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No / Skin pigmentation is a multistep process of melanin synthesis by melanocytes, its transfer to recipient keratinocytes and its degradation. As dyspigmentation is a prominent marker of skin ageing, novel effective agents that modulate pigmentation safely are being sought for both clinical and cosmetic use. Here, a number of plant extracts were examined for their effect on melanogenesis (by melanin assay and Western blotting) and melanin transfer (by confocal immunomicroscopy of gp100-positive melanin granules in cocultures and by SEM analysis of filopodia), in human melanocytes and in cocultures with phototype-matched normal adult epidermal keratinocytes. Mulberry, Kiwi and Sophora extracts were assessed against isobutylmethylxanthine, hydroquinone, vitamin C and niacinamide. Compared with unstimulated control, all extracts significantly reduced melanogenesis in human melanoma cells and normal adult epidermal melanocytes. These extracts also reduced melanin transfer and reduced filopodia expression on melanocytes, similar to hydroquinone and niacinamide, indicating their effectiveness as multimode pigmentation actives.

No / Although we have made significant progress in understanding the regulation of the UVR-exposed epidermal-melanin unit, we know relatively little about how human hair follicle pigmentation is regulated. Progress has been hampered by gaps in our knowledge of the hair growth cycle’s controls, to which hair pigmentation appears tightly coupled. However, pigment cell researchers may have overly focused on the follicular melanocytes of the nocturnal and UVR-shy mouse as a proxy for human epidermal melanocytes. Here, I emphasize the epidermis-follicular melanocyte pluralism of human skin, as research models for vitiligo, alopecia areata and melanoma, personal care/cosmetics innovation. Further motivation could be in finding answers to why hair follicle and epidermal pigmentary units remain broadly distinct? Why melanomas tend to originate from epidermal rather than follicular melanocytes? Why multiple follicular melanocyte sub-populations exist? Why follicular melanocytes are more sensitive to aging influences? In this perspective, I attempt to raise the status of the human hair follicle melanocyte and highlight some species-specific issues involved which the general reader of the pigmentation literature (with its substantial mouse-based data) may not fully appreciate.

Experimentelle Melanin-induzierte Uveitis (EMIU): Modulation der Leukozyten-Endothelzell-Interaktion durch Makrophagendepletion - intravitalmikroskopische Analysen. Einleitung: Die Experimentelle Melanininduzierte Uveitis (EMIU) dient als Modell für eine autoimmune Iridozyklitis und Choroiditis. Die frühe Entzündungsreaktion ist durch eine gesteigerte Leukozyten-Endothel-Interaktion gekennzeichnet. Um die Rolle von Makrophagen bei der Induktion der EMIU zu untersuchen, analysierten wir Veränderungen der Leukozyten-Endothel-Interaktionen in Irisvenolen anästhesierter Ratten nach Makrophagendepletion mit liposomalem Clodronat. Methoden: Die EMIU wurde durch intraperitoneale Injektion einer Emulsion aus 250 µg bovinen Melanosomen in komplettem Freund Adjuvant und Pertussistoxin bei Lewis Ratten induziert. Die Tiere wurden mit 2 ml Clodronat-Liposomen (Clodronat-lip) an den Tagen 2; 1; 4; 6 beziehungsweise 8 nach Immunisierung behandelt. Kontrolltiere erhielten anstelle von Clodronat-lip Leerliposomen (Kontrolle). Für die Intravitalfluoreszenzmikroskopie wurden Leukozyten intravasal mit Rhodamin 6G gefärbt. Anschließend wurden die postkapillären Irisvenolen am 4.; 6.; 8. und 10. Tag untersucht, um die Zahl der rollenden und fest am Endothel adhärenten Leukozyten zu quantifizieren. Weitere Parameter wie Zellzahl und Proteingehalt des Kammerwassers, TNF-alpha und IFN-gamma im Plasma und das Differentialblutbild wurden zur Charakterisierung der Entzündungsreaktion herangezogen. Ergebnisse: Bei makrophagendepletierten Tieren konnten spaltlampenmikroskopisch keine entzündlichen Veränderungen des Vorderabschnittes beobachtet werden. Der prozentuale Anteil rollender Leukozyten war am 8. Tag mit 2 +/- 1.1 vs. 15.2 +/- 1.6; 5.2 +/- 0.5% (Clodronat-lip vs. EMIU; Kontrolle, Mittelwert +/- MSF, ANOVA, p

Many non-human animal species use colour to signal dominance, condition or reproductive status. These signals have not previously been noted in humans. This thesis investigates the effects of skin colouration and pigmentation on the apparent health of human faces. Section 2 showed that individuals with increased fruit and vegetable and carotenoid consumption have yellower skin (Study 1) due to increased carotenoid pigmentation in the skin (Study 2). In Section 3, participants enhanced the redness, yellowness and lightness of the skin portions of colour-calibrated facial photographs to optimise healthy appearance. This suggests roles for blood (red) and carotenoid/melanin (yellow) colouration in providing perceptible cues to health. The contrast between lips and facial skin colour was not found to affect the apparent health of the faces, except in the b* (yellowness) axis, where enhanced facial yellowness caused an apparent blue tint to the lips. In Section 4 participants enhanced empirically-derived oxygenated blood colour more than deoxygenated blood colour to optimise healthy appearance. In two-dimensional trials, when both blood colour axes could be manipulated simultaneously, deoxygenated blood colour was removed and replaced with oxygenated blood colour. Oxygenated blood colouration appears to drive the preference for redness in faces. In Section 5 participants increased carotenoid colour significantly more than they increased melanin colour in both single-axis and two-dimensional trials. Carotenoid colour appears to drive the preference for yellowness in faces. In a cross-cultural study (Section 6), preferences for red and yellow in faces were unaffected by face or participant ethnicity, while African participants lightened faces more than UK participants. A preference for more redness in East Asian faces was explained by this group’s lower initial redness. The thesis concludes that pigments that provide sexually-selected signals of quality in many non-human animal species – carotenoids and oxygenated blood - also provide perceptible cues to health in human faces.

Les neurones exprimant l'hormone de mélano-concentration (MCH) sont observés dans l'hypothalamus postérieur de tous les vertébrés, de la lamproie à l'Homme. Ces neurones sont impliqués dans diverses fonctions comme le cycle veille/sommeil ou la prise alimentaire. Ils forment une population non homogène et au moins deux sous-populations sont reconnues, chez le rat. La première sous-population est composée de neurones nés au 11ème jour de vie embryonnaire (E11) qui projettent massivement sur les régions les plus postérieures du système nerveux central. La seconde est générée à E12/E13 et les neurones la caractérisant projettent sur les régions les plus antérieures du cerveau et expriment le peptide CART (cocaine and amphetamine regulated transcript) et le récepteur NK3 (neurokinine). L'objectif de notre travail était de comprendre l'origine de ces deux sous-populations. Pour cela, nous avons utilisé des approches histologiques, moléculaires et in vitro. Les neurones à MCH sont parmi les premiers neurones à naître et à différencier leur phénotype chimique le long d'une région longitudinale définie par une prolifération intense, appelée " cell cords " par Keyser en 1972. Cette bande longitudinale est caractérisée par l'expression de gènes comme Sonic Hedgehog (Shh), Nkx2.1, Nkx2.2 et a été récemment renommée " diagonale intrahypothalamica " ou ID. La différenciation des neurones à MCH dépend de l'expression du facteur morphogène Shh et ces neurones expriment Nkx2.1 et Nkx2.2, facteurs de transcription régulés positivement par Shh. Les neurones de la première sous-population envoient des projections le long du premier tractus longitudinal à se mettre en place, le tractus postopticus (tpoc). Ceux issus de la deuxième sous-population se différencient concomitamment au développement des régions télencéphaliques et leurs projections changent de direction pour innerver les régions antérieures du cerveau sous la dépendance de protéines de guidage axonal, Nétrine1 et Slit2. Nétrine1 permet d'attirer les axones MCH exprimant le récepteur DCC précocement vers la moelle épinière et plus tardivement vers le télencéphale alors que Slit2 contraint les axones MCH exprimant Robo2 à sortir de l'hypothalamus. L'étude du modèle " MCH " permet de mettre en lumière un changement d'organisation précocement au cours du développement dans l'axe longitudinal du prosencéphale. La bande longitudinale d'expression des facteurs de transcription Shh, Nkx2.2 peut être perçue comme une extension rostrale de la colonne neurogénique médiane déjà décrite chez des espèces d'invertébrés possédant une symétrie bilatérale. Les neurones générés le long de cette colonne le sont très tôt au cours du développement. / Neurons expressing melanin-concentrating hormone (MCH) are observed in the vertebrate posterior hypothalamus, from lampreys to humans. These neurons are involved in various functions such as sleep/wake cycle or food intake. They form a non-homogeneous population and at least two sub-populations are indentified in the rat. The first sub-population is composed of neurons born on the 11th embryonic day (E11) that project heavily on posterior regions of the central nervous system. The second is characterized by neurons born at E12/E13, projecting in anterior regions of the brain and expressing the peptide CART (cocaine and amphetamine Regulated Transcript) and the NK 3 receptor (neurokinin). The aim of this study was to understand the origin of these two sub-populations. For this, we used histological, molecular and in vitro approaches. MCH neurons are among the first neurons to be born and to differentiate their chemical phenotype along a longitudinal region defined by intense proliferation and called " cell cord " by Keyser in 1972. This longitudinal band is characterized by the expression of genes such as Sonic Hedgehog (Shh), Nkx2.1, Nkx2.2 and was recently named " diagonal intrahypothalamica " or ID. Differenciation of MCH neurons depends on expression of the morphogenetic factor Shh and these neurons express Nkx2.1 and Nkx2.2, transcription factors upregulated by Shh. The neurons of the first sub-population send projections along the tractus postopticus (tpoc), which is the first longitudinal tract to develop. Neurons of the second sub-population differentiate concomitantly to the development of the basal forebrain and their projections innervate anterior brain regions. Our results obtained in vitro showed that Netrin1 attracts MCH axons and that this reponse is mediated by DCC. Slit2 repulses MCH axons and this reponse is mediated by the Robo2 receptor. Overall, our study of the development of the MCH system shed light on an organizational change in the longitudinal axis of the forebrain during early development : a primary longitudinal organization characterized by the longitudinal expression of Shh and Nkx2.2 and the path of the tractus postopticus in the diencephalon and mesencephalon. MCH neurons of the first sub-population develop during this stage. Then, as the basal telencephalon extends and expresses Netrin1, the medial forebrain bundle differentiates, inducing a change in the main axis of the forebrain ; meanwhile MCH neurons of the second sub-population appear. MCH sub-populations reflect distinct developmental stages of the forebrain.

A legislação brasileira para cosméticos exige que os apelos mercadológicos desses produtos sejam comprovados. Os testes in vivo utilizando animais para avaliação desta categoria de produtos ou os princípios ativos nela contidos são, atualmente, bastante criticados. O presente trabalho teve como objetivo desenvolver um modelo de cultura de pele humana e avaliar a viabilidade dos melanócitos durante o período de 7 dias. A manutenção do tecido foi avaliada pela observação microscópica após coloração com HE e Masson, os melanócitos ativos pela reação de DOPA e a melanina pela coloração Fontana-Masson. Fragmentos de pele com 2mm2 mantidos em meio de Leibovitz à temperatura ambiente e atmosfera com 95% de ar e 5% de CO2, sofreram menos alterações morfológicas comparados aos mantidos em DMEM à temperatura de 37°C, 5% de CO2, 40% de O2 e 55% de N2. Fragmentos com 20mm2 mantidos em Leibovitz apresentaram alterações semelhantes aos de 2mm2. Células em divisão foram observadas em amostras de pele mantidas em Leibovitz enriquecido com SFB e ácido retinóico. A presença de melanina foi verificada durante todo o período de cultura, bem como a dos melanócitos, que se mostraram DOPA reativos. A radiação UVA/UVB, empregada com a finalidade de verificar se os melanócitos sofriam alguma alteração na atividade e morfologia, não provocou qualquer mudança nestas células, por outro lado induziu uma redistribuição da melanina nos queratinócitos dos fragmentos irradiados. Os resultados obtidos mostraram que é possível manter pele humana em cultura por 7dias, bem como a viabilidade dos melanócitos e sugere ser possível a aplicação do modelo estudado em futuros ensaios de eficácia e segurança de produtos tópicos. / The Brazilian law for cosmetics demands that the marketing of these products should be proofed. The tests in vivo utilizing animals for evaluation of this category of products or the active components in it, are very criticized nowadays. The present work had as an objective to develop a model of culture of human skin, and evaluate the viability of the melanocytes during a period of 7 days. The maintenance of the tissue was evaluated by the microscopic observation after coloring with HE and Masson, the active melanocytes by the DOPA reaction and the melanin by coloring of Fontana-Masson. Fragments of skin with 2mm2 kept in Leibovitz at room temperature and atmosphere with 95 % of air and 5 % of CO2, presented less morphologic alterations than the ones kept in DMEM at the temperature of 37, 5% of CO2, 40% of O2 and 55% of N2. Fragments with 20mm2 kept in Leibovitz presented similar alterations to the 2mm2. Mitotic cells were observed in samples of skin kept in enriched Leibovitz with FBS and retinoic acid. The presence of melanin was verified through all of the period of culture as well as the melanocytes that were DOPA-reactive. The radiation UVA/UVB, used with the aim of verifying if the melanocytes presented any alteration in the activity and morphology, didn\'t provoke any changes in the cells, on the other hand it induced a redistribution of the melanin in the keratinocytes of the irradiated fragments. The results obtained showed that it is possible to keep the human skin in culture for 7 days as well as the viability of melanocytes, and suggests the possibility of application of the studied model in future research on the efficacy and the safety of topic products.

A coloração dos vertebrados deve-se a presença de pigmentos, sintetizados e/ou armazenados em células denominadas células pigmentares cutâneas. A mudança de cor nos vertebrados é principalmente regulada por α-MSH e uma família de enzimas melanossômicas, que incluem tirosinase e as proteínas relacionadas à tirosinase 1 e 2 (TRP-1 e TRP-2, respectivamente). Sua ação está ligada à dispersão dos melanossomos ou síntese de melanina, processos que resultam em escurecimento do animal, enquanto a agregação ou inibição de síntese leva ao seu empalidecimento. Opsinas, como a melanopsina e a rodopsina, além de presentes na retina, podem ser expressas em células pigmentares cutâneas, intermediando foto-respostas de proliferação e de dispersão de melanossomos. O objetivo deste trabalho foi investigar a expressão temporal da rodopsina, tirosinase e do receptor MC1R, bem como os efeitos do tratamento com α-MSH 10-7 M, 10-8 M e 10-9 M por 24 horas sobre esses parâmetros, em melanócitos B16 de Mus musculus, mantidos em escuro constante. Através de PCR em tempo real (quantitativo) demonstrou-se que α-MSH 10-7 M não modula os níveis de mRNA para o receptor MC1R quando comparado com o grupo controle, contudo há uma evidente tendência de redução dos níveis do transcrito. Todavia, na concentração de 10-8 M, observou-se um aumento estatisticamente significativo no nível do transcrito na hora 20 quando comparado ao grupo controle e na concentração de 10-9 M o tratamento mostrou uma diminuição estatisticamente significativa no nível do transcrito entre o grupo controle e o tratado para cada ponto temporal analisado. Para a rodopsina, foi demonstrado que &alpha-MSH 10-7 M modula os níveis do mRNA quando comparado ao grupo controle, mostrando uma diminuição estatisticamente significativa na hora 0 e 16. Na concentração de 10-8 M houve um aumento estatisticamente significativo nos níveis do transcrito na hora 4 quando comparado ao grupo controle. Já, na concentração de 10-9 M, o hormônio induziu um robusto aumento no nível do transcrito quando comparado ao grupo controle para cada ponto temporal analisado. Nossos resultados são pioneiros em demonstrar a modulação de rodopsina por α-MSH, pois não há dados na literatura, seja em retina ou em outros tecidos, que tenham investigado essa ação do hormônio melanotrópico. O mesmo padrão foi observado para a tirosinase, demonstrando uma diminuição estatisticamente significativa na concentração de 10-7 M na hora 0 e um aumento significativo na concentração de 10-8 M na hora 8 e na concentração de 10-9 M na hora 12 e 8. Através de PCR em tempo real (quantitavo) nós demonstramos que α-MSH apresenta uma modulação dose-dependente para o transcrito do mRNA do receptor MC1R, tirosinase e rodopsina, mas não sincronizou a expressão desses genes, que permaneceram arrítmicos / In vertebrates, skin color is given by pigments, synthesized and/or stored in cutaneous pigment cells. The vertebrate color change is mainly regulated by α-MSH and a family of melanosome enzymes, which includes tyrosinase and tyrosinaserelated proteins 1 and 2 (TRP-1 and TRP-2, respectively). α-MSH action is associated with melanosome dispersion or melanin synthesis, processes which lead to skin darkening, whereas melanin aggregation or synthesis inhibition results in skin lightening. Opsins, such as melanopsin and rhodopsin, may be expressed in skin pigment cells, besides being present in the retina, and mediate non visual photoresponses such as cell proliferation and melanosome dispersion. The aim of this study was to investigate the temporal expression of rhodopsin, tyrosinase and the receptor MC1R, as well as the effects of 10-7 M, 10-8 M and 10-9 M α-MSH for 24 hours in Mus musculus B16 melanocytes, kept in constant darkness. Using real time PCR (quantitative) we demonstrated that 10-7 M α-MSH does not modulate MC1R mRNA levels, as compared to the control group, although a tendency to reduction was evident. On the other hand, at the concentration of 10-8 M, we observed a statistically significant increase of the transcript level at the hour 20, as compared to the control group and at the concentration of 10-9 M the treatment showed a statistically significant decrease of the transcript level for each temporal point analyzed. For rhodopsin, we showed that 10-7 M α-MSH modulates mRNA levels, as compared to the control group, demonstrating a statistically significant decrease at the hour 0 and 16. At the concentration of 10-8 M there was a statistically significant increase of transcript levels at the hour 4, as compared to the control group. The hormone at 10-9 M induced a robust increase of the transcript levels, as compared to the control group, for each time point analyzed. Our results are pioneering in demonstrating the regulation of rhodopsin by α-MSH, since there are no data in the literature which report the action of melanotropic hormone on rhodopsin in either the retina or other tissues. Similar pattern was observed for the tyrosinase gene, demonstrating a statistically significant decrease in the concentration of 10-7 M at the hour 0 and a significant increase in the concentration of 10-8 M at the hour 8 and in the concentration of the 10-9 M at the hour 12 and 8. Using real time PCR (quantitative) we demonstrated that α-MSH shows a dose-dependent modulation for mRNA transcripts of the MC1R receptor, tyrosinase and rhodopsin, but the hormone was not able to synchronize the expression of these genes, which remained arhythmic

A área incerto-hipotalâmica (IHy) está envolvida no controle neuroendócrino de fêmeas, com associação de suas células MCHérgicas (hormônio concentrador de melanina). Em ratos, a área pré-óptica medial (MPA), o núcleo periventricular anteroventral (AVPe) e o núcleo arqueado (Arc) são mais densamente inervadas pela IHy em fêmeas do que em machos, sugerindo um dimorfismo sexual das projeções da IHy relevantes para o controle reprodutivo. Nosso objetivo é caracterizar as áreas relacionadas ao controle reprodutivo inervadas pela IHy em camundongos fêmeas, utilizando traçador neuronal anterógrado, analisar a inervação das células GnRH (hormônio liberador de gonadotrofinas) da MPA pela IHy e analisar a inervação do AVPe e Arc pela IHy, usando camundongos Kiss1-hrGFP. Como resultado, observamos que MPA, AVPe e Arc recebem projeções da IHy, assim como áreas relacionadas ao circuito de defesa. Nos animais Kiss1-hrGFP, não encontramos inervação de células KiSS-1 pela IHy. Problemas metodológicos impossibilitaram a análise das projeções da IHy para os neurônios GnRH. / The incerto-hypothalamic area (IHy) is related to the neuroendocrine control of females, involving their MCHergic cells (melanin-concentrating hormone). In rats, the medial preoptic area (MPA), the anteroventral periventricular nucleus (AVPe) and the arcuate nucleus (Arc) are more densely innervated by IHy in females than in males, suggesting a sexual dimorphism of IHy projections relevant to reproductive control. Our aim is to characterize the areas related to reproductive control innervated by IHy in female mice using anterograde neuronal tracer, analyze the innervation of GnRH cells (gonadotropin-releasing hormone) of MPA by IHy and analyze the innervation of AVPe and Arc by IHy using Kiss1-hrGFP mice. As a result, we observed that MPA, AVPe and Arc are innervated by IHy, as well as areas related to the defensive circuit. In Kiss1-hrGFP animals, KiSS-1 cells were not innervated by IHy cells. Methodological problems made it impossible to analyze the projections of IHy to the GnRH neurons.

Melanina é um polímero constituído por uma grande heterogeneidade de monômeros tendo como característica comum a presença de grupos indóis. Por outro lado, a eumelanina produzida pela oxidação enzimática da tirosina é um polímero mais simples constituído principalmente de monômeros 5,6-dihidroxindol (DHI) e de indol-5,6-quinona (IQ). Tirosinase é a enzima chave na produção de melanina, sendo que a sua atividade cinética é medida em função da formação do intermediário dopacroma. Nanopartículas (NPs) de sílica são partículas nanométricas compostas de oxido de silício e são obtidas pelo processo sol-gel desenvolvido por Stöber de hidrólise e condensação de tetraetilortosilicato (TEOS), usando etanol como solvente em meio alcalino. As NPs foram funcionalizadas com 3-Aminopropiltrietoxissilano (ATPES) e depois com glutaraldeído. Este último permitiu a imobilização da tirosinase na superfície da sílica. Caracterizamos as NPs antes e após a reação da enzima, a atividade catalítica da enzima ligada à NP e o mecanismos de formação de melanina na superfície da sílica. As NPs foram caracterizadas por espectrofotometria de absorção e de reflectância, termogravimetria e microscopia eletrônica. A síntese da NP de sílica retornou partículas esféricas com 55nm de diâmetro e a funcionalização da partícula mostrou modificar eficientemente a sua superfície. A imobilização da tirosinase por ligação covalente foi de 99,5% contra 0,5% da adsorção física. A atividade da tirosinase foi caracterizada pela formação de dopacroma. O Km da enzima imobilizada não sofreu alteração em comparação com a tirosinase livre, mas a eficiência catalítica - que considera a eficiência recuperada - foi de apenas 1/3 para a enzima ligada covalentemente, significando que 2/3 das enzimas ligadas não estão ativas. Obtivemos NPs revestidas com melanina a partir de oxidação de tirosina solubilizada em duas preparações: NP com tirosinase ligada covalentemente na superfície e NP funcionalizada com glutaraldeido dispersa em solução de DHI e IQ. O revestimento de melanina foi na forma de um filme fino com espessura ~1,9nm, conferindo perfil de absorção luminosa equivalente ao da própria melanina. Mostramos que o mecanismo de polimerização passa pela oxidação da tirosina pela tirosinase, que gera intermediários oxidados (principalmente DHI e IQ) que vão para solução (mesmo quando a tirosinase está ligada covalentemente na sílica). Estes intermediários ligam-se ao glutaraldeido e a superfície da sílica passa a funcionar como ambiente de polimerização da melanina. / Melanin is a polymer consisting of a large heterogeneity of monomers having as a common feature the presence of indole groups. Contrarily, eumelanin produced by enzymatic oxidation of tyrosine is a simpler polymer consisting mainly of 5,6-dihidroxindol (DHI) and indole-5,6-quinone (IQ) monomers. Tyrosinase is the key enzyme in melanin production, and its kinetic activity is measured by the formation of the intermediate dopacroma. Nanoparticles (NPs) are made of silica nanoparticles of silicon oxide and are obtained by sol-gel method developed by Stöber of hydrolysis and condensation of tetraethylorthosilicate (TEOS), using ethanol as solvent in an alkaline medium. NPs were functionalized with 3-Aminopropyltriethoxysilane (ATPES) and then with glutaraldehyde. The latter allows the immobilization of tyrosinase on the silica surface. We characterized NPs before and after the reaction of the enzyme, the catalytic activity of the enzyme bound to the NP and melanin-forming mechanisms on the silica surface. NPs were characterized by absorption spectrophotometry and reflectance, electron microscopy and thermogravimetric analysis. The synthesis of silica NP returned spherical particles of 55nm diameter and particle functionalization showed efficiently modify its surface. The immobilization of tyrosinase by covalent bond was 99.5% versus 0.5% by physical adsorption. The activity of tyrosinase was characterized by the formation of dopacroma. The Km of the immobilized enzyme did not change compared to the free tyrosinase, but the catalytic efficiency - considering the recovered efficiently - was only 1/3 for the enzyme covalently bound, meaning that 2/3 of the enzymes are not connected active. We obtained melanin coated NPs from tyrosine oxidation in two preparations: NP with covalently bound tyrosinase in the NP surface and NP functionalized with glutaraldehyde dispersed in DHI and IQ solution. The melanin coating was in the form of a thin film with the thickness of ~ 1,9 nm, giving light absorption profile equivalent to that of melanin itself. We showed that the polymerization mechanism involves the oxidation of tyrosine by tyrosinase, which generates oxidized intermediates (especially DHI and lQ) that go into solution (even when tyrosinase is covalently bound to the silica). These intermediates bind the glutaraldehyde and the surface of the silica begins to function as an environment for melanin polymerization.