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.

Fibrillins are modular, disulphide-rich glycoproteins that assemble into microfibrils in the extracellular matrix (ECM). These microfibrils are critical structural elements of many non-elastic and elastic connective tissues. They also regulate the availability of transforming growth factor-β signalling molecules in the ECM. Defects in microfibrils are associated with acquired and inherited connective tissue disorders. In particular, mutations in the human FBN1 gene, which encodes fibrillin-1, are associated with a spectrum of diseases, including Marfan syndrome (MFS). One of the proposed initial steps in microfibril assembly is the interaction between the N- and C-terminal regions of fibrillin monomers. The minimal regions of human fibrillin-1 required for an interaction in vitro were previously identified: the four N-terminal domains, from the fibrillin unique N-terminal (FUN) domain to the third epidermal growth factor-like (EGF) domain (FUN-EGF3), and the three C-terminal calcium-binding EGF-like (cbEGF) domains (cbEGF41-43). Here, fragments corresponding to these regions were produced and shown to interact in pull-down and surface plasmon resonance assays. In addition, the structure of the FUN-EGF3 fragment was determined using nuclear magnetic resonance spectroscopy. This showed the novel structure of the FUN domain and the interdomain interfaces in this region of fibrillin. Combining structural and sequence conservation data may help to identify regions of FUN-EGF3 important for binding to cbEGF41-43. Here, the interaction was probed by site-directed mutagenesis. However, substituting individual residues in FUN-EGF3 with alanine did not abrogate binding to cbEGF41-43. Three MFS-associated residue substitutions were also introduced into the FUN-EGF3 fragment. While they did not abolish the interaction with cbEGF41-43, they did cause misfolding. Two of these substitutions, N57D and W71R, also resulted in the defective secretion of a larger N-terminal fragment by fibroblast cells, suggesting a potential mechanism of disease pathogenesis. Although specific residues involved in the N-C interaction were not identified here, the FUN-EGF3 structure will be vital for understanding the molecular surfaces involved in microfibril assembly and growth factor binding.

A terapia fotodinâmica (TFD) atualmente é considerada uma modalidade terapêutica no fotoenvelhecimento, pois apresenta resposta satisfatória tanto no resultado estético como no tratamento e prevenção das lesões pré-malignas que geralmente acompanham a pele fotoenvelhecida. A destruição seletiva do tecido alvo ocorre através de uma reação química, onde se utiliza um agente fotossensibilizante que é exposto a uma determinada luz com comprimento de onda e na presença de oxigênio produz oxigênio \"singlet\" que é responsável pela citotoxicidade das células proliferativas e conseqüente morte celular. Foram estudadas 13 pacientes com fotoenvelhecimanto clínico que foram submetidas a três sessões de TFD-ALA tópico associado à luz vermelha (630nm) com intervalo quinzenal. Após 21 dias da ultima aplicação 12 pacientes apresentaram melhora do fotonvelhecimento; principalmente na coloração e textura da pele, clareamento das lesões de melanose solar, regressão da queratose actinica e melhora da flacidez. Houve também aumento do tecido colágeno e fibra elástica nas colorações de picrosirius e Weigert-oxona respectivamente. Em relação a modulação do sistema imune houve diminuição da população de linfócitos CD4 e CD8, aumento da interleucina-4 na epiderme e derme, diminuição da TNF&#945; na epiderme e aumento na derme.A população de células de Langerhans e o INF&#947; não apresentaram alterações. A TFD-ALA associada à luz vermelha se mostrou eficaz no tratamento do fotoenvelhecimento clínico, no aumento do colágeno e fibras elásticas e na modulação do sistema imunológico. / PDT is nowadays considered one of the therapies for photo-aging, since it gives a satisfactory response for both, aesthetic result as well as treating and preventing pre-malignous lesions that are a frequent outcome of photo-aged skin. The selective destruction of target tissue occurs through a chemical reaction, by using a photosensitizing agent exposed to a certain light with a wave length which, in the presence of oxygen, produces singlet oxygen responsible for the citotoxidity of the proliferative cells and consequent cellular death. Thirteen pacients with clinical photo-aging have been studied, submitted to three sections of topic ALA-PDT associated with red light (630nm) at fortnight?s intervals.After 21 days of the last treatment, 12 patients showed improved photo-aging, specially regarding skin colour and texture, whitening of the solar melanosis lesions, regression of actinic cheratosis and improvement of flacidity. There also occured an increase of colagenous and elastic fibre tissue in the picrosirius and Weigert-oxona methods respectively.. Regarding the modulation of the immune system, there was a decrease of the lymphocytes CD4 and CD8 population, an increase of interleucin-4 in the epidermis and in the dermis, decrease of TNF-&#945; in the epidermis and increase in the dermis. The Langerhans cells poulation and the INF-&#947; did not present alterations. The ALA-PDT associated to red light was effective for the treatment of clinical photo-aging, for colagenous and elastic fibres increase and in the imune system modulation.

A terapia fotodinâmica (TFD) atualmente é considerada uma modalidade terapêutica no fotoenvelhecimento, pois apresenta resposta satisfatória tanto no resultado estético como no tratamento e prevenção das lesões pré-malignas que geralmente acompanham a pele fotoenvelhecida. A destruição seletiva do tecido alvo ocorre através de uma reação química, onde se utiliza um agente fotossensibilizante que é exposto a uma determinada luz com comprimento de onda e na presença de oxigênio produz oxigênio \"singlet\" que é responsável pela citotoxicidade das células proliferativas e conseqüente morte celular. Foram estudadas 13 pacientes com fotoenvelhecimanto clínico que foram submetidas a três sessões de TFD-ALA tópico associado à luz vermelha (630nm) com intervalo quinzenal. Após 21 dias da ultima aplicação 12 pacientes apresentaram melhora do fotonvelhecimento; principalmente na coloração e textura da pele, clareamento das lesões de melanose solar, regressão da queratose actinica e melhora da flacidez. Houve também aumento do tecido colágeno e fibra elástica nas colorações de picrosirius e Weigert-oxona respectivamente. Em relação a modulação do sistema imune houve diminuição da população de linfócitos CD4 e CD8, aumento da interleucina-4 na epiderme e derme, diminuição da TNF&#945; na epiderme e aumento na derme.A população de células de Langerhans e o INF&#947; não apresentaram alterações. A TFD-ALA associada à luz vermelha se mostrou eficaz no tratamento do fotoenvelhecimento clínico, no aumento do colágeno e fibras elásticas e na modulação do sistema imunológico. / PDT is nowadays considered one of the therapies for photo-aging, since it gives a satisfactory response for both, aesthetic result as well as treating and preventing pre-malignous lesions that are a frequent outcome of photo-aged skin. The selective destruction of target tissue occurs through a chemical reaction, by using a photosensitizing agent exposed to a certain light with a wave length which, in the presence of oxygen, produces singlet oxygen responsible for the citotoxidity of the proliferative cells and consequent cellular death. Thirteen pacients with clinical photo-aging have been studied, submitted to three sections of topic ALA-PDT associated with red light (630nm) at fortnight?s intervals.After 21 days of the last treatment, 12 patients showed improved photo-aging, specially regarding skin colour and texture, whitening of the solar melanosis lesions, regression of actinic cheratosis and improvement of flacidity. There also occured an increase of colagenous and elastic fibre tissue in the picrosirius and Weigert-oxona methods respectively.. Regarding the modulation of the immune system, there was a decrease of the lymphocytes CD4 and CD8 population, an increase of interleucin-4 in the epidermis and in the dermis, decrease of TNF-&#945; in the epidermis and increase in the dermis. The Langerhans cells poulation and the INF-&#947; did not present alterations. The ALA-PDT associated to red light was effective for the treatment of clinical photo-aging, for colagenous and elastic fibres increase and in the imune system modulation.