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.

Instrumentación de referencia para realización automática de ensayos de fiabilidad

En todo proceso de desarrollo de un dispositivo electrónico o equipo cabe la necesidad de evaluar la fiabilidad de sus componentes, es decir, cual es el porcentaje de equipos que tras un determinado periodo de vida mantiene todas sus funcionalidades dentro de especificaciones. La evaluación de la fiabilidad mediante ensayos acelerados es la herramienta que permite una estimación de la vida del dispositivo o equipo de forma previa a su comercialización. La cuantificación de la fiabilidad es crítica para identificar los costos de un determinado periodo de garantía, y para ofrecer a los clientes el nivel de calidad deseado. El objetivo de este Proyecto Fin de Carrera, es el diseño de un sistema automático de instrumentación versátil, para la realización y caracterización de ensayos acelerados, el cual nos sirva para abordar una amplia gama de ensayos con los que evaluar la fiabilidad de los dispositivos electrónicos o equipos. Además del uso industrial donde se evaluará la fiabilidad de forma previa a la comercialización, este sistema se podrá emplear en la docencia de esta área, y fundamentalmente para la realización de ensayos acelerados en investigación de dispositivos electrónicos. La versatilidad de nuestro hardware y aplicación software es un punto a favor, ya que con este sistema de instrumentación se pueden realizar numerosos tipos de ensayos acelerados, sin el problema de tener que cambiar toda la instrumentación, cada vez que se quiera realizar otro ensayo distinto. Los componentes que se elijan para realizar el ensayo acelerado, serán sometidos a un estrés (tensión, corriente, humedad, temperatura…) y se podrá ir observando cómo envejecen, lo que nos permite evaluar la vida del dispositivo en un corto periodo, emulando sus condiciones de trabajo, además de estudiar la fiabilidad también se puede identificar como se degradan sus características principales antes del fallo. El Software utilizado en este Proyecto se ha implementado con un lenguaje de programación gráfico para instrumentación, LabVIEW. La aplicación software se explica de manera muy detallada a lo largo de la memoria, para que su uso y adaptación si fuese necesario no suponga ningún problema para el usuario. En la última parte de esta memoria se encuentra la guía de usuario y un ensayo acelerado planteado como ejemplo. Explicaremos como se han interconectado los equipos a los componentes en los que se va a realizar el ensayo y así se comprobará el correcto funcionamiento del software tomando las medidas necesarias. ABTRACT In all process of development of an electronic device or equipment, we have the need to evaluate the reliability of its components, that is to say, what percentage of equipment that after a certain period of life keeps all of its functionalities within specifications. The evaluation of reliability by means of accelerated tests is the tool that allows an estimation of the lifetime of the device or equipment prior to its marketing. The quantification of reliability is critical to identify the costs of a specific warranty period, and to offer customers the desired quality level. The objective of this Thesis is the design of an automatic very versatile instrument for the realization and characterization of accelerated tests, which will help us to address a wide range of tests to assess the reliability of the devices or electronic equipment. In addition to industrial use where test the reliability before its commercialization, use it can be used in teaching of this area, fundamentally for the realization of accelerated testing in the investigation of electronic devices. The versatility of our hardware and software implementation is a plus, given that this instrumentation system can perform numerous types of accelerated tests, without the problem to have to change everything, every time you want to make another different test. The components that will be chosen to perform the accelerated test, will be subjected to stress (voltage, current, humidity, temperature ...) and you can observe how they age, allowing us to evaluate the life of the device in a short period, emulating their working conditions. In addition to studying the reliability it can also identify how its main characteristics are degraded before failure. The software used in this Thesis has been implemented with a graphical programming language for instrumentation, LabVIEW. This software is explained in great detail throughout the Thesis, so that its use and adaptation, if necessary, will not be a problem for the user. In the last part of this memory we will expose a user guide and test that we have done. We will explain how the equipment has been interconnected to the components in which we are going to perform the test and so we will check the correct operation of the software taking the necessary measures.

Securing implementations of feedback-shift-register-based ciphers using compiler optimizations and co-processors

Los algoritmos basados en registros de desplazamiento con realimentación (en inglés FSR) se han utilizado como generadores de flujos pseudoaleatorios en aplicaciones con recursos limitados como los sistemas de apertura sin llave. Se considera canal primario a aquel que se utiliza para realizar una transmisión de información. La aparición de los ataques de canal auxiliar (en inglés SCA), que explotan información filtrada inintencionadamente a través de canales laterales como el consumo, las emisiones electromagnéticas o el tiempo empleado, supone una grave amenaza para estas aplicaciones, dado que los dispositivos son accesibles por un atacante. El objetivo de esta tesis es proporcionar un conjunto de protecciones que se puedan aplicar de forma automática y que utilicen recursos ya disponibles, evitando un incremento sustancial en los costes y alargando la vida útil de aplicaciones que puedan estar desplegadas. Explotamos el paralelismo existente en algoritmos FSR, ya que sólo hay 1 bit de diferencia entre estados de rondas consecutivas. Realizamos aportaciones en tres niveles: a nivel de sistema, utilizando un coprocesador reconfigurable, a través del compilador y a nivel de bit, aprovechando los recursos disponibles en el procesador. Proponemos un marco de trabajo que nos permite evaluar implementaciones de un algoritmo incluyendo los efectos introducidos por el compilador considerando que el atacante es experto. En el campo de los ataques, hemos propuesto un nuevo ataque diferencial que se adapta mejor a las condiciones de las implementaciones software de FSR, en las que el consumo entre rondas es muy similar. SORU2 es un co-procesador vectorial reconfigurable propuesto para reducir el consumo energético en aplicaciones con paralelismo y basadas en el uso de bucles. Proponemos el uso de SORU2, además, para ejecutar algoritmos basados en FSR de forma segura. Al ser reconfigurable, no supone un sobrecoste en recursos, ya que no está dedicado en exclusiva al algoritmo de cifrado. Proponemos una configuración que ejecuta múltiples algoritmos de cifrado similares de forma simultánea, con distintas implementaciones y claves. A partir de una implementación sin protecciones, que demostramos que es completamente vulnerable ante SCA, obtenemos una implementación segura a los ataques que hemos realizado. A nivel de compilador, proponemos un mecanismo para evaluar los efectos de las secuencias de optimización del compilador sobre una implementación. El número de posibles secuencias de optimizaciones de compilador es extremadamente alto. El marco de trabajo propuesto incluye un algoritmo para la selección de las secuencias de optimización a considerar. Debido a que las optimizaciones del compilador transforman las implementaciones, se pueden generar automáticamente implementaciones diferentes combinamos para incrementar la seguridad ante SCA. Proponemos 2 mecanismos de aplicación de estas contramedidas, que aumentan la seguridad de la implementación original sin poder considerarse seguras. Finalmente hemos propuesto la ejecución paralela a nivel de bit del algoritmo en un procesador. Utilizamos la forma algebraica normal del algoritmo, que automáticamente se paraleliza. La implementación sobre el algoritmo evaluado mejora en rendimiento y evita que se filtre información por una ejecución dependiente de datos. Sin embargo, es más vulnerable ante ataques diferenciales que la implementación original. Proponemos una modificación del algoritmo para obtener una implementación segura, descartando parcialmente ejecuciones del algoritmo, de forma aleatoria. Esta implementación no introduce una sobrecarga en rendimiento comparada con las implementaciones originales. En definitiva, hemos propuesto varios mecanismos originales a distintos niveles para introducir aleatoridad en implementaciones de algoritmos FSR sin incrementar sustancialmente los recursos necesarios. ABSTRACT Feedback Shift Registers (FSR) have been traditionally used to implement pseudorandom sequence generators. These generators are used in Stream ciphers in systems with tight resource constraints, such as Remote Keyless Entry. When communicating electronic devices, the primary channel is the one used to transmit the information. Side-Channel Attack (SCA) use additional information leaking from the actual implementation, including power consumption, electromagnetic emissions or timing information. Side-Channel Attacks (SCA) are a serious threat to FSR-based applications, as an attacker usually has physical access to the devices. The main objective of this Ph.D. thesis is to provide a set of countermeasures that can be applied automatically using the available resources, avoiding a significant cost overhead and extending the useful life of deployed systems. If possible, we propose to take advantage of the inherent parallelism of FSR-based algorithms, as the state of a FSR differs from previous values only in 1-bit. We have contributed in three different levels: architecture (using a reconfigurable co-processor), using compiler optimizations, and at bit level, making the most of the resources available at the processor. We have developed a framework to evaluate implementations of an algorithm including the effects introduced by the compiler. We consider the presence of an expert attacker with great knowledge on the application and the device. Regarding SCA, we have presented a new differential SCA that performs better than traditional SCA on software FSR-based algorithms, where the leaked values are similar between rounds. SORU2 is a reconfigurable vector co-processor. It has been developed to reduce energy consumption in loop-based applications with parallelism. In addition, we propose its use for secure implementations of FSR-based algorithms. The cost overhead is discarded as the co-processor is not exclusively dedicated to the encryption algorithm. We present a co-processor configuration that executes multiple simultaneous encryptions, using different implementations and keys. From a basic implementation, which is proved to be vulnerable to SCA, we obtain an implementation where the SCA applied were unsuccessful. At compiler level, we use the framework to evaluate the effect of sequences of compiler optimization passes on a software implementation. There are many optimization passes available. The optimization sequences are combinations of the available passes. The amount of sequences is extremely high. The framework includes an algorithm for the selection of interesting sequences that require detailed evaluation. As existing compiler optimizations transform the software implementation, using different optimization sequences we can automatically generate different implementations. We propose to randomly switch between the generated implementations to increase the resistance against SCA.We propose two countermeasures. The results show that, although they increase the resistance against SCA, the resulting implementations are not secure. At bit level, we propose to exploit bit level parallelism of FSR-based implementations using pseudo bitslice implementation in a wireless node processor. The bitslice implementation is automatically obtained from the Algebraic Normal Form of the algorithm. The results show a performance improvement, avoiding timing information leakage, but increasing the vulnerability against differential SCA.We provide a secure version of the algorithm by randomly discarding part of the data obtained. The overhead in performance is negligible when compared to the original implementations. To summarize, we have proposed a set of original countermeasures at different levels that introduce randomness in FSR-based algorithms avoiding a heavy overhead on the resources required.

Scientific bases of human-machine communication by voice.

The scientific bases for human-machine communication by voice are in the fields of psychology, linguistics, acoustics, signal processing, computer science, and integrated circuit technology. The purpose of this paper is to highlight the basic scientific and technological issues in human-machine communication by voice and to point out areas of future research opportunity. The discussion is organized around the following major issues in implementing human-machine voice communication systems: (i) hardware/software implementation of the system, (ii) speech synthesis for voice output, (iii) speech recognition and understanding for voice input, and (iv) usability factors related to how humans interact with machines.

Critical feature analysis of a radiotherapy machine

The software implementation of the emergency shutdown feature in a major radiotherapy system was analyzed, using a directed form of code review based on module dependences. Dependences between modules are labelled by particular assumptions; this allows one to trace through the code, and identify those fragments responsible for critical features. An `assumption tree' is constructed in parallel, showing the assumptions which each module makes about others. The root of the assumption tree is the critical feature of interest, and its leaves represent assumptions which, if not valid, might cause the critical feature to fail. The analysis revealed some unexpected assumptions that motivated improvements to the code.

Projected sequential Gaussian processes: flexible interpolation for large data sets

Recently within the machine learning and spatial statistics communities many papers have explored the potential of reduced rank representations of the covariance matrix, often referred to as projected or fixed rank approaches. In such methods the covariance function of the posterior process is represented by a reduced rank approximation which is chosen such that there is minimal information loss. In this paper a sequential framework for inference in such projected processes is presented, where the observations are considered one at a time. We introduce a C++ library for carrying out such projected, sequential estimation which adds several novel features. In particular we have incorporated the ability to use a generic observation operator, or sensor model, to permit data fusion. We can also cope with a range of observation error characteristics, including non-Gaussian observation errors. Inference for the variogram parameters is based on maximum likelihood estimation. We illustrate the projected sequential method in application to synthetic and real data sets. We discuss the software implementation and suggest possible future extensions.

On The Critical Points of some Iteration Methods for Solving Algebraic Equations. Global Convergence Properties

In this work we give su±cient conditions for k-th approximations of the polynomial roots of f(x) when the Maehly{Aberth{Ehrlich, Werner-Borsch-Supan, Tanabe, Improved Borsch-Supan iteration methods fail on the next step. For these methods all non-attractive sets are found. This is a subsequent improvement of previously developed techniques and known facts. The users of these methods can use the results presented here for software implementation in Distributed Applications and Simulation Environ- ments. Numerical examples with graphics are shown.

Business Modeling of the Application Architecture of the Bulgarian Folklore Artery

In an attempt to answer the need of wider accessibility and popularization of the treasury of Bulgarian folklore, a team from the Institute of Mathematics and Informatics at the Bulgarian Academy of Sciences has planned to develop the Bulgarian folklore artery within the national project ―Knowledge Technologies for Creation of Digital Presentation and Significant Repositories of Folklore Heritage‖. This paper presents the process of business modeling of the application architecture of the Bulgarian folklore artery, which aids requirements analysis, application design and its software implementation. The folklore domain process model is made in the context of the target social applications—e-learning, virtual expositions of folklore artifacts, research, news, cultural/ethno-tourism, etc. The basic processes are analyzed and modeled and some inferences are made for the use cases and requirements specification of the Bulgarian folklore artery application. As a conclusion the application architecture of the Bulgarian folklore artery is presented.

Online Access to the Encyclopaedia Slavica Sanctorum

Encyclopaedia Slavica Sanctorum project aims at building a repertoire of medieval and early modern Bulgarian texts for saints in combination with ethnological data and some visual sources. A basic project task is to produce an accessible on-line digital repository of this valuable cultural heritage treasure. The paper presents the Encyclopaedia Slavica Sanctorum environment, its architecture, functional specification, application modeling process and software implementation. The paper also discusses the specifics of the ―Encyclopaedia Slavica Sanctorum‖ project and its knowledge domain. The paper also presents the integration between the Encyclopaedia Slavica Sanctorum and the Bulgarian Iconographical Digital Library, a digital library keeping rare specimens, private collections of Orthodox icons, wall- paintings and other iconographical objects, selected from difficult-to-access storages, distant churches, chapels, and monasteries, objects in a risk environment or unstable conditions.

A Refinement of some Overrelaxation Algorithms for Solving a System of Linear Equations

In this paper we propose a refinement of some successive overrelaxation methods based on the reverse Gauss–Seidel method for solving a system of linear equations Ax = b by the decomposition A = Tm − Em − Fm, where Tm is a banded matrix of bandwidth 2m + 1. We study the convergence of the methods and give software implementation of algorithms in Mathematica package with numerical examples. ACM Computing Classification System (1998): G.1.3.

Manageable Workflows for Processing Parallel Sequencing Data

ACM Computing Classification System (1998): D.2.11, D.1.3, D.3.1, J.3, C.2.4.

Field programmable gate array based target detection and gesture recognition

The move from Standard Definition (SD) to High Definition (HD) represents a six times increases in data, which needs to be processed. With expanding resolutions and evolving compression, there is a need for high performance with flexible architectures to allow for quick upgrade ability. The technology advances in image display resolutions, advanced compression techniques, and video intelligence. Software implementation of these systems can attain accuracy with tradeoffs among processing performance (to achieve specified frame rates, working on large image data sets), power and cost constraints. There is a need for new architectures to be in pace with the fast innovations in video and imaging. It contains dedicated hardware implementation of the pixel and frame rate processes on Field Programmable Gate Array (FPGA) to achieve the real-time performance. ^ The following outlines the contributions of the dissertation. (1) We develop a target detection system by applying a novel running average mean threshold (RAMT) approach to globalize the threshold required for background subtraction. This approach adapts the threshold automatically to different environments (indoor and outdoor) and different targets (humans and vehicles). For low power consumption and better performance, we design the complete system on FPGA. (2) We introduce a safe distance factor and develop an algorithm for occlusion occurrence detection during target tracking. A novel mean-threshold is calculated by motion-position analysis. (3) A new strategy for gesture recognition is developed using Combinational Neural Networks (CNN) based on a tree structure. Analysis of the method is done on American Sign Language (ASL) gestures. We introduce novel point of interests approach to reduce the feature vector size and gradient threshold approach for accurate classification. (4) We design a gesture recognition system using a hardware/ software co-simulation neural network for high speed and low memory storage requirements provided by the FPGA. We develop an innovative maximum distant algorithm which uses only 0.39% of the image as the feature vector to train and test the system design. Database set gestures involved in different applications may vary. Therefore, it is highly essential to keep the feature vector as low as possible while maintaining the same accuracy and performance^

Application of multivariable control using artificial neural networks in a debutanizer distillation column

LOPES, Jose Soares Batista et al. Application of multivariable control using artificial neural networks in a debutanizer distillation column.In: INTERNATIONAL CONGRESS OF MECHANICAL ENGINEERING - COBEM, 19, 5-9 nov. 2007, Brasilia. Anais... Brasilia, 2007

Application of multivariable control using artificial neural networks in a debutanizer distillation column

LOPES, Jose Soares Batista et al. Application of multivariable control using artificial neural networks in a debutanizer distillation column.In: INTERNATIONAL CONGRESS OF MECHANICAL ENGINEERING - COBEM, 19, 5-9 nov. 2007, Brasilia. Anais... Brasilia, 2007

A robotic platform for precision agriculture and applications

Agricultural techniques have been improved over the centuries to match with the growing demand of an increase in global population. Farming applications are facing new challenges to satisfy global needs and the recent technology advancements in terms of robotic platforms can be exploited. As the orchard management is one of the most challenging applications because of its tree structure and the required interaction with the environment, it was targeted also by the University of Bologna research group to provide a customized solution addressing new concept for agricultural vehicles. The result of this research has blossomed into a new lightweight tracked vehicle capable of performing autonomous navigation both in the open-filed scenario and while travelling inside orchards for what has been called in-row navigation. The mechanical design concept, together with customized software implementation has been detailed to highlight the strengths of the platform and some further improvements envisioned to improve the overall performances. Static stability testing has proved that the vehicle can withstand steep slopes scenarios. Some improvements have also been investigated to refine the estimation of the slippage that occurs during turning maneuvers and that is typical of skid-steering tracked vehicles. The software architecture has been implemented using the Robot Operating System (ROS) framework, so to exploit community available packages related to common and basic functions, such as sensor interfaces, while allowing dedicated custom implementation of the navigation algorithm developed. Real-world testing inside the university’s experimental orchards have proven the robustness and stability of the solution with more than 800 hours of fieldwork. The vehicle has also enabled a wide range of autonomous tasks such as spraying, mowing, and on-the-field data collection capabilities. The latter can be exploited to automatically estimate relevant orchard properties such as fruit counting and sizing, canopy properties estimation, and autonomous fruit harvesting with post-harvesting estimations.