Medicago truncatula est une Légumineuse établissant une interaction symbiotique avec une bactérie tellurique de la famille des Rhizobiacées, Sinorhizobium meliloti. Cette interaction induit l’organogénèse racinaire d’un nouvel organe, la nodosité dans laquelle s’établit un microenvironnement propice à la différenciation de S. meliloti en bactéroïde fixateur du diazote atmosphérique. Ce dernier réduit ainsi le N2 atmosphérique en ammonium, assimilé ensuite par la plante hôte. Cette réduction étant très endergonique M. truncatula fournit aux bactéroïdes des substrats carbonés issus de la photosynthèse. Cependant, cette interaction n’est pas pérenne, du fait de la mise en place d’un processus de sénescence ; processus conduisant à la lyse des bactéroïdes et des cellules hôtes végétales. Cependant, à l’heure actuelle, ce processus de rupture symbiotique reste largement méconnu. Afin de mieux caractériser ce processus de sénescence, nous avons développé de nouveaux outils cytologiques permettant par microscopie confocale de suivre in vivo la viabilité, mais également le fonctionnement des bactéroïdes au sein de la cellule hôte végétale. Ces nouvelles approches cytologiques pourraient ainsi offrir de nouvelles perspectives pour une caractérisation plus précise du déroulement du processus de sénescence nodositaire. Dans le cadre de ce travail de thèse, nous avons également cherché à déterminer l’implication de deux cystéines protéases dans la mise en place du processus de sénescence nodositaire. Une des caractéristiques de ce processus de sénescence est une hausse de l’activité protéolytique, notamment des activités cystéine protéases. L’analyse transcriptomique par cDNA-AFLP du processus de sénescence nodositaire (Van de Velde et al. 2006) a pu mettre en évidence 508 gènes différentiellement exprimés dont deux cystéines protéases, MtCP6 et MtVPE. L’analyse spatio-temporelle de MtCP6 et MtVPE, par fusion transcriptionnelle avec le gène rapporteur GUS, a permis de mettre en évidence l’induction de ces deux gènes lors du processus de sénescence nodositaire aussi bien développementale qu’induit par un traitement abiotique ou lors d’une interaction symbiotique non efficace. De plus, nous avons pu démontrer, par génétique inverse, que la diminution de l’expression de ces deux protéases retarde la mise en place du processus de sénescence, alors que leur expression précoce conduit à la promouvoir. Enfin, l’étude par microscopie confocale de la localisation subcellulaire de ces protéases par fusion traductionnelle avec la GFP, démontre leur adressage aux bactéroïdes. Nos données tendent donc à démontrer le rôle clef de MtCP6 et de MtVPE dans le processus de sénescence nodositaire, où ces protéases pourraient participer directement au déclenchement d’une dégradation des bactéroïdes. / Medicago truncatula is a leguminous plant establishing a symbiotic interaction with the bacteria Sinorhizobium meliloti. This symbiosis leads to the de novo development of root nodules involved in biological nitrogen fixation. However, this symbiotic interaction is time limited and an early senescence appears in mature nodule entailing the formation of a senescence zone (zone IV). This degradation process occurs earlier in comparison to senescence of the whole plant. During nodule developmental senescence of plant host cells, a gradual degradation process induces a loss of vacuole and peribacteroid membrane (PBM). But this nodule degradation process still remains to be unravelled. To increase our understanding of the nodule senescence process, we developed new cytologic tools allowing an in vivo assessment of the viability and functioning of bacteroids within plant host cells. Therefore, these new tools provide a new insight of the nodule senescence process which may help for a finer characterization of the nodule senescence. In the M. truncatula model, a previous cDNA-AFLP analysis enlightens an upregulation of several cysteine proteinases during the transition from nitrogen fixing nodule to a senescent one; including an early expression of an SPG31-like peptidase known to be involved in leaf senescence (MtCP6) and a Vacuolar Processing Enzyme described as a plant caspase-like protein (MtVPE) involved in mechanisms similar to hypersensitive response in A. thaliana. In planta spatiotemporal analysis of the expression of these two cysteine proteinases using promoter:reporter gene GUS confirmed their expression during natural senescence at the junction between the nitrogen fixing zone (zone III) and the senescence zone (zone IV). Therefore, to acquire a better insight into the role of these cysteine proteases during the senescence program, we knocked down by RNAi the expression of each gene specifically at the interzone III-IV. Depletion of these transcripts induced a drastic increased of N2 fixation and nodule size. Conversely, overexpression of both genes in the zone III of nodule leads to an extension of the senescence zone. Confocal microscopy images of protein:GFP fusions showed that both proteinases are addressed to bacteroids within plant host cells. Our data revealed that MtCP6 and MtVPE are key players of the nodule senescence process and may be directly involved in symbiosome degradation.

No description available.

Au cours du développement du cancer, la migration des cellules cancéreuse en 3D joue un rôle essentiel dans le processus de dissémination des métastases. L’étude de la migration cellulaire dans des matrices 3D ainsi que les conséquences induites sur cette matrice sont actuellement étudiées par plusieurs équipes de recherche. Notamment, la réorganisation de la matrice extracellulaire et plus précisément les déplacements des fibres de la matrice induits par les forces que la cellule exerce sont des études en plein essor. Nous avons étudié comment les cellules cancéreuses migrent dans des gels 3D en utilisant du collagène et de la fibronectine pour mimer la matrice extracellulaire des tissus. Nous avons utilisé un microscope confocal afin de visualiser le cytosquelette d’actine des cellules en fluorescence et les fibres de collagène en réflexion. Dans ce travail,nous avons utilisé différentes concentrations de collagène et des lignées cellulaires d’invasivités différentes. A partir des films 3D obtenus en microscopie, nous avons déterminé la vitesse et la persistance des cellules cancéreuses en fonction de leur invasivité et de la concentration de collagène. La vitesse augmente avec l’invasivité cellulaire et diminue avec l’augmentation de la concentration en collagène. La persistance ne dépend que de la concentration en collagène et décroit avec celle-ci. Nous avons également calculé les champs de déplacement des fibres de collagène à l’aide d’un programme de corrélation de volume. Nous avons pu étudier ces champs de déplacement en fonction du type de migration de la cellule, de l’invasivité cellulaire et de la concentration en collagène des gels. Nous avons montré que les normes de vecteurs de déplacement augmentent avec l’invasivité cellulaire et diminuent avec l’augmentation de concentration en collagène. Enfin, ces champs de déplacement nous ont permis de déterminer les étapes des migrations mésenchymateuse et amiboïde en 3D. Nous avons découvert 5 étapes pour la migration mésenchymateuse correspondant au repos de la cellule, à la création d’une extension membranaire, à l’adhésion de la cellule aux fibres, au détachement de l’arrière du corps cellulaire afin de permettre à la cellule de migrer et à la dissolution de l’adhésion cellule/fibre. 4 étapes ont été déterminées pour la migration amiboïde et correspondent au repos de la cellule, à la création d’une extension membranaire, au déplacement de la cellule en poussant sur son environnement et à la rotation de la cellule. Ces étapes associées à des champs de déplacement sont en accord avec la littérature et nous avons pu mettre en évidence de nouvelles étapes comme la rotation de la cellule dans la migration amiboïde.Ces résultats permettent de mieux comprendre comment se déroule la migration des cellules cancéreuses dans une matrice extracellulaire. / 3D migration of cancer cells plays an essential role in the dissemination of cells during metastasisin cancer. The behavior of cancer cells migrating in a 3D extracellular matrix and its consequences on themicroenvironment are still currently under investigation. The study of the reorganization of the extracellular matrixfibers and more precisely how the fibers move due to the forces that the cell exerts just start to be investigating.We studied how cancer cells migrate in 3D gels using collagen and fibronectin to mimic the extracellularmatrix. We used confocal microscopy to image the actin cytoskeleton of cells in fluorescence and fibers in reflectionover time. In our studies, we used different collagen concentrations and cell lines with different invasivities. Fromthese 3D movies, we determined cancer cell velocities and persistence as a function of collagen gel concentration aswell as cell invasiveness. The cells velocities increase with invasiveness and decrease with collagen concentration.As for persistence, it decreases with collagen concentration but it do not change with cells invasiveness. We alsocalculated the displacement field of the collagen using a volume correlation program. Using this information, westudied the fibers displacement induced by the cell depending on its migration type, its invasivity and the collagenconcentration. We showed norms of fibers deplacement vectors increase with cell invasiveness and decrease withcollagen concentration. Finally, the displacement fields enabled us to determine the migration steps of mesenchymaland amiboid migrations. We discovered 5 steps in mesenchymal migration : cell rest, creation of extension, adhesionof the cell to the fibers, detachment of the cell rear and dissolution of cell/fibers adhesions. 4 steps have beencharacterized in amiboid migration : cell rest, creation of extension, displacement of the cell by pushing on fibersand rotation of the cell. These steps associated with displacement fields are in agreement with litterature and wehighlighted new steps as the rotation of the cell in amiboid migration.Taken together these results enable us to better understand how the migration of cancer cells takes place in a3D matrix.

Tuberculosis is one of the most prevalent infectious diseases globally due to the successful survival mechanisms displayed by Mycobacterium tuberculosis (Mtb). Mtb primarily infects alveolar macrophages (AMs) and is able to live intracellularly for extended periods of time due to a number of virulence factors which inhibit the antibacterial mechanisms of the AMs. This aspect of the Mtb life cycle means TB treatments suffer from poor bioavailability and efficacy. Additionally, the rise in resistant strains of Mtb means the use of higher doses and the use of alternative second and third line drugs which increase the risk of systemic toxicity. Drug encapsulation is a novel approach that can provide more favourable drug pharmacokinetics and pharmacodynamics. The aim of this project was to develop a liposomal drug delivery system to target Mtb infected alveolar macrophages. The system involved the encapsulation of two drugs; the antibiotic gatifloxacin (GFLX) and Mtb virulence factor inhibitor CV7. The hypothesis was that the two different antibacterial mechanisms would work in synergy and increase the efficacy of the treatment. AM targeting and receptor-mediated endocytic uptake was encouraged by the presence of a ligand attached to the surface of the liposome. Furthermore a pH-sensitive release mechanism was to be incorporated into the liposome to encourage the release of the encapsulated drugs in the vicinity of the intracellular bacteria. The intention was to produce a drug delivery system to enable a TB therapy regime of fewer, lower doses to increase compliance and reduce systemic toxicity by increasing efficacy through improved bioavailability. GFLX was successfully encapsulated using a weak base active loading method. To establish encapsulation efficiency, a homogeneous fluorescence assay able to quantify intra- and extra-liposomal gatifloxacin simultaneously was developed. pH-sensitive release of the payload could be achieved using a pH-sensitive peptide with a novel design based on chimeric structure, namely P3. CV7 was successfully encapsulated using a weak acid active loading method. CV7 liposomes were able to be functionalised by the incorporation of a mannose ligand on the surface of the liposome. An inhibition assay using the target enzyme of CV7, MptpB, was optimised to assess efficacy of liposomally encapsulated and released CV7. Flow cytometry and confocal microscopy studies confirmed that the liposomal formulations were internalised by the target macrophage cell line, J774a.1. Mannose liposomes conveyed superior uptake kinetics. Further confocal microscopy showed that after internalisation the liposomes entered the endolysosomal pathway and colocalised with BCG. A BCG-macrophage infection model was used to determine the intracellular efficacy of the liposomal formulations. Encapsulated CV7 displayed increased efficacy over free CV7, while encapsulation in functionalised liposomes showed better efficacy still. The encapsulation of GFLX did not increase the efficacy of GFLX and synergy between the two drugs was not achieved. In conclusion, the liposomal encapsulation of CV7 increased uptake of the drug by the target cell line and facilitated colocalisation of the drug with the target pathogen thereby increasing efficacy. Such a formulation could potentially increase bioavailability and efficacy in vivo for a more tolerable TB therapy.

Made available in DSpace on 2016-08-17T18:39:46Z (GMT). No. of bitstreams: 1 4853.pdf: 5831443 bytes, checksum: 4e6531437ba418dff23289618e0e9361 (MD5) Previous issue date: 2012-10-25 / Financiadora de Estudos e Projetos / Nonmelanoma skin cancer is the most frequent disease in the world population and it is becoming a public health problem. Due to this fact, new technologies are being tested for the treatment of this disease. Photodynamic Therapy (PDT) is a noninvasive technique with an excellent cosmetic outcome, well tolerated by patients and with good healing results when used for the initial stages of cancer lesions. PDT uses molecular oxygen, light at appropriate wavelengths and the drug photosensitizers to generation of reactive oxygen species such as singlet oxygen responsible for the photoinactivation of target cells. The characteristics of scar tissue after multiple sessions of PDT is not completely understood. It is not known whether multiple sessions of PDT may cause irreversible changes in the treated tissue. Thus, the present study has as its main objective the evaluation of the characteristics of the skin scarred after multiple sessions of PDT. For this study, we used 25 Wistar type male mice. These 25 mice were grouped into 5 subgroups of 5 each: G0, control group, non-subjected to any treatment; G1, exposed to one PDT session and after, there was made a lesion with CO2 laser over the scarred tissue; G2, the group is submitted twice for the same procedure, G3, subjected to 3 times the procedure performed for G1 group; G4, exposed to 4 times the procedure. Once the mice s skin was healed, we performed macroscopic and microscopic evaluations of the histological pieces. Furthermore, we made a microscopic evaluation of collagen fibers and the generation of the second-harmonic through multiphotons microscopy. Macroscopically, it was observed a good healing for the different groups. The aesthetic result showed a decrease after the third and fourth sessions. In the microscopic study, it was observed an conservation of the quantity of sebaceous glands per square millimeter (mm2). The morphology of collagen fibers when evaluated using two-photon excited fluorescence in the confocal microscope, showed thinner fibers with a parallel arrangement and lower density in the G3 e G4 compared to the control group. It was not observed decrease in the generation of second harmonic in histopathology slides. These results suggest that healing in skin after four (4) sessions of PDT/lesion presents alterations in aesthetic and histological of the morphology of collagen fibers that may be related to submitted of the skin in eight (8) consecutive occasions with an incomplete healing between them. / O Câncer de pele não melanoma é o câncer mais frequente da população mundial, se tornando um problema de saúde pública. Devido a essa problemática, novas tecnologias estão sendo testadas para o tratamento desta doença. A Terapia Fotodinâmica (TFD) é uma técnica não invasiva com excelentes resultados estéticos, bem tolerada pelos pacientes e com bons resultados de cura quanto usado para lesões iniciais. Usa o oxigênio molecular, a luz num comprimento de onda adequado e o medicamento fotossensibilizador para formar espécies altamente reativas como o oxigênio singleto responsável pela fotoinativação das células alvo. As características do tecido cicatrizado após múltiplas sessões de TFD não são completamente conhecidas. Ainda não se tem conhecimento se múltiplas sessões de TFD podem ocasionar modificações irreversíveis no tecido tratado. Desta forma, o presente estudo tem como principal objetivo a avaliação das características da pele cicatrizada após múltiplas sessões de TFD. Para este estudo, aprovado pelo comitê de ética e pesquisa foram utilizados 25 ratos machos, da linhagem Wistar, divididos em 5 grupos com 5 animais por cada grupo: G0 controle, não foi submetido a nenhum procedimento; G1, submetidos a uma sessão de TFD na pele do dorso, posteriormente, nesta pele cicatrizada foi feita uma lesão externa com laser de CO2; G2, o grupo passa duas vezes pelo mesmo procedimento explicado anteriormente; G3, o grupo passa três vezes pelo procedimento e para o grupo G4 é repetido os procedimentos em quatro ocasiões. Após a cicatrização dos procedimentos é feita uma avaliação macroscópica, um analise microscópica e uma avaliação da microestrutura das fibras de colágeno e geração do segundo harmônico por microscopia multi-fótons. Macroscopicamente, foi observada uma cicatrização satisfatória para os diferentes grupos tratados se apresentando uma diminuição no resultado estético após a terceira e a quarta sessão. No estudo microscópio, foi evidente uma conservação da quantidade de glândulas sebáceas por mm2. A morfologia das fibras de colágeno apresentaram modificações que foram mais acentuadas na terceira e quarta sessão, se evidenciando fibras de menor espessura, com uma disposição paralela e menor densidade quando comparadas ao grupo controle. Não foi evidente diminuição da geração do secundo harmônico nas lâminas estudadas. A partir destes dados sugere-se que a cicatrização na pele após 4 sessões de TFD/lesão, apresenta modificações no resultado estético, histológico, da morfologia das fibras de colágeno que podem estar relacionadas com acometimento consecutivo da pele em 8 ocasiões com uma cicatrização incompleta entre elas.

O presente estudo objetivou testar experimentalmente a existência da possível correlação entre a penetração de cimento nos túbulos dentinários e a qualidade do selamento. Foram utilizados 60 incisivos centrais superiores humanos que formaram um único grupo experimental. Após a eliminação das porções coronárias, as raízes foram padronizadas em 13 mm de comprimento. A instrumentação dos canais foi realizada no sentido coroa-ápice, e o comprimento de trabalho estabelecido a 1 mm aquém do forame apical. Como solução irrigadora foi empregado o NaOCl a 5,25% e ao final, EDTA a 17%. Em seguida, todos os canais foram obturados com guta-percha e cimento AH Plus marcado com um corante fluorescente. Para determinar a qualidade do selamento das obturações endodônticas, as amostras foram submetidas ao modelo de infiltração de glicose sob pressão. As raízes foram montadas em um dispositivo de dupla-câmara selada para permitir a infiltração da glicose. Como controle negativo foram utilizados 4 dentes hígidos, e como controle positivo, 2 dentes instrumentados porém, não obturados. Foram utilizados 0,75 mL de solução de glicose a 1 mol/L na câmara superior e 0,75 mL de água deionizada na câmara inferior. Os dispositivos foram conectados a um sistema de distribuição de pressão desenvolvido com o objetivo de permitir a infiltração de 32 amostras em uma mesma etapa. A solução de glicose foi forçada apicalmente sob uma pressão de 15 psi durante 1 hora. Uma alíquota de 50 L foi coletada da câmara inferior para quantificar a glicose infiltrada. A concentração de glicose foi determinada através de um método enzimático com o auxílio do Kit Glucose HK e de um espectrofotômetro em um comprimento de onda de 340 nm. Na sequência, as amostras foram desacopladas dos corpos de prova, embutidas em resina epóxi e cortadas em 3 secções transversais. Uma sequência de preparação metalográfica padrão foi realizada para permitir a observação da penetração de cimento nos túbulos dentinários por meio de microscopia confocal e óptica. Os dados obtidos nos 2 experimentos foram cruzados pelo teste de correlação de Spearman, o qual revelou a inexistência de qualquer possibilidade de correlação (r = 0,12). Com base nesses resultados, o presente trabalho concluiu que, dentro das condições experimentais usadas, a quantidade de cimento presente dentro dos túbulos dentinários não teve relação com a qualidade do selamento produzido. / The purpose of the present study was to experimentally test the potential correlation between tubular dentin sealer penetration and sealability. Sixty human maxillary central incisors were selected in order to compose a single experimental group. After removing of crowns, the roots were standardized at 13 mm in length. A crown-down root canal instrumentation was performed 1mm short of the apical foramen (working length). How irrigating solution was used 5.25% NaOCl and final, 17% EDTA. Then, all root canals were filled with gutta percha and AH Plus sealer labeled with a fluorescent dye. To determine the sealability pattern, samples were submitted to glucose leakage model under pressure. Four intact teeth were used as negative control while further 2 instrumented but nonfilled teeth was used as positive control. The roots were mounted in a double-chamber apparatus where 0,75 mL of 1 mol/L glucose solution was placed into the superior chamber and 0,75 mL of deionized water was placed into the inferior chamber. The devices were connected to a pressure distribution system that was developed in order to allow testing 32 specimens at the same experimental stage. The glucose solution was forced apically under a pressure of 15 psi during 1 hour. A sample of 50 L was taken from the inferior chamber and the glucose concentration was measured following an enzymatic reaction using a Glucose HK Kit readed under spectrophotometry (wave-length of 340 nm). After that, the samples were removed from the apparatus, embedded in epoxy resin and cut into 3 sections. Standard metallographic preparation was performed prior the observation of the sealer penetration into dentinal tubules by confocal and optical microscopy. Data provided by the evaluations were submitted to Spearman correlation test which revealed a lack of correlation between the two variables (r = 0.12). Therefore, the present study concluded that there was no correlation between tubular sealer penetration and sealability in non-bonding conventional root-fillings.

In the past two decades, Fluorescence microscopy has imparted tremendous impact in Biology and Imaging. Several super-resolution Fluorescence imaging techniques (e.g. PALM, STED, STORM, 4Pi and structured illumination) have enabled diff raction-unlimited imaging. But high resolution is limited to a depth of few tens of microns. Thus, deep tissue imaging and simultaneous volume imaging have become a highly sought after feature in Fluorescence microscopy. The research work in this thesis address these issues by using spatial filtering techniques to tailor the point spread function (PSF) which uniquely characterizes the optical sys-tem. The advantage of this approach lies in the fact that intricate details about the focal region can be computed and designed with the help of well established theory and experimentation. In particular, this technique was applied to both spherical and cylindrical lenses. The former was used to generate Bessel-like, non-diffracting beams which demonstrated the ability to penetrate deep inside tissue-like media and thereby yielded an imaging depth of nearly 650μm as compared to about 200μm for a state-of-the-art confocal microscope. The latter gave rise to light-sheet and it's extended version that is ideal for planar imaging at large penetration depths. Another development is the generation of multiple light-sheet illumination pattern that can simultaneously illuminate several planes of the specimen. The proposed multiple light-sheet illumination microscopy (MLSIM) technique may enable volume imaging in Fluorescence microscopy. The first two chapters of this thesis are introductory in nature and provides a general overview of the principles of Fluorescence microscopy and three state-of-the-art Fluorescence imaging techniques; namely confocal, multi-photon and light-sheet based microscopy. Confocal microscopes are widely considered as a standard tool for biologists and this discussion shows that even though they have made signi ficant contributions in the fields of biophysics, biophotonics and nanoscale imaging, their inability to achieve better penetration depth has prevented their use in thick, scattering samples such as biological tissue. The system PSF of a confocal microscope broadens as it goes deeper in-side a scattering sample resulting in poor-resolution thereby destroying the very concept of high resolution, noise-free imaging. Additionally, confocal microscopy suffers from in-creased photo-bleaching due to o -layer (above and below the focal plane) excitation and low temporal resolution since it requires point-by-point scanning mechanism. On the other hand, multi-photon microscopy offers several advantages over confocal microscopy such as reduced photo-bleaching and inherent optical sectioning ability, however, it still lacks in providing high temporal resolution. Light-sheet based microscopy have gained popularity in recent years and promises to deliver high spatio-temporal resolution with minimized photo-bleaching. Recently, a considerable amount of research has been dedicated to further develop this promising technique for a variety of applications. The ability to look deeper inside a biological specimen has profound implications. How-ever, at depths of hundreds of microns, several effects (such as scattering, PSF distortion and noise) deteriorates the image quality and prohibits detailed study of key biological phenomenon. Chapter 3 of this thesis describes the original research work which experimentally addresses to this issue. Here, Bessel-like beam is employed in conjugation with an orthogonal detection scheme to achieve imaging at large penetration depth. Bessel beams are penetrative, non-di ffracting and have self-reconstruction properties making them a natural choice for imaging scattering prone specimens which are otherwise inaccessible by other microscopy imaging techniques such as, Widefield, CLSM, 4PI, Structural illumination microscopy and others. In this case such a Bessel-like beam is generated by masking the back-aperture of the excitation objective with a ring-like spatial filter. The proposed excitation scheme allow continuous scanning by simply translating the detection optics. Additionally, only a pencil-like region of the specimen can be illuminated at a given instance thereby reducing premature photobleaching of neighboring regions. This illumination scheme coupled with orthogonal detection shows the ability of selective imaging from a desired plane deep inside the specimen. In such a configuration, the lateral resolution of the illumination arm determines the axial resolution of the overall imaging system. Such an imaging system is a boon for obtaining depth information from any desired specimen layer that includes nano-particle tracking in thick tissue. Experiments performed by imaging the Fluorescent polymer tagged-CaCO3 particles and yeast cell in a tissue-like gel-matrix demonstrates penetration depth that extends up to 650 m. This will advance the field of fluorescence imaging microscopy and imaging. Similar to the ability to observe deep inside a sample, simultaneous 3D monitoring of whole specimens play a vital role in understanding many developmental process in Biology. At present, light-sheet based microscopy is the prime candidate amongst the various microscopy techniques, that is capable of providing high signal-to-background-ratio as far as planar imaging is concerned. Since spatial filtering technique was found to successfully give rise to novel features (such as large penetration depth) in a fluorescence microscope setup, a logical extension would be to implement a similar approach with a light-sheet based microscope setup. These implementations are discussed in Chapter 4 of this thesis where spatial filtering is employed with cylindrical lenses. For facilitating computational and experimental studies, a vectorial formalism was derived to give an explicit computable integral solution of the electric field generated at the focal region of a cylindrical lens. This representation is based on vectorial diffraction theory and further enables the computation of the point spread function of a cylindrical lens. Commonly used assumptions are made in the derivation such as no back-scattering and negligible contribution from evanescent fields. Stationary phase approximation along with the Fresnel transmission coefficients are employed for evaluating the polarization dependent electric field components. Computational studies were carried out to determine the polarization effects and calculate the system resolution. Experimental comparison of light-sheet intensity pro les show good agreement with the theoretical calculations and hence validate the model. This formalism was derived as a first step since it gives the essential understanding of tightly focused E-fields of a high N.A. cylindrical lens systems and thereby helps in further understanding the effect of spatial filtering. As the next step, generation of extended light-sheet for fluorescence microscopy is pro-posed by introducing a specially designed double-window spatial filter at the back-aperture of a cylindrical lens. The filter allows the light to pass through the periphery and center of a cylindrical lens. When illuminated with a plane wave, the proposed filter results in an extended depth-of-focus along with side-lobes which are due to other interferences in the transverse focal plane. Computational studies show a maximum extension of light-sheet by 3:38 times for single photon excitation, and 3:68 times for multi-photon excitation as compared to state-of-art single plane illumination microscopy (SPIM) system and essentially implies a larger field of view. Finally, generation of multiple light-sheet pattern is proposed and demonstrated using a different spatial filter placed at the back aperture of a cylindrical lens. A complete imaging setup consisting of multiple light-sheets for illumination and an orthogonal detection arm, is implemented for volume imaging in fluorescence microscopy. This proposed scheme is a single shot technique that enables whole volume imaging by simultaneously exciting multiple specimen layers. Experimental results confirm the generation of multiple light-sheets of thickness 6:6 m with an inter-sheet spacing of 13:4 m. Imaging of 3 5 m sized fluorescently coated Yeast cells (encaged in Agarose gel-matrix) is per-formed and conclusively demonstrates the usefulness and potential of multiple light-sheet illumination microscopy (MLSIM) for volume imaging. As part of the future scope of the research work presented in this thesis, the Bessel-beam based improved depth microscopy technique may attract applications in particle tracking deep inside tissues and optical injection apart from fluorescence imaging applications. The vectorial formalism derived for cylindrical lens can be used to predict other, complex optical setups involving cylindrical lenses. Extended light-sheet generation proposed in this work by using appropriate spatial filtering with a cylindrical lens, complements the existing and popular selective plane illumination microscopy technique and may facilitate the study of large biological specimens (such as, full-grown Zebra sh and tissue) with high spatial resolution and reduced photobleaching. Finally, the MLSIM technique presented in this thesis may accelerate the field of developmental biology, cell biology, fluorescence imaging and 3D optical data storage.

Este trabalho teve o objetivo de avaliar a capacidade da bactéria Enterococcus faecalis em induzir periodontite apical no modelo experimental canino; verificar se o preparo químico-mecânico afeta a sobrevivência dessa bactéria no sistema de canais radiculares e avaliar o reparo desses dentes por meio de radiografia periapical e tomografia computadorizada cone beam. Dentes com e sem tratamento endodôntico foram avaliados por meio de microscopia confocal de varredura a laser. Para tal, foram utilizados 2 cães. A bactéria Enterococcus faecalis foi inoculada nos canais de 4 pré-molares superiores, 11 prémolares inferiores e 9 incisivos superiores. As câmaras pulpares foram seladas e, após 60 dias, os canais das raízes distais dos pré-molares e de 7 incisivos superiores foram submetidos a tratamento endodôntico em sessão única e os canais das raízes mesiais dos pré-molares e 2 incisivos superiores foram deixados sem tratamento (controle). Amostras microbiológicas foram feitas antes e após o preparo químico-mecânico. O reparo foi avaliado após 6 meses do tratamento mediante radiografias periapicais e por tomografia computadorizada cone beam. A comparação entre as imagens obtidas após o período experimental pelos 2 métodos foi feita por medições da área em mm2 de cada lesão encontrada, utilizando o software ImageTool Os resultados mostraram que a presença de periodontite apical crônica foi verificada em todos os dentes inoculados, independentemente da colonização pela bactéria Enterococcus faecalis ou pela flora mista. O preparo químico-mecânico reduziu significativamente o número de bactérias no interior dos canais radiculares (p<0.05). Os resultados radiográficos e tomográficos demonstraram lesões de menor diâmetro nos dentes tratados endodonticamente em comparação ao grupo controle (p<0.05). A comparação entre os métodos demonstrou diferença estatística entre eles sendo evidenciada áreas radiolúcidas maiores utilizando a tomografia cone beam em comparação com a radiografia periapical (p<0.05). Concluiu-se que a bactéria Enterococcus faecalis induziu periodontite apical crônica similarmente à flora mista; o tratamento endodôntico reduziu o número de bactérias cultiváveis significativamente embora sem relação com o reparo radiográfico. Os dentes tratados em sessão única apresentavam lesões menores em comparação aos não-tratados e as áreas das lesões observadas na TC cone beam foram maiores do que as áreas encontradas nas radiografias periapicais. A microscopia confocal e o método proposto neste estudo se mostraram eficazes para determinar, qualitativamente, a viabilidade bacteriana e a distribuição de ácidos nucléicos bacterianos dentro dos túbulos dentinários. Houve diferença entre a dentina infectada in vitro com o padrão de infecção in vivo, caracterizada pela presença de biofilmes aderidos à parede do canal radicular. / The aims of this study were to evaluate the Enterococcus faecalis ability to induce apical periodontitis in dogs root canals, to verify the bacterias ability to survive to the cleaning and shaping procedures and to assess the healing of the induced apical periodontitis by periapical radiograph and cone beam computed tomography. Also, endodontically treated and non-treated teeth were evaluated by confocal laser scanning microscope. Two mongrel dogs were used in the experiment. Enterococcus faecalis strain was inoculated into the root canals of 4 maxillary premolars, 11 mandibular premolars and 9 maxillary incisors. After 60 days the root canals of the distal roots of the mandibular and maxillary pre-molars and 7 maxillary incisors were endodontically treated. The premolars mesial root canal and 2 maxillary incisors were used as control (no treatment). Microbiologic samples were done after and before the cleaning and shaping procedure. The healing was evaluated after 6 months by periapical radiographs and cone beam computed tomography. The comparison between obtained images after the experimental period by the two methods was done using measures of the lesion area in mm2 with ImageTool software. The results showed the presence of chronic apical periodontitis in every inoculated teeth, with Enterococcus faecalis or mixed infection. The cleaning and shaping procedures reduced the number of bacteria of the root canals (p<0.05). The radiographic and tomographic results showed the lower diameter lesion in endodontically treated teeth than in the control group (p<0.05). The comparison between the methods showed statistical difference and greater radiolucent areas were evident in cone beam computed tomography images (p<0.05). It is possible to conclude that the bacteria Enterococcus faecalis induced chronic apical periodontitis as well as the mixed microflora; the endodontic treatment reduced the number of cultivable bacteria in a significant way, but with no relation to the radiographic healing. The treated teeth in only one session presented smaller lesions in comparison to the non-treated teeth and the lesions area in cone beam computed tomography were bigger than the areas found in periapical radiograph. The confocal microscopy and the proposed method of this study showed to be efficient to determine the bacterial ability and the distribution of bacterial nucleic acids inside the dentin tubules. There was difference between in vitro infected dentin with the in vivo infection pattern, which presents biofilm attached to the root canal walls.

As propriedades fotofísicas e fotoquímicas de cristal violeta (CV) foram investigadas em soluções isotrópicas e verificou-se que solventes com constante dielétrica pequena favorecem a formação do par iônico, já o aumento na viscosidade do meio restringe a movimentação rotacional dos anéis aromáticos, resultando em um aumento no tempo de vida de fluorescência e, portanto no rendimento quântico de fluorescência (&#934;f) (Oliveira 2002). Os experimentos com CV foram conduzidos em micelas reversas do tensoativo aniônico bis-2-etilhexil sulfoccinato de sódio (AOT) em isooctano. A localização interfacial do CV nas micelas reversas de AOT em valores da razão molar entre água e surfactante (W0)pequenos e grandes foram encontrados através da técnica de Ressonância Magnética Nuclear (RMN) de próton e de carbono 13. Utilizando-se espectroscopia UV-Vis identificou-se que pares iônicos de contato estão presentes a valores pequenos de W0 e com o aumento do W0 pares iônicos separados por solventes são as espécies que predominam em solução. A comparação da eficiência de fotodegradação de CV em micelas reversas de AOT em função do W0 indicou que a fotoreatividade é maior em baixos valores de W0 . Este efeito deve estar relacionado à restrição da movimentação dos anéis aromáticos de CV devido ao ambiente restrito no qual este se localiza na micela reversa de OAT a W0 pequenos. A formação de intermediários reativos foi verificada através de Fotólise de Relâmpago a Laser e Emissão no infra-vermelho próximo, indicando a presença de espécies triplete, radical e oxigênio singlete com valor de rendimento quântico menor que 1%. Os produtos de fotólise foram identificados por técnicas cromatográficas e espectroscópicas. Na presença de oxigênio, houve maior formação de cetona de Michler. Com baixa concentração de oxigênio, o produto observável foi leuco-CV. Destes estudos propomos o mecanismo de CV neste meio. Após os estudos com micelas reversas, células cancerosas HeLa foram empregadas para comparar fotoatividade do CV com o azul de metileno (MB). As proporções de CV e MB dentro das células são altas, incorporando 70% e 80% da concentração da solução de incubação, respectivamente. Com o aumento da concentração de MB, um favorecimento da formação de dímero foi identificada. Já CV não sofre agregação nas condições estudadas. Nenhum dos fotossensibilizadores estudados tem um efeito danoso sobre as células HeLa em concentrações abaixo de 10&#181;M. Após irradiação, MB causou uma diminuição de cerca de duas vezes maior na taxa de sobrevivência celular comparado com CV. A formação de formação de oxigênio singlete após incorporação dos fotossensibilizadores foi investigada. Há formação de oxigênio singlete em células incubadas com MB, já com CV a geração de oxigênio singlete é pouco significativa sugerindo um mecanismo radicalar. O processo de morte celular foi estudado por citometria de fluxo e verificou-se que MB induz apoptose depois da irradiação em células HeLa. A absorção de luz por ambos fotossensibilizadores é similar, o que indica que a diminuição na sobrevivência não se deve à diferença de absorção luminosa. As diferenças de sobrevivência observadas com células incubadas com CV e MB e irradiadas foram relacionadas às diferenças das propriedades fotoquímicas destes fotossensibilizadores. A localização celular de CV e MB em células foram caracterizadas por microscopia de fluorescência. Verificou-se que ambos localizam-se em mitocôndrias. O aumento na concentração de CV não alterou o seu perfil de localização. Já para MB ao aumentar a concentração de MB, observa-se que o mesmo localiza-se além das mitocôndrias, em lisossomos. A comparação das propriedades fotoquímicas e de localização foram consideradas para explicar as diferenças de atividade fotodinâmica do CV e do MB em células HeLa / The photophysical and photochemical properties of crystal violet (CV) were investigated in isotropic solutions and it was found that solvents with small dielectric constants favor the formation of the ion pair and that the increase in viscosity of the medium restricts the rotational movement of the aromatic rings, resulting in an increase in fluorescent lifetime and therefore in the fluorescence quantum yield (&#934;f) (Oliveira 2002). CV experiments were conducted in reverse micelles of the anionic tensoactive sodium bis-2-ethylhexyl-sulfosuccinate (AOT) in isooctane. The interfacial localization of CV in the AOT reverse micelles at low and high values of molar ratio between water and surfactant (W0 was found through the proton and carbon 13 Nuclear Magnetic Resonance techniques (NMR). Using UV-Vis spectroscopy, it was identified that contact ion pairs are present in low W0 values and with the increase in the W0 solvent separated ion pairs are the species that predominate in solution. The comparison of the photobleaching efficiency of CV in AOT reverse micelles as a function of W0indicated that the photoreactivity is high with low W0 values. This effect must be related to the restrict environment in which CV is located. The reactive intermediate formation was found through the Laser Flash Photolysis and Near Infra-Red Emission, indicating the presence of triplet, radical and singlet oxygen species with a yield quantum of less than 1%. The photolysis products were identified through the chromatographic and spectroscopic techniques. In the oxygen presence, there was high Michler ketone formation. With the low oxygen concentration, the observable product was leuco-CV. With these studies we hypothesized a mechanism of CV in these proposed media. After the reverse micelles studies, the HeLa cancerous cells were used, in order to compare the CV and methylene blue (MB) photoactivity. The CV and MB proportion inside the cell was high, reaching 70% and 80% of concentration of the incubation solution, respectively. With the increase of MB concentration, a favoring of dimmer formation was identified. CV does not suffer aggregation in the studied conditions. None of the studied photosensitizers has a damaging effect upon the HeLa cells in concentrations below 10&#181;M. After irradiation, MB caused a decrease about twice higher in the cellular survival rate compared to CV. The singlet oxygen formation after the photosensitizer incorporation was investigated. There is a singlet oxygen formation in the cells incubated with MB, though with the CV the singlet oxygen generation is significantly low suggesting the radicalar mechanism. The cellular death process was studied by Fluorescence Activated Cell Sorting and MB-induced apoptosis was found after MB irradiation in HeLa cells. The light absorption by both photosensitizers is similar, which means that the survival decrease is not because of the light absorption difference. The survival differences observed with the cells incubated with CV and MB and irradiated were related to the differences in the photosensitizer photochemical properties. The cellular location of the CV and MB in cells were characterized by fluorescence microscopy. Both photosensitizers are located in mitochondrias. An increase in the CV concentration does not alter its local profile. However, with an increase in the MB concentration, MB was located not only in mitochondrias but also in lysosomes. The comparison of the photochemical and localization properties was considered in order to explain the differences in the photodynamic activity of CV and MB in HeLa cells

Resinas compostas apresentam contração de polimerização e a tensão gerada durante essa polimerização compete com a força adesiva na interface dente/restauração. A técnica incremental e o uso de bases com alta resiliência e módulo de elasticidade próximo ao das estruturas dentárias são técnicas desenvolvidas para tentar diminuir a tensão originada pela contração de polimerização. O presente estudo teve como objetivo analisar a influência do uso de bases de cimento de ionômero de vidro convencional e modificado por resina na qualidade e adaptação marginal na interface dentina/resina composta, após a ciclagem térmica, usando testes de resistência adesiva e análise em microscopia confocal de varredura a laser. Foram confeccionadas cavidades na face oclusal (4,5mm x 3mm x 5mm) de 60 molares humanos extraídos divididos em 6 grupos: 1 e 4 - sistema adesivo (AdperTM Single Bond; 3M ESPE) + resina composta (Filtek Z250; 3M ESPE); 2 e 5 - base de cimento de ionômero de vidro convencional (Ketac Molar Easymix; 3M ESPE) + sistema adesivo + resina composta; e 3 e 6 - base de cimento de ionômero de vidro modificado por resina (Vitrebond; 3M ESPE) + sistema adesivo + resina composta. Os grupos 4, 5 e 6 sofreram um processo de termociclagem com dois banhos (5ºC 55ºC) durante 30 segundos em 5.000 ciclos. Após 24 horas, os dentes foram seccionados em uma máquina de cortes com disco de diamante em espessura de 0,8mm. Uma fatia de cada dente foi separada aleatoriamente para análise em Microscópio Confocal para observação e mensuração de possíveis fendas marginais internas. As demais fatias foram seccionadas para a confecção de palitos (0,8mm X 0,8mm) que foram submetidos a testes de microtração em uma máquina de ensaios universal EMIC. Os resultados de resistência adesiva foram submetidos à análise de variância a um critério (ANOVA) e ao teste t-Student (p< 0,05). A presença de fendas foi avaliada com o teste da razão de verossimilhança ou teste exato de Fisher e os valores de comprimento das fendas foram avaliados pelo teste não paramétrico de Kruskal-Wallis (p<0,05). Não foram observadas diferenças estatisticamente significantes na resistência adesiva em nenhum dos grupos sem termociclagem (G1 19,28 MPa; G2 16,29 MPa; e G3 15,95MPa) ou com termociclagem (G4 19,74 MPa; G5 16,58 MPa; e G6 16,01 MPa). A análise das medidas das fendas revelou não haver diferença estatisticamente significante entre os grupos G1 (1,4µm), G2 (2,88µm) e G3 (4,63µm) e entre os grupos G4 (4,2µm), G5 (12,5µm) e G6 (5,4µm). No entanto, a termociclagem determinou um aumento do tamanho médio das fendas no grupo com base de CIV convencional (G4 12,5µm). A análise da presença ou ausência de fendas mostrou um aumento na porcentagem do número de fendas quando os espécimes foram termociclados, para os grupos sem base e com base de CIV (G1 - 30%; G2 - 25% G3 25%; G4 - 53,33%; G5 70%; e G6 30%). Os resultados mostraram, ainda, não haver relação entre o comprimento e a formação das fendas com a resistência adesiva. Conclui-se, portanto, que o uso de base de cimento de ionômero de vidro modificado por resina mantém mais estável a qualidade da interface adesiva dentina/resina composta após envelhecimento artificial com termociclagem. / Polymerization shrinkage leads to a tension into dentin/resin composite interface that can cause marginal discoloration, poor marginal adaptation, secondary caries and post-operative sensitivity. The incremental restorative technique and the use of a resilient liner with a modulus of elasticity similar to dental structures are techniques used to decrease the shrinkage polymerization tension. The aim of this in vitro study was to analyze the effect of glass-ionomer cement as a liner on the adhesive interface dentin/resin of occlusal restorations after thermocycling aging. Occlusal cavities were prepared sixty human extracted molars, divided into six groups: 1 and 4 with no liner; 2 and 5 glass-ionomer cement (Ketac Molar Easymix); and 3 and 6 resin-modified glass-ionomer cement (Vitrebond). Resin composite (Filtek Z250) was placed after application of adhesive system Adper Single Bond 2. Adhesive system was mixed with fluorescent reagent (Rhodamine B) to allow confocal microscopy analysis. After that, the specimens of groups 4, 5, 6 were thermocycled into 2 baths (5ºC 55ºC) of 30s each in 5.000 cycles. After this period, teeth were sectioned in 0,8mm slices. One slice of each tooth was randomly selected for analysis in Confocal Microscopy. The other ones were sectioned in sticks, which were submitted to micro-tensile test. The results of adhesive strength were analyzed by one way ANOVA and t-Student tests. Gap formation were analysed by Fisher test and the gaps size were analyzed by Kruskal-Wallis test (p<0,05). No statistical difference on adhesive resistance was showed between groups. Confocal Microscopy analysis showed gaps with a higher mean sizes for group 4 (12,5µm) and higger percentage of marginal gaps formation for the thermocycled groups (G1 - 30%; G2 - 25%; G4 - 53,33%; G5 70%). Groups 3 (25%) and 6 (30%) showed the lowest percentage of marginal gap formation. The results revealed that gap formation is not related to adhesive strength. It can be concluded, therefore, that the use of a resin-modified-glassionomer cement liner showed less gap formation on dentin/composite adhesive interface after artificial aging compared to conventional glass ionomer cement liner and restorations with no lining.