The thymus is organised into distinct microenvironments, and trafficking through these regions enables thymocytes to receive essential signals for the generation of a diverse and self-tolerant T-cell repertoire. Thymic epithelial cells (TEC) represent a key stromal cell type during defined stages in T cell development, yet the mechanisms regulating their development are only partly understood. An ontogenetic approach was employed to study stages of cortical thymic epithelial cell (cTEC) development. This study identifies a previously unreported population of cTEC progenitors expressing CD205 and 5T and has defined distinct checkpoints in the development of the cTEC lineage. Furthermore, the importance of thymic crosstalk during specific stages of cTEC development and also the requirement for RANK-RANKL signalling for the development of various medullary thymic epithelial cell (mTEC) subsets has also been defined. Additionally, the importance of chemokine-mediated signalling for the establishment and compartmentalisation of the thymus has been highlighted by employing laser capture microdissection and studying thymus microenvironments in mice deficient for particular chemokine related signalling pathways. Overall this study has provided novel insight into the development of the thymic cortex and will help to understand how these cells become specialised in their ability to support positive selection of developing T cells.

Malignant glioma is the most common and aggressive form of tumours and is usually refractory to therapy. Telomerase and its altered activity, distinguishing cancer cells, is an attractive molecular target in glioma therapeutics. The aim of this thesis was to silence telomerase at the genetic level with a view to highlight the changes caused in the cancer proteome and identify the potential downstream pathways controlled by telomerase in tumour progression and maintenance. A comprehensive proteomic study utilizing 2D-DIGE and MALDI-TOF were used to assess the effect of inhibiting two different regulatory mechanisms of telomerase in glioma. RNAi was used to target hTERT and Hsp90α. Inhibition of telomerase activity resulted in down regulation of various cytoskeletal proteins with correlative evidence of the involvement of telomerase in regulating the expression of vimentin. Vimentin plays an important role in tumour metastasis and is used as an indicator of glioma metastasis. Inhibition of telomerase via sihTERT results in the down regulation of vimentin expression in glioma cell lines in a grade specific manner. While, 9 of 12 glioblastoma tissues (grade IV) showed vimentin to be highly expressed, its expression was absent in lower grades and normal tissues. This suggests that vimentin can be potentially used as a glioma progressive marker. This is the first study to report the potential involvement of telomerase in the regulation of vimentin expression. This study also identified that combination therapy, comprising siRNA targeted towards telomerase regulatory mechanisms and the natural product Epigallocatechin-3-gallate (ECGC), results in decreased cell viability producing comparable results to that of other chemotherapeutic drugs.

Gene expression studies have revealed that there is more than one cellular pathway governing growth inhibition and apoptosis. Mutations in the ras oncogene that(activate ras) are known to lead to the inactivation of genes that are directly involved in these pathways of growth inhibition and apoptosis. Oncogenic activated ras inhibits TGF-P signalling through the down-regulation of RII expression and abrogates apoptotic pathways through down-modulation of PAR-4 gene expression. A majority of pancreatic turnours harbour K-ras point mutations and these mutations dysregulate, the growth inhibition and apoptosis processes. This leads us to hypothesize that K-ras mutant phenotype status in pancreatic turnours will alter the expression of the RII and PAR-4 genes, and would further dysregulate growth inhibitory and apoptotic processes. In this study, the majority of pancreatic turnours showed down-regulation of RII and PAR-4 gene expression. A strong correlation of down-regulation of RII and PAR-4 with K-ras mutational status was observed. In particular, down regulation of PAR-4 correlated with poor survival in patients with pancreatic adenocarcinomas. Blocking the function of oncogenic ras by using a famesyltransferase inhibitor (Frl) restored RII expression and TGF-P signalling, and this caused enhanced sensitivity of cell lines to radiation. The restoration of RH function by FrI was linked to down-modulation of DNA methyltransferase enzyme that is often implicated in hypermethylation of promoters. Over-expression of RII in pancreatic tumour cells led to the restoration of TGF-P signalling and enhancement of radiation sensitivity. Induction of the pro-apoptotic effector gene, bav (bcI-2 family member) by radiation in RII over-expressed pancreatic cancer cells, was found to be a key mechanism involved in radiation sensitivity. Overexpression of PAR-4 sensitized the cells to radiation and this sensitization was linked to down-modulation of radiation induced Bcl-2 protein. Together, these findings strongly suggest that the restoration of function of the key growth inhibitory and cell death genes RII and PAR-4, which are affected by oncogenic ras mutations in pancreatic turnours will restore and enhance cellular responses to radiation induced clonogenic inhibition and apoptosis.