Doctor of Philosophy / Department of Biochemistry / Dolores J. Takemoto / The avascular lens tissue depends on the gap junction channels to facilitate intercellular communication for supplying cells deep within the lens with nutrients and removing waste products of cellular metabolism. In the absence of the protein synthesis machinery in the inner lens fiber cells, the proper regulation of gap junction channels becomes extremely important as disturbance of the lens homeostasis can lead to cataract development. Phosphorylation of gap junction subunit connexin proteins has been shown to play an important channel-modulating role in a variety of tissue. Protein kinase C-[Gamma] (PKC[Gamma]) has been implicated in the phosphorylation of connexins in the lens. Here the role of PKC[Gamma] in the regulation of gap junction coupling in the mouse lens has been investigated. We have compared the properties of coupling in lenses from wild type (WT) and PKC[Gamma] knockout (KO) mice. Western blotting, confocal immunofluorescence microscopy, immunoprecipitation, RT-PCR and quantitative real time PCR were used to study gap junction protein and message expression; gap junction coupling conductance and pH gating were measured in intact lenses using impedance studies. PKC[Gamma]was found to regulate the amount and distribution of Cx43 in the lens. Gap junction coupling conductance in the differentiating fibers (DF) of PKC[Gamma] KO lenses was 34% larger than that of WT. In the mature fiber (MF), the effect was much larger with the KO lenses having an 82% increase in coupling over WT. Absence of PKC[Gamma] in the KO mice also caused abnormal persistence of nuclei in the typical nucleus-free region in the DF. These results suggest a major role for PKC[Gamma] in the regulation of gap junction expression and coupling in the normal lens mediated by phosphorylation of the lens connexins. This becomes very vital in the diabetic lenses which contain a depleted amount of PKC[Gamma] and people suffering from spinocerebellar ataxia type-14 (SCA14) who have a mutated inactive form of PKC[Gamma]. Prolonged exposure of lenses to oxidative stress in these patients can lead to cataract formation. In cultured human lens epithelial cells (HLECs), 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated the depletion of Cx43 protein level via PKC-mediated phosphorylation of Cx43. At the same time Cx46 protein and message levels were upregulated in response to TPA treatment. So, the PKC activator regulates Cx43 and Cx46 in opposing ways. The possible mitochondria localization of Cx46 reported here could help in finding the non-junctional roles for Cx46.

Thesis (PhD)--Stellenbosch University, 2001 / ENGLISH ABSTRACT: The eye is that biological instrument which conveys the light of the external world into the inner world of the mind, wherein we receive the miraculous gift of vision. So precious is this gift, that Science must search for ways to keep this portal clear for the flow of light. Indeed, Science is called upon to “make war upon the bloody tyrant, Time.” (Shakespeare W. Sonnet No. 16). For, in the course of ageing, the lens grows cloudy and cataractous. In this battle between Science and Time, we are fortunate to live in an era in which Science is uncovering the molecular basis for the various obstacles to vision. The question arises, whether or not, the ruinous hand of time can be stayed. Due to unrelenting, progressive lens opacification, most of the elderly are destined to be subjected to loss of vision and with passage of time, even blindness. Globally the cataract surgery rate is inadequate to keep pace with the ever growing demand on financial and human resources created by the cataract problem. An immense challenge therefore is directed to primary eye care: “Can cataract be prevented or can its onset at least be postponed?” This laudable ultimate aim can only be achieved once the etiology of cataractogenesis is well understood. This dissertation seeks to examine two previously unrecognized etiological aspects that, if correctly understood and managed, have the potential to achieve preventive ophthalmological goals that may indeed help to stay the ‘ruinous hand of time’. The first aspect deals with the role of lipids and was examined using a study group of dyslipidemic subjects. The first part of the study concluded that dyslipidemic patients develop cortical lens opacities more frequently and at an earlier age than the normal population, and that cortical lens opacities should be regarded as one of the most reliable clinical signs of dyslipidemia. Furthermore, an extremely strong correlation was found to exist between low HDL Cholesterol levels and the development of opacities. Below a HDL-Cholesterol level of 1,5mmol/l, subjects had more than seven-fold higher risk of falling in the lens opacity subgroup than those with HDLCholesterol levels above 1,5mmol/l. An equally strong correlation was demonstrated between high (>5) LDLHDL ratios and the development of lens opacities. Subjects with a LDL:HDL-C ratio below 5 possessed a 2.35 times greater risk of having lenticular opacities than the group with a LDL:HDL-C ratio greater than 5. The prevention or retardation of dyslipidemia associated lens opacities is therefore possible, provided patients with a genetic predisposition are detected early and their blood lipids managed adequately. The second aspect deals with the relationship between age related cataracts and the acetylation status of the individual. This study compellingly submits that the slow acetylator pheno- and genotype may be regarded as a genetic indicator of risk for age related cataract. The ability accurately to classify a patient genotypically and phenotypically, may henceforth be useful in health counseling since, if an individual is identified as being a slow acetylator, additional preventative and precautionary measures may be taken, i.e. the prevention of UVexposure to the eye and caution with the ingestion of xenobiotics like caffeine, commercial dyes, food preservatives, and drugs. Furthermore, such a finding should be taken into account in the long term therapeutic management of glaucoma, with special regard to carbonic anhydrase inhibitors which are sulphonamide-related drugs and totally dependent on the N-acetyltransferase pathway for metabolism. These drugs may accumulate in the slow acetylator over time and exert toxic effects intra-ocularly, conceivably including cataractogenesis. The search for genetic and metabolic mechanisms that may contribute to human cataractogenesis should be pursued with great enthusiasm. This endeavour may help Science to achieve its primary objective, ablate the effects of Time and really aid in preventing cataracts in man. / AFRIKAANSE OPSOMMING: Die wondergawe van visie word vir ons moontlik gemaak deur die oog wat as biologiese instrument die lig van die buitewereld inlaat na die binnewereld van die brein. So kosbaar is hierdie gawe dat die Wetenskap deurgaans moet poog om die poort oop te hou. Inteendeel, die Wetenskap word gemaan deur Shakespeare in sy Sonnet nommer 16 om “oorlog te maak teen die bloeddorstige tiran, Tyd”. Soos ‘n mens ouer word, word die lens dof en ‘n katarak mag vorm. Ten spyte van hierdie stryd tussen ‘Wetenskap’ en ‘Tyd’ leef ons in die gelukkige era waarin die Wetenskap meer en meer leer van die verskeie obstruksies tot visie. Die vraag ontstaan of die rinnewerende hand van ‘Tyd’ gestuit sal kan word. Vanwee ongenaakbare, progressiewe lensvertroebeling is die meeste bejaardes bestem om aan visie verlies, en met verloop van tyd selfs blindheid, te ly. Die wereldwye katarakchirurgie tempo is nie voldoende om by te hou by die immergroeiende finansiele en mannekrag eise wat deur die katarak probleem gestel word nie. Daar word dus ‘n reuse uitdaging aan primere oogsorg gestel naamlik: “Kan katarakte nie eerder voorkom of die aanvang daarvan ten minste uitgestel word nie?” Hierdie prysenswaardige doelwit kan nie bereik word alvorens die etiologie van kataraktogenese goed verstaan word nie. Hierdie tesis ondersoek twee voorheen onerkende etiologiese aspekte wat, indien hulle korrek verstaan en hanteer word, beslis die potensiaal het om die gemelde voorkomende doelwitte te bereik en sekerlik te kan bydrae om die rinnewerende hand van Tyd te stuit. Die eerste aspek spreek die rol van lipiede aan deur te kyk na 'n studiegroep van dislipidemiese persone. Die eerste deel van die studie kom tot die gevolgtrekking dat dislipidemiese pasiente kortikale lens opasiteite meer dikwels en op ‘n vroeer ouderdom ontwikkel as die normale populasie en dat sulke opasiteite beskou moet word as een van die mees betroubare kliniese tekens van dislipidemie. Daar is ook ‘n baie sterk korrelasie gevind tussen lae HDL cholesterol vlakke en die voorkoms van opasiteite. Persone in die studie met ‘n HDL cholesterol vlak laer as 1,5mmol/l het ‘n sewe keer hoer kans gehad om in die lensopasiteit subgroep te val as die met ‘n HDL cholesterol vlak laer as 1,5mmol/l. ‘n Sterk korrelasie tussen ‘n hoe (>5) LDLHDL verhouding en die voorkoms van lens opasiteite is ook gevind. Persone met ‘n LDLHDL verhouding >5 het ‘n 2.35 maal groter risiko gehad om lensopasiteite te he as die met ‘n LDL:HDL verhouding van <5. Die voorkoming of vertraging van dislipiedemie geassosieerde lens opasiteite is dus moontlik, solank persone met ‘n genetiese geneigdheid daartoe vroeg ontdek en hulle bloedlipiede voldoende beheer word. Die tweede deel van die tesis handel oor die verhouding tussen ouderdoms verwante katarakte en die asetilasie status van die individu. Met oortuiging kom hierdie studie tot die gevolgtrekking dat die stadige asetilator fenoen genotipe as 'n genetiese merker vir ouderdoms verwante katarakte beskou moet word. Die vermoe om ‘n individu beide genotipies en fenotipies akkuraat te klassifiseer mag voorts bruikbaar wees in gesondheidsraadgewing. Indien ‘n individu geTdentifiseer is as ‘n stadige asetileerder, kan addisionele voorsorg maatreels getref word soos bv. die voorkoming van blootstelling van die oog aan UV lig sowel as omsigtigheid met die inname van xenobiotika soos kaffei'ene, kleurstowwe, voedsel preserveermiddels en geneesmiddels. Hierdie bevinding moet ook in berekening gebring word in die langtermyn terapeutiese hantering van gloukoom. Die koolsuuranhidrase inhibitore, dikwels gebruik in die behandeling van gloukoom, is sulfonamied-agtige middels en dus totaal afhanklik van die N-asetieltransferase pad vir hulle metabolisme. Hierdie middels kan ophoop in die stadige asetileerder en gegewe genoeg tyd, bes moontlik toksiese intra-okulere effekte tot gevolg he. Die soeke na genetiese en metaboliese meganismes wat mag bydra tot menslike kataraktogenese behoort nagestreef te word met groot entoesiasme. Hierdie strewe mag dalk net vir die 'Wetenskap' bystaan om sy primere mikpunt te bereik, die effek van ‘Tyd’ te neutraliseer en te help om katarakte werklik te voorkom.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

No description available.

The anterior refracting surface of the eye is the thin tear film that forms on the surface of the cornea. When a contact lens is on worn, the tear film covers the contact lens as it would a bare cornea, and is affected by the contact lens material properties. Tear film irregularity can cause both discomfort and vision quality degradation. Under normal conditions, the tear film is less than 10 microns thick and the thickness and topography change in the time between blinks. In order to both better understand the tear film, and to characterize how contact lenses affect tear film behavior, two interferometers were designed and built to separately measure tear film behavior in vitro and in vivo. An in vitro method of characterizing dynamic fluid layers applied to contact lenses mounted on mechanical substrates has been developed using a phase-shifting Twyman- Green interferometer. This interferometer continuously measures light reflected from the surface of the fluid layer, allowing precision analysis of the dynamic fluid layer. Movies showing this fluid layer behavior can be generated. The fluid behavior on the contact lens surface is measured, allowing quantitative analysis beyond what typical contact angle or visual inspection methods provide. The in vivo interferometer is a similar system, with additional modules included to provide capability for human testing. This tear film measurement allows analysis beyond capabilities of typical fluorescein visual inspection or videokeratometry and provides better sensitivity and resolution than shearing interferometry methods. The in vitro interferometer system has measured the formation and break up of fluid layers. Different fluid and contact lens material combinations have been used, and significant fluid layer properties have been observed in some cases. This dissertation discusses the design of this interferometer along with analysis methods used. Example measurement results of different contact lens are presented highlighting the capabilities of the instrument. This dissertation also provides the in vivo interferometer design, along with the considerations that must be taken when designing an interferometer for on-eye diagnostics. Discussions include accommodating eye movement, design of null optics for a range of ocular geometries, and laser emission limits for on-eye interferometry in general.

Šios disertacijos tikslas – didelio tikslumo matuoklio, skirto šiluminio lęšio lazerių aktyviuosiuose elementuose matavimams sukūrimas, bei įvairių metodų, skirtų šiluminio lęšio įtakos mažinimui tyrimas. Matavimai buvo atliekami Shack‘o ir Hartmann‘o bangos fronto matuokliu, kuris buvo pritaikytas mažų skersinių matmenų (<1x1 mm) šiluminio lęšio matavimams. Pademonstruotas naujos konstrukcijos asferinis kompensatorius iš esmes mažinantis šiluminio lęšio aberacijų įtaką išilginio diodinio kaupinimo lazeriuose. Atlikti šiluminio lęšio aberacijų matavimai buvo panaudoti kompensatoriaus profilio skaičiavimams. Naudojant asferinį kompensatorių, pagamintą plonasienių dangų užgarinimo būdu, generacijos slenkstis sumažėjo daugiau kaip 3 kartus, taip pat ženkliai pagerėjo lazerio pluošto intensyvumo skirstinys. Darbe nagrinėjamas stiprinamų impulsų energijos didinimo būdas pasitelkiant eliptinę rezonatoriaus modą Yb:IAG lazeryje. Šis metodas leidžia kelis kartus padidinti stiprinamų impulsų energiją, išlaikant dirakciškai ribotus erdvinius pluošto parametrus. Taip pat, surasta lazerio rezonatoriaus konfigūracija, leidžianti palaikyti stipriai astigmatišką modą aktyviajame elemente, ir simetrišką modą lazerio išėjime. Pateikiami šiluminio lęšio matavimų rezultatai „atermalinės“ ir Ng orientacijos Yb:KGW kristaluose. Taip pat, parodėme, kad kristalo galų išsigaubimo įtaka Ng orientacijos Yb:KGW aktyviuosiuose elementuose gali siekti 50%. Atlikti tyrimai parodė, kad išbandyta... [toliau žr. visą tekstą] / In this thesis, analysis of thermal effects and various approaches for their mitigation in diode end pumped ultrafast lasers is presented. Experimental investigations were performed by employing Shack-Hartmann wavefront sensor which was adapted for measurements of thermal lens in diode end pumped lasers. During research, operation of high average power, diode-pumped, Nd:YVO4 laser with aspheric aberration corrector was investigated. Actual thermal lens measurements were conducted in order to design properly shaped aberration corrector that was manufactured using a thin film deposition technology. This allows us to conclude that employment of proper thermal lens aberration compensator allowed for laser threshold reducing and ensured improved output beam quality parameter M2 in wide pump power range. Prospects of output pulse energy scaling in diode end-pumped Yb:YAG laser by employing elliptical mode geometry was investigated. During our experiments, maximum average power in of ~ 5.5 W was obtained at repetition rates of 30-100 kHz, while in CW operation mode the 8 W output power was achieved. In spite of strongly astigmatic thermal lens due to optimized cavity design the output beam exhibits high spatial quality: beam quality parameter M2 in both vertical and horizontal plane was close to unity. Detailed comparative study of thermal effects in Yb doped KGW crystals with different orientation was performed. Measurements confirm, that anisotropic optical and thermal properties... [to full text]

In this thesis, analysis of thermal effects and various approaches for their mitigation in diode end pumped ultrafast lasers is presented. Experimental investigations were performed by employing Shack-Hartmann wavefront sensor which was adapted for measurements of thermal lens in diode end pumped lasers. During research, operation of high average power, diode-pumped, Nd:YVO4 laser with aspheric aberration corrector was investigated. Actual thermal lens measurements were conducted in order to design properly shaped aberration corrector that was manufactured using a thin film deposition technology. This allows us to conclude that employment of proper thermal lens aberration compensator allowed for laser threshold reducing and ensured improved output beam quality parameter M2 in wide pump power range. Prospects of output pulse energy scaling in diode end-pumped Yb:YAG laser by employing elliptical mode geometry was investigated. During our experiments, maximum average power in of ~ 5.5 W was obtained at repetition rates of 30-100 kHz, while in CW operation mode the 8 W output power was achieved. In spite of strongly astigmatic thermal lens due to optimized cavity design the output beam exhibits high spatial quality: beam quality parameter M2 in both vertical and horizontal plane was close to unity. Detailed comparative study of thermal effects in Yb doped KGW crystals with different orientation was performed. Measurements confirm, that anisotropic optical and thermal properties... [to full text] / Šios disertacijos tikslas – didelio tikslumo matuoklio, skirto šiluminio lęšio lazerių aktyviuosiuose elementuose matavimams sukūrimas, bei įvairių metodų, skirtų šiluminio lęšio įtakos mažinimui tyrimas. Matavimai buvo atliekami Shack‘o ir Hartmann‘o bangos fronto matuokliu, kuris buvo pritaikytas mažų skersinių matmenų (<1x1 mm) šiluminio lęšio matavimams. Pademonstruotas naujos konstrukcijos asferinis kompensatorius iš esmes mažinantis šiluminio lęšio aberacijų įtaką išilginio diodinio kaupinimo lazeriuose. Atlikti šiluminio lęšio aberacijų matavimai buvo panaudoti kompensatoriaus profilio skaičiavimams. Naudojant asferinį kompensatorių, pagamintą plonasienių dangų užgarinimo būdu, generacijos slenkstis sumažėjo daugiau kaip 3 kartus, taip pat ženkliai pagerėjo lazerio pluošto intensyvumo skirstinys. Darbe nagrinėjamas stiprinamų impulsų energijos didinimo būdas pasitelkiant eliptinę rezonatoriaus modą Yb:IAG lazeryje. Šis metodas leidžia kelis kartus padidinti stiprinamų impulsų energiją, išlaikant dirakciškai ribotus erdvinius pluošto parametrus. Taip pat, surasta lazerio rezonatoriaus konfigūracija, leidžianti palaikyti stipriai astigmatišką modą aktyviajame elemente, ir simetrišką modą lazerio išėjime. Pateikiami šiluminio lęšio matavimų rezultatai „atermalinės“ ir Ng orientacijos Yb:KGW kristaluose. Taip pat, parodėme, kad kristalo galų išsigaubimo įtaka Ng orientacijos Yb:KGW aktyviuosiuose elementuose gali siekti 50%. Atlikti tyrimai parodė, kad išbandyta... [toliau žr. visą tekstą]

Novel net shaping technique Laser Engineered Net shaping™ (LENS) laser based manufacturing solution (Sandia Corp., Albuquerque, NM); Laser can be used to deposit orthopedic implant alloys. Ti-35Nb-7Zr-5Ta (TNZT) alloy system was deposited using LENS. The corrosion resistance being an important prerequisite was tested electrochemically and was found that the LENS deposited TNZT was better than conventionally used Ti-6Al-4V in 0.1N HCl and a simulated body solution. A detailed analysis of the corrosion product exhibited the presence of complex oxides which are responsible for the excellent corrosion resistance. In addition, the in vitro tests done on LENS deposited TNZT showed that they have excellent biocompatibility. In order to improve the wear resistance of the TNZT system boride reinforcements were carried out in the matrix using LENS processing. The tribological response of the metal matrix composites was studied under different conditions and compared with Ti-6Al-4V. Usage of Si3N4 balls as a counterpart in the wear studies showed that there is boride pullout resulting in third body abrasive wear with higher coefficient of friction (COF). Using 440C stainless steel balls drastically improved the COF of as deposited TNZT+2B and seemed to eliminate the effect of “three body abrasive wear,” and also exhibited superior wear resistance than Ti-6Al-4V.

<p>While it is well known that exposure to radiation can result in cataract formation, questions still remain about the presence of a dose threshold in radiation cataractogenesis. Since the exposure history from diagnostic CT exams is well documented in a patient’s medical record, the population of patients chronically exposed to radiation from head CT exams may be an interesting area to explore for further research in this area. However, there are some challenges in estimating lens dose from head CT exams. An accurate lens dosimetry model would have to account for differences in imaging protocols, differences in head size, and the use of any dose reduction methods.</p><p>The overall objective of this dissertation was to develop a comprehensive method to estimate radiation dose to the lens of the eye for patients receiving CT scans of the head. This research is comprised of a physics component, in which a lens dosimetry model was derived for head CT, and a clinical component, which involved the application of that dosimetry model to patient data. </p><p>The physics component includes experiments related to the physical measurement of the radiation dose to the lens by various types of dosimeters placed within anthropomorphic phantoms. These dosimeters include high-sensitivity MOSFETs, TLDs, and radiochromic film. The six anthropomorphic phantoms used in these experiments range in age from newborn to adult.</p><p>First, the lens dose from five clinically relevant head CT protocols was measured in the anthropomorphic phantoms with MOSFET dosimeters on two state-of-the-art CT scanners. The volume CT dose index (CTDIvol), which is a standard CT output index, was compared to the measured lens doses. Phantom age-specific CTDIvol-to-lens dose conversion factors were derived using linear regression analysis. Since head size can vary among individuals of the same age, a method was derived to estimate the CTDIvol-to-lens dose conversion factor using the effective head diameter. These conversion factors were derived for each scanner individually, but also were derived with the combined data from the two scanners as a means to investigate the feasibility of a scanner-independent method. Using the scanner-independent method to derive the CTDIvol-to-lens dose conversion factor from the effective head diameter, most of the fitted lens dose values fell within 10-15% of the measured values from the phantom study, suggesting that this is a fairly accurate method of estimating lens dose from the CTDIvol with knowledge of the patient’s head size.</p><p>Second, the dose reduction potential of organ-based tube current modulation (OB-TCM) and its effect on the CTDIvol-to-lens dose estimation method was investigated. The lens dose was measured with MOSFET dosimeters placed within the same six anthropomorphic phantoms. The phantoms were scanned with the five clinical head CT protocols with OB-TCM enabled on the one scanner model at our institution equipped with this software. The average decrease in lens dose with OB-TCM ranged from 13.5 to 26.0%. Using the size-specific method to derive the CTDIvol-to-lens dose conversion factor from the effective head diameter for protocols with OB-TCM, the majority of the fitted lens dose values fell within 15-18% of the measured values from the phantom study.</p><p>Third, the effect of gantry angulation on lens dose was investigated by measuring the lens dose with TLDs placed within the six anthropomorphic phantoms. The 2-dimensional spatial distribution of dose within the areas of the phantoms containing the orbit was measured with radiochromic film. A method was derived to determine the CTDIvol-to-lens dose conversion factor based upon distance from the primary beam scan range to the lens. The average dose to the lens region decreased substantially for almost all the phantoms (ranging from 67 to 92%) when the orbit was exposed to scattered radiation compared to the primary beam. The effectiveness of this method to reduce lens dose is highly dependent upon the shape and size of the head, which influences whether or not the angled scan range coverage can include the entire brain volume and still avoid the orbit.</p><p>The clinical component of this dissertation involved performing retrospective patient studies in the pediatric and adult populations, and reconstructing the lens doses from head CT examinations with the methods derived in the physics component. The cumulative lens doses in the patients selected for the retrospective study ranged from 40 to 1020 mGy in the pediatric group, and 53 to 2900 mGy in the adult group.</p><p>This dissertation represents a comprehensive approach to lens of the eye dosimetry in CT imaging of the head. The collected data and derived formulas can be used in future studies on radiation-induced cataracts from repeated CT imaging of the head. Additionally, it can be used in the areas of personalized patient dose management, and protocol optimization and clinician training.</p> / Dissertation