Polycyclic aromatic hydrocarbons (PAHs) are organic compounds that are composed by at least two aromatic rings. PAHs can be found in coal and petroleum, but can also be formed from incomplete combustion of for example fossil fuels, tobacco, wood and when smoking food. PAHs has been shown to cause several health risks such as carcinogenic effects, which led to that the U.S Environmental Protection Agency (U.S. EPA) selected 16 PAHs as priority pollutants. Those 16 PAHs is usually analysed when investigating PAH exposure. To analyze dermal exposure of PAHs a tape-stripping technique can be used. The tape-stripping method involves that a tape piece is placed on the skin to absorb the present PAHs and then the tape is removed and the PAHs can be extracted and cleaned-up from the tape. The aim of this study is to optimize a recently elaborated clean-up method for PAHs sampled by the tape-stripping technique. Also, to apply the method and measure the dermal exposure of 16 PAHs among soil remediation workers. Two clean-up methods were evaluated, Florisil SPE columns and deactivated silica (10%). Clean-up using Florisil columns were evaluated using 10 and 12 ml of n-hexane. For elution, poor recoveries were achieved for both elution volumes tested. On the other hand, tests using deactivated silica generated good recoveries for both elution solvents tested (i.e. 4 ml n-hexane:dichloromethane + 4 ml dichloromethane and 8 ml n-hexane). As for the elution solvents, no significant difference could be seen in the recoveries and the mixture of n-hexane and dichloromethane was used for the real samples. The dermal exposure of PAHs for the soil remediation workers were investigated using dermal tapes from the palm and neck of 18 soil remediation workers. Samples from the palm were sampled before and after a working day and there was a small difference between the total PAH concentration before and after a work-shift. For all categories of workers (office staff, machine operators and persons performing sampling) an increase in dermal concentration of PAHs could be observed for ten of the workers, but this increase were highest among the workers active in taking samples at the contaminated site. However, an increase in PAH exposure was not observed for all study participants and possible this is due to hand-washing after toilet visits. Overall, the concentrations of PAHs on the dermal samples from soil remediation workers were low, especially in comparison to other occupations such as chimney sweeps and pavers where PAH exposure is known to exist. The detected PAHs on the dermal tapes corresponded to PAH profiles in soil samples from the site.

Submitted by Laryssa Kataki De Oliveira Veloso (laryssakataki@hotmail.com) on 2018-06-15T13:25:57Z No. of bitstreams: 1 DISSERTAÇÃO LARYSSA FINAL.pdf: 1251310 bytes, checksum: 43a08fca6a961ece3b6833de3671f737 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-06-18T20:28:55Z (GMT) No. of bitstreams: 1 veloso_lko_me_bot.pdf: 1251310 bytes, checksum: 43a08fca6a961ece3b6833de3671f737 (MD5) / Made available in DSpace on 2018-06-18T20:28:55Z (GMT). No. of bitstreams: 1 veloso_lko_me_bot.pdf: 1251310 bytes, checksum: 43a08fca6a961ece3b6833de3671f737 (MD5) Previous issue date: 2018-05-30 / OBJETIVO: Avaliar a eficiência da injeção de ácido hialurônico (AH) ou de soro fisiológico (SF), aplicados no subcutâneo da pálpebra inferior de portadores de ectrópio cicatricial, visando à correção não cirúrgica do mal posicionamento palpebral. MÉTODOS: este foi um estudo prospectivo, intervencionista, envolvendo 23 pálpebras de 15 portadores de ectrópio cicatricial, divididos aleatoriamente em dois grupos: G1 (13 pálpebras) no qual foram feitas quatro aplicações de 4ml de SF (Cloreto de Sódio 0,9%, Equiplex, Goiás, Brasil), associado a 1ml de cloridrato de lidocaína a 2,0% sem vasoconstritor (Xylestesin®, Cristália, São Paulo, Brasil), em intervalos de uma semana entre as aplicações; e G2 (10 pálpebras) que receberam aplicação de 1 ml de AH (Restylane® Lidocaine, Q med, Uppsala, Suécia) em aplicação única. Foram avaliadas as variáveis demográficas dos participantes, as queixas, o grau do ectrópio, o grau de flacidez palpebral, a localização do ectrópio, além de avaliações quantitativas realizadas utilizando-se a fotodocumentação sistematizada dos olhos dos pacientes, 30 dias após a primeira aplicação no G1 e 7 e 30 dias após as aplicações do G2. As imagens obtidas foram transferidas para um computador e avaliadas utilizando-se o programa Image J, avaliando-se a distância da pálpebra inferior até o reflexo corneano (DMR2), distância limbo-margem (LM), ângulo da comissura interna (AI) e externa (AE), área total (AT), lateral (AL) e medial (AM). As áreas a serem avaliadas foram delimitadas por uma linha que une as duas comissuras e outra linha que coincide com a margem da pálpebra inferior. As avaliações foram feitas sem tração e com tração da pálpebra inferior para baixo. Todos os dados foram transferidos para a planilha Excel, sendo realizada a análise comparativa antes e após as aplicações nos dois grupos. RESULTADOS: Os grupos foram semelhantes quanto a idade, sexo, cor da pele e grau do ectrópio. Houve melhora signficativa dos sintomas nos indivíduos de G2 após a aplicação de AH. Apos a injeção, o G1 apresentou redução significativa de AI com e sem tração, AE com tração, LM sem tração e AM com tração. No G2, após as injeções as medidas AI sem tração, AE, DMR2, AT e AM com e sem tração, LM e AL com tração apresentaram alterações significativas (p<0,05). A comparação das medidas realizadas no G2 após sete e 30 dias mostrou estabilidade dos resultados obtidos com a injeção de AH. CONCLUSÃO: A aplicação de AH na pálpebra inferior de portadores de ectrópio cicatricial mostrou melhora parcial do mal posicionamento palpebral. A aplicação de SF não foi efetiva no tratamento do ectrópio cicatricial. / PURPOSE: Evaluate the efficiency of injection of hyaluronic acid (HA) or saline solution (SF) applied to the lower eyelid subcutaneous, aiming at the non-surgical correction of cicatricial ectropion. METHODS: This was a prospective study involving 23 eyelids of 15 patients with cicatricial ectropion, randomly divided into two groups: G1 (13 eyelids) with four applications of 4ml of SF (Sodium Chloride 0.9%, Equiplex, Goiás , Brazil), associated to 1ml of lidocaine hydrochloride 2% without vasoconstrictor (Xylestesin®, Cristália, São Paulo, Brazil), with a one-week interval between applications; and G2 (10 eyelids) which received single application of 1 ml of AH (Restylane® Lidocaine, Q med, Uppsala, Sweden). Demographic variables, complaints, ectropion degree, eyelid flaccidity degree, ectropion location, and quantitative assessments using the systematized photodocumentation of patients eyelids 30 days after the first application in G1 and 7 and 30 days after applications in G2 were studied. The images were transferred to a computer and evaluated using the Image J program and the distance between the lower eyelid margin to corneal reflex (DMR2), limbus margin distance (LM), internal angle (IA) and external angle (EA), total (TA), lateral (LA) and medial areas (MA) were analyzed. The area measurements were delimited by a line between the two commissures and along the lid margin of the lower eyelid. The evaluations were done without traction and with traction downward of the lower eyelid. All data were transferred to the Excel spreadsheet and comparative analysis was performed before and after the applications in the two groups. RESULTS The groups were similar according to age, sex, skin color and degree of ectropion. There was a significant improvement in G2 individuals after the application of AH. After injections G1 presented a significant reduction of AI with and without traction, EA with traction, LM without traction and AM with traction. The G2 after injections showed AI measurements without traction, EA, DMR2, TA and MA with and without traction, LM and LA with traction with significant alterations (p <0.05). The comparison of the G2 measurements obtained after 7 and 30 days showed stability of the results obtained with the AH injection. CONCLUSION: The application of AH in the lower eyelid of patients with cicatricial ectropion resulted in partial improvement of palpebral malposition. The application of SF was not effective in the treatment of cicatricial ectropion.

Made available in DSpace on 2015-05-14T12:59:53Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2558382 bytes, checksum: 1de43c15528cb3827484a1c0362e6f7f (MD5) Previous issue date: 2012-02-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Zizyphus joazeiro Mart. is a plant of the Rhamnaceae family, popularly known as "juazeiro" or "juá", and its leaves and bark are traditionally used by the population of northeastern Brazil as a febrifuge, antiinflammatory, in the treatment of stomach problems, skin diseases and as a hair and teeth cleaning. The wide use of this plant species for medicinal purposes by the population justifies pharmacologycal and toxicological studies to ensure the safety and efficacy of its use. In this sense, a study of its healing activity in experimentally induced excisional skin wounds and antiinflammatory action through the models of paw edema and peritonitis induced carrageenan and a toxicological preclinical acute and subchronic, lasting 21 days of the ethanol extract of stem bark of Z joazeiro after dermal application were proposed. In animal models assessed, Z joazeiro demonstrated positive activity on wound healing, improving their clinical signs. At doses of 35 and 175 mg/kg administered for 7 days, the paw edema and leukocyte migration into the peritoneal cavity after stimulation promoted by carrageenan were reduced. The results obtained through toxicological tests show that the extract is slightly irritating to skin at a dose of 1000 mg/kg, and is capable of inducing changes in water and food intake. When applied for 21 days at doses of 4, 8 and 16mg/kg, the plant caused flaking of the skin application site, changed the water and, food intake and body weight in males and females treated with the higher dose. Changes were found in creatinine levels in both sexes, triglycerides and platelet counts in males. Despite being considered a species with dermatocosmetic properties and low dermal toxicity, its continued use by the population should be done cautiously. / Zizyphus joazeiro Mart. é uma planta da família Rhamnaceae, popularmente conhecida como juazeiro ou juá , sendo suas folhas e cascas tradicionalmente empregadas pela população do nordeste do Brasil como febrífuga, antiiflamatória, no tratamento de problemas gástricos, doenças de pele e como agente de limpeza dos cabelos e dentes. A ampla utilização desta espécie vegetal pela população com fins medicinais justifica a investigação toxicológica farmacológica como segurança e eficácia para sua aplicação. Neste sentido foi proposto um estudo de sua atividade cicatrizante em feridas cutâneas excisionais experimentalmente induzidas tratadas com concentrações de 5, 10 e 20 mg/ml de extrato e ação antiinflamatória através dos modelos de edema de pata e peritonite induzida por carragenina, além de uma avaliação toxicológica pré-clínica aguda e subcrônica, com duração de 21 dias do extrato etanólico das cascas do caule de Z joazeiro nas doses de 4, 8 e 16 mg/kg após aplicação cutânea. Nos modelos animais avaliados, Z joazeiro demonstrou atividade positiva sobre a cicatrização de feridas com melhora de sinais clínicos. Nas doses de 35 e 175 mg/kg administradas durante 7 dias, reduziu o edema de pata e a migração de leucócitos para a cavidade peritoneal após estímulo promovido por carragenina. Os resultados obtidos através de ensaios toxicológicos, mostraram que o extrato é levemente irritante para a pele na dose de 1000 mg/kg e é capaz de induzir alterações na ingestão de água e alimentos. Quando aplicado durante 21 dias nas doses de 4, 8 e 16mg/kg, a planta causou descamação da pele no local de aplicação, alterou a ingestão de água, alimentos e peso corporal em machos e fêmeas tratados com a maior dose. Foram constatadas alterações nos níveis de creatinina em ambos os sexos, triglicerídeos e plaquetas em machos. Apesar de ter sido considerada uma espécie vegetal com propriedades dermatocosméticas e de baixa toxicidade por via dérmica, o uso tópico deve ser realizado com cautela.

Os modelos de reconstrução do microambiente são úteis para investigar as propriedades biológicas dos melanócitos humanos com a matriz e como plataforma para testes de novos fármacos. Existe uma demanda crescente para a utilização de pele e derme reconstruídas em laboratório, em ensaios in vitro de citotoxicidade, viabilidade celular, crescimento celular, irritabilidade e avaliação dos constituintes da matriz extracelular. Caracterizamos, em equivalente dérmico, alguns mecanismos de viabilidade e invasão de melanoma metastático humano quando na presença da matriz, a fim de ampliar o conhecimento a respeito de novas terapias contra o melanoma e entender os mecanismos que favorecem seu potencial invasivo, mimetizando com mais fidelidade o que ocorre in vivo. Através da validação deste modelo, constatamos que ele é essencial para resgatar a fisiopatologia do tumor, pois o melanoma, na presença de equivalente dérmico, torna-se capaz de evadir mecanismos de morte e aumentar a secreção de metaproteinases e citocinas que favorecerão a evolução tumoral. Avaliamos também as propriedades antineoplásicas do ácido clorogênico, que já foram relatadas em um grande número de tumores, porém os mecanismos que levam à sua ação antitumoral ainda não foram bem elucidados. Diante dos trabalhos enfatizando o efeito quimioprotetor dos polifenóis, referentes à sua ação antioxidante em células neoplásicas e com potencial metastático, não há na literatura estudos que comprovem a eficácia do ácido clorogênico sobre células de melanoma metastático humano. Portanto, este estudo visou recriar a estrutura dérmica in vitro e, a partir disso, comparar a resposta de fármacos em células de melanoma cultivada em equivalente dérmico, a fim de avaliar se há modulação diferencial nesse substrato, sugerindo, portanto, um protocolo mais eficaz para a prospecção de fármacos antimelanoma. / The microenvironment reconstruction models are useful for investigating the biological properties of human melanocytes onto the matrix and as platform for testing new drugs. There is a growing demand for the use of reconstructed skin and dermis in the laboratory for in vitro assays of cytotoxicity, viability, cell growth, irritability, and evaluation of the extracellular matrix. We characterize in dermal equivalent some mechanisms for cell viability and invasion of human metastatic melanoma in the presence of matrix, the order to increase knowledge about new therapies against melanoma and to understand the mechanisms that favor its invasive potential, to more closely mimic what occurs in vivo. By means of this model, we find that it is essential to rescue the tumor physiopathology as melanoma, in the presence of dermal equivalent, it is able to evade death mechanisms and increase the expression of metalloproteinases and cytokines which favor the tumor evolution. We also evaluated the antineoplastic properties of chlorogenic acid, that have been reported in a large number of tumors, but the mechanisms that lead to its antitumor action has not been well elucidated. Before the work emphasizing the chemoprotective effect of polyphenols related to its antioxidant action in neoplastic cells and with metastatic potential, there is no literature studies confirming the effectiveness of chlorogenic acid on human metastatic melanoma cells. Therefore, this study aims to rebuild the dermal structure in vitro, and from this, to compare the response to drugs in melanoma cells dermal equivalent cultured in order to evaluate whether modulation differential on the substrate, thus suggesting a protocol more effective for prospecting antimelanoma drugs.

Este trabalho de tese de doutorado baseia-se em um desenvolvimento científico amplamente multidisciplinar combinando-se protocolos, técnicas e ensaios experimentais da área de tecnologia farmacêutica, nanotecnologia, engenharia tecidual e fotobiologia. O trabalho foi realizado em duas etapas subsequentes. Primeiramente realizaram-se os estudos de desenvolvimento e caracterização de um sistema de veiculação de fármacos, o qual foi amplamente avaliado a partir de testes físico-químicos e ensaios de citotoxidade in vitro em cultura de fibroblastos humanos. Na sequencia realizaram-se estudos fotobiológicos combinando-se a aplicação de luz laser de baixa potência e fármaco fotossensível nanoestruturado com modelos teciduais, para avaliação da resposta biológica ao fotoestímulo. Para se mimetizar ao máximo as condições in vivo, construiu-se com sucesso um modelo tridimensional in vitro de pele humana, o qual se apresentou como uma ferramenta ideal para se avaliar a resposta fotobiológica após a indução de processos fotodinâmicos. Avaliou-se também o emprego dos equivalentes dermais como sistemas alternativos em testes de permeação e retenção cutânea in vitro. Os resultados obtidos foram promissores, auxiliando na compreensão dos mecanismos de interação dos processos fotodinâmicos com a matriz extracelular em combinação com a engenharia tecidual, mimetizando assim as condições in vivo da pele humana. Foi possível estabelecer também as bases para novos protocolos disponíveis para testes dermatológicos envolvendo produtos do segmento farmacêutico, cosmecêutico e áreas correlatas. / This work of doctorate thesis bases on a scientific development thoroughly multidiscipline combining protocols, techniques and experimental assays of the pharmaceutical technology, nanotechnology, tissue engineering and photobiology. The work was accomplished in three subsequent stages. Firstly were carried out studies and characterization of a drug delivery system with nanotechnology, which was evaluated thoroughly starting from physical-chemical tests and assays of cytotoxicity in human fibroblast culture. In the sequence were carried photobiological studies combining the application of low-intensity laser and nanosized photosensitizers with tissue models, for evaluation of the biological photostimulation. In mimetic conditions it was developed an in vitro three-dimensional human skin model, as tool to evaluate the photobiological response after the induction of the photodynamic processes and also as alternative for in vitro assays of drug permeation/retention. Results were promising, aiding in the understanding of interaction of extracellular matrix with photodynamic processes. Therefore, the work was based in combination of techniques with application in dermatology, pharmaceutical process, cosmetics and correlate areas.

Com o crescente aumento do mercado cosmético e com o consequente aumento da exposição da população às diversas moléculas que os compõem, se faz fundamental os cuidados no desenvolvimento e na avaliação da segurança de produtos cosméticos. Diferentes ferramentas estão disponíveis para compor a estratégia de avaliação de segurança, sendo uma delas a avaliação da retenção e permeação cutânea in vitro, técnica que não apenas fornece dados para a avaliação da eficácia de princípios ativos, como também permite um aprofundamento na avaliação toxicológica dos ingredientes e produtos cosméticos. Assim como cresce o interesse da população por produtos cosméticos, observa-se também um aumento considerável na procura por produtos contendo ingredientes botânicos, o que torna ainda mais desafiadora a avaliação de segurança. Sendo as isoflavonas de soja bastante consumidas na alimentação e, em alguns casos, aplicadas a cosméticos, e tendo elas propriedades dérmicas interessantes, este trabalho teve como objetivo estudar o perfil de retenção e permeação cutânea in vitro da daidzeína, umas das mais importantes isoflavonas já descritas e estudadas. Para tal, desenvolveu-se uma nanoemulsão cosmética e aplicou-se um extrato botânico de soja rico em daidzeína. Desenvolveu-se e validou-se um método analítico por cromatografia líquida acoplada a detector de arranjo de diodos, e ainda procedeu-se a caracterização da nanoemulsão, a avaliação teórica de segurança de todos os seus ingredientes, além dos estudos de retenção e permeação cutânea in vitro, foco central deste trabalho. Como resultados, teve-se que a técnica de emulsificação à quente seguida de sonicação adotada para o preparo da nanoemulsão foi eficiente, possibilitando a obtenção de uma nanoemulsão pouco polidispersa e com tamanho de partícula médio abaixo de 200 nm. A retenção e permeação cutânea foram dependentes do tempo de aplicação, sendo que a nanoemulsão proporcionou uma maior retenção e menor permeação cutâneas da daidzeína comparado com solução controle no tempo de 6 h. A nanoemulsão desenvolvida mostrou-se adequada para a liberação tópica da daidzeína presente em extrato vegetal. Os testes in vitro de retenção e permeação cutânea são cruciais para esta etapa de desenvolvimento das formulações, quando os aspectos liberação tópica e possível toxicidade sistêmica são avaliados. / With the continuous growth of the cosmetic market, and so the increase in the exposure of the population to the different molecules present on these products, the care in the development and the safety assessment of cosmetics plays an important role. Different tools are available to compose the safety assessment strategy, one of which is the dermal absorption in vitro assay. This technique does not only give information regarding the efficacy of active ingredients, but also allow a deep toxicological evaluation of cosmetic ingredients and products. As the interest of the population on cosmetic products grows, also grows people\'s search for products containing botanical ingredients, fact that turns the safety assessment more challenger. As the soy isoflavones are part of the diet and sometimes used in cosmetic products, and as they present interesting dermal properties, this work aimed to study the profile of skin retention and dermal absorption of the daidzein, one of the most important isoflavones ever described and studied. For that, a cosmetic nanoemulsion was developed, in which a soy botanical extract rich in daidzein was added. An analytical method was developed and validated, based in liquid chromatography coupled with a diode array detector. Besides the in vitro studies of skin retention and dermal absorption, main focus of this project, the characterization of the nanoemulsion and the theoretical safety assessment of all of its ingredients were also performed. Regarding the results, the technique of hot emulsification followed by sonication used for the preparation of the nanoemulsion could be considered efficient based on the fact that it was less polidisperse and with a medium particle size below 200 nm. The skin retention and permeation were dependent on the time of application, and, it was observed that the nanoemulsion has led to greater retention and lesser skin permeation of daidzein incorporated into the nanoemulsion when compared to the control solution after 6 h of application. The nanoemulsion developed showed to be adequate to topical delivery of daidzein that is present in plant extract. In vitro skin retention and skin permeation are critical for this stage of development of the formulations, considering that aspects of topical delivery and systemic toxicity are evaluated.

A classe Chondrichthyes é representada por animais de esqueleto cartilaginoso e calcificação prismática. Eles são divididos em duas subclasses, Elasmobranchii (tubarões e raias) e Holocephali (quimeras), que têm uma história evolutiva de mais de 400 milhões de anos. Devido à grande adaptabilidade destes animais em diferentes habitats, eles desenvolveram sofisticadas habilidades sensoriais, como os sistemas mecanorreceptor da linha lateral e eletrossensorial. A maior ordem de tubarões, a Carcharhiniformes tem a maioria dos representantes ocorrendo no Brasil. Entre eles, destacamos o tubarão-azul (Prionace glauca), pelágico oceânico, espécie de grande porte e cosmopolita, e um pequeno tubarão pelágico costeiro, o tubarão-frango (Rhizoprionodon lalandii), representantes dessa ordem com grande importância econômica pela sua pesca. Dada a diversidade morfológica e funcional dos sistemas sensoriais, em resposta a variações ecológicas e filogenéticas, o objetivo deste estudo foi descrever as estruturas morfológicas da linha lateral e das ampolas de Lorenzini, os dentículos dérmicos e os nervos associados à elas, em P. glauca e R. lalandii, utilizando documentação textual e fotográfica de estruturas macroscópicas, e métodos de microscopia, como microscopias de luz, eletrônica e imunofluorescência. Histologicamente, o neuromasto, principal unidade celular da linha lateral mostrou-se semelhante em ambas as espécies, e em P. glauca, esta célula foi encontrada desde o período intra-uterino. Em P. glauca, foi relatado um novo tipo de canal sensorial associado à linha lateral. A ampola de Lorenzini de R. lalandii revelou-se mais organizada do que em P. glauca, devido à quantidade e estruturação dos alvéolos. Os dentículos dérmicos de R. lalandii são mais especializados em várias regiões, particularmente as que envolvem os poros sensoriais. Em relação às características microscópicas dos nervos, eles se assemelham estruturalmente aos dos vertebrados superiores. Para a imunofluorescência, a colocalização foi realizada por tripla marcação dos núcleos dos nervos (DAPI), com receptor de membrana (P2X7), fibras inibidoras (NOS) e células gliais (GFAP), nos nervos da linha lateral e oftálmico superficial. Em ambas as espécies, a distribuição dos nervos foi uniforme. Em suma, as características estudadas em P. glauca são muito semelhantes às encontradas em R. lalandii, no entanto, devido ao hábito da vida oceânica, outras estratégias sensoriais seriam mais relevantes para a sua sobrevivência neste ambiente. As características microscópicas de R. lalandii demonstraram a relação adaptativa com a ecologia da espécie. Seu estilo de vida costeiro demonstrou maior especialização em certos sistemas sensoriais, contribuindo para melhor adaptação em determinados ambientes, uma vez que a morfologia e a organização dos sistemas sensoriais estão diretamente ligadas ao estilo de vida dos indivíduos. / The class of Chondrichthyes is represented by animals with cartilaginous skeleton with prismatic calcification. They are divided into two subclasses, Elasmobranchii (sharks, skates and rays) and Holocephali (chimaeras), which have an evolutionary history of more than 400 million years. Due to their great adaptability in different habitats, they have developed sophisticated sensorial abilities, such as mechanosensory lateral line and electrosensory systems. The largest order of sharks, the Carcharhiniformes has most of the representatives in Brazil. Among them, we highlight the blue shark (Prionace glauca), oceanic pelagic, large and cosmopolitan specie, and a small coastal pelagic shark, the sharpnose shark (Rhizoprionodon lalandii), species with economical importance. Given the morphological and functional diversity of sensory systems, in response to ecological and phylogenetic variations, the aims of this study is to describe the morphological structures of the lateral line and ampullae of Lorenzini, the dermal denticles and nerves associated with them, in P. glauca and R. lalandii, using textual and photographic documentation of macroscopic structures, and microscopic methods, such as light and electronic microscopies and immunofluorescence. Histologically, the neuromast, main cell unit of lateral line system was similar in both species, and in P. glauca, this cell was found since the intrauterine period. In P. glauca, a new type of sensorial canal associated with the lateral line was reported. The ampullae of Lorenzini of R. lalandii proved to be more organized than in P. glauca, because of the amount and structure of the alveoli. The dermal denticles in R. lalandii were more specialized in various regions, particularly those involving sensory pores. Regarding the microscopic characteristics of the nerves, they resemble structurally to the superior vertebrates. For immunofluorescence, the colocalization was performed by triple labeling the nuclei (DAPI) of the nerves, with a membrane receptor (P2X7), inhibitory fibers (NOS) and glial cells (GFAP) in lateral line and superficial ophthalmic nerves. In both species, the distribution of nerves was uniform. In short, the characteristics studied in P. glauca are very similar to those found in R. lalandii, however, due to the habit of oceanic life, other sensory strategies are most relevant to their survival in this environment. Microscopic features of R. lalandii demonstrated the adaptive relationship with the ecology of the species. Its coastal way of life showed greater specialization in certain sensory and attachments systems, contributing to better adapt in certain environments, since the morphology and organization of sensory systems are directly linked to the lifestyle of individuals.

The discovery of biomarkers for Parkinson's disease (PD) is challenging due to the heterogeneous nature of this disorder, and a poor correlation between the underlying pathology and the clinically expressed phenotype. An ideal biomarker would inform on PD-relevant pathological changes via an easily assayed biological characteristic, which reliably tracks clinical symptoms. Human dermal (skin) fibroblasts are accessible peripheral cells that constitute a patient-specific system, which potentially recapitulates the PD chronological and epigenetic aging history. Here, we compared primary skin fibroblasts obtained from individuals diagnosed with late-onset sporadic PD, and healthy age-matched controls. These fibroblasts were studied from fundamental viewpoints of growth and morphology, as well as redox, mitochondrial, and autophagic function. It was observed that fibroblasts from PD subjects had higher growth rates, and appeared distinctly different in terms of morphology and spatial organization in culture, compared to control cells. It was also found that the PD fibroblasts exhibited significantly compromised mitochondrial structure and function when assessed via morphological and oxidative phosphorylation assays. Additionally, a striking increase in baseline macroautophagy levels was seen in cells from PD subjects. Exposure of the skin fibroblasts to physiologically relevant stress, specifically ultraviolet irradiation (UVA), further exaggerated the autophagic dysfunction in the PD cells. Moreover, the PD fibroblasts accumulated higher levels of reactive oxygen species (ROS) coupled with lower cell viability upon UVA treatment. In essence, these studies highlight primary skin fibroblasts as a patient-relevant model that captures fundamental PD molecular mechanisms, and supports their potential utility to develop diagnostic and prognostic biomarkers for the disease.

Recent occupational health studies have focused on dermal exposure at the hands, but have been unable to accurately express dose without knowing the HSA. There is no standard method to calculate HSA, though some researchers have derived HSA formulas based on dimensions from a Taiwanese population. This research paper describes a shortcut method to estimate the hand surface area (HSA) of a human hand from a two-dimensional hand tracing, and repeated a Taiwanese HSA study in order to explore the viability of its HSA formula in an American university population. A sample of nine adult men and nine adult women, each representing one third of the population percentile in hand length and hand breadth, were selected from a population within the University of South Florida in Tampa, FL. Hand length, breadth, a 2D hand tracing and a 3D light hand scan were collected from each participant. A linear regression was used to analyze the data sets and found a correlation (R=0.94) between 2D HSA and 3D HSA and slope of 2.6 (SD=0.2), with a regression equation of Y=2.6(X). A paired t-test was used to compare the Taiwanese HSA formula data against the 3D HSA. Results found that the Taiwanese data sets were significantly different from the 3D HSA (p<0.001), averaging 57 cm2 less than the 3D HSA. A jackknife analysis was implemented on the 2D HSA hand tracing data, and a paired t-test was performed between the jackknife estimate predictions and 3D HSA. Mean differences were not significantly different (p=0.97), with 0.87 cm2 difference between means. Results indicate that the USF Hand Tracing Method will provide a better estimate of HSA than the Taiwanese method, and can be used as a tool in HSA estimation.

The limited reparative capacity of the skin and the inadequacy of conventional treatments have necessitated the development of tissue engineered skin substitutes. Several substitutes, including Integra Dermal Regeneration Template, are finding increasingly widespread application in the treatment of acute and chronic wounds. To date, these substitutes are unable to fully recreate the functionality and aesthetics of skin prior to injury. This thesis applied an integrated approach combining solution preparation, material fabrication control and biological testing to investigate electrospun silk fibroin (SF) nano-microfibrous scaffolds as potential biomimetic skin substitutes. Further to this, the improvement of the existing Integra scaffold through the incorporation of hyaluronan (HA) was assessed. Through rheological analysis of regenerated SF solutions under shear and extensional deformation a concentration regime transition at 20 wt% SF was identified. Solutions with relaxation times under 0.001 seconds were found to be unsuitable for electrospinning. The incorporation of poly(ethylene oxide) (PEO) was found to significantly increase solution relaxation times and extensional viscosity, making them much more suitable for electrospinning. Solution viscoelastic properties were shown to directly influence electrospun fibre morphology, with increases in viscosity resulting in increases in fibre diameter under stable spinning conditions. The effects of electrospinning parameters on electrospun fibre morphologies were investigated using SF-PEO blended solutions. Increased electrical field, spinneret height and decreased flow rate were found to decrease fibre diameter. In vitro assessment of the attachment, spreading, proliferation, viability and gene expression of primary human dermal fibroblasts (PHDFs) and bone marrow-derived mesenchymal stem cells (BM-MSCs) was conducted. Both PHDFs and BM-MSCs attached and proliferated with greater rapidity on fibres of the smallest diameters (~250-300 nm) with proliferation decreasing as fibre size increased until fibre diameters reached ~1200 nm. Cells were observed to be spread, with multiple attachments between fibres in scaffolds composed of ~250-300 nm diameter fibres. Cells aligned themselves to single fibres in scaffolds composed of fibres greater than 1 micrometre. HA supplementation to Integra resulted in increased proliferation, viability and migration of PHDFs. In ex vivo cutaneous wound healing models, the invasion of Integra was enhanced when scaffolds were supplemented with HA, with increased matrix deposition observed. Optimal supplementation concentrations for in vitro and ex vivo increases in cell proliferation and migration were at 1.5 – 2 mg ml-1 HA. SF electrospun scaffolds facilitated epithelial migration in ex vivo artificial wounds, with the migratory epidermis more closely resembling the structures observed in vivo. Additional preliminary investigations into the efficacy of a paste-form of Integra, Integra Flowable Wound Matrix (IFWM) were performed ex vivo, with cell invasion comparable to the conventional scaffold format. The potential for the incorporation of viable PHDFs and BM-MSCs was also investigated and keratinocyte migration was enhanced in these scaffolds. The results in this thesis provide valuable optimisation information on the development of SF electrospun scaffolds for skin engineering. Additionally, the supplementation of Integra with HA may provide a simple and effective way to enhance the performance of the scaffold in vivo.