The von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is transmitted in an autosomal dominant manner. The disease is characterized by the formation of highly angiogenic tumors in many organs but the main causes of mortality are renal cell carcinomas and hemangioblastomas. Mutations in the VHL protein are responsible for the pathogenesis of the disease. VHL associates with elongin Band C to form the VBC complex. The cullin 2 protein (CUL2) and ring box protein 1 (RBX1) also associate with the VBC complex to form an E3 ubiquitin ligase involved in the ubiquitination and subsequent degradation of the hypoxia inducible transcription factor (HIF2alpha). Mutations in VHL that abrogate its E3 ligase activity lead to increased levels ofHIF2alpha and the subsequent accumulation of pro-proliferative and pro-angiogenic HIF2alpha target genes. VHL also has an important function in the regulation of extracellular matrix (ECM) assembly which is independent of its HIF2alpha regulation pathway. VHL's regulation of ECM assembly was shown to have important consequences for tumor angiogenesis and cell invasion. It was shown to be necessary for the proper assembly of a fibronectin matrix and was most recently found to interact with collagen IV alpha 2 (COL4A2). The aim of this thesis is to further characterize the VHL-COL4A2 interaction. VHL was shown to interact directly and specifically to COL4A2 and is necessary for proper COL4A2 matrix assembly. The association of VHL with COL4A2 appears to be independent of its functions as an E3 ubiquitn ligase and CUL2 was identified as part of the VBC complex that associates with collagen IV (COL4). Furthermore, a strategy to identify the binding site of VHL on COL4A2 has been employed and is in progress. These experiments represent the beginning of investigations into the novel interaction between VHL and COL4A2.

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Este trabalho investigou, pela primeira vez, a influência do CH na modulação do metabolismo e proliferação de fibroblastos dérmicos humanos (FDHs) derivados de áreas fotoprotegida e fotoexposta, cultivados em modelo de monocamada. Além disto, foram investigados os efeitos da suplementação com CH na secreção de colágeno tipo I, em modelo de cultura 3D de equivalente dérmico, derivado de matriz produzida exclusivamente por FDHs. O tratamento com CH não influenciou a proliferação celular dos fibroblastos derivados de ambas as áreas, porém modulou expressivamente o metabolismo dos FDHs cultivados em monocamada, elevando o conteúdo de pró-colágeno I e colágeno I e diminuindo a atividade de metaloproteinases de matriz (MMP) 1 e 2. Concentrações menores de CH foram suficientes para estimular as células de área fotoexposta, sugerindo efeitos mais pronunciados do CH nestas células. Este estudo é uma contribuição importante para compreensão dos efeitos biológicos do CH nas células da pele e viabilidade do seu uso como ingrediente funcional de suplementos alimentares. / This study investigated, for the first time, the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from sun-protected and sun-exposed body sites, cultured in monolayer in vitro model. Moreover, CH effects on the secretion of type I collagen were investigated in dermal equivalent 3D model derived from dermal matrix produced exclusively by HDFs. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of procollagen I and collagen I and decreasing metalloproteinase activity (MMP) 1 and 2. These effects were confirmed in the human dermal equivalent model. Lower concentrations of CH were enough to stimulate sun-exposed-derived HDFs, suggesting more pronounced effect in these cells. This study presents an important contribution to understanding the biological effects of CH in skin cells and viability of its use as a functional ingredient in food supplements.

Este trabalho investigou, pela primeira vez, a influência do CH na modulação do metabolismo e proliferação de fibroblastos dérmicos humanos (FDHs) derivados de áreas fotoprotegida e fotoexposta, cultivados em modelo de monocamada. Além disto, foram investigados os efeitos da suplementação com CH na secreção de colágeno tipo I, em modelo de cultura 3D de equivalente dérmico, derivado de matriz produzida exclusivamente por FDHs. O tratamento com CH não influenciou a proliferação celular dos fibroblastos derivados de ambas as áreas, porém modulou expressivamente o metabolismo dos FDHs cultivados em monocamada, elevando o conteúdo de pró-colágeno I e colágeno I e diminuindo a atividade de metaloproteinases de matriz (MMP) 1 e 2. Concentrações menores de CH foram suficientes para estimular as células de área fotoexposta, sugerindo efeitos mais pronunciados do CH nestas células. Este estudo é uma contribuição importante para compreensão dos efeitos biológicos do CH nas células da pele e viabilidade do seu uso como ingrediente funcional de suplementos alimentares. / This study investigated, for the first time, the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from sun-protected and sun-exposed body sites, cultured in monolayer in vitro model. Moreover, CH effects on the secretion of type I collagen were investigated in dermal equivalent 3D model derived from dermal matrix produced exclusively by HDFs. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of procollagen I and collagen I and decreasing metalloproteinase activity (MMP) 1 and 2. These effects were confirmed in the human dermal equivalent model. Lower concentrations of CH were enough to stimulate sun-exposed-derived HDFs, suggesting more pronounced effect in these cells. This study presents an important contribution to understanding the biological effects of CH in skin cells and viability of its use as a functional ingredient in food supplements.