Keratoconus (MIM#148300), a common bilateral, progressive corneal thinning disorder, is the leading indication for corneal transplantation in the developed world. Keratoconus usually arises in the teenage years and presents a significant health burden in work-age adults. Despite the visual and social impact of keratoconus, our lack of understanding of the molecular pathology of keratoconus is a major obstacle to the development of new therapeutic approaches. This study represents the first reported application of massively parallel sequencing of mRNA (RNA-Seq) to perform whole genome transcriptome analysis of keratoconic keratocytes. Genes-enrichment analysis identified several pathway maps that are disrupted in keratoconic keratocytes and associated with the pathogenesis of keratoconus. Microarray gene expression was used to validate differentially expressed genes identified by RNA-Seq in a global manner, whereas quantitative reverse transcriptase polymerase chain reaction was performed on selected candidate genes. Wnt signalling, TGF-beta signalling. ECM-matrix interactions, oxidative stress and inflammatory- related genes were specifically identified in keratoconic keratocytes and implicated in the pathogenesis of keratoconus. Analysis of individual target genes identified altered expression of both known and novel keratoconus-related genes, in particular, SFRP1, BMP4, CBS, POSTN, C0L11Al, COL4Al, SOD1, IL6, and SP3. Functional analyses and expression profiling of keratoconic keratocytes harbouring a novel heterozygous missense mutation (c.l920G>T; p.Gln640His) in the zinc finger E-box binding gene 1 (ZEB1) was also performed. The mutant ZEB1 protein was stable and localised to the nucleus resulting in an enhanced transcriptional repressor of known ZEB1 targets, involved in epithelialmesenchymal transition and collagen synthesis. This ZEB1 mutation results in a gain-In-function with enhanced transcriptional repression of a number of gene targets associated with keratoconus, corneal thickness and Fuchs endothelial corneal dystrophy. This study has identified a number of molecular targets for keratoconus and provides a significant insight into the gene pathways involved in keratoconus pathogenesis. Further functional studies can build on this evidence to interrogate disease pathogenesis, identify novel genes and develop molecular therapies for keratoconus.

Keratoconus is a bilateral, progressive corneal thinning disorder that is the leading indication for corneal transplantation in the developed world. Keratoconus usually arises in the teenage years and presents a significant health burden in work-age adults. The minimum incidence is 1 in 2000 but it is much more common in some ethnic groups. There is strong evidence for a heritable component in the development of keratoconus. Most studies describe autosomal dominant inheritance, with incomplete penetrance or variable expressivity. The genetic basis of KC provides an opportunity to apply molecular genetic and mapping approaches to elucidate the mechanisms responsible for the pathogenesis of keratoconus. Two main approaches have been applied in this project to investigate the genetic determinants of keratoconus: candidate gene sequencing and genome wide association study using single nucleotide polymorphisms (SNPs) followed by amplicon next generation sequencing of identified candidate genes. As a result of this project mutations in four genes CBS, ZNF469, ZES1 and MIR184 have been implicated in the development of keratoconus. In this study, mutations in ZNF469 have been identified in 11 .8% of keratoconus patients making ZNF469 the most significant genetic factor responsible for keratoconus to date. The identified genes provide further insight into the underlying biochemical processes and signalling pathways involved in keratoconus disease pathogenesis and highlight three main biological processes involved in disease development corneal wound healing (MIR184 and HGF), collagen regulation (ZNF469 and ZEB1) and oxidative stress defence (CBS). No specific treatment exists for keratoconus except to replace the corneal tissue by corneal transplantation when the visual acuity can no longer be corrected by contact lenses. Although photochemical treatments such as collagen cross-linking may prevent progression in selected individuals, they are unlikely to reverse the effects of established disease. Functional studies are required to establish how the identified genes and mutations contribute to the disease development in order to implement diagnostic tests, offer more accurate prognosis and develop novel therapies.

Limbal stem cell deficiency (LSCD) is one of the most prominent causes of cornea related blindness in the world with an incidence of approximately 240 new cases per year in the UK alone. Symptoms of LSCD include reduced vision, pain, watering, redness, photophobia and blepharospasm due to chronic inflammation and breakdown of the corneal epithelium. LSCD mainly results in loss of barrier between the corneal and conjunctival epithelium with overgrowth of conjunctival epithelium over the cornea resulting in corneal vascularisation and scarring. Therefore, LSCD not only causes debilitating blindness depending on the severity of the condition but also leads to long term morbidity. Despite the availability of advanced treatment options for LSCD such as limbal tissue grafting and ex-vivo corneal epithelium transplantation, basic research questions such as the identification of limbal stem cell markers (LSC) for effective isolation of a pure population of LSCs from the corneal epithelium remains unanswered. Anatomical studies on the ocular surface have identified a new structure at the limbus termed Limbal Epithelial Crypt (LEC). It is a solid cord of cells arising from the undersurface of palisades of Vogt and extends into the subepithelial stroma of the limbus. Immunohistochemical and electron microscopic studies have demonstrated the presence of SCs in this region. The primary aim of this research is to confirm the hypothesis that LEC is a corneal SC niche (SCN) and to identify any SC related genes of interest in the LEe. The transcriptional profiling of the ocular surface (OS) epithelial regions was performed with the in-house manufactured spotted oligonucleotide and the Gene 1.0 ST arrays platform. Comparison of the results of these two microarray studies facilitated validation of the results and also contributed to the gene database of the as epithelial regions. Methods Frozen tissue blocks of corneoscleral buttons were cryosectioned to obtain serial tissue sections. Good quality tissue sections comprising of as epithelial regions were transferred onto PALM slides for laser microdissection. Extracted RNA samples were amplified & hybridised to the in-house manufactured 30,000k spotted human oligonucleotide microarray chips and Gene LOST arrays. Primary analysis of raw data of both the microarrays studies was performed to filter and normalise the microarray data for removal of the noise and to smoothen the raw data . Secondary analysis of the data involved statistical methods such as ANOVA, principal component analysis unpaired t-test and Significance Analysis of Microarrays (SAM). A differentially expressed gene list for each as epithelial region was generated with these statistical analysis methods. The gene lists in both the microarray studies were further analysed at tertiary level for pathway analysis with Ingenuity Pathway Analysis and geneontology with Database for Annotation, Visualisation, and Integrated Discovery (DAVID) software respectively. The genes of interest in the microarray studies were validated with quantitative gene expression analysis (qPCR) and immunohistochemistry.

Keratoconus is a bilateral non-inflammatory corneal ectatic disorder characterised by corneal thinning which leads to distortion and progressive impairmient of vision. There are still many unanswered questions relating to its aetiology, clinical course and the factors affecting its progression. Increased visibility of the corneal nerves appears to be an early sign of the disease and there are reports of reduced corneal sensitivity in keratoconic patients. Early detection of keratoconus is of increasing importance, especially in refractive surgery to avoid the possibility of inducing iatrogenic keratectasia. Corneal topographers that can detect subtle anomalies in corneal curvature are being developed and evaluated. The main aim ot this work was to explore the relationship between corneal nerve structure and function in keratoconus using confocai microscopy and aesthesiometry.

- / -

- / -

Limbal stem cell deficiency (LSCD) can be treated successfully using ex vivo limbal epithelial stem cells (LESC) derived from cadaveric donor tissue. However, shortages in such tissues and graft rejection, resulting from inflammation, are persistent issues. The purpose of this study was to optimize current culturing techniques used for LESC transplant tissue, considering expansion and cryopreservation issues surrounding the establishment of a stem cell bank. In addition, a novel anti-inflammatory biomimetic peptide was investigated to address issues surrounding amnion and steroid use in LESC transplantation, inflammation and transplant rejection. Cell cultures derived from Optisol and organ culture stored tissues were examined for optimum growth, characterized by an ability to grow to 70 % to 80 % confluence while maintaining epithelial cell morphology and the presence of positive and negative LESC markers (K3, K19, p63 and PAX-6) as identified by immunocytochemical staining and QRT-PCR. Furthermore, the effect of culture expansion and cryopreservation on stem cell characteristics was examined. A short chain IL-l receptor antagonist peptide was synthesized and characterized using mass spectroscopy (MS), high performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS). Anti-inflammatory properties were investigated using ELISA detection of IL-8, IL-6 and IL- l ~ in keratocytes and LESC following stimulation with either lipopolysaccharide or recombinant human IL- l~. Feasible delivery of cells and peptide on a contact lens was investigated to assess the possibility of an "all in one" graft. Results showed that organ culture stored tissues can provide 100 % successful cell cultures using current techniques in terms of reaching confluence and maintaining LESC morphology and phenotype. Sub-culturing and cryopreservation of cultures however did not produce confluent cell sheets, as required for clinical application. The anti-inflammatory peptide was shown to effectively suppress production of key inflammatory cytokines in LESC and keratocytes by acting as an IL- l receptor antagonist and interrupting the IL- l inflammatory pathway. Binding of the peptide to the contact lens was shown to be possible. Such a scaffold also supported expansion of LESC. However, the 2.7 nmol of peptide bound to the lens did not significantly lower cytokine production. Findings suggest that it is possible to culture adequate numbers of LESC for the initiation of a stem cell bank using current techniques. However, modifications to culturing methods are needed to ensure successful sub-culturing and cryopreservation. The peptide has been shown to be effective in reducing inflammatory cytokine production, providing a possible alternative to steroids. An a11-in-one graft could provide a key development in treating LSCD. However further work is required to optimize the peptide concentration to allow effective inflammation control.

Ο σκοπός της εργασίας αυτής είναι ο προσδιορισμός της ακρίβειας του πάχους του flap που δημιουργείται από τον μικροκερατόμο Moria M2 και του πάχους του κερατοειδή μετεγχειρητικά με την χρήση του σαρωτή υπερήχων υψηλής συχνότητας (η μεγαλύτερης ακρίβειας μέθοδος) και του Orbscan (η πιο δημοφιλής μέθοδος μέτρησης). / The purpose of this study is to evaluate the precision of the flap thickness of Moria M2 Microkeratome by using Artemis Very High Frequency Ultrasound Scanning and the evaluation of the postoperative corneal thickness with Orbscan and Artemis

- / -

Εισαγωγή: η οφθαλμική νεοαγγείωση αποτελεί την πρωταρχική αιτία τύφλωσης για μεγάλο εύρος ασθενειών του οφθαλμού. Ο ακριβής υποκείμενος παθογενετικός μηχανισμός της οφθαλμικής νεοαγγείωσης δεν είναι ακόμη απόλυτα κατανοητός αλλά θεωρείται, ότι η διαταραχή της ισορροπίας μεταξύ αγγειογενετικών και αντί-αγγειογενετικών παραγόντων ευθύνεται, κυρίως για την επαγωγή αγγειογένεσης. Η παρστατίνη είναι ένα πεπτίδιο 41 αμινοξέων που ελευθερώνεται μετά την πρωτεολυτική ενεργοποίηση του PAR1 υποδοχέα και περιέχει μια έντονα υδρόφοβη και μια έντονα υδρόφιλη περιοχή (αλληλουχία αμινοξέων). Πρόσφατα, αποδείχθηκε ότι η παρστατίνη είναι ένας ισχυρός αναστολέας της αγγειογένεσης. Ο σκοπός αυτής της εργασίας ήταν η ανάπτυξη ενός μοντέλου νεοαγγείωσης κερατοειδούς, για την χρήση του στην εκτίμηση της επίδρασης της παρστατίνης στην φλεγμονή και τη νεοαγγείωση του κερατοειδούς και στην διερεύνηση του λειτουργικού ρόλου της δομής της στην νεοαγγείωση. Μέθοδος: για την επαγωγή της νεοαγγείωσης του κερατοειδούς σε Sprague–Dawley ποντίκια, έγινε έγκαυμα, με στειλεούς καλυμμένους 75% με διάλυμα νιτρικού αργύρου και 25% νιτρικού καλίου (κ.β.), στο κέντρο του κερατοειδούς για 4 δευτερόλεπτα. Η παρστατίνη και τα πεπτίδια με την υδρόφοβη και την υδρόφιλη περιοχή της παρστατίνης χορηγήθηκαν υπό τον επιπεφυκότα, αμέσως μετά το χημικό έγκαυμα. Η νεοαγγείωση του κερατοειδούς εκτιμήθηκε την 7η ημέρα μετά το έγκαυμα με τη χρήση ενός ημιαυτόματου προγράμματος που αναπτύχθηκε σε MATLAB 7.5. Η πυκνότητα των νεοαγγείων και η φλεγμονώδης διήθηση επίσης εκτιμήθηκε την 7η ημέρα μετά το έγκαυμα, με ιστοπαθολογική εξέταση λεπτών τομών κερατοειδούς. Αποτελέσματα: η παρστατίνη χορηγούμενη υπό τον επιπεφυκότα ανέστειλε την νεοαγγείωση του κερατοειδούς και 200 μg ήταν η πιο αποτελεσματική δόση (αναστολή 59%). Επιπλέον η παρστατίνη ανέστειλε σημαντικά την φλεγμονώδη διήθηση του κερατοειδούς. Το πεπτίδιο που περιείχε την υδρόφοβη αλληλουχία του μορίου κατέστειλε την νεοαγγείωση με μέγιστη αναστολή 47% στην δόση των 200 μg, ενώ το πεπτίδιο που περιείχε την υδρόφιλη αλληλουχία έδειξε μέγιστη αναστολή 73% επίσης στην δόση των 200 μg. Τα αναγραμματισμένα πεπτίδια δεν έδειξαν κανένα σημαντικό αποτέλεσμα. Συμπεράσματα: η παρστατίνη και ιδιαίτερα το υδρόφιλο τμήμα της, είναι ισχυρός αναστολέας της νεοαγγείωσης του κερατοειδούς και μπορεί να αποτελέσει ισχυρή θεραπευτική στρατηγική για την αντιμετώπιση της οφθαλμικής νεοαγγείωσης / Introduction: Ocular neovascularization is the primary cause of blindness in a wide range of ocular diseases. The exact mechanism underlying the pathogenesis of ocular neovascularization is not yet well understood, but it is believed that the imbalance between angiogenic stimulating factors and angiogenic inhibitors is the major contributor to angiogenesis. Parstatin is a 41-mer peptide formed by proteolytic cleavage on activation of the PAR1 receptor and contains a hydrophobic and a hydrophilic domain. Recently it was demonstrated that parstatin is a potent inhibitor of angiogenesis. The purpose of the study was to develop a model of corneal neovascularization which will be used to investigate the effect of parstatin on corneal inflammation and neovascularization and to evaluate the functional role of its structure on neovascularization. Methods: Corneal neovascularization on Sprague–Dawley rats was induced with cauterization by an applicator stick, coated with 75% silver nitrate and 25% potassium nitrate, to the center of the cornea for 4 seconds. Parstatin and peptides containing the hydrophobic and hydrophilic domains of parstatin were administrated subconjunctival after the chemical burn. Corneal neovascularization was assessed the 7th day after cauterization using a semiautomatic custom software, developed in MATLAB 7.5. Corneal microvessel density and inflammatory cell infiltration was assessed also the 7th day after the burn by histopathology examination of corneal sections. Results: Subconjunctival parstatin inhibited corneal neovascularization, with 200 μg the most effective dose (59% inhibition). In addition parstatin significantly inhibited corneal inflammatory infiltration. The peptide contained the hydrophobic domains of parstatin suppressed corneal neovascularization with maximum inhibition of 47% at 200 μg, whereas the peptide contained the hydrophilic domain exhibit maximum inhibition of 73% at 200 μg. Scrambled peptides were without any significant effect. Conclusion: Parstatin and especially its hydrophilic domain, is a potent antiangiogenic agent of corneal neovascularization and may have clinical potential in the treatment of angiogenesis-related ocular disorders.