The need for anabolic therapies to increase bone formation in difficult orthopaedic circumstances and to treat osteoporosis is an area of intense research focus. There is a current interest in the Wnt signalling pathway as a target for such treatment, with accumulating evidence for a role of this pathway in bone formation. Ectoderm Neural Cortex 1 (ENC1) is a Wnt target gene, not previously studied in bone, which was observed in our laboratory to be up-regulated in an anabolic surgical model of bone formation. The involvement of ENC1 in the differentiation of neuronal and adipocytic cells has previously been reported; therefore, this thesis investigates the expression of ENC1 in cells of the bone and the role of ENC1 during osteoblast differentiation. ENC1 transcript expression was localised to osteoblastic, chondrocytic and osteocytic cells in sections of healing fracture callus and normal mouse bone by in situ hybridisation. The expression of ENC1 was confirmed in differentiating primary osteoblasts and in osteoblastic and osteosarcoma cell lines by quantitative real time PCR and western blotting. ENC1 exists as two protein isoforms of 67 and 57kD in size, which are translated from alternatively spliced ENC1 transcripts. Both isoforms of the protein were detected in differentiating cultures of the pre-osteoblast cell line MC3T3-E1. To address the function of ENC1 in osteoblast differentiation, shRNA knockdown of the endogenous transcript was undertaken in MG63 osteosarcoma cells and in the MC3T3-E1 pre-osteoblastic differentiation model. Stable expression of shRNA targeted to both ENC1 spliceforms resulted in reduced accumulation of alkaline phosphatase positive nodules and alkaline phosphatase transcripts in MG63 cell culture. This reduction was not seen with targeted knockdown of 67kD ENC1 alone. Stable tetracycline-inducible shRNA knockdown targeted to both 57 and 67kD ENC1 isoforms in MC3T3-E1 cells resulted in a significant reduction of Alizarin Red S stained mineralised nodules. When expression of 67kD ENC1 alone was reduced, however, a significant increase in MC3T3-E1 nodule formation was observed. This knockdown had no effect on the expression of early genes involved in osteoblast differentiation Runx2 and osterix, but changes in expression of alkaline phosphatase and osteocalcin mRNA mirrored nodule formation. ENC1 is a member of the BTB-Kelch family of proteins. Some members of this family have recently been found to act as substrate adaptors for the E3 ubiquitin ligase, binding to the cullin 3 component of the complex. These adaptor proteins function to bring a substrate protein within the vicinity of the E2 ubiquitin-conjugating enzyme, thus targeting it for ubiquitination and subsequent proteasomal degradation. The ability of ENC1 to interact with cullin 3 was investigated as a possible mechanism by which it may affect a role in osteoblast differentiation. Full length ENC1 showed robust binding to cullin 3 and weak binding was seen between the N-terminally truncated 57kD isoform and cullin 3. ENC1, therefore, may act as a substrate adaptor protein for the cullin 3 based E3 ubiquitin ligase. These data present ENC1 as a novel candidate protein involved in osteoblast differentiation, and suggest the possible involvement of this protein in proteasomal degradation of a substrate involved in osteoblast differentiation. The ENC1 isoforms and the associated functional pathways thus are possible future therapeutic targets to treat bone loss and enhance or accelerate fracture healing.

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.