Resumo: Os vírus pertencentes ao gênero Begomovirus da família Geminiviridae são transmitidos pela mosca-branca Bemisia tabaci Gennadius, atualmente constituem um dos problemas fitossanitários mais sérios em diversas culturas. A mosca-branca encontra-se disseminada em todas as principais regiões produtoras de hortaliças do Brasil. No estado de São Paulo, em 2005, plantas de pimentão mostrando sintomas de deformação dos frutos, folhas e mosaico foliar, apresentaram-se infectadas por begomovírus. Até então, no Brasil, a infecção de pimentão por vírus deste gênero havia sido verificada apenas nos estados de Pernambuco e Bahia, no ano de 1997. Amostras de plantas de pimentão foram coletadas nos campos de produção localizados nos municípios de Paranapanema, Piraju, Alvinlândia, Ubirajara, Elias Fausto, Mogi Guaçu, Paulínia e Botucatu, no período de novembro de 2004 a maio de 2006, com finalidade de detecção e verificar a variabilidade genética dos begomovírus infectando esta cultura. A detecção de begomovírus foi realizada por meio de PCR com oligonucleotídeos universais e seqüenciamento de parte da região codificadora para a proteína capsidial. Do total de 228 plantas coletadas, foram encontradas 30 amostras positivas de begomovírus, sendo que seqüências de 23 isolados apresentaram uma identidade de nucleotídeos de 98 a 100% com o Tomato severe rugose virus (ToSRV) e dois isolados, um proveniente da região de Alvinlândia e outro de Mogi Guaçu, apresentaram maior identidade (98 e 95% respectivamente) com a provável espécie Tomato yellow vein streak virus (ToYVSV). O DNA-A do isolado ToSRV[PJU] coletado de pimentão Lilac da região de Pirajú apresentou identidade de nucleotídeos de 99% com outro isolado de ToSRV (DQ207749) proveniente de pimenta e 97% com um isolado de ToSRV (AY029750) proveniente de tomate... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The viruses belonging to the genus Begomovirus, of the family Geminiviridae, are transmitted by the whitefly Bemisia tabaci Gennadius, and considered one of the most important fitossanitary problems for several crops. The whitefly is disseminated throughout the country. In São Paulo State, 2005, pepper plants showing fruits deformations, and mosaic on the leaves were infected by begomovirus. Until then in Brazil, the infection of begomovirus in pepper was only reported in 1997, in Pernambuco and Bahia States. Pepper plants were collected in Paranapanema, Piraju, Alvinlândia, Ubirajara, Elias Fausto, Mogi Guaçu, Paulínia and Botucatu, in the period of November of 2004 to May of 2006, with the aim to study the genetic variability of the begomovirus infecting this culture. The begomovirus were detected by PCR using universal oligonucleotídeos and the coat protein region was sequenced. On the total of 228 samples, they were found 30 positive samples of begomovírus, and sequences of 23 isolated presented nucleotides identies of 98 to 100% with Tomato severe rugose virus (ToSRV), while two isolates, one of Alvinlândia and another of Mogi Guaçu, showed identity highest nucleotide (98 and 95% respectively) with the tentative species Tomato yellow vein streak virus (ToYVSV). The nucleotide sequence of the DNA-A of ToSRV[PJU] from pepper cv. Lilac of Pirajú has 99% nucleotide identies with other isolate of ToSRV (DQ207749) collected from pepper and 97% with an isolate of ToSRV (AY029750) from tomato. The sequence of the DNA-B revealed identity of 98% with the DNA-B of ToRMV (AF291706) from Triângulo Mineiro, Minas Gerais and 95% with ToSRV (AY029751). Sap-transmission was verified for Nicandra physaloides and pepper ‘Magda' using infected N. benthamiana as source. By whitefly the virus was transmitted to Lycopersicum esculentum... (Complete abstract click electronic access below) / Orientador: Marcelo Agenor Pavan / Coorientador: Renate Krause Sakate / Banca: Antonio Carlos Maringoni / Banca: Murilo Francisco Zerbini Junior / Banca: Romulo Fujito Kobori / Banca: Valdir Atsushi Yuki / Doutor

Orientador: Katia Cristina Kupper / Banca: Sérgio Florentino Pascholati / Banca: João Martins Pizauro Junior / Banca: Rita de Cássia Panizzi / Banca: Sandra Regina Ceccato Antonini / Resumo: The biocontrol efficiency of Aureobasidium pullulans strain ACBL-77 was evaluated against Geotrichum citri-aurantii, the causal agent of sour rot in citrus, and their interactions were studied in vitro and in vivo. For this purpuse, were evaluated (i) the biocontrol efficiency of A. pullulans ACBL-77 against the pathogen; (ii) the effect of different culture media on the production of yeast cells; (iii) the incorporation of nutritional sources in optimizing the antagonistic activity; (iv) the competition for nutrients between the microorganisms; (v) the effect of nutrients on yeast cell and biofilm production and their relation in the biocontrol of disease; (vi) the survival of yeast in citrus fruits; (vii) the interaction between the microorganisms by scanning electron microscopy; (viii) the effect of A. pullulans applications in the citrus fruits quality; (ix) the effect of ACBL-77 in the activity of the enzymes possibly involved with resistance induction. In the results this study, were verified that in the fruits treated with A. pullulans ACBL-77, preventively, the control percentage was higher than the curative treatment. The potato-dextrose-agar medium promoted the highest amount of yeast cells. The incorporation of micronutrients, such as boric acid, cobalt chloride and ammonium molybdate (1 mM), favoured the antagonistic action of A. pullulans in vitro. Ammonium sulphate 1mM and sucrose 0.5% favoured the yeast during the competition between the microorganisms. However... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The biocontrol efficiency of Aureobasidium pullulans strain ACBL-77 was evaluated against Geotrichum citri-aurantii, the causal agent of sour rot in citrus, and their interactions were studied in vitro and in vivo. For this purpuse, were evaluated (i) the biocontrol efficiency of A. pullulans ACBL-77 against the pathogen; (ii) the effect of different culture media on the production of yeast cells; (iii) the incorporation of nutritional sources in optimizing the antagonistic activity; (iv) the competition for nutrients between the microorganisms; (v) the effect of nutrients on yeast cell and biofilm production and their relation in the biocontrol of disease; (vi) the survival of yeast in citrus fruits; (vii) the interaction between the microorganisms by scanning electron microscopy; (viii) the effect of A. pullulans applications in the citrus fruits quality; (ix) the effect of ACBL-77 in the activity of the enzymes possibly involved with resistance induction. In the results this study, were verified that in the fruits treated with A. pullulans ACBL-77, preventively, the control percentage was higher than the curative treatment. The potato-dextrose-agar medium promoted the highest amount of yeast cells. The incorporation of micronutrients, such as boric acid, cobalt chloride and ammonium molybdate (1 mM), favoured the antagonistic action of A. pullulans in vitro. Ammonium sulphate 1mM and sucrose 0.5% favoured the yeast during the competition between the microorganisms. However, the addition of ammonium sulphate at 1% in the yeast culture stimulated biofilm production and consequently increased the antagonistic activity against the pathogen in vivo. The application of A. pullulans to fruits allowed the better survival of yeast in wounded sites of citrus fruit. The yeast was found to be able to form biofilm on citrus, deforming the ... (Complete abstract click electronic access below) / Doutor

A resistência de plantas daninhas é um grande problema que afeta a produtividade das culturas em nosso país, ao passo que quando é identificada, deve ser estudada visando desenvolver estratégias de manejo que evitem a expansão e seleção em novas áreas, além de seleção de resistência múltipla em áreas onde está presente. Nesta temática o objetivo desta pesquisa foi determinar manejo alternativo ao glyphosato para as espécies Chloris elata e Digitaria insularis no período de entressafra da cultura da soja e identificar características morfológicas da superfície foliar de Chloris elata que possam interferir sobre o controle com herbicidas. Foram estudadas alternativas ao glyphosate para manejo em áreas de sistema de produção soja/milho, em dois experimentos com as plantas daninhas Chloris elata e Digitaria insularis perenizadas e com duas condições de roçada (15 e 30 cm). Foram avaliados o controle visual das plantas daninhas, redução de matéria fresca e seca. Para morfologia das folhas da espécie Chloris elata foi realizada análise histológica com recursos de microscopia eletrônica de varredura, visando caracterizar a morfologia da folha, além de determinar o índice estomático, densidade estomática e densidade de tricomas para as superfícies adaxial e abaxial das folhas, nos estádios 4 e 6 folhas totalmente expandidas. Digitaria insularis se mostrou menos suscetível as técnicas de manejo empregadas para plantas perenizadas, necessitando duas aplicações sequenciais para conter a rebrota dos tratamentos. O uso da maior dose do herbicida haloxyfop associado aos herbicidas glyphosate ou amônio-glufosinato se mostrou eficaz no controle de C. elata e D. insularis. O manejo de roçagem potencializou a ação dos herbicidas, e a planta daninha D. insularis se mostrou mais suscetível a está técnica em relação ao C. elata. Para este manejo a associação da maior dose de haloxyfop ao glufosinato foi eficaz para as duas gramíneas. As alturas de roçagem somente se diferenciaram nas primeiras avaliações. O biótipo resistente de C. elata apresentou menor densidade estomática, no estádio de 4 folhas completamente expandidas e maior densidade de tricomas no estádios de 6 folhas completamente expandidas, o que pode contribuir para a resistência ao glyphosate. Está espécie apresentou formações de cristais de cera que circundam o ostíolo, no estádio de 6 folhas completamente expandidas, podendo estar relacionada a a menor suscetibilidade ao glyphosate desta planta daninha perenizada. / Weed resistance is a major problem that affects crop productivity in our country, whereas when it is identified, it must be studied in order to develop management strategies that avoid expansion and selection in new areas, as well as multiple resistance selection in areas where it is present. In this subject the objective of this research was to determine alternative management of glyphosate for the Chloris elata and Digitaria insularis species during the off - season of the soybean crop and to identify the morphological characteristics of the leaf surface of Chloris elata that could interfere the control with herbicides. It was studied alternatives to glyphosate for management in areas of soybean / maize production system in two experiments with weeds Chloris elata and Digitaria insularis perennial and with two mowing conditions (15 and 30 cm). The visual control of weeds, fresh and dry matter reduction were evaluated. For the morphology of the leaves of the Chloris elata species, a histological analysis was carried out using scanning electron microscopy to characterize the leaf morphology, as well as to determine the stomatal index, stomatal density and trichome density for the adaxial and abaxial surfaces of the leaves, stages 4 and 6 fully expanded leaves. Digitaria insularis was less susceptible to the management techniques used for perennial plants, requiring two sequential applications to contain the regrowth of treatments. The use of the highest dose of the herbicide haloxyfop associated with the herbicides glyphosate or ammonium glufosinate proved to be effective in the control of C. elata and D. insularis. Herbicide management potentiated herbicide action, and D. insularis weed was more susceptible to this technique than C. elata. For this management, the association of the highest dose of haloxyfop to glufosinate was effective for both grasses. The cutting heights differed only in the first evaluations. The resistant C. elata biotype showed lower stomatal density at the stage of 4 fully expanded leaves and greater density of trichomes in the stages of 6 fully expanded leaves, which may contribute to resistance to glyphosate. This species presented waxy crystal formations that surround the ostrich, in the stage of 6 fully expanded leaves, and may be related to the natural tolerance to the glyphosate of this perennial weed.

Os sistemas de lima única constituem a mais recente tecnologia no que se refere ao preparo dos canais radiculares, e vem sendo objeto de estudo sob diversos aspectos. Uma das preocupações referentes aos sistemas automatizados é a manutenção da qualidade e segurança nos preparos quando da reutilização do instrumento. Este estudo teve como objetivo avaliar as alterações de superfície de dois sistemas de lima única - Wave One e One Shape - e seu desempenho através do tempo de preparo no decorrer dos usos. Duzentos condutos de molares inferiores humanos extraídos foram instrumentados com 10 limas Wave One e 10 limas One Shape, acompanhados de irrigação com 10 ml de Hipoclorito de Sódio 2.5%. Cada instrumento foi utilizado por 10 vezes. Os tempos para o preparo completo do canal foram registrados por um cronômetro digital e anotados em tabelas. Para avaliação em microscopia eletrônica de varredura as limas novas foram observadas assim que removidas da embalagem. Após as utilizações, os instrumentos foram limpos, esterilizados em autoclave e observados após 2, 6, 8 e 10 usos. Três avaliadores calibrados observaram as fotomicrografias e registraram de acordo com os scores propostos a presença ou ausência de alterações superficiais. O teste Kappa de concordância entre os avaliadores apresentou maioria de resultados ótimo a moderado (?=5%). Os dados referentes à avaliação das imagens foram submetidos ao teste Wilcoxon-Mann-Whitney com nível de significância de 5% (?=5%). As alterações mais observadas foram aplainamento e microcavidade. O crack foi observado nos dois instrumentos sendo significante a partir do sexto uso para Wave One e do décimo para One Shape. Os dados relativos aos tempos de preparo foram submetidos à análise estatística pelos testes Wilcoxon-Mann-Whitney, ANOVA e Mc-Nemar com nível de significância de 5% (?=5%). Os tempos de preparo registrados para One Shape foram inferiores aos tempos para Wave One. O uso influenciou os tempos de preparo sendo observado um aumento significativo para os dois instrumentos. Um instrumento One Shape fraturou mas a ocorrência não foi estatisticamente significante. / Single-file systems are the most recent technology in regard to the preparation of root canals and have been studied in several ways. One of the concerns related to automated systems is maintaining quality and safety in preparation when the instrument is reused. This study\'s aim was to evaluate the changes on the surface of two single-file systems - Wave One and One Shape - and their performance through preparation time over the uses. Two hundred root canals of extracted human molars were instrumented with 10 Wave One files and 10 One Shape files, followed by irrigation with 10 ml of sodium hypochlorite 2.5%. Each instrument was used 10 times. The time required to complete each root canal was measured by a digital stopwatch and recorded in tables. New unused files were analysed using a scanning electron microscope. After used, the instruments were cleaned and sterilized, then observed after 2, 6, 8 and 10 uses. Three calibrated evaluators observed these SEM images and listed according to the suggested scores the presence or absence of changes on the surface. The Kappa test showed mostly results from moderate to great (? = 5%). The data relating to evaluation of images was submitted to the Wilcoxon-Mann-Whitney test with a significance level of 5% (? = 5%). The most frequent changes were blunt edges and microcavities. Cracking was observed in both instruments being significant in Wave One after the sixth use and in One Shape after the tenth use. Data on preparation times were statistically analysed by the Wilcoxon-Mann-Whitney, ANOVA and Mc-Nemar tests with a significance level of 5% (? = 5%). Regarding the working time taken to shape the canals, One Shape was significantly faster than Wave One. The preparation time was affected by the number of file use increasing significantly on both instruments. A One Shape instrument fractured but the finding was not statistically significant.

O electrospinning (deposição eletrostática) representa um método simples e conveniente para preparar bras poliméricas e bras cerâmicas com interiores sólidos ou ocos, excepcionalmente longos em comprimento, uniformes no diâmetro, variando no intervalo de dezenas de nanômetros a vários micrômetros, com diversicadas composições. Apesar da potencialidade e versatilidade da Microscopia Eletrônica de Varredura existe um grande problema de caracterizar nanobras, já que este processo não é feito automaticamente. Este trabalho apresenta uma nova metodologia para extração automática de medidas em imagens SEM, como uma alternativa eciente em relação aos softwares semi-automáticos propostos na literatura. A metodologia proposta envolve o processamento das imagens SEM usando Morfologia Matemática e os resultados obtidos são discutidos e comparados àqueles processados por Redes Neurais Celulares e pelo método de Otsu. Imagens coletadas do microscópio foram analisadas por técnicas de processamento de imagens para determinação do diâmetro das nanobras. Os resultados mostraram boa concordância entre as metodologias estudadas e o método padrão-ouro indicando que os mesmos podem substituí-lo para constituir uma alternativa automática na caracterização de nanobras. / The electrospinning technique represents a simple and convenient process to provide polymers and ceramics bers with both solid and hollow interiors that are exceptionally long in length, uniform in diameter, varying within the interval of few nanometers to several microns, and diversied in composition. Despite the versatility and potencially the scanning electron microscopy (SEM) there is a serious problem to characterize nanobers, as it is not made automatically. This work presents novel methodology software for automatic measurement in SEM images, as an eficient alternative in comparison with semi-automatic softwares proposed in literature. The proposed methodology involves the image processing SEM using Mathematical Morphology and the obtained results are argued and compared with those processing for Celullar Neural Network and Otsu method. The microscope images were assessed and analysed by imagery processing techniques to determinate diameters of nanobers. The results has shown good agreement between the investigated and gold standard methods suggesting that the same ones can substitute it to constitute an automatic alternative in the nanobers characterization.

Philosophiae Doctor - PhD / The main objective of this study was to advance kinetic performances of formation and decomposition of magnesium hydride by design strategies which include high energy ball milling in hydrogen (HRBM), in combination with the introduction of catalytic/dopant additives. In this regard, the transformation of Mg → MgH2 by high energy reactive ball milling in hydrogen atmosphere (HRBM) of Mg with various additives to yield nanostructured composite hydrogen storage materials was studied using in situ pressure-temperature monitoring that allowed to get time-resolved results about hydrogenation behaviour during HRBM. The as-prepared and re-hydrogenated nanocomposites were characterized using XRD, high-resolution SEM and TEM, as well as measurements of the mean particle size. Dehydrogenation performances of the nanocomposites were studied by DSC / TGA and TDS; and the re-hydrogenation behaviour was investigated using Sieverts volumetric technique.

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).

Thin film arrays of molecules or supramolecules are active subjects of investigation because of their potential value in electronics, chemical sensing, catalysis, and other areas. Scanning probe microscopes (SPMs), including scanning tunneling microscopes (STMs) and atomic force microscopes (AFMs) are commonly used for the characterization and metrology of thin film arrays. As opposed to transmission electron microscopy (TEM), SPMs have the advantage that they can often make observations of thin films in air or liquid, while TEM requires highly specialized techniques if the sample is to be in anything but vacuum. SPM is a surface imaging technique, while TEM typically images a 2D projection of a thin 3D sample. Additionally, variants of SPM can make observations of more than just topography; for instance, magnetic force microscopy measures nanoscale magnetic properties. Thin film arrays are typically two-dimensionally periodic. A perfect, infinite two-dimensionally periodic array is mathematically constrained to belong to one of only 17 possible 2D plane symmetry groups. Any real image is both finite and imperfect. Crystallographic Image Processing (CIP) is an algorithm that Fourier transforms a real image into a 2D array of complex numbers, the Fourier coefficients of the image intensity, and then uses the relationship between those coefficients to first ascertain the 2D plane symmetry group that the imperfect, finite image is most likely to possess, and then adjust those coefficients that are symmetry-related so as to perfect the symmetry. A Fourier synthesis of the symmetrized coefficients leads to a perfectly symmetric image in direct space (when accumulated rounding and calculation errors are ignored). The technique is, thus, an averaging technique over the direct space experimental data that were selected from the thin film array. The image must have periodicity in two dimensions in order for this technique to be applicable. CIP has been developed over the past 40 years by the electron crystallography community, which works with 2D projections from 3D samples. Any periodic sample, whether it is 2D or 3D has an "ideal structure" which is the structure absent any crystal defects. The ideal structure can be considered one average unit cell, propagated by translation into the whole sample. The "real structure" is an actual sample containing vacancies, dislocations, and other defects. Typically the goal of electron and other types of microscopy is examination of the real structure, as the ideal structure of a crystal is already known from X-ray crystallography. High resolution transmission electron microscope image based electron crystallography, on the other hand, reveals the ideal crystal structure by crystallographic averaging. The ideal structure of a 2D thin film cannot be easily in a spatially selective fashion examined by grazing incidence X-ray or low energy electron diffraction based crystallography. SPMs straightforwardly observe thin films in direct space, but SPM accuracy is hampered by blunt or multiple tips and other unavoidable instrument errors. Especially since the film is often of a supramolecular system whose molecules are weakly bonded (via pi bonds, hydrogen bonds, etc.) both to the substrate and to each other, it is relatively easy for a molecule from the film to adhere to the scanning tip during the scan and become part of the tip during subsequent observation. If the thin film array has two-dimensional periodicity, CIP is a unique and effective tool both for image enhancement (determination of ideal structure) and for the quantification of overall instrument error. In addition, if a sample of known 2D periodicity is scanned, CIP can return information about the contribution of the instrument itself to the image. In this thesis we show how the technique is applied to images of two dimensionally periodic samples taken by SPMs. To the best of our knowledge, this has never been done before. Since 2D periodic thin film arrays have an ideal structure that is mathematically constrained to belong to one of the 17 plane symmetry groups, we can use CIP to determine that group and use it for a particularly effective averaging algorithm. We demonstrate that the use of this averaging algorithm removes noise and random error from images more effectively than translational averaging, also known as "lattice averaging" or "Fourier filtering". We also demonstrate the ability to correct systematic errors caused by hysteresis in the scanning process. These results have the effect of obtaining the ideal structure of the sample, averaging out the defects crystallographically, by providing an average unit cell which, when translated, represents the ideal structure. In addition, if one has recorded a scanning probe image of a 2D periodic sample of known symmetry, we demonstrate that it is possible to use the Fourier coefficients of the image transform to solve the inverse problem and calculate the point spread function (PSF) of the instrument. Any real scanning probe instrument departs from the ideal PSF of a Dirac delta function, and CIP allows us to quantify this departure as far as point symmetries are concerned. The result is a deconvolution of the "effective tip", which includes any blunt or multiple tip effects, as well as the effects caused by adhesion of a sample molecule to the scanning tip, or scanning irregularities unrelated to the physical tip. We also demonstrate that the PSF, once known, can be used on a second image taken by the same instrument under approximately the same experimental conditions to remove errors introduced during that second imaging process. The preponderance of two-dimensionally periodic samples as subjects of SPM observation makes the application of CIP to SPM images a valuable technique to extract a maximum amount of information from these images. The improved resolution of current SPMs creates images with more higher-order Fourier coefficients than earlier, "softer" images; these higher-order coefficients are especially amenable to CIP, which can then effectively magnify the resolution improvement created by better hardware. The improved resolution combined with the current interest in supramolecular structures (which although 3D usually start building on a 2D periodic surface) appears to provide an opportunity for CIP to significantly contribute to SPM image processing.

Nanostructures were formed on diced specimens of several silicon carbide polytypes and silicon using electron beam lithography. A general introduction to nanostructure synthesis and electron beam lithography,are presented. A scanning electron microscope was retrofitted with a commercially available electron beam lithography package and an electrostatic beam blanker to permit nanoscale lithography to be performed. A process was first developed and optimized on silicon substrates to expose, poly-methyl-methacrylate (PMMA) resist with an electron beam to make nanoscale nickel masks for reactive ion etching. The masks consist of an array of nickel dots that range in size from 20 to 100 nm in diameter. Several nanoscale structures were then fabricated in silicon carbide using electron beam lithography. The structures produced are characterized by field emission Scanning Electron Microscopy.

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.