Corneal diseases are one of the major causes of visual impairment and blindness worldwide. Used for diagnoses, a laser confocal microscope provides a sequence of images, at incremental depths, of the various corneal layers and structures. From these, ophthalmologists can extract clinical information on the state of health of a patient’s cornea. However, many factors impede ophthalmologists in forming diagnoses starting with the large number and variable quality of the individual images (blurring, non-uniform illumination within images, variable illumination between images and noise), and there are also difficulties posed for automatic processing caused by eye movements in both lateral and axial directions during the scanning process. Aiding ophthalmologists working with long sequences of corneal image requires the development of new algorithms which enhance, correctly order and register the corneal images within a sequence. The novel algorithms devised for this purpose and presented in this thesis are divided into four main categories. The first is enhancement to reduce the problems within individual images. The second is automatic image classification to identify which part of the cornea each image belongs to, when they may not be in the correct sequence. The third is automatic reordering of the images to place the images in the right sequence. The fourth is automatic registration of the images with each other. A flexible application called CORNEASYS has been developed and implemented using MATLAB and the C language to provide and run all the algorithms and methods presented in this thesis. CORNEASYS offers users a collection of all the proposed approaches and algorithms in this thesis in one platform package. CORNEASYS also provides a facility to help the research team and Ophthalmologists, who are in discussions to determine future system requirements which meet clinicians’ needs.

Nous proposons de construire un atlas numérique 3D contenant les caractéristiques moyennes et les variabilités de la morphologie d’un organe. Nos travaux seront appliqués particulièrement à la construction d'un atlas numérique 3D de la totalité de la cornée humaine incluant la surface antérieure et postérieure à partir des cartes topographiques fournies par le topographe Orbscan II. Nous procédons tout d'abord par normalisation de toute une population de cornées. Dans cette étape, nous nous sommes basés sur l'algorithme de recalage ICP (iterative closest point) pour aligner simultanément les surfaces antérieures et postérieures d'une population de cornée vers les surfaces antérieure et postérieure d'une cornée de référence. En effet, nous avons élaboré une variante de l'algorithme ICP adapté aux images (cartes) de cornées qui tient compte de changement d'échelle pendant le recalage et qui se base sur la recherche par voisinage via la distance euclidienne pour établir la correspondance entre les points. Après, nous avons procédé pour la construction de l'atlas cornéen par le calcul des moyennes des élévations de surfaces antérieures et postérieures recalées et leurs écarts-types associés. Une population de 100 cornées saines a été utilisée pour construire l'atlas cornéen normal. Pour visualiser l’atlas, on a eu recours à des cartes topographiques couleurs similairement à ce qu’offrent déjà les systèmes topographiques actuels. Enfin, des observations ont été réalisées sur l'atlas cornéen reflétant sa précision et permettant de développer une meilleure connaissance de l’anatomie cornéenne. / We propose to build a 3D digital atlas which contains the average characteristics and variability of the morphology of an organ. In particular our work consists in the construction of a 3D digital atlas of the entire human cornea including anterior and posterior surfaces. The atlas was built using topographies provided by the Orbscan II system. First, we normalized the given population of corneas using a variant of the ICP (iterative closest point) algorithm for shape registration to fit simultaneously the anterior and posterior surfaces with the anterior and posterior surfaces of a reference cornea. Indeed, we developed a specific algorithm for corneas topographies that considers scaling during registration and which is based on neighborhood search via the Euclidean distance to find the correspondence between points. After that, we built the corneal atlas by averaging elevations of anterior and posterior surfaces and by calculating their associated standard deviations. A population of 100 healthy corneas was used to construct the normal corneal atlas. To illustrate the atlas, we used topographic color maps like those already offered by existing topographic systems. Finally, observations were made on the corneal atlas that reflects its precision and allows to develop a better understanding of corneal anatomy.

L’utilisation de lentilles cornéennes peut servir à améliorer le profil d’administration d’un principe actif dans les yeux. Avec une efficacité d’administration de 5% par l’utilisation de gouttes, on comprend rapidement que l’administration oculaire doit être améliorée. Cette faible administration a donné naissance à plusieurs tentatives visant à fabriquer des lentilles cornéennes médicamentées. Cependant, à cause de multiples raisons, aucune de ces tentatives n’a actuellement été mise sur le marché. Nous proposons dans cette étude, une possible amélioration des systèmes établis par le développement d’une lentille cornéenne à base de 2-(hydroxyéthyle)méthacrylate (HEMA), dans laquelle des microgels, à base de poly N-isopropylacrylamide (pNIPAM) thermosensible encapsulant un principe actif, seront incorporé. Nous avons donc débuté par développer une méthode analytique sensible par HPCL-MS/MS capable de quantifier plusieurs molécules à la fois. La méthode résultante a été validée selon les différents critères de la FDA et l’ICH en démontrant des limites de quantifications et de détections suffisamment basses, autant dans des fluides simulés que dans les tissus d’yeux de lapins. La méthode a été validée pour sept médicaments ophtalmiques : Pilocarpine, lidocaïne, proparacaïne, atropine, acétonide de triamcinolone, timolol et prednisolone. Nous avons ensuite fait la synthèse des microgels chargés négativement à base de NIPAM et d’acide méthacrylique (MAA). Nous avons encapsulé une molécule modèle dans des particules ayant une taille entre 200 et 600 nm dépendant de la composition ainsi qu’un potentiel zêta variant en fonction de la température. L’encapsulation de la rhodamine 6G (R6G) dans les microgels a été possible jusqu’à un chargement (DL%) de 38%. L’utilisation des isothermes de Langmuir a permis de montrer que l’encapsulation était principalement le résultat d’interactions électrostatiques entre les MAA et la R6G. Des cinétiques de libérations ont été effectuées à partir d’hydrogels d’acrylamide chargés en microgels encapsulant la R6G. Il a été trouvé que la libération des hydrogels chargés en microgels s’effectuait majoritairement selon l’affinité au microgel et sur une période d’environ 4-24 heures. La libération à partir de ces systèmes a été comparée à des formules d’hydrogels contenant des liposomes ou des nanogels de chitosan. Ces trois derniers (liposomes, microgels et nanogels) ont présenté des résultats prometteurs pour différentes applications avec différents profils de libérations. Enfin, nous avons transposé le modèle développé avec les gels d’acrylamide pour fabriquer des lentilles de contact de 260 à 340 µm d’épaisseur à base de pHEMA contenant les microgels avec une molécule encapsulée devant être administrée dans les yeux. Nous avons modifié la composition de l’hydrogel en incorporant un polymère linéaire, la polyvinylpyrrolidone (PVP). L’obtention d’hydrogels partiellement interpénétrés améliore la rétention d’eau dans les lentilles cornéennes. L’encapsulation dans les microgels chargés négativement a donné de meilleurs rendements avec la lidocaïne et cette dernière a été libérée de la lentille de pHEMA en totalité en approximativement 2 heures qu’elle soit ou non encapsulée dans des microgels. Ainsi dans cette étude pilote, l’impact des microgels n’a pas pu être déterminé et, de ce fait, nécessitera des études approfondies sur la structure et les propriétés de la lentille qui a été développée. En utilisant des modèles de libération plus représentatifs de la physiologie de l’œil, nous pourrions conclure avec plus de certitude concernant l’efficacité d’un tel système d’administration et s’il est possible de l’optimiser. / The development of corneal contact lenses initially aimed to correct vision troubles but more recently targets to improve administration of ophthalmic drugs. Eye drops from ophthalmic solutions has a poor administration efficiency of 5% or less and is currently the most used method to deliver drugs to the eye. Such administration technique needs to be improved and contact lenses could be the solution according to many opticians. However, no marketed therapeutic contact lenses has been marketed up to date. In this project we have developed a model of a contact lens made of 2-(hydroxyethyl)methacrylate embedding microgels of poly N-isopropylacrylamide (pNIPAM), encapsulating a model drug. We first developed an analytical method capable to quantify simultaneously seven ophthalmic drugs: Pilocarpine, lidocaine, proparacaine, atropine, triamcinolone acetonide, timolol and prednisolone. This method was developed on a HPLC-MS/MS device and was validated according to FDA and ICH criteria. Using this method, we achieved very low detection and quantitation limits with high precision and accuracy in both simulated lachrymal fluids and in rabbit ocular tissues. Each seven drugs was validated using this method. We proceeded with the synthesis of negatively charged microgels of NIPAM using methacrylic acid (MAA) as comonomer. Resulting size were ranging between 200-600 nm and zeta potential was found to increase (absolute value) with temperature. The microgels were used to encapsulate a model molecule, rhodamine 6G (R6G), in different medium and were loaded in the microgel up to 38% (drug loading, DL%). Using Langmuir isotherms to measure affinity and adsorption of R6G, it was found well correlated to MAA content in microgels, suggesting electrostatic interaction was the main parameter for drug loading. Release kinetics was performed using a model hydrogel of acrylamide embedding the R6G-loaded microgels. The measured release was found to follow an affinity-based mechanism for over 4-24 hours. The release kinetics were then compared to a formulation of liposomes and nanogels of chitosan embedded in hydrogel. All formulations exhibited interesting release profiles making them promising systems for different therapeutic applications. Finally, we changed the acrylamide gels for pHEMA designed to reproduce contact lenses containing drug-loaded microgels. The hydrogel composition, in terms of monomer / cross-linker ratio, was first optimized to fit contact lenses properties of 260-340 µm thick contact lenses. We also made use of semi-interpenetrated polyvinylpirrolidone (PVP) in the pHEMA hydrogel matrix to increase its water content. The highest DL% of negatively charged microgels were obtained using lidocaine and were used for release studies, where the total content of lidocaine was released in approximately 2 hours with and without microgels. In the end, this was a pilot study aiming to evaluate the potential of microgel usability in contact lenses. However, the impact of microgels on release was not fully conclusive. Additional studies should be undertaken to achieve a better comprehension and characterization of the release mechanism such as using more eye relevant physiological models. Such studies would provide further insights on the use of such materials for eye drug delivery and its applicability.

Distúrbios na superfície da córnea e nas glândulas lacrimais acometem com frequência os indivíduos com diabetes mellitus. Atualmente não existe tratamento seguro e eficaz para feridas na córnea e o tratamento da síndrome do olho seco (SOS) é predominantemente sintomático. A administração tópica da insulina (INS) é uma estratégia promissora para tratar esses distúrbios, devido à presença de seus receptores na superfície ocular e na glândula lacrimal, e aos seus efeitos metabólicos e mitogênicos. No entanto, os fármacos aplicados topicamente na forma de solução são rapidamente drenados do olho, resultando em uma baixa biodisponibilidade local. O objetivo deste trabalho foi desenvolver formulações contendo INS e avaliar a sua influência no distúrbio das glândulas lacrimais e na regeneração da córnea em ratos diabéticos. Foram desenvolvidas quatro formulações contendo 1 UI/mL de INS: dispersão contendo insulina (DISP INS), dispersão contendo micropartículas quitosana/INS (DISP MP INS), gel termorreversível in situ com INS (Gel INS) e Gel contendo as micropartículas quitosana/INS (Gel MP INS). Também foram produzidas formulações \"brancas\", sem a veiculação do fármaco: dispersão contendo micropartículas sem insulina (DISP MP s/INS); gel termorreversível in situ sem insulina (Gel s/INS). As MP incorporadas nas formulações foram preparadas por spray drying e apresentaram tamanho de 4,0±0,1 ?m, morfologia adequada e grande quantidade de INS (77±6%). Todas as formulações apresentaram pH e osmolalidade compatíveis com o uso ocular. Durante o estudo in vivo, foi realizado o tratamento diário (15 dias) dos animais diabéticos, com as formulações (50 ?L) em ambos os olhos, exceto nos controles positivo (sem diabetes) e controle negativo (diabético não tratado). Todos os animais tratados com INS aumentaram a produção de fluido lacrimal, sendo que, ao término do tratamento, não foi observada diferença estatística entre o controle positivo e aqueles tratados com DISP INS e com Gel MP INS. A INS foi detectada na glândula lacrimal e no globo ocular dos animais tratados com DISP MP INS, Gel INS e Gel MP INS, sendo que a maior concentração de INS foi encontrada nos ratos tratados com Gel MP INS. Ao término do tratamento houve redução da glicemia dos animais tratados com a INS, o que pode sugerir um tratamento coadjuvante em pacientes diabéticos. Estudos de citologia de impressão mostraram que o Gel INS e Gel MP INS foram capazes de aumentar o número de células do epitélio da córnea e melhorar a relação núcleo/citoplasma em relação ao controle negativo. A histologia do globo ocular mostrou que essas duas formulações também melhoraram a espessura do epitélio da córnea, que foi semelhante a dos controles positivos. Conclui-se que a incorporação da INS nos géis e nas micropartículas desenvolvidas desempenhou um efeito positivo no tratamento da síndrome do olho seco e de lesões na córnea. / Disorders in the corneal surface and the lachrymal glands often affect individuals with diabetes mellitus. Nowadays there is no safe and effective treatment for wounds in the cornea and the treatment of dry eye syndrome (DES) is primarily symptomatic. Topical administration of insulin (INS) is a promising strategy for treating these disorders due to the presence of receptors on the ocular surface and lacrimal gland, besides its metabolic and mitogenic effects. However, the drugs applied topically in the form of a solution are quickly drained from the eye, resulting in a low local bioavailability. The aim of this study was to develop formulations containing INS and evaluate their influence on disorders of lacrimal glands and cornea healing in diabetic rats. Four formulations containing 1 IU/mL of INS were developed: dispersion containing insulin (DISP INS), dispersion containing chitosan microparticles/INS (DISP MP INS), in situ forming gel containing INS (Gel INS) and in situ forming gel containing microparticles chitosan/INS (Gel MP INS). \"White\" formulations were also produced without the drug: dispersion containing microparticles without insulin and in situ forming gel without insulin. MP incorporated in the formulations were prepared by spray drying and showed size of 4.0±0.1 ?m, proper morphology and high INS load (77±6%). All formulations exhibited pH and osmolarity compatible with the ocular use. In vivo studies were performed using diabetic rats that were treated daily (15 days) with instillation of the formulations (50 ?L) in both eyes, except in the positive controls (health rats) and negative control (untreated diabetic rats). All animals treated with INS showed an increase in the production of tear fluid. After treatment, no statistical difference was observed between the positive control and those treated with DISP INS and Gel MP INS. The INS was detected in the lacrimal gland of the animals treated with DISP MP INS, Gel INS and Gel MP INS, with the highest concentration of INS found in mice treated with Gel MP INS. After treatment there was a reduction in blood glucose of the animals treated with formulations containing INS, which suggests that developed formulations may be used as an adjuvant treatment in diabetic patients. Impression cytology studies showed that the Gel INS and Gel MP INS were able to increase the number of the corneal epithelium cells and improve nucleus/cytoplasm ratio compared to the negative control. The histology of the eyeball showed that these two formulations have improved the thickness of the corneal epithelium, which was similar to the positive controls. In conclusion, incorporation of INS in the gels and MP developed induced a positive effect in the treatment of dry eye syndrome and corneal wound.

INTRODUÇÃO: O ceratocone é uma doença não inflamatória, sem etiologia definida, caracterizada pelo afilamento estromal e protrusão da córnea. Geralmente esta doença torna-se clinicamente evidente na adolescência. Apesar de possuir sinais clínicos bem conhecidos, a detecção do ceratocone em estádios iniciais pode representar uma tarefa de difícil execução, mesmo quando a videoceratografia computadorizada ou outros métodos são utilizados para avaliar a córnea. Anteriormente, diagnosticar o ceratocone apenas após a identificação de sinais clínicos inequívocos era uma conduta aceitável. Com o advento da cirurgia refrativa porém, a identificação precoce do ceratocone tornou-se um procedimento de vital importância para evitar complicações pós-operatórias. O objetivo principal deste estudo é avaliar o uso de máquinas de vetor de suporte e redes neurais artificiais como métodos auxiliares para identificação de ceratocone e suspeita de ceratocone em exames realizados com o Orbscan II. MÉTODOS: Foram avaliados retrospectivamente dados de 344 pacientes. Os exames selecionados foram classificados em 6 categorias: normal (n=172), astigmatismo (n=89), ceratocone (n=46), ceratocone forma frustra (n=10), suspeita de ceratocone (n=16) e cirurgia refrativa (n=11). Para cada paciente 10 atributos foram obtidos ou calculados a partir de dados fornecidos pelo Orbscan II. O método do holdout e da validação cruzada foram utilizados para encontrar a melhor configuração, treinar e testar os classificadores. Além da acurácia, sensibilidade e especificidade, curvas ROC foram obtidas para cada classificador, e as áreas sob as curvas ROC foram calculadas. RESULTADOS: Os dois classificadores selecionados alcançaram um bom desempenho, com áreas sob as curvas ROC de 0,99. Não houve diferença estatística entre as suas performances. O desempenho dos classificadores foi superior ao desempenho de todos os atributos individuais do Orbscan II. (p<0,05). CONCLUSÃO: Os resultados alcançados sugerem que xi x máquinas de vetor de suporte e redes neurais artificiais podem representar técnicas úteis para a detecção de ceratocone em exames realizados com o Orbscan II. / PURPOSE: Keratoconus is a bilateral and non-inflammatory condition characterized by progressive thinning, protrusion and scarring of the córnea. The disease usually becomes clinically evident at puberty, and its etiology remains unknown. Although it has well-described clinical signs, early forms of the disease may be undetected, even when computer-assisted videokeratography techniques or other methods are used to evaluate the cornea. Prior to the development of refractive surgery, it was considered sufficient to diagnose clinically evident keratoconus. However, given the spread of refractive surgery, a careful differentiation between normal and early keratoconus cases is essential to avoid postoperative complications. This study evaluated the performance of support vector machine and multilayer perceptron neural network, as auxiliary tools to identify keratoconus from Orbscan II maps. METHODS: A total of 344 maps were retrospectively selected and classified into six categories: normal (n=172), astigmatism (n=89), keratoconus (n=46), forme fruste keratoconus (n=10), keratoconus suspect (n=16), and photorefractive keratectomy (n=11). For each map 10 attributes were obtained or calculated from data provided by the Orbscan II. Holdout method and ten-fold cross-validation was used to train and test the classifiers. Besides accuracy, sensitivity and specificity, ROC curves for each classifier were generated and the areas under the curves were calculated. RESULTS: The two selected classifiers provided a good performance and there were no differences between their performances. The area under the ROC curve of the support vector machine and multi-layer perceptron were significantly larger than those for all individual Orbscan II attributes evaluated (p<0.05). CONCLUSION: Overall, our results suggest that support vector machine and multi-layer perceptron classifiers, trained on Orbscan II data, could represent useful techniques for keratoconus detection

No description available.

O presente trabalho trata-se do desenvolvimento de um programa para de análise de imagens de topografia corneana de sistemas comerciais, para ser implementado no topógrafo corneano para Lâmpada de Fenda, em desenvolvimento no Laboratório de Instrumentação Oftálmica - EESC/USP e no Laboratório de Física Oftálmica - FMRP/USP. O programa foi desenvolvido em C++, utilizando a plataforma Windows, e fornece mapas axiais de topografia corneana. O programa foi testado em esferas de calibração e em olhos humanos, apresentando um fator de correlação de 0,9998 para as medidas em esferas e um erro inerente estimado em 3%. Os mapas de topografias axiais em olhos humanos foram comparados com os mapas gerados por sistemas comerciais e o padrão visual de forma e relevo estão em concordância. / This work is about a software for the analisys of corneal topography images provided by commercial available systems to be implemented in a corneal topographer for slit lamps under evelopment at Laboratório de Instrumentação Oftálmica . EESC/USP e no Laboratório de Física Oftálmica . FRMP/USP. The software was developed in Borland C++ Builder for Windows and provides the corneal topography axial maps. The software has been tested in calibration spheres and in human eyes, presenting a correlation factor of 0,9998 for the measurements performed in the spheres and an inherent error of 3%. The axial topographic maps form the exams performe in human eyes have been compared to the axial maps provided by the commercial available system and the visual pattern as well as the relief are in accordance.

皮膚是人體最大的器官，具有多種功能，其中最重要的功能之一就是作為身體內部和外界環境之間的的保護屏障。完整地修復這一保護屏障是創傷癒合和組織再生領域的一個重要內容。本論文探討了人類臍帶被覆上皮細胞 (cord lining epithelial cells, CLECs)作為一種幹細胞來源，可用于表皮重建的潛能. / 本論文的第二章對CLECs的體外分離和增殖進行了詳細地描述。這一類細胞具有較長的染色體端粒，較高的增殖潛能和傳代能力。同時，它們表達上皮幹細胞和多能性幹細胞的標誌性表面抗原。它們還具有多種分化潛能，包括成脂、成骨和成軟骨。然而當皮下異種移植後，它們並不會形成畸胎瘤。 / 本論文的第三章對CLECs的免疫特性進行了評估。結果顯示CLECs不但具有低免疫原性，還具有免疫調節功能。它們表達典型性的一型主要組織相容性複合體(MHC class I)，即人白細胞ABC抗原(HLA-ABC)，但不表達典型性的二型主要組織相容性複合體(MHC class II)，即人白細胞DR抗原(HLA-DR)。它們同時還表達非典型性的MHC class I, 包括人白細胞G抗原和人白細胞E 抗原(HLA-G和HLA-E), 但不表達共激分子(CD40, CD80和CD86)。此外，體外檢測還發現它們表達適度的促炎/抗炎細胞因子和大量的生長因子. / 本論文的第四章對CLECs在表皮重建應用中的潛能進行了考察。結果顯示無論在體外器官培養還是異種移植動物模型中，CLECs都能形成分層的上皮結構，與用表皮細胞構建的分層上皮結構相類似。而且在CLECs構建的皮膚替代物中證實了有表皮分化標誌性抗原的表達。 / 結論：本論文證明了CLECs具有幹細胞樣特性但無致瘤性，具有低免疫原性和表皮分化的可塑性。研究結果支持CLECs在創傷癒合和皮膚再生領域的臨床應用可行性. / The skin is the largest organ in the body and has multiple functions. One of the most important functions is to serve as a protective barrier between the internal and external environments of the body. Restoration of the integrity of this protective barrier is an essential aspect of wound healing and tissue regeneration. In this thesis, the potential of human umbilical cord lining epithelial cells (CLECs) as a source of stem cells with appropriate differentiation capacity for epidermal reconstitution has been explored. / The isolation and propagation of CLECs from human umbilical cord lining epithelium were described in Chapter II. The cells presented a long telomere length and had high proliferative potential and passaging capability. They were also shown to display both epithelial and pluripotent stem cell markers. They were capable of multipotent differentiation, including adipogenesis, osteogenesis and chondrogenesis. However, they didn’t form teratoma after subcutaneous xenotransplantation until 12 weeks. / The immune properties of CLECs in vitro were assessed in Chapter III. The cells were shown to have low immunogenicity but high immunosuppressive function. They expressed classical major histocompatibility complex (MHC) class I antigens (HLA-ABC), but not MHC class II antigen (HLA-DR). They also expressed non-classical MHC class I antigens (HLA-G and HLA-E), but lacked the expression of the co-stimulatory molecules (CD40, CD80 and CD86). Moreover, they expressed moderate pro/anti-inflammatory cytokines and multiple growth factors both in cell supernatants and cell lysates. / The potential of CLECs for epidermal reconstitution was investigated in Chapter IV. In both organotypic culture and xenotransplantation model, CLECs were capable of generating a stratified epithelial structure, which is similar to that constructed by using keratinocytes. Furthermore, the expression of epidermal differentiation markers was verified in CLEC-constructed skin substitutes. / In conclusion, the stem cell-like properties of CLECs have been demonstrated in the present study. In addition to the lack of tumorigenicity, CLECs also have low immunogenicity and significant plasticity in epidermal differentiation. The findings support the potential clinical application of CLECs in wound healing and skin regeneration. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cai, Yijun. / "October 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 114-129). / Abstract also in Chinese. / Abstrac --- p.i / Table of Contents --- p.v / Abbreviations --- p.vii / List of Figures --- p.viii / List of Tables --- p.x / Chapter Chapter I --- Introduction --- p.1 / Skin --- p.3 / Wound healing --- p.6 / Wound regeneration and repair --- p.6 / Recent history of wound treatment --- p.9 / Skin substitutes --- p.11 / Stem cells for wound treatment --- p.14 / Stem cells overview --- p.15 / Adult stem cells --- p.16 / Fetal stem cells --- p.18 / Amniotic membrane derived stem cells --- p.19 / Umbilical cord stem cells --- p.22 / Hypothesis and Specific aims --- p.24 / Chapter Chapter II --- The Isolation and Characterization of the Stem Cell-like Properties of Human Umbilical Cord Lining Epithelial Cells --- p.28 / Introduction --- p.28 / Materials and methods --- p.30 / Results --- p.47 / Discussion --- p.62 / Conclusion --- p.67 / Chapter Chapter III --- The assessment of the Immune Properties of Human Umbilical Cord Lining Epithelial Cells --- p.69 / Introduction --- p.69 / Materials and methods --- p.72 / Results --- p.75 / Discussion --- p.83 / Conclusion --- p.88 / Chapter Chapter IV --- The Investigation of the Potential of Human Umbilical Cord Lining Epithelial Cells for the Epidermal Reconstitution --- p.89 / Introduction --- p.89 / Materials and methods --- p.91 / Results --- p.94 / Discussion --- p.101 / Conclusion --- p.104 / Chapter Chapter V --- Summary and Future Plan --- p.105 / Summary --- p.105 / Future plan --- p.108 / Acknowledgements --- p.113 / References --- p.114 / Appendix --- p.130

A ceratite fúngica (CF) é uma micose ocular oportunística, que tem como sítio de infecção a córnea. Não é uma enfermidade de risco de vida, mas de extremo comprometimento visual e dificuldade terapêutica e, em casos graves, podendo levar à cegueira total ou mesmo a perda do globo ocular. O trabalho objetivou identificar os agentes etiológicos causadores da CF, e descrever os critérios terapêuticos empregados. No período de 1998 a 2011 foram estudados 23 casos de CF diagnosticados no Laboratório de Micologia da Irmandade da Santa Casa de Misericórdia de Porto Alegre / RS. Foram analisados os aspectos demográficos, as doenças de base e os fatores associados ao desenvolvimento da doença, assim como os critérios terapêuticos. No presente estudo, a mediana de idade foi 45 anos, com variação entre 15 e 76 anos, com predomínio do gênero masculino. Os fungos filamentosos figuraram em 78% (18/23) dos casos analisados, sendo o gênero Fusarium spp. o agente etiológico de maior frequência. Concluiu-se que uma compreensão epidemiológica local e a identificação dos fatores de risco, agregados ao diagnóstico micológico precoce e eficaz, são fundamentais na prevenção e correta conduta terapêutica da CF no Rio Grande do Sul, sendo que estas práticas evitarão complicações de perda do globo ocular, melhorando o prognóstico oftalmológico do paciente. / The fungal keratitis is an ocular opportunistic mycosis, which has the cornea as site of infection. Isn’t a life-threatening disease, but with extreme visual impairment and therapeutic difficulty, which can lead to total blindness or even loss of the eyeball, in severe cases. The study aimed to identify the etiologic agents causing fungal keratitis, and describe the therapeutic criteria used. In the period 1998 to 2011 were retrospectively studied 23 cases of fungal keratitis diagnosed at the Mycology Laboratory of Irmandade Santa Casa de Misericórdia Hospital of Porto Alegre / RS. We analyzed the demographics, underlying diseases and the factors associated with disease development, as well as therapeutic criteria. In this study, the median age was 45 years old, ranging between 15 to 76 years old, predominantly male. The filamentous fungus corresponded to 78% (18/23) of the cases analyzed, and Fusarium spp. has been the etiologic agent of highest frequency. It was concluded that an understanding of local epidemiological and identification of risk factors, added to the early and effective mycological diagnosis are essential to prevent and correct therapeutic approach for fungal keratitis in Rio Grande do Sul. These practices will prevent complications of loss of the eyeball, improving the ophthalmological prognosis of the patient.

This thesis presents a novel system that estimates the point-of-gaze (where a person is looking at) remotely while allowing for free head movements and minimizing personal calibration requirements. The point-of-gaze is estimated from the pupil and corneal reflections (virtual images of infrared light sources that are formed by reflection on the front corneal surface, which acts as a convex mirror) extracted from eye images captured by video cameras. Based on the laws of geometrical optics, a detailed general mathematical model for point-of-gaze estimation using the pupil and corneal reflections is developed. Using this model, the full range of possible system configurations (from one camera and one light source to multiple cameras and light sources) is analyzed. This analysis shows that two cameras and two light sources is the simplest system configuration that can be used to reconstruct the optic axis of the eye in 3-D space, and therefore measure eye movements, without the need for personal calibration. To estimate the point-of-gaze, a simple single-point personal calibration procedure is needed. The performance of the point-of-gaze estimation depends on the geometrical arrangement of the cameras and light sources and the method used to reconstruct the optic axis of the eye. Using a comprehensive simulation framework developed from the mathematical model, the performance of several gaze estimation methods of varied complexity is investigated for different geometrical system setups in the presence of noise in the extracted eye features, deviation of the corneal shape from the ideal spherical shape and errors in system parameters. The results of this investigation indicate the method(s) and geometrical setup(s) that are optimal for different sets of conditions, thereby providing guidelines for system implementation. Experimental results with adults, obtained with a system that follows those guidelines, exhibit RMS point-of-gaze estimation errors of 0.4-0.6º of visual angle (comparable to the best commercially available systems, which require multiple-point personal calibration procedures). Preliminary results with infants demonstrate the ability of the proposed system to record infants' visual scanning patterns, enabling applications that are very difficult or impossible to carry out with previously existing technologies (e.g., study of infants' visual and oculomotor systems).