Role of Ectodermal-neural cortex 1 protein in human glioma progression, identification of a peptide internalized by human glioblastoma cells and development of an alternative method to generate growth curves of adherent cultures / Papel da proteína Ectodermal-neural cortex 1 (ENC1) na progressão de glioma humano, identificação de um peptídeo internalizado por células de glioblastoma humano e desenvolvimento de um método alternativo para gerar curvas de crescimento celular

Gliomas are the most common form of primary intracranial malignancy, among which astrocytomas are the most frequent. Ectodermal-cortex protein 1 (ENC 1), also known as Nuclear Restricted Protein/Brain (NRP/B), was first characterized as a protein which interacts with the cytoskeleton by binding to actin through Kelch-like domains, being related to neural fate specification during development of the nervous system. The first chapter of this thesis confirms ENC1 as a tumor suppression properties by a genomic edition approach, analyses ENC1 expression in a set of patient glioma samples and describes the correlation these data with patients survival and progression-free survival, concluding that ENC1 expression may constitute a biomarker for glioma aggressiveness. The second chapter refers to the identification and in vitro characterization of the LHTNELQ peptide, which was selected by the Phage Display method using human glioblastoma cells. This new peptide is able to be internalized by these cells and features as a new tool for the development of glioma therapeutics. The third chapter report an alternative method to generate growth curves of adherent cell cultures, which is based on the CFSE fluorescence decay over time. It is an alternative method to determine growth curves of cultured cells, with smaller variation among technical replicates than that of counting-based methods

Gliomas são a forma mais comum de malignidades primárias intracranianas, dentre os quais os astrocitomas são os mais frequentes. A proteína Ectodermal-neural cortex 1 (ENC1), também conhecida como Nuclear Restricted Protein/Brain (NRP/B), foi primeiramente caracterizada como uma proteína que interage com o citoesqueleto por meio de ligação à actina através de domínios Kelch-like, sendo relacionada com diferenciação neuronal durante o desenvolvimento do sistema nervoso. O primeiro capítulo desta tese descreve confirmação da capacidade supressora tumoral de ENC1 por abordagem de edição genômica, analisa a expressão de ENC1 em um conjunto de amostras de pacientes com gliomas e correlaciona esses dados com tempo de sobrevida geral e sobrevida livre de progressão tumoral nos pacientes, concluindo que a expressão de ENC1 pode ser utilizada como um biomarcador da agressividade do glioma. O segundo capítulo apresenta a identificação e caracterização in vitro do peptídeo LHTNELQ, que foi selecionado pela metodologia de Phage display utilizandose de células de glioblastoma humano. Este novo peptídeo é capaz de internalizar-se nestas células e figura como uma nova ferramenta para o desenvolvimento de estratégias terapêuticas para glioblastomas. No terceiro capítulo propõe-se um método alternativo para gerar curvas de crescimento celular de cultura aderente, o qual é baseado no decaimento da fluorescência do reagente CFSE ao longo do tempo. Tratase de um método alternativo para a determinação de curvas de crescimento de culturas aderentes, com menor variação entre as réplicas técnicas do que os métodos baseados em contagem das células

Effects on HepG2 cells growth of the different domains of genotype 1b hepatitis C virus core proteins / 中华微生物学和免疫学杂志

Objective To study the function of core protein (CORE) of genotype 1b hepatitis C virus (HCV) of different strains (T: derived from tumor tissues; NT: derived from non-tumor tissues; C191: HCV-J6) and different domains (1-172, 1-126, 1-58, 59-126, 127-172 AA) of T CORE in the pathogenesis of HCV infection and to find the therapy target. Methods Different truncated genotype 1b HCV CORE eukaryotic expression plasmids (T, NT, C191) and different domains of T CORE were constructed and transfected to HepG2 cells. Cell apoptosis and necrosis were quantified by flow cytometry. Cell growth curves were observed with real time cell growth instrument. Results COREs from different strains of genotype 1b and different domains of CORE induced cell apoptosis and necrosis, and inhibited HepG2 cell growth at different levels. CORE derived from T induced apoptosis and necrosis and inhibited cell growth higher than that derived NT and C191. N terminal 1-58 AA of CORE derived from T induced cell apoptosis and necrosis and inhibited cell growth higher than any other domains. Conclusion COREs from different strains of genotype 1b HCV and different domains of CORE from the same HCV strain play different roles in their molecular pathogenesis of HCV. Among different domains of CORE, N terminal 1-58 AA might play an important role in its pathogenesis and be one target of gene therapy.