Melanosome transfer between melanocytes and keratinocytes

RESUMO: A pele �� o maior ��rg��o do corpo humano e a sua pigmenta����o �� essencial para a sua colora����o e prote����o contra os efeitos nocivos da radia����o ultravioleta (UV). A pigmenta����o da pele resulta essencialmente de tr��s processos: a s��ntese e o armazenamento de melanina pelos melan��citos, em organelos especializados denominados melanossomas; o transporte dos melanossomas dentro dos melan��citos; e finalmente, a transfer��ncia dos melanossomas para os queratin��citos adjacentes. Nos queratin��citos, a melanina migra para a regi��o perinuclear apical da c��lula para formar um escudo protetor,respons��vel pela prote����o do DNA dos danos causados pela radia����o UV. Os melan��citos est��o localizados na camada basal da epiderme e contactam com 30-40 queratin��citos. Em conjunto, estas c��lulas formam a ���unidade melano-epid��rmica���. Apesar dos processos de s��ntese e transporte de melanina nos melan��citos estarem bastante bem caracterizados, os mecanismos moleculares subjacentes �� transfer��ncia inter-celular de melanina s��o menos conhecidos e ainda controversos. Dados preliminares obtidos pelo nosso grupo, que se basearam na observa����o de amostras de pele humana por microscopia electr��nica, indicam que a forma predominante de transfer��ncia de melanina na epiderme consiste na exocitose dos melanossomas pelos melan��citos e subsequente endocitose da melanina por queratin��citos. Para al��m disso sabe-se que as prote��nas Rab, que controlam o tr��fego membranar, est��o envolvidas em v��rias etapas de pigmenta����o da pele, nomeadamente na biog��nese e no transporte de melanina. Assim, dado o seu papel fundamental nestes processos, question��mo-nos sobre o seu envolvimento na transfer��ncia de melanina. Com este trabalho, propomo-nos a expandir o conhecimento atual sobre a transfer��ncia de melanina na pele, atrav��s do estudo detalhado dos seus mecanismos moleculares, identificando as prote��nas Rab que regulam o processo. Pretendemos tamb��m confirmar o modelo de exo/endocitose como sendo o mecanismo principal de transfer��ncia de melanina. Primeiro, explor��mos a regula����o da secre����o de melanina pelos melan��citos e analis��mos o papel de prote��nas Rab neste processo. Os resultados foram obtidos recorrendo a um m��todo in vitro, desenvolvido previamente no laborat��rio, que avalia a quantidade de melanina segregada para o meio de cultura por espectrofotometria, e ainda por microscopia, contando o n��mero de melanossomas transferidos para os queratin��citos. Atrav��s de co-culturas de melan��citos e queratin��citos, verificou-se que os queratin��citos estimulam a liberta����o de melanina dos melan��citos para o meio extra-celular, bem como a sua transfer��ncia para os queratin��citos. Al��m disso, a prote��na Rab11b foi identificada como um regulador da exocitose de melanina e da sua transfer��ncia para os queratin��citos. De facto, a diminui����o da express��o de Rab11b em melan��citos provocou a redu����o da secre����o de melanina estimulada por queratin��citos, bem como da transfer��ncia desta. Em segundo lugar, para complementar o nosso estudo, centr��mos a nossa investiga����o na internaliza����o de melanina por queratin��citos. Especificamente, usando uma biblioteca de siRNA, explor��mos o envolvimento de prote��nas Rab na capta����o de melanina por queratin��citos. Como primeira abordagem, us��mos esferas fluorescentes como substituto de melanina, avaliando os resultados por citometria de fluxo. No entanto, este m��todo revelou-se ineficaz uma vez que a internaliza����o destas esferas �� independente do recetor PAR-2 (recetor 2 ativado por protease), que foi previamente descrito como essencial na capta����o de melanina por queratin��citos Posteriormente, foi desenvolvido um novo protocolo de endocitose baseado em microscopia, usando melanossomas sem a membrana envolvente (melanocores) purificados do meio de cultura de melan��citos, incluindo um programa inform��tico especialmente desenhado para realizar uma an��lise semi-automatizada. Ap��s internaliza����o, os melanocores acumulam-se na regi��o perinuclear dos queratin��citos, em estruturas que se assemelham ao escudo supranuclear observado na pele humana. Seguidamente, o envolvimento do recetor PAR-2 na capta����o de melanocores por queratin��citos foi confirmado, utilizando o novo protocolo de endocitose desenvolvido. Para al��m disso, a necessidade de quatro prote��nas Rab foi identificada na internaliza����o de melanocores por queratin��citos. A redu����o da express��o de Rab1a ou Rab5b em queratin��citos diminuiu significativamente o n��vel de internaliza����o de melanocores, enquanto o silenciamento da express��o de Rab2a ou Rab14 aumentou a quantidade de melanocores internalizados por estas c��lulas. Em conclus��o, os resultados apresentados corroboram as observa����es anteriores, obtidas em amostras de pele humana, e sugerem que o mecanismo de transfer��ncia predominante �� a exocitose de melanina pelos melan��citos, induzida por queratin��citos, seguida por endocitose pelos queratin��citos. A pigmenta����o da pele tem implica����es tanto ao n��vel da cosm��tica, como ao n��vel m��dico, relacionadas com foto-envelhecimento e com doen��as pigmentares. Assim sendo, ao esclarecer quais os mecanismos moleculares que regulam a transfer��ncia de melanina na pele, este trabalho pode conduzir ao desenvolvimento de novas estrat��gias para modular a pigmenta����o da pele.----------------ABSTRACT: Skin pigmentation is achieved through the highly regulated production of the pigment melanin in specialized organelles, termed melanosomes within melanocytes. These are transported from their site of synthesis to the melanocyte periphery before being transferred to keratinocytes where melanin forms a supra-nuclear cap to protect the DNA from UVinduced damage. Together, melanocytes and keratinocytes form a functional complex, termed ���epidermal-melanin unit���, that confers color and photoprotective properties to the skin. Skin pigmentation requires three processes: the biogenesis of melanin; its intracelular transport within the melanocyte to the cell periphery; and the melanin transfer to keratinocytes. The first two processes have been extensively characterized. However, despite significant advances that have been made over the past few years, the mechanisms underlying inter-cellular transfer of pigment from melanocytes to keratinocytes remain controversial.Preliminary studies from our group using electron microscopy and human skin samples found evidence for a mechanism of coupled exocytosis-endocytosis. Rab GTPases are master regulators of intracellular trafficking and have already been implicated in several steps of skin pigmentation. Thus, we proposed to explore and characterize the molecular mechanisms of melanin transfer and the role of Rab GTPases in this process. Moreover, we investigated whether the exo/endocytosis model is the main mechanism of melanin transfer. We first focused on melanin exocytosis by melanocytes. Then, we started to investigate the key regulatory Rab proteins involved in this step by establishing an in vitro tissue culture model of melanin secretion. Using co-cultures of melanocytes and keratinocytes, we found that keratinocytes stimulate melanin release and transfer. Moreover, depletion of Rab11b decreases keratinocyte-induced melanin exocytosis by melanocytes. In order to determine whether melanin exocytosis is a predominant mechanism of melanin transfer, the amount of melanin transferred to keratinocytes was then assayed in conditions where melanin exocytosis was inhibited. Indeed, Rab11b depletion resulted in a significant decrease in melanin uptake by keratinocytes. Taken together, these observations suggest that Rab11b mediates melanosome exocytosis from melanocytes and transfer to keratinocytes. To complement and extend our study, we of melanin by keratinocytes. Thus, we aimed to explore the effect of depleting Rab GTPases on melanin uptake and trafficking within keratinocytes. As a first approach, we used fluorescent microspheres as a melanin surrogate. However, the uptake of microspheres was observed to be independent of PAR-2, a receptor that is required for melanin uptakecentred our attention in the internalization of melanin by keratinocytes. Thus, we aimed to explore the effect of depleting Rab GTPases on melanin uptake and trafficking within keratinocytes. As a first approach, we used fluorescent microspheres as a melanin surrogate. However, the uptake of microspheres was observed to be independent of PAR-2, a receptor that is required for melanin uptake.Therefore, we concluded that microspheres were uptaken by keratinocytes through a different pathway than melanin. Subsequently, we developed a microscopy-based endocytosis assay using purified melanocores (melanosomes lacking the limiting membrane) from melanocytes, including a program to perform a semi-automated analysis. Melanocores are taken up by keratinocytes and accumulate in structures in the perinuclear area that resemble the physiological supranuclear cap observed in human skin. We then confirmed the involvement of PAR-2 receptor in the uptake of melanocores by keratinocytes, using the newly developed assay. Furthermore, we identified the role of four Rab GTPases on the uptake of melanocores by keratinocytes. Depletion of Rab1a and Rab5b from keratinocytes significantly reduced the uptake of melanocores, whereas Rab2a, and Rab14 silencing increased the amount the melanocores internalized by XB2 keratinocytes. In conclusion, we present evidence supporting keratinocyte-inducedmelanosome exocytosis from melanocytes, followed by endocytosis of the melanin core by keratinocytes as the predominant mechanism of melanin transfer in skin. Although advances have been made, there is a need for more effective and safer therapies directed at pigmentation disorders and also treatments for cosmetic applications. Hence, the understanding of the above mechanisms of skin pigmentation will lead to a greater appreciation of the molecular machinery underlying human skin pigmentation and could interest the pharmaceutical and cosmetic industries.

Fungal communities in archives: assessment strategies and impact on paper conservation and human health

The main results presented in this PhD Dissertation have been published in interna-tional journals included in the Science Citation Index (SCI)

Portuguese banks and the economic adjustment programme: balance sheet adjustments and impact on profitability

Double Degree. A Work Project presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA ��� School of Business and Economics and a Masters Degree in Finance from Louvain School of Management

Light harvesting in solar cells using natural pigments from red fruits adsorbed to mesoporous TiO2

Nature has developed strategies to present us with a wide variety of colours, from the green of leaves to the bright colours seen in flowers. Anthocyanins are between these natural pigments that are responsible for the great diversity of colours seen in flowers and fruits. Anthocyanins have been used to sensitize titanium dioxide (TiO2) in Dye-Sensitized Solar Cells (DSSCs). DSSCs have become one of the most popular research topic in photovoltaic cells due to their low production costs when compared to other alternatives. DSSCs are inspired in what happens in nature during photosynthesis. A primary charge separation is achieved by means of a photoexcited dye capable of performing the electron injection into the conduction band of a wide band-gap semiconductor, usually TiO2. With this work we aimed to synthesize a novel mesoporous TiO2 structure as the semiconductor in order to increase the dye loading. We used natural occurring dyes such as anthocyanins and their synthetic flavylium relatives, as an alternative to the widely used metal complexes of Ru(II) which are expensive and are environmentally unsafe. This offers not only the chance to use safer dyes for DSSCs, but also to take profit of waste biological products, such as wine and olive oil production residues that are heavily loaded with anthocyanin dyes. We also performed a photodegradation study using TiO2 as the catalyst to degrade dye contaminants, such as those from the wine production waste, by photo-irradiation of the system in the visible region of the light spectrum. We were able to succeed in the synthesis of mesoporous TiO2 both powder and thin film, with a high capacity to load a large amount of dye. We proved the concept of photodegradation using TiO2 as catalyst. And finally, we show that wine production waste is a possible dye source to DSSCs application.