<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

Molecular imaging using fluorescence spectroscopy-based techniques is generally inefficient due to the low quantum yield of most naturally occurring biomolecules. Current fluorescence imaging techniques tag these biomolecules chemically or through genetic manipulation, increasing the complexity of the system. A technique capable of imaging these biomolecules without modifying the chromophore and/or its environment could provide vital biometric parameters and unique insights into various biological processes at a molecular level. Pump probe spectroscopy has been used extensively to study the molecular properties of poorly fluorescing biomolecules, because it utilizes the known absorption spectrum of these chromophores. Optical Coherence Tomography (OCT) is an optical imaging modality that harnesses the power of low coherence interferometry to measure the 3-D spatially resolved reflectivity of a tissue sample. We plan to develop a new molecular imaging modality that combines these techniques to provide 3-D, highresolution molecular images of various important biomolecules. The system uses a Fourier domain OCT setup with a modified sample arm that combines the "pump" and "probe" beams. The pump beam drives the molecules from the ground state to excited state and the probe interrogates the population change due to the pump and is detected interferometrically. The pump and the probe beam wavelengths are optimized to maximize absorption at the pump wavelength and maximize the penetration depth at the probe wavelength. The pump-probe delay can be varied to measure the rate at which the excited state repopulates the ground state, i.e., the ground state recovery time. The ground state recovery time varies for different chromophores and can potentially be used to identify different biomolecules. The system was designed and optimized to increase the SNR of the PPOCT signals. It was tested by imaging hemoglobin and melanin samples and yielded encouraging results. Potential applications of imaging hemoglobin using this technique include the mapping of tissue microvasculature and measuring blood-oxygen saturation levels. These applications could be used to identify hypoxic areas in tissue. Melanin imaging can provide means of demarcation of melanoma in various organs such as skin, eye and intestines.

Parasitic nematodes of grazing livestock represent an increasing economic and welfare problem for British agriculture. By investigating specific life-cycle stages of these parasites, it may be possible to identify key molecules or pathways that are required for the survival of the worms, and thus exploit these for future control strategies. It has been shown previously that the third larval stages (L3) of the ovine parasitic nematode Teladorsagia circumcincta produce high levels of transcript for the enzyme GTP-Cyclohydrolase relative to later developmental stages. As the ratelimiting factor in the production of tetrahydrobiopterin, GTP-Cyclohydrolase is required for a number of different biochemical pathways, including those involved in the production of serotonin and melanin. As the L3 do not feed, it can be hypothesised that, if finite resources are being used in the production of transcript encoding this enzyme, then it may be important for survival. In this thesis, a number of approaches were taken to explore the function of GTPCyclohydrolase in the life-cycle development of T. circumcincta. The closely related parasite, Dictyocaulus viviparus, was used as a model organism to explore the role of GTP-Cyclohydrolase and serotonin production with regards to larval arrest, or hypobiosis. This process occurs readily under experimental conditions in D. viviparus, which is not possible with T. circumcincta. Quantitative PCR was used to examine GTP-Cyclohydrolase transcript levels in two different strains of D. viviparus, one that enters larval arrest when exposed to cold conditions and one that does not. No differences were observed between the two strains suggesting that GTP-Cyclohydrolase was unlikely to be involved in hypobiosis. The model nematode, Caenorhabditis elegans, was used to perform functional complementation experiments to assess the role of GTP-Cyclohydrolase in the cuticle, as it has been shown previously that C. elegans GTP-Cyclohydrolase mutants have a ‘leaky cuticle’ and are killed by lower doses of anthelmintics and bleach than the wild-type worms. The T. circumcincta gene for GTP-Cyclohydrolase was able to restore cuticular integrity of C. elegans GTP-Cyclohydrolase-deletion mutants, suggesting that the role played by the protein in both species is similar. In vitro inhibition experiments using a chemical inhibitor of GTP-Cyclohydrolase showed that T. circumcincta larval development was disrupted in the presence of the inhibitor. It was also shown that T. circumcincta L3 that were exposed to sunlight produced melanin, suggesting that the levels of GTP-Cyclohydrolase observed in the preparasitic stages of T. circumcincta may be required for the synthesis of melanin. Together, these data suggest that GTP-Cyclohydrolase is required by the preparasitic stages to survive on pasture. Ultraviolet radiation has been shown previously to be harmful to T. circumcincta L3, so if the melanin production provides protection from this, then it would be crucial for the survival of the pre-parasitic stages.

<p>Dermatopathologists need more reliable tools for analyzing biopsies of lesions that are potentially melanomas and determining the best treatment plan for the patient. Previously inaccessible, the chemical and physical properties of melanin provide insight into melanoma biochemistry. Two-color, near-infrared pump-probe microscopy of unstained, human pathology slides reveals differences in the type of melanins and the distribution of melanins between melanomas and benign nevi. Because the pump-probe response of melanin is resilient to aging, even for hundreds of millions of years, this tool could prove useful in retrospective studies to correlate melanin characteristics with patient outcome, thus eliminating the pathologist's uncertainty from the development of this classification method.</p><p>Pump-probe spectroscopy of a variety of melanin preparations including melanins with varying amounts of metal ions and toxins, those that have been photo-damaged or chemically oxidized, and melanins with a homogeneous size distribution shows that the pump-probe response is sensitive to these chemical and physical differences, not just melanin type as previously hypothesized. When sampling the response at several pump wavelengths, the specificity of this technique is derived from the absorption spectra of the underlying chromophores. Therefore, hyperspectral pump-probe microscopy of melanin could serve as an indicator of the chemical environment in a variety of biological contexts. For example, the melanin chemistry of macrophages suggests that these cells oxidize, homogenize, and compact melanin granules; whereas melanocytes produce heterogeneous melanins.</p> / Dissertation

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-27T18:21:42Z No. of bitstreams: 1 Rodrigo de Almeida Paes.pdf: 4542299 bytes, checksum: fd76ae56b0bcb0f4261dd34bfeb58f26 (MD5) / Made available in DSpace on 2013-09-27T18:21:42Z (GMT). No. of bitstreams: 1 Rodrigo de Almeida Paes.pdf: 4542299 bytes, checksum: fd76ae56b0bcb0f4261dd34bfeb58f26 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A esporotricose é a principal micose subcutânea na América Latina. O estado do Rio de Janeiro constitui uma importante área endêmica de esporotricose zoonótica onde diversas manifestações clínicas incomuns da doença são observadas. Os fatores que podem influenciar no desenvolvimento da esporotricose são o tamanho do inóculo, a profundidade da inoculação traumática, a imunidade do hospedeiro, a virulência e a termotolerância do fungo. Como não há informações disponíveis sobre os fatores de virulência dos isolados de Sporothrix da área endêmica de esporotricose no Rio de Janeiro estudamos 63 isolados obtidos de pacientes desta área endêmica e nove controles de outras localidades, onde a produção de melanina em diferentes condições, termotolerância, produção de protease e urease foram analisadas. Também foi estudada a composição antigênica de oito isolados, sendo sete da área endêmica de esporotricose no Rio de Janeiro e um controle. Os isolados do estudo compreendem as espécies S. brasiliensis (n=59), S. schenckii (n=12) e S. globosa (n=1). A análise de produção de melanina mostrou que, além da já conhecida melanina DHN, as três espécies do complexo Sporothrix estudadas são capazes de produzir outros dois tipos de melanina, utilizando L-DOPA ou L-tirosina como substrato. A produção destes três tipos de melanina foi bastante variado entre os diferentes isolados, sendo influenciada pelo meio de cultivo, temperatura, pH, fonte de carbono e tempo de incubação, sugerindo um controle multifatorial da melanogênese em Sporothrix. A melanina é produzida principalmente nos conídios e nas leveduras, mas quando L-DOPA é adicionado ao meio de cultivo, hifas também podem melanizar. Além disso, a melanina formada a partir de L-tirosina é um composto solúvel que pode recobrir as células fúngicas, sem polimerizar-se na parede celular ao contrário dos outros dois tipos de melanina, conferindo proteção contra luz ultravioleta, fármacos antifúngicos, reativos de oxigênio e de nitrogênio. Também foi verificado que a melanização das diferentes espécies de Sporothrix pode ser influenciada pelo contato com bactérias, principalmente as espécies Pantoea agglomerans e Pseudomonas aeruginosa, vivendo no mesmo ambiente que o fungo. A primeira bactéria inicialmente inibe o crescimento fúngico, porém com sua morte Sporothrix consegue crescer mais e produzir mais melanina do que quando cultivado isoladamente. Já a inibição do crescimento relacionada a P. aeruginosa é maior em S. brasiliensis do que em S. schenckii, o que pode estar relacionado à maior resistência desta espécie a fármacos antifúngicos. A produção de urease também foi diferenciada entre essas duas espécies, podendo a maior produção desta enzima por S. brasiliensis estar relacionada ao elevado número de casos de regressão espontânea desta micose no Rio de Janeiro. Não foi possível correlacionar nenhum fator de virulência, termotolerância ou antígenos de Sporothrix spp. com manifestações clínicas da esporotricose, sugerindo que estas sejam fruto de uma combinação de fatores envolvendo a virulência do fungo e o status imunológico do hospedeiro. / Sporotrichosis is the main subcutaneous mycosis in Latin America. Rio de Janeiro state is an important endemic area of zoonotic sporotrichosis, where several uncommon clinical manifestations of this disease are observed. Factors that may have a role in the development of sporotrichosis are the size of inoculum, the depth of traumatic inoculation, the host immune response status, virulence, and thermotolerance of the fungus. Up to now any information about virulence factors of Sporothrix strains from the endemic sporotrichosis area at Rio de Janeiro is described. We studied 63 strains obtained from patients of this endemic area and nine controls from other places, in which melanin production on different conditions, thermotolerance, protease and urease production wew analyzed. It has been also studied the antigenic composition of eight isolates, seven from the endemic sporotrichosis area at Rio de Janeiro and one control. Strains from this study include the species S. brasiliensis (n=59), S. schenckii (n=12), and S. globosa (n=1). Melanin production analysis showed that, besides the already known DHN melanin, the three studied species from the Sporothrix complex are able to produce other two types of melanin, using L-DOPA or L-tyrosine as substrate. The production of these three types of melanin was quite varied between the different strains, being affected by culture medium, temperature, pH, carbon source and incubation time, suggesting a multifactorial control of Sporothrix melanogenesis. Melanin is mainly produced on conidia and yeast-cells, but when L-DOPA is available on culture medium, hyphae can also melanize. Besides, L-tyrosine derived melanin is a soluble compound that can cover fungal cells, without polymerization on cell wall unlike the other two types of melanin, conferring protection against ultraviolet light, antifungal drugs, oxygen, and nitrogen oxidants. It was also observed that melanization of different Sporothrix species can be influenced by the contact with bacteria living in the same environment than the fungus, especially Pantoea agglomerans and Pseudomonas aeruginosa. Initially, the first bacterium inhibits fungal growth, however, after its death, Sporothrix is able to grow more intensly and to produce more melanin than when cultured alone. The P. aeruginosa-related growth inhibition is higher in S. brasiliensis rather than in S. schenckii, which can be related to the higher resistance of this species to antifungal drugs. Urease production was also differentiated between these two species, and the higher expression of this enzyme by S. brasiliensis might be related to the high number of spontaneous regression cases of this mycosis in Rio de Janeiro. It was not possible to correlate any virulence factor, thermotolerance or antigens of Sporothrix spp. with the clinical manifestations of sporotrichosis, suggesting that they are the result of a combination of factors involving the virulence of the fungus and host immunologic status.

Submitted by JOÃO VITOR PAULIN null (joaovp281@gmail.com) on 2016-04-12T18:10:04Z No. of bitstreams: 1 Paulin, JV - Dissertação.pdf: 8544821 bytes, checksum: 8c1f65d0bedc5cc3413277b0fdc03393 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-04-13T17:18:23Z (GMT) No. of bitstreams: 1 paulin_jv_me_bauru.pdf: 8544821 bytes, checksum: 8c1f65d0bedc5cc3413277b0fdc03393 (MD5) / Made available in DSpace on 2016-04-13T17:18:23Z (GMT). No. of bitstreams: 1 paulin_jv_me_bauru.pdf: 8544821 bytes, checksum: 8c1f65d0bedc5cc3413277b0fdc03393 (MD5) Previous issue date: 2016-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Por mais de 40 anos, a melanina tem despertado cada vez mais atenção como um material funcional para dispositivos orgânicos como células solares, transistores, sensores, engenharia de tecidos, baterias, dispositivos de memória e drug delivery. As melaninas apresentam uma combinação de propriedades físico-químicas únicas com sua biocompatibilidade intrínseca que inspira seu uso em aplicações bioeletrônicas. Contudo, sua aplicação em dispositivos orgânicos é limitada devido a sua baixa solubilidade na maioria dos solventes. Assim, neste trabalho, buscamos obter derivados solúveis de melanina a partir de uma abordagem sintética alternativa utilizando diferentes pressões (4 a 8 atm) oxigênio molecular como agente oxidante em água ou DMSO. Espectroscopia UV-Vis, FTIR, RMN e XPS foram utilizados para caracterização, tendo como referência os materiais obtidos a partir das rotas sintéticas tradicionais. A utilização de pressão de oxigênio acelerou o processo sintético em ambos os casos e foi possível obter uma alta proporção DHICA/DHI. A principal diferença é a obtenção em meio aquoso de um material solúvel em água sem nenhuma funcionalização exótica, enquanto que em solvente orgânico o derivado da melanina, funcionalizado com grupos sulfonatos, apresenta solubilidade em DMSO, DMF e N-metil-2-pirrolidona. Um mecanismo reacional baseado na elevada natureza oxidante do meio sintético, incluindo na decomposição do H2O2 pelo oxigênio é proposto para explicar as alterações estruturais observadas. / For over 40 years, melanin has been focus of increasing attention as a promising functional material for organic devices like solar cells, transistors, sensors, tissue engineering, charge storage, memory devices and drug delivery. Melanins present a combination of unique physical-chemical properties with its intrinsic biocompatibility inspire its use in bioelectronics applications. However, their applicability in organic devices is limited due to its low solubility in most solvents. Thus, in this work, we aim to achieve soluble derivatives of melanin from an alternative synthetic approach using different oxygen pressures (4 to 8 atm) as an oxidizing agent in water or DMSO. UV-Vis spectroscopy, FTIR, RMN and XPS was used to characterize the material using conventional synthesized melanin as a reference. The use of oxygen pressure accelerated the synthetic process and in both cases it was possible to obtain a high DHICA/DHI ratio. The main difference is obtained in aqueous medium a water soluble material without any exotic functionalization, while in organic solvent we have a functionalization with sulfonate group with solubility in DMSO, DMF and N-methyl-2-pyrrolidone. A reaction mechanism based on the higher oxidative nature of the reaction medium including oxygen-induced decomposition of H2O2 is proposed to explain the structural changes observed.

Made available in DSpace on 2014-10-09T12:35:54Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:26Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:35:54Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:26Z (GMT). No. of bitstreams: 0 / Observações feitas pelo próprio autor sugerem potencial ação do Líquido da Castanha de Caju (LCC) na pigmentação da pele, ação esta semelhante a da hidroquinona. O LCC é um líquido contido na casca da castanha de caju, possui característica de resina líquida, bastante viscosa e de odor forte, sua coloração varia de marrom claro, escuro a preto, dependendo do método de extração utilizado, podendo ser denominado de Natural ou Técnico. Este estudo propôs cultivar melanócitos e queratinócitos em cocultura e posteriormente tratálos com LCC. A L-DOPA, agente estimulador da melanogênese, via da produção de melanina, responsável pela pigmentação da pele, foi utilizada na cocultura para avaliar a ação do LCC. A hidroquinona, conhecido inibidor desta via, foi utilizada na cocultura como controle positivo para o LCC, visto que este poderia apresentar ação semelhante a da hidroquinona. Para a utilização do LCC na cocultura, testes de solubilidade do mesmo para posterior dispersão no meio de cultura, foram necessários, bem como a identificação de seu potencial cito e fototóxico in vitro. Para a realização do teste de fototoxicidade foi construída uma câmara específica, atendendo as normas exigidas pelos guias ©ECVAM DB-ALM: INVITTOX protocol e OECD TG-432, sendo esta qualificada por método validado. Os testes realizados com o LCC (natural e técnico) indicaram potencial ação destes na pigmentação da pele, estimulando a proliferação de melanócitos em cocultura. Este perfil apresentado, pelos extratos de LCC, é contrário ao da hidroquinona, e ao esperado inicialmente, sendo necessário aprofundar estes estudos. No entanto, estes resultados são promissores, sugerindo a descoberta de um novo tratamento para hipocromias. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

An intraspecific coat colour polymorphism exists in the genus Acomys. Some populations are dark and others are light-colored. Dark populations live in lava deserts, savannas and in the commensal habitat of Egypt. The subject of this thesis is to compare selected populations of Acomys in behavioral test of forced exploration. They are dark commensal, light secondary non-commensal, light non- commensal, and one dark non-commensal populations. The aim of this research is to find out whether the dark commensal populations of Acomys cahirinus differ in behavior from other populations and whether dark populations differ in behavior from light populations. According to the results, the observed behavioral parameters do not show significant differences between populations. The dark commensal populations of A. cahirinus did not differ significantly from other populations in exploratory behavior. But the dark populations preferred a dark background. No apparent differences in behavior between differently colored populations were found. However, the variability in tested behavior between populations was found. Key words: exploratory behavior, melanin, commensalism, comparative approach, open field test, Acomys

The scientific development of innovative molecular techniques has transformed the approach towards human identification. In forensic casework, the emergence of molecular phenotyping, or phenotypic prediction from DNA, has mitigated some challenges involving the unavailability of references samples for traditional forensic DNA analysis. Molecular phenotyping via SNP analysis can be used as a tool in a forensic setting to predict physical traits, such as hair, skin and eye colour, and provide investigative leads. Several ancestry informative markers (AIMs) have previously been associated with human skin colour in mainly the European and North American population groups, while admixed populations are hardly studied. The present study aims to contribute towards this gap by investigating the relationship between two AIMs (SLC45A2, rs16891982 and SLC24A5, rs1426654) that are typically involved in molecular phenotyping, and melanin index (MI) in the South African (SA) metapopulation (n = 389). The self-reported ancestry, ethnicity and relevant biographic information for each participant was documented and MI was recorded using a dermaspectrophotometer. DNA was extracted from saliva samples and PCR amplification of target regions was performed. Thereafter, SNaPshot® PCR was used to genotype the variants. Significant differences (p < 0.0001) were observed between MI readings and ancestral as well as population census groups. A generalised linear model (GLM) was developed which could accurately predicted the MI readings for each genotype combination within the 95 % confidence interval of the recorded MI readings. Our results suggest that these two markers were consistently associated with MI in the admixed SA population and are thus informative to predict MI in a forensic setting. Finally, this was the first study in a SA context to use SNP analysis for objective MI prediction.