Rozprawy doktorskie na temat „MELANIN PIGMENT”

Melanin is the dark pigment found in most organisms that gives color to the skin, hair, feathers, and eyes of vertebrates. While melanin is known to also be present in the stria vascularis of the mammalian cochlea, its function in the inner ear is still unknown. Some previous studies have indicated that melanin may serve to protect the mammalian ear from hearing loss. Minimal previous research on melanin within the inner ears of fishes has been conducted. In this study, the melanin levels in the inner ears of different color morphs of two fish species (Poecilia latipinna and Cyprinus carpio) were examined, as well as the potential protective role of melanin from acoustical stress. To identify the relationship between fish color morph and inner ear melanin, a spectrophotometric melanin assay was used for dissected inner ears, and transmission electron microscopy (TEM) was used to examine melanosome structure in the crus commune of the inner ears. For each color morph and species, hearing thresholds were quantified before and after sound exposure (150 Hz tone at 165 dB re 1 μPa for 48 h) by measuring auditory evoked potentials (AEPs). Melanin levels were associated with scale color, with black morphs having more inner ear melanin than white or golden morphs. TEM imaging showed that melanosome size varied among color morphs, with black P. latipinna morphs having larger melanosomes than white morphs. Hearing thresholds did not differ significantly among color morphs before sound exposure in either species. However, hearing thresholds post-sound exposure and the associated threshold shifts significantly differed between black morphs and other color morphs in the two species, with black morphs having lower thresholds and exhibiting less hearing loss than the other morphs. This suggests that melanin plays a protective role in the teleost inner ear, similar to what other researchers have found in mammalian models. Teleost fishes may be a new, more efficient model for testing melanin's function in the inner ear and how it interacts with stress from acoustical trauma and ototoxic 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

Muitos vertebrados ectotérmicos ajustam suas cores corporais para serem confundidos com o ambiente, através da migração de pigmentos no interior de cromatóforos, regulada por sistemas neurais e/ou hormonais. Essas mudanças de coloração auxiliam no mimetismo, termorregulação, comunicação social e expressão de comportamentos como excitação sexual, agressividade e medo. Entretanto, cromatóforos de inúmeras espécies respondem diretamente à luz. Estudos sobre a resposta à luz nos melanóforos de Xenopus laevis levaram à descoberta do fotopigmento melanopsina, uma opsina que está presente na retina de todos os grupos de vertebrados, inclusive no homem. Vários hormônios podem regular o processo de mudança de cor nos vertebrados, dentre eles a melatonina, hormônio secretado pela glândula pineal. Este é o principal órgão responsável pela integração do sistema neuroendócrino dos vertebrados ao meio ambiente, traduzindo direta ou indiretamente a informação do fotoperíodo em sinal hormonal, coordenando assim os ritmos fisiológicos circadianos com o meio ambiente. Os objetivos deste trabalho foram: investigar se a expressão gênica das melanopsinas e do receptor de melatonina em melanóforos de Xenopus laevis apresenta variação temporal sob diferentes condições luminosas; verificar se a expressão gênica das melanopsinas e do receptor de melatonina em melanóforos de Xenopus laevis pode ser modulada por melatonina. Dados do trabalho demonstram que as melanopsinas em melanóforos de Xenopus laevis são sincronizadas aos ciclos de claro-escuro, expressando um robusto ritmo ultradiano com período de 16h para Opn4m e um ritmo circadiano com período de 25h para Opn4x. Curiosamente, essa ritmicidade só foi observada quando os melanóforos foram mantidos em ciclos 12C:12E e foram submetidos à troca de meio durante a fase clara do fotoperíodo. A constância na expressão gênica do receptor de melatonina Mel1, quer sob diferentes regimes de luz, quer sob tratamento por melatonina, sugere que esse gene é extremamente estável, não sofrendo alterações ao ser submetido a estímulos exógenos, podendo ser considerado um gene constitutivo. O tratamento com melatonina por 6h na fase clara do fotoperíodo, além de inibir drasticamente a expressão de Opn4x e Opn4m, aboliu a ritimicidade de ambas as melanopsinas. Nossos resultados indicam que os melanóforos de Xenopus laevis possuem um relógio funcional e podem ser caracterizados como relógios periféricos, porém necessitam do ciclo claro-escuro associado à troca de meio para exibirem sua sincronização.
Many ectothermic vertebrates adjust their body color to mimic the environment, through the pigment migration within chromatophores, regulated by neural and / or hormonal systems. These changes in color help in camouflage, thermoregulation, social communication and behaviors such as sexual arousal, agressiveness and fear. However, chromatophores of several species respond directly to light. Studies about light response in melanophores of Xenopus laevis have led to the discovery of the photopigment melanopsin, an opsin that is present in the retina of all vertebrate groups, including man. Various hormones may regulate the process of color change in vertebrates, among them melatonin, hormone secreted by the pineal gland. This is the main organ responsible for the integration of the neuroendocrine system of vertebrates to the environment, translating directly or indirectly the photoperiod information into hormonal signal, thus coordinating physiological circadian rhythms with the environment. The objectives of this work were: to investigate whether the gene expression of melanopsins and melatonin receptor in melanophores of Xenopus laevis exhibited temporal variation under different light conditions; to verify whether gene expression of melanopsins and melatonin receptor in melanophores of Xenopus laevis could be modulated by melatonin. Our data show that melanopsins in melanophores of Xenopus laevis are synchronized to light-dark cycles, expressing a robust ultradian rhythm with a period of 16h for Opn4m and circadian rhythm with a period of 25h for Opn4x. Interestingly, the rhythm was only observed when the melanophores were maintained in 12L: 12D regime and medium change was performed during the fotophase of photoperiod. The constancy in the expression of melatonin receptor Mel1c, either under different light regimes, or under treatment by melatonin, suggesting that this gene is extremely stable, not being altered by exogenous stimulus, and may be considered a constitutive gene. Treatment with melatonin for 6h during the fotophase of the photoperiod, drastically inhibit the expression of Opn4x and Opn4m, and abolished the rhythm of both melanopsins. Our results indicate that melanophores of Xenopus laevis possess a functional clock and can be characterized as peripheral clocks, but they need the light-dark cycle associated with change of medium to exhibit their synchronization.

Diseases caused by rust fungi represent critical constraints to global plant production. A characteristic feature of these pathogens is the striking pigments they produce in one or more spore forms, which give them a rusty appearance. These pigments are thought to protect rust fungi against UV radiation and oxidative stress, and possibly act as virulence factors. The cytoplasmic carotenoid pigment composition and relative abundance of 14 rust species were studied, including Puccinia graminis f. sp. tritici (Pgt), P. striiformis f. sp. tritici, P. triticina, P. hordei, P. psidii, P. thaliae, P. oxalidis, Uromyces viciae-fabae, and Phragmidium rubi-idaei, along with colour mutants of several rust species with white, chocolate, pale yellow, dark brown, black, grey, yellow, yellow-orange, dark red and orange urediniospores. Four carotenoids had been found in rust fungi: phytoene, lycopene, -carotene and -carotene. The carotenoid composition did not vary much within rust species but, Among the 14 species investigated, the ratios of -/-carotene varied from 0.14 to 4.49, which might be used as an additional taxonomic character in rust fungi. Candidate genes for carotenoid biosynthesis in Pgt were identified, cloned and functionally complemented using specifically engineered strains of Escherichia coli. The carotenoid biosynthesis pathway in rust fungi was elucidated, with only two genes, CrtYB and CrtI, catalysing the reactions from GGPP to -carotene. Our understanding of carotenoid pigmentation evolution in rust fungi was improved by phylogenetic analysis. Both CrtYB and CrtI are closely related among rust fungi, other pathogenic fungi, and some aphids. A blackish-brown pigment was extracted from the wall of wild-type Pgt urediniospores. The spectrophotometric analysis results suggested that the spore wall pigment extracted from urediniospores of Pgt resembled the DOPA type melanin. Plausible structures of the fragments from this pigment were tentatively proposed.

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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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Outros
Do fungo Aspergillus nidulans foi isolado um mutante que apresenta producao aumentada do pigmento melanina. Este pigmento, formado pela polimerizacao oxidativa de compostos fenolicos e/ou indolicos, pode estar presente na parede celular ou no meio de cultura. Embora nao seja essencial para o crescimento dos fungos, a melanizacao parece aumentar a capacidade de sobrevivencia das especies em condicoes desfavoraveis, fornecendo protecao contra radiacao UV, altas temperaturas, enzimas hidroliticas, metais pesados e agentes oxidantes. A obtencao deste pigmento, na forma pura, tem despertado interesse biotecnologico devido a sua utilizacao em formulacoes cosmeticas, principalmente pela sua propriedade fotoprotetora e antioxidante. Tendo em vista o potencial farmacologico do pigmento melanina, este trabalho tem como objetivos fazer a caracterizacao estrutural e funcional do pigmento extraido do fungo A. nidulans. Os resultados de espectrometria de massas indicam que a estrutura da melanina do fungo A. nidulans e composta de duas grandes unidades, representadas pelos fragmentos de massas moleculares igual a 573 e 550, com grupamentos diidroxindol e aneis pirrolicos substituidos. Os ensaios de citotoxicidade mostram que a melanina extraida do fungo A. nidulans nao causa danos significativos aos componentes celulares, tanto antes como pos metabolizacao. Os ensaios de mutagenicidade sugerem que a melanina nao apresenta propriedades mutagenicas para as linhagens TA 97a, TA 98, TA 100 e TA 102 de Salmonella thyphimurium. No teste antimicrobiano, observase que a melanina do fungo nao apresenta potencial como antibacteriano frente a Staphylococcus aureus, Escherichia coli e Enterococcus faecalis, quando comparadas com as substancias de referencia, ampicilina e cloranfenicol. Quanto...
A mutant that presents increased production of the melanin pigment was isolated from the Aspergillus nidulans fungus. This pigment, formed by the oxidative polymerization of phenolic and/or indolic compounds, can be present in the cellular wall or culture medium. Although it is not essential for the fungal growth, melanization seems to increase the survival capacity of the species in unfavorable conditions, supplying protections against UV radiation, high temperatures, hydrolytic enzymes, heavy metals and oxidants agents. The attainment of this pigment, in its pure form, has taken biotechnological interest due to its use in cosmetic formulations, mainly for its photoprotector and antioxidant property .Considering the pharmacological potential of the melanin pigment, this study aims to make the structural and functional characterization of the pigment extracted from A. nidulans fungus. The results of mass spectrometry indicate that the melanin structure of the A. nidulans fungus is composed of two large units, represented by the fragments of molecular mass equal to 573 and 550, with dihydroxyindole groups and substituted pyrrolic rings. The cytotoxicity assays have shown that the melanin extracted from the A. nidulans fungus does not cause significant damages to the cellular components, before and after metabolization. The mutagenicity assays suggest that the melanin does not present mutagenic properties for the strains TA 97a, TA 98, TA 100 and TA 102 of Salmonella thyphimurium. In the antimicrobial test, it is observed that the fungal melanin does not present antibacterial potential facing Staphylococcus aureus, Enterococcus faecalis and Escherichia coli, when compared to the reference substances, ampicilin and cloranphenicol. Regarding the immunomodulatory activity, it was verified that the melanin does not stimulate NO release... (Complete abstract click electronic access below)

Orientador: Sandra Regina Pombeiro Sponchiado
Banca: Iracilda Zeppone Carlos
Banca: Valdecir Farias Ximenes
Banca: Jairo Kenupp Bastos
Dagmar Ruth Stach Machado
Resumo: Do fungo Aspergillus nidulans foi isolado um mutante que apresenta producao aumentada do pigmento melanina. Este pigmento, formado pela polimerizacao oxidativa de compostos fenolicos e/ou indolicos, pode estar presente na parede celular ou no meio de cultura. Embora nao seja essencial para o crescimento dos fungos, a melanizacao parece aumentar a capacidade de sobrevivencia das especies em condicoes desfavoraveis, fornecendo protecao contra radiacao UV, altas temperaturas, enzimas hidroliticas, metais pesados e agentes oxidantes. A obtencao deste pigmento, na forma pura, tem despertado interesse biotecnologico devido a sua utilizacao em formulacoes cosmeticas, principalmente pela sua propriedade fotoprotetora e antioxidante. Tendo em vista o potencial farmacologico do pigmento melanina, este trabalho tem como objetivos fazer a caracterizacao estrutural e funcional do pigmento extraido do fungo A. nidulans. Os resultados de espectrometria de massas indicam que a estrutura da melanina do fungo A. nidulans e composta de duas grandes unidades, representadas pelos fragmentos de massas moleculares igual a 573 e 550, com grupamentos diidroxindol e aneis pirrolicos substituidos. Os ensaios de citotoxicidade mostram que a melanina extraida do fungo A. nidulans nao causa danos significativos aos componentes celulares, tanto antes como pos metabolizacao. Os ensaios de mutagenicidade sugerem que a melanina nao apresenta propriedades mutagenicas para as linhagens TA 97a, TA 98, TA 100 e TA 102 de Salmonella thyphimurium. No teste antimicrobiano, observase que a melanina do fungo nao apresenta potencial como antibacteriano frente a Staphylococcus aureus, Escherichia coli e Enterococcus faecalis, quando comparadas com as substancias de referencia, ampicilina e cloranfenicol. Quanto... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: A mutant that presents increased production of the melanin pigment was isolated from the Aspergillus nidulans fungus. This pigment, formed by the oxidative polymerization of phenolic and/or indolic compounds, can be present in the cellular wall or culture medium. Although it is not essential for the fungal growth, melanization seems to increase the survival capacity of the species in unfavorable conditions, supplying protections against UV radiation, high temperatures, hydrolytic enzymes, heavy metals and oxidants agents. The attainment of this pigment, in its pure form, has taken biotechnological interest due to its use in cosmetic formulations, mainly for its photoprotector and antioxidant property .Considering the pharmacological potential of the melanin pigment, this study aims to make the structural and functional characterization of the pigment extracted from A. nidulans fungus. The results of mass spectrometry indicate that the melanin structure of the A. nidulans fungus is composed of two large units, represented by the fragments of molecular mass equal to 573 and 550, with dihydroxyindole groups and substituted pyrrolic rings. The cytotoxicity assays have shown that the melanin extracted from the A. nidulans fungus does not cause significant damages to the cellular components, before and after metabolization. The mutagenicity assays suggest that the melanin does not present mutagenic properties for the strains TA 97a, TA 98, TA 100 and TA 102 of Salmonella thyphimurium. In the antimicrobial test, it is observed that the fungal melanin does not present antibacterial potential facing Staphylococcus aureus, Enterococcus faecalis and Escherichia coli, when compared to the reference substances, ampicilin and cloranphenicol. Regarding the immunomodulatory activity, it was verified that the melanin does not stimulate NO release... (Complete abstract click electronic access below)
Doutor

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Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia)
IPEN/D-MPLO
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia - USP

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Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia)
IPEN/D-MPLO
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo, Sao Paulo

Resumo: Os vertebrados ectotérmicos apresentam um desenvolvido sistema de células com pigmentos melânicos em seu citoplasma, localizado em diversos órgãos e membranas. A dispersão dos grãos de melanina dentro dos melanócitos responde fortemente à exposição ao hormônio estimulante de melanócitos (MSH) e aos hormônios sexuais, promovendo alteração na coloração. Este trabalho buscou avaliar o efeito dos esteróides cipinato de testosterona, 17 β-estradiol e do MSH sobre a pigmentação hepática e testicular de Eupemphix nattereri, mediante análise histológica, e ultra-estrutural. Foram coletados 60 machos adultos na região de São José do Rio Preto, SP. Para cada experimento, dez machos adultos receberam doses subcutâneas contendo 0,5 mg/kg de cipionato de testosterona e 0,5 mg/kg de 17 β-estradiol, dissolvidos em óleo vegetal e, 2,5x10-7 mmol/10g de α-MSH dissolvido em PBS, durante 7 dias, a cada 48h. O grupo controle recebeu apenas óleo vegetal ou PBS. Grupos com cinco animais foram eutanasiados 24h após do término do tratamento, e outros cinco, após 15 dias. Foram realizados estudos morfológicos utilizando microscopia de luz e microscopia eletrônica de transmissão. No tecido hepático foram encontradas células de Kupffer. No interstício testicular observou-se melanócitos, com inúmeros melanossomos. Para o grupo tratado com cipionato de testosterona, foi observado um aumento de aproximadamente 2x na área ocupada pelas células pigmentadas no fígado e 4x nos testículos mediante analise a curto prazo. Para o mesmo tratamento, foi observado aumento de 1,8x no fígado dos animais eutanasiados após 15 dias. Tal resposta pode ser decorrente do aumento da atividade melanossintética, do aumento do número de células, ou ambos. Os animais tratados com 17 β-estradiol e α- MSH não apresentaram alterações na pigmentação hepática e testicular mediante... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Ectothermic vertebrates have a well-developed system of melanin-containing cells, which are distributed throughout several organs and tissues. The dispersion of melanin-containing granules within melanocytes responds to prolonged exposure to high concentration of MSH and sexual hormones inducing color change. The present study aimed to characterize morphological and stereological patterns of pigmented cells in liver and testis of the Eupemphix nattereri under effect of testosterone cypionate, 17 β-estradiol and α-MSH. Sixty adult males were collected in São José do Rio Preto, São Paulo State, Brazil. For each experiment, ten adult males were treated with the following hormones injected in the dorsal lymph sac during seven days: (1) 5 mg/kg dose of testosterone cypionate or (2) 5 mg/kg dose of 17 β-estradiol (dissolved in vegetal oil) or (3) 2,5x10-7 mmol/10g of α-MSH (dissolved in PBS). The control group received only vegetal oil or PBS solution at the same hormonal concentration. Groups of five animals were euthanatized after 24h and the other five, after 15 days. Morphological studies with light and transmission electron microscopy were carried out in the groups. In the hepatic tissue were found Kupffer cells, which are characterized by multivesicle bodies in the cytoplasm and large amount of melanosomes. In the testis, melanocytes-like cells are present in the interstitium. They presented large and irregular aspect and a great amount of intensely pigmented cytoplasm. For the groups treated with testosterone cypionate an increase of approximately 2x in the occupied area by the pigmented cells in the liver and 4x in the testis was observed in the treatment group against the control. The observed response can be due to increase of melanosynthesis Introdução 3 Zieri et al., 2010 activity, increase in the number of pigmented cells, or both. Moreover, for the same treatment... (Complete abstract click electronic access below)
Orientador: Classius de Oliveira
Coorientador: Sebastião Roberto Taboga
Banca: Rejane Maira Góes
Banca: Patrícia Simone Leite Vilamaior
Banca: Selma Maria Almeida Santos
Banca: Wagner José Fávaro
Doutor

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Os vertebrados ectotérmicos apresentam um desenvolvido sistema de células com pigmentos melânicos em seu citoplasma, localizado em diversos órgãos e membranas. A dispersão dos grãos de melanina dentro dos melanócitos responde fortemente à exposição ao hormônio estimulante de melanócitos (MSH) e aos hormônios sexuais, promovendo alteração na coloração. Este trabalho buscou avaliar o efeito dos esteróides cipinato de testosterona, 17 β-estradiol e do MSH sobre a pigmentação hepática e testicular de Eupemphix nattereri, mediante análise histológica, e ultra-estrutural. Foram coletados 60 machos adultos na região de São José do Rio Preto, SP. Para cada experimento, dez machos adultos receberam doses subcutâneas contendo 0,5 mg/kg de cipionato de testosterona e 0,5 mg/kg de 17 β-estradiol, dissolvidos em óleo vegetal e, 2,5x10-7 mmol/10g de α-MSH dissolvido em PBS, durante 7 dias, a cada 48h. O grupo controle recebeu apenas óleo vegetal ou PBS. Grupos com cinco animais foram eutanasiados 24h após do término do tratamento, e outros cinco, após 15 dias. Foram realizados estudos morfológicos utilizando microscopia de luz e microscopia eletrônica de transmissão. No tecido hepático foram encontradas células de Kupffer. No interstício testicular observou-se melanócitos, com inúmeros melanossomos. Para o grupo tratado com cipionato de testosterona, foi observado um aumento de aproximadamente 2x na área ocupada pelas células pigmentadas no fígado e 4x nos testículos mediante analise a curto prazo. Para o mesmo tratamento, foi observado aumento de 1,8x no fígado dos animais eutanasiados após 15 dias. Tal resposta pode ser decorrente do aumento da atividade melanossintética, do aumento do número de células, ou ambos. Os animais tratados com 17 β-estradiol e α- MSH não apresentaram alterações na pigmentação hepática e testicular mediante...
Ectothermic vertebrates have a well-developed system of melanin-containing cells, which are distributed throughout several organs and tissues. The dispersion of melanin-containing granules within melanocytes responds to prolonged exposure to high concentration of MSH and sexual hormones inducing color change. The present study aimed to characterize morphological and stereological patterns of pigmented cells in liver and testis of the Eupemphix nattereri under effect of testosterone cypionate, 17 β-estradiol and α-MSH. Sixty adult males were collected in São José do Rio Preto, São Paulo State, Brazil. For each experiment, ten adult males were treated with the following hormones injected in the dorsal lymph sac during seven days: (1) 5 mg/kg dose of testosterone cypionate or (2) 5 mg/kg dose of 17 β-estradiol (dissolved in vegetal oil) or (3) 2,5x10-7 mmol/10g of α-MSH (dissolved in PBS). The control group received only vegetal oil or PBS solution at the same hormonal concentration. Groups of five animals were euthanatized after 24h and the other five, after 15 days. Morphological studies with light and transmission electron microscopy were carried out in the groups. In the hepatic tissue were found Kupffer cells, which are characterized by multivesicle bodies in the cytoplasm and large amount of melanosomes. In the testis, melanocytes-like cells are present in the interstitium. They presented large and irregular aspect and a great amount of intensely pigmented cytoplasm. For the groups treated with testosterone cypionate an increase of approximately 2x in the occupied area by the pigmented cells in the liver and 4x in the testis was observed in the treatment group against the control. The observed response can be due to increase of melanosynthesis Introdução 3 Zieri et al., 2010 activity, increase in the number of pigmented cells, or both. Moreover, for the same treatment... (Complete abstract click electronic access below)

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The understanding of gene function in the externally visible characteristcs (EVC) expression has several uses in human population evolution studies or in forensic investigations. To this last, some effort has been done to discover an efficient and easy model for prediction of skin and eye color in humans. The obvious advantage of the prediction of such EVCs through the use of DNA is to be incorporated as routine in forensic labs and to be applied to police investigations. In our study we combined the genotyping of eight SNPs in pigment-related genes (rs4778138 - OCA2; rs12913832 - HERC2; rs16891982 - SLC45A2; rs8045560 - MC1R; rs1426654 - SLC24A5; rs2733832 - TYRP1; rs1042602 - TYR; rs916977 - HERC2) with different analytical approaches. Considering this SNP panel we evaluated allele frequencies from HAPMAP and ALFRED data obtained from subjects with High Melanin Content (HMC; from African populations), and Low Melanin Content (LMC; from European populations) and defined the alleles H (to predict HMC subjects) and alleles L (to predict LMC subjects). The cumulative distribution of alleles H and alleles L in two phenotypically different color groups of 134 South Brazilian subjects showed that 82% of HMC subjects (N = 61) had eight or more allele H and 100% of LMC subjects (N = 73) had less than eight allele H, with accuracy value of 96.3%. We performed other analyses using AUC (Area Under the Receiver Operating Characteristic Curve), PGL (Calculation of Pathway Genetic Load), and GP (Genetic Probability) approaches. The AUC was 0.99 in predicting both HMC and LMC phenotypes; PGL showed the eight SNPs panel had 93% of concordance between genotype and HMC or LMC phenotypes; and GP approach showed 91% of concordance between prediction and HMC or LMC phenotypes. Our high-throughput genotyping technology combined with different analytical approaches reached very high accuracy to predict the extreme phenotypes of human pigmentation. We believe this forensic DNA phenotyping (FDP) technique would be particularly useful in cases in which the genetic profiles of crime scenes were not found in the DNA data banks or to help classify degraded cadavers skeletons, or biological clues of dismissed people.
A compreens?o da fun??o e express?o dos genes envolvidos nos tra?os externamente vis?veis (EVC; do ingl?s externally visible characteristics) t?m sido amplamente utilizada em v?rios estudos de evolu??o humana e investiga??es forenses. Para este ?ltimo prop?sito, v?rios esfor?os t?m sido feitos para descobrir um modelo eficiente e f?cil para a predi??o da cor da pele e dos olhos em seres humanos. A vantagem ?bvia da predi??o de tais EVCs, atrav?s da utiliza??o do DNA, ? sua incorpora??o na rotina em laborat?rios forenses, sendo assim aplicada em investiga??es policiais. Em nosso estudo, relacionamos o gen?tipo de oito SNPs em genes relacionados com a pigmenta??o (rs4778138 - OCA2; rs12913832 - HERC2; rs16891982 - SLC45A2; rs8045560 - MC1R; rs1426654 - SLC24A5; rs2733832 - TYRP1; rs1042602 - TYR; rs916977 - HERC2) com diferentes abordagens anal?ticas. Este painel de SNPs considerou as frequ?ncias al?licas obtidas de dados do HapMap e Alfred a partir de indiv?duos com Alto Conte?do de Melanina (HMC; do ingl?s High Melanin Content; a partir de popula??es africanas), e Baixo Conte?do de Melanina (LMC; do ingl?s Low Melanin Content; a partir de popula??es europeias) e definiu os alelos H (para predizer os HMC) e alelos L (para predizer os LMC). A distribui??o cumulativa dos alelos H e L nos dois grupos com caracter?sticas de pigmenta??o fenotipicamente distintas, dos 134 indiv?duos da nossa popula??o, mostrou que 82% dos indiv?duos HMC (N = 61) tinham oito ou mais alelos H e 100% dos indiv?duos de LMC (N = 73) tinham menos de oito alelo H, com o valor de precis?o de 96,3%. Outras abordagens como AUC (do ingl?s; Area Under the Receiver Operating Characteristic Curve), c?lculo de PGL (do ingl?s; Pathway Genetic Load) e GP (do ingl?s; Genetic Probability) foram realizadas. A an?lise AUC foi de 0,99 tanto para a predi??o fenot?pica dos HMC quanto LMC; as an?lises PGL, para o painel com 8 SNPs, teve 93% de concord?ncia gen?tipo-fen?tipo nos HMC ou LMC; e a abordagem GP mostrou 91% de concord?ncia para predi??o dos fen?tipos HMC e LMC. Nossa tecnologia de genotipagem de alto rendimento, combinada com diferentes abordagens anal?ticas, chegou a uma precis?o muito alta para predizer os fen?tipos extremos de pigmenta??o humana. Acreditamos que esta t?cnica de fenotipagem forense pelo DNA (FDP; do ingl?s forensic DNA phenotyping), seria particularmente ?til nos casos em que os perfis gen?ticos de locais de crime n?o fossem encontrados no bancos de dados de DNA ou para ajudar a classificar cad?veres degradados, esqueletos, ou vest?gios biol?gicos de pessoas desaparecidas.

In this research hyperbranched resins containing fatty acid residues like alkyds were synthesized. Dipentaerythritol which has six hydroxyl groups was used as the core molecule, and it was transesterified with (i) castor oil, and (ii) a mixture of castor oil and linseed oil at 240°
C in the presence of sodium hydroxide as catalyst. The resulting molecule, in either case, was then esterified with dimethylol propionic acid at 140°
C in the presence of p-toluene sulfonic acid as catalyst. Melamine-formaldehyde resin was synthesized to be used with the synthesized hyperbranched resins, and it was successfully modified by all hyperbranched resins at a ratio of 3:1. FTIR spectroscopy was used to characterize the hyperbranched resins and the thermal properties were determined by DSC. DSC showed that the hyperbranched resins decomposed between 315-345°
C. The viscosity of the resin that was synthesized by using only castor oil was 3.0 Pa.s and by using 50% linseed oil it was 1.0 Pa.s. When reacted with dimethylol propionic acid, the former&rsquo
s viscosity increased to 7.0 Pa.s, and the second&rsquo
s viscosity increased to 3.7 Pa.s. The hyperbranched resins showed excellent adhesion, gloss, flexibility, and formability. The mixture of hyperbranched resin plus melamine-formaldehyde resin samples had higher hardness values but lower gloss, adhesion and bending resistance values than the hyperbranched resins, and they had good impact and abrasion resistances.

碩士
國立陽明大學
生醫光電工程研究所
92
The purpose of this study was to know whether the melanin in retinal pigment epithelium (RPE) would interfere with retina or not on Raman spectrum; to measure the contents of melanin and to determine the distribution of melanin in RPE. First of all, we tested the Raman spectrum of melanin powder which was synthesized. We got the range of spectrum of melanin powder from 1550cm-1 to 1600cm-1. We tried to measure the Raman spectrum of pig's RPE. The result of spectrum of pig's RPE had the same profile as melanin from 1550cm-1 to 1600cm-1, but the Raman intensity was not equal to melanin. We compared the signal of RPE, sclera, and RPE with sclera. We discover that the spectrum of sclera did not affect the signal of RPE, and we can see that the profile of RPE was the same as that of sclera. We found that the distribution of melanin in RPE didn't present regular model, and the difference of melanin contents was about 800 a.u. on Raman intensity. In the experiment about whether melanin would interfere the retina's Raman signal, we still catch the signal of melanin with and without retina covered on the RPE. The profiles of them were the same but the intensity was not. It would be due to the melanin accumulate at the apical part of RPE cell which surround with photoreceptor outer segment. Therefore, when we make laser light focused on the retina, melanin was easily in the tight focused area. Then we obtain the spectrum of melanin.

A number of fungi produce spalted wood, which is characterized by accumulation of black pigment in fine demarcation lines, often accompanied by discoloration or staining on the wood fibers. Specific spalting fungi were identified by molecular analysis. From a total of 19 isolates and 140 clones studied, 11 fungal species were identified. The two Chlorociboria species from North America were investigated and their anamorphs were unambiguously identified for the first time. Fungal pigment formation under the influence of moisture content and pH variation was investigated in sugar maple, American beech and agar inoculated with spalting fungi. Maximum pigment production occurred at treatment with pH 4.5 for sugar maple and beech inoculated with Trametes versicolor. Xylaria polymorpha produced external pigmentation in beech treated with buffer at pH 5 and sugar maple at pH 4.5. Fungal pigmentation by Trametes versicolor and Xylaria polymorpha was stimulated at low moisture content in both wood species tested. Melanin production by Inonotus hispidus and Polyporus squamosus was stimulated above 22-28% and 34-38% moisture content in beech and in sugar maple respectively. Fomes fomentarius and Polyporus brumalis produced maximum pigmentation in beech at 26 - 41% and in sugar maple at 59 - 96% moisture content. The variation of the moisture content and pH values of wood substrates can stimulate the intensity of pigmentation of specific fungi in wood. To investigate melanin synthesis from a variety of melanin precursors, experimental research on three spalting fungi tested their reaction to catechol and L-Dopa melanin precursors in wood and agar substrate. The results indicate multiple biosynthesis pathways for melanin assembly in Trametes versicolor, Xylaria polymorha and Inonotus hispidus, and catechol produced most pigmentation in all spalting fungi investigated. Microscopic analysis by light, fluorescence, electron and confocal microscopy also indicates a bi- or multi-modal activity of melanin production and assembly by several spalting fungi. Possible variations of melanin assembly were identified based on fungal and wood species. Immunofluorescence and immunogold labelling with Mab 6D2 melanin antibody confirmed the melanin nature of the pigments produced by Oxyporus populinus, Trametes versicolor, Xylaria polymorpha, Fomes fomentarius, and Inonotus hispidus.

no
This study shows that prostaglandins in human FM55 melanoma cells and epidermal melanocytes are produced by COX-1. Prostaglandin production in FM55 melanoma cells was unrelated to that of melanin suggesting that the two processes can occur independently. ¿-Melanocyte stimulating hormone (¿-MSH), which had no effect on melanin production in FM55 cells, stimulated PGD2 production in these cells without affecting PGE2. While cAMP pathways may be involved in regulating PGD2 production, our results suggest that ¿-MSH acts independently of cAMP, possibly by regulating the activity of lipocalin-type PGD synthase. This ¿-MSH-mediated effect may be associated with its role as an immune modulator.
The Wellcome Trust

In recent years particular attention has been focused on the properties of melanin pigments with regard to their association with some pathological conditions and to their controversial role in the response of skin to solar radiation. This is especially true in the case of pheomelanins, typical of red hair phenotype, with red hair pale skin, blue-green eyes and freckles. People exhibiting this phenotype have poor tanning capacity, exhibit a UV-susceptibility trait with high tendency to sunburn and an increased risk for skin tumors and melanoma. On the other hand, eumelanins are commonly believed to be the most important photoprotective factor, even if evidence accumulating over the last decades highlight a much more controversial role of eumelanins in human pigmentation. On these bases, the research work carried out during the PhD course and reported in this thesis was directed at investigating the light-independent effects of purified human hair melanins on keratinocyte cell cultures with particular attention to their pro-oxidant properties and at defining the origin of the broadband absorption spectrum of eumelanin, which underpins their protective shielding effect. Based on the consideration that, besides eumelanin pigments, the entire melanogenic pathway is relevant to melanocyte function, the effect of carboxyl group substituent of indole precursors on eumelanin properties was evaluated and a suitable derivative of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) was prepared to assess the photoprotective properties for potential application in sunscreen formulations. Local excess of pigmentation is one of the most common pigmentary disorder5 whose aesthetically impact has urged the search for efficient strategies for control of skin pigmentation. As a preliminary approach toward the implementation of a novel skin depigmenting agent a conjugate of caffeic acid with dihydrolipoic acid was prepared and tested for its ability to inhibit mushroom tyrosinase activity.

A estética ocupa uma parte significativa do trabalho no âmbito da medicina dentária. A cor da gengiva desempenha um papel importante na estética facial. A pigmentação melánica gengival ocorre em todas as raças, em qualquer idade, não apresentando predileção de gênero. O grau de pigmentação da melanina pode variar de pessoa para pessoa. Os clínicos enfrentam desafios para alcançar uma estética gengival aceitável, bem como para abordar problemas biológicos e funcionais. No entanto, os princípios e a técnica de gestão dos problemas associados à pigmentação da melanina gengival ainda não estão totalmente esclarecidos. Embora a pigmentação clínica da melanina não represente um problema médico, os pacientes podem queixar-se da pigmentação escurecida exagerada da gengiva e requererem terapêutica cosmética. Esta revisão bibliográfica, pretende dar uma informação global sobre o que é a pigmentação gengival e as terapêuticas existentes. Com este propósito, foi feita uma pesquisa utilizando como motores de busca PubMed, Google scholar, B-On e SciELO, pesquisando um total de cerca 200 artigos, escolhendo os 39 que considerei mais relevantes, utilizando como criterio a relevância para o assunto.
Aesthetic occupies a significant part of the work in the field of dental medicine. The colour of gingiva plays an important role in overall aesthetic. Gingival melanin pigmentation occurs in all races, at any age, with no gender preference. The degree of melanin pigmentation may vary from person to person. Clinicians face challenges to achieve an acceptable gingival aesthetic as well as to address biological and functional problems. However, the principles and the management of problems associated with pigmentation of gingival melanin, are not yet fully clarified. Although the clinical pigmentation of melanin does not represent a medical problem, patients may complain of excessive darkened pigmentation of the gums and require cosmetic therapy. This literature review aims to give a global information on what is the gingival pigmentation and existing therapies. For this purpose, a search was performed using PubMed, Google scholar, B-On and SciELO, searching a total of about 200 articles, choosing the 39 that I considered most relevant, using as criteria the relevance to the subject.