Function of C1A barley peptidases and their inhibitors (cystatins) in barley seed germination : Funcion de las peptidasas C1A de cebada y sus inhibidores (cistatinas) en la germinacion de la semilla

Plant proteolysis is a metabolic process where specific enzymes called peptidases degrade proteins. In plants, this complex process involves broad metabolic networks and different sub-cellular compartments. Several types of peptidases take part in the proteolytic process, mainly cysteine-, serine-, aspartyl- and metallo- peptidases. Among the cysteine-peptidases, the papain-like or C1A peptidases (family C1, clan CA) are extensively present in land plants and are classified into catepsins L-, B-, H- and Flike. The catalytic mechanism of these C1A peptidases is highly conserved and involves the three amino acids Cys, His and Asn in the catalytic triad, and a Gln residue which seems essential for maintaining an active enzyme conformation. These proteins are synthesized as inactive precursors, which comprise an N-terminal signal peptide, a propeptide, and the mature protein. In barley, we have identified 33 cysteine-peptidases from the papain-like family, classifying them into 8 different groups. Five of them corresponded to cathepsins L-like (5 subgroups), 1 cathepsin B-like group, 1 cathepsin F-like group and 1 cathepsin H-like group. Besides, C1A peptidases are the specific targets of the plant proteinaceous inhibitors known as phytocystatins (PhyCys). The cystatin inhibitory mechanism is produced by a tight and reversible interaction with their target enzymes. In barley, the cystatin gene family is comprised by 13 members. In this work we have tried to elucidate the role of the C1A cysteine-peptidases and their specific inhibitors (cystatins) in the germination process of the barley grain. Therefore, we selected a representative member of each group/subgroup of C1A peptidases (1 cathepsin B-like, 1 cathepsin F-like, 1 cathepsin H-like and 5 cathepsins L-like). The molecular characterization of the cysteine-peptidases was done and the peptidase-inhibitor interaction was analyzed in vitro and in vivo. A study in the structural basis for specificity of pro-peptide/enzyme interaction in barley C1A cysteine-peptidases has been also carried out by inhibitory assays and the modeling of the three-dimensional structures. The barley grain maturation produces the accumulation of storage proteins (prolamins) in the endosperm which are mobilized during germination to supply the required nutrients until the photosynthesis is fully established. In this work, we have demonstrated the participation of the cysteine-peptidases and their inhibitors in the degradation of the different storage protein fractions (hordeins, albumins and globulins) present in the barley grain. Besides, transgenic barley plants overexpressing or silencing cysteine-peptidases or cystatins were obtained by Agrobacterium-mediated transformation of barley immature embryos to analyze their physiological function in vivo. Preliminary assays were carried out with the T1 grains of several transgenic lines. Comparing the knock-out and the overexpressing lines with the WT, alterations in the germination process were detected and were correlated with their grain hordein content. These data will be validated with the homozygous grains that are being produced through the double haploid technique by microspore culture. Resumen La proteólisis es un proceso metabólico por el cual se lleva a cabo la degradación de las proteínas de un organismo a través de enzimas específicas llamadas proteasas. En plantas, este complejo proceso comprende un entramado de rutas metabólicas que implican, además, diferentes compartimentos subcelulares. En la proteólisis participan numerosas proteasas, principalmente cisteín-, serín-, aspartil-, y metalo-proteasas. Dentro de las cisteín-proteasas, las proteasas tipo papaína o C1A (familia C1, clan CA) están extensamente representadas en plantas terrestres, y se clasifican en catepsinas tipo L, B, H y F. El mecanismo catalítico de estas proteasas está altamente conservado y la triada catalítica formada por los aminoácidos Cys, His y Asn, y a un aminoácido Gln, que parece esencial para el mantenimiento de la conformación activa de la proteína. Las proteasas C1A se sintetizan como precursores inactivos y comprenden un péptido señal en el extremo N-terminal, un pro-péptido y la proteína madura. En cebada hemos identificado 33 cisteín-proteasas de tipo papaína y las hemos clasificado filogenéticamente en 8 grupos diferentes. Cinco de ellos pertenecen a las catepsinas tipo L (5 subgrupos), un grupo a las catepsinas tipo-B, otro a las catepsinas tipo-F y un último a las catepsinas tipo-H. Las proteasas C1A son además las dianas específicas de los inhibidores protéicos de plantas denominados fitocistatinas. El mecanismo de inhibición de las cistatinas está basado en una fuerte interacción reversible. En cebada, se conoce la familia génica completa de las cistatinas, que está formada por 13 miembros. En el presente trabajo se ha investigado el papel de las cisteín-proteasas de cebada y sus inhibidores específicos en el proceso de la germinación de la semilla. Para ello, se seleccionó una proteasa representante de cada grupo/subgrupo (1 catepsina tipo- B, 1 tipo-F, 1 tipo-H, y 5 tipo-L, una por cada subgrupo). Se ha llevado a cabo su caracterización molecular y se ha analizado la interacción enzima-inhibidor tanto in vivo como in vitro. También se han realizado estudios sobre las bases estructurales que demuestran la especificidad en la interacción enzima/propéptido en las proteasas C1A de cebada, mediante ensayos de inhibición y la predicción de modelos estructurales de la interacción. Finalmente, y dado que durante la maduración de la semilla se almacenan proteínas de reserva (prolaminas) en el endospermo que son movilizadas durante la germinación para suministrar los nutrientes necesarios hasta que la nueva planta pueda realizar la fotosíntesis, en este trabajo se ha demostrado la participación de las cisteínproteasas y sus inhibidores en la degradación de las diferentes tipos de proteínas de reserva (hordeinas, albúmins y globulinas) presentes en el grano de cebada. Además, se han obtenido plantas transgénicas de cebada que sobre-expresan o silencian cistatinas y cisteín-proteasas con el fin de analizar la función fisiológica in vivo. Se han realizado análisis preliminares en las semillas T1 de varias líneas tránsgenicas de cebada y al comparar las líneas knock-out y las líneas de sobre-expresión con las silvestres, se han detectado alteraciones en la germinación que están además correlacionadas con el contenido de hordeinas de las semillas. Estos datos serán validados en las semillas homocigotas que se están generando mediante la técnica de dobles haploides a partir del cultivo de microesporas.

Optimal Piecewise Linear Function Approximation for GPU-based Applications

Many computer vision and human-computer interaction applications developed in recent years need evaluating complex and continuous mathematical functions as an essential step toward proper operation. However, rigorous evaluation of this kind of functions often implies a very high computational cost, unacceptable in real-time applications. To alleviate this problem, functions are commonly approximated by simpler piecewise-polynomial representations. Following this idea, we propose a novel, efficient, and practical technique to evaluate complex and continuous functions using a nearly optimal design of two types of piecewise linear approximations in the case of a large budget of evaluation subintervals. To this end, we develop a thorough error analysis that yields asymptotically tight bounds to accurately quantify the approximation performance of both representations. It provides an improvement upon previous error estimates and allows the user to control the trade-off between the approximation error and the number of evaluation subintervals. To guarantee real-time operation, the method is suitable for, but not limited to, an efficient implementation in modern Graphics Processing Units (GPUs), where it outperforms previous alternative approaches by exploiting the fixed-function interpolation routines present in their texture units. The proposed technique is a perfect match for any application requiring the evaluation of continuous functions, we have measured in detail its quality and efficiency on several functions, and, in particular, the Gaussian function because it is extensively used in many areas of computer vision and cybernetics, and it is expensive to evaluate.

Acoustic properties of microperforated panels and their optimizatión by simulated annealing

This thesis investigates the acoustic properties of microperforated panels as an alternative to passive noise control. The first chapters are devoted to the review of analytical models to obtain the acoustic impedance and absorption coefficient of perforated panels. The use of panels perforated with circular holes or with slits is discussed. The theoretical models are presented and some modifications are proposed to improve the modeling of the physical phenomena occurring at the perforations of the panels. The absorption band is widened through the use of multiple layer microperforated panels and/or the combination of a millimetric panel with a porous layer that can be a fibrous material or a nylon mesh. A commercial micrometric mesh downstream a millimetric panel is proposed as a very efficient and low cost solution for controlling noise in reduced spaces. The simulated annealing algorithm is used in order to optimize the panel construction to provide a maximum of absorption in a determined wide band frequency range. Experiments are carried out at normal sound incidence and plane waves. One example is shown for a double layer microperforated panel subjected to grazing flow. A good agreement is achieved between the theory and the experiments. RESUMEN En esta tesis se investigan las propiedades acústicas de paneles micro perforados como una alternativa al control pasivo del ruido. Los primeros capítulos están dedicados a la revisión de los modelos de análisis para obtener la impedancia acústica y el coeficiente de absorción de los paneles perforados. El uso de paneles perforados con agujeros circulares o con ranuras es discutido. Se presentan diferentes modelos y se proponen algunas modificaciones para mejorar la modelización de los fenómenos físicos que ocurren en las perforaciones. La banda de absorción se ensancha a través del uso de capas múltiples de paneles micro perforados y/o la combinación de un panel de perforaciones milimétricas combinado con una capa porosa que puede ser un material fibroso o una malla de nylon. Se propone el uso de una malla micrométrica detrás de un panel milimétrico como una solución económica y eficiente para el control del ruido en espacios reducidos. El algoritmo de recocido simulado se utiliza con el fin de optimizar la construcción de paneles micro perforados para proporcionar un máximo de absorción en una banda determinada frecuencias. Los experimentos se llevan a cabo en la incidencia normal de sonido y ondas planas. Se muestra un ejemplo de panel micro perforado de doble capa sometido a flujo rasante. Se consigue un buen acuerdo entre la teoría y los experimentos.

Diffusion Gradient Temporal Difference for Cooperative Reinforcement Learning with Linear Function Approximation

We introduce a diffusion-based algorithm in which multiple agents cooperate to predict a common and global statevalue function by sharing local estimates and local gradient information among neighbors. Our algorithm is a fully distributed implementation of the gradient temporal difference with linear function approximation, to make it applicable to multiagent settings. Simulations illustrate the benefit of cooperation in learning, as made possible by the proposed algorithm.

Smart Spaces and Smart Objects interoperability Architecture (S3OiA)

The presented work aims to contribute towards the standardization and the interoperability off the Future Internet through an open and scalable architecture design. We present S³OiA as a syntactic/semantic Service-Oriented Architecture that allows the integration of any type of object or device, not mattering their nature, on the Internet of Things. Moreover, the architecture makes possible the use of underlying heterogeneous resources as a substrate for the automatic composition of complex applications through a semantic Triple Space paradigm. Created applications are dynamic and adaptive since they are able to evolve depending on the context where they are executed. The validation scenario of this architecture encompasses areas which are prone to involve human beings in order to promote personal autonomy, such as home-care automation environments and Ambient Assisted Living.

A unified framework for linear function approximation of value functions in stochastic control

This paper contributes with a unified formulation that merges previ- ous analysis on the prediction of the performance ( value function ) of certain sequence of actions ( policy ) when an agent operates a Markov decision process with large state-space. When the states are represented by features and the value function is linearly approxi- mated, our analysis reveals a new relationship between two common cost functions used to obtain the optimal approximation. In addition, this analysis allows us to propose an efficient adaptive algorithm that provides an unbiased linear estimate. The performance of the pro- posed algorithm is illustrated by simulation, showing competitive results when compared with the state-of-the-art solutions.

Confidence: dependencies and their critical role in fostering user acceptance in pervasive applications

Pervasive computing offers new scenarios where users are surrounded by invisible and proactive technology making smart spaces. Although the utility and power of solutions developed using this computer paradigm are proved, there are unresolved problems that hinder their acceptance and inclusion in our private life. Users have problems understanding the operations of a pervasive computing solution, and therefore they should trust that the solution works properly and according to their expectations. Nevertheless, the concept of trust is already framed in a specific use within the ecosystem of applications that can populate a smart space. To take this concept of trust to the whole space, we propose to study and define the concept of confidence. In contrast to the concept of trust, confidence has deeper psychological implications.