Chemotherapy is often administered once every three weeks to allow repopulation of essential normal tissues such as the bone marrow. Repopulation of surviving tumour cells can also occur between courses of chemotherapy and can decrease the efficacy of anticancer treatment. This thesis aims to characterize repopulation, to study the effect of targeted cytostatic agents to inhibit repopulation, and to determine the optimal scheduling of chemotherapy and molecular targeted treatment. The distribution of proliferating and apoptotic cells in human squamous cell carcinoma (A431) xenografts was studied following chemotherapy using fluorescence immunohistochemistry. There was an initial decrease in cell proliferation and in the total functional blood vessels, and an increase in apoptosis observed following treatment with paclitaxel chemotherapy. A rebound in cell proliferation occurred approximately 12 days following treatment, which corresponded with a rebound in vascular perfusion. The effect of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, to inhibit repopulation between courses of chemotherapy was determined using EGFR-overexpressing A431 cells and xenografts. Furthermore, concurrent and sequential schedules of combined chemotherapy and molecular targeted treatment were compared. Gefitinib inhibited the repopulation of A431 cells in culture when administered sequentially between chemotherapy; sequential treatment was more efficacious than concurrent treatment probably because concomitant scheduling rendered quiescent cells less responsive to chemotherapy. However, in vivo studies using chemotherapy in combination with gefitinib or temsirolimus, a mammalian target of rapamycin (mTOR) inhibitor, showed that concurrent scheduling of combined treatment was more effective at delaying regrowth of xenografts than sequential treatment; this was likely due to dominant effects on the tumour microenvironment. The work completed in this thesis has shown that repopulation occurs in A431 xenografts following paclitaxel treatment, and these changes are associated with changes in the tumour vasculature. Repopulation of A431 cells was inhibited by gefitinib administered sequentially with paclitaxel. However, studies in mice showed better inhibitory effects when chemotherapy was given concomitantly with cytostatic agents such as gefitinib or temsirolimus. Our in vivo data highlight the importance of characterizing changes in the tumour microenvironment when determining optimal scheduling of chemotherapy and molecular targeted treatment.

Chemotherapy is often administered once every three weeks to allow repopulation of essential normal tissues such as the bone marrow. Repopulation of surviving tumour cells can also occur between courses of chemotherapy and can decrease the efficacy of anticancer treatment. This thesis aims to characterize repopulation, to study the effect of targeted cytostatic agents to inhibit repopulation, and to determine the optimal scheduling of chemotherapy and molecular targeted treatment. The distribution of proliferating and apoptotic cells in human squamous cell carcinoma (A431) xenografts was studied following chemotherapy using fluorescence immunohistochemistry. There was an initial decrease in cell proliferation and in the total functional blood vessels, and an increase in apoptosis observed following treatment with paclitaxel chemotherapy. A rebound in cell proliferation occurred approximately 12 days following treatment, which corresponded with a rebound in vascular perfusion. The effect of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, to inhibit repopulation between courses of chemotherapy was determined using EGFR-overexpressing A431 cells and xenografts. Furthermore, concurrent and sequential schedules of combined chemotherapy and molecular targeted treatment were compared. Gefitinib inhibited the repopulation of A431 cells in culture when administered sequentially between chemotherapy; sequential treatment was more efficacious than concurrent treatment probably because concomitant scheduling rendered quiescent cells less responsive to chemotherapy. However, in vivo studies using chemotherapy in combination with gefitinib or temsirolimus, a mammalian target of rapamycin (mTOR) inhibitor, showed that concurrent scheduling of combined treatment was more effective at delaying regrowth of xenografts than sequential treatment; this was likely due to dominant effects on the tumour microenvironment. The work completed in this thesis has shown that repopulation occurs in A431 xenografts following paclitaxel treatment, and these changes are associated with changes in the tumour vasculature. Repopulation of A431 cells was inhibited by gefitinib administered sequentially with paclitaxel. However, studies in mice showed better inhibitory effects when chemotherapy was given concomitantly with cytostatic agents such as gefitinib or temsirolimus. Our in vivo data highlight the importance of characterizing changes in the tumour microenvironment when determining optimal scheduling of chemotherapy and molecular targeted treatment.

No description available.

Yes / Implementing the principles of tissue engineering within the clinical management of non-vital immature permanent teeth is of clinical interest. However, the ideal scaffold remains elusive. The aim of this work was to assess the feasibility of decellularising rat dental pulp tissue and evaluate the ability of such scaffold to support stem cell repopulation. Rat dental pulps were retrieved and divided into control and decellularised groups. The decellularisation protocol incorporated a low detergent concentration and hypotonic buffers. After decellularisation, the scaffolds were characterised histologically, immunohistochemistry and the residual DNA content quantified. Surface topography was also viewed under scanning electron microscopy. Biocompatibility was evaluated using cytotoxicity assays utilising L-929 cell line. Decellularised scaffolds were recellularised with human dental pulp stem cells up to 14 days in vitro. Cellular viability was assessed using LIVE/DEAD stain kit and the recellularised scaffolds were further assessed histologically and immunolabelled using makers for odontoblastic differentiation, cytoskeleton components and growth factors. Analysis of the decellularised scaffolds revealed an acellular matrix with histological preservation of structural components. Decellularised scaffolds were biocompatible and able to support stem cell survival following recellularisation. Immunolabelling of the recellularised scaffolds demonstrated positive cellular expression against the tested markers in culture. This study has demonstrated the feasibility of developing a biocompatible decellularised dental pulp scaffold, which is able to support dental pulp stem cell repopulation. Clinically, decellularised pulp tissue could possibly be a suitable scaffold for use within regenerative (reparative) endodontic techniques.

Neste trabalho investigamos o papel do receptor do Fator ativador de Plaquetas (PAF) em diferentes tumores. Observamos que animais PAFR KO são mais resistentes ao crescimento do melanoma B16F10 e do carcinoma TC-1 e apresentaram maior infiltrado de linfócitos CD4+, neutrófilos e de macrófagos M1 do que animais WT. Células de carcinoma humano (C33/SiHa/HeLa/SSC78/ SSC90) expressam PAFR e tiveram sua proliferação in vitro reduzida por um antagonista de PAFR e aumentada pela adição de PAF. A irradiação gama induziu ligantes PAFR. O bloqueio do PAFR durante a radioterapia aumentou a morte induzida pela irradiação. Em modelo de repopulação tumoral observamos que tumores PAFR+ (KBP) tiveram um crescimento acelerado em relação à tumores PAFR- (KBM). Nossos dados sugerem que durante a irradiação ocorre ativação de PAFR nas células tumorais aumentando sua sobrevivência e proliferação. Ao mesmo tempo que ativa PAFR nos macrófagos reprogramando-os para um perfil pró-tumoral. A associação da radioterapia com antagonistas de PAFR pode ser uma estratégia terapêutica promissora. / We investigate the role of the platelet activating factor receptor (PAFR) in tumors. We observed that PAFR KO animals are more resistant to the growth of B16F10 melanoma and TC-1 carcinoma than WT animals. PAFR KO had more infiltration of CD4+ cells, neutrophil and M1 macrophages than WT animals. Human carcinoma cells (C33 / SiHa / HeLa / SSC78 / SSC90) express PAFR and had their in vitro proliferation reduced by a PAFR antagonist and increased by the addition of PAF. Gamma irradiation induced PAFR ligands. Blocking PAFR during radiotherapy increased radiation-induced cell death. In tumor repopulation model, PAFR+ tumors (KBP) had an accelerated growth compared to PAFR- (KBM) tumors. Our data suggest that during irradiation occurs PAFR activation in tumor cells increasing their survival and proliferation. Also, irradiation promote activation of PAFR in macrophages by reprogramming them to a pro-tumor profile. We propose that PAFR represents a possible target for improving the efficacy of radiotherapy through inhibition of tumor repopulation.

The potential of embryonic stem (ES) cells to generate any developmental or adult cell type holds much promise for regenerative medicine and in vitro modelling of development and disease. Haematopoietic stem cells (HSCs) regenerate all lineages of the blood throughout adult life and are essential for the treatment of a vast number of haematalogic disorders. Current sources of HSCs for clinical use and research, including adult bone marrow, peripheral blood stem cells and umbilical cord blood, are limited by the number of HSCs they contain and by the availability of a suitable donor. A system that generates a reliable source of HSCs from ES cells would therefore be an ideal alternative. While much progress has been made in the generation of downstream lineages of the haematopoietic system, progress in the derivation of HSCs capable of long-term self-renewal and multilineage reconstitution from ES cells has been limited. Understanding of the developmental steps leading to HSC emergence in the embryo has been advancing in recent years. In particular, precursors of HSCs (preHSCs) have been isolated from the mouse embryo, characterised and matured into HSCs ex vivo using the specialised conditions of aggregate culture systems (Taoudi et al 2008, Rybtsov et al 2011). We hypothesised that application of the aggregate culture system in the differentiation of ES cells could provide a missing link in the in vitro generation of HSCs. Here I have developed a novel ES cell differentiation system that employs the specialised conditions of the aggregate culture system, after an initial stage of mesoderm differentiation. I show that this system creates an environment for efficient haematopoietic and endothelial progenitor formation and generates cells of a preHSC type I (VE-Cadherin+CD45-CD41lo) and preHSC type II (VE-Cadhein+CD45+) surface phenotype. Notably, the system gives rise to cells that achieve low-levels of haematopoietic repopulation in sublethally irradiated NSG mice. The low-level repopulating cells persist for over 4 months in animals and show both myeloid and lymphoid potential. I identify genes that are expressed in cells of a preHSC II surface marker-phenotype from the E11.5 dorsal aorta, but not in cells of this phenotype from the E11.5 Yolk sac or differentiated ES cells. I also show that enforced expression of Notch downstream target Hes1 in Flk1+ mesoderm during ES cell differentiation does not improve levels of ES-derived repopulation.

No description available.

No description available.