Dry Eye is recognised as one of the most common ocular surface disorders presenting to eye specialists today. The relationship between ocular surface bacteria, tear film function and ocular surface inflammation has been incompletely investigated. Within this study a varied approach to investigate the possible role of ocular surface bacteria in the pathogenesis of DE was undertaken. The research detailed in this thesis was divided into four main areas: diagnosis of normal healthy subjects or DE subjects was established; conjunctival bacterial flora of all subjects was investigated; commensal bacterial interaction with ocular surface cells in vitro was examined; finally, levels of IL-l ~ mRNA expression was investigated in normal and DE subjects. A number of objective and subjective measurements were performed to establish a diagnosis of DE or not. Statistical analysis demonstrated a lack of inter-tear function test correlation and between subjective dry eye assessment measurements. Based on these findings a new diagnostic algorithm was devised to classify study subjects as dry eye or non dry eye. A diagnosis was based on the composite results of four clinical tests and of the 91 subjects recruited, 34 were considered to be DE subjects while the remaining 57 were classified as normal. This study assessed conjunctival flora using routine culture and 16S rDNA PCR with DNA sequencing. Several bacterial species were detected in a number of samples through sequencing while remaining undetectable by culture. A wide variation in the quantity and genus of bacteria was detected between individual dry eye and normal subjects as determined by 16S rDNA PCR and DNA sequencing and routine culture. A number of potentially clinically significant bacteria, not normally associated with the normal ocular surface flora, were identified in both normal and dry eye subjects, including Rhodococcus erythropolis and Klebsiella sp. In vitro model systems of conjunctival epithelial and primary goblet cells were exposed to Staphylococcus epidermidis to assess bacterial invasion and cellular proliferation. Internalisation of S. epidermidis isolated from normal and dry eye subjects by conjunctival epithelial cell line and primary conjunctival goblet cells was demonstrated. Overall, a general decrease in conjunctival epithelial and goblet cell.

Introduction: This PhO study investigates finite objective and subjective clinical outcomes of refractive surgery. Methods: A series of studies was conducted to investigate laser retreatment procedures, a comparison between the OPO-Scan and WaveScan aberrometers, a comparison of aberrometry changes following laser in situ keratomileusis (LASIK) and laser-assisted subepithelial keratectomy (LASEK), internal aberration changes following LASIK, the development of a quality of vision (OoV) questionnaire and the refractive outcomes of the SCHWINO Amaris excimer laser. Results: Good visual and refractive outcomes were found with LASEK retreatment and a high incidence (23%) of epithelial ingrowth occurred following flap-lift LASIK retreatments. In the comparison between the OPO-Scan and WaveScan, medium consistency was found for aberrations z;' and z~ with poor consistency for all other aberrations. In the comparison between LASIK and LASEK, both procedures caused increases to spherical aberration with LASIK additionally causing increases to two fourth-order aberrations and LASEK causing increases to two fifth-order aberrations. Investigations into internal aberrations found LASIK increased internal spherical aberration which attenuated the increase in anterior corneal spherical aberration. A OoV questionnaire was successfully developed utilising Rasch analysis demonstrating good precision, reliability, and internal consistency with good construct validity and test-retest reliability. The SCHWINO Amaris was found to be effective for the treatment of a range of refractive errors. Conclusion: Significant clinical finding were found for laser retreatments and primary treatments with two different laser systems. Aberrations were found to be poorly correlated with the OPO-Scan and the WaveScan. An effective OoV questionnaire was developed which may be used in prospective clinical and research studies. •.

Several clinical and laboratory studies suggest that the niche for corneal epithelial stem cells is located in the corneoscleral limbus. Of particular interest are the palisades of Vogt (POV), which are a series of fibrovascular palisade like structures found predominantly along the superior and inferior limbus. The study was designed to use a relatively new and powerful tool, the laser in vivo confocal microscopy (Rostock corneal module confocal microscope) of scanning laser ophthalmoscope, ) HRTII; to evaluate in details normal anatomical features of the limbus and establish diagnostic features of the defective limbus in patients manifesting clinically with limbal stem cell deficiency (chapter two, three and four). The objective was to be able to diagnose early and late cases of limbal stem cell deficiency without the need fgr invasive methods such ., impression cytology or ocular surface biopsy. Th~ features determined IVCM were validated and correlated with features observed in vitro by histological examination of biopsy specimen. {., Over the last 10 years approximately 25 to 30 patients have undergone limbal stem cell transplantation in the department with at least a one year follow up. The other aim of this study was to evaluate retrospectively and prospectively the outcomes of these procedures (chapter five), to assess the quality of life benefit of the procedures on the patients using a modified National Eye Institute Visual Functioning Questionnaire 25 (NEI II I I I 1 VFQ-25) (chapter six) and to examine the donor eyes and donor sites used for harvesting tissue for limbal transplants both clinically and by confocal microscope (chapter seven). Finally, we hypothesized that gravity may influence the differential migration of cells from the superior and inferior limbic regions, and carried out a simple experiment in vitro which supported this hypothesis (chapter eight). III

This thesis aimed to determine whether Sn expression did not occur on cells other than macrophages and how Sn expression influenced T cell responses responsible for autoimmunity using the EAU mouse model of human ocular inflammation, uveitis.  Sn’s effects were studied on the C57BL/6 genetic background using mice of Sn⁺/⁺ and Sn^-/- genotype.  Mice confirmed to have Sn⁺/⁺ and Sn^-/- genotype and phenotype were immunised with IRBP-20 peptide in adjuvant to induce EAU.  The effects of Sn deletion on EAU were analysed by studying subsequent Thl immune responses and their effects on EAU disease progression. Flow cytometry analysis of the draining lymph nodes (dLN) and spleens of naïve mice identified Sn-expressing cells with phenotype consistent with conventional dendritic cells (DCs) of myeloid and lymphoid types.  These cells expressed DEC205⁺CD8αα-and DEC205⁺CD8αα⁺ phenotype, respectively.  CD8αα⁺ cells were considered to be of lymphoid type rather than plasmacytoid because they expressed CD8αα⁺ at levels similar to T cells and high levels of DEC205 which are typical feature of lymphoid DCs.  DEC205⁺CD8αα^- cells were considered to be myeloid-derived because of their expression of macrophage-like phenotype and DEC205.  DEC205⁺CD8αα^- cells maintained high levels of Sn expression when purified.  They could be propagated in vitro using conditions typical for CD14-derived DCs to yield Sn-expressing cells with allogeneic leukocyte stimulatory capacity that was higher than macrophages which was consistent DCs of this type.  The basal levels of Sn on the two DCs subtypes in naive mice were upregulated in response to immunisation.  Sn upregulation strongly correlated with elevated levels of MHC II and B7.2 expression on the CD11c⁺DEC205⁺CD8αα^- and CD11c⁺DEC205⁺CD8αα⁺ cells.  These changes coincided with the highest level of T cell proliferative responses in immunised Sn⁺/⁺ and Sn^-/^- mice that occurred 6 days post-immunisation.  Supporting the existence of Sn-expressing DCs was the presence of a small subset of Sn⁺ cells with DC morphology that co-expressed MHC II and CD11c in EAU inflammatory lesions.  However, these Sn⁺MHC II⁺ cells comprised a very minor proportion of the total MHC II⁺ cells in the diseased eye.  Thus in addition to macrophages, Sn is also expressed on CD11c⁺DEC205⁺ conventional DCs of myeloid (CD11b⁺CD8αα^-) and lymphoid (CD11b^-CD8αα⁺) type in the dLN, the spleen and the eye and its expression increases following immunisation.

By addressing the vascular features that characterise myopia, this thesis aims to provide an understanding of the early structural changes associated with human myopia and the progression to co-morbidity with age. This thesis addresses three main areas of study: 1. Ocular perfusion features and autoregulatory mechanisms in human myopia; 2. Choroidal thickness at the macular area of myopic eyes; 3. Effect of chronic smoking on the ocular haemodynamics and autoregulation. This thesis demonstrated a reduced resting ocular pulse amplitude and retrobulbar blood flow in human myopia, associated with an apparent oversensitivity to the vasodilatory effects of hypercapnia, which may be due to anatomical differences in the volume of the vessel beds. In young smokers, normal resting state vascular characteristics were present; however there also appeared to be increased reactivity to hypercapnia, possibly due to relative chronic hypoxia. The systemic circulation in myopes and smokers over-reacted similarly to hypercapnia suggesting that physiologic differences are not confined to the eye. Age also showed a negative effect on autoregulatory capacity in otherwise normal eyes. Collectively, these findings suggest that myopes and smokers require greater autoregulatory capacity to maintain appropriate oxygenation of retinal tissue, and since the capacity for such regulation reduces with age, these groups are at greater risk of insufficient autoregulation and relative hypoxia with age.

The aim of this research was to determine the effect of a lutein-based nutritional supplemented on measures of visual function in normal and ARMD-affected eyes. Thirty participants were recruited to the ARMD cohort (aged between 55 and 82 years, mean ± SD: 69.2 ± 7.8) and 46 were recruited into the normal cohort (aged between 22 and 73 years, mean ± SD: 50.0 ± 15.9). Outcome measures were distance (DVA) and near (NVA) visual acuity, contrast sensitivity (CS), photostress recovery time measured with the Eger Macular Stressometer (EMS), central visual function assessed with the Macular Mapping test (MMT), and fundus photography. Reliability studies were carried out for the EMS and the MMT. A change of 14 s is required to indicate a clinically significant change in EMS time, and a change of 14 MMT points is required to indicate a clinically significant change in MMT score. Sample sizes were sufficient for the trial to have 80% power to detect a significant clinical effect at the 5% significance level for all outcome measures in the normal cohort, and for CS in the ARMD cohort. The study demonstrated that a nutritional supplement containing 6mg lutein, 750 mg vitamin A, 250 mg vitamin C, 34 mg vitamin E, 10 mg zinc, and 0.5 mg copper had no effect on the outcome measures over nine or 18 months in normal or ARMD affected participants. The finding that nine months of antioxidant supplementation, in this case, has no significant effect on CS in ARMD-affected participants adds to the literature, and contrasts with previous RCTs, the AREDS and the LAST. This project has added to the debate about the use of nutritional supplementation prior to the onset of ARMD.

Αγγειογένεση καλείται ο σχηματισμός νέων αγγείων από προϋπάρχοντα δίκτυα αιμοφόρων αγγείων σε προηγουμένως ανάγγειες δομές. Η αγγειογένεση συμμετέχει σε μια πλειάδα νοσημάτων (π.χ. καρκίνος, ρευματοειδής αρθρίτιδα) συμβάλλοντας καθοριστικά στην παθογένεια αυτών των καταστάσεων και στην ανάπτυξη επιπλοκών. Στον οφθαλμό, η παθολογική αγγειογένεση στις διάφορες στιβάδες του οργάνου αποτελεί την κύρια αιτία μη αναστρέψιμης απώλειας της όρασης στον Δυτικό κόσμο, καθιστώντας επιτακτική την ανάγκη ανάπτυξης νέων αντι-αγγειογενετικών παραγόντων, ικανών να κατανεμηθούν ενδοφθαλμίως και να αναστείλουν αποτελεσματικά τόσο την εν εξελίξει όσο και την εγκατεστημένη αγγειογένεση. Η Παρστατίνη αποτελεί το ελεύθερο πεπτίδιο 41 αμινοξέων που προκύπτει από την πρωτεολυτική διάσπαση του αμινοτελικού άκρου του ανθρώπινου υποδοχέα PAR1 κατά την ενεργοποίησή του από τη θρομβίνη. Προηγούμενες πειραματικές μελέτες έχουν καταδείξει ότι η Παρστατίνη καταστέλλει την αγγειογένεση στον οφθαλμό και ασκεί καρδιοπροστατευτική δράση σε βλάβες εξ ισχαιμίας/επαναιμάτωσης. Επίσης, έχει δειχθεί ότι τα πεπτιδικά υπομόρια της Παρστατίνης, Π1-26 και Π24-41, παρουσιάζουν εξειδικευμένη δράση και επιδρούν εκλεκτικά στην προστασία του μυοκαρδίου και στην αναστολή της οφθαλμικής νεοαγγειογένεσης, αντιστοίχως. Σύμφωνα με αυτά τα πειράματα, οι δράσεις των Π1-26 και Π24-41 είναι σημαντικά ισχυρότερες από αυτές του μητρικού μορίου, Παρστατίνη. Σκοπός της τρέχουσας μελέτης είναι η διερεύνηση της ιστικής κατανομής in vivo της Παρστατίνης και των αναλόγων-πεπτιδίων (Π1-26, Π24-41) αυτής στον οφθαλμό και στον νεφρό επίμυος. Η Παρστατίνη και τα ανάλογα-πεπτίδιά της (Π1-26, Π24-41) σημασμένα με FITC χορηγήθηκαν υπό τον βολβικό επιπεφυκότα ή ενδοφλεβίως, σε υγιείς αρσενικούς επίμυες. Ακολούθως τα πειραματόζωα θανατώθηκαν σε διαφορετικά χρονικά διαστήματα και οι ιστοί (οφθαλμοί, νεφροί, μυοκάρδιο) απομονώθηκαν χειρουργικά. Κατόπιν κατάλληλης επεξεργασίας, ελήφθησαν τομές ιστών νωπού παρασκευάσματος, πάχους 10 μm, οι οποίες παρατηρήθηκαν σε μικροσκόπιο φθορισμού. Κατά την παρατήρηση των οφθαλμικών τομών με FITC-Παρστατίνη και αυτών με FITC-Π1-26, ισχυρό σήμα φθορισμού ανιχνεύτηκε στις θέσεις χορήγησης και στους περιοφθαλμικούς ιστούς. Δεν παρατηρήθηκε σήμα φθορισμού στις εσώτερες στιβάδες του οφθαλμού. Ειδικότερα για την FITC-Παρστατίνη, η κατανομή του σήματος φθορισμού δεν μεταβλήθηκε σε διαφορετικά χρονικά διαστήματα που κυμάνθηκαν από 30 λεπτά έως 19 ώρες. Συγκριτικά με τα παραπάνω αποτελέσματα, οι αντίστοιχες οφθαλμικές τομές με FITC-Π24-41 ανέδειξαν ισχνό σήμα φθορισμού στις θέσεις χορήγησης και στους περιοφθαλμικούς ιστούς, χωρίς να συνοδεύεται από σήμα φθορισμού στις έσω ανατομικές δομές του οργάνου. Στις τομές νεφρού και μυοκαρδίου, ο ισχυρός αυτοφθορισμός δεν επέτρεψε τη μελέτη της ιστικής κατανομής των πεπτιδίων. Συμπερασματικά, καταδεικνύεται ότι η χορήγηση υπό το βολβικό επιπεφυκότα της Παρστατίνης και των πεπτιδικών αναλόγων της, του Π1-26 και του Π24-41 δεν αποτελεί την ενδεδειγμένη οδό χορήγησης για την ενδοφθάλμια κατανομή των χορηγούμενων ουσιών. / Angiogenesis refers to the formation of new blood vessels from pre-existing vasculature in previously avascular structures. Angiogenesis is an important component of numerous diseases (such as cancer, rheumatoid arthritis etc), contributing in the pathogenesis of these conditions. In the western world, pathological ocular angiogenesis constitutes the major cause of irreversible vision loss. Consequently, this fact gives rise to the need for the development of new anti-angiogenic agents, characterized by successful intraocular delivery and effective inhibition of developing angiogenesis and regression of already established vessels. Parstatin is a 41-amino acid peptide, released through proteolytic cleavage of the human PAR1 receptor N-terminal region upon activation by thrombin. Experimental studies have demonstrated that Parstatin suppresses ocular neovascularization and has a potential therapeutic role in the protection of myocardium by ischaemia and reperfusion injury. In addition, it has been shown that the Parstatin fragments, P1-26 and P24-41, have distinct effects in protecting myocardium and inhibiting ocular neovascularization, respectively. In fact, these effects are more potent than the native Parstatin. The aim of the present study was the in vivo evaluation of tissue-distribution of Parstatin and its peptide fragments (P1-26, P24-41) in rat eyes and kidneys. To address this question, Parstatin and its peptide analogues (P1-26, P24-41) were conjugated with FITC. Pathogen-free male rats received subconjunctival or intravenous injections of the FITC-labeled peptides. Following euthanasia of rats at different time points after the injections were performed, the tissues (eyes, kidneys, hearts) were surgically excised from the respective animals. After treatment, the fresh-frozen tissues were sectioned sagittally into 10 μm thick slices and observed using fluorescence microscope. When sections of eyes treated with FITC-Parstatin and those ones treated with FITC-P1-26 were examined, intensive FITC signal was detected at the sites of administration and at periocular tissues. FITC signal was not observed in internal ocular layers. Regarding FITC-Parstatin, the distribution of specific signal showed no variability during different time intervals which ranged from 30 minutes to 19 hours. As compared to the above results, the respective ocular sections with FITC-P24-41 revealed weak FITC signal at the injection sites and at periocular tissues, accompanied by no detection of intraocular localization. On the other hand, high autofluorescence present in kidney sections and heart sections did not allow the investigation of tissue-distribution of the peptide molecules. In the present study, it appeared that subconjunctival administration of Parstatin and of its peptide fragments, P1-26 and P24-41, is not the appropriate route for the drug delivery into the internal ocular tissues.

Ocular pathology is one of the main health problems worldwide. The number of people with retinopathy symptoms has increased considerably in recent years. Early adequate treatment has demonstrated to be effective to avoid the loss of the vision. The analysis of fundus images is a non intrusive option for periodical retinal screening. Different models designed for the analysis of retinal images are based on supervised methods, which require of hand labelled images and processing time as part of the training stage. On the other hand most of the methods have been designed under the basis of specific characteristics of the retinal images (e.g. field of view, resolution). This compromises its performance to a reduce group of retinal image with similar features. For these reasons an unsupervised model for the analysis of retinal image is required, a model that can work without human supervision or interaction. And that is able to perform on retinal images with different characteristics. In this research, we have worked on the development of this type of model. The system locates the eye structures (e.g. optic disc and blood vessels) as first step. Later, these structures are masked out from the retinal image in order to create a clear field to perform the lesion detection. We have selected the Graph Cut technique as a base to design the retinal structures segmentation methods. This selection allows incorporating prior knowledge to constraint the searching for the optimal segmentation. Different link weight assignments were formulated in order to attend the specific needs of the retinal structures (e.g. shape). This research project has put to work together the fields of image processing and ophthalmology to create a novel system that contribute significantly to the state of the art in medical image analysis. This new knowledge provides a new alternative to address the analysis of medical images and opens a new panorama for researchers exploring this research area.

Σκοπός της μελέτης ήταν ο ποσοτικός προσδιορισμός των ιχνοστοιχείων βαρέων μετάλλων του ψευδαργύρου, καδμίου και χαλκού στο υδατοειδές υγρό ασθενών με ψευδοαποφολίδωση και η μέτρηση του δυναμικού οξειδοαναγωγής στο υδατοειδές αυτών των ασθενών. Υλικό και μέθοδοι: Δείγματα υδατοειδούς υγρού ελήφθησαν κατά την εγχείρηση καταρράκτη από 14 ασθενείς με PEX σύνδρομο (7 άνδρες και 7 γυναίκες) και 21 ασθενείς με καταρράκτη αλλά χωρίς PEX (9 άνδρες και 12 γυναίκες). Τα δείγματα διατηρήθηκαν στους -24C μέχρι την ανάλυση. Τα επίπεδα των τριών μετάλλων και στις δύο ομάδες προσδιορίστηκαν, για πρώτη φορά στην Οφθαλμολογία, με τη μέθοδο της βολταμετρίας με χρήση ηλεκτροδίων υαλώδους γραφίτη με υμένιο υδραργύρου, ενδεικτικού ηλεκτροδίου λευκόχρυσου και αναφοράς Ag/AgCl. Εφαρμόσθηκε η τεχνική της ανοδικής βολταμετρίας απογύμνωσης με τετραγωνικούς παλμούς και έγινε σύγκριση των τιμών ανάμεσα στις δύο ομάδες. Η στατιστική επεξεργασία των αποτελεσμάτων έγινε με το Mann-Whitney test (p<0.05) Στη συνέχεια, κατά τον ίδιο τρόπο, δείγματα υδατοειδούς ελήφθησαν από 13 ασθενείς με PEX (7 άνδρες/6 γυναίκες) και 22 ασθενείς χωρίς PEX (7 άνδρες/15 γυναίκες). Η μέτρηση του δυναμικού οξειδοαναγωγής γινόταν αμέσως μετά την απόκτηση του κάθε δείγματος και για το σκοπό αυτό χρησιμοποιήθηκε για πρώτη φορά στην Οφθαλμολογία, ένα μικροηλεκτρόδιο λευκοχρύσου μαζί με ένα ενσωματωμένο ηλεκτρόδιο αναφοράς Ag/AgCl/KCl. Η στατιστική ανάλυση των αποτελεσμάτων έγινε με το t-test για ανεξάρτητα δείγματα (p<0.05). Αποτελέσματα: O Cu βρέθηκε στατιστικά σημαντικά χαμηλότερος στην PEX ομάδα σε σχέση με τη φυσιολογική ομάδα (p<0.05), ενώ ο Zn και το Cd δεν παρουσίασαν καμία συσχέτιση στο υδατοειδές υγρό ασθενών με ψευδοαποφολίδωση. Το δυναμικό οξειδοαναγωγής ήταν στατιστικά σημαντικά υψηλότερο στο υδατοειδές των ασθενών με PEX σύνδρομο. Συμπεράσματα: Στην παρούσα εργασία εφαρμόζεται για πρώτη φορά, η ανοδική βολταμετρία απογύμνωσης για τον προσδιορισμό στο υδατοειδές υγρό του Zn, Cd και Cu και η μέτρηση του δυναμικού οξειδοαναγωγής στο υδατοειδές. Τόσο τα στατιστικώς σημαντικά χαμηλότερα επίπεδα Cu στο υδατοειδές ασθενών με PEX, όσο και το υψηλότερο δυναμικό οξειδοαναγωγής στο υδατοειδές αυτών των ασθενών ενισχύουν την παθογενετική θεωρία του οξειδωτικού στρες του PEX συνδρόμου. / To determine the Zn, Cd and Cu concentrations in aqueous humor of patients with PEX syndrome and to evaluate the overall oxidative status of the aqueous in this group by measuring the reduction-oxidation (redox) potential in the aqueous samples. Material and methods: Samples from aqueous humor were collected during cataract extraction from 14 patients with PEX syndrome (7 male/7 female) and 21 patients without PEX (9 male/12 female). All samples were stored in -24C until analyzed. The levels of selected trace elements in both groups were assayed for the first time in the field of Ophthalmology, with anodic stripping voltammetry, using the square wave differential pulse technique. In this analytical procedure a glassy carbon mercury film electrode, a platinum indicator and a reference Ag/AgCl electrode were used. Finally the metal levels in all samples were compared between the two groups using the Mann-Whitney test (p<0.05). Similarly, samples were collected during cataract extraction from 13 patients (7 male/6 female) with PEX syndrome and 22 normal patients (7 male/15 female). A platinum electrode together with a reference electrode Ag/AgCl/KCl was used for the first time in Ophthalmology, for the measurement of the apparent redox potential in the aqueous humor. The independent samples t-test was used to compare aqueous redox potential between the study and control group. Statistical significance was set at 0.05. Results: Cu was found significantly lower in the PEX than in the control group (p<0.05), whereas Zn and Cd had no statistical differences between the two groups. The redox potential was found significantly higher in patients with PEX syndrome than in normal patients (p<0.05). Conclusions: In the present study we represent the first successful application of ASV in eye research for the determination of aqueous Zn, Cd and Cu concentrations and the meauserment of aqueous redox potential. The significantly lower Cu concentrations and the higher redox potential in aqeous humor of PEX syndrome support the role of increased oxidative stress in the development of PEX.