Polycomb repressive complex 2 (PRC2) catalyses the methylation of ‘Lys-27’ of histone H3, leading to transcriptional repression of target genes through its catalytic subunit Enhancer of zeste homolog 1/2 (EZH1/2). PRC2 functions as a critical regulator of stem cells in mouse embryonic and adult tissues. However, the role of PRC2 in human skin remains largely unknown. This study investigated the role of PRC2 in human epidermal homeostasis and hair growth. The expression of EZH2 was elevated in differentiating suprabasal layers of the human epidermis. Consistently, EZH1/2 expression and enzymatic activity was upregulated in differentiating primary human keratinocytes (NHEKs) in vitro. Inhibition of EZH2 and Embryonic ectoderm development (EED) in NHEKs stimulated the expression of differentiation-associated genes, therefore leading to their premature differentiation; while inhibition of EZH1/2 reduced cell proliferation and promoted apoptosis. Silencing of EZH2 in NHEKs induced complex changes in gene expression programmes, including the upregulation of terminal differentiation genes, such as Filaggrin. EZH2 expression was downregulated in aged keratinocytes accompanied with upregulation of senescence-associated genes, p16INK4A and p19INK4D, suggesting EZH2 involvement in epidermal aging. In human anagen hair follicle (HF), EZH2 was detected in stem and progenitor cells; and hair matrix keratinocytes. Silencing EZH2 in HFs accelerated anagen-catagen transition and retarded hair growth accompanied by decreased proliferation and increased apoptosis. Silencing EZH2 in outer root sheath keratinocytes resulted in upregulation of p14ARF and K15, suggesting EZH2 involvement in regulating proliferation and stem cell activity. Thus, this study demonstrates that PRC2-mediated repression is crucial for epidermal homeostasis and hair growth. Modulating the activities of PRC2 in skin might offer a new therapeutic approach for disorders of epidermal differentiation and hair growth.

Nowdays there is a plethora of evidence for H2O2-mediated oxidative stress in the epidermis as well as in the system in patients with vitiligo (for review see (Schallreuter, Bahadoran et al. 2008). Xanthine dehydrogenase / xanthine oxidase (XDH / XO) catalyses the oxidative hydroxylation of hypoxanthine to xanthine followed by xanthine to uric acid, the last two steps in purine degradation pathway. Under oxidative conditions, XDH is converted to XO. The reactions catalysed by this enzyme generate H2O2 and O2 ¿- , yielding in the presence of ROS accumulation, allantoin from uric acid. Therefore XO has been considered a major biologic source of oxygen-derived free radicals in many organs. The presence of XO in the human epidermis has not been shown so far. In this study several techniques were utilised to nail the presence and activity of XO in epidermal melanocytes and keratinocytes. The enzyme is regulated by H2O2 in a concentration dependent manner, where concentrations of 10-6M upregulate activity. Importantly, the results showed that the activity of XO is little affected by H2O2 in the mM range. H2O2-mediated oxidation of tryptophan and methionine residues in the sequence of XO yields only subtle alterations in the enzyme active site. These findings are in agreement with enzyme kinetics in the presence of 10-3M H2O2. Since uric acid is the end product of XO activity and this can be oxidised to allantoin by H2O2, we wanted to know whether allantoin is formed in the epidermis of patients with vitiligo. In order to address this issue, we utilised HPLC/mass spectrometry analysis. Analysis of epidermal cell extracts from suction blister tissue identified the presence of allantoin in patients with acute vitiligo, while this product was absent in healthy controls. In conclusion, our results provide evidence for functioning epidermal XO in the human epidermis which 4 can be a major source for the production of H2O2 contributing to oxidative stress in vitiligo. In addition, this thesis also demonstrates for the first time the presence of XO in melanosomes, and we showed that both 7BH4 and 7-biopterin inhibit XO activity in a concentration dependent manner. Moreover, XO has the potential to bind to 6/7BH4 and 6/7-biopterin from the pterin/tyrosinase inhibitor complex. This discovery adds another receptor independent mechanism for regulation of tyrosinase within the melanocyte similar to ¿/ß-MSH as shown earlier (Moore, Wood et al. 1999; Spencer, Chavan et al. 2005). Since the entire epidermis of patients with vitiligo is under H2O2-mediated oxidative stress, oxidative DNA damage would be highly expected. This thesis shows for the first time that epidermal 8-oxoG levels as well as plasma level of this oxidised DNA base are significantly increased in patients compared to healthy controls. We have shown that epidermal cells from patients with vitiligo respond to oxidative DNA damage via the overexpression of p21 and Gadd45¿ leading to a functioning increased short-patch base-excision repair (BER), while increased apoptosis can be ruled out due to lower caspase 3 and cytochrome c response compared to healthy controls. Our results show that patients develop effective DNA repair machinery via hOgg1, APE1 and DNA polymeraseß. Taking into consideration that these patients do not have an increased prevalence for solar-induced skin cancers, our data suggest that BER is a major player in the hierarchy to combat H2O2-mediated oxidative stress preventing ROS-induced tumourigenesis in the epidermis of these patients.

Skin is an organ with a complex structure which plays a crucial role in thebody’s defence against external threats and in maintaining major homeostatic functions. The need for in vitro models that mimic the in vivo milieu is therefore high and relevant with various applications including, among others, penetration, absorption, and toxicity studies. In this context, the choice of the biomaterial that will provide a 3D scaffold to the cultured cells is defining the model’s success. The FN-4RepCT silk is here suggested as a potent biomaterial for skin tissue engineering applications. This recombinantly produced spider silk protein (FN-4RepCT), which can self-assemble into fibrils, creates a robust and elastic matrice with high bioactivity, due to its functionalization with the fibronectin derived RGD-containing peptide. Hence it overcomes the drawbacks of other available biomaterials either synthetic or based on animal derived proteins. Additionally, the FN-4RepCT silk protein can be cast in various 3D formats, two of which are utilized within this project. We herein present a bilayered skin tissue equivalent supported by the FN-4RepCT silk. This is constructed by the combination of a foam format, integrated with dermal fibroblasts and endothelial cells, and a membrane format supporting epidermal keratinocytes. As a result, a vascularized dermal layer that contains ECM components (Collagen I, Collagen III, and Elastin) is constructed and attached to an epidermal layer of differentiated keratinocytes.The protocol presented in this project offers a successful method of evenly integrating cells in the FN-4RepCT silk scaffold, while preserving their ability to resume some of their major in vivo functions like proliferation, ECM secretion, construction of vascular networks, and differentiation. The obtained results were evaluated with immunofluorescence stainings of various markers of interest and further analysed, when necessary, with image processing tools. The results that ensued from the herein presented protocol strongly suggest that the FN-4RepCT silk is a promising biomaterial for skin tissue engineering applications.

No / This work explored the role of skin appendages (shunt route) in passive and iontophoretic drug and liposome penetration. The technique used an epidermis and stratum corneum sandwich from the same skin donor with the additional stratum corneum forming the top layer of the sandwich. Penetration was monitored during occluded passive and iontophoretic (0.5 mA cm-2) delivery of mannitol and estradiol solutions, and ultradeformable liposomes containing estradiol. The shunt route had a significant role during passive penetration of mannitol (hydrophilic compound), but was negligible during penetration of estradiol (lipophilic drug) and liposomes. In iontophoresis, the shunt route significantly contributed to the overall flux of all preparations, being highest for mannitol. However, shunts were not the only pathway for iontophoretic drug delivery and evidence was observed for the creation of new aqueous pathways via disorganization of the intercellular lipid domain of stratum corneum. The skin sandwich technique should prove valuable for general studies on routes of skin penetration.

Os corantes sintéticos são amplamente empregados em indústrias têxteis, de cosméticos, alimentícia, farmacêutica, dentre diversas outras. Entretanto, vários destes compostos apresentam elevada toxicidade intrínseca, e são precursores de intermediários tóxicos e/ou mutagênicos gerados durante a metabolização. Portanto, o emprego de corantes naturais destaca-se como uma alternativa aos sintéticos, na busca por compostos seguros para a saúde humana e ambiental. Neste contexto, no presente trabalho nós investigamos o potencial toxicológico e ecotoxicológico do corante natural eritrostominona (Ery), uma naftoquinona extraída de micro-organismo, utilizando modelos alternativos à experimentação animal. Adicionalmente, foi avaliada a ecotoxicidade do Basic Red 51 (BR51), um corante sintético azo utilizado em indústrias têxteis e de cosméticos, e do Ery degradado (EryD) após a exposição à luz, visando uma alternativa simples para o tratamento de efluentes industriais contendo o corante. As avaliações ecotoxicológicas foram realizadas em diferentes níveis tróficos: nos microcrustáceos Daphnia magna e nos estágios iniciais de desenvolvimento de zebrafish (Danio rerio). As avaliações toxicológicas do Ery foram realizadas em linhagem hepatocelular humana (HepG2) como órgão-alvo de metabolização de xenobióticos, e em epiderme humana equivalente (EHE), um modelo 3D construído com queratinócitos humanos imortalizados (HaCaT), visto que é esperado o contato dérmico devido ao potencial uso como corante de cosmético. Nossos resultados mostraram que o Ery e o BR51 são tóxicos para D. magna e zebrafish. O BR51 induziu alterações na imobilidade, na reprodução, no consumo de oxigênio e no comportamento de D. magna em concentrações até 200 vezes superiores às do Ery capazes de alterar a imobilidade, reprodução e comportamento. Todavia, para embriões e larvas de zebrafish ambos os corantes apresentaram efeitos tóxicos em concentrações próximas, com alterações do desenvolvimento embrionário e do comportamento, indução de efeitos pró-oxidantes e alterações no balanço energético dos organismos. O EryD interessantemente não apresentou nenhum efeito tóxico para os organismos aquáticos, demonstrando que a luz foi capaz de reduzir e/ou inativar a toxicidade da estrutura inicial. Para as linhagens celulares humanas, o Ery foi citotóxico para HepG2, tendo a apoptose como a principal causa de morte celular. O Ery também causou um atraso no ciclo celular de HepG2, em particular na fase da mitose (G2/M), diminuindo a proliferação das células. Por outro lado, o Ery não apresentou potencial genotóxico e mutagênico para HepG2 e não induziu citotoxicidade e genotoxicidade após exposições por períodos curtos em EHE. Em conclusão, o Ery e o BR51 são classificados como tóxicos e muito tóxicos para o ambiente aquático, respectivamente. O Ery também induz efeitos pró-apoptóticos, o qual pode estar ligado à estrutura química das quinonas. No entanto, o Ery apresenta potencial aplicabilidade industrial como um corante eco-friendly, com destaque para a simples e rápida fotodegradação, e como um corante não genotóxico e mutagênico para células humanas. Avaliações adicionais sobre os mecanismos de apoptose devem ser realizadas para assegurar a segurança à saúde humana / Synthetic dyes are widely used by textiles, cosmetic, food and pharmaceutical industries. However, several compounds have high inherent toxicity, and many are precursors of mutagenic and/or carcinogenic intermediates produced during metabolism. Thereby, the use of natural dyes became an alternative to the synthetic ones, in search of safe compounds for human and environmentl health. In this context, we aimed to investigate the toxicological and ecotoxicological potential of the natural dye erythrostominone (Ery), a naphthoquinone compound extracted from microorganism, using alternative methods to animal experimentation. Additionally, it was assessed the ecotoxicity of the Basic Red 51 (BR51), an azo synthetic dye used bu cosmetic and textile industries, and the Ery degraded (EryD) after exposure to light, aiming an easy alternative to industrial effluent treatments containing the dye. Ecotoxicological assessment was performed at different trophic levels: in the microcrutaceans Daphnia magna and in zebrafish (Danio rerio) early life stages. Toxicological assessment of Ery was also performed in human hepatocellular line (HepG2) as target organ of xenobiotic metabolism, and in equivalent human epidermis (EHE), a 3D model constructed with immortalized human keratinocytes (HaCaT), since dermal contact is expected due to potential use as cosmetic dye. Our results showed that Ery and BR51 are toxic for D. magna and zebrafish. BR51 induced alterations in immobility, reproduction, oxygen uptake and behavior of D. magna at concentrations up to 200-fold higher than those of Ery able to impair immobility, reproduction and behavior. However, for zebrafish embryos and larvae, both dyes showed toxic effects at close concentrations, with changes in embryo development and behavior, induction of pro-oxidant effects and changes in the energy balance of organisms. Interestingly, EryD showed no toxic effect on aquatic organisms, demonstrating that light was able to reduce and/or inactivate the toxicity of the initial chemical structure. For human cell lines, Ery showed to be cytotoxic to HepG2, with apoptosis being the main source of cell death. Ery also induced a delay in HepG2 cell cycle, particularly in the mitosis phase (G2/M), decreasing cell proliferation. On the other hand, Ery presented no genotoxic and mutagenic potential for HepG2 and did not induce cytotoxicity and genotoxicity after short-term exposure to EHE. In conclusion, Ery and BR51 are classified as toxic and very toxic to the aquatic environment, respectively. Ery also induces pro-apoptotic effects, which may be linked to the chemical structure of quinones. Therefore, Ery presents potential industrial applicability as an eco-friendly dye, highlighting the easy and rapid photodegradation, and the non-genotoxic and mutagenic effects for human cells. Further assessment of apoptosis mechanisms should be performed to ensure safety to human health

<p>Atopic dermatitis (AD) is a common pruritic skin disease in people and domestic animals that can be severely debilitating and stressful to the patient and the caregiver. The diagnosis of AD requires time consuming and expensive procedures, and treatment is often lifelong at considerable cost. Alterations in the lipid composition of the epidermis are a hallmark of the disease, and these may represent changes caused by the inflammation and defects in the lipid barrier. Liquid chromatography tandem mass spectrometry (LC-MS/MS) and, more recently, untargeted profiling using high-resolution time-of-flight instruments have been used to quantify the lipid composition in skin and other tissues, but these approaches are highly demanding in sample preparation and instrument time. In addition, these methods either detect only a limited number of lipids at the time or the identification of detected mass-to-charge ratio (m/z) is problematic when untargeted profiling is used. New lipidomic approaches that generate lipid profiles in a faster and more efficient manner can lead to a better understanding of these lipid changes. </p><p>The mass spectrometry analytical strategy used in this study, multiple reaction monitoring (MRM)-profiling, rapidly identifies discriminant lipids of the epidermis by flow injection. MRM-profiling is a small molecule accelerated discovery workflow performed in two parts using a triple quadrupole mass spectrometer with electrospray ionization as the ion source. Briefly, the first step consists of discovery experiments based on neutral loss and precursor ion scans to detect lipids in pooled samples by targeting class-specific chemical motifs such as polar heads of phospholipids or sphingoid bases of ceramides. The second step of the MRM-profiling is the screening of individual samples for the transitions detected in the discovery phase. </p><p>We first developed the experimental approach of the MRM-profiling methodology using epidermal samples of mice with AD-like inflammatory skin disease (chronic proliferative dermatitis, cpdm). Subsequently, we investigated lipid changes as the disease in mice progressed from minimal to severe. In order to select the most relevant ions, we utilized a two-tiered filter/wrapper feature-selection strategy. First, we built linear models linking the presence of every lipid monitored to disease stage information. The top 10 lipids, ranked based on η2 effect size, were used to build a predictive elastic-net (E-net) regression model linking the lipid ions detected by MRM-profiling with disease progression. The developed model accurately identified disease stages based on the variations in relative amounts of lipid ions corresponding to phosphatidylcholines, cholesterol esters, and glycerolipids-containing and eicosapentaenoic acid fatty acyl residues. Finally, we investigated the lipid profile of the epidermis in dogs with canine AD using the previously developed methodology. Epidermis from client owned patients and healthy controls were collected. Patients were sampled from affected and unaffected skin avoiding areas with secondary infections and the canine atopic dermatitis extent and severity index (CADESI-4) was recorded. The monitored lipids substantially separated the samples of healthy dogs from atopic dogs and distinguished the affected from the unaffected skin of patients. Samples were grouped into two cohorts for low-score and high-score CADESI-4, the first principal component was able to differentiate the control group from the low and high-score group. Differences in the lipid composition associated with low and high score CADESI-4 were significantly different only after separating the samples by sex of the dogs, demonstrating sexual dimorphism in the lipid changes associated with disease. The compositional data was feature extracted using the CADESI-4 to build linear models that identified oleic acid-containing triacylglycerides, long-chain acylcarnitines and sphingolipids as highly predictive lipids and were subsequently used to construct a predictive E-net regression. The lipid fingerprint obtained from the MRM-profiling was highly correlated (R2=0.89) with the classification of the standardized CADESI-4 score. </p><p>This research showed that changes in the lipid composition of the epidermis can be detected by MRM-profiling in atopic dogs even when the skin looks clinically healthy and that sex is a modifying factor in the lipid profile of canine atopic dermatitis (CAD). We expect that this research leads to a better understanding of the lipid changes in the epidermis during the onset of AD and as the chronic inflammatory process develops. The high prediction rate given by the lipid biomarkers for disease progression identified here by the machine learning strategy provides a potential molecular assessment tool for the diagnosis and monitoring of atopic dermatitis and the patient response to treatment.</p><div><br></div>

Introdução: A dermatite atópica (DA) é uma doença cutânea inflamatória crônica que cursa em surtos. Possui manifestação clínica variável, mas o prurido e a xerose são características frequentes, e pode estar associada a outras manifestações extra-cutâneas de atopia. Pacientes com DA apresentam maior risco de infecções por bactérias e vírus, destacando-se a erupção variceliforme de Kaposi, causada por herpes simples. A DA mostra-se como exemplo de dermatose com comprometimento da barreira cutânea, aliado a disfunção imunológica. São descritas alterações das proteínas da barreira cutânea na DA (filagrina e claudinas ), relacionadas ao maior risco de infecção . Objetivo: Avaliar a expressão de proteínas relacionadas à barreira cutânea como a filagrina, e as claudinas -1 e -4 na pele de pacientes adultos com dermatite atópica, acompanhados no Departamento de Dermatologia da Faculdade de Medicina da Universidade de São Paulo. Métodos: 32 indivíduos com diagnóstico de DA (estabelecido pelos critérios de Hanifin & Rajka) e 23 controles (indivíduos sem DA), maiores de 18 anos, foram submetidos a biópsias cutâneas. Os indivíduos com DA foram biopsiados em dois pontos, tanto na pele lesada, quanto na pele não-lesada. O material obtido foi analisado por imuno-histoquímica, através de marcadores específicos para filagrina, claudina-1 e claudina-4. As lâminas foram digitalizadas pelo Panoramic Scan - 3DHistech - Hungria, e as imagens analisadas pelo software Image Pro Plus 4,5, quanto à intensidade da expressão do marcador. A espessura média da epiderme do local estudado foi também avaliada. O grupo com DA foi também analisado quanto à gravidade da doença (EASI), níveis séricos de IgE e grau de eosinofilia. Resultados: Houve redução da expressão da filagrina na pele de doentes de DA em relação aos controles, tanto na pele com lesão quanto na pele sem lesão. Demonstrou-se correlação inversa na expressão da filagrina, tanto com relação à gravidade da doença quanto à espessura da epiderme. A análise das claudinas -1 e -4 demonstrou redução de ambas na pele dos doentes de DA, mas não houve correlação com a gravidade, espessura da epiderme, níveis de IgE sérica ou eosinofilia. Conclusão: No adulto com dermatite atópica, existe redução da expressão das proteínas relacionadas à barreira cutânea, como a filagrina e as claudinas -1 e -4. A redução da expressão da filagrina relacionou-se inversamente com a gravidade da doença, e com a espessura da epiderme, sugerindo cronicidade das lesões. Houve redução da expressão das claudinas -1 e -4, sem relação com a gravidade da doença, espessura da epiderme, eosinofilia ou com os níveis séricos de IgE / Introduction: Atopic dermatitis (AD) is a chronic, inflammatory dermatosis with ocasional flares. Its clinical features are variable, but pruritus and xerosis are frequent, and the disease may be associated to extracutaneous atopy. Patients with AD have increased risk for bacterial or viral infection, with emphasis on eczema herpeticum due to herpes simplex. AD is an example of a compromised skin barrier, allied to na imune dysfunction. There are reports on efective proteins of the skin barrier (filaggrin and claudins), related to increased risk for infection. Objectives: To evaluate the expression of proteins related to the skin barrier, such filaggrin and claudins-1 and-4 in the skin of adults with AD, followed at the Department of Dermatology, University of Sao Paulo Medical School. Methods: 32 individuals diagnosed as AD, according to Hanifin & Rajka\'s criteria, and 23 non-atopic controls, above the age of 18, were biopsied. Individuals with AD were biopsied in two different sites (lesional and nonlesional skin). The specimens were analyzed by immunohistochemistry through specific markers for filaggrin, claudins 1 and 4. The slides were scanned utilizing Panoramic Scan - 3DHistech - Hungary, and images analyzed by Image Pro Plus 4,5 for the intensity of each marker. The mean epidermal thickness was also evaluated. AD patients were also analyzed for disease severity (EASI), circulating IgE levels and eosinophilia. Results: In lesional and nonlesional skin of AD patients there was a reduced expression of filaggrin, when compared to nonatopic controls. There was an inverse correlation of filaggrin expression with disease severity and epidermal thickness. In the skin of AD individuals, there was reduced expression of claudins 1-and-4, which did not correlate with disease severity, epidermal thickness or eosinophilia. Conclusion: In adults with AD, there is reduced expression of skin barrier proteins, such as filaggrin, claudins 1 and 4. The reduction of filaggrin expression had an inverse correlation with disease severity and epidermal thickness, suggesting disease chronicity. There was reduction of claudins 1 and 4, with no relation with disease severity, epidermal thickness, circulating IgE levels or eosinophilia

Les brûlures cutanées graves dont les lésions profondes et étendues des tissus ne permettent pas d’envisager un traitement classique par autogreffes, bénéficient aujourd’hui de substituts épidermiques autologues pour leur recouvrement. En effet l’ingénierie tissulaire permet l’obtention d’épiderme de culture autologue (CEA) par l’amplification in vitro de kératinocytes à partir d’une petite biopsie de peau saine. Si ces CEAs ont permis la survie de nombreux patients depuis les années 1980, ils n’en restent pas moins largement perfectibles du point de vue de l’esthétique et de la fonctionnalité de l’épiderme et de la jonction dermo-épidermique régénérés mais aussi en termes de coûts et de prévention des zoonoses.Ainsi, ce travail de thèse s’inscrit dans le cadre d’une recherche translationnelle et porte sur l’élaboration d’un épiderme de culture par ingénierie tissulaire en vue d’une application clinique chez les grands brûlés, afin de favoriser la cicatrisation et la régénération épidermique. Ce travail porte également sur l’évaluation non clinique de notre substitut épidermique sur un modèle animal de brûlure cutanée.Après avoir développé un nouveau substitut épidermique sur fibrine de plasma humain (hPBES : human Plasma-Based Epidermal Substitute), nous avons procédé à l’évaluation détaillée de ses caractéristiques phénotypiques et fonctionnelles en comparaison au substitut épidermique de référence Epicel® (Genzyme, US). Nous avons montré des propriétés plus intéressantes en termes de clonogénicité et de caractère souche, de prévention de la différenciation, de prolifération, de potentiel de migration, et de conservation des protéines de la membrane basale avec notre produit par rapport à Epicel®.L’apport de la matrice de fibrine de plasma sur la culture des kératinocytes en comparaison à une matrice de fibrine issue de fibrinogène purifié et à une culture sans matrice a été étudié notamment sous l’angle des facteurs libérés par les matrices. Nous avons observé une amélioration des propriétés des substituts épidermiques par la fibrine : diminution de la différenciation terminale, augmentation du potentiel migratoire et sécrétion de facteurs pro-cicatrisants (GM-CSF et PDGF-BB). En outre, la fibrine de plasma améliore spécifiquement la morphogénèse épidermique, la prolifération kératinocytaire et la clonogénicité.Le procédé de culture de ce nouveau substitut a été adapté aux exigences réglementaires sans altération du potentiel régénératif: remplacement des cellules nourricières murines par des fibroblastes dermiques humains, adaptation du milieu de culture et sécurisation du plasma par traitement à l’amotosalen.Toutes les étapes d’un modèle de brûlure chez le rat nude reproduisant la technique chirurgicale employée pour la greffe de CEA ont été mises au point, mais un rejet du greffon épidermique n’a pas permis l’évaluation d’hPBES sur ce modèle. Nous avons donc évalué le substitut épidermique développé hPBES chez la souris NOD-SCID après greffe au fascia et mis en évidence une bonne prise de greffe épidermique permettant la régénération d’un épiderme humain aux caractéristiques proches d’une peau saine avec une réorganisation de la jonction dermo-épidermique des 2 semaines après greffe. / The deep and large injuries caused by massive burns prevent the use of split-thickness skin autografts. Today, autologous epidermal substitutes constitute an alternative treatment for skin regeneration. In fact tissue engineering allows the production of Cultured Epithelial Autografts (CEA) by in vitro keratinocyte amplification from a small healthy skin biopsy. The clinical use of CEA since the 1980’s has allowed an increase in the survival rate of burnt patients. However, CEAs present numerous drawbacks, among them the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction leading to heavy cosmetic and functional sequelae.This thesis focuses on the bioengineering of epidermal substitute for clinical burn treatment to enhance wound healing and skin regeneration, from a translational research point of view. This work also includes the development of an animal model of third degree burn for the evaluation of epidermal substitute.We first developed a new epidermal substitute cultured on a fibrin matrix from human plasma (hPBES: human Plasma-Based Epidermal Substitute). Then we characterized this new substitute with phenotypical and functional analyses, using as a reference the epidermal substitute Epicel® (Genzyme, US). We observed properties more favorable with hPBES than with Epicel® in terms of clonogenicity, stemness, differentiation level, proliferation, migration potential and basement membrane protein conservation.The influence of the plasma-based fibrin matrix on keratinocytes was studied in comparison with a culture on a fibrin from purified fibrinogen, and with a culture with no matrix. For this study we focused our analysis on the role of the released factors from fibrin matrices during epidermal substitute culture. Both fibrin matrices improved epidermal substitute properties: reduction of terminal differentiation, enhancement of migration potential, secretion of wound healing enhancing factors. Besides, plasma-based fibrin specifically promote epidermal morphogenesis, keratinocyte proliferation and clonogenicity.The culture process was adapted to European regulation requirements without diminishing its regenerative potential: substitution of murine feeder cells by human dermal fibroblasts, adaptation of culture medium and plasma viral inactivation using amotosalen treatment.A burn model including all the surgical steps used in clinics for CEA grating was developed on nude rats. However the evaluation of hPBES on this model could not be achieved due to graft rejection. Therefore we evaluated epidermal regeneration after hPBES graft on acute wounds on NOD-SCID mice. We showed a good graft rate, with the regeneration of a human epidermis close to healthy epidermis with a well-organized dermal-epidermal junction two weeks after hPBES grafting.

INTRODUÇÃO: A alopecia frontal fibrosante (AFF) é uma alopecia cicatricial primária linfocítica descrita em 1994, cuja prevalência vem aumentando rapidamente em todo mundo. A participação de um fator desencadeante ambiental na patogênese da doença é aventada e uma pesquisa recente encontrou uma associação da doença com o uso de cosméticos faciais. Alterações da pele e dos pelos da face e do corpo têm sido descritas em pacientes com AFF nos últimos anos e alguns estudos sugerem que essas alterações possam preceder a perda dos cabelos, indicando o início da doença fora do couro cabeludo. OBJETIVO: Estudar o acometimento da pele e dos pelos na face, pescoço e região anterossuperior do tórax em uma série de pacientes com AFF. MATERIAL E MÉTODOS: A pesquisa constou de três partes. Na primeira parte foram investigadas evidências clínicas e dermatoscópicas do acometimento da pele e dos pelos fora do couro cabeludo em 37 pacientes. A segunda parte do estudo constou da avaliação da espessura epidérmica em biópsias realizadas na face, pescoço e região anterossuperior do tórax de 20 pacientes com AFF e 20 controles. Na terceira parte do estudo foi utilizada microscopia confocal de reflectância a laser \"in vivo\" para comparar a espessura epidérmica e a densidade folicular da pele da linha de implantação frontal de 21 pacientes a de 21 controles. RESULTADOS: O acometimento dos pelos velos da face não se restringiu a linha de implantação fronto-temporal e variou de 30 a 97% dependendo da região estudada, sendo mais frequente quanto mais próximo da linha de implantação frontal do couro cabeludo. Pápulas da face foram encontradas em 60% dos pacientes estudados, localizadas principalmente na região temporal (11/37 casos), seguida pela região malar (10/37 casos) e mento (6/37 casos). Metade dos pacientes (51%) apresentaram lesões hipercrômicas compatíveis com o diagnóstico de líquen plano pigmentoso associado a AFF, acometendo face (18/19 casos), pescoço (7/19 casos) e região anterossuperior do tórax (4/19 casos). As lesões hipercrômicas mostraram-se mais raras em pacientes com fototipo baixo (p=0,022). A espessura da epiderme dos pacientes de AFF não apresentou diferença quando comparada com a dos controles independente da metodologia utilizada. Densidade folicular menor que 3,56 folículos/mm2 na linha de implantação frontal ao exame de microscopia confocal apresentou 90,5% de sensibilidade e 90,5% de especificidade para o diagnóstico de AFF e implicou num risco 90,24 (IC95% 9,5-1132; p < 0,001) vezes maior de ter a doença. CONCLUSÕES: O acometimento dos pelos velos da face é frequente e pode ser detectado de forma rápida e não invasiva pela dermatoscopia. As pápulas da face estão presentes em 60% dos pacientes. As lesões de liquen plano pigmentoso são menos frequentes em pacientes com fototipos baixos. A epiderme dos pacientes de AFF não apresenta uma menor espessura quando comparada com controles pareados por gênero, idade, fototipo e local examinado. A densidade folicular da linha de implantação frontal \"in vivo\" medida através do exame de MCRL apresenta ótima acurácia para o diagnóstico de AFF / INTRODUCTION: Frontal fibrosing alopecia (FFA) is a lymphocytic primary cicatricial alopecia first described in 1994. Its incidence has been rapidly rising worldwide, possibly related to an environmental trigger. The use of facial leave-on creams has been associated with the disease in a recent publication. Vellus follicles involvement and epidermal changes outside the scalp region have been described in FFA patients in the past few years and seem to be an early event in the disease course. OBJECTIVES: To evaluate vellus follicle and epidermal involvement over the facial, neck and upper chest skin in a series of FFA patients. METHODS: This study consisted of three parts. In the first part, prevalence of clinical and dermoscopic findings related to vellus follicle and epidermal involvement in 37 FFA patients was investigated. In part two, epidermal thickness in skin biopsies from 20 FFA patients was compared with 20 control biopsies from the same body site. In the last part, epidermal thickness and follicular density over the frontal hairline were investigated in a group of 21 FFA patients and 21 gender, age and phototype matched controls through \"in vivo\" reflectance confocal microscopy. RESULTS: Vellus follicle involvement in FFA is not restricted to frontal hairline and varies from 30 to 97% according to facial region, with greater frequencies observed on the upper face region. Facial papules were detected in 60% of our patients, most frequently over the temples (11/37 patients), malar (10/37 patients) or chin (6/37 patients) area. Half of our patients (51%) presented hyperchromic lesions compatible with FFA associated lichen planus pigmentosus. Hyperchromic lesions were observed over the face (18/19 patients), but also over the neck (7/19 patients) and upper chest (4/19 patients) skin. Hyperchromic lesions were less frequent in patients with lighter phototypes (p=0.022). Epidermal thickness of FFA patients did not differ from controls both in histology and \"in vivo\" evaluation. Frontal hairline follicular density lower than 3.56 follicles/mm2 on confocal microscopy examination presented 90.5% sensitivity, 90.5% specificity and OR = 90.24 (CI95% 9.5-1132; p < 0.001) for FFA diagnosis. CONCLUSIONS: Facial vellus follicle involvement is frequent and can be easily detected through dermoscopy in most patients. Facial papules are observed in 60% of our patients. Lichen planus pigmentosus lesions are less frequently observed in fair skin patients. Epidermal thinning is not observed in FFA patients when adequate control group is included. Frontal hairline follicular density measured by confocal microscopy has high accuracy for FFA diagnosis

INTRODUÇÃO: A alopecia frontal fibrosante (AFF) é uma alopecia cicatricial primária linfocítica descrita em 1994, cuja prevalência vem aumentando rapidamente em todo mundo. A participação de um fator desencadeante ambiental na patogênese da doença é aventada e uma pesquisa recente encontrou uma associação da doença com o uso de cosméticos faciais. Alterações da pele e dos pelos da face e do corpo têm sido descritas em pacientes com AFF nos últimos anos e alguns estudos sugerem que essas alterações possam preceder a perda dos cabelos, indicando o início da doença fora do couro cabeludo. OBJETIVO: Estudar o acometimento da pele e dos pelos na face, pescoço e região anterossuperior do tórax em uma série de pacientes com AFF. MATERIAL E MÉTODOS: A pesquisa constou de três partes. Na primeira parte foram investigadas evidências clínicas e dermatoscópicas do acometimento da pele e dos pelos fora do couro cabeludo em 37 pacientes. A segunda parte do estudo constou da avaliação da espessura epidérmica em biópsias realizadas na face, pescoço e região anterossuperior do tórax de 20 pacientes com AFF e 20 controles. Na terceira parte do estudo foi utilizada microscopia confocal de reflectância a laser \"in vivo\" para comparar a espessura epidérmica e a densidade folicular da pele da linha de implantação frontal de 21 pacientes a de 21 controles. RESULTADOS: O acometimento dos pelos velos da face não se restringiu a linha de implantação fronto-temporal e variou de 30 a 97% dependendo da região estudada, sendo mais frequente quanto mais próximo da linha de implantação frontal do couro cabeludo. Pápulas da face foram encontradas em 60% dos pacientes estudados, localizadas principalmente na região temporal (11/37 casos), seguida pela região malar (10/37 casos) e mento (6/37 casos). Metade dos pacientes (51%) apresentaram lesões hipercrômicas compatíveis com o diagnóstico de líquen plano pigmentoso associado a AFF, acometendo face (18/19 casos), pescoço (7/19 casos) e região anterossuperior do tórax (4/19 casos). As lesões hipercrômicas mostraram-se mais raras em pacientes com fototipo baixo (p=0,022). A espessura da epiderme dos pacientes de AFF não apresentou diferença quando comparada com a dos controles independente da metodologia utilizada. Densidade folicular menor que 3,56 folículos/mm2 na linha de implantação frontal ao exame de microscopia confocal apresentou 90,5% de sensibilidade e 90,5% de especificidade para o diagnóstico de AFF e implicou num risco 90,24 (IC95% 9,5-1132; p < 0,001) vezes maior de ter a doença. CONCLUSÕES: O acometimento dos pelos velos da face é frequente e pode ser detectado de forma rápida e não invasiva pela dermatoscopia. As pápulas da face estão presentes em 60% dos pacientes. As lesões de liquen plano pigmentoso são menos frequentes em pacientes com fototipos baixos. A epiderme dos pacientes de AFF não apresenta uma menor espessura quando comparada com controles pareados por gênero, idade, fototipo e local examinado. A densidade folicular da linha de implantação frontal \"in vivo\" medida através do exame de MCRL apresenta ótima acurácia para o diagnóstico de AFF / INTRODUCTION: Frontal fibrosing alopecia (FFA) is a lymphocytic primary cicatricial alopecia first described in 1994. Its incidence has been rapidly rising worldwide, possibly related to an environmental trigger. The use of facial leave-on creams has been associated with the disease in a recent publication. Vellus follicles involvement and epidermal changes outside the scalp region have been described in FFA patients in the past few years and seem to be an early event in the disease course. OBJECTIVES: To evaluate vellus follicle and epidermal involvement over the facial, neck and upper chest skin in a series of FFA patients. METHODS: This study consisted of three parts. In the first part, prevalence of clinical and dermoscopic findings related to vellus follicle and epidermal involvement in 37 FFA patients was investigated. In part two, epidermal thickness in skin biopsies from 20 FFA patients was compared with 20 control biopsies from the same body site. In the last part, epidermal thickness and follicular density over the frontal hairline were investigated in a group of 21 FFA patients and 21 gender, age and phototype matched controls through \"in vivo\" reflectance confocal microscopy. RESULTS: Vellus follicle involvement in FFA is not restricted to frontal hairline and varies from 30 to 97% according to facial region, with greater frequencies observed on the upper face region. Facial papules were detected in 60% of our patients, most frequently over the temples (11/37 patients), malar (10/37 patients) or chin (6/37 patients) area. Half of our patients (51%) presented hyperchromic lesions compatible with FFA associated lichen planus pigmentosus. Hyperchromic lesions were observed over the face (18/19 patients), but also over the neck (7/19 patients) and upper chest (4/19 patients) skin. Hyperchromic lesions were less frequent in patients with lighter phototypes (p=0.022). Epidermal thickness of FFA patients did not differ from controls both in histology and \"in vivo\" evaluation. Frontal hairline follicular density lower than 3.56 follicles/mm2 on confocal microscopy examination presented 90.5% sensitivity, 90.5% specificity and OR = 90.24 (CI95% 9.5-1132; p < 0.001) for FFA diagnosis. CONCLUSIONS: Facial vellus follicle involvement is frequent and can be easily detected through dermoscopy in most patients. Facial papules are observed in 60% of our patients. Lichen planus pigmentosus lesions are less frequently observed in fair skin patients. Epidermal thinning is not observed in FFA patients when adequate control group is included. Frontal hairline follicular density measured by confocal microscopy has high accuracy for FFA diagnosis