Made available in DSpace on 2014-06-11T19:29:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-16Bitstream added on 2014-06-13T20:38:35Z : No. of bitstreams: 1 trindade_fz_me_arafo.pdf: 753799 bytes, checksum: 6cf9c912e4ce2332952d19081bfcbfa7 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A utilização da PDT para tratamento de diferentes tipos de infecções, tal como a candidose bucal, tem sido estudada. Entretanto, poucos são os dados científicos que relatam os possíveis efeitos tóxicos dessa terapia. Dessa forma, o objetivo deste estudo foi avaliar os efeitos da irradiação na mucosa bucal de ratos com LED azul (de 460 nm e potência de 200 mW/cm2) em presença do fotossensibilizador (FS) Photogem®, em duas diferentes concentrações (500 mg/L e 1000 mg/L). Para isso, foram utilizados 101 ratos (Rattus Norvegicus Albinus Holtzman) distribuídos em 6 grupos, de acordo com os seguintes tratamentos: Grupo 1 – controle; Grupo 2 – aplicação do FS (500 mg/L); Grupo 3 – aplicação do FS (500 mg/L) e irradiação com LED; Grupo 4 - aplicação do FS (1000 mg/L); Grupo 5 – aplicação do FS (1000 mg/L) e irradiação com LED; e Grupo 6 – irradiação com LED. O FS foi aplicado por 30 minutos (tempo de pré-incubação) e o tempo de irradiação da mucosa foi de 20 minutos (dose de 144 J/cm2). Decorridos os 4 períodos de avaliação propostos (0 dia, 1dia, 3 dias e 7 dias), os animais tiveram a mucosa palatina fotografada para análise macroscópica, sendo então imediatamente sacrificados para remoção cirúrgica do palato e posterior análise em microscopia de luz e de fluorescência. Um mapeamento térmico foi realizado a fim de avaliar a variação de temperatura ocorrida no tecido durante a irradiação com LED. Macroscopicamente, em todos os grupos experimentais e para todos os períodos de avaliação propostos na presente pesquisa, observou-se que a mucosa apresentava-se intacta, com aspecto de normalidade semelhante ao do Grupo 1 (controle). Microscopicamente, alterações teciduais, caracterizadas especialmente por discreta inflamação, puderam ser observadas na mucosa palatina de apenas 4 de um total de 80 animais submetidos a PDT... / The use of PDT has been investigated for the treatment of different types of infection, like oral candidosis. There are, however, few research-based data that report the possible toxic effects of this therapy. Therefore, this study evaluated the effects of irradiating the palatal mucosa of rats with blue LED (460 nm; 200 mW/cm²) in the presence of the photosensitizer Photogem® at two concentrations (500 and 1000 mg/L). Then, 101 rats (Rattus norvegicus albinus Holtzman) were randomly distributed in six groups, according to the treatment performed on the palatal mucosa: Group 1: control; Group 2: Photogem® (500 mg/L); Group 3: Photogem® (500 mg/L) + blue LED; Group 4 - Photogem® (1000 mg/L); Group 5: (1000 mg/L) + blue LED; and Group 6: blue LED. The exposure times to the photosensitizing agent and to the light source were 30 min (pre-incubation time) and 20 min (144 J/cm2 energy density), respectively. At 0, 1, 3 and 7 days posttreatment, the animals had their palatal mucosa photographed for macroscopic analysis and were immediately sacrificed. The palate was removed for further analysis by light and fluorescence microscopy. Thermal mapping was made to evaluate the temperature change occurred in the tissue during LED irradiation. In all experimental groups and periods, the macroscopic analysis revealed intact mucosa with normal aspect similar to that of Group 1 (control). Tissue alterations, characterized primarily by a mild inflammation, were observed microscopically on the mucosa of only 4 out of 80 animals subjected to PDT. Photosensitizer penetration into the treated mucosa was identified by the fluorescence emitted by Photogem® and was limited to the epithelial layer. The thermal mapping revealed a temperature increase from 35 to 41ºC during the 20-min irradiation. In conclusion, under the tested conditions, PDT using Photogem® at 500 and 1000 mg/L concentrations... (Complete abstract click electronic access below)

Neste trabalho estudamos a interação de dois tipos de nanopartículas de prata metálica, obtidas pelo processo de poliol (IQUSP) e pelo método eletrolítico (Khemia®), em células de músculo liso. Um extenso trabalho de caracterização foi realizado, descrevendo a natureza físico-química dessas nanopartículas. Medidas de absorção óptica mostraram que as nanopartículas exibem bandas suaves em torno de 400 nm, região do azul do espectro eletromagnético, devido à ressonância dos plasmons de superfície, evidenciando a tendência à agregação com o tempo. Microscopia eletrônica de transmissão foi realizada para obter as imagens das nanopartículas em micrografias. Histogramas foram construídos para determinar o tamanho das NPs e o índice de polidispersividade. Espectros de EDS foram obtidos para a caracterização química das amostras. Difratogramas de raios X foram obtidos para as AgNPs. Os picos de difração foram indexados e revelaram uma única fase cristalina da prata, com estrutura cúbica e estado de oxidação, Ag0. Com o auxílio desses difratogramas, foram calculados o parâmetro de rede e a distância interplanar dos planos de difração. Utilizando a equação de Scherrer e um ajuste gaussiano dos picos de Ag mostrados nos difratogramas de raios X, foi possível obter o tamanho do cristalito para nanopartículas IQUSP. Experimentos de DLS mostraram distribuição de número monomodal para AgNPs Khemia® e, para AgNPs IQUSP lavadas, distribuição bimodal, estimando-se a distribuição de número e tamanho. Os resultados mostraram que a distribuição dominante é sempre para raios menores, sugerindo partículas menores que se agregam com o tempo e formam maiores dimensões. Resultados de SAXS mostraram que as amostras fornecem boa intensidade de espalhamento. Utilizando modelos teóricos foram calculados o raio médio da distribuição, polidispersividade e raio de giro. Os dados revelaram que as nanopartículas IQUSP possuem um raio maior que as AgNPs Khemia® e não apresentaram agregação. Em contrapartida, AgNPs Khemia® apresentaram maior agregação, com polidispersividade relativa de 72%. Para AgNPs IQ--USP, análises de SAXS forneceram tamanho de partícula comparável a TEM e bastante diferente de DLS. As medidas de SAXS para AgNPs Khemia® mostram diferenças com as medidas de TEM e DLS. Ficou evidente o efeito da agregação, que tem influências desde o preparo das amostras até o tempo de realização das medidas. Testes de citotocixidade e estudos de análise morfológica por microscopia de fluorescência evidenciaram as características citotóxicas de cada nanopartícula. Os resultados apresentados pela análise morfológica realizada com microscopia de fluorescência concordam com os testes de citotoxicidade. AgNPs IQUSP mostraram alta toxicidade até a concentração 9.37 mg/mL, onde as células são apresentadas com fragmentação nuclear. Em concentrações mais baixas, as AgNPs IQUSP exibiram características morfológicas comparáveis ao grupo controle. Por sua vez, AgNPs Khemia® mostram alta toxicidade até a concentração 1.37 mg/mL, com índice IC50 variando na faixa de 1.3 a 6.7 mg/mL. Foi possível observar que concentrações mais altas induzem à fragmentação nuclear, desencadeando processos como apoptose e necrose. Experimentos utilizando a técnica de OMTC demonstraram que as diferentes concentrações de nanopartículas de prata podem modificar a rigidez celular. Isto é evidenciado quando comparamos o grupo controle com os demais grupos, com as diferentes concentrações de NPs. Para concentrações mais altas de nanopartículas, verificou-se um aumento da viscoelasticidade. Os dois tipos de nanopartículas estudadas apresentaram mudanças nas propriedades mecânicas, mas as AgNPs Khemia® apresentaram um maior aumento na viscoelasticidade nas diferentes concentrações de NPs. Essa mudança na viscoelasticidade foi explicada como sendo devido à maior disponibilidade do cálcio, liberado por células apoptóticas, o qual é utilizado no complexo miosina-actina para gerar contração muscular. / In this work we study the interaction of two types of metallic silver nanoparticles, obtained by the polyol process (IQUSP) and the electrolytic method (Khemia®), in smooth muscle cells. An extensive characterization work was carried out, describing the physico-chemical nature of these nanoparticles. Optical absorption measurements showed that nanoparticles exhibit smooth bands around 400 nm, the blue region of the electromagnetic spectrum, due to the resonance of the surface plasmons, evidencing the tendency to aggregate with time. Transmission electron microscopy was performed to obtain images of the nanoparticles in micrographs. Histograms were constructed to determine the size of NPs and the index of polydispersity. EDS spectra were obtained for the chemical characterization of the samples. X-ray diffraction patterns were obtained for the AgNPs. The diffraction peaks have been indexed and showed a single crystal layer of silver, with cubic structure and oxidation state, Ag0. By means of these diffractograms, the network parameter and the interplanar distance of the diffraction planes were calculated. Using the Scherrer equation and a Gaussian fit of the Ag peaks shown in the X-ray diffractograms, it was possible to obtain the crystallite size for IQ-USP nanoparticles. DLS experiments showed monomodal number distribution for Khemia® AgNPs and, for washed IQUSP AgNPs, bimodal distribution, estimating the number and size distribution. The results showed that the dominant distribution is always for smaller rays, suggesting smaller particles that aggregate with time and form larger dimensions. SAXS results showed that the samples provide good scattering intensity. Using the theoretical models, the average radius of the distribution, polydispersity and radius of gyration were calculated. The data revealed that the IQUSP nanoparticles have a larger radius than the Khemia® and did not show aggregation. In contrast, Khemia® AgNPs showed higher aggregation, with 72% relative polydispersity. For IQ-USP AgNPs, SAXS analyzes provided particle size comparable to TEM and quite different from DLS. SAXS measurements for Khemia® AgNPs show differences with TEM and DLS measurements. It was evident the effect of the aggregation that has influences from the sample preparation until the time of performing the measurements. Cytotoxicity tests and morphological analysis studies by fluorescence microscopy evidenced the cytotoxic characteristics of each nanoparticle. The results presented by the morphological analysis performed with fluorescence microscopy agree with the cytotoxicity tests. IQ-USP nanoparticles showed high toxicity up to the concentration of 9.37 mg/mL, where the cells are presented with nuclear fragmentation. At lower concentrations, the IQUSP AgNPs exhibited morphological characteristics comparable to the control group. In addition, Khemia® AgNPs show high toxicity up to the concentration of 1.37 mg/mL, with IC50 in the range of 1.3 to 6.7 mg/mL. It was possible to observe that higher concentrations induce nuclear fragmentation, triggering processes such as apoptosis and necrosis. Experiments using the OMTC technique demonstrated that different concentrations of silver nanoparticles can modify cell stiffness. This is evidenced when we compare the control group with the other groups, with the different concentrations of NPs. For higher concentrations of nanoparticles, there was an increase in viscoelasticity. The two types of nanoparticles studied showed changes in mechanical properties, but Khemia® AgNPs showed a greater increase in viscoelasticity at different concentrations. This change in viscoelasticity was explained to be due to the increased availability of calcium, released by apoptotic cells, which is used in the myosin-actin complex to generate muscle contraction.

A leishmaniose é uma doença tropical negligenciada causada por diferentes espécies do gênero Leishmania que atinge grande parte da população mais pobre do mundo e sua manifestação visceral, que é fatal se não tratada, tem se alastrado atingindo grandes cidades, aumentando o número de pessoas com risco de infecção. Dentre os medicamentos em uso, está o análogo lipídico sintético hexadecilfosfocolina (miltefosina), administrado oralmente, que age nas membranas celulares do parasita e pode induzir apoptose, mas com modo de ação não totalmente esclarecido. O primeiro local de interação desse fármaco é a membrana celular do parasita, sendo importante o conhecimento da sua forma de interação. Neste trabalho examinamos propriedades de diversos modelos de membrana com diferentes composições, levando em consideração o conhecimento existente sobre a composição da membrana plasmática da Leishmania. Assim, as membranas modelo foram vesículas unilamelares grandes e gigantes (LUVs e GUVs), de fosfolipídios puros, de misturas binárias com fosfolipídios e colesterol e ainda misturas ternárias com ceramida, um esterol presente nas membranas de Leishmania. A interação com a miltefosina foi estudada em diferentes intervalos de concentração do fármaco. Como técnicas principais utilizamos a espectroscopia de fluorescência, estática e resolvida no tempo, a espectroscopia de correlação de fluorescência, microscopia de fluorescência e confocal e imagens por tempo de vida de fluorescência. Observou-se que a interação entre o fármaco e as membranas lipídicas ocorre de diferentes formas, dependendo i) da razão molar entre o fármaco e os lipídios; ii) da concentração real do fármaco, se abaixo ou acima da concentração micelar crítica (CMC); iii) da composição do modelo de membrana e da fase lipídica da bicamada. Em concentrações abaixo da CMC, a miltefosina tem efeito de fluidificação das bicamadas, principalmente quando elas se encontram em na fase gel, mas esse efeito é pouco pronunciado na presença de colesterol, pois esse composto protege a bicamada do efeito do fármaco. Em vesículas de misturas ternárias de fosfolipídio, colesterol e ceramida em alta concentração, não há separação de fases, e a presença de 10 mol% de miltefosina promove a formação de domínios de ceramida; nas vesículas em que a ceramida está em concentração molar mais baixa, formando domínios, a separação de fases fica menos evidente com o acréscimo de miltefosina. Em razões de concentração miltefosina/lipídio elevadas, mas ainda abaixo da CMC, observa-se diminuição no tamanho das vesículas, por formação de agregados de fármaco/lipídio com porções da bicamada. Em concentrações acima da CMC, ocorrem efeitos drásticos com solubilização de partes cada vez maiores da bicamada da membrana, e esses efeitos ocorrem em tempos menores quanto maior a concentração de miltefosina. Portanto, de maneira geral, o colesterol protege a bicamada do efeito da miltefosina, mas o fármaco tem efeito pronunciado em modelos de membrana de misturas ternárias contendo ceramida. Os efeitos variam com a concentração da miltefosina, com aumento da fluidez da bicamada em baixas razões fármaco/lipídios, solubilização de pequenas porções da bicamada e diminuição do tamanho das vesículas em razões maiores, mas ainda abaixo da CMC, e acima da CMC, formação de agregados do fármaco com porções dos lipídios da bicamada e fragmentação da membrana. / Leishmaniasis is a complex of diseases part of the neglected tropical diseases caused by several species of the genus Leishmania. It reaches a large part of the poorest people in the world and its visceral form, which is fatal if left untreated, has been spread around big cities, increasing the number of people at risk of infection. Among the used drugs for the treatment, there is the synthetic lipid analogue hexadecylphosphocholine (miltefosine), orally administrated, which acts in the cell membranes and can induce apoptosis like death, but its mechanism of action is not totally clear. The first interactions site of this drug is the cell membrane, and it is important to know its mechanism of interaction. In this work we explore properties of several membrane models with different compositions, taking into account the existent knowledge about the composition of the Leishmania plasma membrane. Therefore, the model membranes were giant and large unilamellar vesicles (GUVs and LUVs), formed from pure phospholipids, binary mixtures of phospholipids and cholesterol and ternary mixtures with ceramide, a sterol present in the Leishmania membranes. The interaction with miltefosine was studied in different intervals of drug concentration. The main techniques used were the steady-state and time-resolved fluorescence spectroscopy, fluorescence correlation spectroscopy, confocal and fluorescence microscopy and fluorescence lifetime imaging. The interaction depends on i) the molar ratio of drug and lipids; ii) the real concentration of the drug, if it is below or above the critical micelle concentration (CMC); iii) the composition of the model membrane and the lipid phase of the bilayer. In concentration below the CMC, miltefosine has an effect of bilayer fluidization, mainly when it is in a more ordered phase, but this effect is less pronounced in cholesterol presence, because this compound protects the bilayers from the drug effect. In vesicles from ternary mixtures of phospholipid, cholesterol and ceramide in high concentration, there is no phase separation, and the presence of 10 mol% of miltefosine promotes ceramide domains formation; in vesicles in which ceramide is in low concentration, forming domains, the phase separation is less evident with miltefosine addition. In high concentration ratio miltefosine/lipids, but below CMC, it is observed a decrease in vesicles size with drug/lipids aggregates formation from portion of the bilayer. In concentrations above the CMC, drastic effects occur, with solubilization of bigger portions of the membrane bilayer, and the effects occur in lower times for higher drug concentration. Therefore, generally, cholesterol protects bilayer from the effect of miltefosine, but the drug has a pronounced effect in model membranes of ternary mixtures containing ceramide. The effects vary with miltefosine concentration, increasing the bilayer fluidity in lower drug/lipid ratio, solubilization of small portions of the bilayer and decrease of vesicles size in higher ratios, but still below CMC, and above CMC, formation of aggregates of the drug with portions of bilayer lipids, and membrane fragmentation.

Os microrganismos endofíticos são aqueles, cultiváveis ou não, que habitam o interior da planta hospedeira sem causar danos aparentes ou estruturas externas visíveis. Essa interação microrganismos-planta é intrínseca a determinadas espécies de plantas e/ou bactérias. Nas últimas décadas os estudos de microrganismos endofíticos têm sido realizados em diversas plantas hospedeiras, sendo esses estudos direcionados principalmente para a diversidade e características benéficas induzidas, inclusive o controle biológico de doenças. A doença causada pelo fungo Ceratocystis fimbriata é considerada emergente no setor florestal. O Brasil está entre os maiores produtores mundiais de eucalipto e a expansão do setor juntamente com o cultivo clonal tem acarretado o aumento da incidência de patógenos. O surgimento de novas doenças exige estudos relacionados tanto a interação do agente patogênico com hospedeiro quanto de todos os componentes do patossistema. Neste contexto, os microrganismos endofíticos têm sido descritos como potenciais controladores biológicos de doenças. Dessa forma, o presente trabalho teve por objetivos avaliar a interação de C. fimbriata com a comunidade bacteriana associada à Eucalyptus sp. Adicionalmente, foi estudada a possível transferência desses endófitos via sementes e o padrão de colonização de Pantoea agglomerans em plântulas. Foi observado que plantas não infestadas por C. fimbriata apresentaram maior densidade bacteriana no rizoplano (20,66 x 104 UFC.cm2 -1 de raiz), enquanto que para a comunidade endofítica, a maior densidade foi observada em plantas infectadas pelo fungo (25,13 x 104 UFC.g-1 de raiz). As análises por ARDRA possibilitaram a obtenção de 8 e 13 ribotipos nas comunidades endofítica de raiz e do rizoplano, respectivamente. Os ribotipos mais freqüentes foram identificados como Bacillus cereus. As análises de diversidade por meio de DGGE das comunidades do rizoplano e endofítica de raiz mostraram que a infestação pelo fungo interfere na colonização de Eucalyptus. Foi observado também que bactérias endofíticas estão presentes no interior de sementes de Eucalyptus spp. em uma densidade de 0,33 a 1,83 X 102 UFC.g-1, para as espécies E. camandulensis e E. urophylla, respectivamente. A densidade bacteriana endofítica de plântulas obtidas de sementes desinfectadas superficialmente variaram entre 0,27 X 102 a 0,87 X 102 UFC.g-1, para E. citriodora e o híbrido E. robusta x E. grandis, respectivamente. Em algumas espécies de Eucalyptus não foram isoladas bactérias endofíticas das sementes e plântulas. Os resultados mostraram que algumas espécies de bactérias endofíticas podem ser transmitidas verticalmente por sementes. P. agglomerans inoculada nas sementes foi capaz de colonizar as plântulas após a germinação da semente, indicando que esta pode ser uma das formas utilizadas pelos microrganismos para colonizar e se estabelecer na planta hospedeira. Assim, os resultados obtidos neste trabalho mostram ainda que possa existir interação entre a presença de C. fimbriata e a comunidade bacteriana endofítica e do rizoplano de Eucalyptus. Foi possível observar também que estas bactérias endofíticas que são transmitidas por meio de sementes, permitindo que plântulas previamente inoculadas com bactérias benéficas possam ser produzidas antes de serem levadas a campo. / The endophytic microorganisms are those, cultivated or not, that inhabit the interior of the plant host without causing apparent damages or visible external structures. This interaction microorganisms-plant is specific to certain species of plants and/or bacteria. In the last few years studies of endophytic microorganisms have been carried out in several plant hosts, being these studies focused mainly to diversity and biotechnological potential, such as biological control of disease. The disease caused by the phytopathogenic fungi Ceratocystis fimbriata is considered emerging by the reforestation companies. Brazil is one of the largest world eucalyptus producers and the increasing of the eucalyptus production associated to clonal reproduction has allowed the increase in pathogen incidence. Studies that evaluate the interaction between pathogens and the microbial community associated to the host plant may allow understanding how disease symptoms come up. Endophytic microorganisms have been described as potential biological control of diseases and therefore, the aims of the present work were to i) study the interaction between C. fimbriata and the bacterial community associated to the Eucalyptus sp.; ii) evaluate the bacterial dissemination by seeds; iii) evaluate the colonization profile of Pantoea agglomerans in seedlings after seed inoculation. It was observed that the highest bacterial density on the rhizoplane (20.66 x 104 CFU.cm2 -1 of root) was observed in C. fimbriata uninfectedplants, while for endophytic community the highest density was observed in C. fimbriata infected plants (25.13 x 104 CFU.g-1 of root). The ARDRA analyses showed that the bacterial community of eucalyptus is composed by 8 and 13 ribotypes on rhizoplane and inside the roots (endophytic), respectively. The most frequent ribotypes were identified as Bacillus cereus. The DGGE analyses of diversity of endophytic and rhizoplane community showed that fungi infection shift the colonization of Eucalyptus associated bacteria. The bacterial community inside Eucalyptus spp. seeds ranged from 0.33 to 1.83 X 102 CFU.g-1, for E. camandulensis and E. urophylla, respectively. After seed germination the endophytic bacterial density in seedlings ranged from 0,27 X 102 to 0,87 X 102 CFU.g-1, for E. citriodora and the hybrid E. robusta x E. grandis, respectively. Although, endophytic bacteria have been isolated from seeds, for some plant species, bacteria were not isolated from seedlings. Also, some bacteria may be vertically transmitted from seed to seedlings, but some is specific for seeds. Seed inoculation of P. agglomerans resulted in seedlings colonized by these bacteria, suggesting that these bacteria could be seed transmitted. The results obtained in the present study show that the fungi C. fimbriata inside the Eucalyptus host can shift the endophytic and rhizoplane bacterial diversity. Also, these endophytic bacteria could be transmitted vertically by seeds, allowing that seeds previously inoculated with beneficial bacteria may result in protected plants before planting in the field.

L’objectif de mon travail de thèse a été de caractériser l’adhésion de vésicules géantes lipidiques et de cellules vivantes. Dans le but d’obtenir des informations quantitatives sur l’adhésion, j’ai développé deux techniques de nanoscopie de fluorescence basées sur la microscopie TIRF (Total Internal Reflection Fluorescence). Cette technique repose sur création d’une onde évanescente à proximité d’une interface. J’ai développé pour cela un montage optique, qui permet de contrôler finement les caractéristiques de l’onde évanescente (longueur d’atténuation, état de polarisation, etc.). L’adhésion des vésicules a été étudiée par nTIRF (TIRF normalisé) : les images TIRF sont normalisées par des images en épi-fluorescence. J’ai pu ainsi caractériser l’adhésion non spécifique (interaction électrostatique) et spécifique (interaction biotine-streptavidine) de vésicules sur différentes surfaces fonctionnalisées. Pour quantifier l’adhésion des cellules, j’ai utilisé l’approche VA-TIRF (TIRF à angle variable). Cette dernière consiste à enregistrer une série d’images en régime évanescent à différents angles d’incidence. Ceci nous a permis d’établir une cartographie des distances entre la membrane ventrale d’une cellule et la surface pour différents comportements d’adhésion initiés sur divers substrats : chimiques ou protéiques. Ces deux techniques permettent de mesurer la distance membrane-surface avec une précision nanométrique, ≈20nm, ce qui est particulièrement adapté à l’étude du processus d’adhésion / The aim of my thesis was to characterize the adhesion of Giant Unilamellar Vesicles and living cells. In order to obtain a quantitative information about the state of the adhesion, I developed two fluorescence nanoscopy techniques based on microscopy TIRF (Total Internal Reflection Fluorescence). This technique consist of creating an evanescent wave in the vicinity of an interface. I developed the experimental setup, which allows an accurate control of characteristics of the evanescent wave (penetration depth, polarization state, etc.). The vesicles adhesion was studied by nTIRF (normalized TIRF). TIRF images are normalized by epi-fluorescence images. I was able to characterize the nonspecific adhesion (electrostatic interaction) and specific adhesion (biotin-streptavidin interaction) of vesicles on different functionalized surfaces. To quantify the adhesion of cells, I used the VA-TIRF approach (variable angle TIRF). The latter is to record a series of images at different angles of incidence in the evanescent regime. This allowed us to map the distances between the ventral membrane of a cell and the surface for different adhesion behaviors initiated on various substrates: chemical or protein. These two techniques permit to measure the membrane surface-distance with the nanometer precision ≈20nm, which is particularly suitable for the study of the adhesion process

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DNA polymerases are a family of molecular machines involved in high-fidelity DNA replication and repair, of which DNA polymerase I (Pol) is one the best-characterized members. Pol is a strand-displacing polymerase responsible for Okazaki fragment synthesis and base-excision repair in bacteria; it consists of three protein domains, which harbour its 5’-3' polymerase, 3’-5’ exonuclease and 5’ endonuclease activities. In the first part of the thesis, we use a combination of single-molecule Förster resonance energy transfer (smFRET) and rigid-body docking to probe the structure of Pol bound to its gapped-DNA substrate. We show that the DNA substrate is highly bent in the complex, and that the downstream portion of the DNA is partly unwound. Using all-atom molecular dynamics (MD) simulations, we identify residues in the polymerase important for strand displacement and for downstream DNA binding. Moreover, we use coarse-grained simulations to investigate the dynamics of the gapped-DNA substrate alone, allowing us to propose a model for specific recognition and binding of gapped DNA by Pol. In the second part of the thesis, we focus on the catalytically important conformational change in Pol that involves the closing of the ‘fingers’ subdomain of the protein around an incoming nucleotide. We make use of the energy decomposition method (EDM) to predict the stability-determining residues for the closed and open conformations of Pol, and test their relevance by site-directed mutagenesis. We apply the unnatural amino acid approach and a single-molecule FRET assay of Pol fingers-closing, to show that substitutions in the stability-determining residues significantly affect the conformational equilibrium of Pol. In the final part of the thesis, we attempt to study Pol in its native environment of the living cell. We make use of the recently developed method of internalization by electroporation, and optimize it for organically labelled proteins. We demonstrate the internalization and single-molecule tracking of Pol, and provide preliminary data of intra-molecular FRET in Pol, both at the single-cell and single-molecule levels. Finally, by measuring smFRET within an internalized gapped-DNA construct, we observe DNA binding and bending by endogenous Pol, confirming the physiological relevance of our in vitro Pol-DNA structure.

Em aplicações biotecnológicas, a estabilização de informações e a expressão gênica em bactérias Gram-negativas são fundamentais. Neste trabalho, analisou-se o efeito de estabilização plasmidial promovido pelo lócus parB (hok/sok) nas bactérias E. coli, C. metallidurans e C. necator transformadas com os plasmídeos pCM2, pBBR1MCS e seus respectivos derivados contendo o lócus parB, pCM3 e pBBPAR. As bactérias transformadas com pCM2 exibiram perda plamidial acentuada após 50 gerações, e as bactérias transformadas com pCM3 e pBBPAR exibiram estabilidade próxima a 100%, após 100 gerações. Também foi avaliada a força relativa de expressão dos promotores hok e sok em relação ao promotor lac, inseridos no plasmídeo pBBEGFP. Por microscopia de fluorescência, foi observado que regulado pelos promotores sok e hok, os transformantes expressaram EGFP. Utilizando citometria de fluxo, foi observado que o promotor hok apresentou maior força de expressão nas bactérias transformantes de E. coli e C. metalliduras; já o promotor sok apresentou maior força de expressão em C. necator. / In biotechnological applications, stabilization of information and gene expression in Gram-negative bacteria are important. In this work, we analyzed the effect of plasmid stabilization promoted by locus parB (hok/sok) in the bacteria E. coli, C. metallidurans and C. necator transformed with the plasmids pCM2, pBBR1MCS, and their derivatives containing the parB locus, pCM3 and pBBPAR. The Bacteria transformed with pCM2 showed a pronounced plasmidial loss after 50 generations, and bacteria transformed with pCM3 and pBBPAR exhibited stability close to 100 % after 100 generations. We also evaluated the relative expression strength of hok and sok promoters relative to the lac promoter, inserted into plasmid pBBEGFP. For fluorescence microscopy, it was observed that regulated by hok and sok promoter, transformants expressed EGFP. Using flow cytometry, it was observed that the hok promoter showed higher expression levels in E. coli and C. metallidurans transformants; as sok promoter showed higher expression level in C. necator.

No presente estudo, a diversidade bacteriana endofítica fixadora de nitrogênio foi pesquisada utilizando métodos microbiológicos e moleculares. Isolaram-se microrganismos de raiz, caule e folha de cana-de-açúcar. Análises por seqüenciamento do 16S rDNA identificaram 150 endófitos. No total, foram identificados 18 gêneros. Destes, apenas 4 estavam presentes em cana-de-açúcar submetida aos 3 tratamentos. A maior diversidade de gêneros foi encontrada em cana sob adubação orgânica: 10 gêneros, em cana sob adubação inorgânica foram 11 gêneros e 8 em cana sem adubação. A maior parte dos gêneros pertence à família Enterobacteriaceae, como Klebsiella, Pantoea e Enterobacter. A enzima endoglicanase foi produzida por 82% dos isolados de cana sob adubação orgânica, (54%) inorgânica e (48%) sem adubação. Quanto à atividade de pectinase: 42%, 60% e 36% foram apresentadas por isolados de cana orgânica, inorgânica e sem adubação, respectivamente. A capacidade de solubilizar fosfatos inorgânicos foi detectada em 76,6% dos isolados, sendo a maior capacidade de solubilização de fosfatos encontrada em bactérias isoladas de cana-de-açúcar sob adubação orgânica (71%), de cana submetida a adubação convencional (78%) e sem adubação (88%). Foram realizados estudos de colonização em plântulas de cana-de-açúcar com 4 endofitos geneticamente modificados (EGMs) capazes de expressar os genes gfp e dsred. A avaliação da colonização na cana pela microscopia de fluorescência, mostrou que os (EGMs) gfp e dsred colonizaram as raízes e caules das plantas inoculadas, sem causar qualquer sintoma de doença. / In the present study, endophytic bacterial diversity has been searched using both microbiologic and molecular methods. Microorganisms were isolated from sugarcane root, shoot and leaf. 150 isolates were identified by 16S rDNA sequencing. 18 genera were found and only 4 were present in sugarcane submitted to the three treatments. The greatest genera diversity was found in sugarcane submited to organic fertlization: 10 genera in sugarcane submitted to inorganic fertilization were found 11 genera and 8 genera in sugarcane without fertilization. Great part of the found genera belongs to the Enterobacteriaceae family: Klebsiella, Pantoea and Enterobacter. Some physiological characteristics were determined in the isolates. Endoglucanase was produced by 82% of the isolates from sugarcane submitted to organic fertilization. Lower activities were found in bacteria isolated from inorganic fertilization and no fertilization, respectively 54% and 48%. As far as pectinase activity is concerned, a percentage of 42%, 60% and 36% was presented by the isolates from organic fertilization, inorganic fertilization and no fertilization, respectively. The hability of phosphate solubilization was detected in 76.6% of the isolates. In sugarcane under organic fertilization a percentage of 71% was found, in bacteria from inorganic fertilization, 78%, and without fertilization, 88%. Plant colonization was determined using sugarcane plantlets inoculated with four genetically modified bactéria (GMEs), able to express the genes gfp and dsred. The colonization was evaluated by fluorescence microscopy, which showed that the endophytic bacteria expressing gfp and dsred genes had invaded roots and shoots from inoculated plants, without causing any disease symptom.

Haloferax volcanii est une archée appartenant au phylum euryarchaeota et à la classe des Halobacteriales. Les mécanismes liés à la réplication et à la réparation chez les archées sont très similaires à ceux rencontrés chez les eucaryotes, faisant d’H. volcanii un des organismes modèle pour l’étude de la réplication et de la biologie des archées, notamment car de nombreux outils génétiques sont disponibles chez cet organisme. De plus, H. volcanii possède la particularité de pouvoir avoir toutes ses origines de réplication supprimées, soulevant beaucoup de questions sur les mécanismes impliqués. Plusieurs hypothèses ont été émises sur la façon dont cette souche initie sa réplication, basées soit sur la dérivation des mécanismes liés à la réparation de l’ADN, soit sur un mécanisme d’initiation de la réplication indépendant des origines. Afin d’étudier ces mécanismes liés à la réplication, j’ai construit une souche d’H. volcanii capable d’incorporer des analogues de la thymidine dans l’ADN lors de sa synthèse grâce à la délétion de gènes impliqués dans la voie de biosynthèse de la thymidine. Des temps de cultures courts de la souche en présence d’un analogue permet son incorporation au niveau des zones actives de réplication pour marquer spécifiquement l’ADN néosynthétisé. L’immunodétection de l’analogue incorporé à l’ADN, en travaillant en cellule entière avec un microscope à fluorescence, permet la localisation de l’ADN néosynthétisé, reflétant ainsi les régions où la réplication est active. Ces analyses révèlent majoritairement 2 à 3 régions de réplication actives dans des cellules en prolifération, sans localisation particulière. Ces régions ont déjà été observées en étudiant la localisation d’une protéine clé de la réplication (RPA2) fusionnée à la protéine verte fluorescente GFP, confirmant sa localisation aux zones actives de réplication. Une étonnante variabilité observée d’une cellule à l’autre et suggère une initiation probabiliste de la réplication. Il est également étonnant de n’observer qu’aussi peu de zones actives de réplication, comparé au fort taux de polyploïdie de cette souche. Se pose alors la question de ce à quoi correspondent ces zones de réplication. Pour cela, j’ai développé chez H. volcanii la technique de peignage moléculaire permettant d’isoler des molécules individuelles d’ADN et révéler spécifiquement les analogues incorporés pour pouvoir déterminer le nombre de copies du chromosome qui sont actives lors de la réplication, ainsi que le nombre d’origines actives sur chacune des copies. J’ai également développé une technique de Time-lapse dans le but de suivre ces régions au cours du temps en observant les divisions cellulaires directement sous le microscope. / Haloferax volcanii is an archaea belonging to the phylum euryarchaeota and the class Halobacteriales. The mechanisms related to replication and repair in archaea are very similar to those found in eukaryotes, making H. volcanii a relevant model organisms for the study of replication and archaeal biology, especially since many genetic tools are available. Interestingly, all replication origins can be removed from the chromosome of H. volcanii, raising many questions about the mechanisms involved. Several hypotheses have been proposed on how this strain initiates its replication, either relying on recombination-dependent replication initiation or an origin-independent mechanism. In order to study these replication-related mechanisms, I have constructed a strain of H. volcanii able to incorporate thymidine analogues into DNA during its synthesis by deleting genes involved in the thymidine biosynthesis pathway. A short-time cultures of the strain in the presence of an analogue allows its incorporation in nascent DNA. By immunodetection of the analog coupled to fluorescence microscopy observation of whole cells, it is possible to investigate the localization of neosynthesized DNA,which reflect the regions where replication is active. These analyses revealed mainly 2 to 3 active replication regions per cell, without any particular location. These regions had already been observed by studying the localization of a key replication protein (RPA2) fused to the fluorescent green protein GFP, confirming its location in active replication areas. A surprising variability in the number of replication foci from one cell to another was observed, suggesting a probabilistic initiation of replication. It is also surprising to observe so few active replication areas compared to the high polyploidy of this strain. This raises the question of what these replication areas correspond to. For further understanding, I developed for H. volcanii molecular combing, to isolate individual DNA molecules and specifically reveal incorporated analogues to determine the number of copies of the chromosome that are being replicated, as well as the number of active origins on each of the copies. I have also developed time-lapse approach to track these regions over time by monitoring cell proliferation directly under the microscope.