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Agradecemos a compreensão. on 2017-06-28T20:48:12Z (GMT) / Submitted by Vinicius Tanajura (vtanajura@hotmail.com) on 2017-07-02T23:28:36Z No. of bitstreams: 1 Dissertação_Vinicius_Tanajura_março_2017.pdf: 1399039 bytes, checksum: 7551679061f33e5a440410c9a4f1e486 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-07-03T15:23:07Z (GMT) No. of bitstreams: 1 tanajura_vs_me_bauru.pdf: 1399039 bytes, checksum: 7551679061f33e5a440410c9a4f1e486 (MD5) / Made available in DSpace on 2017-07-03T15:23:07Z (GMT). No. of bitstreams: 1 tanajura_vs_me_bauru.pdf: 1399039 bytes, checksum: 7551679061f33e5a440410c9a4f1e486 (MD5) Previous issue date: 2017-03-31 / A partir de experiências vividas por meio da prática docente, bem como da análise de registros sobre o ensino e aprendizagem dos conteúdos referentes à Biologia em nível celular, é possível a identificação de diversas dificuldades enfrentadas por docentes e estudantes ao longo desse processo. Tais dificuldades variam desde condições estruturais inadequadas das escolas, até problemas relativos à formação daqueles profissionais, políticas públicas referentes à Educação, ou mesmo obstáculos pedagógicos relacionados às estratégias de ensino comumente adotadas. Assim, muitos desses fatores caracterizam-se como desafios a serem superados pelos sujeitos envolvidos em tal processo. Em vista da importância desses conceitos científicos e do insucesso em seu processo ensino-aprendizagem na escola básica, registrado pelas pesquisas da área, a presente pesquisa teve como objetivo identificar as principais dificuldades e desafios enfrentados por professores(as) de Biologia de escolas públicas de Ensino Médio do Estado de São Paulo, ao ensinarem conceitos biológicos de nível celular. Os dados que compuseram o corpus desta pesquisa foram gerados a partir de entrevistas semiestruturadas com um professor e quatro professoras de Biologia, todos atuantes no Ensino Médio de escolas públicas situadas em duas cidades do interior paulista. A análise do conteúdo dessas entrevistas foi feita sob a perspectiva de Laurence Bardin (1979). Após categorização dos dados, foram identificadas sete categorias referentes à natureza das dificuldades apontadas pelas entrevistas. Os dados revelaram interferências no referido processo oriundas tanto do contexto político-administrativo, como da ação prática docente, que em geral, apresenta-se pouco embasada por resultados de pesquisas científicas da área de Ensino de Ciências e Educação, revelando não ser essa a principal fonte de pesquisa a que professores(as) recorrem pra tentarem superar possíveis obstáculos pedagógicos. Destaca-se também a grande dificuldade imposta pelo caráter dos conceitos estudados e o baixo repertório metodológico e epistemológico para a superação desse tipo de limite, apresentado pelo grupo docente entrevistado. Após essas análises, chama-se a atenção para a urgência em relação a alterações no contexto escolar que resultem em mudanças profundas na qualidade da formação docente e da aprendizagem escolar, especificamente dos conteúdos da Biologia celular. / From the experiences lived through the teaching practice, as well as the analysis of records on the teaching and learning of the contents related to Biology at the cellular level, it is possible to identify several difficulties faced by teachers and students throughout this process. Such difficulties range from inadequate structural conditions in schools, to problems related to the training of those professionals, public policies concerning education, or even pedagogical obstacles related to the teaching strategies commonly adopted. Then, many of these factors are characterized as challenges to be overcome by the subjects involved in such a process. In view of the importance of these scientific concepts and the failure in their teaching-learning process in the basic school, registered by the area's research, This research aimed to identify the main difficulties and challenges faced by Biology teachers of Public Schools of High School in the São Paulo state, teaching biological concepts at the cellular level. The data that composed the corpus of this research were generated from semistructured interviews with a male teacher and four female Biology teachers, all of whom are active in the High School of public schools located in two cities of São Paulo state. The analysis of the content of these interviews was done from the perspective of Laurence Bardin (1979). After categorizing the data, seven categories were identified regarding the nature of the difficulties identified by the interviews. The data revealed interferences in the aforementioned process stemming from both the political-administrative context and the practical teaching action, which in general is not supported by results of scientific research in the area of Science and Education, revealing that this is not the main source of research to which teachers resorts to overcome possible pedagogical obstacles. The great difficulty imposed by the character of the concepts studied and the low methodological repertoire are also highlighted. Epistemological approach to overcome this type of limit, presented by the teacher group interviewed After these analyzes, attention is drawn to the urgency regarding changes in the school context that result in profound changes in the quality of teacher training and school learning, specifically the contents of cellular biology.

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.