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.

Using clustering techniques for intelligent camera-based user interfaces

The area of Human-Machine Interface is growing fast due to its high importance in all technological systems. The basic idea behind designing human-machine interfaces is to enrich the communication with the technology in a natural and easy way. Gesture interfaces are a good example of transparent interfaces. Such interfaces must identify properly the action the user wants to perform, so the proper gesture recognition is of the highest importance. However, most of the systems based on gesture recognition use complex methods requiring high-resource devices. In this work, we propose to model gestures capturing their temporal properties, which significantly reduce storage requirements, and use clustering techniques, namely self-organizing maps and unsupervised genetic algorithm, for their classification. We further propose to train a certain number of algorithms with different parameters and combine their decision using majority voting in order to decrease the false positive rate. The main advantage of the approach is its simplicity, which enables the implementation using devices with limited resources, and therefore low cost. The testing results demonstrate its high potential.

A silviculture-oriented spatio-temporal model for germination in Pinus pinea L. in the Spanish Northern Plateau based on a direct seeding experiment

Natural regeneration in Pinus pinea stands commonly fails throughout the Spanish Northern Plateau under current intensive regeneration treatments. As a result, extensive direct seeding is commonly conducted to guarantee regeneration occurrence. In a period of rationalization of the resources devoted to forest management, this kind of techniques may become unaffordable. Given that the climatic and stand factors driving germination remain unknown, tools are required to understand the process and temper the use of direct seeding. In this study, the spatio-temporal pattern of germination of P. pinea was modelled with those purposes. The resulting findings will allow us to (1) determine the main ecological variables involved in germination in the species and (2) infer adequate silvicultural alternatives. The modelling approach focuses on covariates which are readily available to forest managers. A two-step nonlinear mixed model was fitted to predict germination occurrence and abundance in P. pinea under varying climatic, environmental and stand conditions, based on a germination data set covering a 5-year period. The results obtained reveal that the process is primarily driven by climate variables. Favourable conditions for germination commonly occur in fall although the optimum window is often narrow and may not occur at all in some years. At spatial level, it would appear that germination is facilitated by high stand densities, suggesting that current felling intensity should be reduced. In accordance with other studies on P. pinea dispersal, it seems that denser stands during the regeneration period will reduce the present dependence on direct seeding.

Characterization of underground cellars in the Duero Basin by GNSS, LIDAR and GPR techniques

The underground cellars of the Duero River basin are part of spread and damaged agricultural landscape which is in danger of disappearing. These architectural complexes are allocated next to small towns. Constructions are mostly dug in the ground with a gallery down or "barrel" strait through which you access the cave or cellar. This wider space is used to make and store wine. Observation and detection of the winery both on the outside and underground is essential to make an inventory of the rural heritage. Geodetection is a non-invasive technique, suitable to determinate with precision buried structures in the ground. The undertaken works include LIDAR survey techniques, GNSS and GPR obtained data. The results are used to identify with centimetric precision construction elements forming the winery. Graphic and cartographic obtained documents allow optimum visualization of the studied field and can be used in the reconstruction of the place.

Spatio-temporal filtering with morphological operators for robust cell migration estimation in "in-vivo" images

The understanding of the embryogenesis in living systems requires reliable quantitative analysis of the cell migration throughout all the stages of development. This is a major challenge of the "in-toto" reconstruction based on different modalities of "in-vivo" imaging techniques -spatio-temporal resolution and image artifacts and noise. Several methods for cell tracking are available, but expensive manual interaction -time and human resources- is always required to enforce coherence. Because of this limitation it is necessary to restrict the experiments or assume an uncontrolled error rate. Is it possible to obtain automated reliable measurements of migration? can we provide a seed for biologists to complete cell lineages efficiently? We propose a filtering technique that considers trajectories as spatio-temporal connected structures that prunes out those that might introduce noise and false positives by using multi-dimensional morphological operators.

Impact of fire and post-fire management techniques on soil chemical properties

The effects of fire ( Control burned soil) and two emergency stabilisation techniques (grass Seeding and straw Mulching ) on 20 chemical characteristics were evaluated on 0 – 5 cm top-soils sampled 1, 90, 180 and 365 days after an experimental fi re in a steep shrubland of a temperate-humid region (NW Spain). Most part of pH (in H 2 O and KCl) variance was explained by the sampling date. No clear temporal trends were identi fi able for total soil C and N content, likely due to the large SOM pool in these soils; however, changes on soil δ 13 C were explained by the deposition of 13 C-depleted ashes, followed by its progressive erosion, while those on soil δ 15 N were a consequence of fi re induced N outputs. After the fi re, NH 4 + – N, P, Na, K, Mg, Ca, Mn, Cu, Zn and B concentrations increased, while those of NO 3 − – N, Al, Fe and Co did not vary significantly. Despite a significant decline with time, concentrations of Mg, Ca and Mn at the end of the study were still higher than in unburned soil, while those of K, Cu, Zn and B were similar to the pre-fire levels and those of NH 4 + – N, P and Na were below pre-fire values. Mulching and Seeding treatments for burned soil emergency stabilisation had significant effects on soil δ 15 N and extractable K, Mg and Ca, while data were inconclusive for their possible effects on the extractable Al, Fe and Co

Generating descriptions that summarize geospatial and temporal data

Effective data summarization methods that use AI techniques can help humans understand large sets of data. In this paper, we describe a knowledge-based method for automatically generating summaries of geospatial and temporal data, i.e. data with geographical and temporal references. The method is useful for summarizing data streams, such as GPS traces and traffic information, that are becoming more prevalent with the increasing use of sensors in computing devices. The method presented here is an initial architecture for our ongoing research in this domain. In this paper we describe the data representations we have designed for our method, our implementations of components to perform data abstraction and natural language generation. We also discuss evaluation results that show the ability of our method to generate certain types of geospatial and temporal descriptions.

AquaData and AquaGIS: Two computer utilities for temporal and spatial simulations of water-limited yield with AquaCrop.

The crop simulation model AquaCrop, recently developed by FAO can be used for a wide range of purposes. However, in its present form, its use over large areas or for applications that require a large number of simulations runs (e.g., long-term analysis), is not practical without developing software to facilitate such applications. Two tools for managing the inputs and outputs of AquaCrop, named AquaData and AquaGIS, have been developed for this purpose and are presented here. Both software utilities have been programmed in Delphi v. 5 and in addition, AquaGIS requires the Geographic Information System (GIS) programming tool MapObjects. These utilities allow the efficient management of input and output files, along with a GIS module to develop spatial analysis and effect spatial visualization of the results, facilitating knowledge dissemination. A sample of application of the utilities is given here, as an AquaCrop simulation analysis of impact of climate change on wheat yield in Southern Spain, which requires extensive input data preparation and output processing. The use of AquaCrop without the two utilities would have required approximately 1000 h of work, while the utilization of AquaData and AquaGIS reduced that time by more than 99%. Furthermore, the use of GIS, made it possible to perform a spatial analysis of the results, thus providing a new option to extend the use of the AquaCrop model to scales requiring spatial and temporal analyses.

Predicción espacio-temporal de la irradiancia solar global a corto plazo en España mediante geoestadística y redes neuronales artificiales

El enriquecimiento del conocimiento sobre la Irradiancia Solar (IS) a nivel de superficie terrestre, así como su predicción, cobran gran interés para las Energías Renovables (ER) - Energía Solar (ES)-, y para distintas aplicaciones industriales o ecológicas. En el ámbito de las ER, el uso óptimo de la ES implica contar con datos de la IS en superficie que ayuden tanto, en la selección de emplazamientos para instalaciones de ES, como en su etapa de diseño (dimensionar la producción) y, finalmente, en su explotación. En este último caso, la observación y la predicción es útil para el mercado energético, la planificación y gestión de la energía (generadoras y operadoras del sistema eléctrico), especialmente en los nuevos contextos de las redes inteligentes de transporte. A pesar de la importancia estratégica de contar con datos de la IS, especialmente los observados por sensores de IS en superficie (los que mejor captan esta variable), estos no siempre están disponibles para los lugares de interés ni con la resolución espacial y temporal deseada. Esta limitación se une a la necesidad de disponer de predicciones a corto plazo de la IS que ayuden a la planificación y gestión de la energía. Se ha indagado y caracterizado las Redes de Estaciones Meteorológicas (REM) existentes en España que publican en internet sus observaciones, focalizando en la IS. Se han identificado 24 REM (16 gubernamentales y 8 redes voluntarios) que aglutinan 3492 estaciones, convirtiéndose éstas en las fuentes de datos meteorológicos utilizados en la tesis. Se han investigado cinco técnicas de estimación espacial de la IS en intervalos de 15 minutos para el territorio peninsular (3 técnicas geoestadísticas, una determinística y el método HelioSat2 basado en imágenes satelitales) con distintas configuraciones espaciales. Cuando el área de estudio tiene una adecuada densidad de observaciones, el mejor método identificado para estimar la IS es el Kriging con Regresión usando variables auxiliares -una de ellas la IS estimada a partir de imágenes satelitales-. De este modo es posible estimar espacialmente la IS más allá de los 25 km identificados en la bibliografía. En caso contrario, se corrobora la idoneidad de utilizar estimaciones a partir de sensores remotos cuando la densidad de observaciones no es adecuada. Se ha experimentado con el modelado de Redes Neuronales Artificiales (RNA) para la predicción a corto plazo de la IS utilizando observaciones próximas (componentes espaciales) en sus entradas y, los resultados son prometedores. Así los niveles de errores disminuyen bajo las siguientes condiciones: (1) cuando el horizonte temporal de predicción es inferior o igual a 3 horas, las estaciones vecinas que se incluyen en el modelo deben encentrarse a una distancia máxima aproximada de 55 km. Esto permite concluir que las RNA son capaces de aprender cómo afectan las condiciones meteorológicas vecinas a la predicción de la IS. ABSTRACT ABSTRACT The enrichment of knowledge about the Solar Irradiance (SI) at Earth's surface and its prediction, have a high interest for Renewable Energy (RE) - Solar Energy (SE) - and for various industrial and environmental applications. In the field of the RE, the optimal use of the SE involves having SI surface to help in the selection of sites for facilities ES, in the design stage (sizing energy production), and finally on their production. In the latter case, the observation and prediction is useful for the market, planning and management of the energy (generators and electrical system operators), especially in new contexts of smart transport networks (smartgrid). Despite the strategic importance of SI data, especially those observed by sensors of SI at surface (the ones that best measure this environmental variable), these are not always available to the sights and the spatial and temporal resolution desired. This limitation is bound to the need for short-term predictions of the SI to help planning and energy management. It has been investigated and characterized existing Networks of Weather Stations (NWS) in Spain that share its observations online, focusing on SI. 24 NWS have been identified (16 government and 8 volunteer networks) that implies 3492 stations, turning it into the sources of meteorological data used in the thesis. We have investigated five technical of spatial estimation of SI in 15 minutes to the mainland (3 geostatistical techniques and HelioSat2 a deterministic method based on satellite images) with different spatial configurations. When the study area has an adequate density of observations we identified the best method to estimate the SI is the regression kriging with auxiliary variables (one of them is the SI estimated from satellite images. Thus it is possible to spatially estimate the SI beyond the 25 km identified in the literature. Otherwise, when the density of observations is inadequate the appropriateness is using the estimates values from remote sensing. It has been experimented with Artificial Neural Networks (ANN) modeling for predicting the short-term future of the SI using observations from neighbor’s weather stations (spatial components) in their inputs, and the results are promising. The error levels decrease under the following conditions: (1) when the prediction horizon is less or equal than 3 hours the best models are the ones that include data from the neighboring stations (at a maximum distance of 55 km). It is concluded that the ANN is able to learn how weather conditions affect neighboring prediction of IS at such Spatio-temporal horizons.

Plasma dynamics studies by mass spectrometry techniques

We present temporal information obtained by mass spectrometry techniques about the evolution of plasmas generated by laser filamentation in air. The experimental setup used in this work allowed us to study not only the dynamics of the filament core but also of the energy reservoir that surrounds it. Furthermore, valuable insights about the chemistry of such systems like the photofragmentation and/or formation of molecules were obtained. The interpretation of the experimental results are supported by PIC simulations.

Formal Verification of Temporal Properties for Parametrized Concurrent Programs and Concurrent Data Structures

Los tipos de datos concurrentes son implementaciones concurrentes de las abstracciones de datos clásicas, con la diferencia de que han sido específicamente diseñados para aprovechar el gran paralelismo disponible en las modernas arquitecturas multiprocesador y multinúcleo. La correcta manipulación de los tipos de datos concurrentes resulta esencial para demostrar la completa corrección de los sistemas de software que los utilizan. Una de las mayores dificultades a la hora de diseñar y verificar tipos de datos concurrentes surge de la necesidad de tener que razonar acerca de un número arbitrario de procesos que invocan estos tipos de datos de manera concurrente. Esto requiere considerar sistemas parametrizados. En este trabajo estudiamos la verificación formal de propiedades temporales de sistemas concurrentes parametrizados, poniendo especial énfasis en programas que manipulan estructuras de datos concurrentes. La principal dificultad a la hora de razonar acerca de sistemas concurrentes parametrizados proviene de la interacción entre el gran nivel de concurrencia que éstos poseen y la necesidad de razonar al mismo tiempo acerca de la memoria dinámica. La verificación de sistemas parametrizados resulta en sí un problema desafiante debido a que requiere razonar acerca de estructuras de datos complejas que son accedidas y modificadas por un numero ilimitado de procesos que manipulan de manera simultánea el contenido de la memoria dinámica empleando métodos de sincronización poco estructurados. En este trabajo, presentamos un marco formal basado en métodos deductivos capaz de ocuparse de la verificación de propiedades de safety y liveness de sistemas concurrentes parametrizados que manejan estructuras de datos complejas. Nuestro marco formal incluye reglas de prueba y técnicas especialmente adaptadas para sistemas parametrizados, las cuales trabajan en colaboración con procedimientos de decisión especialmente diseñados para analizar complejas estructuras de datos concurrentes. Un aspecto novedoso de nuestro marco formal es que efectúa una clara diferenciación entre el análisis del flujo de control del programa y el análisis de los datos que se manejan. El flujo de control del programa se analiza utilizando reglas de prueba y técnicas de verificación deductivas especialmente diseñadas para lidiar con sistemas parametrizados. Comenzando a partir de un programa concurrente y la especificación de una propiedad temporal, nuestras técnicas deductivas son capaces de generar un conjunto finito de condiciones de verificación cuya validez implican la satisfacción de dicha especificación temporal por parte de cualquier sistema, sin importar el número de procesos que formen parte del sistema. Las condiciones de verificación generadas se corresponden con los datos manipulados. Estudiamos el diseño de procedimientos de decisión especializados capaces de lidiar con estas condiciones de verificación de manera completamente automática. Investigamos teorías decidibles capaces de describir propiedades de tipos de datos complejos que manipulan punteros, tales como implementaciones imperativas de pilas, colas, listas y skiplists. Para cada una de estas teorías presentamos un procedimiento de decisión y una implementación práctica construida sobre SMT solvers. Estos procedimientos de decisión son finalmente utilizados para verificar de manera automática las condiciones de verificación generadas por nuestras técnicas de verificación parametrizada. Para concluir, demostramos como utilizando nuestro marco formal es posible probar no solo propiedades de safety sino además de liveness en algunas versiones de protocolos de exclusión mutua y programas que manipulan estructuras de datos concurrentes. El enfoque que presentamos en este trabajo resulta ser muy general y puede ser aplicado para verificar un amplio rango de tipos de datos concurrentes similares. Abstract Concurrent data types are concurrent implementations of classical data abstractions, specifically designed to exploit the great deal of parallelism available in modern multiprocessor and multi-core architectures. The correct manipulation of concurrent data types is essential for the overall correctness of the software system built using them. A major difficulty in designing and verifying concurrent data types arises by the need to reason about any number of threads invoking the data type simultaneously, which requires considering parametrized systems. In this work we study the formal verification of temporal properties of parametrized concurrent systems, with a special focus on programs that manipulate concurrent data structures. The main difficulty to reason about concurrent parametrized systems comes from the combination of their inherently high concurrency and the manipulation of dynamic memory. This parametrized verification problem is very challenging, because it requires to reason about complex concurrent data structures being accessed and modified by threads which simultaneously manipulate the heap using unstructured synchronization methods. In this work, we present a formal framework based on deductive methods which is capable of dealing with the verification of safety and liveness properties of concurrent parametrized systems that manipulate complex data structures. Our framework includes special proof rules and techniques adapted for parametrized systems which work in collaboration with specialized decision procedures for complex data structures. A novel aspect of our framework is that it cleanly differentiates the analysis of the program control flow from the analysis of the data being manipulated. The program control flow is analyzed using deductive proof rules and verification techniques specifically designed for coping with parametrized systems. Starting from a concurrent program and a temporal specification, our techniques generate a finite collection of verification conditions whose validity entails the satisfaction of the temporal specification by any client system, in spite of the number of threads. The verification conditions correspond to the data manipulation. We study the design of specialized decision procedures to deal with these verification conditions fully automatically. We investigate decidable theories capable of describing rich properties of complex pointer based data types such as stacks, queues, lists and skiplists. For each of these theories we present a decision procedure, and its practical implementation on top of existing SMT solvers. These decision procedures are ultimately used for automatically verifying the verification conditions generated by our specialized parametrized verification techniques. Finally, we show how using our framework it is possible to prove not only safety but also liveness properties of concurrent versions of some mutual exclusion protocols and programs that manipulate concurrent data structures. The approach we present in this work is very general, and can be applied to verify a wide range of similar concurrent data types.

Determination of the species generated in atmospheric-pressure laser-induced plasmas by mass spectometry techniques

We present temporal information obtained by mass spectrometry techniques about the evolution of plasmas generated by laser filamentation in air. The experimental setup used in this work allowed us to study not only the dynamics of the filament core but also of the energy reservoir that surrounds it. Furthermore, valuable insights about the chemistry of such systems like the photofragmentation and/or formation of molecules were obtained. The interpretation of the experimental results are supported by PIC simulations.

Spatio-temporal distribution of pyrethroids in soil in Mediterranean paddy fields

Purpose The demand of rice by the increase in population in many countries has intensified the application of pesticides and the use of poor quality water to irrigate fields. The terrestrial environment is one compartment affected by these situations, where soil is working as a reservoir, retaining organic pollutants. Therefore, it is necessary to develop methods to determine insecticides in soil and monitor susceptible areas to be contaminated, applying adequate techniques to remediate them. Materials and methods This study investigates the occurrence of ten pyrethroid insecticides (PYs) and its spatio-temporal variance in soil at two different depths collected in two periods (before plow and during rice production), in a paddy field area located in the Mediterranean coast. Pyrethroids were quantified using gas chromatography?mass spectrometry (GC?MS) after ultrasound-assisted extraction with ethyl acetate. The results obtained were assessed statistically using non-parametric methods, and significant statistical differences (p < 0.05) in pyrethroids content with soil depth and proximity to wastewater treatment plants were evaluated. Moreover, a geographic information system (GIS) was used to monitor the occurrence of PYs in paddy fields and detect risk areas. Results and discussion Pyrethroids were detected at concentrations ?57.0 ng g?1 before plow and ?62.3 ng g?1 during rice production, being resmethrin and cyfluthrin the compounds found at higher concentrations in soil. Pyrethroids were detected mainly at the top soil, and a GIS program was used to depict the obtained results, showing that effluents from wastewater treatment plants (WWTPs) were the main sources of soil contamination. No toxic effects were expected to soil organisms, but it is of concern that PYs may affect aquatic organisms, which represents the worst case scenario. Conclusions A methodology to determine pyrethroids in soil was developed to monitor a paddy field area. The use of water fromWWTPs to irrigate rice fields is one of the main pollution sources of pyrethroids. It is a matter of concern that PYs may present toxic effects on aquatic organisms, as they can be desorbed from soil. Phytoremediation may play an important role in this area, reducing the possible risk associated to PYs levels in soil.

Visualization of α9 acetylcholine receptor expression in hair cells of transgenic mice containing a modified bacterial artificial chromosome

The α9 acetylcholine receptor (α9 AChR) is specifically expressed in hair cells of the inner ear and is believed to be involved in synaptic transmission between efferent nerves and hair cells. Using a recently developed method, we modified a bacterial artificial chromosome containing the mouse α9 AChR gene with a reporter gene encoding green fluorescent protein (GFP) to generate transgenic mice. GFP expression in transgenic mice recapitulated the known temporal and spatial expression of α9 AChR. However, we observed previously unidentified dynamic changes in α9 AChR expression in cochlear and vestibular sensory epithelia during neonatal development. In the cochlea, inner hair cells persistently expressed high levels of α9 AChR in both the apical and middle turns, whereas both outer and inner hair cells displayed dynamic changes of α9 AChR expression in the basal turn. In the utricle, we observed high levels of α9 AChR expression in the striolar region during early neonatal development and high levels of α9 AChR in the extrastriolar region in adult mice. Further, simultaneous visualization of efferent innervation and α9 AChR expression showed that dynamic expression of α9 AChR in developing hair cells was independent of efferent contacts. We propose that α9 AChR expression in developing auditory and vestibular sensory epithelia correlates with maturation of hair cells and is hair-cell autonomous.

Temporal dynamics of verbal object comprehension

Knowledge of the stage composition and the temporal dynamics of human cognitive operations is critical for building theories of higher mental activity. This information has been difficult to acquire, even with different combinations of techniques such as refined behavioral testing, electrical recording/interference, and metabolic imaging studies. Verbal object comprehension was studied herein in a single individual, by using three tasks (object naming, auditory word comprehension, and visual word comprehension), two languages (English and Farsi), and four techniques (stimulus manipulation, direct cortical electrical interference, electrocorticography, and a variation of the technique of direct cortical electrical interference to produce time-delimited effects, called timeslicing), in a subject in whom indwelling subdural electrode arrays had been placed for clinical purposes. Electrical interference at a pair of electrodes on the left lateral occipitotemporal gyrus interfered with naming in both languages and with comprehension in the language tested (English). The naming and comprehension deficit resulted from interference with processing of verbal object meaning. Electrocorticography indices of cortical activation at this site during naming started 250–300 msec after visual stimulus presentation. By using the timeslicing technique, which varies the onset of electrical interference relative to the behavioral task, we found that completion of processing for verbal object meaning varied from 450 to 750 msec after current onset. This variability was found to be a function of the subject’s familiarity with the objects.