Thesis (Ph.D.)--University of Wollongong, 2005. / Typescript. EMBARGOED - This thesis is subject to a 12 month embargo (07/03/06 to 07/03/07) and may only be viewed and copied with the permission of the author. For further information please Contact the Archivist. Includes bibliographical references: leaf 236-266.

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A 3- to 5-fold increase in Ca2+ accompanies cataract formation induced by selenite. The mechanism of selenite cataractogenesis involves calcium activation of calpain with subsequent proteolysis within the lens nucleus. This study was undertaken to investigate the biochemical mechanisms that lead to calcium accumulation in these circumstances. The components responsible for rat lens calcium regulation were defined by using either lens membrane vesicle preparations or intact lenses. Both Na+ gradient-dependent Ca2+ uptake and efflux occurred in lens membrane vesicles. Experiments with intact lenses showed that Na + ICa2 + exchange plays an important role in lens calcium regulation. ATP-dependent Ca2+ uptake and Ca2+ -dependent ATP hydrolytic activity have been characterized in lens membrane vesicles. Therefore, both Ca2+ -ATPase and Na + ICa2+ exchange participate in rat lens calcium regulation. Calcium accumulation in lenses treated by selenite may result from either increased influx (via non-selective cation channel), decreased efflux (via Ca2 +-ATPase and Na+ ICa 2+ exchange) or both. The selenite effects on the different components involved in lens calcium regulation were tested. / Ph. D.

"A thesis submitted in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy". / Thesis (PhD) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2007. / Bibliography: p. 243-277. / Introduction -- A convenient synthesis of 30HKG -- Facile synthesis of the UV filter compounds 30HKyn and AHBG -- Synthesis, identification and quantification of novel human lens metabolites -- Modification of bovine lens protein with UV filters and related metabolites -- Effect of UV light on UV filter-treated lens proteins -- Conclusions and future directions. / The kynurenine-based UV filters are unstable under physiological conditions and undergo side chain deamination, resulting in α,β-unsaturated carbonyl compounds. These compounds can react with free or protein bound nucleophiles in the lens via Michael addition. The key sites of the UV filters kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn) modification in human lenses include cysteine (Cys), and to a lesser extent, lysine (Lys) and histidine (His) residues. Recent in vivo studies have revealed that 3-hydroxykynurenine-O-β-D-glucoside (3OHKG) binds to Cys residues of lens crystallins in older normal human lenses. As a result of this binding, human lens proteins become progressively modified by UV filters in an age-dependent manner, contributing to changes that occur with the development of age-related nuclear (ARN) cataract. Upon exposure to UV light, free UV filters are poor photosensitisers, however the role of protein-bound species is less clear. It has been recently demonstrated that Kyn, when bound to lens proteins, becomes more susceptible to photo-oxidation by UV light. Therefore, the investigation of 3OHKG binding to lens proteins, and the effect of UV light on proteins modified with 3OHKG and 3OHKyn, were major aims of this study. As a result of the role of these compounds as UV filters and their possible involvement in ARN cataract formation, it is crucial to understand the nature, concentration and modes of action of the UV filters and their metabolites present in the human lenses. Therefore, an additional aim was to investigate human lenses for the presence of novel kynurenine-based human lens metabolites and examine their reactivity.--As 3OHKG is not commercially available, to conduct protein binding studies, an initial aim of this study was to synthesise 3OHKG (Chapter 2). Through the expansion and optimisation of a literature procedure, 3OHKG was successfully synthesised using commercially available and inexpensive reagents, and applying green chemistry principles, where toxic and corrosive reagents were replaced with benign reagents and solvent-free and microwave chemistry was used. A detailed investigation of different reaction conditions was also conducted, resulting in either the improvement of reaction yields or reaction time compared to the literature method. Applying the same synthetic strategy, and using key precursors from the synthesis of 3OHKG, the UV filters 3OHKyn and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid-O-β-D-glucoside (AHBG), were also successfully synthesised (Chapter 3). / Chapter 4 describes the investigation of both normal and cataractous human lenses in an attempt to identify novel human lens metabolites derived from deaminated Kyn and 3OHKyn (Chapter 4, Part A). Initially, 4-(2-aminophenyl)-4-oxobutanoic acid (AHA), glutathionyl-kynurenine (GSH-Kyn), kynurenine yellow (Kyn yellow), 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid (AHB), glutathionyl-3-hydroxykynurenine (GSH-3OHKyn) and 3-hydroxykynurenine yellow (3OHKyn yellow) were synthesised and human lenses were examined for their presence. AHA and AHB were synthesised from similar precursors to those used in the synthesis of 3OHKG, while the GSH adducts and yellow compounds were synthesised from Kyn and 3OHKyn via base induced deamination. Following isolation and structural elucidation, AHA, AHB and GSH-Kyn were confirmed as novel human lens metabolites. They were quantified in low pmol/mg lens (dry mass) levels in normal and cataractous lenses of all ages, while GSH-3OHKyn, Kyn yellow and 3OHKyn yellow were not detected. In contrast to AHA, the lens metabolites AHB, GSH-Kyn and GSH-3OHKyn were found to be unstable at physiological pH. The spectral properties of these compounds suggest that they may act as UV filters. --Chapter 4 (Part B) also describes the identification and characterisation of a novel human lens UV filter, cysteinyl-3-hydroxykynurenine -O-β-D-glucoside (Cys-3OHKG). An authentic standard was synthesised via Michael addition of cysteine to deaminated 3OHKG. Cys-3OHKG was detected in low pmol/mg lens (dry mass) levels in normal lenses only after the 5th decade of life and was absent in cataractous lenses. Cys-3OHKG showed rapid decomposition at physiological pH. / Chapter 5 describes the identification and quantification of amino acids involved in covalent binding of 3OHKG to lens proteins. Model studies with bovine lens proteins and 3OHKG at pH 7.2 and 9.5 were undertaken. The amino acid adducts were identified via total synthesis and spectral analysis, and subsequently quantified upon acid hydrolysis of the modified lens proteins. Under both pH conditions, 3OHKG was found to react with lens proteins predominantly via Cys residues with low levels of binding also detected at Lys residues. Comparative studies with Kyn (pH 9.5) and 3OHKyn (pH 7.2 and 9.5) resulted in modified lens proteins at Cys residues, with only minor modification at Lys residues at pH 9.5. The extent of modification was found to be significantly higher at pH 9.5 in all cases. His adducts were not identified. 3OHKG-, Kyn- and 3OHKyn-modified lens proteins were found to be coloured and fluorescent, resembling those of aged and ARN cataractous lenses. In contrast, AHB and AHA, which can not form α,β-unsaturated carbonyl compounds, resulted in non-covalent modification of lens proteins. AHB may contribute to lens colouration and fluorescence as further reactions of this material yielded species that have similar characteristics to those identified from 3OHKyn modification. These species are postulated to arise via auto-oxidation of the o-aminophenol moiety present in both 3OHKyn and AHB.--In Chapter 6, the potential roles of 3OHKG and 3OHKyn, and the related species AHA and AHB, in generating reactive oxygen species and protein damage following illumination with UV light was examined. The UV filter compounds were examined in both their free and protein-bound forms. Kyn-modified proteins were used as a positive control. Exposure of these compounds to UV light (λ 305-385 nm) has been shown to generate H2O2 and protein-bound peroxides in a time-dependent manner, with shorter wavelengths generating more peroxides. The yields of peroxides were observed to be highly dependent on the nature of the UV filter compound and whether these species were free or protein bound, with much higher levels being detected with the bound species. Thus, protein-bound 3OHKyn yielded higher levels of peroxide than 3OHKG, with these levels, in turn, higher than for the free UV filter compounds. AHB-treated lens proteins resulted in formation of low but statistically significant levels of peroxides, while AHA-treated lens proteins resulted in insignificant peroxide formation. The consequences of these photochemical reactions have been examined by quantifying protein-bound tyrosine oxidation products (3,4-dihydroxyphenylalanine [DOPA], di-tyrosine [di-Tyr]) and protein cross-linking. 3OHKG-modified proteins gave elevated levels of di-Tyr, but not DOPA, whereas 3OHKyn-modified protein gave the inverse. DOPA formation was observed to be independent of illumination and most likely arose via o-aminophenol auto-oxidation. AHB- and AHA-treated lens proteins resulted in statistically insignificant di-Tyr formation, while a light independent increase in DOPA was observed for both samples. Both reducible (disulfide) and non-reducible cross-links were detected in modified proteins following illumination. These linkages were present at lower levels in modified, but non-illuminated proteins, and absent from unmodified protein samples. / This work has provided an optimised synthetic procedure for 3OHKG and other lens metabolites (Chapters 2 and 3). Four novel lens metabolites have been identified and quantified in normal and cataractous human lenses (Chapter 4). Subsequent experiments, described in Chapter 5, identified the major covalent binding sites of 3OHKG to lens proteins, while AHA and AHB showed non-covalent binding. Further work described in Chapter 6 showed that protein-bound 3OHKG, Kyn and 3OHKyn were better photosensitisers of oxidative damage than in their unbound state. Together, this research has provided strong evidence that post-translational modifications of lens proteins by kynurenine-based metabolites and their interaction with UV light appear, at least in part, responsible for the age-dependent colouration of human lenses and an elevated level of oxidative stress in older lenses. These processes may contribute to the progression of ARN cataract. / Mode of access: World Wide Web. / xxxix, 308 p. ill. (some col.)

The eye lens grows continuously throughout life and changes its shape as the eye changes focus from a distant to a near object (the process of accommodation). These changes are complex because they may affect not only the shape of the lens, but also its refractive index distribution. To date there has been no satisfactory technique for directly and non-invasively measuring these changes. In this study the refractive index distribution through the isolated lens was measured non-invasively using a novel MRI technique. The dependence of the refractive index value of lens tissue on its transverse relaxation rate (R2) was determined empirically from measurements on lens homogenate samples. Using a multi-spin-echo imaging sequence, data were acquired for constructing R2 maps of a central slice through the isolated lens. These R2 maps were transformed to refractive index maps using the empirically determined dependence of refractive index on R2. Using a standard algorithm for ray tracing through gradient index media, the propagation of light rays through the index map were simulated. The optical properties of the lens, such as focal length, were then measured. The technique was validated by also directly measuring the focal length of each lens using laser ray tracing. The subtle changes in refractive index distribution that are responsible for the dramatic change in the optical properties of the isolated lens with age, were observed for the first time. The decrease in surface power of the isolated lens with age accounted only partially for the decrease in total lens power with age, the remainder resulting from a reduction in the gradient of refractive index (GRIN) power. It is likely that this reduction in GFUN power is the mechanism by which the eye maintains emmetropia (good distant vision) with age despite the increasing curvature of its surfaces. The reduction in the GRIN power of the lens was found to be mainly due to a flattening of the refractive index profile in the central region of the lens, accompanied by steepening of the profile near the edge of the lens. In agreement with a previous MRI study of the isolated human eye lens, this study found a decrease in the refractive index of the nucleus with age. However the age related change in this study was not as large and not found to be statistically significant. The results demonstrate that existing simple models for the optics of the eye lens are inadequate to accurately describe its properties. Several more sophisticated models were considered in an attempt to describe better the age-dependent changes that occur in both the power of the lens and its longitudinal aberration. Mathematical modelling was also used to simulate the accommodative process and investigate possible changes in the index distribution of the lens that may occur with accommodation. A preliminary in vivo study was performed aimed at observing the change in the refractive index distribution of the eye lens with age and accommodation. These results demonstrated the feasibility of the technique for in vivo applications and showed that within experimental error there is little change in the central refractive index of the lens with age. However the resolution achievable with standard clinical imaging sequences and signal detection hardware was not optimal for in vivo refractive index mapping of changes in the human eye lens with accommodation. Finally therefore, methods for refining the technique for in vivo applications are discussed which may make it possible to directly and simultaneously measure both the shape and refractive index distribution of the lens with age and accommodation.

Milieu où les ultrasons se propagent environ 10% plus rapidement qu’au sein des tissus environnants, le cristallin est connu pour être la source majeure d’aberrations de phase du milieu oculaire. De fait, l’échographie ophtalmique trans cornéenne est affectée par ses effets qui se manifestent sur les B-scans par une dégradation marquée de la résolution spatiale et du contraste, accompagnée de plus, d’importantes distorsions, particulièrement notables au niveau du fond de l’œil. Face à ce problème et en vue de l’arrivée prochaine de barrettes US HF dans la pratique ophtalmologique, un beamforming adapté a été développé au cours de cette thèse. Basé sur un lancer de rayon à 2 points fixes, il permet le calcul de délais de focalisation qui compensent les aberrations induites par le cristallin, en prenant en compte les effets réfractifs à son interface avec les humeurs. Les résultats obtenus in vitro et ex vivo avec une barrette 20MHz et un échographe de recherche (ECODERM) sont rapportés. / In ophthalmic ultrasonography the crystalline lens is known to be the main source of phase aberration, as ultrasounds (US) propagate about 10% faster than in the surrounding intra-ocular medium. Hence, it impairs significantly both spatial and contrast resolution of axial B-scans, and in addition causes important distortion, especially on the ocular fundus. To deal with this issue and in view of the next coming of US arrays in ophthalmologic practice, we developed in this thesis an adapted beamforming (BF) free from crystalline lens aberrations. It lies on a two point ray tracing approach to compute focusing delays that take into account crystalline lens aberrations including refraction at the interface. Initially developed considering a uniform US velocity in the lens, the adapted BF has been extended to consider the velocity gradient that exists in the real lens. In vitro and ex vivo results obtained with a 20 MHz linear array driven by a US research scanner (the ECODERM) are reported.