Дисертації з теми "Biological optical systems"

Existing optic fibre-bundle based imaging probes have been successfully used to image biological signals from tissue in direct contact with the probe tip (Hirano et al. 1996). These fibre-bundle probe systems employed conventional fluorescence microscopy and thus lacked spatial filtering or a scanned light source, two features used by laser scanning confocal microscopes (LSCMs) to improve signal quality. Improving the methods of imaging tissue in its natural state, deep in-vivo and at cellular resolution is an ever-present goal in biological research. Within this study, a novel (580 μm diameter) optic fibre-bundle direct-contact imaging probe, employing a LSCM, was developed to allow for improved imaging of deep biological tissue in-vivo. The new LSCM/probe system possessed a spatial resolution of 10 μm, and a temporal resolution of 1 msec. The LSCM/probe system was compared to a previously used direct-contact probe system that employed a conventional fluorescence microscope. Quantitative and qualitative data indicated that the LSCM/probe system possessed superior image contrast and quality. Furthermore, the LSCM/probe system was approximately 16 times more effective at filtering unwanted contaminating light from regions below the imaging plane (z-axis). The unique LSCM/probe system was applied to an exploratory investigation of calcium activity of both glial and neuronal cells within the whisker portion of the rat primary somatosensory cortex in-vivo. Fluorescence signals of 106 cells were recorded from 12 female Sprague Dawley rats aged between 7-8 weeks. Fluo-3(AM) fluorophore based calcium fluctuations that coincided with 10 - 14 Hz sinusoidal stimulation of rat whiskers for 0.5-1 second were observed in 8.5% of cells (9 of 106). Both increases and decreases in calcium levels that coincided with whisker stimulation were observed. Of the 8.5 % of cells, 2.8% (3 cells) were categorized as glial and 5.7% (6 cells) as neuronal, based on temporal characteristics of the observed activity. The remaining cells (97 of 106) displayed sufficient calcium-based intensity but no fluctuations that coincided with an applied stimulus. This was partially attributed to electronic noise inherent in the prototype system obscuring potential very weak cell signals. The results indicate that the novel LSCM/probe system is an advancement over previously used systems that employed direct-contact imaging probes. The miniature nature of the probe allows for insertion into soft tissue, like a hypodermic needle, and provides access to a range of depths with minimal invasiveness. Furthermore, when combined with selected dyes, the system allows for imaging of numerous forms of activity at cellular resolution.

La plupart des microscopies impliquées dans l'étude d'échantillons ou de processus biologiques utilise des marqueurs ou des sondes, qui peuvent modifier artificiellement, plus ou moins fortement, les échantillons observés. Afin de proposer une alternative à ces techniques, un microscope haute résolution à plasmons de surface (le SSPM) a été développé. Les plasmons sont des oscillations collectives des électrons libres d'un métal, dont les conditions de résonance sont très sensibles à la variation d'indice diélectrique à la surface de ce métal. L'utilisation d'un objectif à forte ouverture numérique permet la focalisation de la lumière incidente dans une petite zone de l'interface métal/milieu d'observation, et entraîne ainsi la localisation et la structuration de ces ondes. Enfin, un balayage de la surface est réalisé, permettant de détecter les variations locales d'indice diélectrique de l'échantillon. Tout d'abord, nous présentons le principe expérimental du SSPM, mais aussi la modélisation de sa réponse par l'intermédiaire d'une résolution 3D des équations de Maxwell. Dans un deuxième temps, nous étudions la structure des couches minces d'or déposées par évaporation thermique sur des substrats de verre, et utilisées lors des expériences de microscopie SSPM. Puis nous visualisons dans l'air et dans l'eau, des nanoparticules métalliques et diélectriques, de 10 à 200 nm de diamètre, et montrons qu'il est possible de les différencier suivant leur taille ou leur indice diélectrique. Enfin, nous imageons des nucléosomes (complexes nucléoprotéiques d'environ 10 nm de diamètre) non marqués, ainsi que des fibroblastes dont nous résolvons certaines des sous structures
Most of the microscopy techniques used to study biological samples or processes relies on the use of markers or physical probes, which may modify artificially the phenomena considered. So as to propose an alternate to these techniques, a high resolution Scanning Surface Plasmon Microscope (SSPM) has been developed. Plasmons consist in collective oscillations of the free electrons at the surface of a metal. A high numerical aperture objective focuses the incident light on a small area of the metal/observation medium interface, which leads to the localization and the structuring of these waves here. Finally, the local variations of the sample dielectric index are detected while scanning the sample surface. First of all, we present the experimental principle of the SSPM, as well as a modelization of its response thanks to a 3D resolution of the Maxwell's equations. In chapter two, we study the structure of the thin gold films used during the SSPM experiments, after being deposited onto glass substrates by thermal evaporation. We address in the third chapter the problem of imaging in air and in water isolated nanoparticles of different sizes (from 10 to 200 nm of diameter). We show that this method is well suited to visualize such objects and also to discriminate them from their size or the material they are made of (depending on their dielectric index). Finally, we apply in the last chapter the SSPM to the visualization of unlabelled biological samples, such as nucleosomes (nucleoproteic complexes of about 10 nm of diameter) as well as human fibroblasts in which we resolve several subcellular structures (nucleus, nucleolus, cytoskeleton structures)

Thesis (Ph. D.)--University of California, San Diego, 2006.
Title from first page of PDF file (viewed April 4, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 131-146).

Cette thèse aborde les problèmes inverses de contrôle optimal (IOCP) pour trouver les fonctions de coûts pour lesquelles les mouvements humains sont optimaux. En supposant que les observations de mouvements humains sont parfaites, alors que le processus de commande du moteur humain est imparfait, nous proposons un algorithme de commande approximative optimale. En appliquant notre algorithme pour les observations de mouvement humaines collectées: mouvement du bras humain au cours d'une tâche de vissage industrielle, une tâche de suivi visuel d’une cible et une tâche d'initialisation de la marche, nous avons effectué une analyse en boucle ouverte. Pour les trois cas, notre algorithme a trouvé les fonctions de coût qui correspondent mieux ces données, tout en satisfaisant approximativement les Karush-Kuhn-Tucker (KKT) conditions d'optimalité. Notre algorithme offre un beau temps de calcul pour tous les cas, fournir une opportunité pour son utilisation dans les applications en ligne. Pour la tâche de suivi visuel d’une cible, nous avons étudié une modélisation en boucle fermée avec deux boucles de rétroaction PD. Avec des données artificielles, nous avons obtenu des résultats cohérents en termes de tendances des gains et les critères trouvent par notre algorithme pour la tâche de suivi visuel d’une cible. Dans la seconde partie de notre travail, nous avons proposé une nouvelle approche pour résoudre l’IOCP, dans un cadre d'erreur bornée. Dans cette approche, nous supposons que le processus de contrôle moteur humain est parfait tandis que les observations ont des erreurs et des incertitudes d'agir sur eux, étant imparfaite. Les erreurs sont délimitées avec des limites connues, sinon inconnu. Notre approche trouve l'ensemble convexe de de fonction de coût réalisables avec la certitude qu'il comprend la vraie solution. Nous numériquement garanties en utilisant des outils d'analyse d'intervalle
This thesis addresses inverse optimal control problems (IOCP) to find the cost functions for which the human motions are optimal. Assuming that the human motion observations are perfect, while the human motor control process is imperfect, we propose an approximately optimal control algorithm. By applying our algorithm to the human motion observations collected for: the human arm trajectories during an industrial screwing task, a postural coordination in a visual tracking task and a walking gait initialization task, we performed an open loop analysis. For the three cases, our algorithm returned the cost functions which better fit these data, while approximately satisfying the Karush-Kuhn-Tucker (KKT) optimality conditions. Our algorithm offers a nice computational time for all cases, providing an opportunity for its use in online applications. For the visual tracking task, we investigated a closed loop modeling with two PD feedback loops. With artificial data, we obtained consistent results in terms of feedback gains’ trends and criteria exhibited by our algorithm for the visual tracking task. In the second part of our work, we proposed a new approach to solving the IOCP, in a bounded error framework. In this approach, we assume that the human motor control process is perfect while the observations have errors and uncertainties acting on them, being imperfect. The errors are bounded with known bounds, otherwise unknown. Our approach finds the convex hull of the set of feasible cost function with a certainty that it includes the true solution. We numerically guaranteed this using interval analysis tools

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O controle ótimo no mundo biológico tem uma vasta aplicação em incontáveis sistemas os quais influenciam enormemente nossas vidas. Objetiva-se a aplicação desta ferramenta em dois sistemas. O primeiro diz respeito ao controle ótimo de dosagem de drogas no tratamento de pacientes infectados pelo vírus HIV . O modelo de Campello de Souza (1999) é usado para estimar a dosagem de drogas onde a função objetivo é minimizada. Esta função representa um balanço entre os benefícios do tratamento e os efeitos colaterais. A técnica de controle ótimo usada é o Princípio do Máximo de Pontryagin, a qual é simulada através do PROPT-TOMLAB - Matlab Optimal Control System Software em uma versão de demonstração. As simulações objetivam a análise de três diferentes pacientes em dois diferentes cenários. Estes cenários têm como objetivo forçar as variáveis de estado a atingirem valores "normais" a fim de estabilizar a carga viral próximo a uma taxa que seja insignificante e elevar o nível de CD4 do paciente. São simulados tratamentos cedos e tardios. As simulações computacionais compararam diferentes cenários para investigar os parâmetros de incerteza da dinâmica entre o vírus HIV e os linfócitos CD4 e CD8. Os resultados mostram que o controle ótimo permite uma melhor administração entre os efeitos positivos da terapia e os efeitos colaterais, ao invés de se usar dosagens constantes de drogas como na atual prática médica. O segundo sistema descreve a aplicação do controle ótimo, também através do Princípio Máximo de Pontryagin, para controlar o nível de glicose em indivíduos diabéticos usando o modelo matemático desenvolvido por Bergman (1971, 1981). Correlacionam-se dados reais da literatura com o modelo teórico para analisar a robustez do modelo. É também estudada a minimização do funcional objetivo para diminuir os efeitos colaterais e consequentemente melhorar o estado de saúde do paciente. Os resultados mostram os benefícios de se utilizar o controle ótimo para regular a taxa de glicose em pacientes diabéticos

Thesis (M.S.)--University of Missouri-Columbia, 2007.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 7, 2008) Includes bibliographical references.

A sufficiently strong electric field is able to change the cell membrane permeability, forming aqueous pores across it, hence the name electroporation, permitting the passage, otherwise forbidden, of ions and molecules. Since its efficient application in biotechnology and medicine (e. G. Electrochemotherapy), ms/μs-pulse-induced electroporation draws growing interest. Recently, the application of nanosecond pulses has showed electroperturbation on intracellular membranes, opening the way to subcellular manipulations. To date, the mechanisms beyond the electroporation are not still well known. The lack of ultra-rapid and flexible pulsers for cell stimulation on the one hand, and the rapid and subcellular-scale involved dynamics on the other hand, make the investigations complex. In this context, we have designed and realized a compact system providing both the electric pump and the optical probe for electroporation studies. The electric pump consists of a photocommutation-based pulse generator triggered by a sub-nanosecond microchip laser that also provides the optical excitation of the multiplex-CARS microscope used for cell imaging. The main innovation of this system is represented by the sub-nanosecond regime of the common laser source. This choice is justified by the need for synchronizing the nanosecond electrical stimulation with the optical detection. A detailed analysis in the time and frequency domains has been performed in order to verify the whole system efficiency and applicability to nano-electroporation investigations
Un champ électrique suffisamment intense induit des effets sur la membrane cellulaire, notamment la formation des pores qui permettent le passage , autrement interdit, de ions et molécules, d’où le nom électroporation. Grâce à son application à la biotechnologie et à la médecine (électrochimiothérapie), l’électroporation représente un phénomène de grand intérêt. Récemment, des impulsions de l’ordre de la nanoseconde ont étés appliquées, montrant des effets sur les membranes intracellulaires. Les mécanismes qui sont à la base de l’électroporation ne sont pas encore complètement compris. D’une part, il n’y a pas en commerce de générateurs ultra-rapides et flexibles pour une stimulation électrique adaptée. D’autre part, la détection de phénomènes à l’échelle subcellulaire et de dynamiques temporelles rapides résulte très difficile. En ce contexte, nous avons conçu et réalisé un système électro-optique pompe-sonde. Il se compose d’un système optoélectronique dédié à la génération d’impulsions ultracourtes et de forte intensité, et d’une source pour l’imagerie optique non linéaire basée sur la microspectroscopie multiplex-CARS. Les deux sources sont déclenchées par le même laser fonctionnant en régime sub-nanoseconde. Ce régime temporel permet une synchronisation efficace des deux systèmes, mais il nécessite d’une étude approfondie des effets optiques non linéaires qui induisent l’élargissement spectral du faisceau, indispensable pour l’imagerie multiplex-CARS. Une caractérisation dans le temps et en fréquence a été menée afin de vérifier les performances du system entier et son emploi aux études de nano-électroporation

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.
Includes bibliographical references (p. 42-43).
Optical tweezers instruments use laser radiation pressure to trap microscopic dielectric beads. With the appropriate chemistry, such a bead can be attached to a single molecule as a handle, permitting the application of force on the single molecule. Measuring the force applied in real-time is dependent on detecting the bead's displacement from the trapping laser beam axis. Back-focal-plane detection provides a way of measuring the displacement, in two-dimensions, at nanometer or better resolution. The first part of this work will describe the design of a simple and inexpensive position sensing module customized for optical tweezers applications. Single molecule fluorescence is another powerful technique used to obtain microscopic details in biological systems. This technique can detect the arrival of a single molecule into a small volume of space or detect the conformational changes of a single molecule. Combining optical tweezers with single-molecule fluorescence so that one can apply forces on a single molecule while monitoring its effects via single molecule fluorescence provides an even more powerful experimental platform to perform such microscopic studies. Due to the enhanced photobleaching of fluorophores caused by the trapping laser, this combined technology has only been demonstrated under optimized conditions.
(cont.) The second part of this work will describe a straightforward and noninvasive method of eliminating this problem. The study of mechanotransduction in biological systems is critical to understanding the coupling between mechanical forces and biochemical reactions. Due to the recent advances in single molecule technology, it is now possible to probe such mechanisms at the single molecule level. The third and final part of this work will describe a basic mechanotransduction experiment using the well-studied ZIF268 protein-DNA system. An experimental assay and method of analysis will be outlined.
by Peter Lee.
S.M.

L'intensite d'un processus de comptage est une fonction d'un processus etat. On etudie le probleme du filtrage non lineaire d'une fonction du processus etat connaissant une realisation du processus de comptage. La solution est une equation differentielle regissant l'evolution de cette fonction. Pour une intensite lineaire, les equations de tous les moments de l'estimee de l'etat sont ecrites recursivement. Un filtre sous optimal est propose et l'estimation partitionnee des parametres de l'etat est etudiee. L'application de cette theorie concerne l'estimation du debit sanguin cerebral local

People experience the irritating tearing and burning sensation of lachrymatory factor (LF, propanthial S-oxide) when cutting or chopping onion bulbs. LF is produced by lachrymatory factor synthase (LFS) specifically from 1-propenyl sulfenic acid, a breakdown product of trans-1-propenyl-L-cysteine sulfoxide (1-PRENCSO) by alliinase. This thesis describes strategies to produce a tearless onion by using RNA interference (RNAi) silencing. To determine whether a gene silencing cassette can silence lfs gene transcripts from onion (Allium cepa L.), a crop recalcitrant to genetic transformation, a gene silencing assessment system was developed by using a model plant as a host for the gene of interest. Tobacco (Nicotiana tabacum) plants transgenic for LFS enzyme activity from onion were first produced by introducing a CaMV 35S-onion-lfs gene construct. These plants were then subjected to a second transformation with an RNAi construct directed against the lfs gene sequence. LFS enzyme activity assay showed that the transgenic plants, containing both the lfs gene and the RNAi construct, had significantly reduced LFS activity. This observation was supported by Western analysis for the LFS protein and further validated by quantitative RT-PCR analysis that demonstrated a significant reduction in the lfs transcript level in the dual transformants. This work demonstrated that the RNAi construct is a suitable candidate for the development of a tearless onion. This model plant RNAi system has wide reaching applications for assessment and targeting of plant secondary pathway genes, from poorly studied or recalcitrant plant species, that are important in pharmacological, food and process industries. The functional RNAi vector identified in the model system was transformed into onion. Endogenous lfs transcript levels were successfully reduced by up to 43-fold in six transgenic lines. In consequence, LFS enzyme activity was decreased by up to 1573-fold and this observation was supported by Western analysis for the LFS protein. Furthermore, the production of the deterrent LF upon tissue disruption was reduced up to 67-fold. Subjective olfactory assessment of silenced lines indicated that the pungent odour given off by the leaf and bulb material was much reduced compared with that of non-transgenic counterparts, and that this was replaced by a sweeter milder onion odour. A novel colorimetric assay demonstrated that this silencing had shifted the 1-PRENCSO breakdown pathway so that by reducing LFS protein, more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. A consequence of the raised thiosulfinates levels was a marked increase in the downstream production of a non-enzymatically produced zwiebelane isomer that has never previously been identified, and other volatile compounds, di-1-propenyl disulfides and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophenes, which had previously been reported either in small amounts or had not been detected in onions. These raised volatile sulfur compounds provide an explanation for the unique flavour notes of the LF reduced onion and are predicted to have health benefits akin to those found in garlic. These results demonstrated that silencing of LFS enzyme activity by introducing an RNAi construct directed against the lfs gene sequence simultaneously reduced levels of the deterrent LF and increased the desirable thiosulfinates in onions.

As novel engineered nanoparticles such as single-walled carbon nanotubes (SWCNT) are extensively used in nanotechnology due to their superior properties, it becomes critical to fully understand their biodistribution and effect when accidently inhaled. There fore, development of animaging technique which allow longitudinal in vivo follow-up of SWCNT effect based on their intrinsic properties is highly desirable. Non invasive free-breathing hyperpolarized 3He lung MRI protocol was developed complementary to proton systemic MR protocol to allow monitoring SWCNT based on their intrinsic iron impurities after intrapulmonary exposition. Combined toproton lung MRI and ex vivo optical and electron microscopy at different time points, this protocol represents a powerful multimodality imaging techniques which allows a full characterization of the biodistribution and biological impacts of iron containing SWCNT. SWCNT was found to produce granulomatous and inflammatory reactions in a time and dose dependent manner with their bio persistenc eafter intrapulmonary exposition.From biological impact evaluations after intrapulmonary exposition towards biomedical applications, SWCNT hold promise for applications in nanomedicine field with their distinct architecture and their novel physicochemical properties. The biodistribution and pharmacological profile of various well-dispersed pristine and functionalized SWCNT were assessed in blood and target tissues after their intra venous administration by longitudinal in vivo susceptibility weighted MRI and their potential effect on liver metabolism by ex vivo HRMAS 1H NMR. No presence ofacute toxicological effect (variation in liver metabolism) was observed confirmed by the absence of clustering in NMR spectra using Principal Component Analysis (specific biomarkers of toxicity).

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro
Neste trabalho apresentamos as etapas para a utilização do método da Programação Dinâmica, ou Princípio de Otimização de Bellman, para aplicações de controle ótimo. Investigamos a noção de funções de controle de Lyapunov (FCL) e sua relação com a estabilidade de sistemas autônomos com controle. Uma função de controle de Lyapunov deverá satisfazer a equação de Hamilton-Jacobi-Bellman (H-J-B). Usando esse fato, se uma função de controle de Lyapunov é conhecida, será então possível determinar a lei de realimentação ótima; isto é, a lei de controle que torna o sistema globalmente assintóticamente controlável a um estado de equilíbrio. Como aplicação, apresentamos uma modelagem matemática adequada a um problema de controle ótimo de certos sistemas biológicos. Este trabalho conta também com um breve histórico sobre o desenvolvimento da Teoria de Controle de forma a ilustrar a importância, o progresso e a aplicação das técnicas de controle em diferentes áreas ao longo do tempo.
This dissertation presents the steps for using the method of Dynamic Programming or Bellman Optimization Principle for optimal control applications. We investigate the notion of control-Lyapunov functions (CLF) and its relation to the stability of autonomous systems with control. A control-Lyapunov function must satisfy the Hamilton-Jacobi- Bellman equation (H-J-B). Using this fact, if a control-Lyapunov function is known, it is possible to determine the optimal feedback law, in other words, the control law which makes the system globally asymptotically controllable at an equilibrium state. As an application, we present a mathematical model suitable for an optimal control problem of certain biological systems. This dissertation also presents a brief historic about the development of the Control Theory in a way of illustrate the importance and the progress of the control techniques, specially where it can be applied, according to the diverse areas and different times that this techniques were discovered and used.

El presente proyecto de investigación está enfocado al desarrollo de sensores químicos fluoro-cromogénicos, para la detección y determinación de especies químicas de interés biológico, industrial y medioambiental de forma selectiva y con alta sensibilidad. En forma general, se busca el diseñar nuevos sistemas sensores basados en compuestos (receptores) formados por dos unidades: una unidad coordinante que interacciona con el anión a determinar y una unidad generadora de señal que alerta del reconocimiento molecular efectuado. Durante este estudio se están preparando diversas moléculas receptoras funcionalizandas con grupos modificadores de estructura para evaluar su influencia sobre las capacidades de detección y selectividad como receptores de especies específicas en diferentes condiciones y medios. Las diferentes aproximaciones en prueba implican a su vez el diseño y síntesis molecular, así como el análisis de las diferentes señales ópticas producidas en el reconocimiento, con el fin de diseñar sistemas de alta eficacia y eficiencia, y con posibilidades reales de aplicación.
Santos Figueroa, LE. (2014). New approaches for the development of chromo-fluorogenic sensors for chemical species of biological, industrial and environmental interest [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/43216
TESIS
Premiado

Els mecanismes de regulaci o de les c el lules poden ser modelats per controlar i entendre la biologia cel lular. Diferents nivells d'abstracci o s'utilitzen per descriure els processos biol ogics. En aquest treball s'han utilitzat grafs i equacions diferencials per modelar les interaccions cel lulars tant qualitativament com quantitativa. En aquest treball s'han analitzat dades d'interacci o i activitat de diferents organismes, E. coli i S. cerevisiae: xarxes d'interacci o prote na-prote na, de regulaci o de la transcripci o, i metab oliques, aix com per ls d'expressi o gen omica i prote omica. De la rica varietat de mesures de grafs, una variable important d'aquestes xarxes biol ogiques es la distribuci o de grau, i he aplicat eines d'an alisi estad stica per tal de caracteritzar-la. En tots els casos estudiats les distribucions de grau tenen una forma de cua pesada, per o la majoria d'elles presenten difer encies signi catives respecte un model de llei de pot encia, d'acord amb proves estad stiques. D'altra banda, cap de les xarxes podrien ser assignades de forma inequvoca a cap distribuci o testejada. Pel que fa a un nivell m es microsc opic, hem utilitzat equacions diferencials per estudiar la din amica de models de diversos sistemes bioqu mics. En primer lloc, una eina de programari anomenada ByoDyn ha estat creada des de zero. L'eina permet realitzar simulacions deterministes i estoc astiques, analitzar models mitjan cant estimaci o de par ametres, sensibilitat i an alisi d'identi cabilitat, aix com dissenyar optimament experiments. S'ha creat una interf cie web que ofereix la possibilitat d'interactuar amb el programa d'una manera gr a ca, independentment de la con guraci o de l'usuari, permetent l'execuci o del programa en diferents entorns computacionals. Finalment, hem aplicat un protocol de disseny experimental optim en un model multicel lular de l'embriog enesi en vertebrats.
Regulatory mechanisms of cells can be modelled to control and under- stand cellular biology. Di erent levels of abstraction are used to describe biological processes. In this work we have used graphs and di erential equations to model cellular interactions qualitatively and quantitatively. From di erent organisms, E. coli and S. cerevisiae, we have analysed data available for they complete interaction and activity networks. At the level of interaction, the protein-protein interaction network, the tran- scriptional regulatory networks and the metabolic network have been studied; for the activity, both gene and protein pro les of the whole or- ganism have been examined. From the rich variety of graph measures, one of primer importance is the degree distribution. I have applied sta- tistical analysis tools to such biological networks in order to characterise the degree distribution. In all cases the studied degree distributions have a heavy-tailed shape, but most of them present signi cant di erences from a power-law model according to a statistical test. Moreover, none of the networks could be unequivocally assigned to any of the tested distribution. On the other hand, in a more ne-grained view, I have used di erential equations to model dynamics of biochemical systems. First, a software tool called ByoDyn has been created from scratch incorporating a fairly complete range of analysis methods. Both deterministic and stochas- tic simulations can be performed, models can be analysed by means of parameter estimation, sensitivity, identi ability analysis, and optimal ex- perimental design. Moreover, a web interface has been created that pro- vides with the possibility interact with the program in a graphical man- ner, independent of the user con guration, allowing the execution of the program at di erent computational environments. Finally, we have ap- plied a protocol of optimal experimental design on a multicellular model of embryogenesis.

Alzheimer’s disease (AD) is a chronic and progressive, irreversible syndrome that deteriorates the cognitive functions. Official death certificates of 2013 reported 84,767 deaths from Alzheimer’s disease, making it the 6th leading cause of death in the United States. The rate of AD is estimated to double by 2050. The neurodegeneration of AD occurs decades before symptoms of dementia are evident. Therefore, having an efficient methodology for the early and proper diagnosis can lead to more effective treatments. Neuroimaging techniques such as magnetic resonance imaging (MRI) can detect changes in the brain of living subjects. Moreover, medical imaging techniques are the best diagnostic tools to determine brain atrophies; however, a significant limitation is the level of training, methodology, and experience of the diagnostician. Thus, Computer aided diagnosis (CAD) systems are part of a promising tool to help improve the diagnostic outcomes. No publications addressing the use of Feedforward Artificial Neural Networks (ANN), and MRI image attributes for the classification of AD were found. Consequently, the focus of this study is to investigate if the use of MRI images, specifically texture and frequency attributes along with a feedforward ANN model, can lead to the classification of individuals with AD. Moreover, this study compared the use of a single view versus a multi-view of MRI images and their performance. The frequency, texture, and MRI views in combination with the feedforward artificial neural network were tested to determine if they were comparable to the clinician’s performance. The clinician’s performances used were 78 percent accuracy, 87 percent sensitivity, 71 percent specificity, and 78 percent precision from a study with 1,073 individuals. The study found that the use of the Discrete Wavelet Transform (DWT) and Fourier Transform (FT) low frequency give comparable results to the clinicians; however, the FT outperformed the clinicians with an accuracy of 85 percent, precision of 87 percent, sensitivity of 90 percent and specificity of 75 percent. In the case of texture, a single texture feature, and the combination of two or more features gave results comparable to the clinicians. However, the Gray level co-occurrence matrix (GLCOM), which is the combination of texture features, was the highest performing texture method with 82 percent accuracy, 86 percent sensitivity, 76 percent specificity, and 86 percent precision. Combination CII (energy and entropy) outperformed all other combinations with 78 percent accuracy, 88 percent sensitivity, 72 percent specificity, and 78 percent precision. Additionally, a combination of views can increase performance for certain texture attributes; however, the axial view outperformed the sagittal and coronal views in the case of frequency attributes. In conclusion, this study found that both texture and frequency characteristics in combinations with a feedforward backpropagation neural network can perform at the level of the clinician and even higher depending on the attribute and the view or combination of views used.

Fluorescence microscopy is an essential tool in biomedical sciences that allows specific molecules to be visualized in the complex and crowded environment of cells. The continuous introduction of new imaging techniques makes microscopes more powerful and versatile, but there is more than meets the eye. In addition to develop- ing new methods, we can work towards getting the most out of existing data and technologies. By harnessing unused potential, this work aims to increase the richness, reliability, and power of fluorescence microscopy data in three key ways: through standardization, evaluation and innovation. A universal standard makes it easier to assess, compare and analyze imaging data – from the level of a single laboratory to the broader life sciences community. We propose a data-standard for fluorescence microscopy that can increase the confidence in experimental results, facilitate the exchange of data, and maximize compatibility with current and future data analysis techniques. Cutting-edge imaging technologies often rely on sophisticated hardware and multi-layered algorithms for reconstruction and analysis. Consequently, the trustworthiness of new methods can be difficult to assess. To evaluate the reliability and limitations of complex methods, quantitative analyses – such as the one present here for the 3D SPEED method – are paramount. The limited resolution of optical microscopes prevents direct observation of macro- molecules like DNA and RNA. We present a multi-color, achromatic, cryogenic fluorescence microscope that has the potential to produce multi-color images with sub-nanometer precision. This innovation would move fluorescence imaging beyond the limitations of optics and into the world of molecular resolution.

The number of motor proteins attached to cellular cargos is widely believed to influence intracellular transport processes and may play a role in transport regulation. However, to date, investigating the biophysics of multiple-motor dynamics has been challenging since the number of motors responsible for cargo motion is not easily characterized. This work examines the transport properties of structurally-defined motor complexes containing two kinesin-1 motors, from both an experimental and theoretical perspective. Motor complexes were synthesized using DNA as a molecular scaffold and engineered DNA-conjugated protein polymers as linkers to couple motors to scaffolds. After anchoring the motor complexes to a bead their dynamic properties were measured using an automated optical trapping instrument that could be used to perform both static (increasing load) and force-feedback (constant load) optical trapping experiments. Data from these experiments is compared to predictions from a microscopic transition rate model of multiple kinesin dynamics. Together, these studies uncovered that multiple kinesins typically cannot cooperate since the microtubule-bound configuration of a motor complex often prevents both kinesins from sharing cargo loads. Furthermore, multiple-motor behaviors are influenced by the fact that motor complexes display hysteretic force-velocity behaviors when applied loads change rapidly in time. Overall, such behaviors suggest the number of kinesins on a cargo will not be a key determinant of intracellular transport processes, and in turn, will not contribute appreciably to mechanisms that regulate cargo motion. However, this work also provides evidence that processive microtubule motors that are less efficient than kinesin (e.g., dynein) will cooperate productively, produce greater responses to motor number, and may therefore act as a regulator of cargo transport.

The work is evaluated a behaviour of systems utilizing a solar radiation and also looking on concordance rate between a real systems and their dynamics mathematical models. There are compare systems ? biotechnology hall with concentration photobiorectors in Nové Hrady and object of modular greenhouse in Třeboň. The simulation was created in TRNSYS (TRaNsient SYstem Simulation) program and a result from simulation was compare with real data measured in these objects. The original Czech technical components (concentration collector SG1 and passive optical rasters) were implements to TRNSYS like owners module for better describe of monitored objects. It was compared process which running in real time and describes dynamics behaviour of objects, mainly inertia and speed of developed changes. A lot of parameters influence of these processes (lay of accumulation mass, size of translucent area, orientation, shading ...). The first part of thesis consist of describe of measurement and thermal rate in monitored objects. The focus of work in the second one was aim for build of mathematical models of the monitored objects, describe its cardinal functions and technologies. The result is qualification evaluation of energy rates in systems and verifications of mathematical models, so it is mean a compare of calculated and measured values, also correction of parameters inputs to models and recommendation for user of the raster systems.

博士
國立交通大學
光電工程系所
95
Optical tweezers are appropriate for molecular or cell biology research because their properties of non-invasive and non-mechanical contact to biological sample and the trapping force is compatible to lots of biological materials’ forces. In this thesis, we used optical tweezers to study the adhesion between a single Klebsiella pneumoniae type 3 fimbria and collagen IV. Different MrkD adhesin variants on the fimbriae are known to display distinct adherence capability for the bacteria to bind extracellular matrix proteins. For this reason, we measured the adhesive force between different MrkD adhesin variants and collagen IV for the first time. The MrkDv1 adhesin and collagen IV are nearly not adhered. The adhesive force between each of the single fimbria carrying MrkDv2, MrkDv3, and MrkDv4 adhesin variants and collagen IV are 2.03 ± 0.03 pN, 3.79 ± 0.12 pN, and 2.87 ± 0.15 pN, respectively. We also used optical tweezers to measure the adhesive force between integrinαIIbβ3-expressing CHO cells and rhodostomin. The interesting result shows that the adhesive force of wild type and mutant rhodostomin are 4.5 pN and 1,81 pN, respectively. Since a molecular coated bead is usually used in the optical tweezers experiments. The tiny displacement of the bead in the optical trap needs to be precisely detected. In the previous biological experiments, we only measured the 1-D displacement. However, we realized that the 3-D displacements of the bead should be considered. Therefore, we built a single particle tracking (SPT) system which is capable of high spatial and temporal resolutions. Our SPT system has a spatial resolution of 5.5 nm in transverse direction and 11.5 nm in axial direction. If only 2D tracking of the bead is considered, the tracking range can reach 300 nm 300 nm. If the 3D tracking of the bead is needed, the tracking range would reduce to 200 nm 200 nm 200 nm. At last, we setup a dual beam optical tweezers system. At least one optical trap can be arbitrarily moved in the field of view. By delicately designing the methodology, the adhesion or extension experiment of biological materials may only need 2D or 1D tracking of the bead. Thus, the tracking range may increase. We sincerely hope that this dual beam optical tweezers with a SPT system can be applied to more adhesion and extension properties studies in the near future.

"Laser flash spectroscopy of photosystem II" Photosystem II (PS II) of plants and cyanobacteria oxidizes water in a light-powered reaction. Thereby, this protein is the ultimate source of the atmospheric oxygen. The capacity to oxidize water is owed to two properties of PS II: (i) The midpoint potential of the oxidizing chlorophyll moiety is increased by 0.6 V compared to photosystem I or photochemical reaction centers of anoxygenic bacteria, and (ii) the energy requirements of the four steps needed for the tetravalent oxidation of water are adapted to the energy of red light quanta. This thesis deals with two particular aspects, namely: 1. The coupling of the electron transfer from tyrosine Z (YZ) to the primary donor (P680+) to proton transfer, and an inquiry on the role of a positive charge on YZox (plus base cluster) in increasing the oxidizing potential at the catalytic site. 2. The localization of the electron hole, P680+, among the excitonically coupled four inner chlorophyll a molecules, and an estimation of the midpoint potential differences between them. Electron-proton-coupling by YZ This study was carried out with PS II core complexes from spinach or pea with a deactivated (removed) manganese cluster. The reduction of P680+ was investigated as a function of pH by detecting the laser flash induced absorption changes with nanosecond resolution. Two kinetic components were found with different pH-dependence and activation energies. The alteration of kinetic parameters by H/D isotope substitutions or by addition of divalent cations implied two different types of YZ-oxidation: At acidic pH the electron transfer was coupled with proton transfer, whereas in the alkaline region it was more rapid and no longer controlled by proton transfer. The conversion between both mechanisms occured at pH 7.4. This value corresponds either to the apparent pK of YZ itself (i.e. of the hydroxy group of the phenol ring) or to the pK of an acid-base-cluster, which includes YZ. Independent measurements of pH-transients by following the absorption changes of hydrophilic proton indicators corroborated this notion. The data were interpreted as indicating that the phenolic proton of YZ was released into the medium at acidic, but not at alkaline pH. The electron transfer and proton release characteristics of intact, oxygen-evolving PS II resembled those in deactivated samples kept at alkaline pH. We concluded that the electron transfer from YZ to P680+ in the native system was not coupled with proton transfer into the bulk. This has shed doubt on a popular hypothesis on the role of YZ as 'hydrogen abstractor' from bound water. On the other hand, the energetic constraints of water oxidation could be eased by the positive upcharging during oxidation of YZox plus its base cluster. On the localization of the electron hole of P680+ Photooxidation of PS II oxidizes the set of four innermost chlorophyll a molecules giving rise to the only spectroscopically defined species P680+. The deconvolution of difference spectra into bands of pigments is ambiguous. By using photoselective excitation of antennae, i.e. chl a molecules with site specific energies at the long wavelength border of the mean Qy-band, and by polarized detection, it was possible to tag P680+QA-/P680QA and 3P680/P680 difference spectra with a further parameter, the (wavelength-dependent) anisotropy r. Results obtained at liquid nitrogen temperature (77 K) can be clearly interpreted in terms of two chl a monomer bands. The two main components of the P680+QA-/P680QA difference spectrum were marked by two distinct values of the anisotropy and could be interpreted in a straightforward manner: the bleaching of a band at 675 nm belonging to the charged species (chl a+) and an electrochromic blue-shift of a nearby chl a from 684 to 682 nm. The main bleaching band of the 3P680/P680 spectrum (at 77 K) can be apparently attributed to a third (or several) chl a component(s). The analysis of the P680+QA-/P680QA spectrum at cryogenic temperature is compatible with monomeric chl a bands. On the other hand, one could assume a system of excitonically coupled core pigments, as it was recently introduced in the literature on the basis of energy transfer studies ('multimer model'). However, in view of the clear indications for an electrochromic band shift and the location of the bleaching band, which absorbs in a wavelength region of monomeric chl a, one assumption of the 'multimer model' should be questioned. Presumably, the excitonic couplings are rather weak, in particular between each of the two central chl a-molecules (PA/PB) and its respective accessory chl a (BA/BB), because of (i) the distances and (ii) different site energies of the monomeric chromophores. At room temperature, the absorption difference and anisotropy spectra of P680+QA-/P680QA were clearly altered. The anisotropy data indicated that the changes could no longer exclusively be ascribed to thermal broadening of individual bands. The localization of the positive charge on one pigment, analogous to the situation at 77 K, was now unlikely. Hence, the midpoint potential differences between the inner four chlorophyll a molecules were small and were estimated as approximately 15 meV.