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.

Multiplexing QKD systems in Conventional Optical Networks

Current QKD designs try to keep the quantum channel as error free as possible by using a separate physical medium for this purpose. In the most common case, this means the exclusive use of an optical fiber for the quantum channel, precluding its use for any other purpose. In current optical networks, the fiber is the single most expensive element and this poses a major problem from a cost and availability point of view. Sharing the fiber is thus mandatory for the widespread adoption of QKD. The objective of this communication is to propose a general scheme and present some preliminary measurements of a metropolitan area network (MAN) designed to multiplex of the order of 64 addressable quantum channels and the associated QKD classical service signals on a single dark fibre. It uses as much existing components and infraestructure as possible in an attempt to simultaneously lower most of the practical barriers for the adoption of QKD.

Whole-genome genotyping of grape using a panel of microsatellite

The use of microsatellite markers in large-scale genetic studies is limited by its low throughput and high cost and labor requirements. Here, we provide a panel of 45 multiplex PCRs for fast and cost-efficient genome-wide fluorescence-based microsatellite analysis in grapevine. The developed multiplex PCRs panel (with up to 15-plex) enables the scoring of 270 loci covering all the grapevine genome (9 to 20 loci/chromosome) using only 45 PCRs and sequencer runs. The 45 multiplex PCRs were validated using a diverse grapevine collection of 207 accessions, selected to represent most of the cultivated Vitis vinifera genetic diversity. Particular attention was paid to quality control throughout the whole process (assay replication, null allele detection, ease of scoring). Genetic diversity summary statistics and features of electrophoretic profiles for each studied marker are provided, as are the genotypes of 25 common cultivars that could be used as references in other studies.

Estudio acústico de la sala 8 de los cines Kinépolis de Ciudad de la Imagen

Este proyecto consistira en la realization de un estudio aciistico sobre la sala 8 de los cines Kinepolis de Ciudad de la Imagen, que dispone de 408 butacas. Los cines Kinepolis es uno de los mayores complejos multisala de Europa. Cuenta con mas de 9.200 butacas en total distribuidas en 25 salas a las que se accede mediante dos grandes pasillos conectados por el hall. En 1998, ano de su apertura, el complejo recibio el Record Guinness a la sala cinematografica mas grande del mundo, que dispone de 996 butacas. El objetivo de este proyecto es conseguir caracterizar acusticamente una sala de cine a traves de la medicion de parametros acusticos de la sala y de un modelo virtual de la misma. Para llevar a cabo el proyecto, primero se van a realizar tanto una medicion geometrica como acustica de la sala mediante el sistema de medida DIRAC. Los resultados de estas mediciones nos serviran para construir y validar un modelo virtual de la sala real con el software de simulacion EASE. La medicion acustica se va a realizar con el sistema de medicion DIRAC. Este sistema nos dara information sobre una amplia variedad de parametros acusticos. En este proyecto no se va a trabajar con todos ellos, solo con los mas significativos. Estos se describen a continuacion en la introduccion teorica. La medicion geometrica nos va a servir para construir un modelo virtual que tenga las mismas dimensiones que la sala original. Esta medicion la realizaremos mediante un medidor laser y una cinta metrica. Una vez construido el modelo virtual, se procedera a su validacion. Este proceso se realiza ajustando el tiempo de reverberacion del modelo mediante la introduccion de distintos materiales acusticos en las superficies del mismo, de manera que, variando la absorcion de la sala, el tiempo de reverberacion promedio del modelo se asemeje lo mas posible al medido en la sala real. Este proceso tiene como objetivo comprobar que el modelo virtual tiene un comportamiento acustico similar al de la sala real. Es necesario validar adecuadamente el modelo para que las comparaciones y conclusiones sean fiables. Por ultimo, tras la simulacion acustica del modelo, se compararan los resultados simulados con los medidos en la sala. En este proceso se contrastaran algunos de los parametros que guardan relation con el tiempo de reverberacion. De esta manera se verificara si el tiempo de reverberacion es o no un parametro acustico fiable para la validacion de un modelo virtual de una sala de cine. Anteriormente se han realizado proyectos iguales de otras salas de diferente tamano de Kinepolis. El objetivo de realizar el mismo estudio en distintas salas, es comprobar si el tamano de la sala influye en la validacion de los modelos virtuales mediante el tiempo de reverberacion. ABSTRACT. This Project consists on the development of an acoustic research of the movie theater 8 of the Kinepolis complex in Ciudad de la Imagen, Madrid. This room has 408 spots. Kinepolis is one of the biggest multiplex complexes in Europe. It has 9,200 locations disposed in 25 rooms. There are two large corridors which give access to all of theaters. In the middle of the structure, there is the main hall that connects these corridors. In 1998, at the time when the complex was open, it was awarded with the Record Guinness for the biggest theater in the world, which has 996 locations. The target of this project is to successfully characterize the acoustics of a movie theater through reverberation time and a virtual model. In order to reach this goal, in the first place, we are going to perform both, an acoustic and a geometric measurement of the room using DIRAC measurement system. The results of these measures will allow us to build and validate a virtual model of the room, using the simulation software EASE. We are going to use the DIRAC system in order to accomplish the acoustic measure. This operation gives us a huge variety of acoustic parameters. Not all of these are going to be used for this research, only the most significant ones. These are described in the theoretical introduction. The geometric measure is essential to help us to build the virtual model, because the model has to be exactly equal as the real room. This measurement will be performed with an electronic distance meter and a measuring tape. Once the virtual model is finished, it will be proved. This validation process will be realized by adjusting the reverberation time in the model. We will change the walls materials, therefore, the overall absorption of the room will change. We want the model reverberation time resemble to the real one. This practice is going to ensure that the model acoustic performance is close to the real one. In addition, it has to be successfully validate of we want the future comparisons to be reliable. Finally, after the model virtual simulation, we will compare the simulated results with the measure in the room. In this process, we will compare not only the reverberation time, but others parameters that keep relation with the reverberation time. We will verify this way, if the reverberation time is or is not an appropriate acoustic parameter to validate a virtual model of a movie theater. There have been done others similar acoustic researches in different theaters with different sizes. The aim of performing similar researches in different rooms is to determine if the size of the room infers in the validation process.

Red SFN de distribución local de TDT

Este proyecto consiste en el diseño completo, de una red de distribución de TDT, a nivel local, mediante difusión SFN, Single Frequency Network. Este tipo de difusión, tiene la capacidad de difundir los servicios de televisión en una única frecuencia, cubriendo un área, ya sea local o estatal, aprovechando en las zonas de interferencia los rebotes de la señal y así evitar el uso de una frecuencia distinta por cada centro de emisión, todos los que componen un área de cobertura. Para el diseño de la red, se ha optado por diseñar una red IP, mediante distribución multicast, ya que esta es la tecnología imperante a día de hoy, quedando obsoleta ya, la distribución analógica, ya que consume muchos más recursos y por consiguiente mucho más costosa de implementar. El documento se divide en cuatro capítulos. En el primer capítulo se realizará una introducción teórica a las redes de distribución SFN, centrándose en el cálculo de los retardos, punto fundamental en el diseño de este tipo de redes. Se continuará unas nociones básicas de redes IP y el protocolo multicast, en el que se basa el trasporte de la señal. El capítulo dos, se centra en el diseño de la red, desde los centros de producción, donde se generan los programas a emitir, hasta los diferentes centros de difusión que cubrirán todo el área de cobertura requerida, pasando por el centro de multiplexación, donde se sitúa la cabecera que compondrá el múltiplex a difundir. Se describirán los equipos y el diseño de los distintos centros que conforman la red, centros de producción, multiplexación y difusión. A demás se realizará el cálculo de retardo de la señal, necesario en este tipo de redes. Se continuará con el capítulo tres, donde se describirá la configuración de la red, tanto a nivel de equipamiento, como el diseño y asignación IP de toda la red, separando la red de servicio de la red de gestión para una mayor confiabilidad y eficiencia de la red. Se finalizará con la descripción de la gestión de la red, que mediante diferentes herramientas, proporcionan un monitoreado en tiempo real de todo el sistema, dando la posibilidad de adelantarsey previniendo posibles incidencias que, puedan causar alguna deficiencia en el servicio que se entrega al usuario final. ABSTRACT. This project involves the complete design of a network´s TDT distribution, locally, by broadcast SFN (Single Frequency Network). This type of broadcast, has the ability to broadcast television´s services on a single frequency, covering an area, whether local or state, drawing on the interference zones, signal´s rebounds, to avoid the use of a different frequency each broadcast center, all those who make a coverage area. For the design of the network, has been chosen to design an IP network using multicast distribution, since this is the prevailing technology today, as the analogue distribution, consumes more resources and therefore, much more costly to implement. The document is divided into four chapters. In the first chapter you can find a theoretical introduction to SFN distribution networks, focusing on the calculation of delays, fundamental point, in the design of these networks. A basic understanding of IP networks and the multicast protocol, in which the transport of the signal is based, will continue. Chapter two focuses on the design of the network, from production centers, where the programs are created to broadcast, to different distribution centers covering the entire area of coverage required, through the multiplexing center, where the head is located, which comprise the multiplex. Also, the equipment and design of the various centers in the network, production centers, multiplexing center and distribution centers, are described. Furthermore, the calculation of signal delay, necessary in such networks, is performed. We will continue with the chapter three, where the network configuration will be described, both in termsofequipment, such as design IP mapping of the entire network, separating the service network and management network, for increased the reliability and efficiency of the network. It will be completed with the description of the management of the network, using different tools provide real-time monitoring of the entire system, making it possible, to anticipate and prevent any incidents that might cause a deficiency in the service being delivered to final user.

Is Microarray Analysis Really Useful and Sufficient to Diagnose Nut Allergy in the Mediterranean Area?

Background: Component-based diagnosis on multiplex platforms is widely used in food allergy but its clinical performance has not been evaluated in nut allergy. Objective: To assess the diagnostic performance of a commercial protein microarray in the determination of specific IgE (sIgE) in peanut, hazelnut, and walnut allergy. Methods: sIgE was measured in 36 peanut-allergic, 36 hazelnut-allergic, and 44 walnut-allergic patients by ISAC 112, and subsequently, sIgE against available components was determined by ImmunoCAP in patients with negative ISAC results. ImmunoCAP was also used to measure sIgE to Ara h 9, Cor a 8, and Jug r 3 in a subgroup of lipid transfer protein (LTP)-sensitized nut-allergic patients (positive skin prick test to LTP-enriched extract). sIgE levels by ImmunoCAP were compared with ISAC ranges. Results: Most peanut-, hazelnut-, and walnut-allergic patients were sensitized to the corresponding nut LTP (Ara h 9, 66.7%; Cor a 8, 80.5%; Jug r 3, 84% respectively). However, ISAC did not detect sIgE in 33.3% of peanut-allergic patients, 13.9% of hazelnut-allergic patients, or 13.6% of walnut-allergic patients. sIgE determination by ImmunoCAP detected sensitization to Ara h 9, Cor a 8, and Jug r 3 in, respectively, 61.5% of peanut-allergic patients, 60% of hazelnut-allergic patients, and 88.3% of walnut-allergic patients with negative ISAC results. In the subgroup of peach LTP?sensitized patients, Ara h 9 sIgE was detected in more cases by ImmunoCAP than by ISAC (94.4% vs 72.2%, P<.05). Similar rates of Cor a 8 and Jug r 3 sensitization were detected by both techniques. Conclusions: The diagnostic performance of ISAC was adequate for hazelnut and walnut allergy but not for peanut allergy. sIgE sensitivity against Ara h 9 in ISAC needs to be improved.

Whole genome analysis: Experimental access to all genome sequenced segments through larger-scale efficient oligonucleotide synthesis and PCR

The recent ability to sequence whole genomes allows ready access to all genetic material. The approaches outlined here allow automated analysis of sequence for the synthesis of optimal primers in an automated multiplex oligonucleotide synthesizer (AMOS). The efficiency is such that all ORFs for an organism can be amplified by PCR. The resulting amplicons can be used directly in the construction of DNA arrays or can be cloned for a large variety of functional analyses. These tools allow a replacement of single-gene analysis with a highly efficient whole-genome analysis.

Genome scan of human systemic lupus erythematosus: Evidence for linkage on chromosome 1q in African-American pedigrees

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by production of autoantibodies against intracellular antigens including DNA, ribosomal P, Ro (SS-A), La (SS-B), and the spliceosome. Etiology is suspected to involve genetic and environmental factors. Evidence of genetic involvement includes: associations with HLA-DR3, HLA-DR2, Fcγ receptors (FcγR) IIA and IIIA, and hereditary complement component deficiencies, as well as familial aggregation, monozygotic twin concordance >20%, λs > 10, purported linkage at 1q41–42, and inbred mouse strains that consistently develop lupus. We have completed a genome scan in 94 extended multiplex pedigrees by using model-based linkage analysis. Potential [log10 of the odds for linkage (lod) > 2.0] SLE loci have been identified at chromosomes 1q41, 1q23, and 11q14–23 in African-Americans; 14q11, 4p15, 11q25, 2q32, 19q13, 6q26–27, and 12p12–11 in European-Americans; and 1q23, 13q32, 20q13, and 1q31 in all pedigrees combined. An effect for the FcγRIIA candidate polymorphism) at 1q23 (lod = 3.37 in African-Americans) is syntenic with linkage in a murine model of lupus. Sib-pair and multipoint nonparametric analyses also support linkage (P < 0.05) at nine loci detected by using two-point lod score analysis (lod > 2.0). Our results are consistent with the presumed complexity of genetic susceptibility to SLE and illustrate racial origin is likely to influence the specific nature of these genetic effects.

A human model for multigenic inheritance: Phenotypic expression in Hirschsprung disease requires both the RET gene and a new 9q31 locus

Reduced penetrance in genetic disorders may be either dependent or independent of the genetic background of gene carriers. Hirschsprung disease (HSCR) demonstrates a complex pattern of inheritance with ≈50% of familial cases being heterozygous for mutations in the receptor tyrosine kinase RET. Even when identified, the penetrance of RET mutations is only 50–70%, gender-dependent, and varies with the extent of aganglionosis. We searched for additional susceptibility genes which, in conjunction with RET, lead to phenotypic expression by studying 12 multiplex HSCR families. Haplotype analysis and extensive mutation screening demonstrated three types of families: six families harboring severe RET mutations (group I); and the six remaining families, five of which are RET-linked families with no sequence alterations and one RET-unlinked family (group II). Although the presence of RET mutations in group I families is sufficient to explain HSCR inheritance, a genome scan reveals a new susceptibility locus on 9q31 exclusively in group II families. As such, the gene at 9q31 is a modifier of HSCR penetrance. These observations imply that identification of new susceptibility factors in a complex disease may depend on classification of families by mutational type at known susceptibility genes.

Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection

We describe an adaptation of the rolling circle amplification (RCA) reporter system for the detection of protein Ags, termed “immunoRCA.” In immunoRCA, an oligonucleotide primer is covalently attached to an Ab; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, amplification results in a long DNA molecule containing hundreds of copies of the circular DNA sequence that remain attached to the Ab and that can be detected in a variety of ways. Using immunoRCA, analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays in ELISA and microparticle formats. The signal amplification afforded by immunoRCA also enabled immunoassays to be carried out in microspot and microarray formats with exquisite sensitivity. When Ags are present at concentrations down to fM levels, specifically bound Abs can be scored by counting discrete fluorescent signals arising from individual Ag–Ab complexes. Multiplex immunoRCA also was demonstrated by accurately quantifying Ags mixed in different ratios in a two-color, single-molecule-counting assay on a glass slide. ImmunoRCA thus combines high sensitivity and a very wide dynamic range with an unprecedented capability for single molecule detection. This Ag-detection method is of general applicability and is extendable to multiplexed immunoassays that employ a battery of different Abs, each labeled with a unique oligonucleotide primer, that can be discriminated by a color-coded visualization system. ImmunoRCA-profiling based on the simultaneous quantitation of multiple Ags should expand the power of immunoassays by exploiting the increased information content of ratio-based expression analysis.

STRBase: a short tandem repeat DNA database for the human identity testing community

The National Institute of Standards and Technology (NIST) has compiled and maintained a Short Tandem Repeat DNA Internet Database (http://www.cstl.nist.gov/biotech/strbase/) since 1997 commonly referred to as STRBase. This database is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. STRBase consolidates and organizes the abundant literature on this subject to facilitate on-going efforts in DNA typing. Observed alleles and annotated sequence for each STR locus are described along with a review of STR analysis technologies. Additionally, commercially available STR multiplex kits are described, published polymerase chain reaction (PCR) primer sequences are reported, and validation studies conducted by a number of forensic laboratories are listed. To supplement the technical information, addresses for scientists and hyperlinks to organizations working in this area are available, along with the comprehensive reference list of over 1300 publications on STRs used for DNA typing purposes.

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.