Tomography of crystal defects in the electron microscope was first attempted in 2005 by the author and colleagues. This thesis further develops the technique, using a variety of samples and methods. Use of a more optimised, commercial tomographic reconstruction program on the original GaN weak beam dark-field (WBDF) tilt series gave a finer reconstruction with lower background, line width 10-20 nm. Four WBDF tilt series were obtained of a microcrack surrounded by dislocations in a sample of indented silicon, tilt axes parallel to g = 220, 220, 400 and 040. Moiré fringes in the defect impaired alignment and reconstruction. The effect on reconstruction of moiré fringe motion with tilt was simulated, resulting in an array of rods, not a flat plane. Dislocations in a TiAl alloy were reconstructed from WBDF images with no thickness contours, giving an exceptionally clear reconstruction. The effect of misalignment of the tilt axis with systematic row g(ng) was assessed by simulating tilt series with diffraction condition variation across the tilt range of Δn = 0, 1 and 2. Misalignment changed the inclination of the reconstructed dislocation with the foil surfaces, and elongated the reconstruction in the foil normal direction; this may explain elongation additional to the missing wedge effect in experiments. Tomography from annular dark-field (ADF) STEM dislocation images was also attempted. A tilt series was obtained from the GaN sample; the reconstructed dislocations had a core of bright intensity of comparable width to WBDF reconstructions, with a surrounding region of low intensity to 60 nm width. An ADF STEM reconstruction was obtained from the Si sample at the same microcrack as for WBDF; here automatic specimen drift correction in tomography acquisition software succeeded, a significant improvement. The microcrack surfaces in Si reconstructed as faint planes and dislocations were recovered as less fragmented lines than from the WBDF reconstruction. ADF STEM tomography was also carried out on the TiAl sample, using a detector inner angle (βin) that included the first order Bragg spots (in other series βin had been 4-6θ B). Extinctions occurred which were dependent on tilt; this produced only weak lines in the reconstruction. Bragg scattering in the ADF STEM image was estimated by summing simulated dark-field dislocation images from all Bragg beams at a zone axis; a double line was produced. It was hypothised that choosing the inner detector angle to omit these first Bragg peaks may preclude most dynamical image features. Additional thermal diffuse scattering (TDS) intensity due to dilatation around an edge dislocation was estimated and found to be insignificant. The Huang scattering cross section was estimated and found to be 9Å, ten times thinner than experimental ADF STEM dislocation images. The remaining intensity may be from changes to TDS from Bloch wave transitions at the dislocation; assessing this as a function of tilt is for further work. On simple assessment, only three possible axial channeling orientations were found over the tilt range for GaN; if this is typical, dechanneling contrast probably does not apply to defect tomography.

Les microARN sont de petits ARN non codant régulant post-transcriptionnellement l’expression de certains gènes. Du fait de leur fort potentiel régulateur, une modification de leur expression peut conduire à l’apparition de pathologies telles que le cancer ou l’inhibition des mécanismes de défense contre des pathogènes. Notre objectif est de caractériser le rôle de certains miARN dans la formation de cancer gastrique dû à Helicobacter pylori. En effet, cette bactérie peut conduire à l’apparition d’adénocarcinome gastrique et de lymphome du MALT. Sa virulence est essentiellement due à la protéine CagA, injectée dans les cellules de la muqueuse gastrique. Par séquençage à haut débit du contenu en miARN d’une lignée épithéliale gastrique humaine, co-cultivée ou non avec H. pylori, nous avons observé que les niveaux de miR-200b/c sont augmentés par l’infection. Ces miARN sont des inhibiteurs puissants de la transition épithélio-mésenchymateurse (TEM), modification morphologique promotrice d’invasion. Ils ciblent les facteurs de transcription ZEB1/2 avec lesquels ils sont impliqués dans une boucle de rétro-action mutuellement répressive. Le niveau basal élevé de miR-200b/c dans ces cellules réprime totalement ZEB1, tandis que l’infection par H. pylori, sous la dépendance de CagA, promeut une TEM en induisant ZEB1. Paradoxalement, les miR-200b/c sont aussi augmentés lors de l’infection transcriptionnellement. Nous avons pu démontrer que l’augmentation des miR-200b/c dans les cellules infectées a pour rôle de modérer l’induction de ZEB1 via l’activation de NF-kB, constituant ainsi un mécanisme de défense des cellules hôte contre la perte de leur identité épithéliale. / MicroRNA are small noncoding RNA that post-transcriptionally regulate gene expression. Due to their high regulator potential, a change in their expression may lead to the emergence of diseases such as cancer or inhibition of defense mechanisms against pathogens. Our aim is to characterize the role of miRNA in the response of gastric eptithelial cells to Helicobacter pylori (H. pylori). Indeed, H. pylori promote gastric adenocarcinoma and MALT lymphoma. Its virulence is essentially mediated by CagA, injected into cells of the gastric mucosa. Thanks to high throughput sequencing of miRNA content of a gastric epithelial cell line, infected or not with H. pylori: miR-200b and -200c appeared up-regulated upon infection. These miRNA are potent inhibitors of the “epithelial-to-mesenchymal transition” (EMT), a process that drastically alters cell morphology and promotes cell invasion. MiR-200b/c target the transcription factors ZEB1 and ZEB2, with which they are involved in a mutually repressive feedback loop. In basal conditions, the high levels miR-200b/c in gastric epithelial cells totally silence ZEB1 mRNA whereas H. pylori promotes EMT via ZEB1 expression, on the dependence of CagA translocation into host cells. But, paradoxically, miR-200b/c levels were also up-regulated upon infection. The increased miR-200b/c levels in infected cells moderate ZEB1 induction thanks to NF-kB activation and constitute a self-defense mechanism to thwart the loss of their epithelial phenotype upon infection.

This work was performed as part of the MUZIC-2 (Mechanical Understanding of Zirconium Corrosion) collaboration, established with the goal of understanding the mechanism of hydrogen pickup in zirconium alloys. Hydrogen pickup is one of the least understood and most significant degradation mechanisms affecting zirconium alloys in nuclear reactors. These alloys are used as cladding and structural materials in the reactor core, mainly due to their low thermal neutron absorption cross section and excellent corrosion resistance. This project aims to investigate the effect of oxide texture (i.e. the degree of preferred orientation) on the corrosion performance of zirconium alloys. The texture of the oxide is expected to affect the microstructural development of the oxide, the grain boundary distribution and the stress state. It is therefore considered to be one of the most important factors in determining how the corrosion process occurs, and why different alloys exhibit significantly different corrosion performance. It is hoped that this project will add to the current knowledge of the corrosion process, and in particular hydrogen pickup, so that the route of hydrogen through the protective oxide can be identified. This will lead to the development of a new generation of alloys that provide improved oxidation and hydrogen pickup performance, whilst maintaining the required mechanical properties. This work focuses on four zirconium alloys; Zircaloy-4, ZIRLO™, low-Sn ZIRLO™ and Zr-1.0Nb-0.1Fe. The alloys all have different chemical compositions and therefore exhibit different corrosion performance. The macrotexture of the oxide formed on different alloys was measured by glancing angle X-ray diffraction (XRD) and Electron Backscatter Diffraction (EBSD). A fibre texture was formed in all cases, with the (10-3) to (10-5) planes oriented parallel to the metal-oxide interface for the monoclinic phase. The major orientation was found to be independent of alloy chemistry, substrate orientation and oxidation conditions. The monoclinic texture strength was found to be weakened with increasing oxidation temperature, The major orientation of the tetragonal phase was also found to be a fibre texture, with the (001) planes oriented approximately parallel with the interface. Although significant variation from this texture component was observed. It is suggested that the main driving force for oxide texture development is the transformation stress induced by the Zr-ZrO2 transformation. The microtexture of the oxides was measured using two novel techniques, Transmission Kikuchi Diffraction (TKD) and automated crystal orientation mapping with transmission electron microscopy (TEM). The techniques revealed an oxide microstructure consisting of an outer layer of equiaxed grains with a large range of orientations and an inner layer consisting of well-aligned columnar monoclinic grains. This layer of equiaxed grains was observed to form again after the transition in corrosion kinetics. A large fraction of transformation twin boundaries were observed throughout the oxides. Some of these boundaries were observed to surround large monoclinic grains, providing evidence that the tetragonal grains from which they transformed had grown considerably larger than the critical grain size for stabilisation. Without the presence of Sn, larger monoclinic grains were observed to form with a greater degree of preferred orientation, and with a lower fraction of transformation twin boundaries than Sn-containing alloys. In addition, an increased number of well-oriented tetragonal grains was correlated with the presence of Sn. It is therefore concluded that a reduction in Sn will lead to the formation of an oxide microstructure that is more resistant to both oxidation and hydrogen pickup. An analysis of the crystallography and morphology of a ZrO phase present at the metal-oxide interface was also performed. Finally, the observations have been used to construct a model of the corrosion process.

Since its discovery, the one atom thick material graphene has been at the centre of growing interest in two-dimensional materials. Due to its exceptional properties, graphene is a rich topic to explore by physicists, chemists, engineers and materials scientists. In addition to its use in the fundamental research, graphene is also a promising candidate for future electronics, photonics and energy storage devices.The project presented in this thesis was carried out to explore the structure of suspended graphene in particular in order to probe the metal-graphene interaction via Transmission Electron Microscopy, as most graphene applications require interfacing with metals. As the work was based on free standing graphene, graphene layers obtained by mechanical cleavage or growth on a substrate were transferred onto TEM-grids. Therefore, fabrication, suspended sample preparation and identification of graphene layers were first discussed for a better understanding of how to obtain high quality graphene, as this was essential for the rest of the project.Structural, topographic and chemical analysis of pristine suspended graphene layers were investigated in detail via Transmission Electron Microscopy and Scanning Tunnelling Microscopy. The latter technique was also employed for graphene on a substrate along with establishing annealing conditions for residue free graphene.Metal deposited suspended graphene layers were then investigated in the electron microscopes. Different metal behaviours were observed on the graphene surfaces for the same amount of metal evaporation. Generally, metals interact only weakly with graphene as they are not observed on clean (residue free) parts and are mainly clustered. On the other hand, graphene etching has been observed in the presence of metals. The etching was initiated with graphene vacancy formation as a result of the interaction between metal and carbon atoms on clean graphene. Once a vacancy was created, a hole quickly formed and eventually the graphene layers were destroyed. However, those holes created by metals were healed spontaneously either by non-hexagonal or perfect hexagonal rings. The possible etching and healing mechanisms of the suspended graphene were also discussed.

>Magister Scientiae - MSc / There are limited reports on virus population in haloalkaline environments; therefore the aim of this study was to investigate the genetic diversity and biology of bacteriophage communities in these environments. Bacteria were isolated to be used as phage hosts. One bacterium from Lake Magadi and four bacteria from Lake Shala were successfully isolated from sediment samples. A further two Lake Shala bacterial hosts from the IMBM culture collection were also used to isolate bacteriophages. Bacterial isolates were identified to be most closely related to Bacillius halodurans, Halomonas axialensis, Virgibacillus salarius, Bacillus licheniformis, Halomonas venusta, Bacillus pseudofirmus and Paracoccus aminovorans. Bacteriophages were screened using all bacteria against sediment samples from both Lake Shala and Lake Magadi. One phage was identified from Lake Magadi sediments (MGBH1) and two phages from Lake Shala sediments (SHBH1 and SHPA). TEM analysis showed that these phages belong to three different dsDNA phage families; Siphoviridae (MGBH1), Myoviridae (SHBH1) and Podoviridae (SHPA). All phages showed different genome sizes on agarose gel. Due to the small genome size, phage SHPA was chosen for further investigation. Partial, genome sequence analysis showed homology to both bacterial and phage proteins. A further investigation of phage diversity in this environment is essential using metagenomic approaches to understand these unique communities.

Is the nucleolus a sine qua non condition of eukaryotes? The present project starts from this central question to contribute to our knowledge about the origin and the evolution of the cells. The nucleolus is a cryptic organelle that plays a central role in cell function. It is responsible for the orchestration of ribosomal RNA expression, maturation and modification in the regulatory context of cellular homeostasis. Ribosomal expression is undoubtedly the greatest transcriptional and regulatory activity of any cell. The nucleolus is not just a conventional organelle –membrane-limited-, but a magnificent transcriptional puff: a dichotomy between structure and process, form and function. What is the minimum nucleolus? Evolution should bring some light into these questions. Evolutionary cell biology (ECB) has raised increasing attention in the last decades. Is this a new discipline and an historical opportunity to combine functional and evolutionary biology towards the insight that cell evolution underlies organismic complexity? In the post-genomic era, we have developed the potential of combining high throughput acquisition of data with functional in situ and in sillico approaches: integration understood as omics approaches. Can this provide a real consilience between evolutionary and functional approaches? The reduced number of model organisms and cultivation techniques still excludes the majority of the extant diversity of cells from the scope of experimental inquiry. Furthermore, at the conceptual level, the simplification of evolutionary processes in biosciences still limits the conformation of a successful disciplinary link between functional and evolutionary biology. This limits the formulation of questions and experiments that properly address the mechanistic nature of cellular events that underlie microbial and organismic diversity and evolution. Here we provide a critical and comparative review to the historical background of ECB. This project takes the lessons learned from ECB and attempts to find a homologue structure of the eukaryotic nucleolus within the Archaea. We found nanometric structures in S. solfactarius that either are positive to specific nucleolar techniques such as Nucleolar organizer regions NOR silver staining. These is structures are novel and its significance should be revised on the evolutionary cell biology perspective.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23459号 / 理博第4753号 / 新制||理||1681(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 倉田 博基, 教授 島川 祐一, 教授 寺西 利治 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

The purpose of this diploma thesis is to research and compare available wireless communication technologies (frequency, modulation type, transmission speed and consumption). Describe transmission electron microscopes theory and deduce the most sensitive microscope parts to high frequency distortion. Investigate effect of wireless technology on CE standards and come up with, perform and evaluate the influence measurement of chosen technologies on base parameters of transmission electron microscope.

This master thesis deals with conventional methods of bacterial cell analysis, polyhydroxyalkanoates, Raman spectroscopy and electron microscopy in the theoretical part. The production of polyhydroxybutyrate by selected thermophilic bacteria and their analysis by gas chromatography, cryogenic scanning electron microscopy and Raman spectroscopy is described in the experimental part. The chosen sample was analyzed by a transmission electron microscope. Comparing the results from previous mentioned methods it was found that the bacteria Schlegelella thermodepolymerans accumulated the highest amount of PHB. The lowest amount of PHB was obtained by bacteria Rubrobacter xylanophilus. The assumption that the PHB granules formed so-called needle-like plastic deformations during freeze-fracturing was affirmed by cryo-SEM photos analysis. Moreover, it was found that the bacterial cell characterization deduced from microscopic observation of samples corresponded to the description in the literature. TEM provided better resolution photos and in consequence the cells and PHB are more visible. The thesis is also focused on chemical fingerprint analysis of cells by Raman spectroscopy. Several biomolecules were identified by measured Raman spectra for the particular samples.

Résumé : Les instruments de spectrométrie de masse à ionisation secondaire (SIMS) doivent être améliorés afin de satisfaire les exigences et tendances dans de nombreux domaines qui demandent des outils d'analyse pouvant cartographier les échantillons à la fois avec une excellente résolution et une haute sensibilité chimique, mais également avec des temps d’analyse plus court. Les objectifs de cette thèse sont : rechercher à améliorer la résolution en masse des spectromètres de masse à double focalisation en remplaçant le secteur sphérique standard par une nouvelle géométrie sphéroïde ayant de meilleures propriétés de focalisation, et d’étudier la réduction du temps d'analyse en imagerie SIMS, par la preuve de concept du système SIMS à multifaisceaux d'ions. Une comparaison des principales propriétés de focalisation du secteur sphérique, de la géométrie sphéroïde et d’une géométrie de sphéroïde hybride, a été réalisée en utilisant le logiciel SIMION. Une comparaison dans une configuration Nier-Johnson entre le secteur sphérique et le sphéroïde, a montré que le faisceau présente une rotation de 90 ° à la sortie de l'aimant nuisant à la résolution de masse dans la configuration sphéroïde. En ajoutant un élément électrostatique pouvant tourner le faisceau à 90 ° les performances du spectromètre de masse pourrait être amélioré. Toutefois, une comparaison des performances entre les secteurs sphériques et hybrides dans une configuration Mattauch-Herzog a montré que lorsque la double condition de focalisation est optimisée, une meilleure résolution de masse pourrait être obtenue avec la géométrie sphéroïde. Un système multi-faisceau ionique a été étudié pour l'analyse SIMS. La simulation à travers l’optique secondaire d'un Cameca IMS XF de neuf faisceaux a montré une transmission réussie des faisceaux, résultant en neuf points concentrés sur le détecteur plaque à canaux multiples (MCP). La preuve de concept a été achevée expérimentalement dans l'IMS 6F de Cameca, où une ouverture à trous multiples était montée dans la colonne principale, générant 9 et 16 faisceaux de tailles comprises entre 4 µm à 10 µm. Des images d'une grille AlCu ont été obtenues en balayant l'échantillon par le système de multifaisceaux d’ions. Ces résultats montrent que le système multi-faisceau d'ions est une technique possible pour l'imagerie SIMS et qu'en optimisant leur conception les multi-nano-faisceaux d'ions seront une solution permettant de réduire considérablement le temps d'analyse. / Secondary ion mass spectrometry (SIMS) instruments need to be improved in order to satisfy the demands of trends in many fields that require analytical tools that can map samples with both excellent resolution and high-sensitivity chemical information, but also with shorter time of analysis. The objectives of this thesis are: investigate the enhancement of the mass resolution of double focusing mass spectrometers by replacing the standard spherical sector with a novel spheroid geometry which has better focusing properties, and to investigate the reduction of the time of analysis in imaging SIMS by the proof-of-concept of the SIMS multi-ion- beam system.A comparison of the main focusing properties of the spherical sector, the spheroid geometry and a hybrid spheroid geometry, was made using the SIMION software. A comparison in a Nier-Johnson configuration between the spherical sector and the spheroid, showed that the beam presents a rotation of 90° at the exit of the magnet harming the mass resolution in the spheroid configuration. By adding an electrostatic element that can rotate the beam 90° the performance of the mass spectrometer could be improved. However, a comparison of the performances between the spherical and hybrid sectors simulated in a Mattauch-Herzog configuration, showed that when the double focusing condition is properly satisfied, better mass resolution could be achieved with the spheroid geometry.A multi-ion-beam system was investigated for SIMS analysis. A simulation through the secondary optics of a Cameca IMS XF showed successful transmission of nine beams through the optics resulting in nine well focussed spots on the multi channel plate (MCP) detector. The proof-of-concept was completed experimentally in the Cameca IMS 6F, where a multi-hole aperture was mounted in the primary column generating 9 and 16 beams of sizes between 4 μm to 10 μm. Images of an AlCu grid were obtained when t the multi-ion-beam system was scanned over the sample. These results showed that the multi-ion-beam system is a feasible technique for imaging SIMS and by optimizing the design multi-nano-ion-beams will be a solution for reducing drastically the time of analysis.