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.

An Atlas for the future National Park: Las Cumbres de la Sierra de Guadarrama

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Construction d'un Atlas 3D numérique de la cornée humaine par recalage d'images

Nous proposons de construire un atlas numérique 3D contenant les caractéristiques moyennes et les variabilités de la morphologie d’un organe. Nos travaux seront appliqués particulièrement à la construction d'un atlas numérique 3D de la totalité de la cornée humaine incluant la surface antérieure et postérieure à partir des cartes topographiques fournies par le topographe Orbscan II. Nous procédons tout d'abord par normalisation de toute une population de cornées. Dans cette étape, nous nous sommes basés sur l'algorithme de recalage ICP (iterative closest point) pour aligner simultanément les surfaces antérieures et postérieures d'une population de cornée vers les surfaces antérieure et postérieure d'une cornée de référence. En effet, nous avons élaboré une variante de l'algorithme ICP adapté aux images (cartes) de cornées qui tient compte de changement d'échelle pendant le recalage et qui se base sur la recherche par voisinage via la distance euclidienne pour établir la correspondance entre les points. Après, nous avons procédé pour la construction de l'atlas cornéen par le calcul des moyennes des élévations de surfaces antérieures et postérieures recalées et leurs écarts-types associés. Une population de 100 cornées saines a été utilisée pour construire l'atlas cornéen normal. Pour visualiser l’atlas, on a eu recours à des cartes topographiques couleurs similairement à ce qu’offrent déjà les systèmes topographiques actuels. Enfin, des observations ont été réalisées sur l'atlas cornéen reflétant sa précision et permettant de développer une meilleure connaissance de l’anatomie cornéenne.

The Namib Sand Sea digital database of aeolian dunes and key forcing variables

A new digital atlas of the geomorphology of the Namib Sand Sea in southern Africa has been developed. This atlas incorporates a number of databases including a digital elevation model (ASTER and SRTM) and other remote sensing databases that cover climate (ERA-40) and vegetation (PAL and GIMMS). A map of dune types in the Namib Sand Sea has been derived from Landsat and CNES/SPOT imagery. The atlas also includes a collation of geochronometric dates, largely derived from luminescence techniques, and a bibliographic survey of the research literature on the geomorphology of the Namib dune system. Together these databases provide valuable information that can be used as a starting point for tackling important questions about the development of the Namib and other sand seas in the past, present and future.

O uso das tecnologias da informação e comunicação no ensino de geografia: aplicação da página protótipo Desenvolvimento Urbano do atlas municipal escolar de Ourinhos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A Three-Dimensional Stereotaxic MRI Brain Atlas of the Cichlid Fish Oreochromis mossambicus

The African cichlid Oreochromis mossambicus (Mozambique tilapia) has been used as a model system in a wide range of behavioural and neurobiological studies. The increasing number of genetic tools available for this species, together with the emerging interest in its use for neurobiological studies, increased the need for an accurate hodological mapping of the tilapia brain to supplement the available histological data. The goal of our study was to elaborate a three-dimensional, high-resolution digital atlas using magnetic resonance imaging, supported by Nissl staining. Resulting images were viewed and analysed in all orientations (transverse, sagittal, and horizontal) and manually labelled to reveal structures in the olfactory bulb, telencephalon, diencephalon, optic tectum, and cerebellum. This high resolution tilapia brain atlas is expected to become a very useful tool for neuroscientists using this fish model and will certainly expand their use in future studies regarding the central nervous system.

METHODOLOGY FOR AUTOMATICALLY DELIMITING PERMANENT PRESERVATION AREAS ALONG WATER COURSES - THE USE OF GIS IN THE HYDROLOGICAL BASIN OF THE SERGIPE RIVER, BRAZIL

ABSTRACT Permanent Preservation Areas (PPAs) along watercourses have been the focus of numerous studies, not only because of the fragility and ecological relevance of riverine vegetation, but also because of the inefficiency demonstrated in conforming to the legislation protecting it. One of the major difficulties encountered in terms of guaranteeing the effective conservation of these riverside areas is the absence of methodologies that can be used to define them rapidly and accurately without manually determining the widths of the rivers or assigning only uniform linear values for the entire watercourse. The present work sought to develop a spatial analysis methodology capable of automatically defining permanent preservation areas along watercourses using geographic information system (GIS) software. The present study was undertaken in the Sergipe River basin, "considering the river itself and its principal affluents. We used the database of the Digital Atlas of Hydrological Resources (SEMARH/SE), and the delimitations of the PPAs were performed using ArcGIS 10.1 and the XToolPro 9.0 extension. A total of 5,003.82 hectares of Permanent Preservation Areas were delimited along the margins of the rivers analyzed, with a margin of error of <1% in delimiting the widths of the rivers within the entire area considered. The methodology described here can be used to define PPAs efficiently, relatively rapidly, and with very small margins of error, thus representing a technological advance in terms of using GIS for land management.

Analyse de maillages surfaciques par construction et comparaison de modèles moyens et par décomposition par graphes s’appuyant sur les courbures discrètes : application à l’étude de la cornée humaine

Réalisé en cotutelle avec Aix Marseille Université.

AWI Bathymetric Chart of the Weddell Sea, Antarctica (BCWS)

From data collected by RV Polarstern, and additional echosoundings provided by national hydrographic offices, research institutions and the International Hydrographic Organization (IHO) Digital Bathymetric Data Center, the 1:1,000,000 Bathymetric Chart of the Weddell Sea (AWl BCWS) series has been developed. The heterogeneity of bathymetric data and the lack of observations within ice-covered areas required the incorporation of supplementary geophysical and geographical information. A semi-automatic procedure was developed for terrain modeling and contouring. In coastal regions, adjacent sub-glacial information was included in order to model the bathymetry of the transition zone along the Antarctic ice edge. Six sheets of the AWl BCWS series in the scale of 1:1,000,000 covering the southern Weddell Sea from 66°S to 78°S and from 68°W to 0°E were recently completed and included in the 1997 GEneral Bathymetric Chart of the Oceans (GEBCO) Digital Atlas CD-ROM (http://www.gebco.net). On the basis of these six 1:1,000,000 AWl BCWS sheets, a generalized 1:3,000,000-scale bathymetric chart was compiled for the entire southern Weddell Sea.

Atlante digitale della Periegesi di Pausania: metodologia, implementazione e sviluppi

La tesi consiste nella descrizione del complessivo background storico-letterario, archeologico e digitale necessario per la realizzazione di un Atlante digitale dell’antica Grecia antica sulla base della raccolta e analisi dei dati e delle informazioni contenute nella Periegesi di Pausania. Grazie all’impiego degli applicativi GIS, ed in particolare di ArcGIS online, è stato possibile creare un database georiferito contenente le informazioni e le descrizioni fornite dal testo; ogni identificazione di un sito storico è stata inoltre confrontata con lo stato attuale della ricerca archeologica, al fine di produrre uno strumento innovativo tanto per a ricerca storico-archeologica quanto per lo studio e la valutazione dell’opera di Pausania. Nello specifico il lavoro consiste in primo esempio di atlante digitale interamente basato sull’interpretazione di un testo classico attraverso un processo di georeferenziazione dei suoi contenuti. Per ogni sito identificato è stata infatti specificato il relativo passo di Pausania, collegando direttamente Il dato archeologico con la fonte letteraria. Per la definizione di una tassonomia efficace per l’analisi dei contenuti dell’opera o, si è scelto di associare agli elementi descritti da Pausania sette livelli (layers) all’interno della mappa corrispondenti ad altrettante categorie generali (città, santuari extraurbani, monumenti, boschi sacri, località, corsi d’acqua, e monti). Per ciascun elemento sono state poi inserite ulteriori informazioni all’interno di una tabella descrittiva, quali: fonte, identificazione, età di appartenenza, e stato dell’identificazione.

A digital framework to build, visualize and analyze a gene expression atlas with cellular resolution in zebrafish early embryogenesis

A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, using multicolor fluorescence in situ hybridization with nuclear counterstain, requires dedicated computational strategies. To this goal, we designed an original methodological framework implemented in a software tool called Match-IT. With only minimal human supervision, our system is able to gather gene expression patterns observed in different analyzed embryos with phenotypic variability and map them onto a series of common 3D templates over time, creating a 4D atlas. This framework was used to construct an atlas composed of 6 gene expression templates from a cohort of zebrafish early embryos spanning 6 developmental stages from 4 to 6.3 hpf (hours post fertilization). They included 53 specimens, 181,415 detected cell nuclei and the segmentation of 98 gene expression patterns observed in 3D for 9 different genes. In addition, an interactive visualization software, Atlas-IT, was developed to inspect, supervise and analyze the atlas. Match-IT and Atlas-IT, including user manuals, representative datasets and video tutorials, are publicly and freely available online. We also propose computational methods and tools for the quantitative assessment of the gene expression templates at the cellular scale, with the identification, visualization and analysis of coexpression patterns, synexpression groups and their dynamics through developmental stages.

Sentidos e significados da participação democrática através da Internet: uma análise da experiência do Orçamento Participativo Digital

Este trabalho analisa três edições (2006, 2008 e 2011) do Orçamento Participativo Digital (OPD) de Belo Horizonte (MG). Objetiva deslindar os sentidos e significados da participação democrática mediada pela internet nessa experiência. Utiliza-se como método o estudo de caso único, de natureza qualitativa. Os dados foram coletados por meio de observação direta nos sítios do OPD e análise documental. Durante a fase de observação direta foram coletadas mensagens e manifestações dos cidadãos que estavam participando do processo. Tais dados foram tratados com o software Atlas TI 6.0®. Conclui-se que tanto o sentido quanto o significado democrático dessa experiência de participação mediadas pela internet são modificados ao longo das edições, indo de grandes impulsos de esperança democrática até a descrença nessa modalidade de participação digital.