The role of LMX1B and PITX2 in anterior segment development and adult ocular function

Several congenital syndromes associated with anterior segment (AS) anomalies can lead to impaired vision and glaucoma, such as nail-patella syndrome (NPS), caused by mutations in the LIM homeodomain transcription factor LMX1B and Axenfeld-Rieger's syndrome (ARS), caused by mutations in the bicoid-related homeodomain transcription factor PITX2. Targeted mutations in lmx1b and pitx2 and RNA in situ analysis reveal that both genes are required for AS development and are co-expressed within the periocular mesenchyme, suggesting they participate in a shared genetic pathway. Lmx1b homozygous mutants display iris and corneal stroma hypoplasia, and defects in ciliary body formation. In contrast, pitx2 homozygous mutants exhibit a more severe phenotype: the AS chamber, corneal endothelium, and extraocular muscles (EOM) fail to develop. The absence of EOM in pitx2 mutants suggests pitx2 acts upstream of lmx1b, or that other lmx1b family members, such as lmx1a, can compensate for lmx1b function. Lmxla/lmx1b double homozygous mutants have a reduced capacity to generate EOM, implying that lmx1 gene products have a redundant function in EOM development and that lmx1 family members may act downstream of pitx2. However, analysis of pitx2 expression in the AS tissues of lmx1b mutants and reciprocal studies of lmx1b expression in pitx2 mutants indicate that these genes do not function in a simple linear pathway. Instead, lmx1b and pitx2 may regulate a shared set of downstream targets or both genes may work in parallel transcribing unique targets required for a common biological process. Ultrastructural analysis of lmx1b and pitx2 mutant corneas indicates that collagen fibrillogenesis is perturbed, revealing a common role for both genes in the deposition of extracellular matrix. Furthermore, lmx1b/pitx2 double heterozygotes develop corneal opacities not observed in single heterozygotes demonstrating that lmx1b and pitx2 genetically interact. Data suggests that defects in the basement membrane of the corneal endothelium underlie the opacities observed in double heterozygotes. Additionally, double heterozygotes develop anterior synechias that occlude the trabecular meshwork, potentially blocking aqueous humor drainage. These data suggest that lmx1b and pitx2 are responsible for ECM deposition in multiple cell types and imply that such defects may contribute to the glaucomas observed in NPS and ARS patients. ^

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.

Development and Characterization of an in vitro Four-species Anaerobic Dental Biofilm Model

Dental caries is the most common chronic disease worldwide. It is characterized by the demineralization of tooth enamel caused by acid produced by cariogenic dental bacteria growing on tooth surfaces, termed bacterial biofilms. Cariogenesis is a complex biological process that is influence by multiple factors and is not attributed to a sole causative agent. Instead, caries is associated with multispecies microbial biofilm communities composed of some bacterial species that directly influence the development of a caries lesion and other species that are seemingly benign but must contribute to the community in an uncharacterized way. Clinical analysis of dental caries and its microbial populations is challenging due to many factors including low sensitivity of clinical measurement tools, variability in saliva chemistry, and variation in the microbiota. Our laboratory has developed an in vitro anaerobic biofilm model for dental carries to facilitate both clinical and basic research-based analyses of the multispecies dynamics and individual factors that contribute to cariogenicity. The rational for development of this system was to improve upon the current models that lack key elements. This model places an emphasis on physiological relevance and ease of maintenance and reproducibility. The uniqueness of the model is based on integrating four critical elements: 1) a biofilm community composed of four distinct and representative species typically associated with dental caries, 2) a semi-defined synthetic growth medium designed to mimic saliva, 3) physiologically relevant biofilm growth substrates, and 4) a novel biofilm reactor device designed to facilitate the maintenance and analysis. Specifically, human tooth sections or hydroxyapatite discs embedded into poly(methyl methacrylate) (PMMA) discs are incubated for an initial 24 hr in a static inverted removable substrate (SIRS) biofilm reactor at 37°C under anaerobic conditions in artificial saliva (CAMM) without sucrose in the presence of 1 X 106 cells/ml of each Actinomyces odontolyticus, Fusobacterium nucleatum, Streptococcus mutans, and Veillonella dispar. During days 2 and 3 the samples are maintained continually in CAMM with various exposures to 0.2% sucrose; all of the discs are transferred into fresh medium every 24 hr. To validate that this model is an appropriate in vitro representation of a caries-associated multispecies biofilm, research aims were designed to test the following overarching hypothesis: an in vitro anaerobic biofilm composed of four species (S. mutans, V. dispar, A. odontolyticus, and F. nucleatum) will form a stable biofilm with a community profile that changes in response to environmental conditions and exhibits a cariogenic potential. For these experiments the biofilms as described above were exposed on days 2 and 3 to either CAMM lacking sucrose (no sucrose), CAMM with 0.2% sucrose (constant sucrose), or were transferred twice a day for 1 hr each time into 0.2% sucrose (intermittent sucrose). Four types of analysis were performed: 1) fluorescence microscopy of biofilms stained with Syto 9 and hexidium idodine to determine the biofilm architecture, 2) quantitative PCR (qPCR) to determine the cell number of each species per cm2, 3) vertical scanning interferometry (VSI) to determine the cariogenic potential of the biofilms, and 4) tomographic pH imaging using radiometric fluorescence microscopy after exposure to pH sensitive nanoparticles to measure the micro-environmental pH. The qualitative and quantitative results reveal the expected dynamics of the community profile when exposed to different sucrose conditions and the cariogenic potential of this in vitro four-species anaerobic biofilm model, thus confirming its usefulness for future analysis of primary and secondary dental caries.

Isolation and characterization of cDNAs encoding novel homeoproteins from chondrocytes

Bone morphogenesis is a complex biological process. The multistep process of chondrogenesis is the most important aspect of endochondral bone formation. To study the mechanisms which control this multistep pathway of chondrogenesis during embryonic development, I started by isolating cDNAs encoding novel transcriptional factors from chondrocytes. Several such cDNAs encoding putative homeoproteins were identified from a rat chondrosarcoma cDNA preparation. I have been concentrating on characterizing two of these cDNAs. The deduced amino acid sequence of the first homeoprotein, Cart-1, contains a prd-type homeodomain. Northern hybridization and RNase protection analysis revealed that Cart-1 RNAs were present at high levels in a well differentiated rat chondrosarcoma tumor and in a cell line derived from this tumor. Cart-1 transcripts were also detected in primary chondrocytes, but not in numerous other cell types except very low levels in testis. In situ hybridization of rat embryos at different stages of development revealed relatively high levels of Cart-1 RNAs in prechondrocytic mesenchymal cells and in early chondrocytes of cartilage primordia. It is speculated that Cart-1 might play an important role in chondrogenesis. The second putative homeoprotein, rDlx, contains a Distal-less-like homeodomain. rDlx RNAs were also present at high levels in the rat chondrosarcoma tumor and in the cell line derived from this tumor. In situ hybridization of rat embryos revealed high levels of rDlx transcripts in the developing cartilages and perichondria of mature cartilages. rDlx transcripts were also detected in a number of nonchondrogenic tissues such as forebrain, otic vesicles, olfactory epithelia, apical ectodermal ridge (AER) of limb buds, the presumptive Auerbach ganglia of gastrointestinal tract. The unique expression pattern of rDlx suggests that it might play important roles in chondrogenesis and other aspects of embryogenesis. ^

Description and chemical composition of manganese nodules collected in the Pacific Ocean

Chemical and X-ray analyses were performed on the fifteen manganese nodules collected from the Pacific Ocean floor. The results were discussed compared with the previous data on the manganese nodules. Minerals were found to be todorokite, delta-MnO2 and other silicates, montmorillonite, illite, phillipsite and alpha-Si02. Average composition shows that copper is concentrated on the deep sea nodules more than the shallow ones, and that the todorokite rich nodules contain more copper and nickel than the delta-MnO2 rich ones. The analyses of fresh water iron-manganese precipitates by bacterial activity suggest that biological process is one of the important factors on the genesis of the sedimentary iron-manganese deposits, in¬cluding the manganese nodule.

Liver proteome profiling of juvenile Chinese sturgeon (Acipenser sinensis) using GeLC-MS/MS approach

Chinese sturgeon (Acipenser sinensis), mainly distributed in the Yangtze River, has been listed as a grade I protected animal in China because of a dramatic decline in population owing to loss of natural habitat for reproduction and interference by human activities. Understanding the proteome profile of Chinese sturgeon liver would provide an invaluable resource for protecting and increasing the stocks of this species. In this study, we have analyzed proteome profiles of juvenile Chinese sturgeon liver using a one-dimensional gel electrophoresis coupled to LC-MS/MS approach. A total of 1059 proteins and 2084 peptides were identified. The liver proteome was found to be associated with diverse biological processes, cellular components and molecular functions. The proteome profile identified a variety of significant pathways including carbohydrate metabolism, fatty acid metabolism and amino acid metabolism pathways. It also established a network for protein biosynthesis, folding and catabolic processes. The proteome profile established in this study can be used for understanding the development of Chinese sturgeon and studying the molecular mechanisms of action under environmental or chemical stress, providing very useful omics information that can be applied to preserve this species.

Prion crystalization model and its application to recognition pattern

This paper introduces APA (?Artificial Prion Assembly?): a pattern recognition system based on artificial prion crystalization. Specifically, the system exhibits the capability to classify patterns according to the resulting prion self- assembly simulated with cellular automata. Our approach is inspired in the biological process of proteins aggregation, known as prions, which are assembled as amyloid fibers related with neurodegenerative disorders.

Simulación de evolución dirigida de bacteriófagos en poblaciones de bacterias en 2D

The research group is currently developing a biological computing model to be implemented with Escherichia Coli bacteria and bacteriophages M13, but it has to be modelled and simulated before any experiment in order to reduce the amount of failed attempts, time and costs. The problem that gave rise to this project is that there are no software tools which are able to simulate the biological process underlying that com- putational model, so it needs to be developed before doing any experimental implementation. There are several software tools which can simulate most of the biological processes and bacterial interactions in which this model is based, so what needs to be done is to study those available simulation tools, compare them and choose the most appropriate in order to be improved adding the desired functionality for this design. Directed evolution is a method used in biotechnology to obtain proteins or nucleic acids with properties not found in nature. It consists of three steps: 1) creating a library of mutants, 2) selecting the mutants with the desired properties, 3) replicating the variants identified in the selection step. The new software tool will be verified by simulating the selection step of a process of directed evolution applied to bacteriophages. ---ABSRACT---El grupo de investigación está desarrollando un modelo de computación biolóogica para ser implementado con bacterias Escherichia Coli y bacteriofagos M13, aunque primero tiene que ser modelizado antes de realizar cualquier experimento, de forma que los intentos fallidos y por lo tanto los costes se verán reducidos. El problema que dio lugar a este proyecto es la ausencia de herramientas software capaces de simular el proceso biológico que subyace a este modelo de computación biológica, por lo que dicha herramienta tiene que ser desarrollada antes de realizar cualquier implementación real. Existen varias herramientas software capaces de simular la mayoría de los procesos biológicos y las interacciones entre bacterias en los que se basa este modelo, por lo que este trabajo consiste en realizar un estudio de dichas herramientas de simulación, compararlas y escoger aquella más apropiada para ser mejorada añadiendo la funcionalidad deseada para este diseño. La evolución dirigida es un método utilizado en biotecnología para obtener proteínas o ácidos nucleicos con propiedades que no se encuentran en la naturaleza. Este método consiste en tres pasos: 1) crear una librería de mutantes, 2) seleccionar los mutantes con las propiedades deseadas, 3) Replicar los mutantes deseados. La nueva herramienta software será verificada mediante la simulación de la selección de mutantes de un proceso de evolución dirigida aplicado a bacteriofagos.

Conductancia del mesófilo en especies forestales: implicaciones en la respuesta fotosintética a la disponibilidad de agua y luz

La fotosíntesis es el proceso biológico que permite la producción primaria y, por tanto, la vida en nuestro planeta. La tasa fotosintética viene determinada por la ‘maquinaria’ bioquímica y las resistencias difusivas al paso del CO2 desde la atmósfera hasta su fijación en el interior de los cloroplastos. Históricamente la mayor resistencia difusiva se ha atribuido al cierre estomático, sin embargo ahora sabemos, debido a las mejoras en las técnicas experimentales, que existe también una resistencia grande que se opone a la difusión del CO2 desde los espacios intercelulares a los lugares de carboxilación. Esta resistencia, llamada normalmente por su inversa: la conductancia del mesófilo (gm), puede ser igual o incluso superior a la resistencia debida por el cierre estomático. En la presente tesis doctoral he caracterizado la limitación que ejerce la resistencia del mesófilo a la fijación de CO2 en diversas especies forestales y en distintos momentos de su ciclo biológico. En la fase de regenerado, hemos estudiado tres situaciones ambientales relevantes en el mayor éxito de su supervivencia, que son: el déficit hídrico, su interacción con la irradiancia y el paso del crecimiento en la sombra a mayor irradiancia, como puede suceder tras la apertura de un hueco en el dosel forestal. En la fase de arbolado adulto se ha caracterizado el estado hídrico y el intercambio gaseoso en hojas desarrolladas a distinta irradiancia dentro del dosel vegetal durante tres años contrastados en pluviometría. Para cada tipo de estudio se han empleado las técnicas ecofisiológicas más pertinentes para evaluar el estado hídrico y el intercambio gaseoso. Por su complejidad y la falta de un método que permita su cuantificación directa, la gm ha sido evaluada por los métodos más usados, que son: la discriminación isotópica del carbono 13, el método de la J variable, el método de la J constante y el método de la curvatura. Los resultados más significativos permiten concluir que la limitación relativa a la fotosíntesis por la conductancia estomática, del mesófilo y bioquímica es dependiente de la localización de la hoja en el dosel forestal. Por primera vez se ha documentado que bajo estrés hídrico las hojas desarrolladas a la sombra estuvieron más limitadas por una reducción en la gm, mientras que las hojas desarrolladas a pleno sol estuvieron más limitadas por reducción mayor de la conductancia estomática (gsw). Encontramos buena conexión entre el aparato fotosintético foliar y el sistema hídrico debido al alto grado de correlación entre la conductancia hidráulica foliar aparente y la concentración de CO2 en los cloroplastos en distintas especies forestales. Además, hemos mostrado diferentes pautas de regulación del intercambio gaseoso según las particularidades ecológicas de las especies estudiadas. Tanto en brinzales crecidos de forma natural y en el arbolado adulto como en plántulas cultivadas en el invernadero la ontogenia afectó a las limitaciones de la fotosíntesis producidas por estrés hídrico, resultando que las limitaciones estomáticas fueron dominantes en hojas más jóvenes mientras que las no estomáticas en hojas más maduras. La puesta en luz supuso un gran descenso en la gm durante los días siguientes a la transferencia, siendo este efecto mayor según el grado de sombreo previo en el que se han desarrollado las hojas. La aclimatación de las hojas a la alta irradiancia estuvo ligada a las modificaciones anatómicas foliares y al estado de desarrollo de la hoja. El ratio entre la gm/gsw determinó la mayor eficiencia en el uso del agua y un menor estado oxidativo durante la fase de estrés hídrico y su posterior rehidratación, lo cual sugiere el uso de este ratio en los programas de mejora genética frente al estrés hídrico. Debido a que la mayoría de modelos de estimación de la producción primaria bruta (GPP) de un ecosistema no incluye la gm, los mismos están incurriendo en una sobreestimación del GPP particularmente bajo condiciones de estrés hídrico, porque más de la mitad de la reducción en fotosíntesis en hojas desarrolladas a la sombra se debe a la reducción en gm. Finalmente se presenta un análisis de la importancia en las estimas de la gm bajo estrés hídrico de la refijación del CO2 emitido en la mitocondria a consecuencia de la fotorrespiración y la respiración mitocondrial en luz. ABSTRACT Photosynthesis is the biological process that supports primary production and, therefore, life on our planet. Rates of photosynthesis are determined by biochemical “machinery” and the diffusive resistance to the transfer of CO2 from the atmosphere to the place of fixation within the chloroplasts. Historically the largest diffusive resistance was attributed to the stomata, although we now know via improvements in experimental techniques that there is also a large resistance from sub-stomatal cavities to sites of carboxylation. This resistance, commonly quantified as mesophyll conductance (gm), can be as large or even larger than that due to stomatal resistance. In the present PhD I have characterized the limitation exerted by the mesophyll resistance to CO2 fixation in different forest species at different stages of their life cycle. In seedlings, we studied three environmental conditions that affect plant fitness, namely, water deficit, the interaction of water deficit with irradiance, and the transfer of plants grown in the shade to higher irradiance as can occur when a gap opens in the forest canopy. At the stage of mature trees we characterized water status and gas exchange in leaves developed at different irradiance within the canopy over the course of three years that had contrasting rainfall. For each study we used the most relevant ecophysiological techniques to quantify water relations and gas exchange. Due to its complexity and the lack of a method that allows direct quantification, gm was estimated by the most commonly used methods which are: carbon isotope discrimination, the J-variable, constant J and the curvature method The most significant results suggest that the relative limitation of photosynthesis by stomata, mesophyll and biochemistry depending on the position of the leaf within the canopy. For the first time it was documented that under water stress shaded leaves were more limited by a reduction in gm, while the sun-adapted leaves were more limited by stomatal conductance (gsw). The connection between leaf photosynthetic apparatus and the hydraulic system was shown by the good correlations found between the apparent leaf hydraulic conductance and the CO2 concentration in the chloroplasts in shade- and sun-adapted leaves of several tree species. In addition, we have revealed different patterns of gas exchange regulation according to the functional ecology of the species studied. In field grown trees and greenhouse-grown seedlings ontogeny affected limitations of photosynthesis due to water stress with stomatal limitations dominating in young leaves and nonstomatal limitations in older leaves. The transfer to high light resulted in major decrease of gm during the days following the transfer and this effect was greater as higher was the shade which leaves were developed. Acclimation to high light was linked to the leaf anatomical changes and the state of leaf development. The ratio between the gm/gsw determined the greater efficiency in water use and reduced the oxidative stress during the water stress and subsequent rehydration, suggesting the use of this ratio in breeding programs aiming to increase avoidance of water stress. Because most models to estimate gross primary production (GPP) of an ecosystem do not include gm, they are incurring an overestimation of GPP particularly under conditions of water stress because more than half of An decrease in shade-developed leaves may be due to reduction in gm. Finally, we present an analysis of the importance of how estimates of gm under water stress are affected by the refixation of CO2 that is emitted from mitochondria via photorespiration and mitochondrial respiration in light.

Non uniform embedding based on relevance analysis with reduced computational complexity: application to the detection of pathologies from biosignal recordings

Nonlinear analysis tools for studying and characterizing the dynamics of physiological signals have gained popularity, mainly because tracking sudden alterations of the inherent complexity of biological processes might be an indicator of altered physiological states. Typically, in order to perform an analysis with such tools, the physiological variables that describe the biological process under study are used to reconstruct the underlying dynamics of the biological processes. For that goal, a procedure called time-delay or uniform embedding is usually employed. Nonetheless, there is evidence of its inability for dealing with non-stationary signals, as those recorded from many physiological processes. To handle with such a drawback, this paper evaluates the utility of non-conventional time series reconstruction procedures based on non uniform embedding, applying them to automatic pattern recognition tasks. The paper compares a state of the art non uniform approach with a novel scheme which fuses embedding and feature selection at once, searching for better reconstructions of the dynamics of the system. Moreover, results are also compared with two classic uniform embedding techniques. Thus, the goal is comparing uniform and non uniform reconstruction techniques, including the one proposed in this work, for pattern recognition in biomedical signal processing tasks. Once the state space is reconstructed, the scheme followed characterizes with three classic nonlinear dynamic features (Largest Lyapunov Exponent, Correlation Dimension and Recurrence Period Density Entropy), while classification is carried out by means of a simple k-nn classifier. In order to test its generalization capabilities, the approach was tested with three different physiological databases (Speech Pathologies, Epilepsy and Heart Murmurs). In terms of the accuracy obtained to automatically detect the presence of pathologies, and for the three types of biosignals analyzed, the non uniform techniques used in this work lightly outperformed the results obtained using the uniform methods, suggesting their usefulness to characterize non-stationary biomedical signals in pattern recognition applications. On the other hand, in view of the results obtained and its low computational load, the proposed technique suggests its applicability for the applications under study.

The role of the secondary cell wall in plant resistance to pathogens

Plant resistance to pathogens relies on a complex network of constitutive and inducible defensive barriers. The plant cell wall is one of the barriers that pathogens need to overcome to successfully colonize plant tissues. The traditional view of the plant cell wall as a passive barrier has evolved to a concept that considers the wall as a dynamic structure that regulates both constitutive and inducible defense mechanisms, and as a source of signaling molecules that trigger immune responses. The secondary cell walls of plants also represent a carbon-neutral feedstock (lignocellulosic biomass) for the production of biofuels and biomaterials. Therefore, engineering plants with improved secondary cell wall characteristics is an interesting strategy to ease the processing of lignocellulosic biomass in the biorefinery. However, modification of the integrity of the cell wall by impairment of proteins required for its biosynthesis or remodeling may impact the plants resistance to pathogens. This review summarizes our understanding of the role of the plant cell wall in pathogen resistance with a focus on the contribution of lignin to this biological process.

Estudio de la influencia de la relación C/N en la desnitrificación y metanogénesis de aguas residuales en un reactor anaeróbico de lecho fluidizado inverso

La nitrificación-desnitrificación es el proceso biológico tradicional para la remoción de nitrógeno de las aguas residuales (Ruiz G. et al., 2006a), siendo fundamental ya que contribuye a controlar la eutroficación de los cuerpos receptores. Debido al deterioro que sobre la disponibilidad de los recursos han ejercido las actividades antropogénicas, es necesario orientar el tratamiento de las aguas residuales hacia tecnologías que ofrezcan el mayor grado de sustentabilidad, planteando innovaciones en el tratamiento. El presente proyecto de tesis doctoral versa sobre el estudio de la influencia de la relación C/N en la desnitrificación y metanogénesis de aguas residuales urbanas en un reactor anaeróbico de lecho fluidizado inverso (RLFI). Previamente a la realización de las pruebas experimentales de variación de la relación C/N, se llevó a cabo la etapa de arranque del RLFI la cual se inició en modo batch, favoreciendo la formación y adhesión de biopelícula al medio de soporte utilizado (Extendosphere). Después, sobrevino la operación en modo continuo desde una carga volumétrica aplicada (CVA) de 0.5 g DQOs/L⋅d hasta alcanzar 4 g DQOs/L⋅d, carga volumétrica a la cual se logró la plena estabilización del reactor, siendo la alta variabilidad de la concentración de DQOs en el agua residual urbana de alimentación, la principal problemática que ocasionó retrasos en la estabilidad del reactor. A una CVA de 4 g DQOs/L⋅d en estado estacionario, el valor mínimo de eficiencia de remoción de DQOs fue del 32.36% y el máximo de 66.99%. En estas condiciones el porcentaje de metano presente en el biogás producido tuvo un valor medio de 85.57 ± 2.93%, siendo un valor alto comparado con otros porcentajes de metano encontrados en la digestión anaerobia de aguas residuales urbanas. El YCH4 tuvo un valor medio de 0.316 ± 0.110 LCH4/g DQOrem⋅día. Los porcentajes de metanización variaron en el rango de 20.50 a 100%, registrándose un valor medio de 73.42 ± 25.63%. La considerable variabilidad en el porcentaje de metanización se debió principalmente a que se presentaron eventos de lavado de soporte colonizado, lo cual propició que las actividades metabólicas fueran orientadas hacia formación de biopelícula (anabolismo) en vez de estar dirigidas hacia producción de metano (catabolismo). En relación a los ensayos con variación de la relación C/N, se manejaron relaciones DQOs/N-NO3 en el rango de 1.65 a 21.1 g DQOs/g N-NO3. La tasa de remoción anaerobia de DQOs se incrementó con la concentración de sustrato en una relación casi lineal, ajustándose a una cinética de primer orden, lo que regularmente se presenta a concentraciones bajas de sustrato. La eficiencia del proceso de desnitrificación fue por lo regular alta, incrementándose ligeramente con la concentración de DQOs en el influente, con valores en el rango de 73.8 a 99.1%. Por otra parte, la tasa de remoción por metanogénesis se incrementó con la concentración relativa de sustrato (es decir, a mayores relaciones DQOs/N-NO3), siendo más sensitiva la metanogénesis a la concentración relativa de sustrato que la desnitrificación. Conforme aumentó la relación DQOs/N-NO3, la desnitrificación, de ser la ruta metabólica principal de utilización de la materia orgánica (comparada con la metanización), empezó a combinarse con la metanización. De manera evidente, a las relaciones DQOs/N-NO3 probadas, se manifestaron más las actividades desnitrificantes, quedando reflejadas por el alto porcentaje de utilización de la DQOs removida hacia la desnitrificación. La relación experimental DQOs/N-NO3 a la cual se pudiera haber cumplido con el requerimiento de materia orgánica (en términos de DQOs) para la desnitrificación de nitratos en las aguas residuales urbanas tratadas resultó aproximadamente ser igual a 7.1 g DQOs/g N-NO3. A una CVA de 4 g DQOs/L⋅d, se obtuvo un diámetro promedio máximo de soporte colonizado igual a 266.106 ± 69.279 μm aunque, hay que indicarlo, se presentaron fluctuaciones, las cuales se reflejaron también en el espesor de la biopelícula, el cual tuvo un valor máximo de 50.099 μm y un valor promedio de 37.294 ± 11.199 μm. Estas fluctuaciones pudieron deberse a la existencia de corrientes preferenciales dentro del reactor, las cuales no permitieron un acceso equitativo del sustrato a todo el lecho. Nitrification-denitrification is the traditional biological process for nitrogen removal from wastewaters (Ruiz G. et al., 2006a), being fundamental since it contributes to control the eutrophication of the receiving waters. Due to the deterioration that on the availability of the aquatic resources the anthropogenic activities have exerted, it is necessary to orient the treatment of wastewaters towards technologies that offer the greater degree of sustainability, raising innovations in the treatment. This work studied the influence of C/N ratio on denitrification and methanogenesis of urban wastewaters in an inverse fluidized bed reactor (IFBR). Previously to the accomplishment of the experimental tests with variation of C/N ratio, the start up of the IFBR was carried out in batch way, encouraging the formation and adhesion of biofilm to Extendosphere, which it was used as support. The operation in continuous way carried out from an organic loading rate (OLR) of 0.5 g CODs/L ∙ d to 4 g CODs/L ∙ d, when the steady-state was reached. The high variability of the CODs of the urban wastewaters caused delays in the stability of the reactor. Once stationary state was reached, the removal efficiency of CODs ranged from 32.36 to 66.99% to 4 g CODs/L ∙ d. In these conditions the percentage of methane in produced biogas had an average value of 85.57 ± 2.93%, being a high value compared with other studies treating anaerobically urban wastewaters. The YCH4 had an average value of 0.316 ± 0.110 LCH4/g CODrem ∙ d. The percentage of methanisation ranged from 20.50 to 100%, with an average value of 73.42 ± 25.63%. The considerable variability in the methanisation percentage occurred mainly due events of wash-out of colonized support, which caused that the metabolic activities were oriented towards formation of biofilm (anabolism) instead of methane production (catabolism). Concerning the tests with variation of C/N ratio, CODs/NO3-N ratios from 1.65 to 21.1 g CODs/g NO3-N were proved. The CODs anaerobic removal rate increased with the substrate concentration in an almost linear relation, adjusting to a kinetic of first order, which regularly appears to low concentrations of substrate. Efficiency of the denitrification process was regularly high, and it increased slightly with the CODs concentration in the influent, ranging from 73.8 to 99.1%. On the other hand, the CODs removal rate by methanogenesis increased with the substrate relative concentration (e.g., to greater CODs/NO3-N ratios), being more sensitive the methanogenesis to the substrate relative concentration that the denitrification. When the CODs/NO3-N ratio increased, the denitrification, of being the main metabolic route of use of the organic matter (compared with the methanogenesis), began to be combined with the methanogenesis. Definitively, to the proven CODs/NO3-N ratios the denitrification processes were more pronounced, being reflected by the high percentage of use of the removed CODs towards denitrification. The experimental CODs/NO3-N ratio to which it was possible to have been fulfilled the requirement of organic matter (in terms of CODs) for the denitrification of nitrates in urban wastewaters turned out to be approximately 7.1 g CODs/g NO3-N. It was obtained a maximum average diameter of colonized support of 266.106 ± 69.279 μm to 4 g CODs/L ∙ d, although it is necessary to indicate that appeared fluctuations in the thickness of biofilm, which had a maximum value of 50.099 μm and an average value of 37.294 ± 11.199 μm. These fluctuations could be due to the existence of preferential currents within the reactor, which did not allow an equitable access of the substrate to all the bed.

Parametric and structural self-adaptation of embedded systems using evolvable hardware

Los sistemas empotrados han sido concebidos tradicionalmente como sistemas de procesamiento específicos que realizan una tarea fija durante toda su vida útil. Para cumplir con requisitos estrictos de coste, tamaño y peso, el equipo de diseño debe optimizar su funcionamiento para condiciones muy específicas. Sin embargo, la demanda de mayor versatilidad, un funcionamiento más inteligente y, en definitiva, una mayor capacidad de procesamiento comenzaron a chocar con estas limitaciones, agravado por la incertidumbre asociada a entornos de operación cada vez más dinámicos donde comenzaban a ser desplegados progresivamente. Esto trajo como resultado una necesidad creciente de que los sistemas pudieran responder por si solos a eventos inesperados en tiempo diseño tales como: cambios en las características de los datos de entrada y el entorno del sistema en general; cambios en la propia plataforma de cómputo, por ejemplo debido a fallos o defectos de fabricación; y cambios en las propias especificaciones funcionales causados por unos objetivos del sistema dinámicos y cambiantes. Como consecuencia, la complejidad del sistema aumenta, pero a cambio se habilita progresivamente una capacidad de adaptación autónoma sin intervención humana a lo largo de la vida útil, permitiendo que tomen sus propias decisiones en tiempo de ejecución. Éstos sistemas se conocen, en general, como sistemas auto-adaptativos y tienen, entre otras características, las de auto-configuración, auto-optimización y auto-reparación. Típicamente, la parte soft de un sistema es mayoritariamente la única utilizada para proporcionar algunas capacidades de adaptación a un sistema. Sin embargo, la proporción rendimiento/potencia en dispositivos software como microprocesadores en muchas ocasiones no es adecuada para sistemas empotrados. En este escenario, el aumento resultante en la complejidad de las aplicaciones está siendo abordado parcialmente mediante un aumento en la complejidad de los dispositivos en forma de multi/many-cores; pero desafortunadamente, esto hace que el consumo de potencia también aumente. Además, la mejora en metodologías de diseño no ha sido acorde como para poder utilizar toda la capacidad de cómputo disponible proporcionada por los núcleos. Por todo ello, no se están satisfaciendo adecuadamente las demandas de cómputo que imponen las nuevas aplicaciones. La solución tradicional para mejorar la proporción rendimiento/potencia ha sido el cambio a unas especificaciones hardware, principalmente usando ASICs. Sin embargo, los costes de un ASIC son altamente prohibitivos excepto en algunos casos de producción en masa y además la naturaleza estática de su estructura complica la solución a las necesidades de adaptación. Los avances en tecnologías de fabricación han hecho que la FPGA, una vez lenta y pequeña, usada como glue logic en sistemas mayores, haya crecido hasta convertirse en un dispositivo de cómputo reconfigurable de gran potencia, con una cantidad enorme de recursos lógicos computacionales y cores hardware empotrados de procesamiento de señal y de propósito general. Sus capacidades de reconfiguración han permitido combinar la flexibilidad propia del software con el rendimiento del procesamiento en hardware, lo que tiene la potencialidad de provocar un cambio de paradigma en arquitectura de computadores, pues el hardware no puede ya ser considerado más como estático. El motivo es que como en el caso de las FPGAs basadas en tecnología SRAM, la reconfiguración parcial dinámica (DPR, Dynamic Partial Reconfiguration) es posible. Esto significa que se puede modificar (reconfigurar) un subconjunto de los recursos computacionales en tiempo de ejecución mientras el resto permanecen activos. Además, este proceso de reconfiguración puede ser ejecutado internamente por el propio dispositivo. El avance tecnológico en dispositivos hardware reconfigurables se encuentra recogido bajo el campo conocido como Computación Reconfigurable (RC, Reconfigurable Computing). Uno de los campos de aplicación más exóticos y menos convencionales que ha posibilitado la computación reconfigurable es el conocido como Hardware Evolutivo (EHW, Evolvable Hardware), en el cual se encuentra enmarcada esta tesis. La idea principal del concepto consiste en convertir hardware que es adaptable a través de reconfiguración en una entidad evolutiva sujeta a las fuerzas de un proceso evolutivo inspirado en el de las especies biológicas naturales, que guía la dirección del cambio. Es una aplicación más del campo de la Computación Evolutiva (EC, Evolutionary Computation), que comprende una serie de algoritmos de optimización global conocidos como Algoritmos Evolutivos (EA, Evolutionary Algorithms), y que son considerados como algoritmos universales de resolución de problemas. En analogía al proceso biológico de la evolución, en el hardware evolutivo el sujeto de la evolución es una población de circuitos que intenta adaptarse a su entorno mediante una adecuación progresiva generación tras generación. Los individuos pasan a ser configuraciones de circuitos en forma de bitstreams caracterizados por descripciones de circuitos reconfigurables. Seleccionando aquellos que se comportan mejor, es decir, que tienen una mejor adecuación (o fitness) después de ser evaluados, y usándolos como padres de la siguiente generación, el algoritmo evolutivo crea una nueva población hija usando operadores genéticos como la mutación y la recombinación. Según se van sucediendo generaciones, se espera que la población en conjunto se aproxime a la solución óptima al problema de encontrar una configuración del circuito adecuada que satisfaga las especificaciones. El estado de la tecnología de reconfiguración después de que la familia de FPGAs XC6200 de Xilinx fuera retirada y reemplazada por las familias Virtex a finales de los 90, supuso un gran obstáculo para el avance en hardware evolutivo; formatos de bitstream cerrados (no conocidos públicamente); dependencia de herramientas del fabricante con soporte limitado de DPR; una velocidad de reconfiguración lenta; y el hecho de que modificaciones aleatorias del bitstream pudieran resultar peligrosas para la integridad del dispositivo, son algunas de estas razones. Sin embargo, una propuesta a principios de los años 2000 permitió mantener la investigación en el campo mientras la tecnología de DPR continuaba madurando, el Circuito Virtual Reconfigurable (VRC, Virtual Reconfigurable Circuit). En esencia, un VRC en una FPGA es una capa virtual que actúa como un circuito reconfigurable de aplicación específica sobre la estructura nativa de la FPGA que reduce la complejidad del proceso reconfiguración y aumenta su velocidad (comparada con la reconfiguración nativa). Es un array de nodos computacionales especificados usando descripciones HDL estándar que define recursos reconfigurables ad-hoc: multiplexores de rutado y un conjunto de elementos de procesamiento configurables, cada uno de los cuales tiene implementadas todas las funciones requeridas, que pueden seleccionarse a través de multiplexores tal y como ocurre en una ALU de un microprocesador. Un registro grande actúa como memoria de configuración, por lo que la reconfiguración del VRC es muy rápida ya que tan sólo implica la escritura de este registro, el cual controla las señales de selección del conjunto de multiplexores. Sin embargo, esta capa virtual provoca: un incremento de área debido a la implementación simultánea de cada función en cada nodo del array más los multiplexores y un aumento del retardo debido a los multiplexores, reduciendo la frecuencia de funcionamiento máxima. La naturaleza del hardware evolutivo, capaz de optimizar su propio comportamiento computacional, le convierten en un buen candidato para avanzar en la investigación sobre sistemas auto-adaptativos. Combinar un sustrato de cómputo auto-reconfigurable capaz de ser modificado dinámicamente en tiempo de ejecución con un algoritmo empotrado que proporcione una dirección de cambio, puede ayudar a satisfacer los requisitos de adaptación autónoma de sistemas empotrados basados en FPGA. La propuesta principal de esta tesis está por tanto dirigida a contribuir a la auto-adaptación del hardware de procesamiento de sistemas empotrados basados en FPGA mediante hardware evolutivo. Esto se ha abordado considerando que el comportamiento computacional de un sistema puede ser modificado cambiando cualquiera de sus dos partes constitutivas: una estructura hard subyacente y un conjunto de parámetros soft. De esta distinción, se derivan dos lineas de trabajo. Por un lado, auto-adaptación paramétrica, y por otro auto-adaptación estructural. El objetivo perseguido en el caso de la auto-adaptación paramétrica es la implementación de técnicas de optimización evolutiva complejas en sistemas empotrados con recursos limitados para la adaptación paramétrica online de circuitos de procesamiento de señal. La aplicación seleccionada como prueba de concepto es la optimización para tipos muy específicos de imágenes de los coeficientes de los filtros de transformadas wavelet discretas (DWT, DiscreteWavelet Transform), orientada a la compresión de imágenes. Por tanto, el objetivo requerido de la evolución es una compresión adaptativa y más eficiente comparada con los procedimientos estándar. El principal reto radica en reducir la necesidad de recursos de supercomputación para el proceso de optimización propuesto en trabajos previos, de modo que se adecúe para la ejecución en sistemas empotrados. En cuanto a la auto-adaptación estructural, el objetivo de la tesis es la implementación de circuitos auto-adaptativos en sistemas evolutivos basados en FPGA mediante un uso eficiente de sus capacidades de reconfiguración nativas. En este caso, la prueba de concepto es la evolución de tareas de procesamiento de imagen tales como el filtrado de tipos desconocidos y cambiantes de ruido y la detección de bordes en la imagen. En general, el objetivo es la evolución en tiempo de ejecución de tareas de procesamiento de imagen desconocidas en tiempo de diseño (dentro de un cierto grado de complejidad). En este caso, el objetivo de la propuesta es la incorporación de DPR en EHW para evolucionar la arquitectura de un array sistólico adaptable mediante reconfiguración cuya capacidad de evolución no había sido estudiada previamente. Para conseguir los dos objetivos mencionados, esta tesis propone originalmente una plataforma evolutiva que integra un motor de adaptación (AE, Adaptation Engine), un motor de reconfiguración (RE, Reconfiguration Engine) y un motor computacional (CE, Computing Engine) adaptable. El el caso de adaptación paramétrica, la plataforma propuesta está caracterizada por: • un CE caracterizado por un núcleo de procesamiento hardware de DWT adaptable mediante registros reconfigurables que contienen los coeficientes de los filtros wavelet • un algoritmo evolutivo como AE que busca filtros wavelet candidatos a través de un proceso de optimización paramétrica desarrollado específicamente para sistemas caracterizados por recursos de procesamiento limitados • un nuevo operador de mutación simplificado para el algoritmo evolutivo utilizado, que junto con un mecanismo de evaluación rápida de filtros wavelet candidatos derivado de la literatura actual, asegura la viabilidad de la búsqueda evolutiva asociada a la adaptación de wavelets. En el caso de adaptación estructural, la plataforma propuesta toma la forma de: • un CE basado en una plantilla de array sistólico reconfigurable de 2 dimensiones compuesto de nodos de procesamiento reconfigurables • un algoritmo evolutivo como AE que busca configuraciones candidatas del array usando un conjunto de funcionalidades de procesamiento para los nodos disponible en una biblioteca accesible en tiempo de ejecución • un RE hardware que explota la capacidad de reconfiguración nativa de las FPGAs haciendo un uso eficiente de los recursos reconfigurables del dispositivo para cambiar el comportamiento del CE en tiempo de ejecución • una biblioteca de elementos de procesamiento reconfigurables caracterizada por bitstreams parciales independientes de la posición, usados como el conjunto de configuraciones disponibles para los nodos de procesamiento del array Las contribuciones principales de esta tesis se pueden resumir en la siguiente lista: • Una plataforma evolutiva basada en FPGA para la auto-adaptación paramétrica y estructural de sistemas empotrados compuesta por un motor computacional (CE), un motor de adaptación (AE) evolutivo y un motor de reconfiguración (RE). Esta plataforma se ha desarrollado y particularizado para los casos de auto-adaptación paramétrica y estructural. • En cuanto a la auto-adaptación paramétrica, las contribuciones principales son: – Un motor computacional adaptable mediante registros que permite la adaptación paramétrica de los coeficientes de una implementación hardware adaptativa de un núcleo de DWT. – Un motor de adaptación basado en un algoritmo evolutivo desarrollado específicamente para optimización numérica, aplicada a los coeficientes de filtros wavelet en sistemas empotrados con recursos limitados. – Un núcleo IP de DWT auto-adaptativo en tiempo de ejecución para sistemas empotrados que permite la optimización online del rendimiento de la transformada para compresión de imágenes en entornos específicos de despliegue, caracterizados por tipos diferentes de señal de entrada. – Un modelo software y una implementación hardware de una herramienta para la construcción evolutiva automática de transformadas wavelet específicas. • Por último, en cuanto a la auto-adaptación estructural, las contribuciones principales son: – Un motor computacional adaptable mediante reconfiguración nativa de FPGAs caracterizado por una plantilla de array sistólico en dos dimensiones de nodos de procesamiento reconfigurables. Es posible mapear diferentes tareas de cómputo en el array usando una biblioteca de elementos sencillos de procesamiento reconfigurables. – Definición de una biblioteca de elementos de procesamiento apropiada para la síntesis autónoma en tiempo de ejecución de diferentes tareas de procesamiento de imagen. – Incorporación eficiente de la reconfiguración parcial dinámica (DPR) en sistemas de hardware evolutivo, superando los principales inconvenientes de propuestas previas como los circuitos reconfigurables virtuales (VRCs). En este trabajo también se comparan originalmente los detalles de implementación de ambas propuestas. – Una plataforma tolerante a fallos, auto-curativa, que permite la recuperación funcional online en entornos peligrosos. La plataforma ha sido caracterizada desde una perspectiva de tolerancia a fallos: se proponen modelos de fallo a nivel de CLB y de elemento de procesamiento, y usando el motor de reconfiguración, se hace un análisis sistemático de fallos para un fallo en cada elemento de procesamiento y para dos fallos acumulados. – Una plataforma con calidad de filtrado dinámica que permite la adaptación online a tipos de ruido diferentes y diferentes comportamientos computacionales teniendo en cuenta los recursos de procesamiento disponibles. Por un lado, se evolucionan filtros con comportamientos no destructivos, que permiten esquemas de filtrado en cascada escalables; y por otro, también se evolucionan filtros escalables teniendo en cuenta requisitos computacionales de filtrado cambiantes dinámicamente. Este documento está organizado en cuatro partes y nueve capítulos. La primera parte contiene el capítulo 1, una introducción y motivación sobre este trabajo de tesis. A continuación, el marco de referencia en el que se enmarca esta tesis se analiza en la segunda parte: el capítulo 2 contiene una introducción a los conceptos de auto-adaptación y computación autonómica (autonomic computing) como un campo de investigación más general que el muy específico de este trabajo; el capítulo 3 introduce la computación evolutiva como la técnica para dirigir la adaptación; el capítulo 4 analiza las plataformas de computación reconfigurables como la tecnología para albergar hardware auto-adaptativo; y finalmente, el capítulo 5 define, clasifica y hace un sondeo del campo del hardware evolutivo. Seguidamente, la tercera parte de este trabajo contiene la propuesta, desarrollo y resultados obtenidos: mientras que el capítulo 6 contiene una declaración de los objetivos de la tesis y la descripción de la propuesta en su conjunto, los capítulos 7 y 8 abordan la auto-adaptación paramétrica y estructural, respectivamente. Finalmente, el capítulo 9 de la parte 4 concluye el trabajo y describe caminos de investigación futuros. ABSTRACT Embedded systems have traditionally been conceived to be specific-purpose computers with one, fixed computational task for their whole lifetime. Stringent requirements in terms of cost, size and weight forced designers to highly optimise their operation for very specific conditions. However, demands for versatility, more intelligent behaviour and, in summary, an increased computing capability began to clash with these limitations, intensified by the uncertainty associated to the more dynamic operating environments where they were progressively being deployed. This brought as a result an increasing need for systems to respond by themselves to unexpected events at design time, such as: changes in input data characteristics and system environment in general; changes in the computing platform itself, e.g., due to faults and fabrication defects; and changes in functional specifications caused by dynamically changing system objectives. As a consequence, systems complexity is increasing, but in turn, autonomous lifetime adaptation without human intervention is being progressively enabled, allowing them to take their own decisions at run-time. This type of systems is known, in general, as selfadaptive, and are able, among others, of self-configuration, self-optimisation and self-repair. Traditionally, the soft part of a system has mostly been so far the only place to provide systems with some degree of adaptation capabilities. However, the performance to power ratios of software driven devices like microprocessors are not adequate for embedded systems in many situations. In this scenario, the resulting rise in applications complexity is being partly addressed by rising devices complexity in the form of multi and many core devices; but sadly, this keeps on increasing power consumption. Besides, design methodologies have not been improved accordingly to completely leverage the available computational power from all these cores. Altogether, these factors make that the computing demands new applications pose are not being wholly satisfied. The traditional solution to improve performance to power ratios has been the switch to hardware driven specifications, mainly using ASICs. However, their costs are highly prohibitive except for some mass production cases and besidesthe static nature of its structure complicates the solution to the adaptation needs. The advancements in fabrication technologies have made that the once slow, small FPGA used as glue logic in bigger systems, had grown to be a very powerful, reconfigurable computing device with a vast amount of computational logic resources and embedded, hardened signal and general purpose processing cores. Its reconfiguration capabilities have enabled software-like flexibility to be combined with hardware-like computing performance, which has the potential to cause a paradigm shift in computer architecture since hardware cannot be considered as static anymore. This is so, since, as is the case with SRAMbased FPGAs, Dynamic Partial Reconfiguration (DPR) is possible. This means that subsets of the FPGA computational resources can now be changed (reconfigured) at run-time while the rest remains active. Besides, this reconfiguration process can be triggered internally by the device itself. This technological boost in reconfigurable hardware devices is actually covered under the field known as Reconfigurable Computing. One of the most exotic fields of application that Reconfigurable Computing has enabled is the known as Evolvable Hardware (EHW), in which this dissertation is framed. The main idea behind the concept is turning hardware that is adaptable through reconfiguration into an evolvable entity subject to the forces of an evolutionary process, inspired by that of natural, biological species, that guides the direction of change. It is yet another application of the field of Evolutionary Computation (EC), which comprises a set of global optimisation algorithms known as Evolutionary Algorithms (EAs), considered as universal problem solvers. In analogy to the biological process of evolution, in EHW the subject of evolution is a population of circuits that tries to get adapted to its surrounding environment by progressively getting better fitted to it generation after generation. Individuals become circuit configurations representing bitstreams that feature reconfigurable circuit descriptions. By selecting those that behave better, i.e., with a higher fitness value after being evaluated, and using them as parents of the following generation, the EA creates a new offspring population by using so called genetic operators like mutation and recombination. As generations succeed one another, the whole population is expected to approach to the optimum solution to the problem of finding an adequate circuit configuration that fulfils system objectives. The state of reconfiguration technology after Xilinx XC6200 FPGA family was discontinued and replaced by Virtex families in the late 90s, was a major obstacle for advancements in EHW; closed (non publicly known) bitstream formats; dependence on manufacturer tools with highly limiting support of DPR; slow speed of reconfiguration; and random bitstream modifications being potentially hazardous for device integrity, are some of these reasons. However, a proposal in the first 2000s allowed to keep investigating in this field while DPR technology kept maturing, the Virtual Reconfigurable Circuit (VRC). In essence, a VRC in an FPGA is a virtual layer acting as an application specific reconfigurable circuit on top of an FPGA fabric that reduces the complexity of the reconfiguration process and increases its speed (compared to native reconfiguration). It is an array of computational nodes specified using standard HDL descriptions that define ad-hoc reconfigurable resources; routing multiplexers and a set of configurable processing elements, each one containing all the required functions, which are selectable through functionality multiplexers as in microprocessor ALUs. A large register acts as configuration memory, so VRC reconfiguration is very fast given it only involves writing this register, which drives the selection signals of the set of multiplexers. However, large overheads are introduced by this virtual layer; an area overhead due to the simultaneous implementation of every function in every node of the array plus the multiplexers, and a delay overhead due to the multiplexers, which also reduces maximum frequency of operation. The very nature of Evolvable Hardware, able to optimise its own computational behaviour, makes it a good candidate to advance research in self-adaptive systems. Combining a selfreconfigurable computing substrate able to be dynamically changed at run-time with an embedded algorithm that provides a direction for change, can help fulfilling requirements for autonomous lifetime adaptation of FPGA-based embedded systems. The main proposal of this thesis is hence directed to contribute to autonomous self-adaptation of the underlying computational hardware of FPGA-based embedded systems by means of Evolvable Hardware. This is tackled by considering that the computational behaviour of a system can be modified by changing any of its two constituent parts: an underlying hard structure and a set of soft parameters. Two main lines of work derive from this distinction. On one side, parametric self-adaptation and, on the other side, structural self-adaptation. The goal pursued in the case of parametric self-adaptation is the implementation of complex evolutionary optimisation techniques in resource constrained embedded systems for online parameter adaptation of signal processing circuits. The application selected as proof of concept is the optimisation of Discrete Wavelet Transforms (DWT) filters coefficients for very specific types of images, oriented to image compression. Hence, adaptive and improved compression efficiency, as compared to standard techniques, is the required goal of evolution. The main quest lies in reducing the supercomputing resources reported in previous works for the optimisation process in order to make it suitable for embedded systems. Regarding structural self-adaptation, the thesis goal is the implementation of self-adaptive circuits in FPGA-based evolvable systems through an efficient use of native reconfiguration capabilities. In this case, evolution of image processing tasks such as filtering of unknown and changing types of noise and edge detection are the selected proofs of concept. In general, evolving unknown image processing behaviours (within a certain complexity range) at design time is the required goal. In this case, the mission of the proposal is the incorporation of DPR in EHW to evolve a systolic array architecture adaptable through reconfiguration whose evolvability had not been previously checked. In order to achieve the two stated goals, this thesis originally proposes an evolvable platform that integrates an Adaptation Engine (AE), a Reconfiguration Engine (RE) and an adaptable Computing Engine (CE). In the case of parametric adaptation, the proposed platform is characterised by: • a CE featuring a DWT hardware processing core adaptable through reconfigurable registers that holds wavelet filters coefficients • an evolutionary algorithm as AE that searches for candidate wavelet filters through a parametric optimisation process specifically developed for systems featured by scarce computing resources • a new, simplified mutation operator for the selected EA, that together with a fast evaluation mechanism of candidate wavelet filters derived from existing literature, assures the feasibility of the evolutionary search involved in wavelets adaptation In the case of structural adaptation, the platform proposal takes the form of: • a CE based on a reconfigurable 2D systolic array template composed of reconfigurable processing nodes • an evolutionary algorithm as AE that searches for candidate configurations of the array using a set of computational functionalities for the nodes available in a run time accessible library • a hardware RE that exploits native DPR capabilities of FPGAs and makes an efficient use of the available reconfigurable resources of the device to change the behaviour of the CE at run time • a library of reconfigurable processing elements featured by position-independent partial bitstreams used as the set of available configurations for the processing nodes of the array Main contributions of this thesis can be summarised in the following list. • An FPGA-based evolvable platform for parametric and structural self-adaptation of embedded systems composed of a Computing Engine, an evolutionary Adaptation Engine and a Reconfiguration Engine. This platform is further developed and tailored for both parametric and structural self-adaptation. • Regarding parametric self-adaptation, main contributions are: – A CE adaptable through reconfigurable registers that enables parametric adaptation of the coefficients of an adaptive hardware implementation of a DWT core. – An AE based on an Evolutionary Algorithm specifically developed for numerical optimisation applied to wavelet filter coefficients in resource constrained embedded systems. – A run-time self-adaptive DWT IP core for embedded systems that allows for online optimisation of transform performance for image compression for specific deployment environments characterised by different types of input signals. – A software model and hardware implementation of a tool for the automatic, evolutionary construction of custom wavelet transforms. • Lastly, regarding structural self-adaptation, main contributions are: – A CE adaptable through native FPGA fabric reconfiguration featured by a two dimensional systolic array template of reconfigurable processing nodes. Different processing behaviours can be automatically mapped in the array by using a library of simple reconfigurable processing elements. – Definition of a library of such processing elements suited for autonomous runtime synthesis of different image processing tasks. – Efficient incorporation of DPR in EHW systems, overcoming main drawbacks from the previous approach of virtual reconfigurable circuits. Implementation details for both approaches are also originally compared in this work. – A fault tolerant, self-healing platform that enables online functional recovery in hazardous environments. The platform has been characterised from a fault tolerance perspective: fault models at FPGA CLB level and processing elements level are proposed, and using the RE, a systematic fault analysis for one fault in every processing element and for two accumulated faults is done. – A dynamic filtering quality platform that permits on-line adaptation to different types of noise and different computing behaviours considering the available computing resources. On one side, non-destructive filters are evolved, enabling scalable cascaded filtering schemes; and on the other, size-scalable filters are also evolved considering dynamically changing computational filtering requirements. This dissertation is organized in four parts and nine chapters. First part contains chapter 1, the introduction to and motivation of this PhD work. Following, the reference framework in which this dissertation is framed is analysed in the second part: chapter 2 features an introduction to the notions of self-adaptation and autonomic computing as a more general research field to the very specific one of this work; chapter 3 introduces evolutionary computation as the technique to drive adaptation; chapter 4 analyses platforms for reconfigurable computing as the technology to hold self-adaptive hardware; and finally chapter 5 defines, classifies and surveys the field of Evolvable Hardware. Third part of the work follows, which contains the proposal, development and results obtained: while chapter 6 contains an statement of the thesis goals and the description of the proposal as a whole, chapters 7 and 8 address parametric and structural self-adaptation, respectively. Finally, chapter 9 in part 4 concludes the work and describes future research paths.

Medicago truncatula Natural Resistance Associated Macrophage Protein1 is required for iron uptake by rhizobia infected nodule cells

Iron is critical for symbiotic nitrogen fixation (SNF) as a key component ofmultiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule, where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins (e.g. plant leghemoglobin and bacterial nitrogenase). MtNramp1 (Medtr3g088460) is the M. truncatula Natural Resistance-Associated Macrophage Protein family member, with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared with the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II.