Methods for the analysis of multi-scale cell dynamics from fluorescence microscopy images

La embriogénesis es el proceso mediante el cual una célula se convierte en un ser un vivo. A lo largo de diferentes etapas de desarrollo, la población de células va proliferando a la vez que el embrión va tomando forma y se configura. Esto es posible gracias a la acción de varios procesos genéticos, bioquímicos y mecánicos que interaccionan y se regulan entre ellos formando un sistema complejo que se organiza a diferentes escalas espaciales y temporales. Este proceso ocurre de manera robusta y reproducible, pero también con cierta variabilidad que permite la diversidad de individuos de una misma especie. La aparición de la microscopía de fluorescencia, posible gracias a proteínas fluorescentes que pueden ser adheridas a las cadenas de expresión de las células, y los avances en la física óptica de los microscopios han permitido observar este proceso de embriogénesis in-vivo y generar secuencias de imágenes tridimensionales de alta resolución espacio-temporal. Estas imágenes permiten el estudio de los procesos de desarrollo embrionario con técnicas de análisis de imagen y de datos, reconstruyendo dichos procesos para crear la representación de un embrión digital. Una de las más actuales problemáticas en este campo es entender los procesos mecánicos, de manera aislada y en interacción con otros factores como la expresión genética, para que el embrión se desarrolle. Debido a la complejidad de estos procesos, estos problemas se afrontan mediante diferentes técnicas y escalas específicas donde, a través de experimentos, pueden hacerse y confrontarse hipótesis, obteniendo conclusiones sobre el funcionamiento de los mecanismos estudiados. Esta tesis doctoral se ha enfocado sobre esta problemática intentando mejorar las metodologías del estado del arte y con un objetivo específico: estudiar patrones de deformación que emergen del movimiento organizado de las células durante diferentes estados del desarrollo del embrión, de manera global o en tejidos concretos. Estudios se han centrado en la mecánica en relación con procesos de señalización o interacciones a nivel celular o de tejido. En este trabajo, se propone un esquema para generalizar el estudio del movimiento y las interacciones mecánicas que se desprenden del mismo a diferentes escalas espaciales y temporales. Esto permitiría no sólo estudios locales, si no estudios sistemáticos de las escalas de interacción mecánica dentro de un embrión. Por tanto, el esquema propuesto obvia las causas de generación de movimiento (fuerzas) y se centra en la cuantificación de la cinemática (deformación y esfuerzos) a partir de imágenes de forma no invasiva. Hoy en día las dificultades experimentales y metodológicas y la complejidad de los sistemas biológicos impiden una descripción mecánica completa de manera sistemática. Sin embargo, patrones de deformación muestran el resultado de diferentes factores mecánicos en interacción con otros elementos dando lugar a una organización mecánica, necesaria para el desarrollo, que puede ser cuantificado a partir de la metodología propuesta en esta tesis. La metodología asume un medio continuo descrito de forma Lagrangiana (en función de las trayectorias de puntos materiales que se mueven en el sistema en lugar de puntos espaciales) de la dinámica del movimiento, estimado a partir de las imágenes mediante métodos de seguimiento de células o de técnicas de registro de imagen. Gracias a este esquema es posible describir la deformación instantánea y acumulada respecto a un estado inicial para cualquier dominio del embrión. La aplicación de esta metodología a imágenes 3D + t del pez zebra sirvió para desvelar estructuras mecánicas que tienden a estabilizarse a lo largo del tiempo en dicho embrión, y que se organizan a una escala semejante al del mapa de diferenciación celular y con indicios de correlación con patrones de expresión genética. También se aplicó la metodología al estudio del tejido amnioserosa de la Drosophila (mosca de la fruta) durante el cierre dorsal, obteniendo indicios de un acoplamiento entre escalas subcelulares, celulares y supracelulares, que genera patrones complejos en respuesta a la fuerza generada por los esqueletos de acto-myosina. En definitiva, esta tesis doctoral propone una estrategia novedosa de análisis de la dinámica celular multi-escala que permite cuantificar patrones de manera inmediata y que además ofrece una representación que reconstruye la evolución de los procesos como los ven las células, en lugar de como son observados desde el microscopio. Esta metodología por tanto permite nuevas formas de análisis y comparación de embriones y tejidos durante la embriogénesis a partir de imágenes in-vivo. ABSTRACT The embryogenesis is the process from which a single cell turns into a living organism. Through several stages of development, the cell population proliferates at the same time the embryo shapes and the organs develop gaining their functionality. This is possible through genetic, biochemical and mechanical factors that are involved in a complex interaction of processes organized in different levels and in different spatio-temporal scales. The embryogenesis, through this complexity, develops in a robust and reproducible way, but allowing variability that makes possible the diversity of living specimens. The advances in physics of microscopes and the appearance of fluorescent proteins that can be attached to expression chains, reporting about structural and functional elements of the cell, have enabled for the in-vivo observation of embryogenesis. The imaging process results in sequences of high spatio-temporal resolution 3D+time data of the embryogenesis as a digital representation of the embryos that can be further analyzed, provided new image processing and data analysis techniques are developed. One of the most relevant and challenging lines of research in the field is the quantification of the mechanical factors and processes involved in the shaping process of the embryo and their interactions with other embryogenesis factors such as genetics. Due to the complexity of the processes, studies have focused on specific problems and scales controlled in the experiments, posing and testing hypothesis to gain new biological insight. However, methodologies are often difficult to be exported to study other biological phenomena or specimens. This PhD Thesis is framed within this paradigm of research and tries to propose a systematic methodology to quantify the emergent deformation patterns from the motion estimated in in-vivo images of embryogenesis. Thanks to this strategy it would be possible to quantify not only local mechanisms, but to discover and characterize the scales of mechanical organization within the embryo. The framework focuses on the quantification of the motion kinematics (deformation and strains), neglecting the causes of the motion (forces), from images in a non-invasive way. Experimental and methodological challenges hamper the quantification of exerted forces and the mechanical properties of tissues. However, a descriptive framework of deformation patterns provides valuable insight about the organization and scales of the mechanical interactions, along the embryo development. Such a characterization would help to improve mechanical models and progressively understand the complexity of embryogenesis. This framework relies on a Lagrangian representation of the cell dynamics system based on the trajectories of points moving along the deformation. This approach of analysis enables the reconstruction of the mechanical patterning as experienced by the cells and tissues. Thus, we can build temporal profiles of deformation along stages of development, comprising both the instantaneous events and the cumulative deformation history. The application of this framework to 3D + time data of zebrafish embryogenesis allowed us to discover mechanical profiles that stabilized through time forming structures that organize in a scale comparable to the map of cell differentiation (fate map), and also suggesting correlation with genetic patterns. The framework was also applied to the analysis of the amnioserosa tissue in the drosophila’s dorsal closure, revealing that the oscillatory contraction triggered by the acto-myosin network organized complexly coupling different scales: local force generation foci, cellular morphology control mechanisms and tissue geometrical constraints. In summary, this PhD Thesis proposes a theoretical framework for the analysis of multi-scale cell dynamics that enables to quantify automatically mechanical patterns and also offers a new representation of the embryo dynamics as experienced by cells instead of how the microscope captures instantaneously the processes. Therefore, this framework enables for new strategies of quantitative analysis and comparison between embryos and tissues during embryogenesis from in-vivo images.

The conceptus regulates tryptophanyl-tRNA synthetase and superoxide dismutase 2 in the sheep caruncular endometrium during early pregnancy

Copyright © 2015 Elsevier Ltd. All rights reserved. This research project was funded by NHS Grampian R&D (project number RG05/019).

Use of phosphorous mapping and sea-level change data in assessing coastal activity zones and establishing site chronology : A case study from the Icelandic multi-period site of Vatnsfjörður

This paper forms part of Lukasz Mikolajczyk's PhD dissertation, which is supervised by Karen Milek

New lower bounds for the topological complexity of aspherical spaces

Date of Acceptance: 5/04/2015 15 pages, 4 figures

Atlantic salmon (Salmo salar) parr as a model to predict the optimum inclusion of air classified faba bean protein concentrate in feeds for seawater salmon

Peer reviewed

Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs

The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the mfold program package that identified the model that best satisfied the probing data. A long-range structural interaction involving the 5′ and 3′ untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done. The modification data support a very similar intracellular structure. When low reactivity of A and C residues is found in the synthetic RNA, ≈70% of the same sites are relatively more resistant to DMS modification in vivo. A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model. With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.

Local and global attention are mapped retinotopically in human occipital cortex

Clinical evidence suggests that control mechanisms for local and global attention are lateralized in the temporal–parietal cortex. However, in the human occipital (visual) cortex, the evidence for lateralized local/global attention is controversial. To clarify this matter, we used functional MRI to map activity in the human occipital cortex, during local and global attention, with sustained visual fixation. Data were analyzed in a flattened cortical format, relative to maps of retinotopy and spatial frequency peak tuning. Neither local nor global attention was lateralized in the occipital cortex. Instead, local attention and global attention appear to be special cases of visual spatial attention, which are mapped consistently with the maps of retinotopy and spatial frequency tuning, in multiple visual cortical areas.

Tissue engineered perivascular endothelial cell implants regulate vascular injury.

Molecular biomaterial engineering permits in vivo transplantation of cells and tissues, offering the promise of restoration of physiologic control rather than pharmacologic dosing with isolated compounds. We engrafted endothelial cells on Gelfoam biopolymeric matrices with retention of viability, normal growth kinetics, immunoreactivity, and biochemical activity. The production of heparan sulfate proteoglycan and inhibition of basic fibroblast growth factor binding and activity by engrafted cells were indistinguishable from endothelial cells grown in culture. Perivascular implantation of Gelfoam-endothelial cell scaffolds around balloon-denuded rat carotid arteries reduced intimal hyperplasia 88.1%, far better than the isolated administration of heparin, the most effective endothelial mimic compound. In concert with a reduction in intimal area, cell proliferation was reduced by > 90%. To our knowledge, there have been no previous reports of extravascular cell implants controlling vasculoproliferative disease. Tissue engineered cells offer the potential for potent methods of vascular growth regulation and insight into the complex autocrine-paracrine control mechanisms within the blood vessel wall.

Moralidade administrativa: o agente público na democracia do novo milênio

Os altos índices de corrupção no Brasil levaram a criação de algumas leis com o intuito de combater este mal que corrói a democracia e que preocupa todos os países democráticos. Este trabalho busca verificar as novas ferramentas jurídicas, bem como a responder a uma questão. Há mais alguma reforma necessária? Alguns estudos apontam influência de aspectos do sistema eleitoral no índice de corrupção. Se há, que reforma nesse sentido o Brasil poderia adotar? Foram utilizados método dialético e método comparativo, para análise de doutrina e legislação estrangeiras, comparando-as com as nacionais, e método indutivo para a análise de dados. Foram feitos grandes avanços legislativos, mas há que discutir o que pode ser aprimorado para criar um ambiente menos fértil à corrupção e tornar mais eficazes os mecanismos de controle dos agentes públicos.

Agro-estratégias através do tempo

No início dos anos 2000 consolidou-se o quadro de significativas alterações e ajustes nas estratégias das organizações agrícolas. Destacam-se: a consolidação das organizações, a internacionalização dos sistemas de base agrícola, a inovação presente em processos e produtos e de natureza organizacional, a introdução da variável socioambiental, e a adoção de estratégias de transparência. A cooperação pode exigir investimentos especializados, e os incentivos para a sua realização dependem de mecanismos de controle de custos de transação. Com a presença de incerteza no ambiente econômico e nas transações, a flexibilidade planejada visa eventuais ajustes em face de eventos inesperados. Arranjos institucionais complexos (leia-se, contratos) são observados como forma de responder a necessidades apontadas. Além de confiança, reputação, e mecanismos relacionais, a evolução dos mecanismos sociais por trás dos contratos de sociedade é algo a ser desenvolvido. O presente estudo propõe que as cooperativas agrícolas podem desenvolver mecanismos de governança que geram uma competência adaptativa para enfrentar eventos inesperados. O presente estudo explorou uma visão retrospectiva de estratégias adotadas por cooperativas brasileiras. Assumiu-se aqui uma nova vertente analítica da \"História de Negócios\" e suas implicações voltadas ao sistema agroindustrial. Como diretriz de método, foram seguidas as etapas de identificação das principais estratégias relatadas nos estudos de casos escolhidos, sobre cooperativas, desenvolvidos entre 1991 e 2002. Na sequência compararam-se as estratégias com as diretrizes apresentadas no capítulo teórico. Admite-se que as estratégias que implicam em maiores investimentos em ativos específicos tendem a tornarem mais rígidos os arranjos e dificultam a plasticidade, ou adaptação, das cooperativas agrícolas - onde naturalmente as mudanças ocorrem de forma mais lenta - frente a choques ou eventos externos.

Application of multi-sensor advanced DInSAR analysis to severe land subsidence recognition: Alto Guadalentín Basin (Spain)

Multi-sensor advanced DInSAR analyses have been performed and compared with two GPS station measurements, in order to evaluate the land subsidence evolution in a 20-year period, in the Alto Guadalentín Basin where the highest rate of man-induced subsidence (> 10 cm yr−1) of Europe had been detected. The control mechanisms have been examined comparing the advanced DInSAR data with conditioning and triggering factors (i.e. isobaths of Plio-Quaternary deposits, soft soil thickness and piezometric level).

O direito ao próprio direito: modelos latino-americanos de autonomia política indígena

O objetivo deste trabalho é saber se o direito indigenista, como denominarei o direito estatal que diz respeito aos povos indígenas, reconhece a legitimidade do direito indígena, como denominarei o direito produzido pelos povos indígenas, nas experiências colombiana, boliviana e brasileira. A escolha da Bolívia se justifica pelo fato de as Constituições recentes deste país e do Equador serem consideradas um novo marco do constitucionalismo pluralista ao refundarem suas ordens buscando superar a ausência indígena constituinte. Já a Colômbia se destaca entre os países que, sob a influência recente do Convênio 169, incorporaram expressamente o pluralismo jurídico em suas Constituições. A jurisprudência produzida pela Corte Constitucional do país a respeito do direito indígena é considerada exemplar e inspiradora dos desenvolvimentos mais recentes na Bolívia. O trabalho está voltado para dois aspectos do tema: a autonomia jurisdicional, ou a capacidade para julgar conflitos conforme as normas e procedimentos próprios, e os mecanismos de controle de tais decisões. A metodologia do trabalho abrange revisão bibliográfica, seleção e análise documental de decisões judiciais e textos legais. Argumento que a acomodação de autonomias políticas e ordens jurídicas de diferentes culturas depende da criação de meta-instituições e metarregras que solucionem conflitos e promovam a coordenação entre os direitos, permitindo que os grupos se relacionem de maneira equitativa, controlem a dinâmica de suas identidades culturais e se sintam parte de uma mesma comunidade política. A prática das instituições brasileiras, no entanto, está muito mais voltada a aplicar o direito estatal aos índios do que a exercer controle sobre o direito indígena, o que indica que o paradigma da assimilação prevalece sobre eventuais concepções multiculturais de Estado e sociedade, ainda que o direito legislado apresente regras que reconhecem o pluralismo jurídico. Em outras palavras, as instituições estatais enxergam os indígenas como pessoas que percorrem o caminho da incapacidade jurídica à capacidade plena à medida em que se familiarizam com a cultura dominante, e não como pessoas que podem transitar entre diferentes ordens jurídicas. Por outro lado, a experiência recente de países latino-americanos que se abriram ao pluralismo jurídico mostra um caminho difícil e repleto de questões em aberto. As que mais se destacam são a possibilidade de violações de direitos humanos por autoridades indígenas e a tensão entre centralização política e autonomia política. Em relação ao primeiro caso, o aspecto crucial é saber quem deve julgar as violações e sob quais critérios, além de evitar decisões culturalmente enviesadas. Já o segundo caso depende da superação de traços autoritários relacionados ao governo central e da predominância das estruturas estatais já consolidadas, tanto no nível central quanto no nível local, sobre as instituições mantidas pelos povos indígenas. Ainda há um descompasso entre o discurso constitucional de igualdade entre as ordens jurídicas e a prática de subordinação das ordens indígenas às instâncias estatais.

Technical report of the Commission on Obscenity and Pornography /

Includes bibliographical references and index.

Germ cells enter meiosis in a rostro-caudal wave during development of the mouse ovary

Germ cells in the mouse embryo remain undifferentiated until about 13.5 days post-coitum (dpc), when male germ cells enter mitotic arrest and female germ cells enter meiosis. The molecular signals and transcriptional control mechanisms governing the differential fate of germ cells in males and females remain largely unknown. In order to gain insights into the behavior of germ cells around this period and into likely mechanisms controlling entry into meiosis, we have studied by wholemount in situ hybridization the expression pattern of two germ cell-specific markers, Oct4 and Sycp3, during mouse fetal gonad development. We observed a dynamic wave of expression of both genes in developing ovaries, with Oct4 expression being extinguished in a rostro-caudal wave and Sycp3 being upregulated in a corresponding wave, during the period 13.5-15.5 dpc. These results indicate that entry into meiosis proceeds in a rostro-caudal progression, in turn suggesting that somatically derived signals may contribute to the control of germ cell entry into meiosis in developing ovaries. (C) 2004 Wiley-Liss, Inc.

Novel strategies in feedforward adaptation to a position-dependent perturbation

To investigate the control mechanisms used in adapting to position-dependent forces, subjects performed 150 horizontal reaching movements over 25 cm in the presence of a position-dependent parabolic force field (PF). The PF acted only over the first 10 cm of the movement. On every fifth trial, a virtual mechanical guide (double wall) constrained subjects to move along a straight-line path between the start and target positions. Its purpose was to register lateral force to track formation of an internal model of the force field, and to look for evidence of possible alternative adaptive strategies. The force field produced a force to the right, which initially caused subjects to deviate in that direction. They reacted by producing deviations to the left, into the force field, as early as the second trial. Further adaptation resulted in rapid exponential reduction of kinematic error in the latter portion of the movement, where the greatest perturbation to the handpath was initially observed, whereas there was little modification of the handpath in the region where the PF was active. Significant force directed to counteract the PF was measured on the first guided trial, and was modified during the first half of the learning set. The total force impulse in the region of the PF increased throughout the learning trials, but it always remained less than that produced by the PF. The force profile did not resemble a mirror image of the PF in that it tended to be more trapezoidal than parabolic in shape. As in previous studies of force-field adaptation, we found that changes in muscle activation involved a general increase in the activity of all muscles, which increased arm stiffness, and selectively-greater increases in the activation of muscles which counteracted the PF. With training, activation was exponentially reduced, albeit more slowly than kinematic error. Progressive changes in kinematics and EMG occurred predominantly in the region of the workspace beyond the force field. We suggest that constraints on muscle mechanics limit the ability of the central nervous system to employ an inverse dynamics model to nullify impulse-like forces by generating mirror-image forces. Consequently, subjects adopted a strategy of slightly overcompensating for the first half of the force field, then allowing the force field to push them in the opposite direction. Muscle activity patterns in the region beyond the boundary of the force field were subsequently adjusted because of the relatively-slow response of the second-order mechanics of muscle impedance to the force impulse.