"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.)

This diploma thesis deals with the creation of engineering design of holder of projector lens. The thesis is created in cooperation with department of development in Varroc Lighting Systems, s.r.o. Initial part provides a brief description of the development of lighting technology used in car´s headlamps. The following part describes solutions currently used in clamping the lens in holder. The main part of the thesis focuses on the design of holder of projector lens itself. Projector lenses testing provide critical values of cohesive failure of the lens material, which cannot be exceed during the process of lens fitting and its subsequent clamping. In the final part the engineering design is verified and optimized using finite element analysis.

This dissertation proposes, studies and experimentally demonstrates novel liquid crystal (LC) optics to solve challenging problems in RF and photonic signal processing, freespace and fiber optic communications and microscopic imaging. These include free-space optical scanners for military and optical wireless applications, variable fiber-optic attenuators for optical communications, photonic control techniques for phased array antennas and radar, and 3-D microscopic imaging. At the heart of the applications demonstrated in this thesis are LC devices that are non-pixelated and can be controlled either electrically or optically. Instead of the typical pixel-by-pixel control as is custom in LC devices, the phase profile across the aperture of these novel LC devices is varied through the use of high impedance layers. Due to the presence of the high impedance layer, there forms a voltage gradient across the aperture of such a device which results in a phase gradient across the LC layer which in turn is accumulated by the optical beam traversing through this LC device. The geometry of the electrical contacts that are used to apply the external voltage will define the nature of the phase gradient present across the optical beam. In order to steer a laser beam in one angular dimension, straight line electrical contacts are used to form a one dimensional phase gradient while an annular electrical contact results in a circularly symmetric phase profile across the optical beam making it suitable for focusing the optical beam. The geometry of the electrical contacts alone is not sufficient to form the linear and the quadratic phase profiles that are required to either deflect or focus an optical beam. Clever use of the phase response of a typical nematic liquid crystal (NLC) is made such that the linear response region is used for the angular beam deflection while the high voltage quadratic response region is used for focusing the beam. Employing an NLC deflector, a device that uses the linear angular deflection, laser beam steering is demonstrated in two orthogonal dimensions whereas an NLC lens is used to address the third dimension to complete a three dimensional (3-D) scanner. Such an NLC deflector was then used in a variable optical attenuator (VOA), whereby a laser beam coupled between two identical single mode fibers (SMF) was mis-aligned away from the output fiber causing the intensity of the output coupled light to decrease as a function of the angular deflection. Since the angular deflection is electrically controlled, hence the VOA operation is fairly simple and repeatable. An extension of this VOA for wavelength tunable operation is also shown in this dissertation. A LC spatial light modulator (SLM) that uses a photo-sensitive high impedance electrode whose impedance can be varied by controlling the light intensity incident on it, is used in a control system for a phased array antenna. Phase is controlled on the Write side of the SLM by controlling the intensity of the Write laser beam which then is accessed by the Read beam from the opposite side of this reflective SLM. Thus the phase of the Read beam is varied by controlling the intensity of the Write beam. A variable fiber-optic delay line is demonstrated in the thesis which uses wavelength sensitive and wavelength insensitive optics to get both analog as well as digital delays. It uses a chirped fiber Bragg grating (FBG), and a 1xN optical switch to achieve multiple time delays. The switch can be implemented using the 3-D optical scanner mentioned earlier. A technique is presented for ultra-low loss laser communication that uses a combination of strong and weak thin lens optics. As opposed to conventional laser communication systems, the Gaussian laser beam is prevented from diverging at the receiving station by using a weak thin lens that places the transmitted beam waist mid-way between a symmetrical transmitter-receiver link design thus saving prime optical power. LC device technology forms an excellent basis to realize such a large aperture weak lens. Using a 1-D array of LC deflectors, a broadband optical add-drop filter (OADF) is proposed for dense wavelength division multiplexing (DWDM) applications. By binary control of the drive signal to the individual LC deflectors in the array, any optical channel can be selectively dropped and added. For demonstration purposes, microelectromechanical systems (MEMS) digital micromirrors have been used to implement the OADF. Several key systems issues such as insertion loss, polarization dependent loss, wavelength resolution and response time are analyzed in detail for comparison with the LC deflector approach. A no-moving-parts axial scanning confocal microscope (ASCM) system is designed and demonstrated using a combination of a large diameter LC lens and a classical microscope objective lens. By electrically controlling the 5 mm diameter LC lens, the 633 nm wavelength focal spot is moved continuously over a 48 [micro]m range with measured 3-dB axial resolution of 3.1 [micro]m using a 0.65 numerical aperture (NA) micro-objective lens. The ASCM is successfully used to image an Indium Phosphide twin square optical waveguide sample with a 10.2 [micro]m waveguide pitch and 2.3 [micro]m height and width. Using fine analog electrical control of the LC lens, a super-fine sub-wavelength axial resolution of 270 nm is demonstrated. The proposed ASCM can be useful in various precision three dimensional imaging and profiling applications.

Automotive radars have gained considerable interest in recent years for applications of road safety for vulnerable road users. The use of multipleinput and multiple-output (MIMO) technology in automotive radar has helped in realising a virtual aperture greater than the physical aperture of the antenna which has reduced the size of the overall radar module. But increasing the number of MIMO channels for greater angular resolution can introduce increased computational complexity, processing time and latency. A new type of radar using the multiple input multiple steered output (MIMSO) radar can alleviate these concerns by replacing the angle-FFT with beamforming by means of a lens. A double-layer lens with a beamforming layer and a radiating layer with a radiating aperture on the 2-D footprint of the lens is proposed as an antenna system for this new radar technique. This work focuses on the radiating aperture which has been realised as a travelling wave planar slotted array in gap waveguide technology due to its benefit of low losses and ease of manufacturing. A ridged gap waveguide is chosen for the reduction of the waveguide size and to avoid the appearance of grating lobes in the visible range for large scan angles. The planar slotted array is synthesised in the travelling wave configuration and reflection cancelling notches are used in the ridge to cancel the reflections from the slots. The aperture is chosen to be of a circular shape for a compact design and to maximise aperture efficiency. The planar array is verified with a full-wave simulation with a bandwidth of 76 to 81 GHz and a realised gain of 27.7 dBi at the centre frequency. The array can be scanned up to ±50◦ with a scan loss of 2.4 dBi. / Fordonsradarer har fått stort intresse under de senaste åren för tillämpningar av trafiksäkerhet för utsatta trafikanter. Användningen av MIMO-teknik (multipleinput och multiple-output) i bilradar har hjälpt till att realisera en virtuell bländaröppning som är större än antennens fysiska bländaröppning, vilket har minskat storleken på den totala radarmodulen. Men att öka antalet MIMOkanaler för större vinkelupplösning kan introducera ökad beräkningskomplexitet, bearbetningstid och latens. En ny typ av radar som använder MIMSO-radarn (multiple input multiple steered output) kan lindra dessa problem genom att ersätta vinkel-FFT med strålformning med hjälp av en lins. En dubbelskiktslins med ett strålformande skikt och ett strålande skikt med en strålande bländare på linsens 2D-fotavtryck föreslås som ett antennsystem för denna nya radarteknik. Detta arbete fokuserar på strålningsöppningen som har realiserats som en plan slitsad array i gap-vågledarteknologi på grund av dess fördel med låga förluster och enkel tillverkning. En vågledare med räfflade gap väljs för att minska vågledarstorleken och för att undvika uppkomsten av gitterlober i det synliga området för stora avsökningsvinklar. Den plana uppsättningen syntetiseras i den vandringsvågkonfigurationen och reflektionsupphävande skåror används i åsen för att eliminera reflektionerna från slitsarna. Bländaren är vald för att ha en cirkulär form för en kompakt design och för att maximera bländareffektiviteten. Planar arrayen verifieras med en helvågssimulering med en bandbredd på 76 till 81 GHz och en realiserad vinst på 27, 7 dBi vid mittfrekvensen. Arrayen kan skannas upp till ±50◦ med en skanningsförlust på 2, 4 dBi.

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Die komplexe Analysen großer Datensätze stellt in der Informationsvisualisierung eine zunehmende Herausforderung dar. Mit Hilfe von Magic Lenses wird die teilweise unübersichtliche Visualisierung dieser Datensätze lokal manipuliert und vereinfacht. Dabei spielen besonders der Umfang an Filterfunktionen und wie sie verändert werden können eine Rolle. Die Vorteile eines Multitouchdisplays im Vergleich zu herkömmlicher Maus- oder Keyboardinteraktion kommen dabei zum Einsatz. Es treten jedoch stetig Probleme bei der Adaption bisheriger Menüdesigns auf. Da Magic Lenses über Menüs parametrisiert werden sollen, stellt sich die Frage welche Menü- und Interaktionsarten besser dazu geeignet sind. In dieser Arbeit wird ein Multitouch-basiertes Kontextmenü für Magic Lenses evaluiert. Es befindet sich direkt am Linsenrand und ist kompakt jedoch neuartig. Es wird die Konkurrenzfähigkeit zu einem speziell für die Studie entworfenen und implementierten klassischen, globalen Menü getestet. Dieses ist für Nutzer vertrauter, aber distanziert sich von der Linse. Die Ergebnisse werden anschließend unter quantitativen und qualitativen Punkten zusammengefasst und ausgewertet. Es zeigt sich, dass klassische Menüs mit Touchinteraktion performanter waren. Das Multitouch-basierte Kontextmenü war allerdings bei den Probanden beliebter und wurde für die Arbeit mit Linsen bevorzugt. / The complex analysis of huge data sets is an increasing challenge in information visualization. With the help of emph{Magic Lenses} the somtimes confusing visualization of those data sets is being locally manipulated and simplified. Especially the amount of filter functions and how they can be altered matter. The advantages of multitouch displays in comparison to conventional mouse and keyboard interaction are used. However, there occur continual problems with the adaption of existing menu designs. Since emph{Magic Lenses} have to be parameterized with those menus the question arises which kind of menu and interaction are more fitting. In this work we evaluate a multitouch based context menu for emph{Magic Lenses}. It is located directly at the lens and compact but new to users. We test its competitiveness to a classical global menu specifically designed and implemented for this study. Users are more familiar with it but it is distanced from the lens. Finally, the results are summarized and analyzed under quantitative and qualitative points. It turns out that classical menus with touch interaction performed best. The multitouch-based context menu though was more popular with probands and was preferred for the work with lenses.

O objetivo do estudo foi analisar a cápsula e o epitéilo subcapsular cristaliniano (ESC) de pacientes submetidos à capsulotomia curvilínea contínua (CCC) utilizando o corante azul de tripano (AT) a 0,1%, através de microscopia óptica (MO), da técnica TUNEL, de imunohistoquímica e de microscopia eletrônica transmissão (MET). Realizamos um estudo prospectivo, controlado e randomizado utilizando 30 amostras de cápsulas e ESC obtidos de pacientes após CCC durante cirurgia de facectomia. Essas amostras foram divididas em dois grupos (15 espécimes cada) um utilizando o AT (grupo experimental) no ato cirúrgico e o outro sem o uso do corante (grupo controle). As cápsulas e o ESC destes grupos foram fixados e processados para análises estruturais posteriores com técnicas de MO de rotina, técnica TUNEL para detecção de morte celular por apoptose, imunohistoquímica para analisar a expressão da beclina-1 (um marcador de morte celular por autofagia), além de análise ultraestrutural por meio da MET. Foram realizadas análises morfométricas das imagens de microscopia após captura e digitalização, utilizando o programa Image Pró Plus (Cybernetics®, USA). Foram encontrados resultados positivos para a expressão de morte celular por apoptose e por autofagia no grupo submetido ao uso do AT, enquanto que no grupo controle os resultados foram negativos. As análises através da MET do ESC mostraram alterações em células coradas com o AT, incluindo ruptura mitocondrial, dilatação das cisternas do retículo endoplasmático, aumento da elétrondensidade citoplasmática e nuclear, e alteração no perfil nuclear. Os resultados estatísticos obtidos pelo teste de Mann-Whitney a partir da morfometria obtida de micrografias, demonstraram diferenças morfológicas significativas entre os grupos estudados, tanto nas dimensões dos maiores eixos nucleares, quanto na relação perímetro/área do núcleo celular (p=0,03). Em relação à espessura da cápsula, do epitélio subcapsular e do conjunto dessas estruturas obtidas a partir da MO de rotina não houve diferença estatisticamente significativa entre os grupos (p= 0,1). O AT provoca no ESC toxicidade celular com sinais indicativos de morte celular. Observamos nos aspectos morfológicos e moleculares morte celular tanto pelo mecanismo de apoptose quanto de autofagia. A partir destes achados podemos sugerir que a ação do AT, talvez possa ajudar a prevenir ou reduzir a opacificação da cápsula posterior do cristalino no período pós-operatório das facectomias / The purpose of this study was to evaluate the effect of trypan blue (TB) 0.1% staining on lens epithelial cells (LECs) and capsules of patients undergoing capsulorhexis using routine optical microscopy (OM), TUNEL technique, immunohistochemistry and transmission electron microscopy (TEM). In a prospective controlled and randomized study we evaluated 30 samples of capsules with LECs obtained after capsulorhexis during cataract surgery. Samples were randomly assigned to one of two groups (15 specimens each), one submitted to TB (experimental group) during the surgery and the other without the dye (control group). The capsule and the LECs of both groups were fixed and processed for later structural analysis with routine optical microscopy, immunohistochemistry for beclin-1 expression (a marker of cell death by autophagy), and the TUNEL technique to detect apoptosis, in addition to ultra-structural analysis by TEM. Morphometrical analysis were performed by using the Image Pro Plus software (Cybernetics®, USA). In the TB-stained group we have found positive results for the expression of cell death by autophagy and apoptosis while in the control group the results were negative. Analysis of LEC by TEM showed abnormalities in TB-stained cells including mitochondrial disruption, dilation of the endoplasmic reticulum cisterns, increased cytoplasmic and nuclear electron density and abnormalities in the nuclear profile. Statistical analysis using the Mann-Whitney test on morphometric data from micrographies showed significant morphologic differences between the two groups, both regarding longest nuclear axis difference and the ratio between the total nuclear perimeter and the cell area (p=0.03). No statistically significant difference was observed in capsule thickness, the LEC and the grouping of these two structures obtained from routine OM (p=0.1). Trypan blue is toxic to LECs, and cause abnormalities indicative of cell death. We observed molecular and morphologic aspects of cell death both by the mechanism of apoptosis and autophagy. Our findings lend support to the hypothesis that staining with 0.1% TB can help prevent or reduce the incidence of posterior capsule opacification following cataract surgery

<p>A previously designed lens system is constructed and characterized for possible application to engine diagnostics and 3D reconstructions of combusting flows. Since optical engines cannot be operated at the same conditions as a production engine, optical access to a production engine, with minimum changes in the engine geometry is necessary for developing a better understanding of the in-cylinder processes. The application of a probe designed to fit in the pressure transducer port of a diesel engine was demonstrated in this work. Measurement of various optical parameters established the ability of the lens system to have a good resolution over the entire stroke length of the cylinder. The temperature analysis of the probe and lens system confirmed its ability to withstand the high-temperature conditions in the engine cylinder head. The lens system design was coupled with imaging fibers to transmit images from the image plane of the probe to a high-speed camera located at a safe distance from the combusting environment. Due to the robust design of the probe and its compatibility with an imaging fiber, the probe was identified to be a good alternative as an inexpensive lens system for tomography in challenging environments. To validate its use, 3D reconstruction of a sewing pin using a range of views was demonstrated. Parameters affecting the reconstructions were identified and optimized to obtain high-quality reconstructions. </p>

Transforming growth factor beta (TGF-β) has been implicated in anterior subcapsular cataract (ASC) development. In the first section of this thesis, an in-vitro rat lens model was used to determine the role of matrix metalloproteinases during TGF-β-induced ASC. In the second part, an in-vivo TGF-β transgenic and Smad3 knockout model was used to examine the role of Smad3 signaling pathway in TGF-β-induced ASC development. Lastly, an in-vitro rat lens epithelial explant culture model was used to investigate the potential role of heat shock proteins (Hsps) in TGF-β-induced epithelial-mesenchymal transition (EMT). Optical, morphological and molecular changes were analyzed in theses studies. Results from cultured rat lenses show a significant increase of back vertex distance variability (decrease of sharpness and focus) during ASC development. Inhibition of MMPs eliminated the TGF-β-induced plaque formation. Similarly, the overexpression of TGF-β1 in transgenic mouse lenses leads to ASC formation and a decrease in lens optical quality in comparison to wild-type lenses, while TGF-β1/Smad3-/- (null) lenses show diminished TGF-β-induced effects. The plaques formed in the TGF-β1/Smad3-/- lenses are substantially smaller than in the TGF-β1/Smad3+/+ lenses. The morphological and molecular changes of TGF-β2/FGF-2 treated rat lens epithelial explants are similar to those found in the TGF-β2 treated rat lenses and transgenic TGF-β1 mouse lenses. Heat shock treatment prior to TGF-β treatment significantly reduced the effects of EMT in rat LECs. In conclusion, MMP inhibition prevented TGF-β-induced ASC formation whereas heat shock treatment and the absence of Smad3 protein expression only reduced the severity of TGF-β-induced effects.

Transforming growth factor beta (TGF-β) has been implicated in anterior subcapsular cataract (ASC) development. In the first section of this thesis, an in-vitro rat lens model was used to determine the role of matrix metalloproteinases during TGF-β-induced ASC. In the second part, an in-vivo TGF-β transgenic and Smad3 knockout model was used to examine the role of Smad3 signaling pathway in TGF-β-induced ASC development. Lastly, an in-vitro rat lens epithelial explant culture model was used to investigate the potential role of heat shock proteins (Hsps) in TGF-β-induced epithelial-mesenchymal transition (EMT). Optical, morphological and molecular changes were analyzed in theses studies. Results from cultured rat lenses show a significant increase of back vertex distance variability (decrease of sharpness and focus) during ASC development. Inhibition of MMPs eliminated the TGF-β-induced plaque formation. Similarly, the overexpression of TGF-β1 in transgenic mouse lenses leads to ASC formation and a decrease in lens optical quality in comparison to wild-type lenses, while TGF-β1/Smad3-/- (null) lenses show diminished TGF-β-induced effects. The plaques formed in the TGF-β1/Smad3-/- lenses are substantially smaller than in the TGF-β1/Smad3+/+ lenses. The morphological and molecular changes of TGF-β2/FGF-2 treated rat lens epithelial explants are similar to those found in the TGF-β2 treated rat lenses and transgenic TGF-β1 mouse lenses. Heat shock treatment prior to TGF-β treatment significantly reduced the effects of EMT in rat LECs. In conclusion, MMP inhibition prevented TGF-β-induced ASC formation whereas heat shock treatment and the absence of Smad3 protein expression only reduced the severity of TGF-β-induced effects.