Embora exista considerável progresso na área de biossensores, ainda é primordial aprimorar muitos desses dispositivos. Este trabalho tem por objetivo contribuir para o contínuo crescimento dos biossensores a base de enzimas e de moléculas de DNA, sendo dois biossensores eletroquímicos descritos. O primeiro trata-se de um biossensor enzimático utilizando tirosinase (Tyr) imobilizada por intermédio de cistamina (CYS) e glutaraldeído (GA) para a detecção de compostos fenólicos. Eletrodos de carbono impressos (SPCE) foram modificados com nanobastões de ouro (AuNRs) em filme de poli(amido amina) (PAMAM) geração 4 para o favorecimento da transferência direta de elétrons (DET) entre o eletrodo e o sítio ativo da enzima. Para caracterizar os AuNRs e AuNRs-PAMAM, espectroscopia de absorção no UV-Visível, espalhamento de luz dinâmico (DLS) e microscopia eletrônica de varredura (SEM) foram empregadas. As etapas do biossensor foram estudadas por voltametria cíclica e linear, amperometria e microscopia de força atômica (AFM) na presença de dois analitos: catecol (CAT) e dopamina (DA). A faixa linear para CAT foi de 2,8 a 30,3 μmol L-1 com um limite de detecção (LD) de 1,0μmol L-1 enquanto para a DA, a faixa linear foi de 27,8 a 448,7 μmol L-1 e LD de 10,0 μmol L-1. Além de apresentar ótima resposta frente a possíveis interferentes, o sensor também mostrou excelente desempenho em amostras reais, que atrelados aos testes de repetibilidade e reprodutibilidade mostram a estabilidade e acurácia do biossensor. O segundo sensor, trata-se de um sensor de DNA impedimétrico em eletrodo de ouro para a detecção da mutação c.68_69del relacionada à predisposição ao câncer de mama. A imobilização da sequência de captura (SH-ssDNA) no eletrodo ocorreu pela ligação ouro-enxofre (Au-S) e o modelo de hibridização escolhido foi a hibridização direta. O genossensor distinguiu eficientemente entre as sequências alvo (tarDNA) e não-complementar (ncsDNA), apresentando faixa linear de 1,0 a 200,0 nmol L-1 e LD de 0,14 nmol L-1. Os resultados sugerem que ambos biossensores têm potencial e que as estratégias propostas são promissoras para o desenvolvimento de outros biossensores. / Despite a considerable progress in the area of biosensors, it is still crucial to improve most of these sensors. This study aims to contribute to the ongoing growth of enzyme- and DNA-based biosensors, being described two electrochemical biosensors. The first one is an enzyme-based biosensor with immobilized Tyrosinase (Tyr), through cystamine (CYS) and glutaraldehyde (GA), for detection of phenolic compounds. Screen-printed carbon electrodes (SPCE) were modified by gold nanorods (AuNRs) stabilized with poly(amide amine) PAMAM generation 4 to facilitate direct electron transfer (DET) between electrode and enzyme active site. AuNRs and AuNRs-PAMAM were characterized using UV-Visible absorption spectroscopy, dynamic light scattering (DLS) e scanning electron microscopy (SEM). Biosensor stages were studied by cyclic and linear voltametry, amperommetry and atomic force microscopy (AFM) and tested agains two analytes: catechol (CAT) and dopamine (DA). Detection limit (LD) for CAT is 1 μmol L-1 and linear range from 2.8 to 30.3 μmol L-1, for DA, LD is 10.0 μmol L-1 and linear range 27.8 to 448.7 μmol L-1. Besides, the biosensor shows great response in the presence of interferents, it also had an excellent performance in real samples that along with repeatability and reproducibility tests indicate stability and accuracy of the biosensor. The second sensor is an impedimetric DNA sensor prepared on gold electrode to detect c.68_69del mutation related to breast cancer predisposition. Capture sequence (HS-ssDNA) immobilization occurred due to gold-sulfur bond (Au-S) and direct hybridization was the chosen hybridization model. The genosensor was able to distinguishing between target sequence (tarDNA) and non-complementary sequence (ncsDNA) and linear range and LD were found to be 1.0 to 200.0 nmol L-1 and 0.14 nmol L-1, respectively. Results suggest both biosensors have potential and proposed strategies are promising for other biosensors development.

No description available.

Der monoklonale Antikörper SM5-1 wurde in Vorarbeiten mittels eines Immunisierungsprotokolls (Kooperation Prof. Guo, Cleveland, USA) von einer humanen Melanom-Zell-Linie gewonnen. In der vorliegenden Arbeit wurde an nicht-melanozytären benignen Geweben, 16 Nävi, 84 nicht-melanozytären Tumoren sowie 745 Melanomproben das Färbeverhalten von SM5-1 mittels Immunhistochemie charakterisiert. SM5-1 wurde zunächst mit HMB-45 und anti-S100 an 250 primären und 151 metastasierten Melanomen und in einer zweiten Stichprobe mit Antikörpern gegen Melan-A/MART-1 (A103) und Tyrosinase (T311) an 101 primären und 243 metastasierten Melanomen verglichen. Es zeigte sich, dass SM5-1 für normale Zellen negativ ist. SM5-1, anti-S100 und HMB-45 färbten 100% der Nävi und 97 - 99% der 250 primären Melanome. Während SM5-1 und anti-S100 96% der 151 Melanommetastasen korrekt identifizierten, waren für HMB-45 nur 83% der Proben positiv. Alle HMB-45 negativen Metastasen reagierten mit SM5-1. Weder SM5-1 noch HMB-45 färbten nicht-melanozytäre Tumore, wohingegen der unspezifischere Antikörper anti-S100 21 von 84 dieser Tumore färbte. Insgesamt wurde beim primären und metastasierten Melanom für SM5-1 eine Sensitivität von 98%, für HMB-45 von 93% und für anti-S100 von 97% beobachtet. Im Vergleich der Antikörper SM5-1, A103 und T311 färbten SM5-1 92,4%, A103 82,9% und T311 71,2% der insgesamt 344 primären und metastasierten Melanome. SM5-1 zeigte innerhalb einer Tumorzellpopulation ein homogeneres Färbeverhalten und eine höhere Färbeintensität bei den 243 Melanommetastasen als A103 und T311. Der monoklonale Antikörper SM5-1 zeigte gegenüber den gebräuchlichen Antikörpern erhebliche Vorteile bei der immunhistochemischen Beurteilung des Melanoms, insbesondere bei dessen Metastasen. Somit kann er als Marker der ersten Wahl bei der immunhistochemischen Diagnostik von Melanomen empfohlen werden. Um die Rolle des durch SM5-1 erkannten Antigens zu verstehen, sind jedoch noch weitere Untersuchungen notwendig. / Antibodies such as HMB-45 and anti-S100 protein have been widely used as markers of malignant melanoma. Using a subtractive immunization protocol in preliminary works (cooperation Prof. Guo, Cleveland, USA), the monoclonal antibody SM5-1 was generated from a mouse model of human melanoma. The immunhistochemical staining of SM5-1 was studied in paraffin-embedded specimens of normal non-melanocytic tissue, melanocytic nevi of the skin (n = 16), non-melanocytic neoplasms (n = 84) and 745 melanomas. 250 primary melanomas and 151 metastases were compared with HMB-45 and anti-S100 staining. Further 101 primary melanomas and 243 metastases were compared with Melan-A/MART-1 (A103) and tyrosinase (T311). Staining of normal cells for SM5-1 was found to be negative. SM5-1, anti-S100 and HMB-45 reacted with nevi and 97 - 99% of 250 primary melanomas. Whereas SM5-1 and anti-S100 showed a high degree of positive staining in 96% of 151 metastases, only 83% reacted with HMB-45. All HMB-45-negative melanoma metastases were found to be positive for SM5-1. Whereas neither SM5-1 nor HMB-45 stained any of 84 specimens from non-melanocytic neoplasms, anti-S100 was positive in 21/84. Altogether SM5-1 has a sensitivity of 98%, HMB-45 of 93% and anti-S100 of 97% for primary and metastatic melanomas. SM5-1 stained 92,3%, A103 82,9% and T311 71,2% of 344 primary and metastatic melanomas. SM5-1 showed a stronger and more homogeneous reactivity as A103 und T311 in metastases (n = 243). All tested antibodies had a comparable staining intensity for primary melanomas (n = 101). The monoclonal antibody SM5-1 appears to have advantages for the immunhistochemical analysis of melanoma over currently available antibodies, especially for melanoma metastases. Therefore it is useful as a first line reagent in immunohistochemistry of melanoma. Further studies are needed to elucidate the exact nature of the antigen recognized by SM5-1.

Se ha desarrollado un nuevo método de inmovilización de tirosinasa de champiñón, basado en la adsorción de esta enzima a un éster cinámico de un carbohidrato fotoentrecruzado, con muy buenos resultados. Se han obtenido inmovilizados de tirosinasa directamente a partir de extractos de champiñón. Se han estudiado los parámetros que afectan a la inmovilización (pH, tiempo y concentración de enzima), las características de tirosinasa inmovilizada (cantidad de enzima inmovilizada, actividad enzimática, pH y temperatura óptimos de reacción, estabilidad térmica, operacional y al almacenado), las propiedades cinéticas (constante de Michaelis, velocidad máxima y constante catalítica) de tirosinasa inmovilizada sobre distintos soportes al actuar sobre diversos sustratos y la estereoespecificidad de tirosinasa inmovilizada sobre un soporte quiral y otro no quiral respecto a los isómeros L y D y racémicos. Los conocimientos adquiridos se han aplicado con éxito a la producción de diversos o-difenoles como L-dopa, a partir de sus correspondientes monofenoles. / A new mushroom tyrosinase immobilization method consisting of tyrosinase adsorption on a photocrosslinked cinnamic carbohydrate ester has been developed with very good results. Immobilized tyrosinase was obtained directly from a mushroom extract. We studied the parameters affecting the immobilization process (pH, time and enzyme concentration), the properties of immobilized tyrosinase (quantity of immobilized enzyme, enzymatic activity, optimum pH and reaction temperature; thermal, operational and storage stability), kinetic properties (Michaelis constant, maximum steady-state rate and catalytic constant) of tyrosinase immobilized on several supports and acting over several substrates, and tyrosinase stereospecificity immobilized on a chiral and a nonchiral support acting on L and D isomers and racemics. The knowledge acquired was used to successfully produce several o-diphenols, such as L-dopa, from the corresponding monophenols.

No description available.

Le mélanome est un cancer rare de la peau qui se développe à partir des cellules responsables de la pigmentation : les mélanocytes. Depuis quelques années, nous observons une augmentation significative du nombre de personnes atteintes par cette maladie, de mauvais pronostic et très agressive (132 000 nouveaux cas chaque année dans le monde). Le taux de mortalité de ce cancer est très élevé en raison de la rapidité de propagation des cellules cancéreuse dans d’autres régions du corps. En France, on remarque un nombre relativement élevé de cas détectés, plus particulièrement en Bretagne. La tumeur se présente comme une tâche dont l’apparence est très proche du grain de beauté. Si elle est détectée suffisamment tôt, un prélèvement suffit à la guérison et les risques de récidives sont très faibles. En revanche, une fois que les métastases se propagent, les chances de survie à long terme sont très faibles. Malgré les récentes avancées en thérapie ciblée, les traitements du mélanome métastatique restent encore limités. En partenariat avec le groupe Malakoff Médéric, nous développons actuellement un système optique pour la détection précoce du mélanome cutané. Celui-ci est basé sur les propriétés de polarisation de la lumière. La thèse présente la conception du système, de l’étude de faisabilité jusqu’à l’étape finale d’étalonnage. Nous proposons également différents tests sur des échantillons de simulation. Ces derniers nous permettent de démontrer la corrélation qu’il existe entre les effets de polarisation et les modifications biologiques en cours lors du développement du mélanome. Cette étude préliminaire nous prépare aux expérimentations sur de vrais échantillons. / Melanoma is a rare cancer that develops from the pigmentation cells of the skin. Recently, we notice a significant increase in the number of people affected by this aggressive disease with a poor prognosis (132 000 new cases each year worldwide). The mortality rate of this cancer is very high, which is due to the rapid spread of cancerous cells to other parts of the body. In France, there is a relatively high number of cases detected, especially in Britanny. The tumor is a spot which looks like a mole. If detected early, a levy is sufficient to healing and the risk of recurrence is very low. However, once metastasis spread, the long-term prognosis is very low. Despite recent advances in targeted therapy, the treatments of metastatic melanoma are still limited. In partnership with the Malakoff Mederic group, we are currently developing an optical system for early detection of cutaneous melanoma. It is based on the polarization properties of light. The PhD focuses on the design of the system, from the feasibility study to the final calibration. We also present various tests on samples of simulation. These allow us to demonstrate the correlation between the effects of polarization and biological changes during the development of melanoma. This preliminary study prepares us to experiments on real samples.

Nowdays there is a plethora of evidence for H₂O₂-mediated oxidative stress in the epidermis as well as in the system in patients with vitiligo (for review see (Schallreuter, Bahadoran et al. 2008). Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the oxidative hydroxylation of hypoxanthine to xanthine followed by xanthine to uric acid, the last two steps in purine degradation pathway. Under oxidative conditions, XDH is converted to XO. The reactions catalysed by this enzyme generate H₂O₂ and O₂̇⁻, yielding in the presence of ROS accumulation, allantoin from uric acid. Therefore XO has been considered a major biologic source of oxygen-derived free radicals in many organs. The presence of XO in the human epidermis has not been shown so far. In this study several techniques were utilised to nail the presence and activity of XO in epidermal melanocytes and keratinocytes. The enzyme is regulated by H₂O₂ in a concentration dependent manner, where concentrations of 10-6M upregulate activity. Importantly, the results showed that the activity of XO is little affected by H₂O₂ in the mM range. H₂O₂-mediated oxidation of tryptophan and methionine residues in the sequence of XO yields only subtle alterations in the enzyme active site. These findings are in agreement with enzyme kinetics in the presence of 10-3M H₂O₂. Since uric acid is the end product of XO activity and this can be oxidised to allantoin by H₂O₂, we wanted to know whether allantoin is formed in the epidermis of patients with vitiligo. In order to address this issue, we utilised HPLC/mass spectrometry analysis. Analysis of epidermal cell extracts from suction blister tissue identified the presence of allantoin in patients with acute vitiligo, while this product was absent in healthy controls. In conclusion, our results provide evidence for functioning epidermal XO in the human epidermis which 4 can be a major source for the production of H₂O₂ contributing to oxidative stress in vitiligo. In addition, this thesis also demonstrates for the first time the presence of XO in melanosomes, and we showed that both 7BH4 and 7-biopterin inhibit XO activity in a concentration dependent manner. Moreover, XO has the potential to bind to 6/7BH4 and 6/7-biopterin from the pterin/tyrosinase inhibitor complex. This discovery adds another receptor independent mechanism for regulation of tyrosinase within the melanocyte similar to α/ß-MSH as shown earlier (Moore, Wood et al. 1999; Spencer, Chavan et al. 2005). Since the entire epidermis of patients with vitiligo is under H₂O₂-mediated oxidative stress, oxidative DNA damage would be highly expected. This thesis shows for the first time that epidermal 8-oxoG levels as well as plasma level of this oxidised DNA base are significantly increased in patients compared to healthy controls. We have shown that epidermal cells from patients with vitiligo respond to oxidative DNA damage via the overexpression of p21 and Gadd45α leading to a functioning increased short-patch base-excision repair (BER), while increased apoptosis can be ruled out due to lower caspase 3 and cytochrome c response compared to healthy controls. Our results show that patients develop effective DNA repair machinery via hOgg1, APE1 and DNA polymeraseß. Taking into consideration that these patients do not have an increased prevalence for solar-induced skin cancers, our data suggest that BER is a major player in the hierarchy to combat H₂O₂-mediated oxidative stress preventing ROS-induced tumourigenesis in the epidermis of these patients.

Considerando o melanoma a forma mais agressivas de câncer de pele e mais resistente aos tratamentos convencionais, o presente estudo teve como objetivo avaliar a atividade de um novo complexo de platina (II) com derivado de hidantoína (CX42) em células de melanoma humano e murino. Foram utilizados também para comparação células da pele (fibroblastos, queratinócitos e melanócitos) e os compostos cisplatina (CIS) e complexo de hidantoína isolado (NN10). Ensaios de viabilidade, ciclo e morte celular foram realizados. Investigou-se também a atividade da enzima tirosinase, principal enzima que regula a síntese de melanina durante o processo conhecido como melanogênese. Como resultados, obteve-se a diminuição da viabilidade celular e parada de ciclo na fase G0/G1 nas células de melanoma, principalmente na linhagem murina B16F10, frente ao composto CX42. Em células B16F10, foi possível observar estímulo na melanogênese, com aumento da atividade da enzima tirosinase. O CX42 apresentou uma atividade com efeito citostático nas células de melanoma, não sendo observado efeito citotóxico nas células da pele. Ainda, com a prévia estimulação da melanogênese, o CX42 apresentou-se mais efetivo, aumentando a morte celular em 40% por apoptose. Portanto, conclui-se que o CX42 diminuiu a viabilidade celular e seu efeito foi mais intenso quando as células apresentavam-se pigmentadas, demonstrando indução da tirosinase e da morte celular, atributo importante para potenciais novos fármacos anti-melanoma. / Since melanoma is one of the most aggressive forms of skin cancer and more resistant to conventional treatments, the present study aimed to evaluate the activity of a new platinum complex (II) with hydantoin derivative (CX42) in melanoma cells human and murine. Skin cells (fibroblasts, keratinocytes and melanocytes) were used as control and the compounds cisplatin (CIS) and hydantoin compound alone (NN10) were also evaluated. Viability assays, cell cycle analysis and cell death characterization were performed. Likewise, the activity of tyrosinase, the key enzyme that regulates melanin synthesis during the process known as melanogenesis, was also investigated. The results demonstrated a reduction in cell viability and cell cycle arrest in G0/G1 phase of murine melanoma B16F10 treated with CX42. In B16F10 cells, it was also observed melanogenesis stimulation with increased activity of tyrosinase. The CX42 showed a selective cytostatic effect on melanoma cells, however no toxic effects were observed in skin cells. In addition, with prior stimulation of melanogenesis, the CX42 demonstrated to be more effective, increasing apoptosis cell death in 40%. Therefore, the results demonstrated that CX42 decreased cell viability and the intensity of its effect is associated with cell pigmentation, demonstrating an association between tyrosinase high activity and induction of cell death which is an important attribute for new potential anti - tumoral drugs.

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

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.