<p>Melanin is a biological pigment that is ubiquitous in nature and generally produced within melanosomes, specialized organelles. Typically, melanin is categorized into two distinct classes, based on color and molecular precursor: eumelanin (brown-black) and pheomelanin (yellow-red). Whereas much is known regarding the molecular precursors to the two pigments, an understanding of their resulting molecular structure remains elusive. Despite this lack of knowledge, several functions are attributed to the pigments, including photoprotection and photosensitization. Epidemiological data for skin and ocular cancers have observed an increased incidence for increased relative concentrations of pheomelanin. Furthermore, eumelanin is generally identified as photoprotective and antioxidant, whereas pheomelanin is generally identified as photoreactive and pro-oxidant. This thesis describes the photophysical properties of the naturally-occuring melanin pigments and presents new insights into their roles within the context of skin and ocular cancers.</p><p> Photoemission electron microscopy provides a unique opportunity to probe the complex photoproperties of melanins contained within intact melanosomes isolated from tissues of bovine and human eyes. Photoionization threshold potentials characteristic of eumelanin and pheomelanin have been determined and are used to investigate the molecular architecture of the pigments within the melanosome. Furthermore, a novel approach to photoemission electron microscopy is used to obtain the first direct measurements of the absorption coefficients from intact melanosomes. </p><p> Human iridal stroma melanosomes are comprised of both eumelanin and pheomelanin in various ratios according to iris color; dark brown and blue-green iris melanosomes are characterized by a eumelanin:pheomelanin ratio of 14.8 and 1.3, respectively. Despite the significant difference in the overall pigment composition, a common eumelanin surface photoionization threshold is obtained for both melanosomes. This data indicates that within the melanosome, the phototoxic pheomelanin pigment is encased by eumelanin. This structure mitigates the adverse photochemical properties of pheomelanin. However, damage to the eumelanic exterior and or significant reduction in the amount of eumelanin present could compromise the protective ability of eumelanin, providing mechanisms for exposure of pheomelanin and consequently contributing to oxidative stress.</p><p> The absorption spectra of intact melanosomes of varying melanin compositions were determined over the spectral range from 244 to 310 nm. The absorption spectra of eumelanic melanosomes are similar regardless of monomer composition or embryonic origin. Furthermore, the absorption spectra of melanosomes containing a mixture of pigments were similar to those containing pure eumelanin, arguing that the absorption properties of the melanosome are maintained regardless of increased pheomelanin composition. Therefore, the correlation between epidemiological data and the eumelanin:pheomelanin ratio is not predicted to be a reflection of the melanosome's decreased ability to attenuate biologically relevant wavelengths, but instead is predicted to be a reflection of the different photoreactivities of the melanin pigments contained within.</p> / Dissertation

Skin pigmentation is an important issue in most cultures. Until recently we have not understood the most important elements of pigmentation regarding detailed chemical structure. The synthesis of melanin is very complex, and although core enzymes, other important proteins, and parts of the melanin structure have been identified much information in this context awaits disclosure. The function of the melanocyte and the deposition of melanin pigments into the keratinocytes are very important in the protection against UV light. Melanin pigments consist of high-molecular structures often described as brown to black eumelanin and yellow to red pheomelanin. Eumelanin is photoprotective, whereas pheomelanin is believed to be carcinogenic after UV radiation. There is strong evidence that people of fair complexion with freckles who tan poorly are at higher risk of developing melanoma. These people have a higher pheomelanin to eumelanin ratio in their skin. Melanoma, one of the most widely spread cancers, is derived from melanocytes. There is accumulating evidence that pigment constitution is highly involved in the development of melanoma. We found that patients with advanced melanoma secrete substantial amounts of pigment structures into the urine, in particular those with diffuse melanosis. In subsequently performed experiments we purified these pigments and subjected the product to chemical degradation by either hydrogen peroxide oxidation or hydriodic hydrolysis. Several new chromatographic methods were developed for the structural analysis of these products. Structural analysis of new chromatographic peaks was performed. In conclusion, complex pheomelanin structures as well as low molecular weight pigments and free benzothiazoles have been identified in the urine of patients with melanoma and diffuse melanosis. The present thesis provides new insight into melanogenesis and melanoma progression. This opens the doorway to further approaches to the investigation of melanins and can help to understand fundamental problems about the structure and biosynthesis of natural melanins.

Coat color and patterning phenotypes have been extensively studied as a model for advancing our understanding of the relationship between genetic and phenotypic variation. In this thesis, genes of relevance for pigment cell biology were investigated. The dissertation is divided in two parts. Forward genetics was used in the first part (Paper I and II) to identify the genes controlling the Silver and Sex-linked barring loci in chicken. In the second part, reverse genetics was employed to create a mouse line in which the PMEL17 protein is inactivated (Paper III). In Paper I, we report five mutations in SLC45A2 causing plumage color variants in both chicken and Japanese quail. Normal function of the SLC45A2 gene has previously been shown to be essential for the synthesis of both red/yellow pigment (pheomelanin) and brown/black pigment (eumelanin) in numerous species, including humans. The major discovery in this paper is the specific inhibition of pheomelanin in Silver chickens, whilst null mutations at this locus cause an almost complete absence of both pheomelanin and eumelanin. In Paper II, we report that Sex-linked barring in chickens is controlled by the CDKN2A/B tumor suppressor locus. The locus encodes two proteins, INK4B and ARF. The genetic analysis indicates that missense mutations in ARF or mutations in the promoter region of the ARF transcript are causing Sex-linked barring. In previous studies, mutations inactivating the CDKN2A/B tumor suppressor locus, have been shown to be responsible for familiar forms of human melanoma. Here we propose that these mutations in chicken CDKN2A/B cause the premature cell death of melanocytes as opposed to the cell proliferation and tumor growth associated with loss-of-function alleles in humans. In Paper III, we created a mouse line in which the PMEL17 protein is inactivated. Missense mutations in the gene encoding PMEL17 have previously been shown to be associated with reduced levels of eumelanin in epidermal tissues in several vertebrate species. The knockout mice are viable, fertile, and display no obvious developmental defects. The eumelanosomes within the melanocytes of these mice are spherical in contrast to the cigar-like shaped eumelanosomes present in wild-type animals. PMEL17 protein inactivation has only a subtle diluting effect on the coat color phenotype in four different genetic backgrounds. This suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations.

Pigmentation plays a protective role against damage caused by ultraviolet (UV) irradiation. Humans with fair skin and light hair have a higher susceptibility to UV-induced DNA damage that can lead to the development of skin cancers. The melanocytes found in the skin and hair follicles depend on different signaling molecules for their proper development and pigment production. α-Melanocyte Stimulating Hormone (α-msh) binds to the Melanocortin 1 receptor (Mc1r) to regulate pigment production and the switch between eumelanin and pheomelanin. Lethal yellow mice (Ay) overexpress the agouti signaling protein, which inhibits the binding of α-msh, resulting in a yellow coat color phenotype. Endothelin 3 (Edn3) encodes for a ligand involved in melanocyte development by regulating the differentiation, proliferation and migration of melanocyte precursors. A tetracycline inducible transgenic mouse in which Edn3 was placed under the keratin 5 promoter (K5-tTA;TRE-Edn3-lacZ) displays a hyperpigmentation phenotype due to the accumulation of melanocytes in the skin and an increase in hair pigment. Comparative analysis of dorsal hairs from Ay and Ay; K5-tTA;TRE-Edn3-lacZ mice using high performance liquid chromatography showed that transgenic Edn3 expression significantly increased both eumelanin and pheomelanin. No significant difference in the number of follicular melanocytes between Edn3 transgenic and non-transgenic mice was evidenced by immunofluorescence using an antibody against Tyrosinase related protein 1. Gene expression analysis of hair follicles showed that Edn3 upregulates the expression of melanogenic genes. Deactivation of transgenic Edn3 is possible with doxycycline (dox) treatment. To test if transgenic Edn3 expression is required to rescue and maintain a dark pigmentation phenotype in Ay mice, dox was administered during embryonic and postnatal development to manipulate transgenic Edn3 expression. Results showed that transgenic Edn3 expression is required to maintain a dark pigmentation phenotype after birth but is independent of a developmental requirement. Transgenic Edn3 expression in Mc1re/e mice also resulted in a darkened coat color. Our results indicate that the paracrine expression of Edn3 from keratinocytes is capable of generating and maintaining a dark coat color by the regulation of melanogenic genes independent of Mc1r signaling. The results of this study may open new approaches to the treatment of hypopigmentation disorders.