In the diagnosis of dry eye, an evaluation of tear fluid volume is an important parameter. The tear menisci hold approximately 75-90% of the overall tear fluid volume and serve as reservoirs, supplying tears to the pre-corneal tear film. The measurement of the anterior curvature radius of the tear meniscus (TMR) is an indicator of tear film volume and when it is performed non-invasively has been found to have good dry eye diagnostic accuracies. Optical coherence tomography and meniscometry are existing techniques that can measure TMR non-invasively. These techniques have not found wide application among clinicians, either because they are not commercially available or they are too expensive. This PhD describes a series of experiments that investigated the development, evaluation and application of a new instrument for non-invasive tear meniscus measurement. From the results of these studies, it can be concluded: A Portable Digital Meniscometer (PDM) was developed. This consists of an application tool for the iPod-touch, a slit-lamp holder for the iPod-touch and an image analysis software for TMR calculation. A simple iPod-touch or an iPhone mounted on a commercially available digital slit-lamp can be used to project a grid of black and white lines on the tear meniscus. Using the principal of reflective meniscometry, the radius of the lower tear meniscus can be non-invasively measured. This newly developed instrument is a simple, mobile and useful device for measuring tear meniscus radius, and therefore tear volume, and is suitable for use by clinicians. The newly developed PDM was evaluated in vitro and in vivo. It produced accurate and reliable measurements and provided similar values for the tear meniscus radius, in human studies, to the existing video-meniscometer. PDM and OCT measurements of the TMR were significantly correlated. Since with the PDM no image calibration is needed, it seems to be a quick and non-invasive technique for evaluation of tear fluid quantity. The PDM appears to measure the radius of the central section of the tear meniscus The PDM was able to non-invasively measure alterations in TMR and TMH along the lower lid. The flatter TMR and higher TMH at the nasal and temporal locations may be caused by the LIPCOF degree of the underlying conjunctiva. To avoid any interference by LIPCOF, it is recommended that TMR and TMH be measured along the lower lid margin below the pupil centre. Furthermore, the PDM was able to usefully detect changes in TMR following the instillation of artificial tears. The difference in residence time is likely to reflect the different viscosity and Newtonian properties of these drops. An overload with a large drop may result in initial increased blink rate. Blink rate at baseline is significantly related to dry eye symptoms.

This thesis presents the results and conclusions of experiments designed to extend the current models for the origin of corneal transparency. The cornea is the transparent window at the front of the eye, which is responsible not only for the majority of refraction of light that enters, but also the protection against damage, infection and mechanical stress. The property of transparency is only realised by corneal and lens tissue in the human body. In the cornea, it has long been suspected to be caused by the precise arrangement of the fibrils of collagen that are contained within the central layer, the stroma, regulated by the sulphated proteoglycans (PG) that keep fibril spacing within acceptable boundaries. These models are consistent and give a complete description of the reasons for the transparency of a stroma that is entirely acellular. However, it is well known that the stroma is not acellular, and that the short-range order that is critical for transparency would necessarily be disturbed by the cells of the stroma, the keratocytes, which are at least an order of magnitude thicker than the maximum allowed range. Originally, an acellular stroma was considered to be a reasonable approximation due to the perceived sparsity of the cells, but more recent measurements have cast doubt upon this, and explanations have begun to focus on the properties of the cells themselves. One such property would be their refractive index (RI). If the cells could match their own RI to that of their surroundings then they would not scatter and hence would not cause a loss in transparency. This research attempts to measure that RI and by comparison with previously calculated values for the RI of the extra-cellular matrix, attempts to quantify the scale of the scattering that any mismatch would cause, using theoretical models based on both Mie scattering and finite-difference time-domain methods. In addition to models of healthy corneas, this thesis also provides results and conclusions drawn from studies of pathological corneas and discussions of how the pathology, and the treatments, can cause initial losses in transparency. The first such study concerned a cornea afflicted with keratoconus, a disorder of as yet unknown origin that causes the weakening of the corneal tissue, leading to a characteristic cone-shaped cornea, which had been treated with a full penetrating keratoplasty (PK) transplant before being donated. The study was conducted using the techniques of electron microscopy and x-ray diffraction, to both qualitatively and quantitatively analyse the properties of the fibrils and their spacing. This was done on both the original sections of diseased tissue and the donated sections, in order to investigate the idea of keratoconus recurring in previously healthy donated tissue. Any such discovery could provide evidence that keratoconus is not an entirely inherited disorder. The structural properties of the observed scar, that was present as a direct result of the PK procedure that was carried out decades before, were also investigated using the same methods. This investigation was designed to provide insights into the priorities of wound healing in the cornea, and whether any appreciable change in fibril spacing could account for the observed loss of transparency. The final study presented here is a novel tomographic reconstruction of a feature of the disease macular corneal dystrophy (MCD). MCD is a genetic disorder that affects the sulphation of the PG keratan sulphate. MCD gives rise to a multitude of abnormalities but one that has not been fully investigated is the apparent presence of areas within the stroma entirely populated by PGs, with no collagen present. This study attempts to reconstruct three-dimensional views of these lakes, as well as stromal lamellae, in order to investigate the interactions between free PGs and between PGs and collagen in MCD.

Purpose: To i) investigate the stimulus characteristics of the Belmonte OPM pneumatic aesthesiometer in vitro and in vivo; ii) explore the relationship between corneal sensitivity threshold (CST), spontaneous eye blink rate (SEBR), ocular surface temperature (OST) and tear film quality, using the non-contact aesthesiometer (NCCA), and iii) investigate the relationship between heating and cooling of the ocular surface on CST (with the NCCA). Methods: Stimulus flow rate (airflow meter), air stimulus volume (air capture) and force (weight scales) were recorded in vitro. Stimulus temperature was measured in vitro (using thermocouples) and in vivo (via a thermal infrared camera). SEBR, CST, OST, NIBUT and lipid pattern of the tear film were recorded. CST (using NCCA), OST and tear film quality were obtained at baseline, and after increasing and decreasing OST, using heated goggles or cooling gel mask, respectively, until OST baseline level was reached again. OST was recorded during stimulus presentation at CST, immediately after heating and cooling of the ocular surface. Conclusions: Stimulus characteristics measured in vitro showed little variability and may therefore be considered as reliable. Stimulus temperature recorded in vivo showed that a change in stimulus flow rate produced a small, but statistically significant effect on OST. Consequently, this air stimulus may not be exclusively ‘mechanical’, and may provoke an additional response from temperature sensitive C fibres. A moderate correlation between corneal sensitivity and blink frequency was established, and a strong correlation between tear film quality, SEBR and OST was obtained, emphasising that ocular surface condition represents one important trigger for the initiation of a blink. After both an increase and a decrease of OST, a greater localised OST decrease was required at CST, indicating that heating or cooling of the ocular surface may affect the nervous activity / sensitivity of cold thermoreceptors in the superficial cornea.

No description available.

No description available.

Children with delayed reading skills and/or poor academic achievement are increasingly being referred to Eye Care Professionals with suspected eye movement/“tracking” difficulties. However, current clinical techniques are highly subjective, poorly controlled, and relatively imprecise. It is therefore reasonable to suggest that Eye Care Professionals face challenges in recognising and diagnosing genuine eye movement disorders, and consequently, fail to support and/or manage these children. The principal aim of the studies described in this thesis was to characterise eye movements in children with learning related difficulties who are frequently considered to be at risk of eye movement disorders. Using a novel child-friendly method, we have shown that, in general, eye movement characteristics in children with reading/learning related difficulties are not different from those in typically developing age-matched children when compared as a group. The findings also showed that when eye movement characteristics in children with reading/learning related difficulties were compared on an individual basis, some of these children had eye movement parameters outside their age-matched norms. Further, our results suggested that children whose eye movements were outside their age-matched norms, generally corresponded to those who had specific, more complex and global difficulties (e.g. dyspraxia, general developmental delay). In conclusion, the studies presented in this thesis suggest that there is an association between specific learning difficulties and eye movement disorders, but challenge the view that eye movement disorders can be found in isolation in children with delayed reading/academic performance. Finally, based on the sum of results obtained, simple actionable guidelines are proposed to improve the examination of eye movements in clinical practice in order to recognise genuine eye movement disorders.

This thesis explored some aspects of the relationship between structural progression of the glaucomatous optic nerve head (ONH) and functional progression of the visual field. Sixty-one individuals with a longitudinal series of ONH images were manually identified from a database of approximately 2800 individuals attending a hospital glaucoma clinic. The ONH images obtained from the various photographic sources were equalized, for each individual, in terms of ONH size. Custom-software was designed to enable the viewing of consecutive and chronologically different ONH image-pairs under monoscopic and stereoscopic conditions, with and without sequential flicker. The efficacy, for the identification of progressive glaucomatous loss, amongst the 61 individuals, of the four viewing techniques was qualitatively evaluated by two ophthalmologists. Stereo-flicker identified the largest number of cases of progression, although little agreement was present between the two ophthalmologists. The digital characteristics of the ONH images from 27 of the 61 individuals enabled quantitative digital stereo-planimetry. A weak positive curvilinear association was present, at baseline, between the reduction in the neuroretinal rim area and the outcomes of perimetry, including residual retinal ganglion cell (RGC) count. However, little agreement was again present between the two ophthalmologists. Little association was present with either ophthalmologist between progressive structural damage and functional damage. A separate manual search of 1000 individuals with glaucoma archived in ‘Open eyes’ identified 112 individuals with a minimum of 5 visual field examinations over a minimum of 5 years. The outcomes at each stimulus location of the differential light sensitivity, expressed in decibels (dB), and of the residual RGC count, against time to follow-up, were compared using univariate linear regression analysis. In general, residual RGC count identified progression, in terms of a greater statistical significance and/ or of more stimulus locations, at an earlier stage of the disease than sensitivity expressed in dB.

Cross-linked Actin Networks (CLANs) were first identified in bovine trabecular meshwork (BTM) cells in response to steroid treatment. Since then, work within our group has identified CLANs in human TM (HTM) cells and human tissue ex vivo. An alteration in the cytoskeleton of TM cells has been associated with changes in outflow facility and is a promising therapeutic target in glaucoma research. CLANs which are believed to make TM cells more rigid, were found in increase numbers in tissue from glaucomatous donors, but were also present in tissue from non-glaucomatous donors. This would indicate that CLAN formation is not merely a steroid response but may be triggered by changes within the outflow system. The current work set out to identify CLAN inducing agents present within the normal outflow system and questioned whether age-related changes within the anterior chamber could influence CLAN number in a similar manner to glaucoma. Investigating the influence of aqueous humor (AH) and some of its constituent growth factors on CLAN formation in both BTM and HTM cells revealed that AH itself was capable of inducing CLANs and that the main CLAN inducing agent present was TGF-β2. Conversely mitogenic growth factors (FGF and HGF) did not increase CLAN incidence and implicated that CLAN incidence was linked to cell shape change linked and therefore to function. In vitro analysis of CLANs in reference to donor age revealed that TGF-β2 could induce a higher percentage of CLANs in HTM cells from older donors compared to HTM cells derived from younger donors. Ex vivo analysis run in parallel demonstrated that CLAN incidence increased with increasing donor age. In order to identify how age-related stress within the TM environment influenced CLAN incidence cells were treated with hydrogen peroxide. In BTM cells pre-treatment for 1 hour was sufficient to significantly increase CLAN incidence. In further experiments it would seem that CLANs are not associated with apoptotic cells nor with senescent cells. The findings of this study indicate that CLANs can form when no glaucomatous pathology is detected and that changes in the extracellular environment can influence their formation. This is especial evident in older tissue.

Over expression of lens epithelium derived growth factor (LEDGF) is described in a number of different tumours. In addition, self-antibodies against this protein are detectable in patients with lymphomas and chronic lymphocytic leukemia (CLL). Alternative splicing of LEDGF results in a number of different isoforms, and thus the initial aim of this thesis was to determine the variability in LEDGF isoform expression in a cohort of CLL cases. Utilising RT-PCR, it is shown that CLL cells variably express four different LEDGF isoforms. The study was then extended to quantitate the expression levels of these four isoforms in individual CLL cases, and to compare them to normal B cells. RT-qPCR analysis showed that the longest of these isoforms, p75, is significantly over expressed, whilst the shortest form, p52bΔE6 is significantly under expressed in CLL, compared to normal B cells. All four isoforms of LEDGF are generally over expressed in CD38 positive CLL cases compared to those that are deficient for surface expression of this molecule. Alternative splicing occurs as a result of the inclusion or exclusion of different exons within the mRNA. This process is governed by the splicing machinery, of which Splicing Factor 3B, subunit 1 (SF3B1) is a critical component. Recently, it has been shown that mutations within SF3B1 are recurrent in CLL. Such mutations impact on the splicing of a significant array of genes within the cell. One of the genes predicted to be affected is LEDGF. Therefore, the mutational status of SF3B1 was examined in the same cohort of CLL cases and correlated with the expression of the LEDGF isoforms. As well as the commonly reported mutations in SF3B1, a novel nonsense mutation was identified. Although not statistically significant, cases with unmutated SF3B1 have higher levels of all isoforms of LEDGF and a possible explanation for this is discussed. That two of the major isoforms of LEDGF (p75 and p52) have differing functions within the cell is well documented and can be explained by virtue of their participation in distinct protein complexes. In order to identify components of the complexes generated by the four LEDGF isoforms identified in this study, stable cell lines exogenously expressing these isoforms were generated. Each of the isoforms was ‘double-tagged’, and a purification protocol optimised that would allow successful characterisation of the complex associated with each isoform. The results provide useful insights and provoke thought for the role of alternative splicing in the altered phenotype of cancer cells and its contribution as an additional epigenetic mechanism in normal and malignant gene regulation.

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