No description available.

Acanthamoeba castellanii, a causative agent of Acanthamoebic Keratitis (AK) is usually associated with contact lens wear. The present drug regimen induces encystment. Hence there is a need for identification of effective drug targets. This thesis demonstrates the potential of the cellulose biosynthesis pathway, shikimate pathway and methionine biosynthesis pathway as effective drug targets. These biochemical pathways are absent in human hosts. A partially coding gene of cellulose synthase, the ultimate enzyme in the cellulose biosynthesis pathway was amplified from A. castellanii Neff strain. Gene knock down studies showed that the cellulose synthase gene is necessary for A. castellanii (Neff strain) encystation. 2, 6-Dichlorobenzonitrile, which was shown previously to inhibit encystation failed to inhibit in our laboratory conditions. The cellulose biosynthetic pathway could be a potential adjuvant to the present drug regimen. The role of the shikimate pathway, seven enzyme mediated pathway was characterised during encystation and excystation using a known inhibitor, glyphosate [N-(phosphonomethyl) glycine]. Glyphosate inhibited encystation of A. castellanii Neff strain. Glyphosate also inhibited the growth of a recently isolated T4 clinical isolate of A. castellanii. Glyphosate was not effective in inhibiting excystation. The complete coding sequence of seven enzymes of the shikimate pathway- 3- deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, chorismate synthase (CS), 3-dehydroquinate synthase (DHQS), 5-enolpyruvylshikimate- 3-phosphate (EPSP) synthase, 3-dehydroquinate dehydratase (DHQD), shikimate 5- dehydrogenase (SDH) and shikimate kinase (SK) have been amplified from A. castellanii Neff and T4 clinical isolate eDNA. The molecular arrangement of the seven enzymes in A. castellanii is novel and not reported in any other organism. A. castellanii Neff strain and the T4 clinical strain possess a four functional AROM like structure encoding DHQS, EPSP synthase, SK and DHQD. CS and DAHP are transcribed by mono functional genes. A. castellanii Neff and the T4 clinical isolate possess a bi-functional novel gene arrangement of SDH and phosphoribo anthranilate synthase (PRAI). PRAI is part of the tryptophan biosynthesis pathway. The shikimate pathway provides a potentially new therapeutic anti-Acanthamoebic target. Herein, is also demonstrate the potential of the methionine biosynthesis pathway as a new drug target. A de novo methionine biosynthesis pathway is present in A. castellanii. Although methionine synthesis is present in human hosts, A. castellanii uses different enzymes to synthesise methionine when compared to human hosts. A complete coding gene of methionine synthase - the ultimate enzyme involved in methionine biosynthesis has been amplified from A. castellanii Neff and T4 clinical isolate cDNA. A. castellanii Neff and T4 clinical isolate growth assays in the absence of methionine and cysteine showed the presence of de novo synthesis of methionine present in A. castellanii. The methionine biosynthetic pathway could be a potential drug target.

No description available.

Autosomal dominant optic atrophy (DOA) classically presents with bilateral, symmetric visual failure in early childhood, with the pathological hallmark being the selective loss of retinal ganglion cells (RGCs). In the first population-based epidemiological study of DOA, we were able to estimate its minimum prevalence at 1 in 35,000 in the North of England. In independent case series from Northern Europe and North America, the majority of families with DOA harboured pathogenic OPA1 mutations (50.0-57.6%), and large-scale OPA1 rearrangements were present in only a small subgroup (11.1-12.9%). We also confirmed that OPA3 mutations were very rare in non-syndromal DOA cases. Visual deterioration was observed in over half (54.2-67.4%) of all patients during long term follow-up, and the rate of visual decline varied markedly both between and within families. In a large multi-centre study of 104 OPA1-positive patients from 45 independent families, we established that additional neuromuscular complications are common in OPA1 disease, affecting up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and chronic progressive external ophthalmoplegia (CPEO) from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. Patients with these syndromal disease variants (DOA+) had a worse visual prognosis, and this was associated with a more pronounced reduction in retinal nerve fibre layer thickness compared to patients with pure DOA. Interestingly, there was a two- to three-fold increased risk of developing DOA+ features with missense OPA1 mutations and those located within the GTPase domain.

Non-motor symptoms such as dementia and visual hallucinations are key determinants of long-term outcome and quality of life in Parkinson’s disease (PD). Attempting to understand these issues better was the motivation behind this thesis. A major aim of the study was to characterise the visual symptoms experienced by patients with PD and PD dementia, focussing not just on complex visual hallucinations, whose prognostic implications are already well-described, but also on a range of other visual symptoms including illusory misperceptions, sensations of passage and presence and double vision. A major objective was to define key measures of visual exploration strategy during visuocognitive assessment and examine the link between strategy, cognition and visual and motor symptoms. We also set out to examine the utility of retina-specific visual assessment techniques to define the potential role of retinal dysfunction in visual impairment and symptomatology. A major finding of this study was that not all visual symptoms share a common pathophysiological basis. Our results argue in favour of splitting hallucinations into separate phenomenological groups in order to better define causation and predictive value in future longitudinal studies. In addition, exploration strategy on a variety of visual tasks was demonstrated to be significantly less efficient in subjects with perceptual difficulties, providing insight into the interaction between cognition and eye movements in PD. Retinal structure, as assessed by optical coherence tomography, was not significantly altered in PD and our results would caution against the use of this technique as a disease biomarker until more is known about the limitations of this method. Finally, our neurophysiological assessment hints at the retina as the site of diminished visual acuity in PD despite there being no striking differences in central and peripheral retinal responses between control and PD subjects.

Background: Glaucoma is a major cause of blindness world-wide. There is a need for methods to identify individuals at risk of developing glaucoma, so that early treatment can prevent visual loss. Genetic screening tests offer the prospect of pre-symptomatic diagnosis of at risk individuals. There is now strong evidence that a number of different genes are associated with glaucoma susceptibility. Mutations in the PITX2 homeobox transcription factor gene disrupt normal development of the anterior segment and cause overt structural abnormalities. It is possible that, as yet undetected mutations/polymorphisms in PITX2 may produce subtle and undetected abnormalities in anterior segment structure and function that could predispose to glaucoma. Purpose: The aim of this thesis is two fold: 1. Screening for the presence of single nucleotide polymorphisms in PITX2 gene in a cohort of 100 unrelated primary open angle glaucoma/ ocular hypertension patients, 10 Posterior embryotoxon subjects and 100 age and ethnically matched controls to establish the mutation spectrum. 2. Identification, phenotyping and recruitment for genetic studies of primary open angle glaucoma patients with strong family history of glaucoma. Materials and methods: 1. 100 primary open angle glaucoma patients and 60 age and ethnically matched controls were enrolled in the study. Patients and controls were phenotyped and a blood sample for DNA extraction collected. PITX2 exon-specific primers were used to PCR amplify patient and control DNA. Direct sequencing was used to screen for sequence alterations in the entire coding sequence of PITX2 gene. Concurrently, polymorphic sites reported in the PITX2 gene were identified from the NCBI and Ensemble databases and the frequency of polymorphic sites was investigated. The SHEsis and UNPHASED software packages were used for statistical analysis. 2. Patients diagnosed with glaucoma and strong family history were identified from Glaucoma Unit at Sunderland Eye Infirmary, phenotyped and enrolled in the study. The pedigrees were constructed and interested relatives enrolled in the study and phenotyped. A sample of blood for DNA extraction was collected from all people enrolled in the study. Results: 1. Direct sequencing did not identify any sequence variation in the coding region. 26 PITX2 polymorphic sites were identified from the internet databases, including five in the coding sequence. Sixteen non coding SNPs were confirmed within our study group and SNP frequencies were examined. None of the coding sequence SNPs was identified in our cohort, demonstrating a high degree of sequence conservation. Also, none of the SNPs confirmed in this study group showed an increased frequency in the primary open angle glaucoma group compared with the control group. 2. Thirty-three pedigrees were identified with strong family of glaucoma during the time allowed for patient recruitment. Of these, twenty-two agreed to take part in the study. Thirteen pedigrees are presented in this study, mostly demonstrating autosomal dominant inheritance. Conclusion: There is ample evidence to suggest that genetics play an important role in unravelling the pathogenesis of glaucoma. Identification and recruitment of patients for genetic studies is an essential step and the role of the clinician in this process is paramount. Also, developmental glaucoma genes are an important group of genes to be screened in primary open angle glaucoma/ocular hypertension patients, as they may play a role in the pathogenesis of this preventable blinding disease.

Diabetic retinopathy is the most common microvascular complication of diabetes mellitus and the leading cause of blindness in persons from age 20 to 74. The relative risk of blindness in persons with diabetes has been reported to be 5.2 times the risk of those without diabetes. The fundus abnormalities described in diabetic retinopathy result from structural damage to the microvasculature wall with subsequent leakage or as a result of retinal ischaemia with secondary overproduction of vascular growth factors. Several clinical and screening classifications schemes have been developed to categorize and quantify the severity of each of the retinopathic features based on the degree of retina involvement. The ultimate goals of these classification schemes have been to provide a system by which the natural history of the disease and the risk of progression of retinopathy and visual loss can be identified and the subsequent response to interventions can be evaluated to improve patient care. The present strategies for dealing with diabetic retinopathy address retinopathy that is already established. However, recent studies - supported by computer based imaging analysis – have focused on changes in retinal vascular caliber and demonstrated various associations with increased risk of diabetes and predicted the onset of microvascular retinal complications. This suggests that other structural and geometrical parameters might also be utilised, which can provide more information regarding the retinal vascular network. Few studies have reported different changes in retinal vascular geometry with age, systemic hypertension and peripheral vascular diseases. The objective of this thesis is to analyse the retinal vascular geometrical features in normal subjects and evaluate its role in diabetic subjects with different stages of diabetic retinopathy. For this purpose, a semi-manual vascular analysis technique is designed to measure and analyse the different retinal vascular geometrical parameters and ratios. The developed technique performance and precision is compared to other available manual and semi-manual vascular analysis techniques.The various sources of variability in retinal geometrical measurements are then evaluated, including observer’s measurement errors, variations in image capture, and potential short term changes in the subjects’ vascular geometrical features. The second step of this work is to perform a detailed analysis of the retinal vascular geometry in normal subjects, including the topographic distribution of different geometrical measurements across the fundus, the effect of different demographic and clinical factors, and the stability of measurements between both eyes. The next step evaluates the retinal vascular geometry in diabetic subjects with different grades of diabetic retinopathy to determine any changes of geometrical features with advancement of retinopathic stages. The results demonstrate significant associations of changes in vascular structural and geometrical features with increased stages of diabetic retinopathy. Finally, the predictive value of retinal vascular geometry analysis and its practical role on the individual level is analysed for a sample of subjects who progressed from no retinopathy to proliferative retinopathy as compared to a sample of subjects with no sign of progression. The preliminary results suggest that geometrical changes trend can be detected on the individual level with progression of diabetic retinopathy and those differences can be noted between progressors and non-progressors at baseline. In conclusion, this thesis describes novel retinal vascular geometrical markers indicative of establishment of advancing diabetic retinopathy, together with a potential predictive role in determining risk of future progression to proliferative retinopathy.

No description available.

There has been interest in ocular temperature for over a century, with the publication of a limited amount of literature over that time. This thesis reports on the development and utilisation of a system for the measurement of ocular surface temperature based on an advanced infrared detector. The development of a suitable protocol for the infrared imaging and temperature measurement of the eye was established. Results from the analysis of the ocular temperature of 98 normal subjects indicated that the temperature at the centre of the cornea was 31.68 ± 0.60°C (mean ± SD), and the mean ocular surface temperature (MOST) was 32.10 ± 0.53°C. This value rose with increasing room and oral temperature (both p< 0.0001) and decreased with corneal thickness (p< 0.05) and age (p< 0.01). Ninety five per cent of the subjects had an inter-ocular difference in MOST within 0.53°C. There was a positive relationship between the variation in temperature across the ocular surface and the distance between the corneal apex and a coronal plane through the limbus (p< 0.001). The effect on eye temperature of changes in the ocular blood, nerve and tear supplies was studied. Three of nine patients (33%) with unilateral inflammatory disease had ocular temperature outside the established normal limits. For the group of patients as a whole, the temperature of the affected eye was warmer than the fellow (32.40 ± 0.81°C compared with 32.10 ± 0.70°C; p< 0.05). In a further study, there was a significant negative relationship between the degree of stenosis of the carotid artery and eye temperature (p< 0.001). In the examination of two patients with Horner's syndrome, one demonstrated a difference in eye temperature (0.61°C) outside normal limits (0.53°C), with the affected eye being warmer.

Retina is one of the key features of the human eye. Analysis of retinal images is essential for early detection and diagnosis of pathologies. This thesis deals with retinal image processing and analysis of colour fundus images. The main contributions of this thesis are: • A geometrical and morphological analysis of the retinal vasculature. Starting from a binary image of the vascular network, the analogy between the vasculature and the structure of a graph was explored, yielding a matrix representation of blood vessels. Properties of each graph edge (corresponding to vessels) were computed, such as length, width, area, curvature, branching angles. This analysis resulted in developing a novel tortuosity measure taking width information into account, as well as a novel approach to the detection of the retinal main vein. • Context-based detection of a reference frame of the retina, by detecting the mai'n retinal landmarks: the optic disk, the macula, an approximation of the main blood vessel. Our detection algorithms introduce a technique based on plausible detection and constraint satisfaction, which provides several optic disk candidates among the brightest image regions, and several macula candidates among the darkest image regions, and fits two half parabolas centered at the optic disk candidate by minimizing a cost function. The actual landmarks are chosen among the candidate landmarks that satisfy several constraints imposed by the anatomy of the retina. • A preliminary architecture for a fuzzy diagnosis system for retinal pathologies. The system inputs are several symptoms related to a certain disease, and the output is a grading of the disease.An example of a fuzzy diagnosis system was presented for diabetic retinopathy. Keywords: image processing, retina, optic disk, macula, main vessel, tortuosity, diagnosis, fuzzy, main vein, vascular morphology.