京都大学 / 新制・課程博士 / 博士(医学) / 甲第24482号 / 医博第4924号 / 新制||医||1063(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩田 想, 教授 萩原 正敏, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Melanin is a ubiquitous biological pigment found in bacteria, fungi, plants, and animals. It has a diverse range of ecological and biochemical functions including display, evasion, photoprotection, detoxification, and metal scavenging. Two forms of melanin produced from different molecular precursors are present in nature - eumelanin (dark brown-black in color) and pheomelanin (orange-red in color). Both eumelanin and pheomelanin are complex highly cross-linked biopolymers that are found intertwined with proteins, lipids, and metal ions in nature. </p><p> </p><p>Recent reports have used morphological evidence to suggest the presence of melanin in the fossil record. These studies have been met with criticism due to their lack of chemical evidence to support melanin identification. This dissertation describes chemical approaches to unambiguously verify the presence of melanin in the fossil record and characterize the ancient pigment. It also explores the limitations for the survival of melanin in the fossil record and the possibility that melanin acts as a protective matrix to preserve other biomolecules that are embedded in the pigment. </p><p>Melanin has unique chemical signatures that are commonly used to characterize and compare the pigment of modern organisms. We applied these chemical approaches to the study of fossil pigmentation. Analysis of the black pigmentation of two > 160 million year old (Mya) Jurassic cephalopod ink sacs provided the first conclusive evidence for eumelanin in the fossil record. The preserved fossil eumelanin was then compared to modern cephalopod eumelanin from Sepia officinalis. Using these chemical approaches we found that fossil eumelanin was chemically and morphologically identical to S. officinalis eumelanin. </p><p> Although there is mounting chemical evidence for the presence and preservation of melanin in the fossil record, there is very little data constraining its long-term survival. We applied the analytical approaches designed to study fossil melanins and techniques used to study fossil sediments to compare the fossil inks from three deposits of similar age and lithology, but different maturation histories. Specifically, two ~ 180 Mya fossil ink sacs from a site that has entered the oil window in Holzmaden, Germany were compared to the previously analyzed fossil inks from two less mature sites in southern England. The chemistry of eumelanin was shown to alter at the onset of the oil window regardless of the age of the specimen. The decrease in surviving melanin was accompanied by an increase in the relative abundance of organic macromolecular material (kerogen), but no consistent change in melanin morphology. </p><p> Finally, the role of melanin as a matrix that enhances the preservation of other biomolecules in the fossil record was considered. Proteins, commonly associated with melanin in modern organisms, were discovered in the aforementioned fossil ink sacs during full-scale chemical analysis. The amino acid profile of the protein in each fossil specimen was determined with an amino acid analyzer and compared to the amino acid profile the protein in modern S. officinalis. Statistical analysis of the amino acid distributions indicated that there is no significant difference between the amino acid profile of modern and fossil melanins. In order to verify the ancient origin of the amino acids in the fossil ink sacs, the ratio of D/L amino acid isomers was determined. While the proteins of living organisms consist of only L-amino acids, post-mortem the amino acids slowly convert from L to D form until they reach equilibrium (D/L = 1). This process is called racemization. The amino acids in the fossil ink sacs were racemized, which suggests their ancient origin. This marks the oldest determination of protein in a fossil system and provides evidence that the longevity of proteins may be enhanced when associated with melanin.</p> / Dissertation

Im Mittelpunkt dieser Arbeit stehen Untersuchungen zur Bildung des Pigments Neuromelanin, das die Ursache für die dunkle Farbgebung der humanen Substantia nigra pars compacta ist. Eine Beteiligung von Neuromelanin an den pathobiochemischen Ereignissen bei Parkinson-Krankheit erklärt das klinische Interesse an Neuromelanin. Die Untersuchungsmöglichkeiten von Neuromelanin sind limitiert: einerseits ist eine chemische Strukturaufklärung aufgrund der Unlöslichkeit dieses amorphen Polymers kaum zu bewerkstelligen, andererseits wird mangels geeigneter biologischer Testsysteme ein Einblick in die Biogenese von Neuromelanin verwehrt. Zurzeit wird die Bildung von Neuromelanin anhand der beiden konkurrierenden Hypothesen als Autoxidation von Dopamin oder durch Beteiligung eines Enzyms („Tyrosinase-Konzept“) erklärt. In dieser Arbeit wurden beide hypothetischen Ansätze bearbeitet, wobei einer enzymatischen Biogenese von Neuromelanin die Präferenz gegeben wird. Zur globalen Untersuchung von Neuromelanin-Granula wurde nun erstmals eine Isolierung der Pigment-haltigen Organelle vorgestellt, die die Basis für eine umfassende Proteomanalyse mittels 1-D-SDS-PAGE und ESI-Tandem-Massenspektrometrie bildete. Mit diesem methodischen Ansatz wurden ingesamt 73 Proteinen identifiziert. Diese waren vor allem lysosomalen Proteinen zuordenbar, z.B. charakteristischen Membranproteinen (LAMP-1), sämtlichen Proteasen, Proteinen des Metabolismus von (Glyco-)Lipiden und Glycoproteinen, aber auch Proteinen des Cytosols und des vesikulären Verkehrs. Entscheidend war die Anwesenheit von Proteinen des Endoplasmatischen Reticulums (ER); Calnexin gilt als ein melanogenes Chaperon, das nicht in Lysosomen vorkommt, dagegen aber in Lysosomen-verwandten Organellen. Im Vergleich mit bereits existierenden Proteinprofilen von Lysosomen und Lysosomen-verwandten Organellen zeigten die in Neuromelanin-Granula identifizierten lysosomalen Proteine und Proteine des ER, dass diese Organellen der humanen Substantia nigra keine konventionellen Lysosomen sind, sondern mit hoher Wahrscheinlichkeit der Gruppe der Lysosomen-verwandten Organellen zuzuordnen sind. / This work focusses on the formation of the polyphenolic pigment neuromelanin, which accounts for the dark colour of the human substantia pars compacta. A contribution of neuromelanin to the patho-biochemical events in Parkinson’s disease is suggested, which is the reason for the clinical interest in this pigment. The analytical approaches to investigate neuromelanin are limited: on the one hand, an elucidation of the chemical structure is hardly possible to accomplish due to the insolubility of this amorphous polymer, on the other hand, the lack of appropriate biological test systems prevents insight into the biogenesis of neuromelanin. Currently the formation of neuromelanin is addressed by two competing hypotheses either by autoxidation of dopamine (“autoxidative concept”), or by contribution of an enzyme (“tyrosinase concept”). This work covered both hypothetical approaches, but the concept of an enzymatic biogenesis of neuromelanin is preferred. Aiming at a global analysis of these pigmented organelles, the isolation of neuromelanin granules was introduced for the first time, which formed the basis for an extensive proteomic analysis by 1-D SDS-PAGE and ESI tandem mass spectrometry. Subsequently, the analyses by mass spectrometry facilitated the identification of 73 proteins, most of which were attributable to lysosomal proteins, e.g. characterisitcal membrane proteins (LAMP-1), various proteases, proteins engaged in the metabolism og (glycol)-lipids and glycoproteins, but also cytosolic proteins and proteins involved in vesicular traffic. Intriguingly, there were proteins identified which originate form the endoplasmic reticulum (ER), most notably calnexin, a melanogenic chaperone, which is not found in lysosomes but in lysosome-related organelles. By comparison of yet existing protein profiles of lysosomes and lysosome-related organelles it became clear that neuromelanin granules from the human substatia nigra are no conventional lysosomes, but should rather be attributed to the group of lysosome-related organelles.

The dematiaceous (melanised) fungus Lomentospora (Scedosporium) prolificans is a life-threatening opportunistic pathogen of immunocompromised humans, resistant to anti-fungal drugs. Melanin has been shown to protect human pathogenic fungi against antifungal drugs, oxidative killing and environmental stresses. To determine the protective role of melanin in L. prolificans to oxidative killing (H2O2), UV radiation and the polyene anti-fungal drug amphotericin B, targeted gene disruption was used to generate mutants of the pathogen lacking the dihydroxynaphthalene (DHN)-melanin biosynthetic enzymes polyketide synthase (PKS1), tetrahydroxynapthalene reductase (4HNR) and scytalone dehydratase (SCD1). Infectious propagules (spores) of the wild-type strain 3.1 were black/brown, whereas spores of the PKS-deficient mutant ΔLppks1::hph were white. Complementation of the albino mutant ΔLppks1::hph restored the black-brown spore pigmentation, while the 4HNR-deficient mutant ΔLp4hnr::hph and SCD-deficient mutant ΔLpscd1::hph both produced orange-yellow spores. The mutants ΔLppks1::hph and ΔLp4hnr::hph showed significant reductions in spore survival following H2O2 treatment, while spores of ΔLpscd1::hph and the ΔLppks1::hph complemented strain ΔLppks1::hph:PKS showed spore survivals similar to strain 3.1. Spores of the mutants ΔLp4hnr::hph and ΔLpscd1::hph and complemented strain ΔLppks1::hph:PKS showed spore survivals similar to 3.1 following exposure to UV radiation, but survival of ΔLppks1::hph spores was significantly reduced compared to the wild-type strain. Strain 3.1 and mutants ΔLp4hnr::hph and ΔLppks1::hph:PKS were resistant to amphotericin B while, paradoxically, the PKS1- and SCD1-deficient mutants showed significant increases in growth in the presence of the antifungal drug. Melanin was shown to play no role in the protection of the pathogen from immune cell recognition and killing by alveolar macrophages, with similar degrees of engulfment, and spore viabilities, of mutant and wild-type strains after phagocytosis. Contrary to expectations, the albino PKS-deficient mutant was significantly more virulent than the melanised wild-type strain during pathogenicity studies in the invertebrate infection model Galleria mellonella, with levels of virulence restored to near wild-type levels in the complemented strain ΔLppks1::hph:PKS. Taken together, the results presented in this thesis show that melanin protects L. prolificans from UV radiation and from oxidative killing by H2O2, consistent with its survival in extreme environmental habitats. However, melanin was 3 not found to play a role in the resistance of the pathogen to the antifungal drug amphotericin B, to protect the fungus from immune cell recognition or killing by alveolar macrophages, or to its pathogenicity.

Die gängigen therapeutischen Behandlungsmethoden für die verschiedensten Krebserkrankungen zeigen nach wie vor Mängel bezüglich der Effizienz sowie zahlreiche Nebenwirkungen während und nach der Behandlung. Maßgeblich für diese Defizite ist die teilweise geringe Sensitivität der meisten konventionellen diagnostischen Systeme und damit einhergehend die oftmals zu späte Identifikation entarteter Gewebsbereiche. Zur Lösung dieser Problematik bieten onkolytische Vaccinia-Viren einen Ansatz, sowohl die Effizienz der Therapie wie auch die Diagnostik zu verbessern. In beiden Fällen sind die Tumorzell-spezifische Vermehrung der Viren und die Möglichkeit entscheidend, die Viren als Vektorsystem zur Expression therapeutischer oder diagnostischer Fremdgenkassetten zu nutzen. Um ein auf Vaccinia-Virus-basierendes Reportersystem zum diagnostischen Nachweis von Krebszellen mittels Tiefengewebs-Tomographie bereit zu stellen, wurden die für die murine Tyrosinase (mTyr) und das Tyrosinase-Helferprotein 1 (Tyrp1) kodierenden Gene in das Genom eines onkolytischen Vaccinia-Virus inseriert. Die Tyrosinase ist das Schlüsselenzym der Melaninsynthese. Bereits die solitäre Expression der Tyrosinase führt in der transformierten Zelle zur Melaninproduktion. Das Tyrosinase-Helferprotein 1 ist an der Prozessierung und Stabilisierung der Tyrosinase beteiligt. Bereits in verschiedenen Studien konnte gezeigt werden, dass Melanin als Reportermolekül für die Magnetresonanz sowie für die multispektrale optoakustische Tomographie einsetzbar ist. Es wurde deswegen angestrebt, die Kombination aus dem therapeutischen Potential des onkolytischen Vaccinia-Virus und der diagnostischen Anwendung des Melanins als Reporter auszunutzen. Sämtliche in dieser Arbeit aufgeführten rekombinanten Vaccinia-Viren (rVACV) wurden von der Firma Genelux Corporation zur Verfügung gestellt und in dieser Arbeit hinsichtlich der therapeutischen Effizienz und des diagnostischen Potentials untersucht. In ersten Zellkultur-Versuchen wurde anhand verschiedener konstitutiv melanogener rVACV-Konstrukte festgestellt, dass die Kombination aus dem Vaccinia-Virus-spezifischen synthetic early/late Promotor und dem Enzym Tyrosinase (GLV-1h327) bzw. den Enzymen Tyrosinase und Tyrosinase-Helferprotein 1 (GLV-1h324) die höchste Melaninsynthese-Rate zeigte. Anschließend wurde mittels der Bestimmung der spektralen Absorption und der Enzymaktivität der viral kodierten Melanin synthetisierenden Enzyme sowie mikroskopischer Analysen gezeigt, dass es mit diesen auf 8 Vaccinia-Virus-basierenden melanogenen Reportersystemen möglich ist, die Melaninsynthese in nicht-melanogenen Zellen zu induzieren. Anhand elektronenmikroskopischer Untersuchungen in Zellkultur und ex vivo konnte gezeigt werden, dass die nach rVACV-Infektion stattfindende Melaninsynthese in den Lysosomen der Wirtszelle abläuft. Eine Analyse der atomaren Zusammensetzung des viral vermittelten Melanins ergab, dass es sich um eine Mischform aus Eu- und Phäomelanin handelt. Dieser Melanin-Mix ähnelte dem Melanin aus Haut und Augen, jedoch lagen an Melanin-gebundene Metallionen in erhöhtem Maß vor... / The common therapeutic approaches available to treat various cancers still have deficiencies concerning their efficiency and side effects. Decisive for these deficiencies is the poor sensitivity of most of the conventional diagnostic systems and associated with that the late, sometimes too late, identification of tumorous tissue. The oncolytic vaccinia virus is an opportunity to enhance the efficiency of both the therapy and the diagnosis of tumor tissue. In both cases, the specificity for tumor cells and the simplicity of inserting foreign gene cassettes into the viral genome are key factors. The genes encoding murine tyrosinase (mTyr) and tyrosinase related protein 1 (Tyrp1) were inserted into the genome of an oncolytic vaccinia virus. Tyrosinase is the key enzyme during melanogenesis and expression of this enzyme alone can lead to melanin production. Tyrp1 is involved in processing and stabilizing tyrosinase. Various studies have demonstrated melanin to be an excellent reporter molecule for MRI (magnetic resonance imaging) and MSOT (multispectral optoacoustic tomography). Therefore, melanogenic oncolytic vaccinia viruses were constructed to combine the therapeutic potential of this virus with diagnostic abilities of melanin. All recombinant vaccinia viruses (rVACV) mentioned were kindly provided by Genelux Corporation. In this thesis, we tested for their therapeutic efficiency and diagnostic potential. Initial cell culture experiments have shown that the combination of vaccinia virus-specific synthetic early/late promoter and the melanogenic enzyme tyrosinase (GLV-1h327) or the enzymes mTyr and Tyrp1 (GLV-1h324) have the highest melanin synthesis rate. It was observed that the infection of non-melanin producing cells with an rVACV-based melanogenic reporter system resulted in melanogenesis. Electron microscopy showed that the viral associated melanogenesis is localized in the lysosomes of the infected host cell. The atomic composition analysis of the virus-mediated melanin molecules revealed a mixture of eu- and pheomelanin. The virus-associated melanin is comparable to that in the skin and eye but showed higher amounts of melanin-bound metal ions. ...

LIM and SH3 protein 1 (LASP1) is a nucleocytoplasmic scaffolding protein. LASP1 interacts with various cytoskeletal proteins via its domain structure and is known to participate in physiological processes of cells. In the present study, a detailed investigation of the expression pattern of LASP1 protein in normal skin, melanocytic nevi and melanoma was carried out and the melanocyte–specific function of LASP1 was analyzed. LASP1 protein was identified in stratum basale of skin epidermis and a very high level was detected in nevi, the benign tumor of melanocyte. In the highly proliferative basal cells, an additional distinct nuclear localization of the protein was noted. In different tumor entities, an elevated LASP1 expression and nuclear localization, correlated positively with malignancy and tumor grade. However, LASP1 level was determined to be very low in melanoma and even reduced in metastases. Melanoma is distinguished as the first tumor tested to date – that displayed an absence of elevated LASP1 expression. In addition no significant relation was observed between LASP1 protein expression and clinicopathological parameters in melanoma. The epidermal melanin unit of skin comprises of melanocytes and keratinocytes. Melanocytes are specialized cells that synthesize the photo protective coloring pigment, melanin inside unique organelles called melanosomes. The presence of LASP1 in melanocytes is reported for the first time through this study and the existence was confirmed by immunoblotting analysis in cultured normal human epidermal melanocyte (NHEM) and in melanoma cell lines, along with the immunohistostaining imaging in normal skin and in melanocytic nevi. LASP1 depletion in MaMel2 cells revealed a moderate increase in the intracellular melanin level independently of de novo melanogenesis, pointing to a partial hindrance in melanin release. Immunofluorescence images of NHEM and MaMel2 cells visualized co-localization of LASP1 with dynamin and tyrosinase concomitant with melanosomes at the dendrite tips of the cells. Melanosome isolation experiments by sucrose density gradient centrifugation clearly demonstrated the presence of LASP1 and the melanosome specific markers tyrosinase and TRP1 in late stage melanosomes. The study identified LASP1 and dynamin as novel binding partners in melanocytes and provides first evidence for the existence of LASP1 and dynamin (a protein well–known for its involvement in vesicle formation and budding) in melanosomes. Co-localization of LASP1 and dynamin along the dendrites and at the tips of the melanocytes indicates a potential participation of the two proteins in the membrane vesicle fission at the plasma membrane. In summary, a possible involvement of LASP1 in the actin–dynamin mediated membrane fission and exocytosis of melanin laden melanosome vesicles into the extracellular matrix is suggested. / LIM und SH3 protein 1 (LASP1) ist ein nukleozytoplasmatischen Gerüstprotein. LASP1 interagiert über seine Domänenstruktur mit verschiedenen Zytoskelettproteinen und ist an physiologischen Prozessen wie Migration und Zellproliferation beteiligt. In der vorliegenden Studie wurde eine detaillierte Untersuchung des Expressionsmusters von LASP1 in normale Haut, Nävi und Melanom durchgeführt und die Melanozyten-spezifische Funktion des Proteins analysiert. LASP1 konnte durch immunhistologische Färbungen im Stratum basale der Epidermis und in hoher Konzentration in Nävi (gutartige Tumore der Melanozyten) nachgewiesen werden, während die Expression in Melanom und Metastasen sehr gering ist. Auch wurde kein signifikanter Zusammenhang zwischen der LASP1 Proteinexpression in Melanomen und den klinisch-pathologischen Parametern bei 58 Patienten festgestellt. Dies steht im Gegensatz zu allen bisher getesteten Tumoren (u.a. Brust, HCC und Medulloblastom), bei der eine erhöhte LASP1 Expression in den Tumoren beobachtet wurde, die mit dem Tumorgrad korrelierte. Die hochproliferativen Basalzellen der Epidermis bestehen aus Keratinozyten und Melanozyten und weisen, im Gegensatz zu Nävi und Melanomzellen, eine deutliche Kernlokalisation des LASP1 Proteins auf. Melanozyten sind spezialisierte Zellen, die das UV-Schutzfarbpigment Melanin in einzigartigen Organellen, genannt Melanosomen, synthetisieren. Die Expression von LASP1 in diesen Melanozyten konnte zum ersten Mal in dieser Studie nachgewiesen werden. Immunfluoreszenzaufnahmen und Western Blots mit kultivierten normalen menschlichen epidermalen Melanozyten (NHEM) und Melanom-Zelllinien bestätigen die LASP1 Expression. Funktionelle Experimente mit LASP1 depletierten Zellen zeigen eine erhöhte Melaninkonzentration, die unabhängig von der de novo Melanogenese ist. Immunfluoreszenaufnahmen visualisieren eine Ko-Lokalisation von LASP1 mit Tyrosinase an den Melanosomen in den Zellausläufern von pigmentierten MaMel2 Zellen. Eine Auftrennung der einzelnen Melanosomenstadien durch Saccharose-Dichtegradienten-Zentrifugation erlauben die Detektion von LASP1 mit Dynamin, TRP1 und Tyrosinase in späten Stadium IV Melanosomen. Die vorliegende Studie liefert den ersten Beweis für die Existenz von Dynamin (einem für die Vesikelbildung essentiellem Protein) in Melanosomen und identifiziert Dynamin als neuartigen Bindungspartner von LASP1 in Melanozyten. Die Ko-Lokalisierung LASP1 und Dynamin entlang der Dendriten und in den Spitzen der Melanozyten weist auf eine mögliche Beteiligung beider Proteine an der Melanosomen-Vesikel-Abspaltung an der Plasmamembran hin. Zusammenfassend lässt sich sagen, dass LASP1 an der Actin-Dynamin vermittelten Exocytose von Melanin-beladenen Melanosomvesikeln in die Extrazellulärmatrix beteiligt ist.

In this Medical Physics M.Sc. project, a near-infrared (NIR) fluorescence imaging system was built for in-vivo diagnosis and evaluation of pigmented skin abnormalities and diseases. Light coming from a 785 nm diode laser is coupled into a ring light guide to uniformly illuminate the skin surface with a field-of-view (FOV) of 25 mm diameter. The diffuse reflectance and emitted fluorescence photons are collected by an NIR-sensitive CCD camera, with computer-controlled filter switch to select between reflectance mode and fluorescence mode. Both reflectance and fluorescence images of skin disorders were obtained with an exposure time of 2 seconds. The results show that cutaneous melanin in pigmented skin disorders emits higher NIR autofluorescence (AF) than surrounding normal tissue. This finding challanged the conventional concept that melanin is a non-fluorescence substance. The developed NIR autofluorescence imaging method also provided a new and direct way to characterize cutaneous melanin and can potentially be used for evaluation and diagnosis of pigmented skin diseases and skin cancers, such as melanoma.

In this Medical Physics M.Sc. project, a near-infrared (NIR) fluorescence imaging system was built for in-vivo diagnosis and evaluation of pigmented skin abnormalities and diseases. Light coming from a 785 nm diode laser is coupled into a ring light guide to uniformly illuminate the skin surface with a field-of-view (FOV) of 25 mm diameter. The diffuse reflectance and emitted fluorescence photons are collected by an NIR-sensitive CCD camera, with computer-controlled filter switch to select between reflectance mode and fluorescence mode. Both reflectance and fluorescence images of skin disorders were obtained with an exposure time of 2 seconds. The results show that cutaneous melanin in pigmented skin disorders emits higher NIR autofluorescence (AF) than surrounding normal tissue. This finding challanged the conventional concept that melanin is a non-fluorescence substance. The developed NIR autofluorescence imaging method also provided a new and direct way to characterize cutaneous melanin and can potentially be used for evaluation and diagnosis of pigmented skin diseases and skin cancers, such as melanoma.

La première partie de la thèse était de définir et de valider une méthode d'extraction de cellules épidermiques la plus simple, la plus efficace et la plus applicable à des petits échantillons, et évitant les réactifs animaux. Dans cette étude, TrypLPM, une enzyme de dissociation cellulaire exempte d'origine animale, a remplacé la dispase et la trypsine comme enzyme destinée à l'extraction des mélanocytes. Ce produit pourrait simplifier le protocole d'extraction, éliminer les contaminants pathogènes potentiels de l'animal et diminuer la contamination par les kératinocytes pendant l'extraction et la culture des mélanocytes. L'efficacité d'extraction a été jugée sur les critères suivants: séparation épiderme / derme, fonctionnalité des mélanocytes extraits (prolifération, synthèse de la mélanine).La deuxième partie de la thèse était d'explorer l'interaction entre l'épiderme et le derme dans l'hyperpigmentation, en se focalisant sur la protéine Dickkopf 1 (Dkkl) qui est sécrétée par le fibroblaste dermique et codée par le gène Dkkl. Dkkl est un inhibiteur de la voie de signalisation Wnt, qui en isolant le co-récepteur LRP5 / 6, empêche la formation du complexe Frizzeled-Wnt­LRP5 / 6. Nous avons émis l'hypothèse que TGF-β1 participe au développement du lentigo solaire en réduisant l'expression de Dkkl dans les fibroblastes par la voie p38 MAP kinase, ce qui conduit à une activation de la cascade de signalisation Wnt / β-caténine. Cette hypothèse a été confirmée dans le modèle cellulaire 2D de fibroblastes issus de lentigo et de peau normale adjacente, et également dans le modèle de peau 3D irradiée aux UV, en étudiant l'expression génique et la sécrétion protéique de Dkk 1. / The first part of the thesis was to define and validate an epidermal cell extraction method being the simplest, the most effective and applicable to smaller samples and avoiding animal reagents. In this study, TrypLE™, an animal origin-free cell dissociation enzyme, replaced dispase and trypsin as dissociated enzyme for melanocytes extraction. This product could simplify extraction protocol, eliminate potential pathogenic contaminants from animal and decrease keratinocytes contamination during extraction and subculture of melanocytes. The extraction efficiency was judged on the following criteria: separation epidermis / dermis, functionality of the extracted melanocytes (proliferation, melanin synthesis).The second part of the thesis was to explore the role of cross-talking between epidermis and dermis in hyperpigmentation, focused on Dickkopf-related protein l(Dkkl) which is secreted by dermal fibroblast and encoded by Dkkl gene is an antagonistic inhibitor of the Wnt signaling pathway, by isolating the LRP5/6 co-receptor and preventing the Frizzeled-Wnt-LRP5/6 complex formation. We hypothesized that TGF-βl mediates development of solar lentigo by reducing Dkkl expression in fibroblasts through the p38 MAP kinase pathway, which leads to an activation of the Wnt/ β-catenin signaling cascade. This hypothesis was confirmed in the fibroblast of lentigo and adjacent normal skin, also in the UV A-irradiation 3D skin mode! by testing of Dkkl secretion in protein level and Dkkl expression in RNA level.

Made available in DSpace on 2014-10-09T12:50:17Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:58:47Z (GMT). No. of bitstreams: 1 10828.pdf: 923135 bytes, checksum: 56ecab1b373cf9b110daef6cebfdb5ea (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia - USP