Apesar de ser bem conhecido que a radiação ultravioleta (UV) causa danos na pele, já foram descritos alguns efeitos benéficos desta radiação, como por exemplo a sensação de bem estar proporcionado pela exposição a luz. Na pele, o triptofano (Trp) é metabolizado a compostos biologicamente ativos, e acredita-se que a síntese de serotonina (SER), um dos metabólitos do Trp, na fototerapia seja parte dos mecanismos de remissão da depressão de pacientes com desordem de humor sazonal. Ainda, a radiação UV induz diretamente a expressão e atividade de TDO, enzima que catalisa a transformação de Trp em quinurenina (KYN), em bactérias, efeito que ainda não havia sido estudado em células humanas. Com isso, o objetivo deste trabalho foi avaliar a habilidade da radiação UV-A em modular a expressão de enzimas envolvidas nas rotas catabólicas do Trp em melanócitos e melanomas. Para isso foi realizada a padronização das condições de radiação UV-A através de ensaio de viabilidade celular, e então definimos condições que não levaram a morte das células (1,5 J/cm2 para melanócitos e 3 e 6 J/cm2 para melanomas). A radiação UV-A aumentou a expressão de IDO e TDO nos melanomas, enzimas que favorecem a progressão tumoral, e não só essas enzimas, houve aumento da expressão de KYNU e KMO, que também estão envolvidas na progressão tumoral. Além disso, a expressão de AANAT e HIOMT, responsáveis pela produção de melatonina (MLT), foi maior nos melanomas após 48 horas da radiação, enquanto a enzima INMT teve sua expressão aumentada em todos os tempos. O aumento de INMT em melanomas é muito interessante e podemos relacionar ao bem estar proporcionado pela exposição à luz, já que o produto dessa enzima, DMT, é conhecido por proporcionar essa sensação, porém o aumento observado na expressão de IDO e TDO em melanomas, indica um efeito nocivo da luz associado a produção de moléculas que estão fortemente ligadas aos processos de progressão e imunoescape tumoral. Os melanócitos parecem possuir menor susceptibilidade a radiação UV-A, pois as únicas enzimas que tiveram expressão aumentada após a radiação foram a KMO e a TPH1, e apesar da enzima TPH1 ter a expressão aumentada, esse aumento foi modesto o que nos levou a pensar que outras células da pele possam ter um papel mais relevante na produção de SER ou então outras condições de radiação. / Although it is well known that ultraviolet (UV) radiation causes skin damage, it was already described some benefits to this radiation, for instance the well-being feelings provided by light exposure. In the skin, tryptophan (Trp) is metabolized to biologically active compounds, and it is believed that the synthesis of serotonin, one of tryptophan metabolites, in phototherapy is part of mechanisms of remission of depression in patients with seasonal mood disorder. Moreover, in bacteria the UV radiation directly induces the expression and activity of TDO, the enzyme that catalyzes the metabolization of Trp to kynurenine (KYN). This effect has not been studied in human cells yet. Therefore, the objective of this work was to evaluate if UV-A radiation modulates the expression of enzymes involved in melanocytes and melanoma Trp metabolism. For this aim, we standardized the UV-A radiation conditions through cell viability assay, and then we defined the better conditions to avoid cell death (1,5 J/cm2 to melanocytes and 3 and 6 J/cm2 to melanomas). The UV-A radiation increased the expression of IDO and TDO in melanomas, enzymes that contribute to tumor immune- escape. The expression of KYNU and KMO also increased, and these enzymes are also involved with some types of tumors progression. Furthermore, the expression of AANAT and HIOMT, responsible for melatonin (MLT) production, was higher in melanomas after 48 hours of radiation while INMT had an increased expression at all times. The increase of INMT by melanomas is very interesting and can be related to the well being provided by exposure to light, since the product of this enzyme, DMT, is known to provide this sensation. However, the observed increased of expression of IDO and TDO in melanomas indicates a harmful effect of light associated with the production of molecules linked to tumor progression and immune-escape processes. The melanocytes appear to be less susceptible to UV-A radiation, because only the enzymes KMO and TPH1 had their expression increased after radiation, and for TPH1 this effect was relatively small. Thus, we believe that other skin cells may have a more relevant role in SER production or other radiation conditions.

Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

La pigmentation de la peau résulte en grande partie de la présence de mélanine dans l’épiderme. Ce pigment est synthétisé par les mélanocytes puis transféré aux kératinocytes pour assurer une fonction photoprotectrice. Le transfert de mélanosomes nécessite une reconnaissance cellulaire entre mélanocytes et kératinocytes. L’interaction entre lectines et glycanes joue un rôle dans cette reconnaissance et peut constituer une cible d’intérêt pour le développement de produits à activité dépigmentante. Par ailleurs, les conditions du microenvironnement cutané, telles que le taux d’oxygène, sont cruciales pour l’homéostasie tissulaire. Les objectifs de ce travail de thèse, réalisé dans le cadre du projet FUI Glycoskin I, ont été : l’étude de la reconnaissance cellulaire entre mélanocytes et kératinocytes à travers l’interaction lectine-glycane, la caractérisation des mélanosomes sécrétés par les mélanocytes et la mise au point d’une méthode d’évaluation du transfert des mélanosomes aux kératinocytes pour tester l’activité de phosphoconjugués. D’autre part, nous avons étudié l’effet du taux d’oxygène sur le processus de mélanogénèse et sur l’interaction lectine-glycane. Nos résultats ont permis d’élucider les profils glycaniques et lectiniques à la surface des mélanocytes et des kératinocytes et de sélectionner des phosphoconjugués potentiellement inhibiteurs du transfert de mélanosomes aux kératinocytes. Nous avons mis au point un modèle d’évaluation du transfert de mélanosomes aux kératinocytes afin de tester l’effet inhibiteur des phosphoconjugués. Nous avons identifié un phosphosaccharide inhibiteur de la reconnaissance entre mélanosomes et kératinocytes. Par ailleurs, ce projet constitue la première étude de la pigmentation en physioxie. Nous avons montré qu’un travail en physioxie induit des modulations des profils glycaniques et lectiniques, ainsi qu’une stimulation de la mélanogénèse. Ces résultats montrent l’importance de se placer en physioxie lors de l’étude de la mélanogénèse in vitro afin de se rapprocher au maximum des conditions physiologiques du microenvironnement cutané lors de l’évaluation de composés actifs. / Skin pigmentation is mostly due to the presence of melanin in the epidermis. This pigment is produced by melanocytes and transferred to keratinocytes, to play a photoprotective role. Melanosome transfer requires cellular recognition between melanocytes and keratinocytes. Lectin-glycan interaction plays a role in this phenomena and can be an interesting target for developing depigmenting products. Besides, the cutaneous microenvironment conditions, such as oxygen level, are crucial for tissular homeostasis. The aims of this work, as part of the Glycoskin I FUI project, were : to study cellular recognition between melanocytes and keratinocytes through lectin-glycan interaction, to characterize melanosomes released from melanocytes and to develop a method for the evaluation of melanosome transfer to keratinocytes in order to assess phosphoconjugate activity. Also, we studied the effect of oxygen level on melanogenesis and lectin-glycan interaction. Our results allowed to elucidate lectin and glycan profiles on the surface of melanocytes and keratinocytes and to select phosphoconjugates potentially able to inhibit melanosome transfer. We developed a method to assess melanosome transfer in order to test phosphoconjugates inhibiting effect. We identified one phosphosaccharide able to inhibit melanocytes-keratinocytes recognition. Furthermore, this project is the first study of pigmentation in physioxia. We showed that physioxia induces modulations of lectin and glycan profiles and stimulated melanogenesis. These results show the importance of physioxia conditions when studying melanogenesis in vitro to approach cutaneous physiological microenvironment when evaluating active compounds.

Genetic interactions that underlie developmental processes such as cell differentiation and pattern formation are complex and difficult to elucidate. Neural Crest (NC) cells and their derivatives offer an optimal system in which to probe for these complex interactions as they acquire different cell fates and constitute a variety of structures. The transcription factors Sox10 and Pax3 as well as the transmembrane receptor Endothelin receptor b (Ednrb) are temporally and spatially co-expressed early in NC cells and mutations in these genes lead to similar hypopigmentation phenotypes due to a reduced number of NC-derived melanocyte precursors, the melanoblasts. The goal of this study was to establish whether Sox10 and Ednrb or Pax3 and Ednrb interact to promote normal murine melanocyte development. Crosses of Sox10 or Pax3 with Ednrb heterozygous mutants showed that the double heterozygous hypopigmentation phenotype was significantly more pronounced than phenotypes of single heterozygotes, implying that a synergistic interaction exists between Sox10 and Ednrb and Pax3 and Ednrb. This interaction was further explored by the attempt to rescue the Sox10 and Pax3 hypopigmentation phenotypes by the transgenic addition of Ednrb to melanoblasts. Pigmentation was completely restored in the Sox10 and partially restored in the Pax3 mutant mice. The comparison of the number of melanoblasts in transgenic and non-transgenic Sox10 mutant embryos showed that the transgenic rescue occurred as early as E11.5, a critical time for melanoblast population expansion. Cell survival assays indicated that the rescue was not due to an effect of the transgene on melanoblast survival. A novel phenotype arose when studying the interaction between Ednrb and Pax3. Newborns appeared normal but by 3.5 weeks of age, the affected pups were smaller than normal littermates and developed a dome-shaped head; some also developed thoracic kyphosis. Affected pups were dead by 4 weeks of age: 80% were Pax3Sp/+ and 75% were female. When compared to normal littermates, affected mice had brains with enlarged 4th ventricles and more glia while skeletal staining showed kyphosis, wider rib cages and pelvic differences. An epistatic interaction resulting from the mixing of genetic backgrounds that is exacerbated in the presence of Pax3 heterozygosity is suspected.

Objective: The aim of this study was to investigate inherent in vitro permeability, metabolism, and cytotoxicity of idebenone - an active used to protect skin as an anti-aging agent -and compare it to idebenone linoleate. Methods: Idebenone and idebenone linoleate were investigated in pig ear skin and melanoma (B16: F10 mouse) cells. Diffusion experiments were conducted at 37 °C (bath temperature) using Franz diffusion cells. Authentic metabolite samples were synthetically prepared. Samples were analyzed using liquid chromatography-mass spectrometry/mass spectrometry. Cell viability was determined via the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results: Idebenone was shown to permeate across viable porcine ear tissue; there was no evidence that idebenone linoleate permeated across porcine ear tissue after 4 h. Idebenone was metabolized to idebenone acid in both pig ear and mouse melanocytes; only minor idebenone linoleate metabolism was observed. Idebenone displayed delayed in vitro toxicity (via MTT assay) in melanocytes, while idebenone linoleate displayed no such in vitro toxicity. Conclusions: The in vitro metabolism and cytotoxicity results suggest that metabolic activation of idebenone is the likely culprit that activates the skin irritation mechanism via idebenone in vivo usage. An idebenone ester (e.g. idebenone linoleate) appears to provide a superior in vitro safety profile over idebenone.

During regeneration, cells must coordinate proliferation and differentiation to rebuild tissues that are lost. Understanding how source cells execute the regeneration process has been a longstanding goal in regenerative biology with implications in wound healing and cell replacement therapies. Melanocytes are pigment-producing cells in the skin of vertebrates that can be lost during hair graying, injury and disease-related depigmentation. Melanoma is an aggressive skin cancer that develops from melanocytes, and it is hypothesized that melanoma cells have properties that are similar to melanocyte stem cells. To gain insight into melanocyte regeneration we set out to identify the source of regeneration melanocytes in adult zebrafish and the path through which progenitor cells reconstitute the pigment pattern. Using targeted cell ablation and single cell lineage-tracing analyses we identified that a majority of regeneration melanocytes arise through direct differentiation of mitfa-expressing progenitor cells. Concurrently, other mitfa-expressing cells divide symmetrically to generate additional mitfa-positive progenitors, thus maintaining regeneration capability. Using reporter assays and drug studies, we found that Wnt signaling gets turned on in progenitor cells during regeneration and Wnt inhibition after melanocyte ablation blocks regeneration. Based on our finding that Wnt signaling is active in differentiated melanocytes but not in the progenitor cells, we explored the role of Wnt signaling in tumor initiation. We found that approximately half of the melanomas are Wnt silent, and overexpression of dkk1b, a negative regulator of canonical Wnt signaling, accelerates melanoma onset. This work defines an unappreciated contribution by direct differentiation in melanocyte regeneration and suggests a broader role for this process in the maintenance of epithelial sheets. This study also identifies a shared pathway between melanocyte progenitors and melanoma cells, which could be applicable to other cancers.

No description available.

Cutaneous malignant melanoma is the deadliest form of skin cancer with a rising incidence rate. Epidemiological studies show exposure to ultraviolet radiation (UVR) cause 80% of melanomas. However, the underlying molecular mechanisms by which UVR promotes melanomagenesis are unclear. The mutagenic properties of UVR are incontrovertible; however, well-studied driver mutations of melanomagenesis (BRAF V600E and NRAS Q61L/R) do not bear UVR signature mutations and so the role UVR mutations play in the early initiation of melanoma remains controversial. This highlights the gap in knowledge of the initial critical molecular mechanisms of UVR-induced melanomas and warrant investigating non-mutational mechanisms as causal factors of UVR-induced melanomagenesis. Aberrant DNA methylation is a signature of melanoma and regulates expression of important tumor suppressors. While epigenetic dysregulation is an important aspect of melanoma etiology, it has never been investigated in the context of UVR. We hypothesize that these initial UVR-induced DNA methylation changes may sensitize a field of melanocytes to acquiring subsequent complementary spontaneous and/or UVR-induced genetic mutations and render them susceptible to melanomagenesis. My preliminary data demonstrate that UVR can modulate DNA methylation in melanocytes and suggests a pigment dependent mechanism. UVR-induced DNA methylation changes in highly pigmented melanocytes primarily in intergenic regions as areas of active transcription were protected from 5’mC changes. Additionally, UVR induced long-term transcriptional changes in both dark and light pigmented melanocytes suggesting multiple epigenetic mechanisms being altered. Evaluation of the protein regulation of the enzymes involved in writing or erasing 5’mC point towards a dysregulation in TET2. Further work is needed to determine if changes in TET2 could contribute to the observed methylation changes. To determine if these methylation changes had any significance to melanoma development, they were compared to the skin cutaneous melanoma cohort in the TCGA database which found a modest correlation in UVR-induced methylation changes and those found in melanoma patients. Interestingly, 5’mC at UVR-sensitive sites was prognostic of patient survival. A highly pigmented human melanoma cell line was UV-irradiated to see if DNA methylation can also be affected in transformed cells; however, no changes were observed. This suggests UV-induced methylation contributes to early changes in melanoma development and/or other relevant physiological changes within the melanocytes. Altogether, these data identify a novel non-mutation mechanism by which UVR may contribute of melanomagenesis. / Cancer Biology & Genetics

No description available.

Objective: Aortic valve (AV) leaflets rely on a precise extracellular matrix (ECM) microarchitecture for appropriate biomechanical performance. The ECM structure is maintained by valvular interstitial cells (VICs), which reside within the leaflets. The presence of pigment produced by a melanocytic population of VICs in mice with dark coats has been generally regarded as a nuisance, as it interferes with histological analysis of the AV leaflets. However, our previous studies have shown that the presence of pigment correlates with increased mechanical stiffness within the leaflets as measured by nanoindentation analyses. In the current study, we seek to better characterize the phenotype of understudied melanocytic VICs, explore the role of these VICs in ECM patterning, and assess the presence of these VICs in human aortic valve tissues. Approach and Results: Immunofluorescence and immunohistochemistry revealed that melanocytes within murine AV leaflets express phenotypic markers of either neuronal or glial cells. These VIC subpopulations exhibited regional patterns that corresponded to the distribution of elastin and glycosaminoglycan ECM proteins, respectively. VICs with neuronal and glial phenotypes were also found in human AV leaflets and showed ECM associations similar to those observed in murine leaflets. A subset of VICs within human AV leaflets also expressed dopachrome tautomerase, a common melanocyte marker. A spontaneous mouse mutant with no aortic valve pigmentation lacked elastic fibers and had reduced elastin gene expression within AV leaflets. A hyperpigmented transgenic mouse exhibited increased AV leaflet elastic fibers and elastin gene expression. Conclusions: Melanocytic VIC subpopulations appear critical for appropriate elastogenesis in mouse AVs, providing new insight into the regulation of AV ECM homeostasis. The identification of a similar VIC population in human AVs suggests conservation across species.