Orientador: Maria Rosa Bet de Moraes Silva / Banca: Maria de Lourdes Veronesi Rodrigues / Banca: Eduardo Melani Rocha / Resumo: Avaliar e comparar córneas de coelhos com implante de anel de FERRARA® com e sem revestimento de condroitin sulfato quanto à presença de hiperemia, secreção conjuntival, edema de córnea, vascularização corneana e extrusão do anel intraestromal e espessuras central e temporal da córnea. Pelo estudo histopatológico avaliar e comparar entre os dois grupos o número de camadas de células epiteliais sobre o anel, presença de alterações histopatológicas no estroma corneano, membrana de Descemet e endotélio. Foram estudados 30 coelhos albinos da raça Norfolk divididos em 2 grupos experimentais cada qual formado por 15 coelhos em cujas córneas do olho direito foram implantados o anel de FERRARA® clássico (G1) e anel de FERRARA® revestido por condroitin sulfato (G2). Os olhos esquerdos formaram o grupo controle. Em todos os grupos foram avaliados parâmetros clínicos: paquimetria central e temporal, hiperemia conjuntival, secreção, edema de córnea, vascularização e extrusão do anel. Foram também avaliados parâmetros morfológicos: número de camadas do epitélio, o estroma corneano, a membrana de Descemet e o endotélio. Os parâmetros clínicos foram avaliados em 3 M1 (1º dia pós-operatório), M2 (30º dia pós-operatório e M3 (60º dia pósoperatório). Os parâmetros morfológicos foram analisados no M3. As alterações encontradas foram descritas e comparadas através dos testes t de Student, Mann- Whitney, Wilcoxon para amostras independentes; Teste exato de Fisher, Teste Qui-quadrado, Teste de McNemar para amostras dependentes, conforme o parâmetro. Quanto à espessura central da córnea ocorreu um aumento estatísticamente significante entre os momentos antes e após a implantação nos dois grupos e aumento na espessura temporal apenas no G1. Os demais parâmetros clínicos não...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: To evaluate and compare rabbit corneas with chondroitin-sulfatecoated and uncoated Ferrara ring implants as regards the presence of hyperemia, conjunctival secretion, corneal edema, corneal vascularization and extrusion of the intrastromal ring as well as central and temporal corneal thickness. To evaluate and compare the number of epithelial cell layers on the ring and the presence of histopathological alterations in the corneal stroma, Descemet's membrane and endothelium between the two groups by means of a histopathological investigation. Thirty albinal Norfolk rabbits were studied in 2 experimental groups. Each group comprised 15 rabbits whose right-eye cornea received a classic Ferrara ring implant in group 1 (G1) and a Ferrara ring coated by chondroitin sulfate in Group 2 (G2). Their left eyes formed the control group. The following clinical parameters were evaluated in all groups: central and temporal pachymetry, conjunctival hyperemia, secretion, corneal edema, vascularization and ring extrusion. Morphological parameters were also evaluated as follows: number of epithelial layers, corneal stroma, Descemet's membrane and endothelium. The clinical parameters were evaluated at 3 moments: M1 (first post-operative day), M2 (30th post-operative day) and M3 (60th post-operative day). The morphological parameters were analyzed at M3. The alterations found were described and compared by Student's t, Mann- Whitney's and Wilcoxon's tests for independent samples and by Fisher's exact, Chi-Square and McNemar's tests for dependent samples, according to the different parameters. As regards central corneal thickness, a statistically significant increase was found between the moments prior to and after implantation in the two groups. Temporal thickness increase was observed only in G1.The other clinical parameters showed no significant ...(Complete abstract, click electronic access below) / Mestre

The purpose of this thesis was to investigate the effect of corneal swelling on the accuracy of Goldmann tonometry estimates of intraocular pressure (IOP). In the first experiment, central corneal thickness (CCT, ultrasonic pachymetry), IOP (Goldmann tonometry) and corneal curvature (keratometry) was measured in one eye of 25 subjects every two hours for 24 hours, except for 8 hours overnight (no measurements taken), and for the first two hours after awakening (measurement frequency 20 minutes). CCT (+20.1??10.9 pm) and IOP (+3.1??2.4 mmHg) peaked on eye opening, and then decreased at a similar rate (r=0.967, p<0.001) for the next two hours. Corneal swelling may have influenced the accuracy of Goldmann IOP measurements during this time. In the second and third studies, the CCT, IOP and corneal curvature were measured in both eyes of two groups of 25 subjects before and after the induction of corneal swelling, resulting from two hours of monocular closed eye contact lens wear. The increase in IOP was correlated to the increase in CCT at a rate of 0.33 to 0.48 mmHg per 10 pm, which signified an overestimation error in Goldmann IOP measurement. However, the change in IOP could not be accounted for solely by the change in CCT. In the fourth study, CCT, IOP and corneal curvature were used in conjunction with the Orssengo-Pye algorithm to determine the range of Young's modulus in the normal population, which was 0.29??0.06 MPa. Physiological variations in Young's modulus had a similar effect on Goldmann tonometry to CCT. In the fifth study, the data collected for studies 2 and 3 was used to calculate the Young's modulus changes associated with corneal swelling, again with the assistance of the Orssengo-Pye algorithm. No systematic change in Young's modulus was recorded after contact lens wear, but the model suggested that corneal biomechanical changes were responsible for the remainder of the change in IOP. All experimental results were combined to develop a model to calculate the diurnal variation of Goldmann IOP errors. The likely error in IOP due to overnight corneal swelling was 0.6 to 1.4 mmHg, which may explain as much as 45% (1.4 mmHg) of the 3.1 mmHg diurnal variation of IOP. In summary, small amounts of corneal swelling were shown to have a clinically significant impact on the accuracy of Goldmann tonometry. This may interfere with the measurement of the diurnal variation of IOP, particularly if measurements are taken prior to the resolution of overnight corneal swelling.

The Herpes simplex virus (HSV) is a virus that commonly infects the skin, and mucous membrane of the mouth, genitalia, and the eye. HSV-1 is the strain that is most commonly associated with corneal infections, and it is the most frequent cause of corneal blindness in North America [1]. Currently no cure is available, and many limitations are characterized by the currently available synthetic antiviral drugs, which suggest the need for other potential drug alternatives and delivery strategies. Anti-microbial peptides are naturally occurring peptides that are potent killers of a broad range of micro-organisms, including bacteria, fungi, and viruses [2]. AMPs are known to be a key component of the innate immune response at the human ocular surface. The human cathelicidin-derived AMP, LL-37, expressed in human corneal epithelial cells provides a wide range of protection against viral pathogens such as HSV-1 [3]. My thesis research addressed the design and recombinant production of hybrid AMP sequences containing LL-37 with the potential ability to form chemical or physical associations with a Collagen scaffold material, such as those used in current artificial cornea constructs to address the need for alternative anti-viral drugs. Three fusion proteins were tested, and compared for feasible design anti-microbial peptide expression and purification in E. coli. It was illustrated that the thioredoxin and SUMO fusion systems are good candidates for successful recombinant production of active designed peptides. The point-mutated LL-37 sequence was successfully expressed and purified using the thioredoxin fusion system. It was demonstrated that this modified LL-37 was effective against HSV-1 infection. The SUMO system was used to express the bio-functional LL-37 containing a collagen-binding sequence. Further work is required to address issues regarding recombinant AMP production, such as increasing enzymatic cleavage efficacy, and minimizing proteolytic degradation or modification.

The Herpes simplex virus (HSV) is a virus that commonly infects the skin, and mucous membrane of the mouth, genitalia, and the eye. HSV-1 is the strain that is most commonly associated with corneal infections, and it is the most frequent cause of corneal blindness in North America [1]. Currently no cure is available, and many limitations are characterized by the currently available synthetic antiviral drugs, which suggest the need for other potential drug alternatives and delivery strategies. Anti-microbial peptides are naturally occurring peptides that are potent killers of a broad range of micro-organisms, including bacteria, fungi, and viruses [2]. AMPs are known to be a key component of the innate immune response at the human ocular surface. The human cathelicidin-derived AMP, LL-37, expressed in human corneal epithelial cells provides a wide range of protection against viral pathogens such as HSV-1 [3]. My thesis research addressed the design and recombinant production of hybrid AMP sequences containing LL-37 with the potential ability to form chemical or physical associations with a Collagen scaffold material, such as those used in current artificial cornea constructs to address the need for alternative anti-viral drugs. Three fusion proteins were tested, and compared for feasible design anti-microbial peptide expression and purification in E. coli. It was illustrated that the thioredoxin and SUMO fusion systems are good candidates for successful recombinant production of active designed peptides. The point-mutated LL-37 sequence was successfully expressed and purified using the thioredoxin fusion system. It was demonstrated that this modified LL-37 was effective against HSV-1 infection. The SUMO system was used to express the bio-functional LL-37 containing a collagen-binding sequence. Further work is required to address issues regarding recombinant AMP production, such as increasing enzymatic cleavage efficacy, and minimizing proteolytic degradation or modification.

Thesis (M. Med. Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (46-49).

The proper organization and differentiation of the anterior segment is pivotal for normal eye development. Neural crest-derived POM cells are key contributors to correct anterior segment formation, differentiating to form the monolayered corneal endothelium. Mice with homozygous null mutations in the forkhead transcription factor gene, Foxc1, fail to develop a proper corneal endothelium stabilized by adherens junctions, with the endothelium adhering to the lens, preventing anterior chamber separation. The aim of this study was to evaluate the interaction between Foxc1 and the adherens junction protein, N-cadherin, as well as an associated gene, Msx1, during key stages in corneal endothelium development. Foxc1 was over-expressed in E12.5 and E13.5 POM cells and qPCR was carried out to determine the effect of Foxc1 on N-cadherin and Msx1 gene expression. Data showed over-expression of Foxc1 in wildtype E12.5 and E13.5 POM cells to cause significant fluctuations in N-cadherin and Msx1 expression (p < 0.05). POM cells were then transfected with a Foxc1 knock-down plasmid or the Foxc1 overexpression plasmid to evaluate the effect of Foxc1 on N-cadherin protein expression by Western blot analysis, however, these results were inconsistent with the gene expression analyses with no significant differences in N-cadherin expression detected. N-cadherin protein expression and localization was then further assessed by means of immunocytochemistry (ICC) and confocal microscopy in monolayer and hanging-drop POM cell cultures. Both qPCR and confocal microscopy data showed consistency, indicating increased amounts of N-cadherin in E12.5 cells relative to E13.5 cells, with membrane-bound N-cadherin showing a clear lattice-work pattern in hanging drop culture. Foxc1 over-expression/knock-down studies on E12.5 and E13.5 POM cells together suggest that N-cadherin is transcriptionally regulated by Foxc1 and that Foxc1 has a threshold level at which it is able to exert control over N-cadherin in POM cells. Foxc1 expression is therefore essential in establishing N-cadherin adhesion junctions in the corneal endothelium. Preliminary data also suggests that Msx1 may directly interact with Foxc1 in POM cells, however, further studies must be undertaken to verify and establish the effects of Foxc1/N-cadherin/ Msx1 interaction in the development of a cohesive, integrated corneal endothelium and functional anterior segment. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

Corneal endothelial development is an intricate process driven by finely tuned gene expression. Its formation is necessary for the continued normal development of the anterior segment of the eye. The presence of an inductive lens able to secrete factors such as TGFβ2 as well as the expression of Foxc1 and Pitx2 is essential to corneal endothelial development, as in the absence of any of these; the corneal endothelium fails to form. Corneal endothelial development begins as peri-ocular mesenchyme (POM) cells migrate into the space between the lens and surface ectoderm at E11.5. From E12.5, these cells begin to transition from a mesenchymal to an epithelial/endothelial (MET) phenotype, differentiating into a monolayered endothelium by E15 characterised by inter-cellular junctions. To study the initial process of development, immortalised POM cell lines from E12.5 and E13.5 embryos were used. Expression of the key genes, the transcription factors, Foxc1 and Pitx2 and two genes involved in EMT/MET, Slug and Tsc22, were analysed at these stages to establish the developmental norm. The effect of the lens on these expression levels was then determined. To establish whether TGFβ2 is the lens secreted signal responsible for gene expression changes, cells were subjected to TGFβ2 treatment. In all these experiments, the role of Foxc1 in regulating gene expression was determined by Foxc1 overexpression and knockdown. The effect of the lens on cellular proliferation and on the expression and cellular arrangement of N-cadherin, a junction protein was also determined. The results showed that, at E12.5, the lens downregulates Foxc1 and Pitx2 expression, is a potent inducer of Tsc22 expression and is required for maintaining Slug levels. TGFβ2 was shown to play a role in Foxc1 and Pitx2 downregulation. Analysis suggests that Tsc22 expression is responsive to lens signals, but that TGFβ2 is not the signal responsible for its downregulation between E12.5 and E13.5. The lens has no effect on Slug expression in the presence of Foxc1, but when Foxc1 is silenced, Slug is induced. Thus, Foxc1 plays a crucial regulatory role in Slug expression. At E13.5, as differentiation is initiated, Foxc1 expression remains responsive to the lens and to TGFβ2. Pitx2 expression is still induced by the lens but, at this stage, TGFβ2 does not play a part in Pitx2 regulation suggesting involvement of other unknown lens secreted signals. Other lens secreted signal/s were also shown to downregulate Tsc22 and Slug at this stage. The lens was implicated in MET as it was shown to have an effect on N-cadherin localisation in 3-dimensional culture. E12.5 Spheroids exposed to E6 lenses formed a distinct lattice arrangement of N-cadherin compared to the uniform distribution in control cells. Although the 13.5 control cell aggregates also showed a lattice framework, it was more pronounced in the lens treated cells. The transcriptional role of Foxc1 was determined by overexpression and knockdown experiments where Foxc1 overexpression and knockdown upregulated Tsc22 and downregulated Pitx2 and Slug at E12.5. At E13.5, Pitx2 was downregulated and Slug was upregulated in response to aberrant expression of Foxc1. This was illustrative of the sensitivity these genes have to Foxc1 expression during development. It is known that the presence of a functioning lens and Foxc1 are essential for proper development of the corneal endothelium, which in turn is necessary for normal eye development. The understanding of the precise molecular mechanisms required for corneal endothelial development and the processes requisite for cell proliferation and differentiation has important consequences for providing further insight into the pathophysiology of anterior segment dysgenesis and glaucoma. Previous studies suggest that stem-cell like qualities are conferred in cells undergoing EMT. Such an investigation may lead to application in regenerative medicine such as the bioengineering of corneal tissue. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

No description available.

The Herpes simplex virus (HSV) is a virus that commonly infects the skin, and mucous membrane of the mouth, genitalia, and the eye. HSV-1 is the strain that is most commonly associated with corneal infections, and it is the most frequent cause of corneal blindness in North America [1]. Currently no cure is available, and many limitations are characterized by the currently available synthetic antiviral drugs, which suggest the need for other potential drug alternatives and delivery strategies. Anti-microbial peptides are naturally occurring peptides that are potent killers of a broad range of micro-organisms, including bacteria, fungi, and viruses [2]. AMPs are known to be a key component of the innate immune response at the human ocular surface. The human cathelicidin-derived AMP, LL-37, expressed in human corneal epithelial cells provides a wide range of protection against viral pathogens such as HSV-1 [3]. My thesis research addressed the design and recombinant production of hybrid AMP sequences containing LL-37 with the potential ability to form chemical or physical associations with a Collagen scaffold material, such as those used in current artificial cornea constructs to address the need for alternative anti-viral drugs. Three fusion proteins were tested, and compared for feasible design anti-microbial peptide expression and purification in E. coli. It was illustrated that the thioredoxin and SUMO fusion systems are good candidates for successful recombinant production of active designed peptides. The point-mutated LL-37 sequence was successfully expressed and purified using the thioredoxin fusion system. It was demonstrated that this modified LL-37 was effective against HSV-1 infection. The SUMO system was used to express the bio-functional LL-37 containing a collagen-binding sequence. Further work is required to address issues regarding recombinant AMP production, such as increasing enzymatic cleavage efficacy, and minimizing proteolytic degradation or modification.

The purpose of this thesis was to investigate the effect of corneal swelling on the accuracy of Goldmann tonometry estimates of intraocular pressure (IOP). In the first experiment, central corneal thickness (CCT, ultrasonic pachymetry), IOP (Goldmann tonometry) and corneal curvature (keratometry) was measured in one eye of 25 subjects every two hours for 24 hours, except for 8 hours overnight (no measurements taken), and for the first two hours after awakening (measurement frequency 20 minutes). CCT (+20.1??10.9 pm) and IOP (+3.1??2.4 mmHg) peaked on eye opening, and then decreased at a similar rate (r=0.967, p<0.001) for the next two hours. Corneal swelling may have influenced the accuracy of Goldmann IOP measurements during this time. In the second and third studies, the CCT, IOP and corneal curvature were measured in both eyes of two groups of 25 subjects before and after the induction of corneal swelling, resulting from two hours of monocular closed eye contact lens wear. The increase in IOP was correlated to the increase in CCT at a rate of 0.33 to 0.48 mmHg per 10 pm, which signified an overestimation error in Goldmann IOP measurement. However, the change in IOP could not be accounted for solely by the change in CCT. In the fourth study, CCT, IOP and corneal curvature were used in conjunction with the Orssengo-Pye algorithm to determine the range of Young's modulus in the normal population, which was 0.29??0.06 MPa. Physiological variations in Young's modulus had a similar effect on Goldmann tonometry to CCT. In the fifth study, the data collected for studies 2 and 3 was used to calculate the Young's modulus changes associated with corneal swelling, again with the assistance of the Orssengo-Pye algorithm. No systematic change in Young's modulus was recorded after contact lens wear, but the model suggested that corneal biomechanical changes were responsible for the remainder of the change in IOP. All experimental results were combined to develop a model to calculate the diurnal variation of Goldmann IOP errors. The likely error in IOP due to overnight corneal swelling was 0.6 to 1.4 mmHg, which may explain as much as 45% (1.4 mmHg) of the 3.1 mmHg diurnal variation of IOP. In summary, small amounts of corneal swelling were shown to have a clinically significant impact on the accuracy of Goldmann tonometry. This may interfere with the measurement of the diurnal variation of IOP, particularly if measurements are taken prior to the resolution of overnight corneal swelling.