O cristalino de vertebrados se origina a partir da invaginação do ectoderme que recobre a vesícula óptica. A invaginação epitelial em diversos modelos é causada pela constrição celular apical, mediada pela contração apical de actina e miosina II e regulada pela GTPase RhoA. Neste trabalho nós investigamos se a invaginação do cristalino em embriões de galinha ocorre devido à constrição celular apical e se este evento é controlado por RhoA. Actina filamentosa e miosina II são expressas na porção apical do cristalino durante a invaginação. Quando a polimerização de actina é inibida por Citocalasina D, o cristalino não invagina, sugerindo que a constrição celular apical poderia contribuir para a invaginação do cristalino. RhoA também é expressa durante o desenvolvimento do cristalino, mas a inibição de RhoA, por eletroporação da forma dominante-negativo, não impediu a invaginação do placóide do cristalino, não alterou a distribuição de miosina II na porção apical do cristalino nem sua ativação, indicando que a invaginação do cristalino independe de RhoA. / Vertebrate lens derives from invagination of the ectoderm that overlies optic vesicles. Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase RhoA. Here we investigate the possibility that chick lens placode invagination could also be driven by apical cell constriction and controlled by RhoA. We show that actin and myosin II are expressed at lens apical side during lens invagination. Actin polymerization inhibition by in ovo Cytochalasin D treatment prevents lens placode invagination, suggesting that lens placode invagination could be driven by apical cell constriction. RhoA GTPase is also expressed at apical portion of lens placode and during lens invagination. However, when we overexpressed by electroporation the dominant-negative RhoA in the pre-lens ectoderm invagination was not affected. Furthermore, dominant-negative RhoA didnt affect myosin II apical localization nor myosin II phosphorilation, indicating that in lens invagination this process is not regulated by GTPase RhoA.

No presente estudo investigou-se amostras de plasma com DNA livre circulante (DNA LC) por meio da técnica Varredura Z. Esta é uma técnica eficiente na determinação de parâmetros de diferentes materiais, tais como cristais líquidos, ferrofluidos e compostos biológicos. Esta experiência é realizada através da focalização de um feixe laser de perfil gaussiano numa amostra. Na medida em que a amostra se aproxima do foco da lente, a intensidade do feixe aumenta e alcança seu valor máximo no ponto focal, então diminui para pontos distantes do foco. Na região próxima ao ponto focal se amplificam os fenômenos não-lineares. Recentemente foi demonstrado que níveis elevados de DNA LC no plasma ocorrem com frequência em pacientes com vários tipos de câncer, podendo ser utilizados para discriminar pacientes com malignidade de pessoas saudáveis. As amostras de DNA LC, submetidas ao experimento Varredura Z, forneceram respostas ópticas devido ao fenômeno de lente térmica. Os resultados revelaram que a amplitude de lente térmica das amostras extraídas do plasma de pacientes com malignidade difere daquela de doadores sãos. A técnica Varredura Z se mostrou mais vantajosa em relação a outras biológicas porque revelou uma maior diferença entre os grupos estudados e tem o caráter de detectar mudanças estruturais no DNA LC. / In the present study plasma samples with cell-free DNA were investigated by means of the Z-Scan technique. This is a powerfull technique in determining parameters of different materials, such as liquid crystals, ferrofluids and biological compounds. This experiment is performed by focusing a Gaussian profile laser beam on a sample. As the sample approaches the focus of the lens, the intensity of the beam increases and reaches its maximum value at the focal point, then decreases to points distant from the focus. In the region near the focal point non-linear phenomena are amplified. It has recently been demonstrated that high levels of plasma cell-free DNA occur frequently in patients with various cancers and can be used to discriminate patients with malignancy from healthy donors. The cell-free DNA samples, submitted to the Z-Scan experiment, provided optical responses due to the thermal lens phenomenon. The results revealed that the thermal lens amplitude of samples extracted from the plasma of patients with malignancy differs from that of healthy donors. The Z-Scan technique was more advantageous than other biological ones because it revealed a greater difference between the studied groups and has the character of detecting structural changes in cell-free DNA.

Hintergrund: RGD-Peptide hemmen kompetitiv die Adhäsionsmoleküle von Linsenepithelzellen (LEC). Ziel unserer Untersuchungen war es herauszufinden, ob diese Peptide in der Lage sind, auch nach Kurzzeitinkubation eine suffiziente Inhibition der Adhäsion bzw. eine Ablösung adhärenter Zellen und damit eine ausreichende Prävention des Nachstars zu bewirken. Außerdem wurde überprüft, ob das von uns verwendete RGD-Peptid eine Toxizität für die Hornhaut aufweist. Material und Methoden: Kulturen boviner und humaner LEC, boviner Hornhautendothelzellen, humane und bovine Linsenkapselexzidate und humane explantierte Hornhäute wurden verwendet. Wir untersuchten die Inhibition der Adhäsion und die Ablösung konfluenter LEC-Layer mittels des zyklischen RGD-Peptids cRGDDFV (Inkubationszeiten von 1 Stunde bzw. 5-7 Tagen und Konzentrationen von 10-4 M, 10-3 M und 2x10-3 M wurden angewandt). Ergebnisse: Wir fanden nach nur einstündiger Inkubation in der Kulturschale eine Adhäsionsinhibition von 48% für bovine LEC und von 100% für humane LEC. Die Differenz zwischen Kontrollpeptid und cRGDDFV war statistisch signifikant (p / Purpose: RGD-peptides competitively inihibit adhesion molecules of the lens epithelial cells (LEC). The purpose of our study was to investigate whether this peptide could be able to inhibit adhesion sufficiently after short term incubation resp. to detach adherent cells and so to prevent posterior capsule opacification (PCO). Also there was proofed if there is any toxicity for the cornea. Methods: Cultures of bovine and human LEC, bovine cornea endothelial cells, humane and bovine fragments of the lens capsule and explanted humane corneas were used. The inhibition of adhesion and the detachment of confluent LEC-layer by the cyclic RGD-peptide cRGDDFV were studied (incubation time was 1 hour resp. 5-7 days and concentrations of 10-4, 10-3 M and 2x10-3 M were used). Results: After one hour incubation time in a culture dish inhibition of adhesion was 48% for bovine LEC resp. 100% for humane LEC. There was a statistically significant difference between the control-peptide-group and cRGDDFV (p

Neste trabalho, mostramos que fluxos de Reeb dinamicamente convexos em um espaço lenticular $L(p, 1)$, $p>1$, admite uma órbita periódica de Reeb especial $P$ que é o binding de uma decomposição em livro aberto racional, com páginas tipo-disco tal que cada página é uma seção global. O índice de Conley-Zehnder da $p$-ésima iterada de $P$ é $3$. Como corolário, o fluxo de Reeb possui duas ou infinitas órbitas periódicas. Este resultado aplica-se ao Hamiltoniano de Hénon-Heiles, cujo fluxo restrito a energia baixa possui $Z_3$-simetria e define um fluxo de Reeb em $L(3, 1)$. Devido a $Z_4$-simetria aplicamos nosso resultado ao problema lunar de Hill regularizado. Na segunda parte deste trabalho investigamos a existência de uma folheação $3-2^3$ em níveis de energia no sistema Hamiltoniano de Hénon-Heiles, para energia logo acima da crítica. Provamos que certa região de interesse é uma hipersuperfície de contato. Provamos também que o fluxo de Reeb possui uma órbita periódica $Z_3$ simétrica, cujo índice de Conley-Zehnder é $3$ e possui número de auto-enlaçamento $-1$. / We show that a dynamically convex Reeb flow on a lens space $L(p, 1)$, $p>1$ admits a special closed Reeb orbit $P$ which is the binding of a rational open book decomposition with disk-like pages so that each page is a global surface of section. The Conley-Zehnder index of the $p$-th iterate of $P$ is $3$. As a corollary, the Reeb flow has $2$ or infinitely many closed Reeb orbits. This result applies to the Hénon-Heiles Hamiltonian whose flow restricted to low energy levels has $Z_3$-symmetry and descends to $L(3,1)$. Due to a $Z_4$-symmetry we also apply our results to Hill\'s lunar problem. In the second part of this work we investigate the existence of a $3-2^3$ foliation on energy levels of the Hénon-Heiles Hamiltonian, for energies above the critical one. We show that some region is of contact-type and the Reeb flow has a $Z_3$-symmetric periodic orbit, whose Conley-Zehnder is $3$ and has self-linking number $-1$.

Le poisson Astyanax mexicanus présente, au sein de la même espèce, plusieurs populations de poissons de rivières (SF) et de poissons de grottes aveugles (CF). Chez les poisons cavernicoles aveugles, les yeux se développent presque normalement pendant l’embryogenèse. Mais 24 heures après la fécondation (hpf), quand l’embryon éclot, le cristallin entre en apoptose, ce qui déclenche la dégénérescence progressive de l’œil entier. Mon projet de thèse visait à comprendre le mécanisme conduisant à l’apoptose du cristallin, jusqu’alors totalement incompris, en partant du postulat selon lequel le défaut devait avoir lieu pendant les stades précoces du développement du cristallin. Le cristallin se développe à partir d’une placode, un épaississement de l’ectoderme au stade neurula. Toutes les placodes, qui donnent naissance à des organes des sens de la tête, sont issues du champ panplacodal, situé à la bordure de la plaque neurale antérieure à 10 hpf. Nous avons comparé la régionalisation de ce champ chez les deux morphes, par hybridations in situ de gènes marqueurs des différentes placodes. Chez le CF, le territoire présomptif du cristallin est réduit à 10 hpf, et le cristallin est plus petit à tous les stades étudiés. D’autre part, la placode olfactive est étendue, et donne naissance à un épithélium olfactif plus large chez le CF. Les modifications de taille de ces deux placodes pourraient être le résultat évolutif d’un « trade-off » entre ces deux composantes sensorielles. La régionalisation modifiée du champ panplacodal chez le CF est due au moins partiellement à des différences spatiales et temporelles d’expression des molécules de signalisation Shh, Fgf, et peut-être Bmp4.Nous avons pensé que la petite taille du cristallin pouvait être la cause directe de son entrée en apoptose, par un défaut d’effet de communauté. Nous avons réalisé une ablation laser partielle des cellules précurseurs du cristallin à 12-14 hpf chez l’embryon SF, mimant ainsi la taille du cristallin CF. L’apoptose dans le petit cristallin des larves SF à 60 hpf n’a pas été augmentée, ce qui montre que la petite taille n’est pas suffisante pour induire l’apoptose.L’apoptose du cristallin pourrait aussi provenir de défauts de morphogenèse ou d’un problème de lignage cellulaire. Nous utilisons donc l’imagerie biphoton in vivo sur des embryons SF et CF, de 10 à 24 hpf, préalablement injectés au stade une cellule avec des ARNm de H2B-mCherry et Ras-GFP pour marquer les noyaux et les membranes. Les premiers résultats sur les poissons de surface montrent que nous pouvons suivre à rebours les cellules du cristallin de la fin du film jusqu’au champ panplacodal, et étudier la morphogenèse et les divisions.La différenciation du cristallin est également affectée chez le CF : au moins 5 cristallines, qui sont des protéines structurales du cristallin, ne sont pas exprimées correctement chez le CF, d’après des hybridations in situ et des qPCR. Cependant, le rôle fonctionnel de deux de ces modifications d’expression a été testé, et individuellement, elles n’expliquent pas le phénotype apoptotique. Nous émettons l’hypothèse qu’une combinaison de défauts d’expression de plusieurs cristallines serait à l’origine de l’apoptose du cristallin CF. Enfin, et plus largement, les forces évolutives qui ont conduit à la perte de l’œil chez Astyanax mexicanus ne sont pas encore comprises. Par une étude d’évolution moléculaire à l’échelle du transcriptome nous avons identifié des mutations fixées entre SF et CF, et avons pu mettre en évidence une accumulation de mutations dans des « gènes d’yeux » chez les CF. Cela suggère un relâchement de la pression de sélection sur ces gènes, peut-être devenus inutiles dans l’obscurité. De même, les séquences des cristallines de CF paraissent accumuler des mutations fixées à un taux élevé vu leur bas niveau de polymorphisme. / The fish Astyanax mexicanus presents, within the same species, several populations of river-dwelling surface fish (SF) and blind cave-living fish (CF). In blind cavefish, the eyes first develop almost normally during embryogenesis. But 24 hours after fertilization (hpf), when the embryo hatches, the lens enters apoptosis, which triggers the progressive degeneration of the entire eye. My thesis project aimed at understanding the mechanism leading to lens apoptosis, which was so far unknown. We reasoned that the defect(s) should take place during the early stages of lens development. The lens develops from a placode, a thickening of the ectoderm at the neurula stage. All placodes, giving rise to sense organs of the head, originate from the “panplacodal” field, located at the border of the anterior neural plate at 10 hpf. We compared the patterning of the panplacodal field in the 2 morphs, using in situ hybridizations for placodal marker genes. In CF, the lens placode territory is reduced at 10 hpf, and the lens is smaller at all stages examined. Conversely, the olfactory placode is enlarged, and gives rise to a bigger olfactory epithelium in CF. The modifications in size of these two placodes could result evolutionarily from a trade-off between these two sensory components. Developmentally, the modified patterning of the panplacodal field in CF is at least partly due to the spatial and temporal differences in the expression of Shh and Fgf (and perhaps Bmp4) signaling molecules.We hypothesized that the small size of the lens could be the direct cause of its apoptosis, through a lack of community effect. We performed partial laser ablation of lens precursor cells at 12-14hpf in surface fish (thereby mimicking the CF lens size). Apoptosis in the resulting small lens of SF larvae at 60hpf was not enhanced, showing that small size is not sufficient to induce apoptosis. Lens apoptosis could also result from morphogenesis defects or from a problem in cell lineage. We are performing two-photon live imaging, from 10 to 24 hpf, of SF and CF embryos previously injected at the one cell stage with H2B-mCherry and Ras-GFP mRNAs to label nuclei and membranes. First results on surface fish show that we can back-track lens cells to the panplacodal field, and follow morphogenesis and divisions. Lens differentiation is also affected in cavefish: at least 5 crystallins, which are lens structural components, are not expressed correctly in CF, based on in situ hybridization and qPCR data. However the functional role of two of these expression modifications / losses was tested and, individually, they don’t seem to explain the apoptosis phenotype. We propose that a combination of several crystallins expression defects would explain CF lens apoptosis.Finally, and more globally, evolutionary forces that led to eye loss in Astyanax mexicanus are not yet understood. Through a transcriptome-wide molecular evolution approach, we identified fixed mutations in transcripts between SF and CF, and we could show an accumulation of mutations in “eye genes” in CF. This suggests that the selection is relaxed on these genes, that have maybe become useless in the dark. Similarly, CF crystallin sequences seem to accumulate fixed mutations at a high rate, considering their low polymorphism level.

Les matériaux artificiellement structurés que sont les cristaux photoniques sont couramment utilisés pour leurs propriétés dispersives. Leur constante diélectrique varie périodiquement à l'échelle de la longueur d'onde selon deux ou trois directions avec un contraste d'indice suffisamment élevé. La relation de dispersion ω = ω(k) qui résulte de cette variation périodique a la forme d'une structure de bande à l'intérieur de laquelle il existe des bandes interdites photoniques où la propagation du champ électromagnétique est interdite. En dehors de ces bandes, i.e. dans les bandes photoniques, se trouvent les propriétés de dispersion des cristaux photoniques.Le but de ce travail de thèse est de concevoir, de fabriquer et de caractériser des dispositifs à cristal photonique à gradient. Ces dispositifs ont été conçus de façon à s'appliquer dans les domaines allant des micro-ondes à l'optique. Nous avons conçu des dispositifs à partir de cristaux photoniques dont les propriétés dispersives les rendent analogues à des milieux linéaires, homogènes et isotropes (LHI). À la maille élémentaire de ces cristaux photoniques LHI, nous avons appliqué un gradient pour réaliser des lentilles à gradient 1D. Des résultats importants concernant la conception, la fabrication et la caractérisation expérimentale d'une lentille plate à gradient d'indice fonctionnant dans la bande X des micro-ondes sont reportés. Celle lentille focalise une onde plane incidente et collimate l'onde émise par une source ponctuelle situés dans son plan focal. Si cette lentille constitue en soi un démonstrateur et valide la démarche mise en œuvre pour la concevoir, ses applications potentielles concernent particulièrement les antennes. Nous réalisons également plusieurs lentilles à gradient 2D dont des lentilles de Lüneburg et Half Maxwell Fisheye; leurs applications aux antennes sont importantes. Nous nous intéressons aussi à la réalisation de lentilles optiques à gradient d'indice dites « SELFOC® ». Dans le but de confirmer les propriétés dispersives remarquables qui ont été mises en évidence, nous avons poursuivi dans ce sens en revisitant une expérience classique qui met en évidence l'existence des ondes évanescentes : celle du « double prisme à angle droit ». Nous mettons également en évidence le phénomène de « réflexion totale frustrée » ainsi que le décalage, découvert par Goos et Hänchen, que subit l'onde réfléchie sur le dioptre. Ce sont ces deux points — réflexion totale frustrée et effet Goos-Hänchen — que nous vérifions dans le cas de cristaux photoniques LHI. / Artificially structured materials that are photonic crystals are commonly used for their dispersive properties. Their dielectric constant varies periodically across the wavelength in two or three directions with a sufficiently high index contrast. The resulting dispersion relation ω = ω(k) of the periodic variation has the form of a band structure within which there are photonic bandgaps in which the propagation of the electromagnetic field is prohibited. Outside of these bands, i.e. in the photonic band, there are the dispersion properties of the photonic crystals.The aim of this thesis is to design, fabricate and characterize graded photonic crystal devices. These devices were designed to be applied in areas ranging from microwaves to optics. We designed devices from photonic crystals with dispersive properties which make them similar to linear, homogeneous and isotropic media (LHI). In the unit cell of the LHI photonic crystal, we applied a gradient to achieve 1D graded lenses. Important results regarding the design, manufacturing and experimental characterization of a flat lens GRIN operating in X-band microwaves are deferred. This lens focuses an incident plane wave and collimates the wave emitted by a point source located in its focal plane. If this lens is itself a demonstrator and validates the approach implemented for the design, its potential applications particularly concern antennas. We also carry several 2D graded lenses including Lüneburg and Half Maxwell Fisheye lenses; their applications to the antennas are important. We are also interested in making optical graded index lenses called "SELFOC®".In order to confirm the remarkable dispersive properties that have been identified, we continued in that direction by revisiting a classic experiment that highlights the existence of evanescent waves: the "double right angle prism". We also highlight the phenomenon of "frustrated total internal reflection" and the shift discovered by Goos and Hänchen suffered by the reflected wave on the interface. It's these two points – frustrated total internal reflection and Goos-Hänchen effect - that we check in the case of LHI photonic crystals.

Introdução: A partir de relatos casos de opacifiação da lente intraocular acrílica hidrofílica Ioflex (Mediphacos, Belo Horizonte, Brasil), muitas vezes utilizada em mutirões de catarata para pessoas carentes, avaliamos a frequência das complicações oculares relacionadas com o implante desta lente quatro anos após a cirurgia, bem como seus efeitos a longo prazo na eficiência da cirurgia de catarata. Métodos: Foi selecionada amostra aleatória com 102 indivíduos submetidos à cirurgia de catarata com implante da lente intraocular Ioflex, em campanha comunitária no interior de Pernambuco, Brasil, quatro anos após a cirurgia. Um total de 31 pacientes não foi localizado e treze (18,3%) não compareceram para o exame. Assim, 58 pacientes (87 olhos) foram examinados. Para o cálculo da frequência da opacificação da cápsula posterior e para a avaliação da eficiência da cirurgia da catarata, foi selecionado apenas um olho de cada paciente. Os sujeitos foram submetidos a exame oftalmológico completo. Pacientes com redução da acuidade visual decorrentes das complicações pós-operatórias foram encaminhados para tratamento. O custo desses eventuais tratamentos foi estimado para análise da eficiência da cirurgia de catarata com a lente Ioflex. Para avaliação dos índices de opacificação da cápsula posterior as variáveis foram expressas por média e desvio padrão. Foi usado o teste t de Student\'s para análise de diferenças entre amostras independentes e pareadas. Resultados: Seis olhos (7%) apresentaram opacificação da lente intraocular. Nenhuma doença ou fator ambiental foi relacionado à opacificação da lente. A opacificação da cápsula posterior foi detectada em 39 (67%) olhos, sendo 24 (41,3%) olhos submetidos ao tratamento com laser para recuperação da acuidade visual. O custo total do tratamento das complicações pós-operatórias representou 6,3% do orçamento cirúrgico inicial. O custo adicional foi de $9,03 dólares por paciente para manter a boa acuidade visual. Conclusão: A lente intraocular acrílica hidrofílica Ioflex apresentou elevada frequência de complicações pós-operatórias tardias relacionadas à própria lente, reduzindo, a longo prazo, a eficácia e eficiência da cirurgia de catarata / Introduction: Based on reports of Ioflex hydrophilic acrylic intraocular lens opacification (Mediphacos, Belo Horizonte, Brazil), used in cataract surgeries for underserved communities, we evaluated the ocular complications related to the intraocular lens implant, four years after surgery, as well as its effects on the visual efficiency of long-term cataract surgery. Methods: A random sample of 102 patients that underwent cataract surgery and Ioflex intraocular lens implantation, in a community campaign in the rural area from Pernambuco, Brazil, four years after surgery. A total of 31 patients could not be located and 13 (18.3%) were not present for examination. Thus, 58 patients (87 eyes) were available for examination. In order to calculate the frequency of posterior capsule opacification and to evaluate the efficiency of cataract surgery, only one eye was selected from each patient. The subjects underwent a complete ophthalmological examination. Patients with reduced visual acuity due to postoperative complications were referred for treatment. The costs of these treatments were estimated for the analysis of the efficiency of the cataract surgery with the Ioflex lens in the long term period. For the evaluation of posterior capsule opacification rates the variables were expressed by means and standard deviation. The Student\'s t test was used for analysis the differences between independent and paired samples. Results: Six eyes (7.0%) had intraocular lens opacification. No disease or environmental factor could be related to intraocular lens opacification. Posterior capsule opacification was detected in 39 (67%) eyes, and 24 (41.3%) eyes were submitted to laser treatment for visual acuity recovered. The total cost of postoperative complications treatment represented 6.3% from the initial budget of the whole patients group evaluated. The additional costs to maintain a good visual outcome was USD $9.03 per patient with Ioflex lens implantation. Conclusion: The Ioflex hydrophilic acrylic intraocular lens had high rates of postoperative complications related to the intraocular lens itself, decreasing effectiveness efficiency in long-term cataract surgery

Neste trabalho, mostramos que fluxos de Reeb dinamicamente convexos em um espaço lenticular $L(p, 1)$, $p>1$, admite uma órbita periódica de Reeb especial $P$ que é o binding de uma decomposição em livro aberto racional, com páginas tipo-disco tal que cada página é uma seção global. O índice de Conley-Zehnder da $p$-ésima iterada de $P$ é $3$. Como corolário, o fluxo de Reeb possui duas ou infinitas órbitas periódicas. Este resultado aplica-se ao Hamiltoniano de Hénon-Heiles, cujo fluxo restrito a energia baixa possui $Z_3$-simetria e define um fluxo de Reeb em $L(3, 1)$. Devido a $Z_4$-simetria aplicamos nosso resultado ao problema lunar de Hill regularizado. Na segunda parte deste trabalho investigamos a existência de uma folheação $3-2^3$ em níveis de energia no sistema Hamiltoniano de Hénon-Heiles, para energia logo acima da crítica. Provamos que certa região de interesse é uma hipersuperfície de contato. Provamos também que o fluxo de Reeb possui uma órbita periódica $Z_3$ simétrica, cujo índice de Conley-Zehnder é $3$ e possui número de auto-enlaçamento $-1$. / We show that a dynamically convex Reeb flow on a lens space $L(p, 1)$, $p>1$ admits a special closed Reeb orbit $P$ which is the binding of a rational open book decomposition with disk-like pages so that each page is a global surface of section. The Conley-Zehnder index of the $p$-th iterate of $P$ is $3$. As a corollary, the Reeb flow has $2$ or infinitely many closed Reeb orbits. This result applies to the Hénon-Heiles Hamiltonian whose flow restricted to low energy levels has $Z_3$-symmetry and descends to $L(3,1)$. Due to a $Z_4$-symmetry we also apply our results to Hill\'s lunar problem. In the second part of this work we investigate the existence of a $3-2^3$ foliation on energy levels of the Hénon-Heiles Hamiltonian, for energies above the critical one. We show that some region is of contact-type and the Reeb flow has a $Z_3$-symmetric periodic orbit, whose Conley-Zehnder is $3$ and has self-linking number $-1$.

O formato, posição e alinhamento corretos dos componentes oculares são definidos através de uma série de mudanças morfológicas complexas durante sua embriogênese. A retina se origina de células da vesícula óptica do tubo neural enquanto as células precursoras do cristalino surgem do ectoderma que reveste o ápice da vesícula óptica. Este ectoderma é delimitado molecularmente como pre-placoidal, sofre uma série de eventos morfogênicos durante o seu desenvolvimento inicial para formar o placóide do cristalino e, posteriormente, a vesícula do cristalino. O placóide do cristalino surge a partir do espessamento do ectoderma pre-placoidal. Posteriormente, o placóide invagina para formar a vesícula do cristalino. Durante a invaginação do placóide, as células da ectoderme que circundam o placóide (células periplacodais) também se movem para fechar a abertura do cristalino que invaginou e reconstruir o ectoderma da superfície. Aqui, nos concentramos em dois processos do desenvolvimento do olho. Nós investigamos o papel da matriz extracelular no espessamento do placóide do cristalino e a dinâmica da emissão de protrusões de membrana pelas células periplacodais durante a invaginação do placóide. A matriz extracelular desempenha papel relevante na morfogênese placodal. Por exemplo, a Fibronectina na matriz extracelular entre a vesícula óptica e o ectoderma pré-placoidal é necessária para a formação de placóide do cristalino. No entanto, a dinâmica da arquitetura de Fibronectina durante a formação do placóide é desconhecida. Assim, nosso primeiro objetivo aqui foi investigar a arquitetura da Fibronectina e da Laminina, dois importantes componentes da matriz extracelular, durante o espessamento do placóide do cristalino através de imagens confocais em 3D. Nossos dados sugerem que um padrão de Fibronectina e Laminina difuso e pontuado é restrito à região do placóide. Este padrão é mantido durante o espessamento e invaginação do placóide. Encontramos um padrão similar de Laminina na região do placóide de embrião de camundongo, sugerindo a conservação desta arquitetura neste contexto. Também demonstramos que a inibição mediada por Noggin (inibidor da sinalização de BMP), que interrompe o desenvolvimento do olho, afeta a organização da Fibronectina e da Laminina, sugerindo que a sinalização de BMP regula a organização da matriz extracelular durante o desenvolvimento do placóide do cristalino. Nosso segundo objetivo foi analisar a emissão de 5 protrusões celulares finas por células periplacodais correlacionando com o movimento de invaginação. Aqui, nós investigamos a dinâmica e composição do citoesqueleto dessas protrusões para entender sua função durante o desenvolvimento do olho. Observamos uma grande quantidade de protrusões em células periplacodais de embriões de galinha e de camundongo. Nossos resultados de quantificação com protrusões de embriões de galinha não mostraram correlação entre comprimento e direção de emissão ou com meia-vida. Nós também analisamos a diversidade na composição do citoesqueleto, uma vez que encontramos protrusões positivas para Cofilina e Tubulina. Estes dados sugerem uma população heterogênea de protrusões finas de membrana periplacodais. Finalmente, também identificamos essas protrusões em outras superfícies ectodérmicas de embriões de galinha e de camundongo, sugerindo que elas desempenham um papel no desenvolvimento de ectoderme superficial. / The correct shape, position and alignment of optic components are defined through a series of complex morphological changes during the embryogenesis of the eye. The retina originates from the neural tube´s optic vesicle while the lens precursor cells arise from the ectoderm that overlie the apex of the optic vesicle. This ectoderm is molecularly delimited as preplacodal and undergoes a series of morphogenic events during its initial development to form the lens placode and subsequently the lens vesicle. The lens placode arises from the thickening of the pre-placodal ectoderm. Subsequently, the placode invaginates to form the vesicle of the lens. During the invagination of the placode, the ectodermal cells that surround the placode (peri-placodal cells) also move to close the opening of the lens that invaginated and reconstruct the surface ectoderm. Here we focus on two processes of eye development. We investigated the role of the extracellular matrix in the lens placode thickening and the dynamics of the emission of membrane protrusions by the peri-placodal cells during the lens placode invagination. The extracellular matrix plays a relevant role in placodal morphogenesis. For example, Fibronectin in the extracellular matrix between the optic vesicle and the preplacodal ectoderm is required for the formation of lens placode. However, the dynamics of the Fibronectin architecture during placode formation is unknown. Thus, our first aim here was to investigate the architecture of Fibronectin and Laminin, two important components of the extracellular matrix, during thickening of lens placode through confocal 3D images. Our data suggest that both Fibronectin and Laminin present a diffuse and punctate pattern restricted to the placodal region. This pattern is maintained during thickening and invagination of the placode. We found a similar pattern of Laminin in the placodal region of the mouse embryo, suggesting the conservation of this architecture in this context. We also demonstrate that Noggin-mediated inhibition of BMP signalling, which disrupts the development of the eye, affects the organization of Fibronectin and Laminin, suggesting that BMP signalling regulates the organization of the extracellular matrix during the lens placode development. Our second objective was to analyse the emission of thin cellular protrusions by peri-placodal cells correlating with the lens invagination movement. Here we investigated the dynamics and 3 composition of the cytoskeleton of these protrusions to understand their function during the development of the eye. We observed a large number of protrusions in peri-satellite cells of chicken and mouse embryos. Our quantification results with chicken embryo protrusions showed no correlation between length and direction of emission or with half-life. We also analysed the diversity in the composition of the cytoskeleton, and we found protrusions positive for Cofilin and Tubulin. These data suggest a heterogeneous population of periplacodal protrusions. Finally, we have also identified these protrusions on other ectodermal surfaces of chicken and mouse embryos, suggesting that they play a role in the development of surface ectoderm.

The ongoing merger of the digital and optical components of the modern microscope is creating opportunities for new measurement techniques, along with new challenges for optical modelling. This thesis investigates several such opportunities and challenges which are particularly relevant to biomedical imaging. Fourier optics is used throughout the thesis as the underlying conceptual model, with a particular emphasis on three--dimensional Fourier optics. A new challenge for optical modelling provided by digital microscopy is the relaxation of traditional symmetry constraints on optical design. An extension of optical transfer function theory to deal with arbitrary lens pupil functions is presented in this thesis. This is used to chart the 3D vectorial structure of the spatial frequency spectrum of the intensity in the focal region of a high aperture lens when illuminated by linearly polarised beam. Wavefront coding has been used successfully in paraxial imaging systems to extend the depth of field. This is achieved by controlling the pupil phase with a cubic phase mask, and thereby balancing optical behaviour with digital processing. In this thesis I present a high aperture vectorial model for focusing with a cubic phase mask, and compare it with results calculated using the paraxial approximation. The effect of a refractive index change is also explored. High aperture measurements of the point spread function are reported, along with experimental confirmation of high aperture extended depth of field imaging of a biological specimen. Differential interference contrast is a popular method for imaging phase changes in otherwise transparent biological specimens. In this thesis I report on a new isotropic algorithm for retrieving the phase from differential interference contrast images of the phase gradient, using phase shifting, two directions of shear, and non--iterative Fourier phase integration incorporating a modified spiral phase transform. This method does not assume that the specimen has a constant amplitude. A simulation is presented which demonstrates good agreement between the retrieved phase and the phase of the simulated object, with excellent immunity to imaging noise.