The cavity within the transmembrane staphylococcal α-hemolysin (αHL) pore is roughly a sphere of diameter ~45 Ã (volume ~32,600 Ã 3). The alpha-hemolysin gene was modified to introduce exogenous polypeptide sequences between positions 105 and 106 of αHL. These modified αHLs were assembled either by themselves or with wild-type (W) subunits to form stable homoheptamers and heteroheptamers, respectively. First, the ability to accommodate Gly/Ser-rich polypeptide sequences in the central cavity was tested. Concatemerized Gly/Ser-containing sequences ("loops", L; L(10n + 5), n = 0 to 21) were inserted by genetic approaches. Detailed analysis of bilayer recordings and electrophoretic migration patterns of assembled pores indicate that the upper capacity of the cavity is ~175 amino acids. Then two different polypeptides were placed in the cavity to introduce novel functional properties to the αHL pore. By introducing tandem repeats of elastin-like polypeptide sequences (VPGGG), αHL pores (E101W6) that featured a temperature-responsive gating mechanism were obtained. The temperature-dependent properties of E101W6 pores were monitored by single-channel current recording in planar lipid bilayers. The amplitude and the frequency of the transient blockades increased as the temperature increased, while their duration decreased. The hydrophobic collapse of the inserted ELP loop is proposed for the source of the observed sigmoidal two-state transition for normalized closed states of E101W6 pores. Lastly, an αHL pore was designed to detect proteins from the cis side of the membrane. The heat-stable protein kinase inhibitor (PKI) sequence was inserted into the mid-position of the Gly/Ser loop, which was generated by previous project (L105 construct). The heteromeric pore with the PKI-containing loop (P1151W6) was able to detect cAMP-dependent protein kinase catalytic subunit (PKA) at single molecular level. These engineered αHL pores provide numerous possibilities as tools for drug delivery, cryopreservation, or molecular sensing.

100Gbit/s transmission data rate is very attractive for the next generation Ethernet transport systems, and this kind of high-speed channel based WDM systems is very attractive for constructing cost-effective optical transport networks. However, it is quite difficult to achieve such high bit-rate by using the conventional amplitude shift keying (ASK) technology. For the subcarrier modulation of the orthogonal frequency division multiplexing (OFDM) scheme, it is possible to use various modulation formats. It is relatively easy to use such an advanced format in the microwave domain compared to the optical domain. Therefore, it is possible to increase the spectral efficiency relatively easily by using the OFDM technology. In this master thesis, 100 Gbit/s WDM system with OFDM is discussed. The performance of 8 channels 100Gbit/s OFDM system was evaluated. The simulation result showed that the BER of all channels were below 10-3 after 500 km transmission, and the performance could be improved by using the FEC. This result shows the possibility of 100Gbit/s transmission system using the OFDM technology. The major concern of WDM-PON system is the cost. For WDM-PON system, it needs several light sources for downstream signals and upstream signals. For the practical implementation of WDM-PON, it is essential to develop a low-cost light source in the optical network unit (ONU). In order to develop a simple efficient ONU, a promising solution is re-modulated the downstream signal from central office (CO) as the upstream signal at ONU using reflective semicounductor optical amplifier (RSOA) so that there is no need to setup an additional light source at the ONU side. For the conventional fiber-to-the-home (FTTH) system, the modulation format for both downstream signal and upstream signal is intensity modulation with direct detection (IM/DD). The demerit of the IM/DD scheme for the FTTH system is that the performance of the re-modulated upstream signal is limited by the interference of the downstream signal. In order to overcome this issue, the IM/DD format of downstream should be replaced by some different formats such as differential phase shift keying (DPSK). As there is no amplitude modulation for the DPSK downstream signal, the re-modulated signal will not be affected by the downstream signal. Therefore, this master thesis is focusing on comparing the re-modulated signal quality for both IM/DD downstream and DPSK downstream. The experimental results shows that the upstream signal of the DPSK downstream case shows better performance than the IM/DD downstream case, and the performance difference of the re-modulated signal is 17dB.

Thesis (M.S.)--Marshall University, 2003. / Title from document title page. Document formatted into pages; contains 68 p. Includes bibliographical references (p. 56-58).

This study involved the determination of the effects of binocular viewing on contrast sensitivities in 11 normal subjects and in different categories of amblyopes. These were simple anisometropic amblyopes (n=9), micro-esotropic amblyopes with anomalous BSV (n=6), esotropic amblyopes with anomalous BSV (n=3) esotropic without BSV 9n=5), exotropic amblyopes without BSV (n=2) and a group of non-amblyopic strabismics (non-amblyopic esotropes without BSV (n=4); non-amblyopic exotropes without BSV (n=2).An ophthalmic examination was carried out on all individuals. The examination procedures undertaken comprised determination of the visual acuity, subjective refraction, the results of which were confirmed by retinoscopy, and assessment of uniocular fixation patterns. The state of BSV, the direction and magnitude of the angle of deviation, the amplitude of accommodation and pupillary diameter were also determined. The subjects were accordingly placed into the appropriate groups on the basis of the basis of the results of the ophthalmic examination. Measurement of uniocular and binocular contrast sensitivities in response to stationary vertical sinusoidal grating patterns were undertaken. The stimulus display consisted of a Tektronix 5103 cathode ray tube (CRT) with a screen subtense of 2 degrees. Mean contrast threshold values were measured for monocular and binocular viewing over the range of spatial frequencies studied which varied between 8c/deg to 40c/deg depending on the group being examined. The conclusions reached were, first, in individuals with BSV (normal or anomalous), binocular enhancement of contrast sensitivities occurred. However, strabismic amblyopes without BSV and non-amblyopic strabismics without BSV did not exhibit enhanced binocular contrast sensitivities; on the contrary, binocular contrast sensitivities were reduced compared to those obtained through the better eye. Furthermore, when bifoveal stimulation was effected, a further reduction in binocular contrast sensitivity occurred. This study has thus shown that binocular contrast sensitivities are augmented compared with monocular contrast sensitivities when BSV is present, but are decreased when BSV is absent. Furthermore, correction of the angle of squint in strabismics, whether BSV is present or not, further reduces the binocular contrast sensitivities.

A literature review revealed a lack of systematic research investigating standards of clinical practice within optometry. Three main approaches have been used to evaluate the content of clinical consultations: abstraction of clinical records, clinical vignettes and unannounced standardised patients. The aim of this thesis was to obtain an objective insight into the content of optometric eye examinations using these three approaches. In the first scenario, the SP presented for a private eye examination as a 20 year-old myope, complaining of recent onset headaches. The presence of headache was detected in 98% of cases. 22% asked at least four of the eight questions appropriate for primary care headache investigation and 69% of practitioners asked the patient to seek a medical opinion regarding the headaches. The second SP presented as a 44 year-old patient of African racial origin for a private eye examination having experienced recent difficulty with her near vision. 95% of optometrists visited carried out optic disc assessment and tonometry and 35% of optometrists carried out all of these tests. 6% advised the SP of the increased POAG risk in those of African racial descent. The third SP presented for a private eye examination as a 59 year-old patient, with recent onset flashing lights in one eye in the dark. The presence of photopsia was proactively detected in 87% of cases. 35% asked four of the seven questions appropriate for identifying the nature of the flashing lights. 66% recommended dilated fundoscopy to be carried out by either themselves or by another eyecare practitioner. 29% of optometrists asked the patient to seek a second opinion regarding the photopsia. SP encounters are an effective way of measuring clinical care within optometry. Substantial differences exist between different practitioners in the duration and depth of their clinical investigations. This is not surprising, since practitioners are individuals with different levels of experience and therefore variations in approach are inevitable. This highlights the fact that not all eye examinations are the same. The findings of optometric consultations for record abstraction mirror the findings in other healthcare disciplines: clinical records are an imperfect representation of the content of a clinical consultation. Clinical records are subject to a recording bias leading to both under- and over-estimation of the care provided due to the presence of false negatives and false positives. It was proven that clinical vignettes can be easily administered and are a cost-effective way of assessing levels of clinical care and can therefore be used in a great variety of settings. Different methods of measuring clinical care capture different elements of clinical practice and are prone to different biases. A three-way comparison of standardised patient, clinical record cards and computerised vignettes showed that clinical records are an imperfect representation of the content of an optometric clinical consultation as they tend to under-estimate actual care provided, while vignette scores tend to over-estimate clinical performance.

In the UK, 20-50% of older people have undetected reduced vision and in most of these cases the poor vision can be readily corrected by new spectacles and/or cataract surgery. It is often assumed that older people with vision loss will have regular eye examinations so that these problems can be detected, but for many older people this assumption is wrong. One approach to improving the take-up of eyecare services is to carry out vision screening of older people in the community to raise awareness of the need for professional eyecare. The present study aimed to investigate which tests would be most appropriate to screen for correctable visual loss in the older population and to incorporate these tests in a screening tool that would be effective yet simple to administer. The present research sought to investigate whether computerised techniques would be an effective method to screen vision in older people. In Study 1, a computerised vision screener was used to test 180 older people in South London. All participants also received a full, ‘gold standard’, eye examination. Significant cataract was present in 32%, correctable refractive error in 39%, and overall 58% had at least one of these forms of correctable visual problems. The computerised vision screener was able to detect these conditions in about 80% of cases. In Study 2, 200 participants were screened using a revised version of the computerised vision screener. A new flipchart vision screener including the main tests from the computer vision screener was also investigated. 31% of participants in Study 2 had significant cataracts, 30% had correctable refractive errors, and 51% had at least one of these conditions. The computer screener and flipchart tool were both good at detecting significant cataract and refractive errors. About 80% of cases of visual loss due to these problems or due to AMD could be detected with either of the screening tools. Using a pragmatic operational criterion, the screening tools detect about 94% of cases who might be considered by an optometrist to be in need of an eye examination (either overdue or reduced visual acuity). Glaucoma is a difficult disease to diagnose and it was found, as expected, that neither screening instrument was very good at detecting glaucoma. The results showed that the best single test to use for screening of visual loss is HCVA which provides both a high sensitivity (77%) and specificity (73%). Greater sensitivity (80%) is achieved when high contract acuity, low contrast acuity and near acuity are used in combination. Greater specificity (77%) can be achieved by using low contrast acuity alone. It is concluded that vision screening does not replace the need for professional eyecare, but acts as a tool to better inform the public of the need for regular eyecare.

Age-related cataract is the commonest cause of treatable blindness in the world today. It is an ever-increasing problem with prolonged life-expectancy and a burgeoning elderly population. Although successfully treated by modem, sophisticated cataract surgery, the resources needed to provide a surgical set-up and the prolonged training time required to produce a competent surgeon can challenge the available finances of developed economies and result in unacceptable waiting times while they may simply be unaffordable in developing economies and result in high prevalence of blindness. Surgical treatment, although effective in dramatically reversing blindness, is associated with a small risk of sight-threatening complications. Against this background, it is worth considering an alternative, simpler, medical means of treating cataracts that is easily administered and equally effective. Calpains are Ca2+dependent intracellular proteases and several of these enzymes are believed to participate in cataractogenesis. Calpain 2 is the major isoform of calpain involved in human cataractogenesis and its activation appears to be investigate the mechanism of lipid activation of calpains as an essential link towards understanding the molecular and cellular dynamics of cataractogenesis in vivo. A clear picture of the enzymatic events in cataractogenesis will form the basis of drug development to cause selective enzyme inhibition. In chapter 2, atomic absorption spectroscopy shows that the progressive uptake of extra-lenticular Ca2+ by porcine lenses correlates with increases in levels of lens opacification with 8.0 umoles Ca2+ (gm wet lens weight)-1 corresponding to cataract occupying approximately 70% of the lens cell volume. This degree of cataract was reduced by approximately 40%, when a calpain inhibitor, SJA6017 (final concentration 0.8 uM), was included in the extra-lenticular medium, therefore suggesting that the observed porcine opacifications involve the Ca2+ mediated activation of calpains and that cataract could be retarded by the topical administration of calpain inhibitors. In chapter 3, DWIH (Depth weighted insertion hydrophobicity) analysis shows the small subunit of several calpain 2 isozymes to each posess a segment with the potential to form a lipid / membrane interactive a-helix (DWIH values circa 7.0). Extended hydrophobic moment analysis shows these segments to be potential oblique orientated a-helix formers (< uH > circa 0.5). This potential is confirmed by hydropathy plot and graphical analyses, which show each a-helix to possess a significant N -> C hydrophobicity gradient. It is suggested that the lipid / membrane activation of calpain 2 may involve oblique angled membrane penetration by an ahelical segment in domain V of the enzyme. In chapter 4, VP1, a peptide homologue of this domain V segment, is shown to be strongly haemolytic (half-maximal lyric dose = 1.45 mM). FTIR conformational analysis shows VP1 to adopt a-helical structure (20% - 65% of primary structure) in the presence of lipids. These levels are maximal in the presence of anionic lipid (65% of primary structure) and monolayer studies show the peptide to exhibit high levels of anionic lipid monolayer penetration with surface pressure changes (A SP) of 5-6 mN m-1 at 30 mN m-1, which are reduced by approximately 40% ± 15% in the presence of 100 mM NaCl. It is suggested that membrane penetration by the domain V a-helix of calpain 2 may proceed via electrostatic interactions and snorkelling, involving associations between an arginine residue located in the polar face of this a-helix and anionic membrane lipid. It has been suggested that lipid / membranes may modulate calpain 2 activity by lowering the enzyme's in vivo Ca2 requirements. In chapter 5, colorimetric assay of calpain 2 shows that the enzyme requires 4 mM Ca2+ for 100% proteolytic activity, as defined by this assay. In the presence of 1 mM Ca2+, negligible calpain 2 proteolysis is detected but at this level of Ca2+, in the presence of either Dimyristoyl phosphatidylinositol(DMPI), Dimyristoyl phosphatidylserine(DMPS), Dimyristoyl phosphatidylcholine(DMlPC) or Dimyristoyl phosphätidylethanolamine(DMPE), calpain 2 shows proteolytic activity which ranges between 37% and 77% of the protein's full enzymatic activity. The large subunit of calpain 2 (LS-calpain 2) is proteolytical]y active in the absence of the calpain 2 small subunit and it has been suggested that this latter subunit is dispensable for the lipid activation of calpain 2. LS-calpain 2 is assayed under conditions corresponding to those used here for calpain 2 assay and is shown to require the presence of 6 mM Ca 2 for 100% enzymatic activity. These Ca2+ levels are unaffected by the presence of either: DMPI, DMPS, DMPC or DMPE, and based on these combined results, it is suggested that the lipid activation of calpain 2 requires the presence of the small subunit. In addition, it is shown that when compared to zwitterionic lipid under corresponding conditions anionic lipid induces an approximate twofold enhancement of calpain 2 proteolytic activity (70% - 77% as compared to 37% - 49%) and a similar enhancement in its average rates of lipid monolayer interaction (ASP = 1.5 x 10 mN M-1 sec-1 at 10 mN M-1 as compared to ASP = 5.0 x 10-4 mN M-1 sec-1 at 10 mN M-1). It is suggested that calpain 2 may posess an electrostatically driven preference for anionic lipid, which contributes to lowering the enzyme's Ca2+ requirement for activation. In chapter 6, these combined data are discussed in relation to a model for the lipid activation of calpain 2 and proposals for future work are presented.

Aims: The cornea is a tough, transparent tissue providing the primary refractive element of the eye. The stroma consists of specially arranged collagen required for corneal transparency. Correct stromal hydration is important in the maintenance of transparency, a feature controlled by the endothelial cells on the posterior surface of the cornea. The aims of this research were firstly to investigate the morphology of corneal endothelial cells and their expression of the sodium bicarbonate cotransporter during avian embryonic development and secondly, to clarify the effect of disease, surgery and drugs on the posterior cornea including in particular the corneal endothelium. Methods: The corneal endothelial cell morphology and posterior stroma were examined using transmission electron microscopy to determine the ultrastructure of the cells and collagen fibril arrangement in the stroma in all results chapters. Immunohistochemistry and A-scan ultrasonography were employed to identify the presence of the Na+HCO3- cotransporter and to determine the thickness changes in embryonic chick cornea, respectively. Electron tomography was also used to determine the collagen arrangement in Descemet’s membrane. Results: The expression of the Na+HCO3- cotransporter was identified in the endothelial layer of the embryonic chicks at all stages imaged. Central corneal thickness increased in the initial stages of development before a plateau between the E12-E15 developmental period followed by a steady thickness decrease. The ultrastructure of Descemet’s membrane was determined using electron tomography of transverse and en face resin embedded sections from which a model was produced. Polygonal and elongated structures were observed with proteoglycans present at the intermodal regions of the collagenous structures. The polygonal lattice visualised in en face sections appeared to be composed of stacked globular domains which were integrated into the collagen type VIII model. Predominant changes in the Col8a2 knock-in mouse models were observed in the posterior cornea. Differences included increased proteoglycans at the Descemet’s endothelial interface, dilated rough endoplasmic reticulum and focal posterior oedema. This animal model exhibits features similar to those seen in the human form of early-onset Fuchs’ endothelial corneal dystrophy, unlike previous models reported. The final chapter is concerned with regeneration of the corneal endothelial cells. Tissue from posterior corneal surgery examined using electron microscopy revealed the presence of the host endothelial cells and fibrous tissue at the interface in non-Descemet’s membrane stripping automated endothelial keratoplasty and interface haze in Descemet’s membrane stripping automated endothelial keratoplasty. However, these features did not appear to interfere with the adhesion of the graft nor the clarity. Finally, ultrastructural analysis of Rho-kinase inhibited cells showed cells with typical morphology when compared with the untreated group Conclusions: 1) The Na+HCO3- cotransporter is present in the embryonic cornea. It is possible that the cotransporter is involved in the developmental stages and probably the thickness changes we observe during this period. 2) The ultrastructure of Descemet’s membrane appears to be composed of stacked globular domains arranged in a polygonal lattice alongside more elongated structures interspersed with proteoglycans within the internodal regions. 3) Our studies have helped validate Col4a2 mice as a promising Fuchs’ endothelial corneal dystrophy model. 4) Our investigation into posterior corneal surgery revealed ultrastructural changes that occur post-surgery at the graft interface.

The work presented in this thesis is a collection of papers from research spanning over 25 years. The research commenced whilst the candidate was employed in the Tennent Institute of Ophthalmology and later the Department of Anatomy at the University of Glasgow and continued in the School of Anatomy & Human Biology, The University of Western Australia. The research focuses on the biology of immune cells, primarily dendritic cells (DC), macrophages and mast cells, in the context of various components of the eye, including the aqueous outflow pathways, iris, cornea, ciliary body, choroid and retina, and the supporting tissues (lids and conjunctiva). The studies are broad in the sense that they deal with the role of these cells in development (such as removing the vascular networks around the developing lens), their normal homeostasis and function (distribution, phenotype, turnover and functional activity) and their role in models of a number of eye diseases. The findings are important in understanding the pathogenesis of diseases including bacterial keratitis, anterior uveitis, autoimmune uveoretinitis (endogenous posterior uveitis), toxoplasmic retinochoroiditis, age-related macular degeneration and ocular allergic responses, namely any ocular disease with an immune-mediated pathology. Many of the findings in the enclosed papers were firsts in the field and have shaped our understanding of ocular inflammatory disease. In part the success of some these studies was due to the novel method of performing immunostaining on tissue wholemounts dissected from small rodent eyes. These preparations provided unique ‘plan’ views of the complex networks of DCs and macrophages in the iris and choroid which previously had not been appreciated. In addition, the wholemount approach used in the characterisation and study of immune cells in delicate eye tissues, were applied to related studies of the meninges and choroid plexus of the brain. These studies were amongst the first to fully characterise distinct DC and macrophage networks in the pia, arachnoid, dura and choroid plexus. The research presented in the more recent publications have utilised a range of transgenic, knock-out, congenic and chimeric mouse models to elucidate the function of immune cells in the eye.

Glaucoma is a progressive disease of the optic nerve that can result in irreversible loss of visual function and impairment in everyday visual tasks. The experimental studies described in this thesis primarily aim to investigate the performance of people with glaucoma on search and other visual tasks whilst simultaneously monitoring eye movements, making comparison with age-related visually healthy people. In an experiment focussing on visual search, a patient group (n=30) took significantly longer on average to find a target in images of everyday scenes than controls (n=30). Furthermore, comparison of eye movements made by the participants during this task revealed there was a statistically significant reduction (6%) in saccade rate in the patients compared to the controls, and that saccade rate correlated with performance. Similar differences in eye movements were observed when the same groups passively viewed a selection of images in a slideshow. A bivariate contour ellipse (BCE) analysis revealed that, on average, patients viewed smaller regions of the images compared to the controls. Eye movement differences between patients and controls were also examined in a different cohort of people with glaucoma (n=14) and visually healthy controls (n=22) whilst they watched a selection of Hazard Perception Test driving films. Saccade rate of the patients was found to increase by 9%, though results from the BCE analysis suggested the average size of viewing area was similar in both groups. Finally, a novel interview-based study of 50 people with glaucoma provides evidence that patients do not perceive their visual field defect as a black ‘tunnel’ effect, or as ‘black patches’, but more like blurred regions: this finding may, for example, impact on how glaucomatous visual field loss is depicted in patient information about the condition. In conclusion, the results from this thesis show how visual loss from glaucoma influences how patients perceive and react to their visual environment. The principal findings from the studies described in this thesis also show, for the first time, that eye movement analysis could provide a window into the functional deficits associated with glaucoma.