Over the past three decades in cellular neuroscience there has been a shift towards the view of the 'tripartite synapse', where, astrocytes -- as well as the pre-synapse and post-synapse -- are involved in synaptic signalling. The migration of astrocytes to form branched networks in the brain is, therefore, of great interest in understanding brain development and neuronal function. Migration is a complex interplay between cytoskeletal reorganisation and cell mechanical stiffness. In order to improve understanding of this process, correlative measurements of cytoskeletal organisation and mechanical stiffness are required. To investigate astrocyte migration a technique combining atomic force microscopy (AFM) with stimulated emission depletion (STED) microscopy was developed. First a custom STED microscope was developed. To facilitate the design of this system the theoretical performance of a range of STED techniques (cw-STED, time-gated STED, pulsed STED and RESOLFT) were compared, identifying that pulsed STED theoretically has the highest photon efficiency. A pulsed STED microscope, which uses adaptive optics, was then designed, developed and characterised. The microscope was found to achieve resolutions below 50 nm. The STED microscope was combined with a commercial AFM to study live cells. Using the recently developed SiR-actin and SiR-tubulin dyes and AFM probes optimised for live cell mechanical property studies, images of the actin and tubulin cytoskeleton were correlated with AFM topography and mechanical stiffness measurements. It was found that, in astrocytes, actin contributes significantly both to astrocyte stiffness and topography. Investigations of migrating cells showed differences in actin organisation and mechanical stiffness between the basis and leading edge of migration. A further study was performed, investigating the effects of the gap-junction protein connexin30, which is expressed during the early stages of brain development, on migration. This protein was found to inhibit the actin reorganisation and mechanical stiffness changes observed in basal conditions. Overall the combination of mechanosensitive AFM measurements with advanced microscopy, such as super-resolution, on live cells is a promising approach which will enable a range of investigations, for instance when studying cell structural remodeling during brain development or tumorigenesis.

Made available in DSpace on 2014-06-11T19:33:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-04-03Bitstream added on 2014-06-13T19:04:03Z : No. of bitstreams: 1 arruda_em_dr_rcla.pdf: 9662595 bytes, checksum: 0ed9158bd4d58632c075670ed3c2f2ba (MD5) / A pesquisa analisa os eventos deposicionais, esculturadores do relevo, na bacia do Ribeirão Entupido, Complexo Alcalino do Passa Quatro, Estado de São Paulo, a partir do estudo dos depósitos recentes da área. Além da análise geomorfológica na referida unidade espacial, a pesquisa buscou estabelecer a idade absoluta de deposição de algumas unidades sedimentares estruturadoras do relevo e os mecanismos envolvidos na sua gênese. Neste contexto, utilizou-se datações por Luminescência Opticamente Estimulada (LOE) e Termoluminescência (TL). Os depósitos sedimentares foram estudados a partir da abordagem morfoestratigráfica. O cruzamento de dados morfoesculturais e morfoestruturais e de datação absoluta permitiu estabelecer quais mecanismos, atuantes ao longo do último milhão de anos, comandaram a morfogênese na bacia em foco. A determinação da morfogênese permitiu definir a susceptibilidade do relevo às mudanças ambientais, inclusive às intervenções antrópicas. Os depósitos sedimentares apresentaram idades vinculadas ao Pleistoceno Superior (61.500 anos) e Holoceno (650 anos) representando, portanto, eventos diferenciados no desenvolvimento e localização deste ambientes. A complexidade da paisagem na bacia do Ribeirão Entupido envolve justamente esta interdigitação de materiais de idades diversas que compõem o mesmo ambiente de deposição, interferindo conjuntamente a tectônica e as condições climáticas, a priori, na espacialização dos depósitos e nas características dos sedimentos. / This research of doctor’s grade analyze the depositional and sculptural events, at stream Entupido basin, Passa Quatro Alkalin Complex, Estate of São Paulo, from the study of recent deposits.Beyond the geomorphologycal analysis at referred spacial unit, this research tried to establish the absolute age of some sedimentary units that structure the relief and the mechanism involved in its genesis. The dating method of Optically Stimulated Luminescence (OSL) Thermoluminescence (TL) was used in that context.The sedimentary deposits were studied by morphostratigraphic approach. The crossing of mophosculptural and moprostructural data and absoluting dating allow establish wich mechanism, acting through the last million years, commanded the morphogenesis process at the basin.The determination of morfogenesis allowed define the susceptilidy of relief to ambiental changes, including antropic intervention. The sedimentary deposits show ages linked at Up Pleistocene (61.500 years) and Holocene (650 years), showing, therefore, differenced events in development and localization of this environment.The landscape complexity in stream Entupido basin, involves precisely this interdigitation of materials and different ages that compose the same depositional environment, interfering jointly tectonic and climatic conditions, in prior, at specialization of deposits and sedimentary characteristics.

Ce travail de thèse présente une combinaison unique d'imagerie X par contraste de phase avec la fluorescence X pour des échantillons biologiques étudiés par nanosonde par fluorescence X excitée par le rayonnement synchrotron. Les récents développements dans ce domaine ouvrent la possibilité d'une imagerie chimique quantitative à l'échelle sub-cellulaire. Ceci a été rendu possible par l'utilisation d'un outil unique qui est la station de nanoimagerie X ID22NI de l'ESRF qui permet de délivrer un faisceau sub-100 nm avec un très haut flux à haute énergie entrainant une sensibilité très haute, de l'ordre de quelques centaines d'atomes pour différents éléments (Fe, Cu, Zn…). Le couplage des informations issues de l'imagerie X par contraste de phase (masse surfacique de la cellule) et de la fluorescence X (masse surfacique des éléments chimiques) a pu être obtenu pour la première fois donnant accès à une cartographie des éléments chimiques constituant les cellules et de leurs fractions massiques absolues associées. Dans l'immédiat, il n'a été possible d'étudier des cellules qui ont été congelées rapidement puis lyophilisées, cependant, une nouvelle ligne de nanoimagerie, NINA, en construction à l'ESRF, fonctionnera comme un cryomicroscope et permettra l'analyse 2D/3D d'échantillons biologiques ou non congelés hydratés. L'extension de l'imagerie chimique 2D présentée dans ce travail à une imagerie 3D représente une importante avancée pour bon nombre de problématiques scientifiques en biologie. Une des limitations de ce type d'analyse est celle des dommages radio-induits à la suite de l'irradiation de l'échantillon par un haut flux de particules ionisantes. Il existe que peu ou pas d'étude sur les effets de la nanoanalyse par fluorescence X sur les cellules lyophilisées. Nous avons combiné l'imagerie de phase à l'imagerie par fluorescence X ce qui nous permis de conclure à une rétractation des structures cellulaires accompagnée d'une volatilisation des éléments du fait de l'irradiation lors de l'analyse par fluorescence X. Ces aspects ont été confortés par des analyses utilisant une technique complémentaire non-synchrotron de microscopie ionique en transmission et à balayage (STIM). Plus important encore, nous apportons ainsi un outil rapide et non-destructif pour la cellule (imagerie X de phase) qui permet de corriger la perte de masse due à la volatilisation d'éléments légers (C, H, O, N) de la matrice cellulaire. Cette démarche permet de fiabiliser l'analyse quantitative de la composition chimique cellulaire. Cette approche sera précieuse pour corriger ces effets de perte de masse lors de futures analyses tomographiques de cellules entières congelées hydratées. Nous avons également contribué à l'étude de distribution intracellulaire de nouvelles nanoparticules d'or ou de platine fonctionnalisées. Nous avons pu exploiter les données issues de la fluorescence X pour estimer le nombre de nanoparticules et la taille des clusters internalisés au sein des cellules. Toutefois, des expériences dédiées pour des analyses sur un plus grand nombre de cellules auxquelles l'imagerie X par contraste de phase serait menée en parallèle permettraient surement de préciser plus finement ces aspects quantitatifs sur le nombre de nanoparticules intracellulaires. Dans l'ensemble ce travail ouvre la possibilité d'une imagerie chimique quantitative absolue sub-cellulaire en 2D ou 3D avec la perspective d'imagerie corrélative avec de nombreuses techniques complémentaires notamment la microscopie électronique à transmission pour l'ultrastructure, la microscopie de fluorescence pour la localisation de proteines d'intérêts et d'autres techniques d'analyses chimiques telles le NanoSIMS ou le nano-PIXE. / This work presents some recent developments in the field of hard X-ray imaging appliedto biomedical research. As the discipline is evolving quickly, new questions appear andthe list of needs becomes bigger. Some of them are dealt with in this manuscript.It has been shown that the ID22NI beamline of the ESRF can serve as a proper experimentalsetup to investigate diverse aspects of cellular research. Together with its highspatial resolution, high flux and high energy range the experimental setup providesbigger field of view, is less sensitive to radiation damages (while taking phase contrastimages) and suits well chemical analysis with emphasis on endegeneous metals (Zn, Fe,Mn) but also with a possibility for for exogoneous one’s like these found in nanoparticles(Au, Pt, Ag) study.Two synchrotron-based imaging techniques, fluorescence and phase contrast imagingwere used in this research project. They were correlated with each other on a numberof biological cases, from bacteria E.coli to various cells (HEK 293, PC12, MRC5VA,red blood cells).The explorations made in the chapter 5 allowed preparation of more establishedand detailed analysis, described in the next chapter where both techniques, X-ray fluorescenceand phase contrast imaging, were exploited in order to access absolute metalprojected mass fraction in a whole cell. The final image presents for the first timetrue quantitative information at the sub-cellular level, not biased by the cell thickness.Thus for the first time a fluorescence map serves as a complete quantitative image of acell without any risk of misinterpretation. Once both maps are divided by each otherpixel by pixel (fluorescence map divided by the phase map) they present a completeand final result of the metal (Zn in this work) projected mass fraction in ppm of dryweight. For the purpose of this calculation the analysis was extended to calibration(non-biological) samples. Polystyrene spheres of a known diameter and known densityworked very well here and allowed validation of the presented method. Different images(phase map, AFM, STIM) and profiles were compared and statement on the high accuracyof phase contrast imaging for the thickness/structures determination was made.The result on true metal projected mass fraction represents a first step to an absolutesub-cellular analysis and certainly can be improved to even closer reflect on reality.All the measurements were taken on freeze-dried cells. Thus the result is in ppm ofdry weight. In fact the measurement would have even deeper meaning if it was madeon hydrated cells. For the moment this is not possible with the existing setup of theID22NI beamline but will be possible in the future with a new beamline devoted tonano science - NINA (Nano-Imaging and Nano-Analysis). The new beamline will befurnished with a cryostage and X-ray imaging will be made on frozen-hydrated samples.Nevertheless the analysis presented in this manuscript is of undeniable importance toboth the biomedical community and to the ESRF team engaged in the NINA development.To answer the problems of cell irradiation both imaging techniques were exploitedagain. Repeating the phase contrast imaging after the fluorescence scanning allowedto show the changes induced by radiation damage during X-ray fluorescence scan. Thechanges were not only clearly visible but could be as well quantified. Together with thenumerical evaluation of damages, the dose delivered to a cell during the experiment was calculated as well. To complete the picture, a different non synchrotron-basedimaging technique, STIM, was used and compared. It is the first time that phase contrastimaging is used to monitor radiation damage effects during X-ray fluorescencemicroscopy experiments.

La dispersion est définie comme le mouvement d'un individu entre le site de naissance et le premier site de reproduction ou entre deux sites de reproduction. La dispersion se traduit par des échanges d'individus et des flux de gènes entre les populations et est donc reconnue comme un trait d'histoire de vie clé de part son rôle déterminant sur de nombreux processus écologiques et évolutifs comme la dynamique ou la génétique des population, la répartition spatiale des espèces ou encore la capacité des espèces à faire face aux changements brutaux induits par les activités humaines. Pourtant les conséquences de la dispersion en terme de valeur sélective individuelle restent mal connues malgré leur importance dans l'évolution de la dispersion. Le but de cette thèse est d'identifier plus précisément les conséquences de la dispersion en terme de valeur sélective individuelle en utilisant à la fois des approches corrélative et expérimentale dans une population sauvage de passereaux migrateurs, le gobe mouche à collier (Ficedula albicollis). Grâce à des données à long terme comprenant plus de 20 ans de suivi, des différences entre les individus dispersants et philopatriques ont pu être mises en évidence à la fois à l'échelle de la vie des individus et à l'échelle annuelle, celle de l'événement de reproduction. Les résultats mettent en évidence des effets de la dispersion dépendant à la fois des conditions et du phénotype des individus et soulignent donc le fait que la balance entre les coûts et les bénéfices est le résultat d'interactions subtiles entre l'environnement et les caractéristiques de l'individu. D'autre part, l'expérience de dispersion forcée a permis de démontrer clairement l'existence de coûts liés à l'établissement dans un environnement non familier que seuls certains individus sont capables de surmonter. Enfin, l'absence de différence dans les décisions majeures de reproduction une fois les individus établis, suggère que la dispersion doit majoritairement être adaptative, une fois les coûts de l'installation surmontés / Dispersal is commonly defined as the movement of an individual from its natal or previous breeding site to a new breeding site. Because dispersal involves movements of individuals and genes among populations, it is widely recognized as a key life history trait with strong effects on many ecological and evolutionary processes, such as population dynamics and genetics but also species spatial distribution or response to brutal environmental variations induced by human activities. Yet, the consequences of dispersal in terms of individual fitness remain poorly understood despite their crucial importance in the understanding of the evolution of dispersal. The aim of this PhD is to get better insights in the fitness consequences of dispersal using both correlative and experimental approaches at different scales, i.e. annual and lifetime scales, in a wild patchy population of migratory passerine bird, the collared flycatcher (Ficedula albicollis). Using a long term data set encompasses more than 20 years of data, differences between dispersing and philopatric individuals were showed both at a lifetime and annual scale. The results highlighted strong phenotypic- and condition-dependent effects of dispersal and underline that the balance between the costs and benefits of dispersal is likely to be the result of subtle interactions between environmental factors and individuals’ phenotype. Moreover, the experiment of forced dispersal demonstrated that dispersal might entail costs linked with settlement in a new habitat, which only some individuals may overcome. Nevertheless, the absence of difference in major fitness related decisions after settlement suggests that dispersal is mostly adaptive for individuals overcome such costs

This thesis is dealing with application of Correlative Probe and Electron Microscopy. All measurements were carried out by atomic force microscope LiteScope which is designed especially to be combined with electron microscopes. Advantages of Correlative AFM/SEM Microscopy are demonstrated on selected samples from field of nanotechnology and material science. Application of the correlative imaging was proposed and then realized particularly in case of low-dimensional structures and thin films. Further, this thesis deals with the possibility of combining Correlative AFM/SEM Microscopy with other integrated techniques of an electron microscope such as Focused Ion Beam and Energy Dispersive X-rays Spectroscopy.

High pressure on the development of new magnetic materials and their miniaturization also emphasizes the development of new analytical techniques. This diploma thesis deals with the development and demonstration of correlated magnetic force and electron microscopy, which is a promising tool for the characterization of magnetic nanomaterials. The first part of this thesis describes the fundamental physics of micromagnetism with a focus on cylindrical nanofibers. The following pages describe optic, probe, electron, and synchrotron methods for mapping the magnetic properties of materials. The next part describes magnetic domain wall motion in cylindrical nanowires performed as a part of a more extensive material study. The last part of the thesis describes the development of correlated magnetic force and electron microscopy on LiteScope device. A production of magnetic probes was designed and successfully tested. Probes were fabricated by focused electron beam-induced deposition from the Co2(CO)8 precursor. Further, the developed correlated microscopy is demonstrated on a multilayer PtCo sample, magnetic cylindrical nanofibers, NiFe vortex structures, and FeRh metamagnetic nano-islands.

The diploma thesis deals with an assessing selected indicators of a KAVALE company s. r. o. using time series analysis. Issues of financial analysis, analysis of period lines, regressive analysis and correlative analysis are described in a theoretical part. Practical part is focused on statistic analysis of selected indicators of financial analysis. Its aim is to reveal dependance between indicators and prediction of their future development for the next two periods of time. Comparison of the analyzed company with an average sector and with two rival companies is also part of the practical section. On the basis of these analyzes I recommend proposals to improve the financial situation of the company.

Le système de sécrétion type VII (T7SS) présent chez les mycobactéries comporte cinq loci, nommés ESX-1 à ESX-5, chacun possédant leurs propres fonctions. Le système ESX-3, le plus conservé entre les espèces de mycobactéries, participe au transport du fer et à la sécrétion de protéines dont la protéine EsxH. EsxH interagit avec le système ESCRT dans le macrophage et contribue à la persistance de M. tuberculosis (Mtb) dans l’hôte. Afin de mieux comprendre le mécanisme du T7SS, nous avons surexprimé le locus ESX-3 de Mtb dans les organismes recombinants M. smegmatis et M. marinum. Nous avons purifié un coeur protéique partiel du complexe ESX-3 par chromatographie liquide de protéine rapide (FPLC) et déterminé différentes structures in vitro qui pourraient représenter son dynamisme par une analyse des particules individuelles. Nous avons localisé le complexe ESX-3 aux pôles chez M. marinum par microscopie de fluorescence (fLM) et microscopie super-résolution d’illumination de structure (SR-SIM). Finalement, nous avons reconstruit un modèle in vivo 3D de ce complexe en jumelant une technique de corrélation entre la microscopie par localisation photoactivée (PALM) et la tomographie électronique en conditions cryogéniques (cryo-ET). En jumelant nos structures in vitro et notre modèle in vivo, nous discutons d’un mécanisme possible du complexe ESX-3. Ces analyses pourront aussi supporter le criblage d’inhibiteurs potentiels pour traiter les infection mycobactériennes. / A novel, type VII secretion system (T7SS) was recently discovered in Mycobacteria. Five subsystems, called ESX-1 to ESX-5, have been identified, with the ESX-3 being the most conserved. The ESX-3 system is essential for growth and pathogenesis, and has been implicated in iron transport and secretion of effector proteins into the host. The secreted proteins are shown to prevent phagosome maturation by interacting with the ESCRT machinery of the macrophage. To characterize the structure and mechanism of secretion employed by the T7SS, we use the ESX-3 system from Mycobacterium tuberculosis (Mtb) and recombinantly express it in M. smegmatis and M. marinum cells. By combining Fast Protein Liquid Chromatography (FPLC) and Single Particle electron microscopy analysis, we have reconstructed several in vitro models that contain at least three of the ESX-3 cluster proteins and may represent the dynamic nature of the core complex. Using fluorescent light microscopy (fLM) and super-resolution structure illumination microscopy (SR-SIM), we localized the ESX-3 complex to the lateral pole in M. marinum cells. We also used super-resolution photoactivated localization microscopy (PALM) in correlation with cryo-electron tomography (cryo-ET) of whole M. marinum cells to localize and reconstruct an in vivo 3D model of the ESX-3 secretion system. By combining the single particle reconstructions and the cryotomography data, we discuss a possible mechanism of ESX-3 secretion. Such analysis may support future inhibitor screens to prevent mycobacterial infections.

Uppsatsen är en retoriskt studie av T.S. Eliots begrepp objektiva korrelat; ett litterärt begrepp som, enligt Eliot, var det konstfulla sättet att framställa känslor på. Känslan skulle framställas i dikt genom att en objektiv framställning, Detta innebar att känslan skulle ha en konkret motsvarighet i dikten, exempelvis skulle den inkapslas i ett objektiv, en situation eller händelsekedja. Detta skulle i sin tur omedelbart väcka känslan hos läsaren. Uppsatsens syfte är att undersöka och visa att det objektiva korrelatet förutsätter en retorisk kommunikationsmodell. Detta görs mot bakgrunden till att det fanns en modernistisk uppfattning om retoriken som skadligt, något som litteraturen skulle befrias ifrån. För att uppfylla syftet undersöks text-, författare-, och läsarfunktionen, vilka kan förstås som viktiga delar i en kommunikationsmodell.   Studien visar att begreppet delar flera likheter med och förstås utifrån flera retoriska teorier, exempelvis Chaïm Perelmans auditoriebegrepp, Mats Rosengrens definition av doxa och Wayne C. Booths begrepp telling och showing. Det objektiva korrelatet kan förstås som en poetisk argumentation som ämnar påverka läsarna, men vars funktion också är avhängig publiken då författaren måste förhålla sig till de kollektiva referenspunkter som publiken håller gemensamt. Studien visar att de kommunikationsparameterar som undersöks är av retorisk karaktär och såldes förutsätter begreppet en retorisk kommunikationsmodell.

Direction Finding (DF) algorithms are used by the Swedish Defence Research Agency (FOI) in the context of electronic warfare against radio. Parallelizing these algorithms using a Graphics Processing Unit (GPU) might improve performance, and thereby increase military support capabilities. This thesis selects the DF algorithms Correlative Interferometer (CORR), Multiple Signal Classification (MUSIC) and Weighted Subspace Fitting (WSF), and examines to what extent GPU implementation of these algorithms is suitable, by analysing, implementing and evaluating. Firstly, six general criteria for GPU suitability are formulated. Then the three algorithms are analyzed with regard to these criteria, giving that MUSIC and WSF are both 58% suitable, closely followed by CORR on 50% suitability. MUSIC is selected for implementation, and an open source implementation is extended to three versions: a multicore CPU version, a GPU version (with Eigenvalue Decomposition (EVD) and pseudo spectrum calculation performed on the GPU), and a MIXED version (with only pseudo spectrum calculation on the GPU). These versions are then evaluated for angle resolutions between 1° and 0.025°, and CUDA block sizes between 8 and 1024. It is found that the GPU version is faster than the CPU version for angle resolutions above 0.1°, and the largest measured speedup is 1.4 times. The block size has no large impact on the total runtime. In conclusion, the overall results indicate that it is not entirely suitable, yet somewhat beneficial for large angle resolutions, to implement MUSIC using GPU computing.