The entire epidermis of patients with vitiligo exhibits accumulation of up to 10-3M concentrations of hydrogen peroxide (H2O2) (Schallreuter, Moore et al. 1999). Over the last decade our group and others have focused on the effect of H2O2-mediated oxidative stress on the function of many proteins and peptides due to oxidation of target amino acid residues in their structure including L-methionine, L-tryptophan, L-cysteine and seleno cysteine (Rokos, Beazley et al. 2002; Gillbro, Marles et al. 2004; Hasse, Kothari et al. 2005; Schallreuter, Chavan et al. 2005; Spencer, Chavan et al. 2005; Chavan, Gillbro et al. 2006; Elwary, Chavan et al. 2006; Gibbons, Wood et al. 2006; Schallreuter, Bahadoran et al. 2008; Shalbaf, Gibbons et al. 2008; Wood, Decker et al. 2009). Moreover, it was shown that patients with vitiligo possess up regulated wild type functioning p53 protein in their skin (Schallreuter, Behrens- Williams et al. 2003). The reason behind this up regulation has remained unclear (Schallreuter, Behrens-Williams et al. 2003). Therefore the aim of this thesis was to get a better understanding of these puzzling data. Along this project different techniques have been used including Western blot, dot blot, immuno precipitation, immuno fluorescence, EMSA and computer modelling. In this thesis we confirmed the previous result on up regulation of p53 in vitiligo and we showed that p90MDM2, the master regulator for p53 protein is not different in patients and healthy controls. Therefore we decided to test for expression of p76MDM2 which mediates the inhibition of p90MDM2-p53 binding. Our results show for the first time the presence and over expression of p76MDM2 protein in vitiligo compared to 3 healthy individuals. This result could provide an explanation, why up regulated p53 is not degraded in this disease. Since epidermal H2O2 accumulation has been extensively documented in vitiligo, we wanted to know whether other ROS could also contribute to the overall oxidative stress in this scenario. Therefore we turned our interest to nitric oxide (NO) and its possible effects on p53 protein. In order to elucidate this role in more detail, the expression levels of epidermal nitric oxide synthesase (iNOS) and the oxidation product of NO and O2 - i.e peroxynitrite (ONOO-) were investigated. Our data revealed over expression of iNOS and nitrated tyrosine residues, the foot print for ONOO-. Moreover, we show for the first time the presence of abundant nitration of p53 protein in vitiligo. In addition using purified p53 from E. coli strain (BL21/DE3) and mutant p53 protein from HT-29 cells (colon cancer cells), we show that nitration takes place in a dose and time dependent manner. On this basis we investigated the effect of both H2O2 and ONOO- on p53-DNA binding capacity employing EMSA, since this is the most acceptable technique to follow the binding between proteins and DNA. Our results revealed that ONOO- abrogated p53-DNA binding capacity at concentrations >300 ¿M, meanwhile oxidation of p53 protein with H2O2 at the same concentrations does not affect binding capacity. Importantly, a much higher p53- DNA binding capacity was observed after exposure to both ONOO- and H2O2. Taken together, p53 is regulated by both ROS (H2O2) and RNS (ONOO-). Next we identified the presence of phosphorylated and acetylated p53 in vitiligo. Phosphorylation of ser 9 and ser 15 residues of the protein are associated with over expressed ATM protein kinase, while acetylation of lys 373, 382 residues correlates with increased PCAF expression. We show that up regulated p53 is associated with over expressed p21 (cyclin dependent kinase inhibitor 1) and induced PCNA 4 expression. Hence, we can conclude that p53 in patients with vitiligo is up regulated, activated and functional. Finally we show up regulated BCL-2 supporting the long voiced absence of increased apoptosis in vitiligo. Given that patients with vitiligo have no increased risk for solar induced skin cancer and increased photo damage (Calanchini-Postizzi and Frenk 1987; Westerhof and Schallreuter 1997; Schallreuter, Tobin et al. 2002), despite the presence of increased DNA damage as evidenced by increased 8-oxoG levels in the skin and in the plasma, our findings suggest that both p53 and PCNA provide a powerful machinery to mediate DNA repair via hOgg1, APE1 and DNA polymerase ß (Shalbaf 2009). On this basis it is tempting to conclude that DNArepair is the overriding mechanism to combat oxidative stress in this disease. / Egyptian government; Institute for Pigmentary Disorders in association with the EM Arndt University of Greifswald, Germany.

Vitiligo is a skin disease with 2 % prevalence in a worldwide population. It is characterised by loss or decrease in activity of epidermal melanocytes, which lead to skin and hair depigmentation. It has negative impact on psyche, social relationships of patients and reduces the protection of the organism against UV radiation. One of the treatment methods is autologous transplantation of melanocytes or suspension of melanocytes with keratinocytes. Use of the biocompatible membrane, which allows the cultivation of these cells with resulting transplantation on the depigmented lesion, could improve treatment and make it more efficient. The main goal of this work was to create the biocompatible membrane from nanofiber layers of polyvinylalcohol (PVA) which can stand as a carrier for cell transplants in vitiligo therapy. PVA scaffolds were prepared by electrostatic spinning and later on modified by cold methane plasma (CH4) for lowering their hydrofility. Samples of modified nanofiber carriers were analysed according to their physical and chemical characteristics (visualization fiber morphology by SEM, XPS and surface Zeta potential analysis and contact angle). Consequently, adhesion, proliferation and metabolic activity of cultivating mice cell lines of melanocytes (Melan-a) and keratinocytes (XB2) were examined...

Le vitiligo est une dermatose inflammatoire caractérisée par une perte progressive des mélanocytes de la lame basale de l’épiderme. Cette pathologie reste à ce jour orpheline de traitement efficace. Toutefois, les mécanismes qui sont liés à la perte des mélanocytes restent débattus et impliquent un détachement des mélanocytes de la lame basale ou leur mort cellulaire par apoptose. Nous avons montré au sein de notre équipe l’implication des lymphocytes T CD8+ effecteurs mémoires dans la pathogénie du vitiligo. Ces populations produisent des niveaux élevés de deux cytokines inflammatoires qui sont, le TNF-α (tumor necrosis factor) et l’IFN-γ (interféron γ), suggérant ainsi un rôle majeur de ces deux cytokines dans la pathogénie du vitiligo. L’objectif de mon projet de thèse est d’étudier les mécanismes immunologiques impliqués dans la perte du mélanocyte au cours du vitiligo. Ainsi, nous avons observé dans les zones péri-lésionnelles de vitiligo ou lésionnelles de psoriasis, une localisation suprabasale des mélanocytes et une anomalie de l’expression de la E-cadhérine dans l’épiderme, protéine majeure impliquée dans l’attachement des mélanocytes. Nous avons également montré l’absence de la mort cellulaire par apoptose des mélanocytes au niveau cutané chez les patients atteints de vitiligo et/ou de psoriasis. En se basant sur ces observations, nous nous sommes donc intéressés à évaluer les effets combinés du TNF-α et de l’IFN-γ sur l’adhésion des mélanocytes. Nos résultats ont montré que les deux cytokines combinés diminuent l’expression du gène codant la Ecadhérine et entrainent probablement la redistribution de cette protéine. De plus, nous avons observé que ces deux cytokines en combinaison altèrent l’expression de la E-cadhérine dans un modèle d’épidermes reconstruits pigmentés in vitro. Cette altération était associée à une augmentation des niveaux de la E-cadhérine soluble (sE-cad) au niveau des surnageants de culture. D’une manière intéressante, nous avons montré que ces deux cytokines induisent l’expression kératinocytaire de la métalloprotéase 9 (MMP9) dont l’action est connue pour cliver la E-cadhérine sous sa forme soluble, participant ainsi au détachement des mélanocytes. Des taux élevés de MMP9, mais également de la sE-cad sont retrouvés dans le sérum des patients atteints de vitiligo. L’inhibition de la MMP9 dans des modèles in vitro et in vivo empêche les effets combinés du TNF- α et de l’IFN-γ sur le détachement des mélanocytes permettant leur stabilisation à la lame basale. Par ailleurs, comme nous avons montré que la survie des mélanocytes n’était pas altérée dans le vitiligo, nous nous sommes intéressés à évaluer l’action combinée du TNF-α et de l’IFN-γ sur la fonction, le phénotype des mélanocytes et leur production des médiateurs inflammatoires. Nous avons montré que ces deux cytokines en combinaison inhibent l’expression des gènes mélanocytaires (MITF, TYR, DCT) et favorisent l’induction des chimiokines CXCL9 et CXCL10, de la cytokine inflammatoire TNF-α et de la molécule d’adhésion ICAM-1, suggérant un rôle majeur des mélanocytes dans la promotion de l’inflammation. Enfin, considérant que la voie de signalisation du récepteur de l’IFN-γ est dépendante de la voie JAK/STAT, nous avons étudié l’impact de l’inhibition de cette voie dans les effets induits dans nos modèles, et avons montré au niveau de l’expression des gènes, une amélioration des gènes associés à la fonction mélanocytaire et une inhibition de ceux associés à l’inflammation. L’ensemble de nos résultats mettent en évidence un nouveau mécanisme pour expliquer la perte des mélanocytes et identifie MMP9 ainsi que les inhibiteurs de JAK comme des cibles thérapeutiques prometteuses permettant ainsi de mieux comprendre les mécanismes physiopathologiques au cours du vitiligo et d’établir un lien direct entre immunité, facteurs solubles inflammatoires et perte des mélanocytes au cours du vitiligo. / Vitiligo is a chronic inflammatory skin disorder characterized by a progressive loss of melanocytes. This stigmatizing disease has a major social impact and no real effective therapies have been reported so far. However, the mechanisms leading to melanocyte disappearance remain debated and include melanocyte detachment and/or death. The role of the immune response has now been well described, implying CD8+ effector memory T cells that produce high levels of inflammatory cytokines as TNF-α (tumor necrosis factor) and IFN-γ(interferon γ), suggesting the involvement of these two cytokines in the pathogenesis of vitiligo. Thus, the aim of this project is to study the interplay between the inflammatory response characterizing vitiligo disease and melanocyte loss. We first observed that melanocytes are located in suprabasal layers of the epidermis in perilesional skin of vitiligo and lesional skin of psoriasis patients, which was associated with an altered expression of E-cadherin, a major protein involved in melanocyte attachment to the basal membrane. Such suprabasal melanocytes did not undergo apoptosis. Based on these observations, we next investigated the combined effects of TNF-α and IFN-γ on melanocyte adhesion. We showed that these two cytokines decrease E cadherin gene expression and probably induce a redistribution of E-cadherin. In addition, these two cytokines in combination altered the expression of E-cadherin in reconstructed human pigmented epidermis in vitro. This finding was associated with increased levels of soluble E-cadherin in culture supernatants. Furthermore, TNF-α and IFN-γ induced the production of matrix metalloproteinase 9 (MMP9) by keratinocytes, leading to the cleavage of the E-cadherin. Inhibition of MMP9 prevents the combined effects of TNF-α and IFN-γ on melanocyte detachment and led to their stabilization to the basal membrane of epidermis in vitro and in vivo models. Since we demonstrated that melanocyte survival is not impaired in vitiligo, we assessed the impact of these two cytokines on melanocyte function, phenotype and inflammation. We demonstrate that the combination of TNF-α and IFN-γ inhibits the expression of genes involved in melanocyte function (MITF, TYR, DCT) and promote the induction of the chemokine ligands CXCL9 and CXCL10, the inflammatory cytokine TNF-α and adhesion molecule ICAM-1, suggesting an important role of melanocytes in the promotion of inflammation. Lastly, considering that the signaling pathway of IFN-γ involves activation of the JAK / STAT pathway, we studied the impact of the inhibition of that pathway in our models. Our results show that the JAK inhibition suppressed the effects of TNF-α and IFN-γ on melanocyte function, on the release of pro-inflammatory mediators and led to the melanocyte stabilization to the basal membrane of epidermis. All of our results highlight a new mechanism to explain the loss of melanocytes and identify MMP9 and JAKs as promising therapeutic targets to better understand the physiopathological mechanisms during vitiligo and establish a direct link between immunity, soluble factors. Inflammation and loss of melanocytes during vitiligo.

O objetivo do presente trabalho foi avaliar a ação melanogênica das furanocumarinas, psoraleno e bergapteno assim como do extrato seco das raízes de Brosimum gaudichaudii Trécul, contendo 1,2 % m/m de psoraleno, e 5,2 % m/m de bergapteno. A toxidade celular in vitro foi avaliada pelo teste de incorporação do vermelho neutro em quatro linhagens de células diferentes (fibroblastos-L929, queratinócitos-HaCat, Melanoma-B16F10, e melanócitos-Melam-A) e apresentou resposta dose dependente tanto para os fármacos puros quanto para o extrato de B. gaudichaudii. Quando expostas à radiação ultravioleta do tipo A e B houve aumento da toxicidade, proporcional a dose de radiação. A mutagenicidade e genotoxicidade realizas pelos ensaios de micronúcleo e cometa, mostrou que os compostos são genotóxicos e mutagênicos em doses >= 150 ?g.mL-1.A síntese de melanina in vitro realizada em cultura de melanoma B16F10 foi dependente da dose e do tempo de exposição aos fármacos e UV. Nas concentrações máximas usadas (48 ?g.mL-1 de psoraleno, 104 ?g.mL-1 de bergapteno e 0,5 mg.mL-1 de extrato) o psoraleno aumentou a produção de melanina em 26 %, o bergapteno em 69 %, e o extrato de B.gaudichaudii em 163 %. Quando utilizado mistura equivalente 6 ?g.mL-1 de psoraleno e 26 ?g.mL-1 de bergapteno a presente em 0,5 mg.mL-1 de extrato a produção de melanina foi de 61%. A atividade da tirosinase em culturas de B16F10, tratadas com 20 ?g.mL-1 de psoraleno ou bergapteno, quando comparada ao grupo não tratado aumentou em 13% a atividade enzimatica , quando associados foi de 37%, e 0,5 mg.mL-1 de extrato em 54,1%. Com o ensaio de microdiálise in vivo observou-se que os fármacos são rapidamente absorvidos pela pele e distribuídos no plasma. Ambos os composto apresentaram um cinética linear . O estudo de produção de melanina in vivo confimou que a radiação estimula a produçao de melanina assim como o extrato de B.Gaudichaudii, e quando associados (PUVA) a sintese de melanina foi 143% maior em comparação ao controle negativo. Os resultados destre trabalho mostrou a capacidade de pgmentaçao dos fármacos assim como do B.Gaudichaudii, revelando ainda o sinergismo entre psoraleno e bergapteno. / The objective of this study was to evaluate the melanogenic action of furanocoumarins, namely, psoralen and bergapten, as extract of Brosimum gaudichaudii Trécul containing 1.2% m / m (psoralen), 5.2% m / m (bergapten) . The cellular toxicity (in vitro) was evaluated by neutral red incorporation test in four different cell lines (fibroblasts, L929, keratinocytes, HaCaT, melanoma, B16F10, and melanocyte- Melam-A) and showed dose dependent response to both extracted compounds, as well as the whole extract of B. gaudichaudii. The ultraviolet radiation type A and B increased the toxicity associated with the compounds and severity of toxicity was proportional to the radiation dose. The mutagenicity and genotoxicity evaluated by the micronucleus test and comet showed that the compounds are genotoxic and mutagenic compounds at concentrations >= 150 ?g/mL. The in vitro melanin synthesis, performed in melanoma B16F10, was dose, duration and UV dependent. In the maximum concentration used (48 ?g/mL psoralen, 104 ?g/mL bergapten and 0.5 mg.mL-1 extract) psoralen increased melanin synthesis by 26%, bergapten by 69% and the B.gaudichaudii extract by 163%. When administered as an equivalent mixture of 6 ?g/mL psoralen and 26 ?g/mL of bergapten to 0.5 mg/mL of extract, melanin synthesis was increased by 61%. The enzymatic activity of tyrosinase in B16F10 cultures treated with 20 ?g/mL of psoralen or bergapten, when compared to non-treated group, increased by 13%; the increases were 37% and 54.1% in the two-compound mixture and 0.5 mg/mL of whole extract. The in vivo microdialese test in rats showed that the drugs are quickly absorbed through the skin and distributed in plasma. Both compound exhibited linear kinetics. The in vivo evaluation also showed that melanin production was stimulated by UV radiation as well as B.Gaudichaudii extract. When combined with PUVA, melanin synthesis was 143% higher compared to the negative control

Para verificar a contribuição da audiometria em altas freqüências (9 a 20 kHz) e das emissões otoacústicas - produto de distorção para a identificação de alterações auditivas em indivíduos com desordem pigmentar do tipo vitiligo, 30 indivíduos portadores de vitiligo foram avaliados audiologicamente, e comparados a um grupo controle. Tanto para as altas freqüências (9 a 20 kHz) como para as emissões otoacústicas foram observadas diferenças significantes entre os grupos, que não foram evidenciadas na audiometria convencional (0,25 a 8 kHz). Os resultados sugerem prejuízo nas funções auditivas para o grupo com desordem pigmentar do tipo vitiligo. / In order to verify the contribution of the high frequency audiometry (9 to 20 kHz) and distortion product- otoacoustic emissions for the identification of auditory impairments in individuals with pigmentary disorder type vitiligo, 30 individuals with vitiligo were evaluated, and compared with a control group. As much for the high frequencies (9 the 20 kHz) as for the otoacoustic emissions, significant differences were observed between the groups, that were not evidenced in the conventional audiometry (0,25 the 8 kHz). The results suggest damage in the auditory functions for the group with pigmentary disorder type vitiligo

Submitted by Filipe dos Santos (fsantos@pucsp.br) on 2017-03-27T10:22:27Z No. of bitstreams: 1 Augusta Renata Almeida do Sacramento.pdf: 9888290 bytes, checksum: fadb890d0459ba8b85c16f2db87c194a (MD5) / Made available in DSpace on 2017-03-27T10:22:27Z (GMT). No. of bitstreams: 1 Augusta Renata Almeida do Sacramento.pdf: 9888290 bytes, checksum: fadb890d0459ba8b85c16f2db87c194a (MD5) Previous issue date: 2017-03-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The proposal of this thesis, based on psychosomatic theoretical literature, was to present the perception of women with vitiligo about a group psychological intervention on their coexistence with the disease. The participants were evaluated in two moments, with the use of the following instruments: semi-structured interview and group psychological intervention. The intervention was carried out in five encounters, with a sample of 12 women, of the State of Sergipe, divided into Group A (n=8) and B (n=4), aged between 20 and 72 years old, who live with the disease in the range of 5 to 58 years old. The intervention included the use of three resources: word, body, and art. The data were analyzed and grouped into five main themes: 1) perception of self; 2) perception of the disease; 3) perception of the relationship with the disease; 4) perception of friendly, lovely and family relations; 5) perception of group psychological clinical intervention. The overall results indicate, for all participants, different expressions of psychological suffering associated with the illness by vitiligo, pointing to a not recognized grieving process. Women complain that there is no social validation of clinical condition presented and, at the same time, it does not seem to allow the personal recognition of losses arising with the pathology. There was either the need for guidance to the family because one realizes that living with vitiligo has the potential to affect not only the people with the disease, but also their family members, in addition to the love and friendship relational contexts. We observed that the intervention allowed the participants to express the anguish and to recognize the limitations and defensive behaviors due to the illness, being perceived by them in a positive way, therefore, revealing itself as a useful resource in the care of patients with vitiligo, striving for the development of resiliency. Bring these issues to debate can contribute to the minimization of individual and collective effects, by the reduction of discrimination and stigmatization associated with the diagnosis, which can contribute to a more satisfying prognosis and improvements to the quality of life of the public / A proposta desta tese, sustentada no referencial teórico da psicossomática, foi apresentar a percepção de mulheres com vitiligo acerca de uma intervenção clínica psicológica grupal na sua convivência com a doença. As participantes foram avaliadas em dois momentos, com a utilização dos seguintes instrumentos: entrevista semiestruturada e intervenção clínica psicológica grupal. A intervenção foi realizada em cinco encontros, com uma amostra de 12 mulheres, do estado de Sergipe, divididas em grupo A (n=8) e B (n=4), com idades entre 20 e 72 anos, que convivem com a doença no intervalo de 5 a 58 anos. A intervenção contou com a utilização de três recursos: palavra, corpo e arte. Os dados foram analisados e agrupados em cinco eixos temáticos: 1) percepção de si; 2) percepção da doença; 3) percepção da relação com a doença; 4) percepção das relações de amizade, amorosas e familiares; 5) percepção da intervenção clínica psicológica grupal. Os resultados gerais indicam, para todas as participantes, diferentes expressões de sofrimento psicológico associadas ao adoecimento por vitiligo, que apontam para um processo de luto não reconhecido. As mulheres se queixam de que não há uma validação social da condição clínica apresentada e, ao mesmo tempo, isso parece não possibilitar o reconhecimento pessoal das perdas advindas com a patologia. Verificou-se, ainda, a necessidade de orientações à família, pois se percebe que a vivência com vitiligo tem o potencial de afetar não somente as pessoas com a doença, mas também seus familiares, além dos contextos relacionais amorosos e de amizade. Observamos, igualmente, que a intervenção permitiu às participantes a expressão de angústias e o reconhecimento de limitações e comportamentos defensivos devido à doença, sendo percebida por elas de forma positiva, revelando-se, portanto, como um útil recurso no cuidado a pacientes com vitiligo, com vistas ao desenvolvimento da resiliência. Trazer ao debate essas questões pode colaborar com a minimização de efeitos individuais e coletivos, pela redução da discriminação e estigmatização associadas ao diagnóstico, o que pode contribuir para um prognóstico mais satisfatório e melhorias para a qualidade de vida desse público

La couleur de la peau résulte de la synthèse et de la distribution de la mélanine dans l’épiderme et les poils. La mélanine est un pigment produit par les mélanocytes, des cellules dérivées de la crête neurale. Les mélanocytes transfèrent la mélanine aux kératinocytes environnants et forment l'unité épidermique de mélanisation constituée d’un mélanocyte pour 40 kératinocytes. Les interactions entre les mélanocytes et les kératinocytes sont assurées principalement par la E-CADHÉRINE, une protéine responsable de la formation de jonctions adhérentes entre deux cellules adjacentes. L’ancrage de ses jonctions au cytosquelette est assuré par l’intermédiaire de la β-CATÉNINE. Le rôle de ces protéines n’est pas simplement adhésif, elles interviennent aussi dans de nombreux processus développementaux et assurent le maintien de l’architecture de l’épiderme. De plus, β-CATÉNINE est une protéine centrale de la voie de signalisation WNT/β-CATÉNINE essentielle dans la formation du lignage mélanocytaire. Lors de cette thèse, nous nous sommes intéressés au rôle des protéines E-CADHÉRINE et β-CATÉNINE, d’une part dans l’homéostase des mélanocytes et d’autre part dans le développement embryonnaire d’un type particulier de mélanocytes, ceux peuplant l’extrémité des membres. Dans la première partie, nous avons étudié l’implication de ces protéines dans une leucodermie circonscrite acquise, le Vitiligo. Dans cette pathologie, les mélanocytes disparaissent, générant des zones de peau dépigmentées. L’une des hypothèses invoquées est un défaut adhésion des mélanocytes et leur élimination de la lame basale vers les couches supérieures de l’épiderme. Nous avons montré que la présence de E-CADHÉRINE et β-CATÉNINE à la membrane est altérée dans les mélanocytes Vitiligo avant leur disparition de l’épiderme, au niveau de la peau pigmentée des patients. L’altération de E-CADHÉRINE et β-CATÉNINE à la membrane des mélanocytes est corrélée à une localisation suprabasale de ces cellules et une perturbation de l’unité épidermique de mélanisation. A l’aide de systèmes de peaux reconstruites in vitro et de souris déficientes en E-CADHÉRINE dans les mélanocytes, nous avons démontré un rôle essentiel de E-CADHÉRINE pour l’adhésion des mélanocytes à la lame basale de l’épiderme en présence de stress mécanique et oxydatif, deux facteurs aggravants de la dépigmentation dans le Vitiligo. Nous proposons que l’altération de E-CADHÉRINE est un événement précoce dans la pathologie du Vitiligo qui reste silencieux jusqu’à ce que des stress mécaniques ou oxydatifs accélèrent la perte des mélanocytes (Wagner et al., 2015).Dans la deuxième partie, nous nous sommes intéressés aux rôles des protéines E-CADHÉRINE et β-CATÉNINE dans le développement embryonnaire des mélanocytes. Nous avons examiné la possibilité de générer des mélanoblastes à partir des dérivés des cellules de crêtes neurales qui emprunteraient la voie ventrale de migration et non la voie dorso-latérale classiquement décrite. Nous avons montré que les mélanoblastes produits à partir de la voie ventrale dérivent de cellules précurseurs se spécifiant à E14 en mélanoblastes ou en cellules de Schwann. A l’aide d’un mutant gain de fonction de β-CATÉNINE, nous avons mis en évidence que l’activation de la signalisation de β-CATÉNINE induit la spécification des mélanocytes au détriment des cellules de Schwann. La E-CADHÉRINE n’intervient pas dans la spécification des mélanoblastes issus de la voie ventrale mais est impliquée dans l’expansion des mélanoblastes issus de la voie dorso-latérale au niveau des membres.Ces résultats démontrent un rôle critique de E-CADHÉRINE et β-CATÉNINE dans l’homéostase des mélanocytes dans des conditions de stress et dans la régulation du développement des mélanocytes. / Skin pigmentation results from the synthesis and the distribution of melanin by melanocytes. Melanocytes are neural crest derived cells that produce and transfer melanin to their surrounding keratinocytes. One melanocyte makes contacts with approximately 40 keratinocytes, forming the so-called epidermal melanin unit. Adhesion between melanocytes and keratinocytes is mediated by the adhesive protein E-CADHERIN, which is responsible for the formation of adherens junctions. These junctions are anchored to the cytoskeleton via β-CATENIN. The main function of adhesive proteins is to form cell-cell junctions and to maintain epidermal architecture. β-CATENIN is a central component of the WNT signalling pathway, which is implied in the development of the melanocyte lineage. During this PhD we were interested in the potential roles of E-CADHERIN and β-CATENIN proteins first in melanocyte homeostasis and second in melanocyte development in the mouse limb.In the first part of this PhD project, we studied the role of these proteins in an acquired leuco-derma: the Vitiligo disease. In this disease, depigmented areas appears in the skin due to melano¬cyte loss. One hypothesis for this loss is a defect in adhesive proteins of melanocytes, leading to melanocyte detachment and loss. We examined pigmented skin biopsies of patients with or without Vitiligo and observed that membranous staining of E-CADHERIN and β-CATENIN is absent from, or discontinuously distributed across melanocyte membranes of Vitiligo patients long before clinical lesions appeared. The abnormal distribution of E-CADHERIN correlated with lower melanocyte numbers in the basal epidermal layer and higher melanocyte numbers in the suprabasal layer. Using reconstructed human epidermis and mouse models with defective E-CADHERIN expression in melanocytes, we showed that E-CADHERIN is required for melanocyte adhesiveness to the basal layer under oxidative and mechanical stress. These observations establish a link between pre-clinical, cell-autonomous defects in Vitiligo melanocytes and known environmental stressors accelerating disease onset. Our results implicate a primary predisposing skin defect affecting melanocyte adhesiveness, which under stress conditions, leads to the disappearance of melanocytes and clinical Vitiligo (Wagner et al., 2015).In the second part of this PhD project, we examined the role of these two proteins E-CADHERIN and β-CATENIN in the development of melanoblasts from the ventrally migrating pathway in contrast to the laterally migrating pathway previously described. We observed that ventrally migrating melanoblasts arose from precursors specified at E14 in melanoblasts or Schwann cells. Using a β-CATENIN gain of function mouse model, Tyr::Cre ; bcatΔex3 we observed that β-CATENIN signalling activation induced melanoblast specification at the expense of Schwann cells. We also demonstrated that E-CADHERIN loss in melanocytes (Tyr::Cre ; EcadF/F) decreased dorso-laterally migrating melanoblast expansion in the limb. Taken together, these results point to a critical role for E-CADHERIN and β-CATENIN in maintaining melanocyte homeostasis under stress conditions and regulating melanocyte development.

O RNA de interferência (RNAi) é um processo envolvido com o silenciamento pós-transcricional de genes, sendo elucidado por moléculas de RNAs de dupla fita de 21-25 nucleotídeos, os small interfering RNA (siRNA), que ocorre naturalmente em uma ampla variedade de animais, plantas e microrganismos. Esse processo têm demonstrado potencial utilização para o tratamento de doenças em que se observa a super-expressão de genes, pois apresentam diversas vantagens como possibilidade de se utilizar esse mecanismo regulatório apenas pelo conhecimento da sequência do gene terapêutico, menores efeitos tóxicos e alta especificidade. Contudo, o principal desafio consiste no desenvolvimento de vetores seguros e eficazes que permitem a utilização do siRNA como terapia, visto que permitem a proteção do siRNA contra degradação enzimática, apresentam meia-vida prolongada na corrente sanguínea e propiciam um efetivo escape endossomal. Nesse sentido, nanopartículas de cristais líquidos associados ao polímero catiônico polietilenoimina (PEI) foram avaliadas como potenciais vetores não-virais para o siRNA específico para a proteína-1 relacionada à tirosinase (TRP-1), como alternativa para o tratamento tópico do vitiligo. Para isso, os cristais líquidos contendo o PEI foram complexados ao siRNA e avaliados quanto à estrutura líquido cristalina por microscopia de luz polarizada e por difração de raios-X a baixo ângulo (SAXS), tamanho de partícula/polidispersividade, potencial zeta e eficiência de complexação. A citotoxicidade dos sistemas foi avaliada em melanócitos melan-A pelo ensaio do MTT e por citometria de fluxo e, a avaliação da transfecção celular foi realizada por microscopia de fluorescência e por citometria de fluxo. Os diferentes sistemas contendo o polímero PEI apresentaram estruturas líquido cristalinas de fase hexagonal e de fase lamelar pela análise por SAXS, no entanto, a análise sob microscopia de luz polarizada, demonstrou estruturas de fase hexagonal, lamelar e isotrópica. A análise do tamanho de partícula mostrou a presença de sistemas nanoestruturados que foram capazes de se complexar ao siRNA na concentração de 10 ?M. Os estudos em cultura de célula demonstraram a maior viabilidade das células melan-A após tratamento com as nanodispersões líquido cristalinas formadas por monoleína (MO), ácido oleico (AO) e PEI, em relação ao polímero catiônico PEI em sua forma livre. Em relação à transfecção celular por microscopia de fluorescência e por citometria de fluxo foi possível observar a elevada eficiência de transfecção das nanodispersões líquido cristalinas formadas pelo sistema MO:AO:PEI. Resultados de inibição da expressão da proteína TRP-1 foram observadas em células melan-A por Western Blotting, após administração das nanodispersões líquido cristalinas associadas ao siRNA-TRP-1 específico. As nanodispersões liquido cristalinas estudas também proporcionaram maior liberação de siRNA na pele em modelo animal. Esses resultados, demonstram a potencial utilização desses sistemas para a terapia anti-sense de doenças cutâneas como o vitiligo, representando assim, uma importante contribuição para a terapia gênica tópica desta doença / The RNA interference (RNAi) is a process involved with the post-transcriptional gene silencing being elucidated by double-stranded RNA molecules of 21-25 nucleotides, the small interfering RNA (siRNA) that occurs naturally in a wide variety of animals, plants and microorganisms. This process has shown potential use for the treatment of diseases in which there is overexpression of genes, as they offer several advantages such as the possibility of using this regulatory mechanism just by knowing the sequence of the therapeutic gene, lower toxicity and high specificity. However, the main challenge is to develop safe and effective vectors that enable the use of siRNA as a therapy, since they allow the protection of siRNA against enzymatic degradation, have prolonged half-life in the bloodstream and provide an effective endosomal escape. Accordingly, liquid crystal nanoparticles associated with the cationic polymer polyethylenimine (PEI) were evaluated as potential non-viral vectors for specific siRNA for the protein related to tyrosinase-1 (TyRP-1) as an alternative for the topical treatment of vitiligo. For this, the liquid crystals containing PEI were complexed to siRNA and evaluated for liquid crystalline structure by polarized light microscopy and X-ray diffraction (SAXS), particle size / polydispersity index, zeta potential and complexation efficiency. The cytotoxicity of the systems was evaluated by MTT assay and flow cytometry in melan-A melanocytes and the evaluation of cellular uptake was performed by fluorescence microscopy and flow cytometry. The different systems containing the polymer PEI exhibited liquid crystalline structures of hexagonal and lamellar phases by SAXS analysis, however, the analysis under polarized light microscopy showed liquid crystalline structures of hexagonal phase, lamellar and isotropic. The analysis of particle size showed the presence of nanostructured systems that were capable of complexing to the siRNA at concentration of 10 ?M. Studies in cell culture demonstrated a higher viability of melan-A cells after treatment with the liquid crystalline nanodispersions formed by monolein (MO), oleic acid (OA) and PEI in relation to the cationic polymer PEI in its free form. Regarding cellular uptake by fluorescence microscopy and flow cytometry was observed the high efficiency in uptake melan-A cells mediated by liquid crystalline nanodispersions formed by system MO:OA:PEI. Results inhibition of the expression of TyRP-1 protein were observed by Western Blotting in melan-A cells, after administration of liquid crystalline nanodispersions associated with specific siRNA-TyRP-1. The liquid crystalline nanodispersions evaluated also provided greater release of siRNA in the skin in an animal model. These results demonstrate the potential use of these systems for antisense therapy of skin diseases such as vitiligo, thus representing, an important contribution to the topical gene therapy for this disease

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-01-13T12:38:13Z No. of bitstreams: 2 Tese - Kênia Alves Pereira Lacerda - 2016.pdf: 1930787 bytes, checksum: d2d35012cd70eaa30ae8e03b674c7055 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-16T11:01:34Z (GMT) No. of bitstreams: 2 Tese - Kênia Alves Pereira Lacerda - 2016.pdf: 1930787 bytes, checksum: d2d35012cd70eaa30ae8e03b674c7055 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-16T11:01:34Z (GMT). No. of bitstreams: 2 Tese - Kênia Alves Pereira Lacerda - 2016.pdf: 1930787 bytes, checksum: d2d35012cd70eaa30ae8e03b674c7055 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-12-06 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Vitiligo is a skin disease that affects about 1% of the population worldwide. This disease does not cause physical incapacity, but due to its disfiguring appearance, the carriers can present psychological disturbances and impairment in the quality of life. The pathogenesis has not yet been clarified. Recently, evidence has suggested that some gene polymorphisms are associated with risk of developing vitiligo. This study aimed to investigate the Glu298Asp polymorphism of the endothelial nitric oxide synthase gene (eNOS) in a group of patients with vitiligo and to compare the frequencies of the genotypes with a control group from the center-west region of Brazil, as well as assess the quality of life and the perception of stress In individuals with vitiligo and to compare the perception of stress with non-carriers. First study included 51 individuals with vitiligo and 50 controls. The whole blood samples were obtained by collection of peripheral venous blood, then DNA extraction was performed. The genotyping of the Glu298Asp polymorphism was performed by PCR-RFLP analysis. The research on the association of the polymorphism of the eNOS Glu298Asp gene with vitiligo did not present a significant difference in the distribution of genotypes and in the distribution of allelic frequencies between the carriers and the control group. However, in the analysis of the association between the genotypes and the variables location of vitiligo and skin color in the carriers, a higher proportion of TT genotypes was observed in individuals with 100% unisegmentar vitiligo and GT in patients with vitiligo located 68.3% (p = 0.028 ). Also, in the dominant model, a significantly higher proportion of GG genotype was observed in individuals of black color 34.5%, while a higher frequency of moderate-color individuals with GG + TT genotype was observed 54.5% (p = 0.035 ). In the second study, the sample consisted of 102 participants divided into two groups, with vitiligo and without vitiligo. We used the Dermatology Life Quality Index questionnaires and the Perceived Stress Scale. The mean quality of life score for patients with vitiligo was 4.7 ± 5.8, demonstrating a mild impairment in the patients. There was a significant association between quality of life and black skin color (β = 5.64, p <0.001), pattern of involvement of exposed vitiligo (β = 5.22, p <0.001) and perceived stress (β = 0,22 p = 0.033). The perceived stress assessed between the groups presented a mean of 20.7 ± 6.0 and 17.8 ± 7.0 for case and control, respectively. Vitiligo patients presented a high perception of stress when compared to the control group (β = 3.12, p = 0.022). The results pointed out a high perception of stress in vitiligo patients, evidencing that the fact of having vitiligo increases the level of stress. It is known that several factors, such as the genetic profile and ethnicity of the analyzed population, can influence the complex susceptibility to the disease, therefore, more studies are needed to elucidate the role of eNOS gene polymorphism in the pathogenesis of vitiligo. / O vitiligo é uma doença da pele que afeta cerca de 1% da população em todo o mundo. Esta doença não causa incapacidade física, mas devido à sua aparência desfigurante, os portadores podem apresentar distúrbios psicológicos e comprometimento na qualidade de vida. A patogênese ainda não foi esclarecida. Recentemente, evidências sugeriram que alguns polimorfismos de genes estão associados com risco de desenvolvimento do vitiligo. Este estudo objetivou investigar polimorfismo Glu298Asp do gene óxido nítrico sintase endotelial (eNOS) em um grupo de pacientes com vitiligo e comparar as frequências dos genótipos com um grupo controle da região centro oeste do Brasil, também avaliar a qualidade de vida e a percepção de estresse em indivíduos portadores de vitiligo e comparar a percepção de estresse com os não portadores. Primeiro estudo, incluiu 51 indivíduos com vitiligo e 50 pessoas controles. As amostras de sangue total foram obtidas por coleta de sangue venoso periférico, em seguida, foi realizado a extração do DNA. A genotipagem do polimorfismo Glu298Asp foi realizada por análise de PCR-RFLP. A investigação sobre a associação do polimorfismo do gene eNOS Glu298Asp com vitiligo, não apresentou diferença significativa na distribuição dos genótipos e na distribuição das frequências alélicas entre os portadores e grupo controle. Porém, na análise da associação entre os genótipos e as variáveis localização do vitiligo e cor da pele nos portadores, verificou maior proporção de genótipos TT em indivíduos com vitiligo unisegmentar 100% e GT em portadores com vitiligo localizado 68,3% (p = 0,028). Ainda, no modelo dominante, verificou-se uma proporção significativamente maior de genótipo GG em indivíduos da cor negra 34,5%, enquanto se verificou maior frequência de indivíduos da cor morena moderada com genótipo GG+TT 54,5% (p = 0,035). No segundo estudo, a amostra foi composta por 102 participantes divididos em dois grupos, com vitiligo e sem vitiligo. Utilizou-se os questionários Dermatology Life Quality Index e a Perceived Stress Scale. O escore médio de qualidade de vida para os portadores de vitiligo foi de 4,7 ± 5,8, demonstrando um comprometimento leve nos portadores. Verificou-se associação significativa entre qualidade de vida e cor da pele negra (β = 5,64; p < 0,001), padrão de envolvimento do vitiligo exposto (β = 5,22; p < 0,001) e estresse percebido (β = 0,22; p = 0,033). O estresse percebido avaliado entre os grupos apresentou média de 20,7±6,0 e 17,8±7,0 para caso e controle, respectivamente. Portadores do vitiligo apresentaram uma percepção elevada de estresse quando comparado ao grupo controle (β = 3,12; p = 0,022). Os resultados, apontaram uma percepção elevada de estresse nos portadores de vitiligo, evidenciando que o fato de possuir vitiligo aumenta o nível de estresse. Sabe-se que vários fatores, tais como o perfil genético e etnia da população analisada, podem influenciar no complexo susceptibilidade à doença, portanto, mais estudos são necessários para elucidar o papel do polimorfismo do gene da eNOS na patogênese do vitiligo.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-09-06T12:14:24Z No. of bitstreams: 2 Dissertação - Amanda Rodrigues Miranda - 2016.pdf: 3245490 bytes, checksum: 810f33abf110260d76f828e568843cfe (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-06T13:30:11Z (GMT) No. of bitstreams: 2 Dissertação - Amanda Rodrigues Miranda - 2016.pdf: 3245490 bytes, checksum: 810f33abf110260d76f828e568843cfe (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-06T13:30:11Z (GMT). No. of bitstreams: 2 Dissertação - Amanda Rodrigues Miranda - 2016.pdf: 3245490 bytes, checksum: 810f33abf110260d76f828e568843cfe (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-12-03 / Vitiligo is a chronic disease of skin, acquired and idiopathic, characterized by circumscribed depigmented macules or patches. The treatment is still a challenge and don´t have an effective treatment that may improve all patients. The traditional psoralen photosensitizers are options for the treatment of vitiligo. In the Brazilian cerrado is popularly known action of Brosimum gaudichaudii Trécul (BGT), or mamacadela, which is an herbal psoralen. The goal of this study was evaluate the efficacy, safety and tolerability of BGT in adult patients with vitiligo, in topical and systemic presentations. A randomized controlled trial, double-blind and placebo-controlled was conducted. Of the 58 selected patients, 33 were randomized, 17 in the treatment group (G1) and 16 in the placebo group (G2). These remained until the end of the study 10 patients in G1 and G2 in 6 patients. The interventions were 800 mg / day of medication per orally and use of the ointment for 22 weeks, together with graded and daily photoexposure. The effectiveness of the BGT was assessed using the Vitiligo Area Score Index (VASI), applied by a dermatologist at all clinic visits. Already the safety and tolerability were assessed by laboratory tests before and during treatment, and observation of adverse events. All patients were classified with generalized vitiligo. The group that received the medication (G1) improved significantly VASI in intra-group evaluation (p <0.05) and also a higher proportion of repigmentation compared to G2. The VASI don´t have association with the analysis variables (gender, age, skin type, previous treatment, education, emotional stress, time of diagnosis, treatment adherence) (p> 0.05). Adherence to photoexposure was irregular in both groups, and don´t have statistically significant relationship with changes of VASI (p > 0.05). Laboratory tests showed no significant changes. Adverse events were mild and transient and the most common being erythema, and occurred predominantly in G1 patients. Therefore, it can be concluded that the BGT is effective in the treatment of vitiligo, with safe and well tolerated in the long term. / O vitiligo é dermatose crônica adquirida, idiopática, que se manifesta por manchas acrômicas e bem delimitadas. Seu tratamento ainda é um desafio e não existe até o momento intervenção completamente eficaz. Os psoralenos são fotossensibilizantes tradicionais utilizados para o tratamento do vitiligo. No cerrado brasileiro é popularmente conhecida a ação da planta mamacadela (Brosimum gaudichaudii Trécul (BGT)) devido a presença de derivados psoralênicos em sua composição . O presente estudo teve como objetivo avaliar a eficácia, segurança e tolerabilidade de formulações farmacêuticas contendo extrato de raízes de BGT em pacientes adultos portadores de vitiligo, nas apresentações tópica e sistêmica. Foi realizado ensaio clínico randomizado, duplo-cego e placebo-controlado. Dos 58 pacientes selecionados, 33 foram randomizados, sendo 17 no grupo 1 (G1), de tratamento, e 16 no grupo 2 (G2), placebo. Destes, permaneceram até o final do estudo 10 pacientes no G1 e 6 pacientes no G2. Foram administrados 800 mg/dia da medicação, via oral, além da pomada, durante 22 semanas, associados a fotoexposição diária e gradativa. A eficácia do BGT foi avaliada através do Vitiligo Area Score Index (VASI), aplicado por dermatologista em todas as visitas clínicas. Já a segurança e tolerabilidade foram avaliadas pela realização de exames laboratoriais, antes e durante o tratamento, além da observação de eventos adversos. Todos os pacientes foram classificados com vitiligo generalizado. O grupo que recebeu a medicação (G1) apresentou melhora significativa do VASI na avaliação intragrupos (p<0,05) e também maior proporção de repigmentação quando comparado ao G2. O VASI não demonstrou associação com as variáveis de análise (sexo, idade, fototipo, tratamento prévio, escolaridade, estresse emocional, tempo de diagnóstico, adesão ao tratamento) (p>0,05). A adesão à fotoexposição foi irregular em ambos os grupos e não apresentou relação estatisticamente significante com as alterações do VASI (p>0,05). Em consonância, os exames laboratoriais não apresentaram alterações significativas. Os eventos adversos foram leves e transitórios, sendo mais comum o eritema, e ocorreram predominantemente nos pacientes do G1. Portanto, pode-se concluir que o BGT é eficaz no tratamento do vitiligo, sendo seguro e bem tolerado a longo prazo.