de AERIS con los de GAINS para un escenario de emisiones determinado. El análisis reveló un buen nivel de correspondencia en las medias y en las distribuciones probabilísticas de los conjuntos de datos. Las pruebas de verificación que fueron aplicadas a AERIS sugieren que los resultados son suficientemente consistentes para ser considerados como razonables y realistas. En conclusión, la principal motivación para la creación del modelo fue el producir una herramienta confiable y a la vez simple para el soporte de las partes involucradas en la toma de decisiones, de cara a analizar diferentes escenarios “y si” con un bajo coste computacional. La interacción con políticos y otros actores dictó encontrar un compromiso entre la complejidad del modeladomedioambiental con el carácter conciso de las políticas, siendo esto algo que AERIS refleja en sus estructuras conceptual y computacional. Finalmente, cabe decir que AERIS ha sido creado para su uso exclusivo dentro de un marco de evaluación y de ninguna manera debe ser considerado como un sustituto de los modelos de calidad del aire ordinarios. ABSTRACT Improving air quality is an eminently inter-disciplinary task. The wide variety of sciences and stakeholders that are involved call for having simple yet fully-integrated and reliable evaluation tools available. Integrated AssessmentModeling has proved to be a suitable solution for the description of air pollution systems due to the fact that it considers each of the involved stages: emissions, atmospheric chemistry, dispersion, environmental impacts and abatement potentials. Some integrated assessment models are available at European scale that cover each of the before mentioned stages, being the Greenhouse Gas and Air Pollution Interactions and Synergies (GAINS) model the most recognized and widely-used within a European policy-making context. However, addressing air quality at the national/regional scale under an integrated assessment framework is desirable. To do so, European-scale models do not provide enough spatial resolution or detail in their ancillary data sources, mainly emission inventories and local meteorology patterns as well as associated results. The objective of this dissertation is to present the developments in the design and application of an Integrated Assessment Model especially conceived for Spain and Portugal. The Atmospheric Evaluation and Research Integrated system for Spain (AERIS) is able to quantify concentration profiles for several pollutants (NO2, SO2, PM10, PM2.5, NH3 and O3), the atmospheric deposition of sulfur and nitrogen species and their related impacts on crops, vegetation, ecosystems and health as a response to percentual changes in the emissions of relevant sectors. The current version of AERIS considers 20 emission sectors, either corresponding to individual SNAP sectors or macrosectors, whose contribution to air quality levels, deposition and impacts have been modeled through the use of source-receptor matrices (SRMs). Thesematrices are proportionality constants that relate emission changes with different air quality indicators and have been derived through statistical parameterizations of an air qualitymodeling system (AQM). For the concrete case of AERIS, its parent AQM relied on the WRF model for meteorology and on the CMAQ model for atmospheric chemical processes. The quantification of atmospheric deposition, impacts on ecosystems, crops, vegetation and human health has been carried out following the standard methodologies established under international negotiation frameworks such as CLRTAP. The programming structure isMATLAB ® -based, allowing great compatibility with typical software such as Microsoft Excel ® or ArcGIS ® Regarding air quality levels, AERIS is able to provide mean annual andmean monthly concentration values, as well as the indicators established in Directive 2008/50/EC, namely the 19th highest hourly value for NO2, the 25th highest daily value and the 4th highest hourly value for SO2, the 36th highest daily value of PM10, the 26th highest maximum 8-hour daily value, SOMO35 and AOT40 for O3. Regarding atmospheric deposition, the annual accumulated deposition per unit of area of species of oxidized and reduced nitrogen as well as sulfur can be estimated. When relating the before mentioned values with specific characteristics of the modeling domain such as land use, forest and crops covers, population counts and epidemiological studies, a wide array of impacts can be calculated. When focusing on impacts on ecosystems and soils, AERIS is able to estimate critical load exceedances and accumulated average exceedances for nitrogen and sulfur species. Damage on forests is estimated as an exceedance of established critical levels of NO2 and SO2. Additionally, AERIS is able to quantify damage caused by O3 and SO2 on grapes, maize, potato, rice, sunflower, tobacco, tomato, watermelon and wheat. Impacts on human health aremodeled as a consequence of exposure to PM2.5 and O3 and quantified as losses in statistical life expectancy and premature mortality indicators. The accuracy of the IAM has been tested by statistically contrasting the obtained results with those yielded by the conventional AQM, exhibiting in most cases a good agreement level. Due to the fact that impacts cannot be directly produced by the AQM, a credibility analysis was carried out for the outputs of AERIS for a given emission scenario by comparing them through probability tests against the performance of GAINS for the same scenario. This analysis revealed a good correspondence in the mean behavior and the probabilistic distributions of the datasets. The verification tests that were applied to AERIS suggest that results are consistent enough to be credited as reasonable and realistic. In conclusion, the main reason thatmotivated the creation of this model was to produce a reliable yet simple screening tool that would provide decision and policy making support for different “what-if” scenarios at a low computing cost. The interaction with politicians and other stakeholders dictated that reconciling the complexity of modeling with the conciseness of policies should be reflected by AERIS in both, its conceptual and computational structures. It should be noted however, that AERIS has been created under a policy-driven framework and by no means should be considered as a substitute of the ordinary AQM.

Development of a hand-gesture recognition system for human-computer interaction

The aim of this Master Thesis is the analysis, design and development of a robust and reliable Human-Computer Interaction interface, based on visual hand-gesture recognition. The implementation of the required functions is oriented to the simulation of a classical hardware interaction device: the mouse, by recognizing a specific hand-gesture vocabulary in color video sequences. For this purpose, a prototype of a hand-gesture recognition system has been designed and implemented, which is composed of three stages: detection, tracking and recognition. This system is based on machine learning methods and pattern recognition techniques, which have been integrated together with other image processing approaches to get a high recognition accuracy and a low computational cost. Regarding pattern recongition techniques, several algorithms and strategies have been designed and implemented, which are applicable to color images and video sequences. The design of these algorithms has the purpose of extracting spatial and spatio-temporal features from static and dynamic hand gestures, in order to identify them in a robust and reliable way. Finally, a visual database containing the necessary vocabulary of gestures for interacting with the computer has been created.

A modern human pattern of dental development in Lower Pleistocene hominids from Atapuerca-TD6 (Spain)

The study of life history evolution in hominids is crucial for the discernment of when and why humans have acquired our unique maturational pattern. Because the development of dentition is critically integrated into the life cycle in mammals, the determination of the time and pattern of dental development represents an appropriate method to infer changes in life history variables that occurred during hominid evolution. Here we present evidence derived from Lower Pleistocene human fossil remains recovered from the TD6 level (Aurora stratum) of the Gran Dolina site in the Sierra de Atapuerca, northern Spain. These hominids present a pattern of development similar to that of Homo sapiens, although some aspects (e.g., delayed M3 calcification) are not as derived as that of European populations and people of European origin. This evidence, taken together with the present knowledge of cranial capacity of these and other late Early Pleistocene hominids, supports the view that as early as 0.8 Ma at least one Homo species shared with modern humans a prolonged pattern of maturation.

Development of an epithelium-specific expression cassette with human DNA regulatory elements for transgene expression in lung airways

The efficient expression of therapeutic genes in target cells or tissues is an important component of efficient and safe gene therapy. Utilizing regulatory elements from the human cytokeratin 18 (K18) gene, including 5′ genomic sequences and one of its introns, we have developed a novel expression cassette that can efficiently express reporter genes, as well as the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, in cultured lung epithelial cells. CFTR transcripts expressed from the native K18 enhancer/promoter include two alternative splicing products, due to the activation of two cryptic splice sites in the CFTR coding region. Modification of the K18 intron and CFTR cDNA sequences eliminated the cryptic splice sites without changing the CFTR amino acid sequence, and led to enhanced CFTR mRNA and protein expression as well as biological function. Transgenic expression analysis in mice showed that the modified expression cassette can direct efficient and epithelium-specific expression of the Escherichia coli LacZ gene in the airways of fetal lungs, with no detectable expression in lung fibroblasts or endothelial cells. This is the first expression cassette which selectively directs lung transgene expression for CFTR gene therapy to airway epithelia.

From cell death to embryo arrest: Mathematical models of human preimplantation embryo development

Human preimplantation embryos exhibit high levels of apoptotic cells and high rates of developmental arrest during the first week in vitro. The relation between the two is unclear and difficult to determine by conventional experimental approaches, partly because of limited numbers of embryos. We apply a mixture of experiment and mathematical modeling to show that observed levels of cell death can be reconciled with the high levels of embryo arrest seen in the human only if the developmental competence of embryos is already established at the zygote stage, and environmental factors merely modulate this. This suggests that research on improving in vitro fertilization success rates should move from its current concentration on optimizing culture media to focus more on the generation of a healthy zygote and on understanding the mechanisms that cause chromosomal and other abnormalities during early cleavage stages.

Tumor necrosis factor-related apoptosis-inducing ligand in T cell development: Sensitivity of human thymocytes

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a recently identified member of the tumor necrosis factor cytokine superfamily. TRAIL has been shown to induce apoptosis in various tumor cell lines, whereas most primary cells seem to be resistant. These observations have raised considerable interest in the use of TRAIL in tumor therapy. Yet little is known about the physiological function of TRAIL. This is particularly the case in the immune system, where TRAIL has been suggested by some to be involved in target cell killing and lymphocyte death. We have developed a panel of mAbs and soluble proteins to address the role of TRAIL in lymphocyte development. These studies demonstrate activation-induced sensitization of thymocytes to TRAIL-mediated apoptosis and expression of the apoptosis-inducing TRAIL receptors. However, with the use of several model systems, our subsequent experiments rule out the possibility that TRAIL plays a major role in antigen-induced deletion of thymocytes. In contrast to thymocytes, there is no up-regulation of TRAIL receptors in peripheral T cells on activation, which remain resistant to TRAIL. Thus, susceptibility to TRAIL-induced apoptosis is controlled differently by central and peripheral T cells.

A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans

The Deleted in AZoospermia (DAZ) genes encode potential RNA-binding proteins that are expressed exclusively in prenatal and postnatal germ cells and are strong candidates for human fertility factors. Here we report the identification of an additional member of the DAZ gene family, which we have called BOULE. With the identification of this gene, it is clear that the human DAZ gene family contains at least three members: DAZ, a Y-chromosome gene cluster that arose 30–40 million years ago and whose deletion is linked to infertility in men; DAZL, the “father” of DAZ, a gene that maps to human chromosome 3 and has homologs required for both female and male germ cell development in other organisms; and BOULE, a gene that we propose is the “grandfather” of DAZ and maps to human chromosome 2. Human and mouse BOULE resemble the invertebrate meiotic regulator Boule, the proposed ortholog of DAZ, in sequence and expression pattern and hence likely perform a similar meiotic function. In contrast, the previously identified human DAZ and DAZL are expressed much earlier than BOULE in prenatal germ stem cells and spermatogonia; DAZL also is expressed in female germ cells. These data suggest that homologs of the DAZ gene family can be grouped into two subfamilies (BOULE and DAZL) and that members of the DAZ family evolved from an ancestral meiotic regulator, Boule, to assume distinct, yet overlapping, functions in germ cell development.

The synthesis, discovery, and development of a highly promising class of microtubule stabilization agents: Curative effects of desoxyepothilones B and F against human tumor xenografts in nude mice

We have evaluated two synthetic epothilone analogues lacking the 12,13-epoxide functionality, 12,13-desoxyepothilone B (dEpoB), and 12,13-desoxyepothilone F (dEpoF). The concentrations required for 50% growth inhibition (IC50) for a variety of anticancer agents were measured in CCRF-CEM/VBL1000 cells (2,048-fold resistance to vinblastine). By using dEpoB, dEpoF, aza-EpoB, and paclitaxel, the IC50 values were 0.029, 0.092, 2.99, and 5.17 μM, respectively. These values represent 4-, 33.5-, 1,423- and 3,133-fold resistance, respectively, when compared with the corresponding IC50 in the parent [nonmultiple drug-resistant (MDR)] CCRF-CEM cells. We then produced MDR human lung carcinoma A549 cells by continuous exposure of the tumor cells to sublethal concentrations of dEpoB (1.8 yr), vinblastine (1.2 yr), and paclitaxel (1.8 yr). This continued exposure led to the development of 2.1-, 4,848-, and 2,553-fold resistance to each drug, respectively. The therapeutic effect of dEpoB and paclitaxel was also compared in vivo in a mouse model by using various tumor xenografts. dEpoB is much more effective in reducing tumor sizes in all MDR tumors tested. Analysis of dEpoF, an analog possessing greater aqueous solubility than dEpoB, showed curative effects similar to dEpoB against K562, CCRF-CEM, and MX-1 xenografts. These results indicate that dEpoB and dEpoF are efficacious antitumor agents with both a broad chemotherapeutic spectrum and wide safety margins.