Chloroplasts divide by a constrictive fission process that is regulated by FtsZ proteins. Given the importance of photosynthesis and chloroplasts in general, it is important to understand the mechanisms and molecular biology of chloroplast division. An FtsZ gene is known to be of prokaryotic origin and to have been transferred from a symbiont's genome to host genome via lateral transfer. Subsequent duplication of the initial FtsZ gene gave rise to the FtsZ1 and FtsZ2 genes and protein families in eukaryotes. These proteins co-localize mid-chloroplast to form the Z-ring. Z-ring assembly initiates chloroplast division, and it serves as a scaffold for other chloroplast division proteins. Little is known, however, about the FtsZ protein subunit turnover within the Z-ring, the effects of accessory proteins on Z-ring turnover assemblies, as well as the in vivo ultrastructure of the Z-ring in plants. To investigate the Arabidopsis thaliana FtsZ subunit turnover rate within the Z-ring, a section of the Z-ring in the chloroplasts of living plants expressing fluorescently tagged FtsZ1 or FtsZ2 proteins was photobleached and the recovery rate was monitored. The results show that the fluorescence recovery half times for the FtsZ1 and FtsZ2 proteins are 117s and 325s, respectively. This is significant as these data mirror their differences in GTP hydrolysis rates. To elucidate in vivo structure and ultrastructure of the Z-ring, a protocol was established that maintained fluorescence during high pressure freezing, freeze substitution and low temperature embedding. Afterwards, a correlative microscopy approach was employed to visualize and identify fluorescently labeled puncta, circular structures, at the light microscopy level. These puncta were further resolved as mini-rings using optical microscopy eXperimental (OMX) superresolution microscopy. Electron microscopy (EM) analysis imaged mini-rings and filament assemblies comprised of dense subunits. Electron tomography (ET) showed mini-rings composed of protofilaments.

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Submitted by PIETRO CARELLI REIS DE OLIVEIRA CALTABIANO null (pietrocarelli@hotmail.com) on 2017-01-10T01:33:15Z No. of bitstreams: 1 Caltabiano_Pietro_Tese_2016.pdf: 23652706 bytes, checksum: 3e2ad76601dc03b6f3704911772a5005 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-12T19:31:25Z (GMT) No. of bitstreams: 1 caltabiano_pcro_dr_guara.pdf: 23652706 bytes, checksum: 3e2ad76601dc03b6f3704911772a5005 (MD5) / Made available in DSpace on 2017-01-12T19:31:25Z (GMT). No. of bitstreams: 1 caltabiano_pcro_dr_guara.pdf: 23652706 bytes, checksum: 3e2ad76601dc03b6f3704911772a5005 (MD5) Previous issue date: 2016-11-25 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Ferramentas de processamento e análise digital de imagens foram desenvolvidas com a finalidade de avaliar a evolução da textura morfológica e cristalográfica da microestrutura da liga de alumínio 7075 sob diferentes níveis de deformação plástica por compressão uniaxial. Amostras da liga de alumínio 7075-T6 passaram por um processo de recozimento pleno seguido de um estágio de compressão uniaxial, obtendo níveis de deformações entre 25 e 65%. As microestruturas das amostras foram avaliadas em função dos parâmetros morfológicos dos precipitados, da reorientação dos planos cristalográficos dos grãos e da orientação das subestruturas formadas durante o processo de deformação. Para a caracterização foram utilizadas técnicas de difração de raios-X, microscopia eletrônica, microscopia óptica utilizando técnicas de polarização linear e circular, microscopia correlativa e processamento digital de imagens. Os resultados de difração de raios-X indicaram uma reorientação do plano cristalográficos (200) para o (220) após a deformação, e as técnicas de microscopia eletrônica identificaram precipitados de Mg2Si, Al7Cu2Fe e Al6(FeCu) na liga. A análise morfológica dos precipitados indicou uma maior fragmentação dos precipitados devido à maior ativação do plano (331) a partir de 39% de deformação. Por meio do processamento de imagens foi encontrada uma tendência de correlação entre os planos cristalográficos e a fração de área das fases, enquanto que os parâmetros morfológicos das subestruturas formadas durante o processo de deformação permitiram avaliar apenas qualitativamente o nível de encruamento das amostras. / Digital image processing and analysis tools were developed to perform the AA 7075 crystallographic and morphologic texture evaluation under different level of plastic deformation by uniaxial compression. Samples of AA 7075-T6 were submitted to full annealing process followed by uniaxial compression, thus obtaining deformations between 25 and 65% of thickness. The samples microstructure evaluation was performed considering: precipitates morphological parameters, crystallographic lattices reorientation and deformation substructure orientation. The characterization technics were: X-ray diffraction, electron microscopy, optical microscopy with polarization light, correlative microscopy and digital images processing. Xray diffraction results showed that the crystallographic plane (200) was reoriented to (220) after compression. The EDS analysis identified precipitates of Mg2Si, Al7Cu2Fe e Al6(FeCu). The precipitates morphological analysis showed an increase in fragmentation due to plane (331) at 39% of deformation. The digital image process of the samples etched with Barker reagents indicated a correlation between area fraction and the diffraction peaks, and the deformation substructures analysis made viable a qualitative characterization of the hardening process. / FAPESP: 2011/00403-2

La métastase peut être considérée comme le produit final d’un processus à la fois biologique mécanique et chimique où les cellules cancéreuses disséminent dans l’organisme pour envahir un nouvel organe à distance en s’établissant dans un nouvel microenvironnement tissulaire. Bien que les métastases soient la principale cause de décès liée au cancer, les principaux mécanismes impliqués dans ce processus restent à élucider. La communauté scientifique manque de techniques d’imagerie adaptées pour disséquer avec la plus haute résolution possible le comportement des cellules tumorales in vivo. Par conséquent, le but principal de ma thèse a été de développer une approche d’imagerie intravitale non-invasive appliquée à la souris. Cette approche a été inclue dans le développement d’un protocole de microscopie corrélative intravitale permettant l’étude de cellules tumorales à différentes échelles dans leur environnement naturel. Ce protocole a été utilisé dans l’étude de cellules invasives tumorales uniques dans l’oreille et le cerveau de la souris. Le but était de décrire les détails des protrusions cellulaires ainsi que les interactions cellule-matrice lors de l’invasion et l’intravasation de cellules cancéreuses. / Metastasis can be considered as the end product of a multistep bio-mechano-chemical process where cancer cells disseminate to anatomically distant organs and home and establish themselves in a new tissue microenvironment. Although metastasis is the leading cause of cancer-related death, the main cellular mechanisms enabling this process remain to be elucidated. Importantly, the scientific community lacks adapted imaging technologies to accurately dissect, at the highest resolution possible, tumor cell behavior in vivo. Therefore, the main goal of my PhD thesis was to develop an intravital and non-invasive imaging approach to track tumor progression in the living mouse. This approach was included in the development of an intravital Correlative Light and Electron Microscopy protocol allowing to track tumor cells at different scales in their natural environment. It was used to study single invasive tumor cells in the mouse ear and brain and to describe the details of cell protrusions and cell-matrix interactions during invasion and intravasation of cancer cells.

abstract: AA 7XXX alloys are used extensively in aircraft and naval structures due to their excellent strength to weight ratio. These alloys are often exposed to harsh corrosive environments and mechanical stresses that can compromise their reliability in service. They are also coupled with fasteners that are composed of different materials such as Titanium alloys. Such dissimilar metal contact facilitates galvanic and crevice corrosion, which can further reduce their lifetimes. Despite decades of research in the area, the confluence of mechanical, microstructural, and electrochemical aspects of damage is still unclear. Traditionally, 2D and destructive methods have often been employed to study the corrosion and cracking behavior in these systems which can be severely limiting and lead to inaccurate conclusions. This dissertation is aimed at comprehensively studying the corrosion and cracking behavior of these systems using time-dependent 3D microstructural characterization, as well as correlative microscopy. The microstructural evolution of corrosion in AA 7075 was studied using a combination of potentiodynamic polarization, X-ray Computed Tomography (XCT) and Transmission X-ray Microscopy (TXM). In both experiments, a strong emphasis was placed on studying localized corrosion attack at constituent particles and intergranular corrosion. With an understanding of the alloy’s corrosion behavior, a dissimilar alloy couple comprising AA 7075 / Ti-6Al-4V was then investigated. Ex situ and in situ x-ray microtomography was used extensively to investigate the evolution of pitting corrosion and corrosion fatigue in AA 7075 plates fastened separately with Ti-6Al-4V screws and rivets. The 4D tomography combined with the extensive fractography yielded valuable information pertaining the preferred sites of pit initiation, crack initiation and growth in these complex geometries. The use of correlative microscopy-based methodologies yielded multimodal characterization results that provided a unique and seminal insight on corrosion mechanisms in these materials. / Dissertation/Thesis / Doctoral Dissertation Materials Science and Engineering 2020

Les éléments chimiques métalliques tels que Fe, Cu ou Zn sont présents en quantité infime dans le cerveau. Le rôle de ces métaux traces dans les fonctions neuronales, telles que la transmission synaptique et les processus de mémorisation, reste encore largement à élucider. En outre, une dyshoméostasie des métaux est retrouvée dans de nombreuses neuropathologies, telles que maladie d’Alzheimer, de Parkinson ou sclérose latérale amyotrophique. Les mécanismes d’interaction des métaux traces au niveau neuronal sont difficiles à décrire faute de méthodes analytiques possédant une résolution et une sensibilité adaptées pour en déterminer la distribution à l’échelle synaptique. La spectrométrie de fluorescence-X synchrotron (SXRF) est une méthode d’analyse chimique multi-élémentaire permettant de décrire la distribution quantitative de ces éléments à l’échelle subcellulaire avec une résolution nanométrique (40 nm avec la ligne ID16A du synchrotron ESRF) et une très haute sensibilité. Afin d’interpréter avec précision les résultats d’imagerie chimique, nous avons développé un protocole pour corréler l’imagerie nano-SXRF avec la microscopie super résolutive de déplétion par émission stimulée (STED) permettant ainsi la corrélation des distributions des métaux avec celle de protéines cibles en super résolution. Nous avons marqué les microtubules et l’actine-F de neurones primaires d’hippocampe de rat puis imagé le cytosquelette par microscopie STED avant de déterminer par spectrométrie SXRF les distributions des éléments P, S, Fe, Cu et Zn. Nous avons ainsi mis en évidence la colocalisation du Zn et des microtubules au niveau dendritique ainsi qu’une localisation du Cu essentiellement dans le cou des épines dendritiques riches en F-actine, et une distribution du Fe sous forme de points très localisés dans les dendrites. Ces résultats ont révélé le rôle essentiel du Zn dans l’architecture du cytosquelette des neurones d’hippocampe et la méthode d’imagerie corrélative développée ouvre de nouvelles perspectives pour l’étude des dyshoméostasies des métaux dans les maladies neurodégénératives. / Metallic chemical elements such as Fe, Cu or Zn are present in minute quantities in the brain. The role of these trace metals in neuronal functions such as synaptic transmission and memory processes remains largely unclear. Moreover, metal dyshomeostasis is found in many neuropathologies, such as Alzheimer’s disease, Parkinson’s or amyotrophic lateral sclerosis. The interaction mechanisms of trace metals at the neuronal level are difficult to describe because of the lack of analytical methods with appropriate resolution and sensitivity to determine metal distribution at the synaptic level. Synchrotron X-ray fluorescence spectrometry (SXRF) is a multielemental chemical analysis method for describing the quantitative distribution of these elements at the sub-cellular scale with a nanometric resolution (40 nm at ESRF beamline ID16A ) and a very high sensitivity. In order to accurately interpret chemical imaging results, we have developed a protocol to correlate nano-SXRF imaging with stimulated emission depletion super resolution microscopy (STED), allowing the correlation of metal distribution and that of target proteins in super resolution. We labeled microtubules and F-actin of primary rat hippocampal neurons and imaged the cytoskeleton by STED microscopy before determining the distributions of P, S, Fe, Cu and Zn by SXRF spectrometry. We evidenced the colocalization of Zn and microtubules at the dendritic level and a localization of Cu mainly in the neck of dendritic spines rich in F-actin, and a distribution of Fe in the form of very localized points in the dendrites. These results highlight the crucial role of Zn in cytoskeleton architecture of hippocampal neurons and the developed correlative imaging method opens new perspectives for the study of metal dyshomeostasis in neurodegenerative diseases.

The efficacy Biological wastewater treatment process is largely dependent on the formation of microbial flocs and settleability before the water is released into the environment. Settleability and flocculation are reliant upon stable physicochemical parameters. Extracellular polymeric substance constituents dictate physicochemical parameters of flocs. The fluctuation of these constituents within mixed microbial flocs is poorly studied. A novel aspect of this research was the use of CLSM data to get a semi- quantitative assessment of the constituents within mixed microbial flocs. Wastewater treatment flocs were characterized for eubacterial ecology, physicochemical properties, and they were visualized through correlative microscopy. It was observed that the microbial communities from the three sampling sites exhibited significant variability in numerous physicochemical properties. Overall, these results provide a first step to examine micro-localization of physicochemical properties, architecture and processes within flocs that may help better understand the causes of floc- related inefficiencies in biological wastewater treatment. / Canadian Hemophilia Society

Cette thèse porte sur la définition d'un schéma de recalage automatique en microscopie corrélative 2D et 3D, en particulier pour des images de microscopie optique et électronique (CLEM). Au cours des dernières années, la CLEM est devenue un outil d'investigation important et puissant dans le domaine de la bio-imagerie. En utilisant la CLEM, des informations complémentaires peuvent être collectées à partir d'un échantillon biologique. La superposition des différentes images microscopiques est généralement réalisée à l'aide de techniques impliquant une assistance manuelle à plusieurs étapes, ce qui est exigeant et prend beaucoup de temps pour les biologistes. Pour faciliter et diffuser le procédé de CLEM, notre travail de thèse est axé sur la création de méthodes de recalage automatique qui soient fiables, faciles à utiliser et qui ne nécessitent pas d'ajustement de paramètres ou de connaissances complexes. Le recalage CLEM doit faire face à de nombreux problèmes dus aux différences entre les images de microscopie électronique et optique et leur mode d'acquisition, tant en termes de résolution du pixel, de taille des images, de contenu, de champ de vision et d'apparence. Nous avons conçu des méthodes basées sur l'intensité des images pour aligner les images CLEM en 2D et 3D. Elles comprennent plusieurs étapes : représentation commune des images LM et EM à l'aide de la transformation LoG, pré-alignement exploitant des mesures de similarité à partir d'histogrammes avec une recherche exhaustive, et un recalage fin basé sur l'information mutuelle. De plus, nous avons défini une méthode de sélection robuste de modèles de mouvement, et un méthode de détection multi-échelle de spots, que nous avons exploitées dans le recalage CLEM 2D. Notre schéma de recalage automatisé pour la CLEM a été testé avec succès sur plusieurs ensembles de données CLEM réelles 2D et 3D. Les résultats ont été validés par des biologistes, offrant une excellente perspective sur l'utilité de nos développements. / This thesis is concerned with the definition of an automated registration framework for 2D and 3D correlative microscopy images, in particular for correlative light and electron microscopy (CLEM) images. In recent years, CLEM has become an important and powerful tool in the bioimaging field. By using CLEM, complementary information can be collected from a biological sample. An overlay of the different microscopy images is commonly achieved using techniques involving manual assistance at several steps, which is demanding and time consuming for biologists. To facilitate and disseminate the CLEM process for biologists, the thesis work is focused on creating automatic registration methods that are reliable, easy to use and do not require parameter tuning or complex knowledge. CLEM registration has to deal with many issues due to the differences between electron microscopy and light microscopy images and their acquisition, both in terms of pixel resolution, image size, content, field of view and appearance. We have designed intensity-based methods to align CLEM images in 2D and 3D. They involved a common representation of the LM and EM images using the LoG transform, a pre-alignment step exploiting histogram-based similarities within an exhaustive search, and a fine mutual information-based registration. In addition, we have defined a robust motion model selection method, and a multiscale spot detection method which were exploited in the 2D CLEM registration. Our automated CLEM registration framework was successfully tested on several real 2D and 3D CLEM datasets and the results were validated by biologists, offering an excellent perspective in the usefulness of our methods.

Předložená diplomová práce se zabývá korelativním přístupem multimodální analýzy struktur prokovování s různým rozlišením. Výzkum je součástí mezinárodního projektu týkajícího se charakterizace poruch zmíněných struktur, které jsou implementovány v polovodičových zařízeních. Kombinace korelativní mikroskopie a tomografie technikami NanoXCT, FIB-SEM (EDS), FIB-SIMS a AFM byla navržena k zavedení opakovatelného pracovního postupu. Tomografie fokusovaným iontovým svazkem je metoda přesného odprašování v řezech, která mimo jiné v každém průřezu získává cenné snímky s vysokým rozlišením (FIB-SEM) nebo mapy chemického složení (FIB-SIMS). Následující transformace obrazu umožňuje identifikaci defektů jako funkci hloubky ve struktuře. Práce dále věnuje pozornost metodám sjednocení obrazů za účelem optimální prezentace získaných dat.

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.