Age-related macular degeneration (AMD) and inherited macular diseases (IMD) are retinal disorders that can cause blindness through atrophy of the retinal pigment epithelium (RPE) or choroidal neovascularisation (CNV). RPE transplantation in severe forms of neovascular AMD has been performed with promising short-term outcomes. However, this approach has not been evaluated in atrophic types of AMD or IMD. Furthermore, the long-term outcomes of photoreceptors cell function rescue by RPE reconstruction in neovascular AMD is unknown. Current surgical techniques are complex with associated high complication rates. Therefore, other treatment approaches to reconstruct the RPE are required. This thesis aims to examine whether long-term photoreceptor cell function rescue can be achieved through RPE reconstruction by investigating the outcomes of autologous RPE transplantation or full macular translocation in AMD and IMD. A further aim is to determine the feasibility of a new approach to reconstruct the RPE using human embryonic stem cell (hESC). A prospective study of autologous RPE-choroid grafts in 9 patients with atrophic macular disease secondary to AMD or IMD demonstrated that submacular RPE graft can support retinal function and fixation. However, there was a high surgical and post-operative complication rates and the overall visual acuity and reading ability declined. Long-term follow-up demonstrated that the graft can maintain retinal function for over 2 years in some patients. A retrospective review of long-term outcomes following autologous RPE-choroid grafts and full macular translocation in 12 and 40 patients with neovascular AMD, respectively, showed that rescue of retinal function beyond 2 years is possible. A visual acuity of 6/12 was achieved and maintained for over 2 years in 8% and 15% of patients who had patch graft and translocation, respectively. However, overall visual acuity outcomes were limited by delayed post-operative complications such as recurrent CNV and cystoid macular oedema. A prospective porcine experiment showed that subretinal implant of hESC derived-RPE was feasible and human donor cell can survive in vivo for up to 6 weeks. However, there was significant loss of the hESC-RPE which may have occurred intra-operatively or during the first 2 weeks post-operatively. Macrophages were noted at the site of the graft suggesting some inflammatory and immunological responses to the human cells, polyester substrate or surgical trauma. The work in this thesis has provided the proof of principle that reconstruction of the RPE can maintain retinal function in atrophic and neovascular macular diseases over the long-term. A novel approach using hESC-RPE on an artificial substrate may be a more feasible and safer alternative to current clinical techniques of RPE reconstruction.

Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness in those over 50 years old in Western countries. It is a late-onset, neurodegenerative retinal disease, which is characterised by extracellular deposits containing amyloid beta peptides (Aβ) on the Bruch’s membrane. In half of AMD cases, polymorphisms in the gene encoding complement factor H (CFH) are associated with susceptibility to the disease. The aims of this thesis were; (1) to identify sites of Aβ accumulation in mouse ageing eye and macrophage up-regulation, (2) to investigate the effects of immunotherapy targeting Aβ as a potential treatment for AMD, (3) to examine how pathogens trigger retinal disease in CFH mice, (4) to determine whether the strategy of inhibiting complement component C3 (C3) and complement activation is beneficial or detrimental in CFH mice. I show that Aβ deposition increases with age and is accumulated on photoreceptor outer segment and on Bruch’s membrane. Systemic administration of an antibody targeting Aβ improved retinal pathology, by decreasing deposits and reducing the activation of C3. I also show that genetic mutation or polymorphism is not the only factor triggering the onset of AMD but also environmental factors such as pathogen load are also critical. C3 deficiency resulted in Aβ deposition and photoreceptor cell loss along with failure to activate macrophages, supporting a beneficial, neuroprotective role of C3 in the retina. Collectively these data show that inflammation is one factor that forms an umbrella for the onset and progression of AMD. However, inflammation is not always a negative phenomenon. Aβ deposition and pathogen load are factors that will trigger an inflammatory response in tissue and therefore ways to regulate inflammatory responses to physiological levels and subsequently removing the factor causing the inflammation without affecting the homeostasis of the tissue will be a step forward in treating AMD.

The MD will primarily examine the role of surgery in the management of the wet form of age related macular degeneration (AMD) and secondarily for specific inherited macular dystrophies. It is postulated that in the early stages of wet AMD and other sub-foveal disorders involving choroidal neovascular membranes (CNV), photoreceptor loss is relatively limited with the disease confined to the sub-foveal layers, namely the choriocapilliaris-Bruch membrane-retinal pigment epithelium (RPE) interface. At this stage the retina is affected functionally and reversibly but with time the damage becomes permanent and irreversible. As such a critical window of opportunity exists to: 1. Salvage function from the existing photoreceptor pool before fibrovascular proliferation causes marked ‘irreversible’ photoreceptor loss 2. Treat any visual loss that may be due to secondary and potentially ‘reversible’ factors such as sub-foveal fluid and haemorrhage and 3. Mechanically restore normal anatomy. Previous attempts at sub-macular surgery have been associated with the loss of RPE in the area of the CNV during removal that secondarily causes degeneration of photoreceptors. Consequently, different innovative surgical approaches, including 360-degree macular translocation and full thickness autologous RPE transplantation, are under investigation for the management of sub-foveal CNV. The rationale of surgery in both techniques is to effectively restore the choriocapilliaris-Bruch’s-RPE interface beneath the foveal photoreceptors and rescue function before fibrovascular proliferation causes marked ‘irreversible’ photoreceptor loss. Pilot studies have been established to: 1. Examine the surgical feasibility and the anatomical and functional outcomes for each procedure 2. Investigate the pathophysiology of the underlying disease processes. In addition, a number of parameters will be investigated to evaluate the quality of recovery of vision. This will include assessing fixation stability, reading ability, histopathological studies and electrophysiological correlates.

Leukocyte migration from the blood vessel, across the vascular wall and into the tissue underneath occurs in both an inflammatory response as well as immunosurveillance. During this process multiple adhesive interactions occur between leukocytes and vascular endothelial cells (ECs). The EC itself is rendered compliant to transmigration following inside-out signalling in response to leukocyte adhesion altering the activity of a number of different cellular components including the actin cytoskeleton, Rho GTPases and various protein kinases. For this, adhesion to endothelial intercellular adhesion molecule-1 (ICAM-1/CD54) is particularly important and the focus of this study. Indeed, previous work in our lab has shown that endothelial ICAM-1 signalling controls lymphocyte diapedesis by modulating interendothelial VE-cadherin (VEC) junction phosphorylation via a pathway involving calcium, AMP kinase and nitric oxide synthase (eNOS). In this study, I have investigated if ICAM-1-mediated endothelial mitogen-activated protein (MAP) kinases activation played a role during lymphocyte transmigration across brain microvascular ECs. All three MAP kinases, namely ERK, JNK and p38, were found to be activated in response to ICAM-1 engagement, however only JNK was important for lymphocyte transmigration. Significantly, specific neutralisation experiments using small-molecule inhibitors or dominant-negative plasmids inhibiting JNK resulted in inhibition of transmigration. Activation of JNK required Src, Rho GTPase, MKK7 and protein kinase C (PKC), with all these components also found to be important for lymphocyte transmigration. I further demonstrate that this novel pathway led to the phosphorylation of the actin-associated protein paxillin and its association with VEC. Ultimately this triggered VEC internalisation, suggesting that adherens junction modulation is an important element during transendothelial leukocyte migration. Furthermore, this also suggests that at least two endothelial signalling pathways (JNK-paxillin and eNOS-VEC) converge and cooperate to regulate ICAM-1-mediated lymphocyte transmigration.

Carcinomas begin with a single transformed cell in an otherwise normal epithelial monolayer. To study the nature of interactions between a transformed cell and its neighbours at this initial stage of tumourigenesis a tetracycline-inducible system driving expression of a constitutively active form of oncogenic Ras (RasV12) was established in MDCK epithelial cells by the Fujita laboratory. Upon interaction with normal cells, RasV12 cells most commonly undergo apical extrusion from an epithelial monolayer. In order to identify proteins and pathways involved in interactions between normal and transformed cells, I have performed several biochemical screens described in this thesis. Firstly, I have shown that Hsp90β, identified previously in a 2D gel screen, is increased in RasV12 cells surrounded by normal neighbours in a non-cell-autonomous fashion. By using inhibitors and a dominant negative form of Hsp90, I have shown that upregulation of this chaperone is most likely a part of a stress response delaying extrusion of transformed cells. Secondly, I have performed a screen for tyrosine phosphorylated proteins and identified myosin IE and plectin as modified in mixed cultures of normal and transformed cells. Finally, I have undertaken quantitative mass spectrometry of phosphorylated peptides using SILAC labelling to assess changes in RasV12 cells upon their interaction with normal cells. In this screen I have found that an actin anticapping protein, VASP, is phosphorylated on serine 239 in RasV12 cells interacting with normal cells. This modification is known to inhibits its related to actin function. I have shown that depletion of VASP in RasV12 cells results in their enhanced extrusion from normal monolayers, most likely due to their compromised attachment. The phosphorylation on serine 239 may be an early step in extrusion, contributing to disassembly of focal adhesions and stress fibres in transformed cells. I have also studied the role of another protein identified in the SILAC screen, MRCKβ, and shown that its depletion results in enhanced extrusion of RasV12 cells from normal monolayers.

Previous studies into glaucoma in animal models have concentrated on the retinal ganglion cell (RGC) degeneration associated with the disease. However it is of interest also to examine any changes that occur in the rest of the retina and visual system, including the brain areas associated with vision, to gain a better understanding of the disease and to have better knowledge for designing treatments. This study looks at two rat models of RGC degeneration which are currently used in our lab for treatment studies. These models are the Morrison method of glaucoma and a model where an intravitreal injection of Aβ1-42 oligomers is given to cause RGC apoptosis. To investigate functional changes in the visual system from the retina to the central visual areas of these animals we have employed electrophysiological techniques at different time points after model induction. Using electroretinography (ERG) and histology of the retina we found that the RGC degeneration in the OHT model of glaucoma is also accompanied by 16 weeks after surgery by a dysfunction of the outer retina. However in the Aβ oligomer model only the RGCs were affected in the retina by 16 weeks after injection. We also found changes in the synaptic transmission from the RGCs to the superior colliculus (SC) in both models using in vitro SC slice electrophysiology. This included changes in the group III metabotropic glutamate receptor (mGluR) modulation of synaptic transmission from the RGCs to the SC and NMDA receptor contribution to synaptic transmission in the SC. The results from this study suggest that it will be necessary in the future to study the mechanisms of changes in cells other than just the RGCs in the retina and changes that occur to the central visual areas due to RGC degeneration in greater detail. It has also highlighted the need for the development of neuroprotective strategies and has added further importance for studies looking at earlier changes in and diagnosis of diseases where RGC degeneration is involved.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterised by the selective death of motor neurons. Mutations in the SOD1 gene lead to protein misfolding and cause a dominant familial form of ALS (fALS). The accumulation of misfolded proteins in proteinaceous inclusions is a prominent pathological feature in ALS and other related neurological diseases such as Alzheimer’s disease, Huntington’s disease and Parkinson’s disease. The degradation of misfolded proteins by the proteasome involves the cooperation of molecular chaperones and ubiquitin chain recognition factors. The molecular chaperone Homo sapiens DnaJ 1(HSJ1) combines the ability to bind misfolded proteins with a J domain that stimulates substrate loading onto heat shock protein 70 (Hsp70) and ubiquitin interaction motifs to bind ubiquitylated chaperone clients, and has previously been shown to protect neurons against polyglutamine mediated protein aggregation. Therefore, the effect of HSJ1 on mutant SOD1 misfolding and aggregation was studied in vitro and in vivo. An in vitro model of SOD1 aggregation was developed and showed that the accumulation of mutant SOD1 inclusions was ameliorated in the presence of HSJ1a or HSJ1b. To test if this anti-aggregation effect of HSJ1 was capable of protecting motor neurons in vivo, a panel of HSJ1 transgenic mice were crossed with G93A SOD1 mutant mice. SOD1 mutant mice over expressing human HSJ1a had a significantly greater number of surviving motor units than control mutant SOD1 mice at a late stage of disease progression. In contrast, SOD1 mice over-expressing HSJ1b were not found to differ in motor unit number from control SOD1 mice. HSJ1 knock-out animals were also investigated. Interestingly, HSJ1-/- mice had fewer motor units in the extensor digitorum longus (EDL) muscle than WT mice at 120 days of age and also at 60 days of age (young adults). SOD1 mice with HSJ1 knocked out showed that although at 120 days of age SOD1 HSJ1-/- mice were of a similar strength to SOD1 mice, fewer motor units survived in the EDL muscle. The work presented in this thesis suggests that HSJ1 may be important for the maintenance of motor neurons.

Ginkgo biloba has been advocated as a neuroprotective agent for several years in glaucoma. In this study, immunohistochemistry was used to identify known potential molecular targets of Ginkgo biloba related to retinal ganglion cell (RGC) apoptosis in experimental glaucoma, including amyloid precursor protein (APP), Aß, cytochrome c, caspase-3 and tumor necrosis factor receptor-1 (TNF-R1). Furthermore, using apoptotic inducers related to mechanisms implicated in glaucoma, namely Dimethyl sulphoxide (DMSO), ultraviolet C (UVC) and Sodium Azide (NaN3), the effects of the terpenoid fraction of Ginkgo biloba (Ginkgolide A, Ginkgolide B and Bilobalide) were investigated separately in cultured retinal ganglion cells (RGC-5). Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and morphological analysis of DMSO treated RGC-5 was performed using Hoechst 33342 stain. Immunohistochemistry showed a strong inverse correlation between Aß and APP in ocular hypertension (OHT) animals, with APP and Aß accumulation peaking at 1 and 12 weeks after intraocular pressure (IOP) elevation respectively. Cytochrome c and TNF-R1 expression peaked at 3 weeks, and active caspase 3 activity at 12 weeks after IOP elevation. 1% DMSO, UV40, 1mM NaN3 and 50μM Aβ25-35 dose dependently reduced RGC-5 survival at 24 hours by 27%, 20%, 35% and 27% respectively. These effects were inhibited by Ginkgolide A, Ginkgolide B and Bilobalide in different assays at different levels. In these experiments, all three compounds showed a dose-related response although some intrinsic toxicity was observed with Ginkgolide A. Ginkgolide B had the most profound neuroprotective effects in the majority of assays at a concentration range of 0.5-5μg/ml, whereas Ginkgolide A and Bilobalide had variable activity. Although the effect of simultaneous administration of all three fractions was not assessed, work in this thesis suggest that Ginkgolide B can be neuroprotective to RGCs in preventing apoptosis and cell death, therefore may be of use as a neuroprotective strategy in glaucoma management.

A flickering stimulus can appear brighter or of different colour than a steady stimulus of the same spectral composition and equal time-averaged intensity. The change in appearance is consistent with the distortion product of a nonlinearity in the human visual system. This nonlinearity is used for non-invasive dissection of the human visual system into prenonlinearity and post-nonlinearity linear stages, which affect the amplitude of the sinusoidal signals that they process. A five-channel Maxwellian system was used to generate cosine-windowed, amplitude-modulated, sinusoidally-flickering stimuli. The subjects adjusted the maximum modulation of the signal in order to set the threshold for the perception of flicker and colour or brightness change. The pre-nonlinearity filter was tested by varying the carrier frequency between 5 Hz and 60 Hz and the post-nonlinearity filter was measured by varying the amplitude-modulation frequency between 0.25 Hz and 5 Hz. The L- and M-cone pathways are separately measured by using a new combination of the silent-cone-substitution technique and the sandwich model. The distortion product at the output of the nonlinearity is measured by using a new matching method for measuring the colour and brightness changes. The results suggest that the pre-nonlinearity filter is band-pass and shows substantial adaptation with light intensity that can explain most of the adaptation in the visual pathways. The post-nonlinearity filter is low-pass and shows little or no adaptation with light intensity. The input-output function of the nonlinearity can be described as an expansive, quadratic function that rapidly saturates at high input levels.

Modulation of wound healing is required to inhibit scar formation after trabeculectomy. Mitomycin C and 5-fluorouracil (5-FU) are the anti-proliferative drugs currently used in the clinic. Their effectiveness is limited due to their toxicity and suboptimal local tissue pharmacokinetics caused by rapid clearance within the subconjunctival space. Therefore, prolonged release of anti-scarring agents is needed. Ilomastat is a matrix metalloproteinase inhibitor that has been shown to moderate favorably the wound healing response after surgery. However, due to rapid local tissue clearance, several injections are necessary in a clinically relevant animal model. The thesis describes the development and investigation of implantable ocular tablets for wound healing modulation after trabeculectomy. A tablet would be placed in the subconjunctival space called a bleb, which is caused by the outflow through the fistula made during surgery. While the bleb volume varies, the tablet is thought to dissolve in non-sink conditions. Tablet dissolution was evaluated in vitro using a flow chamber that mimics the liquid outflow conditions of the bleb. The in vitro release studies suggest that tablet dissolution would act to prolong drug release in comparison with injections. Tablet dissolution in the subconjunctival space is affected by many factors including the physical/chemical properties of the drug and tablet, temperature, the volume and flow rate of the dissolution media, and the surface area of the tablet. The efficacy of an excipient-free ilomastat tablet was evaluated using a clinically validated rabbit model. The tablet dissolution was examined using molecular dynamics simulations to consider the molecular interactions of ilomastat and 5-FU undergoing dissolution in non-sink conditions. A mathematical model was also developed to describe the dissolution of excipient-free tablets in the non-sink conditions with 94% confidence. In conclusion, we have established methods to investigate and simulate dissolution of excipientless tablets in non-sink conditions within the subconjunctival space.