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Exposure of the skin to ultraviolet radiation (UVR) results clinically in the formation of deep wrinkles and mottled pigmentation and histologically, in a vast remodelling of the dermal extracellular matrix (ECM), in particular the elastic fibre network. Fibrillin microfibrils and fibulin-5 are early biomarkers of photoageing, where a loss of these fibres from the dermal epidermal junction is apparent. A study by our group showed that isolated fibrillin microfibrils and fibronectin which are rich in amino acids which absorb energy from UVR (UV-chromophores) are susceptible to UVR-induced damage, whilst UV-chromophore poor collagen type I is not. This research, with other earlier studies, indicates that acellular mechanism may work in tandem with cell-mediated up-regulation of matrix metalloproteinases (MMPs) in the progression of photoageing. This thesis aims to: i) test whether acellular mechanisms of photoageing are a result of direct photon absorption and/or the photodynamic production of reactive oxygen species (ROS); ii) assess the functional consequences of UVB degradation on the susceptibility of fibrillin microfibrils to MMPs and; iii) assay whether ECM proteins are differentially susceptible to solar simulated radiation (SSR) or UVA (315-400nm) alone using physiologically relevant doses of irradiation. Isolated proteins were exposed to UVB (280-315nm) in depleted-O2 conditions and in the presence of deuterium oxide. Depleted-O2 conditions decreased and deuterium oxide conditions increased UVR-induced degradation. Isolated proteins also show a similar pattern of degradation when exposed to H2O2 as an exogenous source of ROS. These results indicate that ROS play an important role in the differential degradation of dermal proteins. MMPs-3 and -9 are both upregulated in the skin after exposure to UVR and have the ability to degrade elastic fibre components. After exposure to UVB, damaged fibrillin microfibrils become more susceptible to degradation by both MMPs-3 and -9. Chromophore-rich fibrillin microfibrils and fibronectin are susceptible to degradation by both SSR and UVA alone, whereas chromophore-poor collagens type I and VI and tropoelastin are not. These results support our previous findings that amino acid composition of proteins is a good indicator of their relative susceptibility to UV-induced damage with a physiologically relevant irradiation system. In conclusion this work shows that ROS are an important mediator of acellular mechanisms of photoageing and that amino acid composition is a good indication of relative susceptibility of proteins to both ROS and UVR. The ability to predict ROS-susceptible proteins also has wider implications for human ageing as a whole.

Context: Squamous cell carcinoma (SCC) is the second most common skin cancer. Ultraviolet radiation (UVR) exposure, its most important risk factor, has mostly been investigated in cross-sectional study designs. This study presents a comprehensive and longitudinal examination of determinants of SCC, including photoageing. Objective: To examine the determinants of SCC and its precursor condition of photoageing. Above all, the objective was to investigate the interplay of phenotypic traits; occupation and leisure-time sun exposure patterns; and personal exposures, in particular, tobacco smoking and life course sun exposure, upon the risk of SCC and photoageing. Setting and Design: This investigation formed part of a large community-based, long-term cohort study of skin cancer. The Nambour Skin Cancer Study (forthwith, the Nambour Study) began in 1986 and concluded in 2007. In 1986 a random sample of 2095 people (aged 20-69 years) from Nambour, Queensland participated in a skin cancer survey. In 1992, a 5 year field trial to assess the preventive actions of sunscreen and beta-carotene was initiated (n=1621). Regular full skin examinations were conducted to determine the presence of skin cancer and actinic skin damage. In 1994, participants detailed their life course sun exposure (n=1290). After the trial ended in 1996, participants continued to complete regular questionnaires and ascertainment of skin cancers continued to 2007. Participants: The participants were 1339 unselected adults aged 25 to 75 years who had taken part in the Nambour Study in 1992 and consented to the follow-up study. Methods: Life course sun exposure hours were estimated from questionnaires and the approximate UVR exposure for Nambour (latitude 26S) was determined. Descriptive analyses examined patterns of exposure within the population. Factors influencing the severity of photoageing were also investigated. Informed by these analyses, relative risks were calculated for determinants of SCC and population attributable risk percentage (PAR%) for key modifiable risk factors. To investigate tobacco smoking as a risk factor for SCC, systematic review and meta-analysis were performed. Exposure measures: Pigmentary phenotype, estimated UVR exposure, tobacco smoking, sun-related behaviours, e.g. sunscreen use. Outcome measures: Incident and histologically proven SCC of the skin from 1992 to 2007 was the main outcome assessed. Photoageing, assessed clinically and micro-topographically (Beagley and Gibson scale), was an intermediate outcome measure and an objective measure of cumulative sun exposure in the final SCC analysis. Results: Examination of self-reported UVR revealed mean annual exposures were highest in early life and older adulthood (older than 60 years.) Women reported spending less time in the sun than men in all stages of life (p<0.05) and the more sun-sensitive the person’s skin type, the less sun exposure was reported at each life stage. The role of tobacco smoking in cutaneous SCC was reviewed in the published literature and a small positive association was noted in the meta-analysis. However, few studies had adjusted, or adjusted adequately, for sun exposure. Within the Nambour Study, with adjustment for age, sex, skin phenotype, lifetime sun exposure, current and former smoking had no association with SCC (RR 1.2, 95%CI 0.7, 2.0 and RR 1.1, 95%CI 0.8, 1.5, respectively compared with lifelong non-smokers). In this study population, with moderate to severe photoageing at study baseline, increasing age, male sex, a sun-sensitive phenotype were found to increase the odds of more severe actinic damage (p<0.05). High or very high UVR exposure in adulthood predicted a greater severity (OR 2.2, 95%CI 1.3, 4.0). Finally, the determinants of SCC were examined. Increasing age (4% increase per year of life, 95%CI 3% to 5%), male sex (RR 1.4, 95%CI 1.1, 1.9) and fair skin (RR 4.7 95%CI 2.0, 11.4) were associated with SCC. Having light eye colour and fair or red hair also significantly increased SCC risk. Recalled life course sun exposure overall was not found to be associated with SCC. Signs of actinic damage at baseline were, however, very strongly associated with SCC. Recent sun exposure, defined as that reported in the period (1-2 years) before the occurrence of SCC or for those unaffected at the end of the study, was also examined. A strong positive association was found between high recent exposure and SCC (RR 2.1, 95%CI 1.3, 3.3). PAR% estimates of prominent modifiable risk factors for SCC suggested considerable potential for reduction in incidence for at-risk populations if recent sun exposure were reduced. Conclusions: Subjective measures of solar UVR exposure and objective measures of photoageing varied according to personal and phenotypic factors. The interplay between risk factors observed here highlight the need to control for confounding in investigating solar factors as causes of skin cancer. Although SCC occurred on the background of high cumulative UVR exposure, which was best determined with objective rather than recalled measures, recent UVR exposure was also important. Self-reported recent exposure being less subject to recall bias than reported life course exposure may have partly influenced this, but the impact of UVR acting as a tumour initiator and promoter is also likely to explain the relation of SCC to sun exposure in the recent past.

Photoaged skin caused by chronic ultraviolet radiation (UVR) is characterised clinically with hyperpigmentation, coarse skin texture and deep wrinkles; the worst outcome is skin cancer. Histological investigation of the alteration within major extracellular matrices (ECM; elastic fibres, fibrillar collagens) is essential study to understand the cellular effect on skin structure from UVR. This thesis used an acute dose of radiation to examine in humans in vivo the effect of UVR on ECM components before assessing whether a dietary intervention could protect skin from UVR damage. Green tea catechins (GTCs) have anti-oxidant properties and may be an interesting option as a systemic photoprotection agent. Hence this thesis assesses: 1) the effect of acute irradiation of skin on dermal ECM damage to see whether it mimics the changes observed in photoageing and; 2) whether dietary supplementation with GTC will provide dermal ECM protection. UV-induced change in elastic fibre network. Initially, the effect of two different UV light sources on elastic fibre protein (elastic fibres, fibrillin-rich microfibrils and fibulin-2 and -5 microfibrils) remodelling was performed. The effect of ultraviolet B vs full-spectrum solar simulated radiation (SSR) were investigated in a small sample of healthy Caucasian volunteers (n = 6 per group). At 24 hour after 3× MED irradiation, Weigert's resorcin–fuchsin stained elastic fibres showed a significant reduction regardless of irradiation protocol (UVB, P<0.01; SSR P<0.05). Specific components were identified by immunohistochemistry; a significant reduction in fibrillin-rich microfibrils (FRM) was observed in UVB-irradiated skin (P<0.05), whilst fibulin-5-positive microfibrils were only affected by SSR (P<0.05). The data revealed, therefore, differential effects on UV wavelength on ECM remodelling. SSR, the more physiologically relevant light source was used in subsequent studies Supplement effect in SSR-induced damage in elastic fibre. Fifty healthy volunteers were recruited to this randomised control trial to investigate whether GTC can protect skin from photodamage. Volunteers were randomized to GTC (1080 mg plus 100 mg vitamin C; n=25) or placebo (maltodextrin; n = 25) daily for 12-weeks with compliance assessed biochemically in urine samples. Of the n = 50 recruited, 44 volunteers completed the study. In baseline, UVR challenge resulted in a significant remodeling of the cutaneous elastic fiber system (P<0.001), particularly fibulin-2 and fibulin-5-positive microfibrils at 24-hr after 3×MED irradiation. In post-supplementation, fibulin-5 positive microfibrils were protected from UVR remodeling (% staining, mean ± SE; no UV, 18.1±0.89; UVR, 17.1±0.61; P=0.30) whilst no protection was seen in the placebo group (no UVR, 19.41±0.79; UVR, 17.69±0.61; P<0.05). Supplement effect in SSR-induced damage in collagenous matrix. In the identical experiment, collagenous matrices including synthesis of procollagen I was also examined as fibrillar collagens are the major ECM components providing strength within dermis. The fibrillar collagen and newly synthesised procollagen I were stained by Picrosirius red and immunohistochemistry respectively. At baseline, acute irradiation significantly reduced papillary dermal fibrillar collagens (P<0.001) and induced deposition of newly synthesised pro-collagen I (P=0.02). In post-supplementation, GTC enhanced the deposition of thin collagen fibres in the dermis. Whilst placebo showed no effect on the altered organisation of fibrillar collagens or deposition of pro-collagen I following the irradiation challenge, GTC protected the organisation of fibrillar collagens in the papillary dermis (P=0.97).This novel in vivo human study may be used to recapitulate elastic fibre and collagen changes associated with photoageing and may be useful for dissecting out the mechanisms underlying extracellular matrix damage in response to chronic sunlight exposure. Furthermore, in a randomized control trial, dietary GTC protected fibulin-5 microfibrils and collagen fibres in the papillary dermis from UV-mediated degradation. The mechanism by which this protection occurs requires further study.

The objective of this study is to characterize the mechanical and adhesive properties of polyvinylbutyral and determine the effect of plasticizer content and photoageing on these properties. This data would be used to understand how the performance of laminated safety glass is related to the choice of adhesive interlayer. Samples of PVB plasticized with 0 to 40% butyl benzyl phthalate by weight were analyzed using a single strain rate tensile test, dynamic mechanical thermal analysis and a 1350 peel test. Two regimes of behavior were identified. PVB with low plasticizer contents, up to 10%, acts as a glassy polymer at room temperature. At higher plasticizer concentrations, greater than 20%, the material becomes elastomeric, with increased ductility and decreased moduli. The adhesive strength of each of the formulations was similar if compared at their respective glass transition temperatures. At room temperature, the heavily plasticized formulations had good adhesive strength whereas the less plasticized formulations readily debonded from the glass. These results suggest that PVB plasticized with between 20 and 40% plasticizer content is highly suitable as an interlayer material. UV radiation exposure was used to simulate the effect of sunlight on PVB. Forty micron thick samples were irradiated for up to 1000 hours of exposure time and analyzed using FTIR spectroscopy. It was determined that photo degradation had occurred in the form of a breakdown of the acetal ring structure in the vinyl butyral function. Similar treatment of 0.75 mm thick samples produced no major degradation in the mechanical or adhesive properties of the material. This is an unexpected result that conflicts with the obvious degradation discovered during FTIR spectroscopic testing. This is most likely due to the increased thickness of the mechanical samples and corresponding lo\ver UV dosage per polymer chain. / Master of Science

Ce travail de thèse s’inscrit dans les thèmes de la photodégradation et de la biodégradation de polymère pouvant être ou non d’origine renouvelable. Il a pour principal objectif d’étudier la durabilité photochimique de différents polymères ou mélanges de polymères utilisés en particulier dans l’agriculture et donc soumis à un vieillissement climatique. La structure chimique des polymères est un des principaux paramètres susceptible d’influencer la photodégradation. Un ensemble de (co)polyesters comportant des unités aliphatiques, cycliques et / ou aromatiques a été sélectionné dans le but d’exprimer une relation structure / photodurabilité. Le photovieillissement des matériaux a été réalisé en conditions naturelles et en conditions accélérées. Des mécanismes de photo-oxydation ont été proposés pour chacun des matériaux à partir de l’évolution des propriétés viscoélastiques traduisant celle de la structure macromoléculaire. Dans ce contexte, l’étude de la durabilité de ces matériaux respectueux de l’environnement doit s’intéresser à des systèmes extrêmement complexes dont chaque constituant est susceptible d’évoluer. Cette caractéristique exige de mettre au point une méthodologie permettant de déterminer la composition d’un biocomposite et d’en suivre les modifications en cours de vieillissement en même temps que l’évolution de la structuration de ces matériaux. / This thesis is part of the subject photodegradation and biodegradation of polymer that can be or not from renewable resources. Its main objective is to study the photochemical durability of various polymers or polymer blends used particularly in agriculture and therefore subject to weathering. The chemical structure of polymers is one of the main parameters that could influence the photodegradation. A set of (co)polyesters containing units aliphatic, cyclic and / or aromatic have been selected in order to express the relationships structure / photodurability. The photoageing of materials was carried out under natural and accelerated conditions. Photo-oxidation mechanisms have been suggested for each material based on the evolution of the viscoelastic properties reflecting the macromolecular structure. In this context, the study of the durability of these ecofriendly materials should address in highly complex systems in which each component is subject to change. This feature requires the development of a methodology to determine the composition of a biocomposite and monitor the changes during ageing at the same time as changing the structure of these materials.