Hypomelanosis, a severe reduction in pigmentation, is a widespread phenomenon which affects many different vertebrate species, including humans and chickens. The cause(s) of various forms of hypomelanosis is (are) not known. The aim of this study was to determine the cause of hypomelanosis in a breed of white chickens (White Plymouth Rock x Pile Game). It was hoped that this hypomelanotic breed may provide insight into the etiopathogenesis of certain human hypomelanotic disorders, such as vitiligo and albinism. To determine whether melanocytes are present in the hypomelanotic skin, two melanocyte-specific assays were carried out, in situ DOPA histochemistry and a sensitive radiometric assay for tyrosinase. The results show that active tyrosinase was present in 8, 9 and 10 day skins. However, unlike normal black skin, the level of tyrosinase did not increase with age, suggesting that the melanocytes either die or that they do not continue to synthesise tyrosinase. Ultrastructurally, these melanocytes appeared to be morphologically normal and they did not show signs of premature degeneration. Unlike black chick melanocytes, however, they contained very few premelanosomes and fully melanised melanosomes were never observed, suggesting that hypomelanosis results from the arrested development (melanisation) of melanosomes in vivo. Two different experiments were carried out to determine whether this blockage in melanogenesis is intrinsic in the melanocyte or whether it is caused by extrinsic environmental factors. The outcome of these studies were conflicting: 1) In culture, white chick neural crest cells produced pigment, suggesting that the melanocyte is not defective. However, ultrastructural examination of these cultured melanocytes showed that they contained a large proportion of partially melanised melanosomes. 2) Black chick neural crest cells migrated into white skin explants and contributed towards pigment in the developing feathers, suggesting that the white chick tissue environment is also not defective. The results hint that hypomelanosis in the white chicks is caused by the interaction of at least two genetic defects: an intrinsic mutation of the melanocyte, as well as an extrinsic mutation in the melanocyte environment that, in combination, exert an inhibitory influence on melanin synthesis. This study shows that, in situ, white chick melanocytes share some features with ty-pos albino melanocytes and may be representative of this pigmentary disorder. White Plymouth Rock x Pile Game chicks may also be useful as a model for the multi-faceted disorder, vitiligo.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The neural crest (NC) is a multipotent embryonic cell type derived from the ectoderm during neurulation giving rise to a variety of cell lineages such as neurons, glia and pigment cells. Most genes associated with the correct initiation, differentiation and migration of the neural crest have been found through reverse genetics. Similarities between neural crest development and some features of cancer progression are remarkable. For instance, it has been suggested that some cancer types recapitulate NC processes in an unregulated manner such as epithelial-mesenchymal transition or active cell migration throughout the body to form distant metastases. However, to date very little is known about initiators and drivers that direct neural crest cell migration to specific target sites. The Medaka mutant hirame represents an interesting melanocyte specific migration defect on the yolk sac caused by a loss of functional Yes-associated protein (YAP). Medaka hirame mutants were initially studied for their profound changes in body morphology. Genomic mapping identified the causal mutation as a nonsense point mutation within the first WW domain in the Yes-associated protein 1 (YAP1), causing translation of a dysfunctional YAP protein. YAP is a downstream transcriptional co-activator of the recently discovered and evolutionarily conserved Hippo pathway. Alterations within Hippo signalling are linked to cell survival, proliferation and abnormal tissue overgrowth. We demonstrate that hirame melanocyte precursors (melanoblasts) are initially present in normal abundance, but show an early migration defect with a lack of melanoblasts on the yolk sac, and corresponding accumulation in the lateral parts of the body. Subsequently, we observe an overall decline in differentiated melanocyte numbers during late stage embryogenesis. We designed an overexpression cassette linking enhanced GFP to either wild type or a mutated activated version of YAP and present evidence that it can efficiently rescue the melanocyte defect after injection of mRNA into one-cell stage embryos. Furthermore, analysis of the yolk sac anatomy via transmission electron microscopy indicates that a fraction of yolk membrane cells undergo apoptosis and we propose that this may contribute to the establishment of altered environmental cues leading to abnormal melanoblast migration onto the yolk sac. Injection of yap mRNA directly into the yolk sac however, failed to rescue melanoblast patterning. To advance our study, we isolated and characterised a 3.6 kb Medaka dopachrome tautomerase (Dct) promoter fragment, and used it to drive expression of enhanced green fluorescent protein (eGFP) in vivo. We generated germline transgenics with this construct that showed lineage-specific expression of eGFP within early migrating melanoblasts, a phenotype that is maintained in differentiated melanocytes throughout embryogenesis. In addition, using this promoter we overexpressed our egfp-yap fusion cassette and established transgenic lines to assess the cell autonomy of YAP within the melanocyte lineage. However, no fluorescent signal could be detected in the latter transgenics, necessitating future experimentation to properly characterise these lines. Finally, we analysed a range of neural crest markers to examine the extent of the neural crest defects in hirame mutants. In addition to the melanocyte phenotype, we identified a dramatic reduction in xanthophore numbers, although early leucophore development appears unaffected. We also observed a decreased number of dorsal root ganglia in the peripheral nervous system as well as smaller and partly ectopic cranial neural crest ganglia populations within the epibranchial arches. The characterisation of a novel Medaka melanocyte specific promoter as well as additional novel NC markers will be widely applicable and useful to the wider Medaka research community as a tool for the study of neural crest related mechanisms during development.

Magister Chirurgiae Dentium - MChD / Oral physiological pigmentation presents with great variability with respect to sites, forms, patterns and contrasts in colour. Knowledge of the existence of pigmented lesions and their significance remained unclear for both the general public and oral clinicians alike. The possibility of malignant transformation of some pigmented lesions makes them important to monitor and biopsy. The prevalence of physiological pigmentation is unknown for the defined population group in this study. The results will be beneficial as part of a larger multicentre study with South Africa (Feller et al, 2015). Methodology: A cross sectional analytical study of patients that attended the University of the Western Cape Oral Health centres for routine treatment was conducted. After obtaining informed consent, patients were screened and asked a series of questions using a standardized questionnaire. From these completed questionnaires a prevalence relating to oral physiological pigmentation was determined. Oral physiological pigmentation did not have a male or female predominance in this study population group, but was associated with increased age. Oral pigmentation seemed to be well represented after 18 years of age. Patients were not usually aware of the pigmented gingiva unless being made aware off it.

Thesis (Ph. D.)--University of Hong Kong, 2004. / Also available in print.

Made available in DSpace on 2014-10-09T12:24:06Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:47Z (GMT). No. of bitstreams: 1 01002.pdf: 6118336 bytes, checksum: 2606c025b057757950e77424968c6b47 (MD5) / Dissertacao (Mestrado) / IEA/D / Instituto de Biociencias, Universidade de Sao Paulo - IB/USP

Made available in DSpace on 2014-10-09T12:24:06Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:47Z (GMT). No. of bitstreams: 1 01002.pdf: 6118336 bytes, checksum: 2606c025b057757950e77424968c6b47 (MD5) / Dissertacao (Mestrado) / IEA/D / Instituto de Biociencias, Universidade de Sao Paulo - IB/USP

Le vitiligo généralisé est une leucodermie acquise qui touche 0,5 à 1% de la population mondiale et qui résulte d’une perte progressive des mélanocytes.Le mécanisme à l’origine de la perte des mélanocytes au cours du vitiligo reste obscur et la destruction des mélanocytes n’a jamais été observée.De nombreuses hypothèses ont été avancées pour expliquer la disparition des mélanocytes : une susceptibilité génétique, l’auto-immunité, la théorie neurale et la théorie oxydative.Notre équipe a développé une théorie intégrée qui reprend ces différents mécanismes. Cette théorie considère que le vitiligo est lié au détachement des mélanocytes et à leur élimination à travers l’épiderme ou mélanocytorrhagie. Dans cette théorie, un défaut d’adhésion des mélanocytes est le facteur prédisposant au vitiligo.L’interaction des mélanocytes avec les kératinocytes environnants et avec la membrane basale est médiée par les intégrines et les cadhérines. L’expression de l’intégrine et de la E-cadhérine n’est pas modifiée dans le vitiligo généralisé.En 2006, Fukunaga-Kalabis et al. montrent que l’attachement des mélanocytes à la membrane basale est en partie dû à DDR1 qui est sous le contrôle de la protéine CCN3. Ils ont observé que l’inhibition de CCN3 induit le détachement des mélanocytes.Récemment, des variants génétiques de DDR1 ont été observés chez des patients d’origine ethnique différente atteints de vitiligo. Nous avons décidé d’étudier d’une part l’expression de CCN3 au niveau de la peau lésionnelle et non lésionnelle de patients atteints de vitiligo et d’autre part l’impact de l’inhibition de CCN3 et de DDR1 au niveau de mélanocytes utilisés pour des reconstructions épidermiques. Nos résultats in vitro et in vivo suggèrent que CCN3 est impliqué dans la physiopathologie du vitiligo. / Common generalized vitiligo is an acquired hypopigmentation which is found in 0, 5-1% of individuals world-wide and which results in progressive loss of melanocytes.The mechanism underlying the elimination of melanocytes in vitiligo remains unclear and melanocyte destruction has never been clearly demonstrated in non segmental vitiligo. Various hypotheses have been put forward to explain the disappearance of melanocytes in vitiligo: genetic susceptibility, autoimmunity, neural and impared redox status. We previously proposed a new theory that integrates those pathomechanisms. This theory considers vitiligo as a disease caused by the chronic detachment and transepidermal loss of melanocytes named melanocytorrhagy. In this theory, the defective adhesion of melanocytes is the predisposing factor.Interactions between melanocytes and the basement membrane are mediated by integrins and interactions between melanocytes and keratinocytes are mediated by cadherins in association with β-catenin. But integrin expression is not affected in NSV and a normal expression of E-cadherin in lesional and non lesional vitiligo skin is observed.In 2006, Fukunaga-Kalabis et al. reported that attachment of melanocytes to basal lamina is in part due to DDR1, which is under control of CCN3. They have observed that inhibition of CCN3 induces the detachment of melanocytes.Recently, DDR1 genetics variants have been associated with vitiligo in patients of different ethnic origin. We have decided to study in parallel the expression of CCN3 and DDR1 in lesional and non lesional skin of vitiligo patients and the impact of inhibition of CCN3 and DDR1 in melanocytes on their behaviour in reconstructed epidermis.In conclusion, our in vivo and in vitro data suggest that CCN3 is implicated in vitiligo etiology.

No description available.

Tyrosinase (E.C. 1.14.18.1) is a key enzyme in the biosynthesis of melanin. The control of melanin sythesis was explored in skin melanocytes of the following strains; wild type (C57BL/6J-C/C) (which maximally synthesize melanin at normal mammalian body temperature, Himalayan (C57BL/6J-cʰ/cʰ) (which maximally synthesize melanin at temperatures below 37°C) and albino (Balb c-c/c) (a mutant which does not synthesize melanin) The effect of a-MSH on tyrosinase activity was initially investigated. A skin culture tyrosinase assay that made it possible to measure the effect of α-MSH on the activity of this enzyme in vitro was first developed. It was found that α-MSH activated the wild type and Himalayan tyrosinase in a dose-dependent manner and that this activation did not require the de novo synthesis of new enzyme. The role of glycosylation on the wild type and particularly the Himalayan tyrosinase activity was next investigated. The results do not support, but are not in conflict with the theory that the Himalayan tyrosinase is inherently underglycosylated. Translation and transcription as additional control mechanisms of tyrosinase activity was finally investigated. The correlation between the levels of tyrosinase activity, abundance of the enzyme and the synthesis of tyrosinase mRNA in wild type, Himalayan and albino mice was determined. It was shown that the levels of newly synthesized tyrosinase and tyrosinase mRNA transcripts were higher in the wild type than in the Himalayan skin. This could account for the reduced tyrosinase activity in the Himalayan mutant at normal body temperature. Low levels of tyrosinase mRNA were found in the albino skin though there was no immunodetectable enzyme in this tissue.