Submitted by TATIANA MARIA GONÇALVES null (tati_unesp@hotmail.com) on 2016-04-01T01:09:37Z No. of bitstreams: 1 Dissertação TATIANA HOMOLOGAÇAO (2) pdf.pdf: 2109094 bytes, checksum: 0a330bb6e84a7dbf2b6ff2f896c7189d (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-04-05T14:49:04Z (GMT) No. of bitstreams: 1 goncalves_tm_me_bot.pdf: 2109094 bytes, checksum: 0a330bb6e84a7dbf2b6ff2f896c7189d (MD5) / Made available in DSpace on 2016-04-05T14:49:04Z (GMT). No. of bitstreams: 1 goncalves_tm_me_bot.pdf: 2109094 bytes, checksum: 0a330bb6e84a7dbf2b6ff2f896c7189d (MD5) Previous issue date: 2016-02-19 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introdução: denomina-se presbifonia o conjunto de alterações nos padrões vocais consequentes ao envelhecimento da laringe, podendo cursar com sintomas de disfonia, voz fraca, trêmula e baixa. Estudos histológicos e imunohistoquímicos da presbilaringe demonstram atrofia do epitélio, da lâmina própria e do músculo vocal, além de aumento de fibras colágenas e diminuição de fibras elásticas e das proteínas não fibrosas da matriz extracelular. Os estudos de microscopia eletrônica da presbilaringe são escassos e podem acrescentar detalhes ultraestruturais importantes e auxiliar na compreensão da fisiopatologia da presbifonia. Objetivos: descrever os achados de microscopia eletrônica de varredura e transmissão da prega vocal senil. Casuística e métodos: Foram removidas 16 laringes humanas durante necrópsia e distribuídas em dois grupos: controle (n-8; idade 30 - 50 anos; 6F e 2M) e idoso (n-8; idade 75- 92 anos; 6F e 2M). As porções medianas de ambas as pregas vocais foram dissecadas, fixadas em glutaraldeído 2,5% e preparadas para exames de microscopia eletrônica de varredura e transmissão. A espessura do epitélio foi medida nas fotografias de microscopia eletrônica de varredura, com aumentos semelhantes, utilizando-se o programa de morfometria digital Scandium. Resultados: Microscopia eletrônica de varredura: Grupo controle: epitélio composto por 5 a 7 camadas de células sobrepostas, raras células em descamação, e discreta ondulação. Lâmina própria com rede uniforme de fibras colágenas e elásticas, correndo paralelamente à membrana basal. Grupo idosos: epitélio atrófico, composto por 2 a 3 células, maior número de células em descamação, junções intercelulares demarcadas por sulcos profundos. As medidas da espessura do epitélio foram menores nos grupos de idosos do que nos controles (22163.91 ± SD 14590.41nm versus 41783.73 ± SD 2139.314 nm, respectivamente). Lâmina própria com rede densa de fibras colágenas e elásticas formando emaranhados e com distribuição irregular. Nas camadas profundas as fibras colágenas formavam verdadeira carapaça fibrótica e rígida. Microscopia eletrônica de transmissão: Grupo Controle: epitélio íntegro, com células justapostas e desmossomos entre as junções intercelulares. Membrana basal contínua e uniforme e lâmina própria contendo fibras colágenas e elásticas em arranjo frouxo e regular, e alguns fibroblastos de diferentes formatos. Grupo Idosos: células epiteliais distanciadas umas das outras e junções intercelulares alargadas. Membrana basal sem alterações, lâmina própria com predomínio de fibroblastos de formato alongado e citoplasma contendo vacúolos. Na lâmina própria havia densa rede de fibras colágenas, na qual alguns fibroblastos estavam mergulhados. Conclusões: neste estudo de microscopia eletrônica foram identificadas algumas alterações estruturais restritas às laringes dos idosos, tanto no epitélio como na lâmina própria, algumas delas com provável participação dos fibroblastos, o que reforça a importância de estudos adicionais voltados a essas células, tanto ultraestruturais como moleculares, por serem os principais precursores dos componentes da matriz extracelular. / Introduction: Presbyphonia is called the set of changes in vocal patterns consequent aging of the larynx, which can present with symptoms of dysphonia, weak voice, trembling and low. Histological and immunohistochemical studies of presbylarynx show atrophy of the epithelium, lamina propria and the vocal muscle, and increase of collagen fibers and diminution of elastic fibers and non-fibrous proteins of the extracellular matrix. The studies of electron microscopy of presbylarynx are scarce and can add significant ultrastructural details contribute to further understanding of the pathophysiology of presbyphonia. Objectives: To describe the findings of scanning and transmission electron microscopy of senile vocal folds. Methods: 16 human larynx were removed during autopsy and distributed into two groups: control (n-8; age 30-50 years; 6F and 2M) and elderly (n-8, age 75- 92 years; 6F and 2M). The median portions of the right and left vocal folds were dissected, fixed in 2.5% glutaraldehyde and prepared for examination using scanning and transmission electron microscopy respectively. The thickness of the epithelium was analyzed in the pictures of the scanning electron microscopy with similar magnification, using the Scandium morphometric digital program. Results: Scanning electron microscopy: Control Group: epithelium composed of 5-7 layers of overlapping cells, rare cells in flaking and slight ripple. Lamina propria with uniform network of collagen and elastic fibers running parallel to the basement membrane. Group Elderly: atrophic epithelium, 2 to 3 cells, with more flaking cells and intercellular junctions marked by deep grooves. The epithelial thickness was lower in elderly than controls (22163.91 ± SD 14590.41nm versus 41783.73 ± SD 2139.314 nm, respectively). Lamina propria with dense network of collagen and elastic fibers forming tangles with irregular distribution. In the deep layers of collagen fibers formed shell true fibrotic and stiff. Transmission electron microscopy: Control Group: intact epithelium, with overlapping cells and desmosomes between intercellular junctions. Continuous and smooth basement membrane and lamina propria containing collagen and elastic fibers in loose and regular arrangement, and fibroblasts of different formats. Group Elderly: epithelial cells apart from one another enlarged intercellular junctions. Basement membrane unchanged lamina propria predominantly with fibroblasts with elongated shape and cytoplasm containing vacuoles. In the lamina propria was dense network of collagen fibers, in which some fibroblasts were plunged. Conclusions: In this study of electron microscopy identified some structural changes restricted to the larynx of the elderly in both the epithelium and lamina propria, some of them with possible participation of fibroblasts. This reinforces the importance of additional studies, both ultrastructural and molecular, facing fibroblasts, because these are the main precursors of extracellular matrix components. / FAPESP: 2014/13741-1

Made available in DSpace on 2014-06-11T19:33:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-03-22Bitstream added on 2014-06-13T20:25:02Z : No. of bitstreams: 1 cavassim_r_dr_arafo.pdf: 4752194 bytes, checksum: dce6b5a310de662c347f9a2267e4dbb2 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O tratamento periodontal engloba o processo de raspagem e alisamento radicular, podendo ser realizado por diferentes meios que além de remover o cálculo dental, produzem diferentes características na superfície radicular como ranhuras e concavidades e também a formação de smear layer. O uso de agentes químicos é proposto na literatura para remover essa smear layer, e descontaminar a superfície radicular, aumentando assim as chances de formação de nova inserção conjuntiva. O objetivo do Estudo 1 foi avaliar, por meio de microscopia eletrônica de varredura, a influência de diferentes concentrações, modos e tempos de aplicação de ácido cítrico na biomodificação de superfícies radiculares submetidas à raspagem e alisamento radicular. Neste estudo, 270 amostras foram divididas em 6 grupos (45 amostras/grupo): soro fisiológico (controle), ácido cítrico (0.5%, 1%, 2%, 15% e 25%), com tempos de 1, 2 ou 3 minutos para cada grupo, nos modos de aplicação: a) aplicação passiva (bolinha de algodão); b) fricção suave (pincel); c) fricção vigorosa (bolinha de algodão), com renovação da solução a cada 30 segundos. As amostras foram submetidas à desidratação em concentrações crescentes de álcool etílico e HMDS, sendo em seguidas metalizadas e levadas para observação em microscopia eletrônica de varredura. Um examinador treinado, calibrado (kappa=0,93) e cego avaliou as fotomicrografias obtidas. A análise estatística foi realizada utilizando-se os testes de Kruskal- Wallis e Dunn. No estudo 2, investigou-se a influência da biomodificação radicular associada a diferentes meios de instrumentação na adesão de coágulo e elementos sanguíneos. Cento e cinquenta dentes afetados periodontalmente foram divididos em 5 grupos: Grupo I, instrumentação com curetas; Grupo II, instrumentação com curetas removendo cálculo superficial; Grupo III, remoção... / Periodontal treatment encompasses the scaling and root planning process and can therefore be carried out by various means. In addition to removing dental calculus, the root instrumentation produces different characteristics in the radicular surface as the formation of concavities and smear layer. The use of chemical agents is proposed in the literature to remove the smear layer, and detoxify the radicular surface, thereby increasing the chances of forming new connective attachment. The aim of the Study 1 was to assess, through scanning electron microscopy, the influence of different concentrations, application methods and application times of citric acid in the root conditioning after scaling and root planning. Two hundred seventy (270) samples were equally divided into six groups (n=45) for treatment with saline solution (control) and five different concentrations of citric acid (0.5, 1, 2, 15, and 25 percent). Three acid application methods were used (passive, brushing, and burnishing) as well as three application periods (1, 2, and 3 minutes). A previously trained, calibrated (kappa score = 0.93), and blind examiner subsequently scored scanning electron micrographs (SEMs) of the samples. Statistical analyses were performed by using Kruskal-Wallis and Dunn’s post-hoc tests. In Study 2, was investigated the influence of root conditioning associated with different modes of instrumentation in the adhesion of clot and blood elements. One hundred and fifty periodontally affected teeth were divided into five groups: Group I, instrumentation with curettes; Group II, instrumentation with curettes removing the superficial calculus; Group III, ultrasonic scaler with removal of the superficial calculus; Group IV, ultrasonic scaler for removal of the superficial calculus and after that, instrumentation with curettes; Group V, calculus surface. These five groups were further divided... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:33:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-12-11Bitstream added on 2014-06-13T19:04:23Z : No. of bitstreams: 1 ishi_ep_dr_arafo.pdf: 1080830 bytes, checksum: b4ee579b120082701f882275e1467551 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A remoção da smear layer e a exposição da matriz colágena da dentina de superfícies radiculares desprovidas de sua inserção conjuntiva tem o potencial de auxiliar o tratamento e/ou a regeneração periodontal. O objetivo deste estudo in vitro foi avaliar, por meio de microscopia eletrônica de varredura, a remoção da smear layer e a exposição da matriz colágena da dentina produzidas pela aplicação de cloridrato de tetraciclina (TTC-HCl). O cemento radicular das amostras foi removido com fresas diamantadas, processo seguido de raspagem e alisamento radicular com cureta. As 450 amostras foram divididas em 10 grupos: controle (soro fisiológico) e os que compreendiam a aplicação de TTCHCl nas concentrações: 10mg/ml, 25mg/ml, 50mg/ml, 75mg/ml, 100mg/ml, 125mg/ml, 150mg/ml, 200mg/ml e 250mg/ml. Todos os grupos receberam essa aplicação de diferentes formas (passiva, pincel e fricção) e em diferentes tempos (1, 2 e 3 minutos). A avaliação foi feita por um examinador treinado, calibrado e que não conhecia o grupo ao qual as amostras pertenciam, utilizando o índice de Sampaio (1999) modificado para este estudo. Os dados foram analisados pelos testes de Kruskal- Wallis e de Dunn. As concentrações de 50mg/mL e 75mg/mL aplicadas por fricção e por pincelamento foram as mais efetivas na remoção de smear layer e na exposição de colágeno. O modo de aplicação passivo mostrou-se inferior aos demais (p=0,0001) com relação aos mesmos parâmetros, assim como o tempo de aplicação de 1 minuto também foi inferior em relação aos demais (p=0,002). Concluiu-se que as concentrações de 50mg/mL e 75mg/mL aplicadas por pincelamento ou fricção durante 2 ou 3 minutos foram as mais efetivas. / Smear layer removal and collagen fibers exposure may improve periodontal treatment and regeneration. This in vitro study assessed smear layer removal and collagen fibers exposure after application of tetracycline hydrochloride (TTC-HCl) on root surfaces by means of scanning electron microscopy. Root cementum was removed with diamond burs followed by scaling and root planning. Four hundred and fifty samples were divided into 10 groups: control (application of saline) and application of TTC-HCl 10mg/ml, 25mg/ml, 50mg/ml, 75mg/ml, 100mg/ml, 125mg/ml, 150mg/ml, 200mg/ml and 250mg/ml. The TTCHCl application was performed in all groups in 3 different ways (passive, brushing and burnishing) and in 3 different times (1, 2 and 3 minutes). A previously trained, calibrated and blind examiner evaluated the photomicrographs using the Sampaio index (1999) modified for this study. Statistical analysis was performed by Kruskal-Wallis and Dunn tests. The 50mg/mL and 75mg/mL concentrations applied by burnishing or brushing were the most effectives in smear layer removal and collagen fibers exposure. Both, the passive mode of application (p=0.0001) and 1-minute time of application (p=0,002) were the less effectives. In conclusion, the 50mg/mL and 75mg/mL concentrations applied by brushing or burnishing during 2 or 3 minutes were the most effectives.

Made available in DSpace on 2014-06-11T19:34:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-01-23Bitstream added on 2014-06-13T21:06:56Z : No. of bitstreams: 1 nozaki_dn_dr_botfca.pdf: 1542379 bytes, checksum: 590b28b89a75aa5cd0e92315447c603f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / 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... / 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)

Metallophthalocyanines and metal nanoparticles were successfully synthesized and applied for the electrooxidation of amitrole, nitrite and hydrazine individually or when employed together. The synthesized materials were characterized using the following techniques: predominantly scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), electrochemistry and scanning electrochemical microscopy (SECM). Different electrode modification methods were used to modify the glassy carbon substrates. The methods include adsorption, electrodeposition, electropolymerization and click chemistry. Modifying the glassy carbon substrate with MPc (electropolymerization) followed by metal nanoparticles (electrodeposition) or vice versa, made a hybrid modified surface that had efficient electron transfer. This was confirmed by electrochemical impedance studies with voltammetry measurements having lower detection potentials for the analytes. This work also describes for the first time the micropatterning of the glassy carbon substrate using the SECM tip. The substrate was electrografted with 4-azidobenzenediazonium salt and then the click reaction was performed using ethynylferrocene facilitated by Cu⁺ produced at the SECM tip. The SECM imaging was then used to show the clicked spot.

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abstract: The remarkable conservation of molecular and intra-/inter-cellular pathways underpinning the fundamental aspects of sugar partitioning in two evolutionarily divergent organisms – a non-vascular moss Physcomitrella patens and a vascular cereal crop Oryza sativa (rice) – forms the basis of this manuscript. Much of our current knowledge pertaining to sugar partitioning in plants mainly comes from studies in thale cress, Arabidopsis thaliana, but how photosynthetic sugar is loaded into the phloem in a crop as important as rice is still debated. Even less is known about the mechanistic aspects of sugar movement in mosses. In plants, sugar either moves passively via intercellular channels called plasmodesmata, or through the cell wall spaces in an energy-consuming process. As such, I first investigated the structure of plasmodesmata in rice leaf minor vein using electron tomography to create as of yet unreported 3D models of these channels in both simple and branched conformations. Contrary to generally held belief, I report two different 3D morphotypes of simple plasmodesmata in rice. Furthermore, the complementary body of evidence in arabidopsis implicates plasma membrane localized Proton Pyrophosphatase (H+-PPase) in the energy-dependent movement of sugar. Within this wider purview, I studied the in situ ultrastructural localization patterns of H+-PPase orthologs in high-pressure frozen tissues of rice and physcomitrella. Were H+-PPases neo-functionalized in the vascular tissues of higher plants? Or are there evolutionarily conserved roles of this protein that transcend the phylogenetic diversity of land plants? I show that H+-PPases are distinctly expressed in the actively growing regions of both rice and physcomitrella. As expected, H+-PPases were also localized in the vascular tissues of rice. But surprisingly, H+-PPase orthologs were also prominently expressed at the gametophyte-sporophyte junction of physcomitrella. Upon immunogold labeling, H+-PPases were found to be predominantly localized at the plasma membrane of the phloem complexes of rice source leaves, and both the vacuoles and plasma membrane of the transfer cells in the physcomitrella haustorium, linking H+-PPases in active sucrose loading in both plants. As such, these findings suggest that the localization and presumably the function of H+-PPases are conserved throughout the evolutionary history of land plants. / Dissertation/Thesis / 3D MODEL OF SIMPLE PLASMODESMATA / 3D MODEL OF COMPLEX PLASMODESMATA / MODELING SIMPLE PLASMODESMATA IN IMOD / MODELING COMPLEX PLASMODESMATA IN IMOD / Doctoral Dissertation Biology 2016

abstract: Operando transmission electron microscopy (TEM) is an extension of in-situ TEM in which the performance of the material being observed is measured simultaneously. This is of great value, since structure-performance relationships lie at the heart of materials science. For catalyst materials, like the SiO2-supported Ru nanoparticles studied, the important performance metric, catalyst activity, is measured inside the microscope by determining the gas composition during imaging. This is accomplished by acquisition of electron energy loss spectra (EELS) of the gas in the environmental TEM while catalysis is taking place. In this work, automated methods for rapidly quantifying low-loss and core-loss EELS of gases were developed. A new sample preparation method was also established to increase catalytic conversion inside a differentially-pumped environmental TEM, and the maximum CO conversion observed was about 80%. A system for mixing gases and delivering them to the environmental TEM was designed and built, and a method for locating and imaging nanoparticles in zone axis orientations while minimizing electron dose rate was determined. After atomic resolution images of Ru nanoparticles observed during CO oxidation were obtained, the shape and surface structures of these particles was investigated. A Wulff model structure for Ru particles was compared to experimental images both by manually rotating the model, and by automatically determining a matching orientation using cross-correlation of shape signatures. From this analysis, it was determined that most Ru particles are close to Wulff-shaped during CO oxidation. While thick oxide layers were not observed to form on Ru during CO oxidation, thin RuO2 layers on the surface of Ru nanoparticles were imaged with atomic resolution for the first time. The activity of these layers is discussed in the context of the literature on the subject, which has thus far been inconclusive. We conclude that disordered oxidized ruthenium, rather than crystalline RuO2 is the most active species. / Dissertation/Thesis / Doctoral Dissertation Materials Science and Engineering 2016

abstract: Integrated oxide/semiconductor heterostructures have attracted intense interest for device applications which require sharp interfaces and controlled defects. The research of this dissertation has focused on the characterization of perovskite oxide/oxide and oxide/semiconductor heterostructures, and the analysis of interfaces and defect structures, using scanning transmission electrom microscopy (STEM) and related techniques. The SrTiO3/Si system was initially studied to develop a basic understanding of the integration of perovskite oxides with semiconductors, and successful integration with abrupt interfaces was demonstrated. Defect analysis showed no misfit dislocations but only anti-phase boundaries (APBs) in the SrTiO3 (STO) films. Similar defects were later observed in other perovskite oxide heterostructures. Ferroelectric BaTiO3 (BTO) thin films deposited directly onto STO substrates, or STO buffer layers with Ge substrates, were grown by molecular beam epitaxy (MBE) in order to control the polarization orientation for field-effect transistors (FETs). STEM imaging and elemental mapping by electron energy-loss spectroscopy (EELS) showed structurally and chemically abrupt interfaces, and the BTO films retained the c-axis-oriented tetragonal structure for both BTO/STO and BTO/STO/Ge heterostructures. The polarization displacement in the BTO films of TiN/BTO/STO heterostructures was investigated. The Ti4+ atomic column displacements and lattice parameters were measured directly using HAADF images. A polarization gradient, which switched from upwards to downwards, was observed in the BTO thin film, and evidence was found for positively-charged oxygen vacancies. Heterostructures grown on Ge substrates by atomic layer deposition (ALD) were characterized and compared with MBE-grown samples. A two-step process was needed to overcome interlayer reaction at the beginning of ALD growth. A-site-rich oxide films with thicknesses of at least 2-nm had to be deposited and then crystallized before initiating deposition of the following perovskite oxide layer in order to suppress the formation of amorphous oxide layers on the Ge surface. BTO/STO/Ge, BTO/Ge, SrHfTiO3/Ge and SrZrO3/Ge thin films with excellent crystallinity were grown using this process. Metal-insulator-metal (MIM) heterostructures were fabricated as ferroelectric capacitors and then electrically stressed to the point of breakdown to correlate structural changes with electrical and physical properties. BaTiO3 on Nb:STO was patterned with different top metal electrodes by focused-ion-beam milling, Au/Ni liftoff, and an isolation-defined approach. / Dissertation/Thesis / Doctoral Dissertation Materials Science and Engineering 2018

L’adressage des protéines vers leur correct emplacement est crucial pour la cellule. L’information d’adressage est fournie sous la forme d’une séquence signale par le polypeptide lui-même. Chez Escherichia coli, les protéines membranaires sont adressées vers la membrane de façon co-traductionnelle via la particule de reconnaissance du signal (SRP) tandis que les protéines sécrétées suivent la voie de translocation post-traductionnelle caractérisée par les protéines SecB et SecA qui sont impliquées dans le processus d’adressage. Ces deux voies convergent au niveau du canal de translocation des protéines SecYEG. Chose intéressante, SecYEG a la possibilité de recruter les domaines accessoires SecDF-YajC et YidC et ainsi former le complexe holotranslocon (HTL). La recherche actuelle sur la translocation des protéines se concentre principalement sur la structure et fonction du canal de translocation des protéines hétérotrimérique bactérien SecYEG qui est conservé. Peu de choses sont connues concernant la structure et la fonction des composants additionnels SecD, SecF et YidC formant la machinerie de translocation et qui sont essentiels pour E. coli. Ceci est dû principalement à l’absence d’un complexe holotranslocon SecYEG-DF-YidC (HTL) recombinant purifié. En conséquence, une analyse biophysique et structurale minutieuse de ce large complexe transmembranaire composé de sept sous-unités est toujours en suspens.En utilisant un nouveau système d’expression pour des complexes multi-protéiques basé sur la recombinaison de vecteur chez E. coli, nous avons avec succès surproduit l’holotranslocon SecYEG-DF-YajC-YidC et son sous-complexe composé de SecDF-YajC-YidC (DFYY). Nous avons également réussi à solubiliser avec l’aide de détergents et à purifier ces complexes. L’holotranslocon purifié a ensuite été utilisé afin de caractériser de façon biochimique le complexe et de déterminer la structure de l’holotranslocon. Premièrement, le complexe HTL semble être plus compétent pour l’insertion co-traductionnelle des protéines membranaires comparé à SecYEG isolé. Concernant la translocation post-traductionnelle d’une protéine de la membrane externe à tonneau β, dépendante de la présence de SecA et d’ATP, l’influence de la force proton motrice sur ce processus est augmentée. De plus, la présence du domaine accessoire semble améliorer l’attachement du ribosome au translocon. En utilisant des cellules déplétées de SecDF et YajC, nous avons identifié des substrats possibles de HTL qui doivent maintenant être confirmés et analysés manière plus approfondie par des expériences de translocation in vitro.Par la suite, nous avons résolu la structure de l’holotranslocon par cryo-microscopie électronique (ME) et analyse des particules isolées. En comparant les reconstructions de ME8du complexe HTL avec le sous-complexe de domaine accessoire SecDF-YajC-YidC, nous avons été capable de localisé le complexe principal SecYEG. La structure de HTL par cryo-ME a pu être affinée jusqu’à une résolution de 10.5 Å. Cette structure permet le placement des structures à haute résolution disponibles de SecYEG, SecDF et YidC afin de générer un modèle quasi-atomique de l’holotranslocon. Les jeu de données ainsi obtenus sont volumineux et souffrent d’un taux élevé de « faux positifs », probablement dû à des réactions de réticulation inter-complexe. C’est pourquoi ils nécessitent une évaluation minutieuse et les résultats intéressants devraient être confirmés par une méthode indépendante. Dans le futur, des études structurales du complexe ribosome-HTL par cryo-ME ainsi qu’une reconstitution de HTL dans des nanodisques vont être menées pour révéler la conformation de HTL en cours de translocation dans un environnement plus physiologique. Des études biochimiques complémentaires sur le mécanisme de co- et post-translocation par HTL et son spectre substrats abordent la question du rôle physiologique de l’holotranslocon dans la cellule. / Targeting of proteins to their proper location in the cell is crucial to the cell. The targeting information is provided in form of a signal sequence by the polypeptide itself. In Escherichia coli, membrane proteins are targeted co-translationally via the signal recognition particle (SRP) to the membrane whereas secretory proteins follow the post-translational translocation pathway characterized by the proteins SecB and SecA involved in the targeting process. Both pathways converge at the protein-conducting channel SecYEG. Interestingly, SecYEG has the possibility to recruit accessory domains SecDF-YajC and YidC, forming the holotranslocon (HTL) complex. Current research on protein translocation mostly focuses on the structure and function of the conserved bacterial heterotrimeric protein conducting channel SecYEG. Not much is known about the structure and function of the additional components of the translocation machinery SecD, SecF and YidC which are essential for E. coli. This is largely due to the lack of a purified, recombinant SecYEG-DF-YidC holotranslocon (HTL) complex. Accordingly, a thorough biophysical and structural analysis of this large, seven-membered transmembrane complex is still pending.Using a new recombineering-based vector system for expression of multi-protein complexes in E. coli, we successfully over-produced the SecYEG-DF-YajC-YidC holotranslocon and its subcomplex consisting of SecDF-YajC-YidC (DFYY). We also succeeded in detergent-solubilising and purifying these complexes. The purified holotranslocon was used to biochemically characterize the complex and to determine the structure of the holotranslocon. First of all, the HTL seems to be more competent for co-translational membrane proteins insertion compared to SecYEG alone. Regarding the post-translational translocation of a β-barrel outer-membrane protein, driven by SecA and ATP, the proton-motive force dependence of this process is increased. Furthermore, the presence of the accessory domains seems to enhance the binding of the ribosome to the translocon. By using cells depleted of SecDF and YajC, we identified possible HTL-substrates which have to be confirmed and further analyzed yet by in vitro translocation experiments.Subsequently, we solved by cryo-electron microscopy (EM) and single particle analysis the structure of the holotranslocon. By comparing the EM reconstructions of the HTL complex with the subcomplex of the accessory domains SecDF-YajC-YidC, we were able to localize the core complex SecYEG. The HTL cryo-EM structure could be refined to a resolution of 10.5 Å. This structure allows the placement of the available high–resolution crystal structure of SecYEG, SecDF, and YidC to generate a quasi-atomic model of the holotranslocon.6In order to confirm our quasi-atomic model, we made use of different crosslinking- and mass spectroscopy-based approaches (CLMS) to characterize the protein-protein interactions within the holotranslocon complex. These CLMS data sets are large and suffer from a high rate of ‘false positives’, possibly caused by inter-complex crosslinks. Thus, they need to be carefully evaluated and interesting fits should be confirmed by an independent method. In the future, structural studies of the ribosome-HTL complex by cryo-EM together with reconstitution of the HTL into nanodiscs will be undertaken to reveal the conformation of the actively translocating HTL in a more physiological environment. Additional biochemical studies on the molecular mechanism of co- and post-translocation by HTL and its substrate spectrum are addressing the question about the physiological role of the holotranslocon in the cell.