This thesis concerns the experimental application of the technique of optical sectioning in the aberration-corrected scanning transmission electron microscope (STEM). Another aim was to perform optical sectioning experiments on the still relatively new scanning confocal electron microscope (SCEM). To test the feasibility of this technique, experiments were performed on a variety of samples to measure the achievable depth response. Deconvolution methods were explored in an attempt to further improve the depth response. Finally, some of the first optical sectioning experiments were performed in the SCEM using both elastic and inelastically scattered electrons. The results showed a clear need to investigate confocal electron microscopy due to the missing cone problem for incoherent imaging in the STEM. This is particularly evident when imaging objects of greater width than the STEM probe. Confocal electron microscopy using inelastic electrons appeared to be a promising imaging mode for the future with this thesis consisting of early work in the field.

Canola (Brassica napus) contributes $19.3 billion dollars to the Canadian economy each year as a result of its oil- and protein-rich seeds. These economically important seed products are produced in highest concentration in the embryo. Embryo development is supported nutritionally and structurally by the maternal subregions, which include the inner (ISC) and outer distal seed coat (OSC), the chalazal seed coat (CZSC), and the chalazal proliferating tissue (CPT). Research on the maternal seed subregions is limited to the SC as a result of its accessibility; the embedded CZSC and CPT subregions have yet to be characterized in canola. Using light and transmission electron microscopy, I found the CZSC and CPT to be anatomically distinct and experience profound changes throughout seed development. To understand these changes at the RNA level, laser microdissection and RNA sequencing were used to profile these subregions spatially and temporally from the ovule to mature green stage of seed development. Employing vigorous bioinformatics analyses, I found that the maternal subregions are transcriptomically distinct and possess unique RNA populations. From here I began to elucidate the biological processes operating within the maternal subregions. As a whole, the maternal subregions appear to have a critical role in transporting nutrients to the filial subregions as well as in coping with oxidative stress produced during these energy-rich processes. Additionally, using CanEnrich, I was able to generate predictive transcriptional circuits regulating the biological processes occurring within the maternal seed. This research has produced the most comprehensive dataset on the canola seed to date and will provide a valuable resource for research on seed development as well as seed improvement. / October 2016

The processing and properties of novel polymer-based nano-composite (PNC) dielectrics for capacitor applications has been studied. PNCs were fabricated via a vacuum based deposition technique and their micro/nano-structure, chemical and dielectric properties investigated. After process development and optimisation, co-deposited Al and nylon-6 PNCs had a dielectric constant k∼7 at an approximate Al volume fraction of 0.3 that agreed with analytical predictions if it was assumed that the Al transformed to an oxide in-situ and/or after deposition. The significant effect of absorbed water vapour and temperature on PNC dielectric properties was revealed using different types of post-deposition heat treatment. Alternately-deposited PNCs consisting of Al or Ag 2-20 nm layers sandwiched between nylon-6 layers were fabricated in which the overall PNC Al or Ag volume fraction was controlled by varying the nominal Al or Ag layer thickness. Ag layers comprised of discrete nano-islands that produced a nano-capacitor network effect that increased k to ∼11. In the case of Al layers, when the layer thickness was ≥ 5 nm, corresponding to a nominal volume fraction of 0.1, Al (core)-oxide (shell) nanoparticles were formed and the PNC dielectric constant increased to ∼19. The detailed nano-structure of the core-shell particles was studied using various types of transmission electron microscopy (TEM), and the elevations in dielectric constant ascribed to multiple-interface polarisation effects dependent on the formation of the core-shell structure. PNCs based on alternate deposition of Ti sandwiched in nylon-6, and then both Ti and Ag in nylon-6 were also fabricated, with k reaching ∼73 for Ag+Ti/nylon-6 PNCs. As well as Ti-based core (metal)-shell (oxide) particles, the Ag volume fraction was sufficiently high in the 10 nm nylon-6 layers to again form a nano-capacitor network that contributed to the overall device capacitance and effective dielectric constant. Again, various types of high magnification TEM were critical in resolving the Ti-based core-shell structure and its role in high-k behaviour. The vacuum-based alternate deposition technique has been developed to offer ease of operation, reliability, flexibility and applicability to chemically different filler and matrix systems in the fabrication of high-k PNC based capacitors, in which high-k performance relies critically on the formation of core (metal)-shell (oxide) particles in both Al and Ti based systems.

No description available.

Septinas são GTPases consideradas como um novo componente do citoesqueleto. Essas proteínas interagem entre si para formar heterocomplexos filamentosos e estruturas de alta ordem que são importantes para a citocinese e uma variedade de outros processos celulares. Existem muitos aspectos mecânicos dessas proteínas que não são totalmente compreendidos, incluindo a forma como os heterocomplexos se agrupam corretamente. Em humanos, há 13 genes que codificam septinas, classificadas em quatro grupos quanto à similaridade em relação à estrutura primária. O complexo hexamérico SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 foi o melhor caracterizado, com uma estrutura cristalina resolvida à 4 Å. Segundo às Regras de Kinoshita, as septinas desse complexo podem ser substituídas nesse arranjo por outras pertencentes ao mesmo grupo. Neste trabalho utilizamos a técnica de microscopia eletrônica de transmissão com análise de partícula única para estudar dois complexos de septinas. Um dos complexos estudados neste projeto é formado por septinas humanas, para as quais atualmente não há informações estruturais disponíveis. O complexo SEPT5-SEPT6-SEPT7 foi expresso heterólogamente em E. coli e purificado em alta concentração salina para evitar a polimerização. A análise de partículas únicas de imagens por contrastação negativa mostrou a presença de partículas alongadas de aproximadamente 25 nm de comprimento, compostos por seis monômeros, como esperado. Com o objetivo de localizar a posição da SEPT5 no complexo, foi realizada uma fusão com MBP (Maltose Binding Protein) e imunomarcação com anticorpo monoclonal anti-SEPT5, concluindo que a SEPT5 está localizada na extremidade do complexo hexamérico. Porém, a SEPT5 pertence ao mesmo grupo da SEPT2, que foi relatada estar localizada no centro do hexâmero. Este resultado possibilitou uma nova discussão sobre a maneira que as septinas formam os complexos e, como a sensibilidade à concentração salina está relacionada com a fragilidade da interface NC, análogo ao observado em complexos de Saccharomyces cerevisiae. Um complexo de Ciona intestinalis incluindo a SEPT2, SEPT6, SEPT7 e SEPT9, também expresso heterólogamente em E. coli, foi preparado por contrastação negativa. A análise de partícula única das imagens coletadas mostrou um heterocomplexo aparentemente hexamérico, embora fosse esperado um octâmero devido à presença das quatro septinas diferentes, sendo uma pertencente à cada um dos quatro grupos. Os resultados deste trabalho proporcionaram um avanço na compreensão da formação de heterocomplexos de septinas e como essas proteínas interagem umas com as outras nesta montagem. / Septins are GTPases that appear to be a novel component of the cytoskeleton. These proteins interact with each other to form filamentous heterocomplexes and high order structures which are important for cytokinesis and a variety of other cellular processes. There are many mechanistic aspects of these proteins that are not fully understood, including how the heterocomplexes correctly assemble. In humans, there are 13 genes encoding septins, classified in four groups based on primary structure. The SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 hexameric complex was the best characterized, with a crystalline structure solved at 4 Å. According to Kinoshita´s Rules, the septins of this complex can be replaced in this arrangement by others belonging to the same group. In this work, we used transmission electron microscopy with single particle analysis to study two septin complexes. One of the complexes studied in this project is composed of three human septins, for which there is currently no structural information available. The SEPT5-SEPT6-SEPT7 complex was heterologously expressed in E. coli and purified at high salt concentration to avoid polymerization. Single particle analysis of negatively stained samples showed the presence of elongated particles of approximately 25 nm in length. To locate SEPT5 in the complex, a fusion with MBP (Maltose Binding Protein) and immunoblotting with anti-SEPT5 monoclonal antibody was performed, concluding that SEPT5 is located at the end of the hexameric complex. However, SEPT5 belongs to the same group as SEPT2, which was reported to be located in the center of the hexamer. This result allowed for a new discussion on the way that septins form heterocomplexes and also, on how the sensitivity of the NC interface in related to salt concentration, analogous to that observed in the heterocomplex of Saccharomyces cerevisiae. A Ciona intestinalis complex including SEPT2, SEPT6, SEPT7 and SEPT9, also expressed heterologously in E. coli, was prepared by negative staining. The single particle analysis of the collected images showed an apparently hexameric heterocomplex, although an octamer was expected due to the presence of the four different septins, one belonging to each of the four groups. The results of this work represent advances in the understanding of the formation of septin heterocomplexes and how these proteins interact with each other during assembly.

Os primatas neotropicais, ou primatas do novo mundo, são pertencentes a um grupo diverso e variado de animais, com características anatômicas, comportamentais e taxonômicas próprias, que os diferem dos primatas do velho mundo. Estima-se que representem 31% dos primatas não-humanos do planeta. Têm sofrido com as altas taxas de crescimento populacional e perturbação antrópica resultante do desflorestamento, agricultura e criação de gado. Com o intuito de reverter esta situação e maximizar o potencial reprodutivo das espécies, o desenvolvimento de biotecnologias associadas à reprodução tem sido amplamente justificado nos dias de hoje, por representarem importantes ferramentas reprodutivas, contribuindo para a preservação da biodiversidade e manutenção da diversidade genética. Assim, o objetivo do presente estudo foi padronizar a colheita de sêmen de Alouatta guariba clamitans por eletroejaculação por via retal com posterior análise do sêmen fresco, procedimentos nunca descritos para a espécie, e adaptar o método de preparo de sêmen de Leontopithecus chrysomelas, Alouatta caraya e Alouatta guariba clamitans para microscopia eletrônica de transmissão, permitindo a comparação da ultraestrutura dos espermatozoides das três espécies e apresentando resultados também inéditos na literatura científica. Para este estudo, foram utilizados quatro exemplares de L. chrysomelas, dois exemplares de A. caraya e dois exemplares de A.g. clamitans, machos adultos mantidos em cativeiro. L. chrysomelas tiveram o sêmen colhido por vibroestimulação peniana, e A. caraya e A.g. clamitans foram submetidos à eletroejaculação por via retal. Após a obtenção dos ejaculados, o sêmen foi avaliado quanto ao volume, pH, concentração, motilidade, integridade de membrana plasmática, integridade de membrana acrossomal e morfologia, e posteriormente preparado para microscopia eletrônica de transmissão. A análise dos 12 espermatozoides das três espécies estudadas revelou que todos apresentam os componentes básicos descritos para o espermatozoide humano cabeça, colo e cauda, formada pelas peças intermediária, principal e terminal com variações na forma e tamanho de cada componente. As diferenças mais significativas encontradas entre os espermatozoides comparados foram extensão de acrossomo, comprimento, largura e organização de peça intermediária. O presente estudo atingiu os objetivos propostos, apresentando resultados inéditos na literatura científica / The neotropical primates, also known as primates of the new world, are a diverse and varied group of animals, with unique anatomical and taxonomic characteristics, as well a unique behaviour, that distinguish those from the old world primates. It is estimated that these present 31% of the non-human primate population of the planet. High taxes of populational growth and anthropological disturbance have been undergoing due to deforestation, agriculture and cattle farms. In order to revert this situation and maximize the reproductive potential of the species, the development of biotechnologies associated to the reproduction are widely justified nowadays, for representing important reproductive tools, contributing to the preservation of the biodiversity and maintenance of genetic diversity. Hence, the purpose of this study was to collect Alouatta guariba clamitans semen by rectal electroejaculation then analyze the fresh semen, procedures never before described for the species, and adapt the preparation method of Leontopithecus chrysomelas, Alouatta caraya and Alouatta guariba clamitans semen for electron transmission microscope, allowing the comparison of the spermatozoa ultrastructure from the three species and also presenting new results to the scientific literature. For this study, it was used four specimens of L. chrysomelas, two specimens of A. caraya and two specimens of A.g. clamitans, adult males kept in captivity. L. chrysomelas had the semen taken by penine vibratory stimulation, and A. caraya and A.g. clamitans were submitted to rectal electroejaculation. After the semen was obtained, it was evaluated regarding the volume, pH, concentration, motility, sperm membrane integrity, acrosome integrity and morphology, and then prepared for electron transmission microscopy. The analysis of the spermatozoa of the three studied species revealed that all present the basic components described for the human spermatozoa - head, neck region and tail, formed by middle, principal and terminal pieces - with variations in 14 shape and size of each component. The most significant differences found between the compared spermatozoa were the acrosome extension and middle piece length, thickness and organization. The present study attained the proposed goals, presenting unprecedented results for the scientific literature

Titanium nitride (TiN) films are widely applied as diffusion barrier layers in microelectronic devices. The continued miniaturization of such devices not only poses new challenges to material systems design, but also puts high demands on characterization techniques. To gain understanding of diffusion processes that can eventually lead to failure of the barrier layer and thus of the whole device, it is essential to develop routines to chemically and structurally investigate these layers down to the atomic scale. In the present study, model TiN diffusion barriers with a Cu overlayer acting as the diffusion source were grown by reactive magnetron sputtering on MgO(001) and thermally oxidized Si(001) substrates. Cross-sectional transmission electron microscopy (XTEM) of the pristine samples revealed epitaxial, single-crystalline growth of TiN on MgO(001), while the polycrystalline TiN grown on Si(001) exhibited a [001]-oriented columnar microstructure. Various annealing treatments were carried out to induce diffusion of Cu into the TiN layer. Subsequently, XTEM images were recorded with a high-angle annular dark field detector, which provides strong elemental contrast, to illuminate the correlation between the structure and the barrier efficiency of the single- and polycrystalline TiN layers. Particular regions of interest were investigated more closely by energy dispersive X-ray (EDX) mapping. These investigations are completed by atom probe tomography (APT) studies, which provide a three-dimensional insight into the elemental distribution at the near-interface region with atomic chemical resolution and high sensitivity. In case of the single-crystalline barrier, a uniform Cu-enriched diffusion layer of 12 nm could be detected at the interface after an annealing treatment at 1000 °C for 12 h. This excellent barrier performance can be attributed to the lack of fast diffusion paths such as grain boundaries. Moreover, density-functional theory calculations predict a stoichiometry-dependent atomic diffusion mechanism of Cu in bulk TiN, with Cu diffusing on the N-sublattice for the experimental N/Ti ratio. In comparison, the polycrystalline TiN layers exhibited grain boundaries reaching from the Cu-TiN interface to the substrate, thus providing direct diffusion paths for Cu. However, the microstructure of these columnar layers was still dense without open porosity or voids, so that the onset of grain boundary diffusion could only be found after annealing at 900 °C for 1 h. The present study shows how to combine two high resolution state-of-the-art methods, TEM and APT, to characterize model TiN diffusion barriers. It is shown how to correlate the microstructure with the performance of the barrier layer by two-dimensional EDX mapping and three-dimensional APT. Highly effective Cu-diffusion barrier function is thus demonstrated for single-crystal TiN(001) (up to 1000 °C) and dense polycrystalline TiN (900 °C).

In dieser Arbeit werden die grundlegenden Wachstumsprozesse von Ga2O3 , mittels Transmissionselektronenmikroskopie analysiert. Dazu gehört die Untersuchung des heteroepitaktischen Wachstums von Galliumoxidschichten welche mittels Molekularstrahlepitaxie (molekular beam epitaxy MBE), der gepulsten Laser Abscheidung (pulsed laser deposition (PLD)) und der metallorganischen Gasphasenepitaxie (metalorganic vapor phase epitaxy (MOVPE)) auf (0001) orientierte Saphir Substraten abgeschieden wurden. Heteroepitaktisches Wachstum von Ga2O3 auf Saphir (0001) erfolgt bis zu einer Dicke von 3 Monolagen pseudomorph als α-Ga2O3 welches durch die Gitterfehlpassung zwischen Galliumoxid und dem Saphire Substrat induzierte Gitterverspannung stabilisiert wir. Weiterhin, im Fokus der Arbeit stehend, wird das homoepitaktische Wachstum von Galliumoxid auf (100) orientierten Galliumoxidsubstraten untersucht. Neben den Besonderheiten des Schichtwachstums, die sich aus den eingesetzten metallorganischen Präkursoren und Sauerstoffquellen ergeben, wird die Schichtstruktur in Abhängigkeit der typischen Wachstumsparameter (Wachstumstemperatur, Wachstumsrate, Kammerdruck und Fehlorientierung des Substrates) analysiert. Dabei wird gezeigt das homoepitaktischen Wachstum auf (100) orientiertem, β-Ga2O3, mittels MOVPE, die kristalline Perfektion der gewachsenen Schichten stark von den verwendeten Präkursoren (Trimethylgallium (TMGa) und Triethylgallium (TEGa) als metallorganische Ausgangsstoffe und H2 O oder purer Sauerstoff als Oxidant) und den chemischen Prozessen an der Oberfläche bestimmt wird. Des Weiteren wird die Entstehung von Zwillingslammelen in abhähngigkeit der Fehlorientierung untersucht. Durch die Einführung von vorbestimmten Fehlorientierungswinkeln der Substrate ist es möglich das Entstehen der Zwillingslamellen zu verhindern, und ein Stufenflusswachstum zu generieren. Durch die Anwendung eines Ratengleichungsansatzes ist es möglich die experimentell beobachteten Dichten an Zwillingslamellen zu erklären und einen Diffusionskoeffizienten zu bestimmen. (i) Heteroepitaktisches Wachstum von Ga2O3 auf Saphir (0001) erfolgt bis zu einer Dicke von 3 Monolagen pseudomorph als alpha-Ga2O3. Oberhalb dieser Schicht wächst relaxiertes ß-Ga2O3 in Form von 3 Rotationsdomänen auf. Die Stabilisation der dünnen alpha-Ga2O3 Schicht wird, durch die Gitterfehlpassung zwischen Galliumoxid und dem Saphire Substrat induzierte Gitterverspannung bewirkt. (ii) Beim homoepitaktischen Wachstum auf (100) orientiertem ß-Ga2O3 mittels MOVPE wird die kristalline Perfektion der gewachsenen Schichten stark von den verwendeten Präkursoren (Trimethylgallium (TMGa) und Triethylgallium (TEGa) als metallorganische Ausgangsstoffe und H2O oder purer Sauerstoff als Oxidant) und den chemischen Prozessen an der Oberfläche bestimmt. Während beim Wachstum mittels TMGa und O2 vorwiegend polykristalline Schichten entstehen, ergeben sich beim Wachstum mittels TMGa und H2O sowie TEGa und O2 geschlossenen epitaktische Schichten. Dieser signifikante Unterschiede lässt sich durch die unterschiedlichen Reaktionswege der Ausgangsstoffe sowie durch die katalytische Wirkung der (100) Flächen des ß-Ga2O3 erklären. (iii) Die Perfektion, mittels MOVPE gewachsener, homoepitaktischer Schichten, auf (100) orientierten Substraten, ist stark von der Fehlorientierung des Substrates bestimmt. Schichten die auf Substraten mit geringen Fehlorientierungen abgeschieden werden (< 2° bei Wachstumstemperaturen < 850°C) sind durch eine hohe Dichte an Zwillingslamellen gekennzeichnet. Die Entstehung der Zwillingslamellen ist ein Resultat eines Doppelpositionierungsprozesses der Atome auf der Oberfläche der Wachstumsebene. Durch die Einführung von vorbestimmten Fehlorientierungswinkeln der Substrate ist es möglich das Entstehen der Zwillingslamellen zu verhindern, und ein Stufenflusswachstum zu generieren. Durch die Anwendung eines Ratengleichungsansatzes, welcher die konkurrierenden Prozesse des Einbaus von Atomen in Oberflächenstufen sowie der Nukleation und des Wachstum von zweidimensionalen Inseln beschreibt, ist es möglich die experimentell beobachteten Dichten an Zwillingslamellen zu erklären und einen Diffusionskoeffizienten zu bestimmen, Dieser ist um zwei Größenordnungen geringer als bei klassischen Halbleitern, wie z. B. GaAs. In dieser Arbeit werden die grundlegenden Wachstumsprozesse von Ga2O3 , mittels Transmissionselektronenmikroskopie analysiert. Dazu gehört die Untersuchung des heteroepitaktischen Wachstums von Galliumoxidschichten welche mittels Molekularstrahlepitaxie (molekular beam epitaxy MBE), der gepulsten Laser Abscheidung (pulsed laser deposition (PLD)) und der metallorganischen Gasphasenepitaxie (metalorganic vapor phase epitaxy (MOVPE)) auf (0001) orientierte Saphir Substraten abgeschieden wurden. Heteroepitaktisches Wachstum von Ga2O3 auf Saphir (0001) erfolgt bis zu einer Dicke von 3 Monolagen pseudomorph als α-Ga2O3 welches durch die Gitterfehlpassung zwischen Galliumoxid und dem Saphire Substrat induzierte Gitterverspannung stabilisiert wir. Weiterhin, im Fokus der Arbeit stehend, wird das homoepitaktische Wachstum von Galliumoxid auf (100) orientierten Galliumoxidsubstraten untersucht. Neben den Besonderheiten des Schichtwachstums, die sich aus den eingesetzten metallorganischen Präkursoren und Sauerstoffquellen ergeben, wird die Schichtstruktur in Abhängigkeit der typischen Wachstumsparameter (Wachstumstemperatur, Wachstumsrate, Kammerdruck und Fehlorientierung des Substrates) analysiert. Dabei wird gezeigt das homoepitaktischen Wachstum auf (100) orientiertem, β-Ga2O3, mittels MOVPE, die kristalline Perfektion der gewachsenen Schichten stark von den verwendeten Präkursoren (Trimethylgallium (TMGa) und Triethylgallium (TEGa) als metallorganische Ausgangsstoffe und H2 O oder purer Sauerstoff als Oxidant) und den chemischen Prozessen an der Oberfläche bestimmt wird. Des Weiteren wird die Entstehung von Zwillingslammelen in abhähngigkeit der Fehlorientierung untersucht. Durch die Einführung von vorbestimmten Fehlorientierungswinkeln der Substrate ist es möglich das Entstehen der Zwillingslamellen zu verhindern, und ein Stufenflusswachstum zu generieren. Durch die Anwendung eines Ratengleichungsansatzes ist es möglich die experimentell beobachteten Dichten an Zwillingslamellen zu erklären und einen Diffusionskoeffizienten zu bestimmen. Des Weiteren wird das Wachstum im Mischsystem (InxGa1-x)2O3 untersucht und gezeigt das Indium als grenzflächenaktive Substanz wirken kann. / In this work we study the basic growth processes of epitaxial Ga2O3 films, by means of transmission electron microscopy. We investigate the heteroepitaxial growth of thin layers Ga2O3 on the (0001) plane of sapphire grown by molecular beam epitaxy (MBE), pulsed laser deposition (PLD) and metal organic vapor phase epitaxy (MOVPE). Furthermore, we will focus on the homoepitaxial growth on the (100) plane by MOVPE. Beside the peculiarities of the layer growth dependence on different metal organic precursors and oxygen sources, we investigate the influence of typical growth parameters (i.e. growth temperature, growth-rate, chamber pressure and miscut angle of the substrate) on the layer morphology. Incase of heteroepitaxial growth of β-Ga2O3 on (0001) plane of sapphire, independent of the growth method, the formation of a 3 monolayer thick α-Ga2O3 layer is observed, which is stabilized through strain, as a result of the lattice mismatch between sapphire and α-Ga2O3. In case of homoepitaxial growth by MOVPE on (100) oriented β-Ga2O3, the crystalline quality of the grown layer strongly depends on the used precursor (tri-methyl-gallium (TMGa) or tri-ethyl-gallium (TEGa) as metal precursor and H2O or pure oxygen as oxidant) and the chemical processes on the surface, respectively. Further on is the crystalline perfection of homoepitaxial layers grown by MOVPE on (100) oriented β-Ga2O3 substrates strongly dependent on the miscut-angle of the substrates. Layer grown on substrate with a small miscut-angle (< 2°) show high amount of twin lamella. These twin lamella are a result of a possible double positioning mechanism of ad-atoms on the growth surface. By introducing appropriate miscut-angles of the substrate it is possible to suppress the formation of these twin lamellae, and enable step flow growth. By applying a rate equation approach, describing the competing processes of incorporation of ad-atoms at kink sites or nucleation and growth of two dimensional island, it is possible to quantitatively reproduce the experimentally observed twin lamella densities and to determine a surface diffusion coefficient of the ad-atoms. Furthermore, in case of the alloy system (InxGa1-x)2O3, it is shown that indium can act as an surfactant, by increasing the surface diffusion.

L'utilisation récente des nanoparticules (NPs) comme vecteurs pour l'imagerie et l'adressage d'agents thérapeutiques en nano-médecine nécessite la compréhension de leurs mécanismes d'internalisation et de transport au niveau des barrières biologiques. Dans ce contexte, l'objectif de cette étude est de caractériser l'interaction et la transcytose de NPs de silice fluorescentes en fonction de leur taille (15, 50 et 100 nm) dans un modèle in-vitro de barrière endothéliale pulmonaire humaine. L'internalisation et le transport trans-endothélial des NPs a été analysé quantitativement à l'échelle nanométrique par microscopie électronique à transmission (MET) combinée à de la stéréologie. Un transport trans-endothélial a été observé pour toutes les tailles de NPs. Néanmoins, l'analyse de la distribution intracellulaire révèle une tendance à l'accumulation dans les voies de dégradation cellulaires pour les NPs de 50 et 100 nm. Cette accumulation est moindre pour les NPs de 15 nm. L'internalisation des NPs a également été analysée par cytométrie en flux et MET en présence de différents inhibiteurs de l'endocytose dans le but d'identifier leurs voies d'internalisation. En fonction de la taille des NPs, les mécanismes d'endocytose varient, suggérant une dépendance du transport trans-cellulaire à certains mécanismes d'endocytose. L'internalisation des NPs de 15 nm par la voie d'endocytose cavéole dépendante pourrait ainsi expliquer l'efficacité de leur transport du côté basal. Les méthodologies développées pour l'étude du transport trans-cellulaire des NPs de silice peuvent être appliquées à l'étude de NPs synthétiques plus complexes ou de NPs biologiques, telles que les lipoprotéines de basse-densité, et ce dans un contexte pathologique. / The recent use of nanoparticles (NPs) as carriers for imaging and delivery of therapeutics agents in nanomedecine involves understanding their endocytosis and transcytosis mechanisms at biological barriers. In this context, the aim of this study was to characterize the interaction and transcytosis of fluorescent silica NPs in function of their size (15, 50, and 100 nm) in an in-vitro model of human pulmonary endothelial barrier. NPs internalization and trans-endothelial transport has been quantitatively analyzed at nanometer resolution using transmission electron microscopy (TEM) combined with stereology. Trans-endothelial transport has been observed for each size of NPs. However cellular distribution analysis shows an accumulation in the cellular degradation pathways for 50 nm and 100 nm NPs. Whereas 15 nm NPs are less accumulated. NPs uptake was also analyzed by flow cytometry and TEM in the presence of different inhibitors to decipher NPs internalization pathways. Depending on NPs size, the involved endocytosis pathways were different, suggesting a dependency of trans-cellular transport toward endocytic mechanisms. The specific internalization of 15 nm NPs by the caveolin dependant pathway could explain the efficacy of their release at the basal side. Techniques developed for the study of the trans-cellular transport of silica NPs can also be applied to more complex synthetic NPs or biological NPs, such as low-density lipoproteins, in a pathological context.

The deformation mechanisms operating in diamond-like carbon (DLC) coatings on (100) and (111) Si, has been investigated. The effect of coating thickness, indenter geometry, substrate orientation and deposition technique on the deformation of DLC coatings and the underlying substrate was studied by undertaking nanoindentation followed by subsurface microstructural characterization. Uncoated (111) Si was also investigated for comparison. The observed microstructural features were correlated to the indentation response of the coatings and compared with simulation studies, as well as observations on uncoated Si. In uncoated (111) Si, phase transformation was found to be responsible for the discontinuities in the load-displacement curves, similar to (100) Si. However, slip was activated on {311} planes instead of on {111} planes. Moreover, the density of defects was also significantly lower and their distribution asymmetric. The coatings were adherent, uniformly thick and completely amorphous. The load-displacement curves displayed several pop-ins and a pop-out, the indentation loads for the first pop-in and the pop-out depending primarily on the thickness of the coating. The coatings exhibited localized compressive deformation in the direction of loading without any through-thickness cracks. The extent of this localized deformation increased with indentation load. Hardness and thickness of the coatings and the geometry of the indenter influenced the magnitude of compressive strains. Harder and thinner coatings and a blunt indenter exhibited the minimum degree of deformation. Densification by rearrangement of molecules has been suggested as the mechanism responsible for plastic compression. At indentation loads corresponding to the first pop-in, (100) and (111) silicon substrates initially deformed by <111> and <311> slip respectively. Higher indentation loads caused phase transformation. Therefore, unlike in uncoated Si, dislocation nucleation in the Si substrate has been proposed as the mode responsible for the first pop-in. Subsequent pop-ins were attributed to further deformation by slip and twinning, phase transformation and extensive cracking (median and secondary cracks) of the substrate. The pop-out, however, was ascribed to phase transformation. Extensive deformation in the substrate, parallel to the interface, is attributed to the wider distribution of the stress brought about by the DLC coating. Good correlation was obtained between the nanoindentation response, microstructural features and simulation studies.