Innovations of wide-field optical-sectioning fluorescence microscopy : toward high-speed volumetric bio-imaging with simplicity

Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere.

The concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths.

Among existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often result in certain degrees of photo-damages because of the high focal intensity at the scanning point. In order to overcome such an issue, several wide-field optical-sectioning techniques have been proposed and demonstrated, although not without introducing new limitations and compromises such as low signal-to-background ratios and reduced axial resolutions. As a result, single-point-scanning optical-sectioning techniques remain the most widely used instrumentations for volumetric imaging of living biological systems to date.

In order to develop wide-field optical-sectioning techniques that has equivalent optical performance as single-point-scanning ones, this thesis first introduces the mechanisms and limitations of existing wide-field optical-sectioning techniques, and then brings in our innovations that aim to overcome these limitations. We demonstrate, theoretically and experimentally, that our proposed wide-field optical-sectioning techniques can achieve diffraction-limited optical sectioning, low out-of-focus excitation and high-frame-rate imaging in living biological systems. In addition to such imaging capabilities, our proposed techniques can be instrumentally simple and economic, and are straightforward for implementation on conventional wide-field microscopes. These advantages together show the potential of our innovations to be widely used for high-speed, volumetric fluorescence imaging of living biological systems.

Isolation of the murine interleukin 2 gene and characterization of its regulatory architecture

Interleukin 2 (IL2) is the primary growth hormone used by mature T cells and this lymphokine plays an important role in the magnification of cell-mediated immune responses. Under normal circumstances its expression is limited to antigen-activated type 1 helper T cells (T_H1) and the ability to transcribe this gene is often regarded as evidence for commitment to this developmental lineage. There is, however, abundant evidence than many non-T_H1 T cells, under appropriate conditions, possess the ability to express this gene. Of paramount interest in the study of T-cell development is the mechanisms by which differentiating thymocytes are endowed with particular combinations of cell surface proteins and response repertoires. For example, why do most helper T cells express the CD4 differentiation antigen?

As a first step in understanding these developmental processes the gene encoding IL2 was isolated from a mouse genomic library by probing with a conspecific IL2 cDNA. The sequence of the 5' flanking region from + 1 to -2800 was determined and compared to the previously reported human sequence. Extensive identity exists between +1 and -580 (86%) and sites previously shown to be crucial for the proper expression of the human gene are well conserved in both sequence location in the mouse counterpart.

Transient expression assays were used to evaluate the contribution of various genomic sequences to high-level gene expression mediated by a cloned IL2 promoter fragment. Differing lengths of 5' flanking DNA, all terminating in the 5' untranslated region, were linked to a reporter gene, bacterial chloramphenicol acetyltransferase (CAT) and enzyme activity was measured after introduction into IL2-producing cell lines. No CAT was ever detected without stimulation of the recipient cells. A cloned promoter fragment containing only 321 bp of upstream DNA was expressed well in both Jurkat and EL4.El cells. Addition of intragenic or downstream DNA to these 5' IL2-CAT constructs showed that no obvious regulatory regions resided there. However, increasing the extent of 5' DNA from -321 to -2800 revealed several positive and negative regulatory elements. One negative region that was well characterized resided between -750 and -1000 and consisted almost exclusively of alternating purine and pyrimidines. There is no sequence resembling this in the human gene now, but there is evidence that there may have once been.

No region, when deleted, could relax either the stringent induction-dependence on cell-type specificity displayed by this promoter. Reagents that modulated endogenous IL2 expression, such as cAMP, cyclosporin A, and IL1, affected expression of the 5' IL2-CAT constructs also. For a given reagent, expression from all expressible constructs was suppressed or enhanced to the same extent. This suggests that these modulators affect IL2 expression through perturbation of a central inductive signal rather than by summation of the effects of discrete, independently regulated, negative and positive transcription factors.

Visual pattern recognition network: Its training algorithm and its optoelectronic architecture

A visual pattern recognition network and its training algorithm are proposed. The network constructed of a one-layer morphology network and a two-layer modified Hamming net. This visual network can implement invariant pattern recognition with respect to image translation and size projection. After supervised learning takes place, the visual network extracts image features and classifies patterns much the same as living beings do. Moreover we set up its optoelectronic architecture for real-time pattern recognition. (C) 1996 Optical Society of America

Caretta caretta (Loggerhead Sea Turtle). Nest Architecture.

Researchers compared nest architecture in loggerhead sea turtles at natural beaches in Florida, USA and Brazil to determine how similarities and differences in female morphology and reproductive output in these two populations are reflected in the structure of the nest.

4th European Conference on Energy Efficiency and Sustainability in Architecture and Planning : Donostia-San Sebastián, 1-3 Julio 2013

XXXII Cursos de Verano de la UPV/EHU

5th European Conference on Energy Efficiency and Sustainability in Architecture and Planning : Donostia-San Sebastián, 7-9 Julio 2014

XXXIII Cursos de Verano de la UPV/EHU

Distributed Optimal Control of Cyber-Physical Systems: Controller Synthesis, Architecture Design and System Identification

The centralized paradigm of a single controller and a single plant upon which modern control theory is built is no longer applicable to modern cyber-physical systems of interest, such as the power-grid, software defined networks or automated highways systems, as these are all large-scale and spatially distributed. Both the scale and the distributed nature of these systems has motivated the decentralization of control schemes into local sub-controllers that measure, exchange and act on locally available subsets of the globally available system information. This decentralization of control logic leads to different decision makers acting on asymmetric information sets, introduces the need for coordination between them, and perhaps not surprisingly makes the resulting optimal control problem much harder to solve. In fact, shortly after such questions were posed, it was realized that seemingly simple decentralized optimal control problems are computationally intractable to solve, with the Wistenhausen counterexample being a famous instance of this phenomenon. Spurred on by this perhaps discouraging result, a concerted 40 year effort to identify tractable classes of distributed optimal control problems culminated in the notion of quadratic invariance, which loosely states that if sub-controllers can exchange information with each other at least as quickly as the effect of their control actions propagates through the plant, then the resulting distributed optimal control problem admits a convex formulation.

The identification of quadratic invariance as an appropriate means of "convexifying" distributed optimal control problems led to a renewed enthusiasm in the controller synthesis community, resulting in a rich set of results over the past decade. The contributions of this thesis can be seen as being a part of this broader family of results, with a particular focus on closing the gap between theory and practice by relaxing or removing assumptions made in the traditional distributed optimal control framework. Our contributions are to the foundational theory of distributed optimal control, and fall under three broad categories, namely controller synthesis, architecture design and system identification.

We begin by providing two novel controller synthesis algorithms. The first is a solution to the distributed H-infinity optimal control problem subject to delay constraints, and provides the only known exact characterization of delay-constrained distributed controllers satisfying an H-infinity norm bound. The second is an explicit dynamic programming solution to a two player LQR state-feedback problem with varying delays. Accommodating varying delays represents an important first step in combining distributed optimal control theory with the area of Networked Control Systems that considers lossy channels in the feedback loop. Our next set of results are concerned with controller architecture design. When designing controllers for large-scale systems, the architectural aspects of the controller such as the placement of actuators, sensors, and the communication links between them can no longer be taken as given -- indeed the task of designing this architecture is now as important as the design of the control laws themselves. To address this task, we formulate the Regularization for Design (RFD) framework, which is a unifying computationally tractable approach, based on the model matching framework and atomic norm regularization, for the simultaneous co-design of a structured optimal controller and the architecture needed to implement it. Our final result is a contribution to distributed system identification. Traditional system identification techniques such as subspace identification are not computationally scalable, and destroy rather than leverage any a priori information about the system's interconnection structure. We argue that in the context of system identification, an essential building block of any scalable algorithm is the ability to estimate local dynamics within a large interconnected system. To that end we propose a promising heuristic for identifying the dynamics of a subsystem that is still connected to a large system. We exploit the fact that the transfer function of the local dynamics is low-order, but full-rank, while the transfer function of the global dynamics is high-order, but low-rank, to formulate this separation task as a nuclear norm minimization problem. Finally, we conclude with a brief discussion of future research directions, with a particular emphasis on how to incorporate the results of this thesis, and those of optimal control theory in general, into a broader theory of dynamics, control and optimization in layered architectures.

6th European Conference on Energy Efficiency and Sustainability in Architecture and Planning

[ES]Esta obra recoge las comunicaciones seleccionadas para el 6º Congreso Europeo sobre Eficiencia Energética y Sostenibilidad en Arquitectura, organizado por el grupo de investigación Calidad de Vida en Arquitectura de la Universidad del País Vasco/Euskal Herriko Unibertsitatea. El congreso, que se celebra en el marco de los XXXIV Cursos de Verano de la UPV/EHU, aborda en esta cuarta edición el tema “Ciudades en riesgo: resiliencia y redundancia”. Alrededor de este tema general se desarrollan cinco ponencias magistrales, a cargo de Margaretha Breil (Centro Euro-Mediterráneo para el Cambio Climático), Cristina Garzillo Leemhuis (ICLEI), Ignasi Fontanals (OptiCits), Juan Carlos Barrios Montenegro (Global Action Plan) y Manuel Valdés López (Ajuntament de Barcelona). Además, 24 comunicaciones seleccionadas por el comité científico presentarán trabajos de investigaciones actuales en las sesiones orales y póster. Es objetivo paralelo del congreso es fortalecer las líneas de investigación en eficiencia energética y sostenibilidad de los grupos de investigación y formación de la UPV/ EHU comprometidos con esta propuesta, con objeto de colaborar en el reforzamiento de la I D i en su ámbito de conocimiento y apoyar la apuesta específica de los Gobiernos Central y Vasco, así como de otras instituciones nacionales e internacionales respecto a las actividades de I D i en las materias relacionadas con el cambio climático, la eficiencia energética y la sostenibilidad ambiental [ENG] This work contains the selected abstracts of the 6th European Conference on Energy Efficiency and Sustainability in Architecture and Planning, organized by the research group Quality of life in Architecture of the University of the Basque Country. The conference is part of the XXXIV Summer Courses of the UPV/EHU and deals, in its fourth edition, with the topic “Cities at risk: resilience and redundancy”. Around this general theme there are five invited speakers: Margaretha Breil (Euro-Mediterranean Centre for Climate Change), Cristina Garzillo Leemhuis (ICLEI), Ignasi Fontanals (OptiCits), Juan Carlos Barrios Montenegro (Global Action Plan) y Manuel Valdés López (Barcelona City Council). 24 abstracts additional have been selected by the scientific committee that offer actual research works in presentations and posters. The purpose of the conferences is to strengthen the investigation lines in energy efficiency and sustainability of the research and education groups of the University of the Basque Country (UPV/EHU) involved, with the purpose of collaborating in the reinforcement of the I D i in its field of knowledge, and support the specific projects of the Central and Basque Governments, as well as other national and international institutions related to the I Di activities in similar fields of climate change, energy efficiency and environmental sustainability.

Sentidos e significados sobre o choro das crianças nas creches públicas do Município de Juiz de Fora/MG

O objetivo desta tese consistiu em saber se os sentidos compartilhados e os significados construídos sobre o choro das crianças nas creches públicas do município de Juiz de Fora, durante as sessões reflexivas com as coordenadoras/diretoras, produzem espaços de reflexão teórica sobre as práticas capazes de criar uma prática de reflexão. Pelo tipo de problema formulado optei por trabalhar a perspectiva metodológica a partir do paradigma crítico de pesquisa, concretizado na modalidade de pesquisa crítica de colaboração, com enfoque sócio-histórico-cultural. Isto se justifica porque busco compreender o lugar do choro das crianças nas práticas das coordenadoras/diretoras, procurando identificar se a consciência das ações institucionalizadas pode produzir mudanças nas práticas no interior das creches. O corpus discurso da tese constitui-se de 10 sessões reflexivas, cujos dados produzidos foram organizados e analisados a partir da perspectiva teórico-metodológica dos Núcleos de Significação de Aguiar e Ozella (2006). O campo teórico está circunscrito no diálogo entre Vigotski, Bakhtin e Wallon. Isso porque estes autores contribuíram, sobremaneira, para a discussão sobre sentido, significado; linguagem e consciência, zona de desenvolvimento proximal, além do estudo da emoção, em especial, sobre a manifestação do choro da criança no contexto da creche. A arquitetura dos Núcleos de Significação revelou a necessidade de aprofundar questões relacionadas aos diversos olhares para o choro da criança; estratégias para lidar com o choro a partir da visão das coordenadoras/diretoras; o controle/descontrole/não controle do choro; a inter-relação do choro, da creche e da família e, finalmente, a reflexão teórico-prática como possibilidade de reverberação no cotidiano da creche. As análises confirmaram três premissas básicas: a) que a emoção constitui-se como o primeiro recurso de interação com o outro, que antecede a própria representação simbólica e por isso é valioso o aprofundamento deste assunto em cursos de formação; b) quando tornamos a cena vivida mais clara, essa clareza pode trazer elementos para outras possíveis intervenções, para outros possíveis diálogos sobre o choro da criança. A perspectiva de transformação pode acontecer exatamente no diálogo entre o cotidiano, a história e espaços de reflexão; c) que o fundamental, nos contextos de formação, é não focalizar apenas o conteúdo a ser transmitido sem possibilidade de reflexão sobre o próprio contexto a que se destina porque é pelo possível distanciamento e necessário estranhamento das práticas rotineiras, esporadicamente ou quase nunca questionadas, que a reflexão e a crítica se estabelecem.

Cidade e processos educativos: CIEPs e PEU Bairro-Escola no caminho das Cidades Educadoras

Esta pesquisa parte da Carta das Cidades Educadoras para avaliar como o espaço urbano das periferias pode também ser usado como espaço para a educação e construção crítica dos alunos para não se configurar, no imaginário construído, apenas como espaços de exclusão e violência. O estudo aborda duas iniciativas no estado do Rio de Janeiro como paradigma: uma, anterior ao conceito de? Cidades Educadoras?, os Centros Integrados de Educação Pública, e outra, posterior e atual, o PEU Bairro-Escola de Nova Iguaçu. Os CIEPs foram utilizados como paradigma arquitetônico, principalmente por terem sido espaços diretamente pensados para o Programa Político Pedagógico da época e também por terem, ao longo de todos esses anos após sua criação, passado por sérias modificações em seu estado físico e em seu PPP, o que possibilitou uma reavaliação de sua posição como equipamento educacional e cultural. O PEU Bairro-Escola, e seus respectivos projetos, em aplicação no município de Nova Iguaçu, revelam uma iniciativa pública de reestruturação e requalificação urbanística de periferia, avaliando a relação ensino-aprendizagem dos discentes, dentro e fora dos edifícios escolares, procurando elencar os principais novos territórios onde estão acontecendo as ações educativas nas periferias.