Orientadores: Ricardo Della Coletta, Tuula Anneli Salo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-28T03:47:30Z (GMT). No. of bitstreams: 1 Rodrigues_PriscilaCampioni_D.pdf: 16816198 bytes, checksum: b4b1451b61822bf0cb3a2b1af00fb915 (MD5) Previous issue date: 2015 / Resumo: Fascina e plectina são proteínas estruturais e funcionais do citoesqueleto com atividades envolvidas na modulação da motilidade celular. Embora a expressão alterada destas proteínas tenha sido descrita em alguns tipos de câncer, sendo consideradas como possíveis biomarcadores tumorais, estudos avaliando a contribuição de fascina e plectina no carcinoma espinocelular (CEC) oral são escassos. Em estudo prévio, nós demonstramos que os níveis de fascina e plectina foram significantemente maiores no CEC oral em comparação com tecido oral normal. O objetivo deste estudo foi compreender o papel de fascina e plectina na tumorigênese oral. Visando esse objetivo, 1) nós inicialmente confirmamos a superexpressão de fascina e plectina em linhagens celulares e amostras tumorais em comparação à tecidos normais por qRT-PCR e imuno-histoquímica, 2) analisamos a expressão imuno-histoquímica de fascina e plectina em amostras orais que caracterizam a evolução histopatológica do CEC oral, 3) realizamos a análise da distribuição imuno-histoquímica dessas proteínas em 113 amostras de CEC oral e correlacionamos com as características clínico-patológicas, de recorrência e sobrevida dos pacientes e 4) neutralizamos estavelmente a expressão da fascina nas linhagens celulares derivadas de CEC oral SCC-15 e HSC-3 e realizamos ensaios de adesão, migração e invasão celular. Nossos resultados confirmaram que a expressão de fascina e plectina são significantemente maiores no CEC oral em comparação com a mucosa oral normal (fascina p<0,0001; plectina p<0,0001). Em linhagens celulares, as expressões de fascina e plectina foram também significantemente maiores em células de CEC oral em comparação a linhagem de queratinócitos não-tumorigênicos (fascina p<0,0001 para SCC-4, SCC-9, SCC-25, SCC-9 ZsGreen LN-1, SCC-9 e HSC-3 e p<0,001 para ZsGreen LN-2 e plectina p<0,0001 para SCC-4, SCC-15, SCC-25, SCC-9 ZsGreen LN-1, SCC-9 ZsGreen LN-2 e p<0,001 para SCC-9 e HSC3). Quanto à progressão do CEC oral, fascina demonstrou uma expressão significantemente maior nas amostras de displasia e CEC quando comparada com o grupo das hiperplasias fibrosas com epitélio normal (p<0,0001), enquanto que a expressão de plectina foi significantemente maior nos CECs em comparação as hiperplasias fibrosas e displasias (p<0,0001), mas não entre as hiperplasias fibrosas e displasias. A superexpressão de fascina foi associada com um pior prognóstico, como revelado por uma menor sobrevida global (p=0,0005) e sobrevida específica (p=0,001). A neutralização de fascina nas linhagens derivadas de CEC oral foi capaz de aumentar significantemente a adesão à fibronectina (HSC-3 p<0,0001 e SCC-15 p<0,0001), diminuir a migração (HSC-3 p<0,0001 e SCC-15 p p<0,0001) e a invasão celular em ambas as linhagens(HSC-3 p<0,0001 e SCC-15 p p<0,0001) quando comparadas aos controles. Em conclusão, os resultados deste estudo sugerem que a proteína fascina é um marcador prognóstico independente para sobrevida global e sobrevida específica da doença em pacientes com CEC oral e revelam que fascina é capaz de regular eventos importantes para o desenvolvimento e progressão do CEC oral. Embora a expressão de plectina tenha sido maior em CEC oral comparado com mucosa oral normal, nenhuma correlação com sobrevida foi observada / Abstract: Fascin and plectin are structural and functional proteins of the cytoskeleton with activities involved in cell motility. Although the expression of these proteins has been described in some cancers, being considered as potential tumor biomarkers, studies evaluating the contribution of fascin and plectin in oral squamous cell carcinoma (OSCC) are limitted. In a previous study, we demonstrated that the levels of fascin and plectin are significantly higher in OSCCs compared to normal oral tissues. The aim of this study was to characterize the role of fascin and plectin in oral tumorigenesis. To achive this purpose: 1) we initially confirmed the overexpression of fascin and plectin in cell lines and oral samples of normal and tumor tissues by qRT-PCR and immunohistochemistry, 2) we analyzed the immunohistochemical expression of fascin and plectin in oral samples featuring the histopathological evolution of OSCC 3) we performed the analysis of those proteins in 113 OSCCs and correlated with the clinical and pathological features, recurrence and survival of patients, and 4) we stably neutralized the expression of fascin in OSCC cell lines SCC-15 and HSC-3 and performed adhesion, cell migration and invasion assays. Our results confirmed that the expressions of fascin and plectin are significantly higher in OSCC compared to normal oral mucosa. In cell lines, fascin and plectin expression was also significantly higher in OSCC cells compared with non-tumorigenic keratinocytes. Regarding OSCC progression, fascin showed a significantly higher expression in dysplasia and OSCC samples compared to fibrous hyperplasia containing normal oral epithelium, whereas plectin immunoexpresion was higher in OSCCs compared to fibrous hyperplasia and samples with dysplasia but not between fibrous hyperplasia and samples with dysplasia. The overexpression of fascin was associated with worse prognosis, as revealed by shortened overall survival and disease-specific survival. Fascin silencing in cell lines derived from OSCC increased significantly the adhesion to fibronectin and decreased cell migration and invasion in both cell lines when compared to controls. In conclusion, the results of this study suggest that fascin is an independent prognostic marker for overall survival and disease-specific survival in patients with OSCC and reveal that fascin regulate important events associated with OSCC development and progression. Although plectin expression was higher in OSCCs compared to normal oral mucosa, no correlation with survival was observed / Doutorado / Patologia / Doutor em Estomatopatologia

The muscle fibre cytoskeleton of skeletal and heart muscle cells is composed mainly of intermediate filaments (IFs), that surround the myofibrils and connect the peripheral myofibrils with the sarcolemma and the nuclear membrane. Desmin is the first muscle specific IF protein to be produced in developing muscles and is the main IF protein in mature muscles. In skeletal muscle, desmin is particularly abundant at myotendinous and neuromuscular junctions. In the heart an increased amount of desmin is found at intercalated discs and in Purkinje fibres of the conduction system. Interactions between the IFs themselves, and between IFs and other structures such as Z-discs and the sarcolemma, are mediated by intermediate filament associated proteins (IFAPs). A transgenic mice model, which lacks the desmin gene have been developed to study the function of desmin. In these mice, morphological abnormalities are observed in both heart and skeletal muscles. Similar defects have been observed in human myopathies, caused by different mutations in the desmin gene. In the present thesis, skeletal and heart muscles of both wild type and desmin knock-out (K/O) mice have been investigated. Furthermore the cytoskeletal organisation in skeletal muscles from human controls and from a patient with desmin myopathy was examined. In the desmin K/O mice, no morphological alterations were observed during embryogenesis. These mice postnatally developed a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. Ruptures of the sarcolemma appear to be the primary event leading to muscle degeneration and fibrosis both in cardiac and affected skeletal muscles. In the heart the muscle degeneration gave rise to calcifications, whereas in skeletal muscles regeneration of affected muscle was seen. In mature wild type mice, the IF proteins synemin and paranemin, and the IFAP plectin were present together with desmin at the myofibrillar Z-discs, the sarcolemma, the neuromuscular junctions and the myotendinous junctions. Nestin was only found in these junctional regions. In desmin K/O mice, all four proteins were detected at neuromuscular and myotendinous junctions. The normal network of synemin and paranemin were not observed, whereas the distribution of plectin was preserved. In normal human muscles, synemin, paranemin, plectin and αB-crystallin were colocalised with desmin in between the myofibrils, at the sarcolemma and at myotendinous and neuromuscular junctions. In the human desmin myopathy, the distribution of desmin varied considerably. A normal pattern was seen in some fibres areas, whereas other regions either contained large subsarcolemmal and intermyofibrillar accumulations of desmin or totally lacked desmin. Nestin, synemin, paranemin, plectin and αB-crystallin also exhibited an abnormal distribution. They were often aggregated in the areas that contained accumulations of desmin. In cultured satellite cells from the patient, a normal network of desmin was present in early passages, whereas aggragates of desmin occurred upon further culturing. In the latter, also the nestin network was disrupted, whereas vimentin showed a normal pattern. αB-crystallin was only present in cells with a disrupted desmin network. Plectin was present in a subset of cells, irrespective of whether desmin was aggregated or showed a normal network. From the present study it can be concluded that an intact desmin network is needed to maintain the integrity of muscle fibres. Desmin may be an important component in the assembly of proteins, which connect the extrasarcomeric cytoskeleton with the extracellular matrix.

No description available.