α-MSH immunoreactive peptides were fractionated and characterized in rat and human brain and rat pituitary by reversed phase high pressure liquid chromatographic techniques. α-MSH and deacetylated α-MSH were two major naturally existing peptides in both brain and pituitary gland. Subsequent experiments examined the roles of these two peptides in neuronal function. The α-MSH was clearly more effective than deacetylated α-MSH in improving performance on a visual discrimination task after intraperitoneal administration and in inducing excessive grooming after intraventricular administration. The difference in behavioral potency may be explained by the fact that α-MSH was much more resistant to peptidase degradation than was deacetylated α-MSH. N-acetylation of α-MSH may be an effective regulatory process for modulating the behavioral potency of the secretory product of α-MSH-containing pituitary cells and neurons.

Les ultraviolets A (UVA) sont carcinogènes et produisent des espèces réactives de l'oxygène (ERO). Le récepteur à la mélanocortine de type 1 (MC1R) est un récepteur couplé aux protéines G (RCPG) qui est impliqué dans la mélanogénèse et dans l'inflammation cutanée. Certains variants du gène sont associés à un risque accru de mélanomes et de carcinomes cutanés. Le MC1R est exprimé surtout dans les mélanocytes mais son expression peut être induite par les UV in vitro dans les kératinocytes et in vivo dans la peau. Le récepteur MC1R est activé par l'α-MSH. L'objectif de ce travail de thèse a été d'étudier les effets du récepteur MC1R sur le stress oxydatif induit par les UVA dans des lignées kératinocytaires humaines HaCaT exprimant le récepteur MC1R ou son variant non fonctionnel Arg151Cys. Nous avons montré que la production d'ERO intracellulaire induite par les UVA est fortement inhibée dans les cellules HaCaT-MC1R et que cette inhibition est renforcée en présence d'α-MSH. L'inhibition du stress oxydatif induit par les UVA dans les cellules transfectées par le MC1R est en partie dépendante de la phosphorylation de la sous-unité activatrice, NoxA1 de la NADPH oxydase. Le traitement des cellules HaCaT-MC1R par un inhibiteur du récepteur au facteur de croissance épidermique (EGFR) restaure l'habilité de ces cellules à induire un stress oxydatif après irradiation UVA. Ces résultats montrent que l'activité constitutive du récepteur MC1R dans des kératinocytes pourrait inhiber le stress oxydatif induit par les UVA via des mécanismes dépendants de l'AMPc et de l'EGFR. / Ultraviolet A (UVA) radiations are responsible for deleterious effects, mainly due to reactive oxygen species (ROS) production. Alpha-melanocyte stimulating hormone (α-MSH) binds to Melanocortin-1 Receptor (MC1R) in melanocytes to stimulate pigmentation and modulate cutaneous inflammatory responses. MC1R may be induced in keratinocytes after UV exposure. To investigate the effect of MC1R signaling on UVA-induced ROS (UVA-ROS) production, we generated HaCaT cells that stably express human MC1R (HaCaT-MC1R) or the Arg151Cys (R151C) non- functional variant (HaCaT-R151C). We then assessed ROS production immediately after UVA exposure and found that: (1) UVA-ROS production was strongly reduced in HaCaT-MC1R but not in HaCaT-R151C cells compared to parental HaCaT cells; (2) this inhibitory effect was further amplified by α-MSH treatment of HaCaT-MC1R cells before UVA exposure; (3) after UVA irradiation, NoxA1 phosphorylation was increased i n HaCaT-MC1R compared to HaCaT and HaCaT-R151C cells. Inhibition of PKA in HaCaT-MC1R cells resulted in a marked increase of UVA-ROS production; (4) the ability of HaCaT-MC1R cells to produce UVA-ROS was restored by inhibiting epidermal growth factor receptor (EGFR) or extracellular signal-regulated kinases (ERK) activity before UVA exposure. Our findings suggest that constitutive activity of MC1R in keratinocytes may reduce UVA-induced oxidative stress via EGFR and cAMP-dependent mechanisms.

Orientador: Hélio Amante Miot / Resumo: A patogênese do melasma, especialmente o papel dos queratinócitos e fibroblastos no desenvolvimento e manutenção da doença não é bem compreendida. Alterações dérmicas como dano estrutural à zona de membrana basal, melanócitos em pêndulo, elastose solar, celularidade, proliferação vascular, além da expressão de mediadores inflamatórios, fatores de crescimento, expressão epitelial de melanocortina e receptores dos hormônios sexuais; sugerem interação entre a unidade epidermo-melânica e a derme na fisiopatologia do melasma. A pigmentação melânica da pele pode ser estimulada por diferentes vias de sinalização, sendo a radiação ultravioleta, citocinas dérmicas e inflamação epidérmica, os modelos mais usuais. Neste estudo, objetivamos comparar a morfologia nuclear e a textura da cromatina entre queratinócitos basais no melasma facial e pele adjacente, investigar a ativação das diferentes vias de estímulo à pigmentação, além do envolvimento da derme, com foco na zona de membrana basal e colágeno, no melasma facial. Para a sua execução, foram coletados pares de biópsias faciais (2mm) de mulheres adultas com melasma e de pele adjacente (<2 cm). Processaram-se para coloração de PAS e picrosirius red; imunofluorescência para p53, p38, IL- 1α, αMSH, MC1R e COX2; imunoistoquímica para Melan-Acontracorada com PAS; Microscopia Eletrônica de Transmissão, além de cultura primária de fibroblastos para real-timePCR array e marcação para SA-β-gal. Foram avaliados: núcleos de quer... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The melasma pathogenesis, specially the role of keratinocytes and fibroblast in the disease development and maintenance are not completely understood. Dermal alterations such as basal membrane structural damage, pendulous melanocytes, solar elastosis, cellularity, vascular proliferation, in addition to the expression of inflammatory mediators, grow factors, epithelial melanocortin and sexual hormones receptors, suggest there is an interaction between the epidermal melanin unit and dermis in melasma physiopathology. The melanin skin pigmentation can be stimulated by different signaling pathways. UVR, dermal cytokines and epidermal inflammation are the most common models. These study aims to compare the nuclear morphology and chromatin texture in basal keratinocytes between melasma and adjacent skin, to investigate the activation of different pigmentation signaling pathways, and the dermal involvement, focusing on basal membrane zone and collagen. Therefore, facial skin biopsies (2 mm) from women were taken from melasma and normal skin (<2 cm apart) and processed for PAS and picrosirius red; immunofluorescence for p53, p38, IL-1α, αMSH, MC1R and COX2, immunohistochemistry for Melan-A counterstaining with PAS, and transmission electronic microscopy. Furthermore, primary fibroblast culture for real-timePCRarray and SA-β-gal staining. The nuclei of basal keratinocytes were evaluated as nuclear morphometry and chromatin texture; the fluorescence intensity was quantified in the epid... (Complete abstract click electronic access below) / Doutor

Submitted by GABRIELLI BRIANEZI null (gabrielli.brianezi@hotmail.com) on 2016-02-26T15:36:13Z No. of bitstreams: 1 20160224 Tese Gabrielli Brianezi.pdf: 3073309 bytes, checksum: 8fd0793265926aa1a58aea12aad689a6 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-02-26T20:04:11Z (GMT) No. of bitstreams: 1 brianezi_g_dr_bot.pdf: 3073309 bytes, checksum: 8fd0793265926aa1a58aea12aad689a6 (MD5) / Made available in DSpace on 2016-02-26T20:04:11Z (GMT). No. of bitstreams: 1 brianezi_g_dr_bot.pdf: 3073309 bytes, checksum: 8fd0793265926aa1a58aea12aad689a6 (MD5) Previous issue date: 2016-01-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A patogênese do melasma, especialmente o papel dos queratinócitos e fibroblastos no desenvolvimento e manutenção da doença não é bem compreendida. Alterações dérmicas como dano estrutural à zona de membrana basal, melanócitos em pêndulo, elastose solar, celularidade, proliferação vascular, além da expressão de mediadores inflamatórios, fatores de crescimento, expressão epitelial de melanocortina e receptores dos hormônios sexuais; sugerem interação entre a unidade epidermo-melânica e a derme na fisiopatologia do melasma. A pigmentação melânica da pele pode ser estimulada por diferentes vias de sinalização, sendo a radiação ultravioleta, citocinas dérmicas e inflamação epidérmica, os modelos mais usuais. Neste estudo, objetivamos comparar a morfologia nuclear e a textura da cromatina entre queratinócitos basais no melasma facial e pele adjacente, investigar a ativação das diferentes vias de estímulo à pigmentação, além do envolvimento da derme, com foco na zona de membrana basal e colágeno, no melasma facial. Para a sua execução, foram coletados pares de biópsias faciais (2mm) de mulheres adultas com melasma e de pele adjacente (<2 cm). Processaram-se para coloração de PAS e picrosirius red; imunofluorescência para p53, p38, IL- 1α, αMSH, MC1R e COX2; imunoistoquímica para Melan-Acontracorada com PAS; Microscopia Eletrônica de Transmissão, além de cultura primária de fibroblastos para real-timePCR array e marcação para SA-β-gal. Foram avaliados: núcleos de queratinócitos da camada basal quanto à morfometria nuclear e textura da cromatina; quantificada a intensidade de fluorescência nos compartimentos da epiderme e derme; avaliadas organelas citoplasmáticas e zona de membrana basal; além do colágeno dérmico e densidade de melanócitos: totais e em pêndulo. Em relação à pele adjacente sem melasma: núcleos de queratinócitos basais da epiderme com melasma facial apresentaram índices morfométricos e textura da cromatina alterados; houve aumento da expressão epitelial de αMSH e MC1R, sem diferença quanto ao p53, p38, IL1α e COX2; houve maior número de organelas citoplasmáticas e melanossomas em estágios de maturação maiores nos queratinócitos e melanócitos; áreas danificadas na zona de membrana basal com presença de microvesículas adjacentes à membrana basal; maior número de melanócitos em pêndulo; colágeno dérmico desestruturado. Entre os fibroblastos, houve maior marcação para SA-β-gal e expressão alterada dos genes COL4A1, IL6, ESR2, DKK3, CCL2, WIF1, WNT3A, HGF, IL1B, MMPs 1-7-9 no melasma. As alterações encontradas nos queratinócitos, na derme e zona de membrana basal, sugerem que o fenótipo do melasma pode resultar de alterações em toda unidade epidermo-melânica, não somente de uma hipertrofia dos melanócitos, as vias inflamatórias da epiderme e dependentes de p53 não se mostraram proeminentes, além de ser identificado um possível papel do processo de reparo/dano da derme e da senescência de fibroblastos na patogênese da doença. / The melasma pathogenesis, specially the role of keratinocytes and fibroblast in the disease development and maintenance are not completely understood. Dermal alterations such as basal membrane structural damage, pendulous melanocytes, solar elastosis, cellularity, vascular proliferation, in addition to the expression of inflammatory mediators, grow factors, epithelial melanocortin and sexual hormones receptors, suggest there is an interaction between the epidermal melanin unit and dermis in melasma physiopathology. The melanin skin pigmentation can be stimulated by different signaling pathways. UVR, dermal cytokines and epidermal inflammation are the most common models. These study aims to compare the nuclear morphology and chromatin texture in basal keratinocytes between melasma and adjacent skin, to investigate the activation of different pigmentation signaling pathways, and the dermal involvement, focusing on basal membrane zone and collagen. Therefore, facial skin biopsies (2 mm) from women were taken from melasma and normal skin (<2 cm apart) and processed for PAS and picrosirius red; immunofluorescence for p53, p38, IL-1α, αMSH, MC1R and COX2, immunohistochemistry for Melan-A counterstaining with PAS, and transmission electronic microscopy. Furthermore, primary fibroblast culture for real-timePCRarray and SA-β-gal staining. The nuclei of basal keratinocytes were evaluated as nuclear morphometry and chromatin texture; the fluorescence intensity was quantified in the epidermis and dermis; cytoplasm organelles and basal membrane zone were evaluated; in addition to dermal collagen and melanocytes density: total and pendulous. Regarding adjacent healthy skin, the melasma skin showed alterations in morphometric index and chromatin texture; there was greater epithelial expression of αMSH and MC1R, but without difference for p53, p38, IL1α and COX2; cytoplasm organelles and melanossomas in higher maturity were in greater number in keratinocytes and melanocytes; there were commoner damaged areas in basal membrane zone, presence of microvesicules adjacent to basal membrane; and higher number of pendulous melanocyte; dermal collagen was less structured. There were greater SA-β-gal staining and altered expression of COL4A1, IL6, ESR2, DKK3, CCL2, WIF1, WNT3A, HGF, IL1B, MMPs 1-7-9 genes in melasma fibroblasts.The alterations presented in keratinocytes, dermis and basal membrane zone suggest the melasma phenotype can result from alteration in entire epidermal melanin unit, not just hypertrophic melanocytes, the epidermal inflammatory pathways and p53 dependents do not show prominence, in addition to the possible role of the repair/ damage process identified at dermis and fibroblast senescence in the melasma pathogenesis. / FAPESP: 12/09233-5 / FAPESP: 12/05004-1