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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.

Identification of Wnt-1l0-catenin responsive genes in mouse mammary epithelial cells

Kenny, Paraic A. January 2002 (has links)
The Wnt/β-catenin signal transduction pathway plays a central role in metazoan development, controlling such diverse processes as cell growth, proliferation and organogenesis. Wnt-1 was initially identified as a mammary oncogene. Mutations in other components of this signal transduction pathway - APC, β-catenin and Axin - have been implicated in the initiation and progression of an increasing number of human malignancies. Aberrant activation of this pathway leads to the inappropriate expression of target genes such as c-MYC and CYCLIN D1. Elucidation of additional target genes of this pathway will shed further light upon the mechanisms underlying the malignant phenotype. As an in vitro model of Wnt/β-catenin signalling in the mouse mammary gland, mammary epithelial cell lines were generated in which this pathway could be activated in a tetracycline-dependent manner. The transcriptional consequences of aberrant Wnt/β-catenin signalling were then investigated using cDNA microarrays. Using this approach, 70 genes have been identified as being potential transcriptionally upregulated targets of the pathway and a similar number have been shown to be repressed. Similar experiments were performed in a colorectal cancer cell line lacking functional APC. An analysis of a representative sample of these differentially expressed genes is presented here. The use of human tumour tissue microarrays containing 300 tumour samples from a variety of tissues was validated as an approach to test the relevance of candidate genes.

Identification of markers for relapse and candidate genes for involvement in the progression of stage I non-seminomatous testicular germ cell tumours

Gilbert, Duncan January 2009 (has links)
Testicular germ cell tumours (TGCT) are the commonest solid tumour to affect young adult males and are increasing in incidence for reasons poorly understood. 60% of the non-seminomatous (NS) subtype present with stage I disease (clinically localised to the testes). Although apparently cured by surgery alone, up to 50% will subsequently relapse through the presence of micrometastases. Adjuvant chemotherapy reduces the relapse rate to 2%, but is associated with acute toxicity and long-term side effects (second cancers and an increased risk of cardiovascular disease) and represents unnecessary treatment for at least half of these patients. Histological evidence for vascular invasion is currently used as an indicator of subsequent relapse but is inaccurate and therefore improved prognostic markers are urgently required. In order to identify genomic regions associated with likelihood of relapse, full tiling path resolution BAC array comparative genomic hybridisation was used to characterise formalin fixed, paraffin embedded material from 32 stage I NS, 15 of which were known to subsequently relapse. Published expression microarray data from TGCT was then used to identify gene expression patterns from the genomic regions identified. This identified candidate genes as markers of relapse as well as supporting the importance of RAS and PI3 kinase signalling in TGCT. In addition, the expression levels of known mediators of metastatic dissemination were identified as further candidates for involvement in TGCTs. Genomic data demonstrated loss of 22q12.2 (containing DRG1 and PIK3Ip1) and gain of 22q13.32 (BRD1) being associated with relapse and a cluster of cytokines on 17q12 associated with vascular invasion. These genes were investigated using immunohistochemistry on a tissue microarray constructed from 83 stage I NS with known clinical outcomes. Candidates selected on the basis of the expression data and their potential functional role were VEGF-A, MMP2, MMP9 and the chemokine CXCL12 and its receptor CXCR4. Variable rates of expression of VEGF-A, MMP2 and MMP9 were identified though no correlations with subsequent clinical behaviour found. Universal expression of the chemokine receptor CXCR4 by TGCT was demonstrated, with autocrine expression of the ligand CXCL12 associated with a reduced rate of subsequent relapse (p=0.003) indicative of a significant prognostic marker. This could be explained by low intratumoural expression of CXCL12 mediating migratory behaviour. Consistent with this hypothesis, functional work using TGCT cell lines demonstrated CXCR4 mediated migration towards CXCL12 gradients, with concurrent activation of ERK1/2. Based on this study, work to validate CXCL12 as a clinically useful prognostic marker in stage I NS is warranted and currently underway using samples from patients treated and monitored in a clinical trial setting.

Pre-clinical and clinical evaluation of apoptosis targeting drugs

Dean, Emma January 2009 (has links)
Lung cancer is the leading cause of cancer deaths amongst both men and women and is commonly divided into two histological groups, non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). Despite advances in early diagnosis and treatment, chemotherapy provides poor response rates and rare complete remissions, warranting the development of novel therapeutic strategies. Evasion of apoptosis is a hallmark of cancer, allowing malignant cells to survive in hostile micro-environments and to resist chemotherapy with the potential for continued proliferation. This thesis evaluates four novel agents, primarily in lung cancer, in both the pre-clinical and early clinical settings which target key molecules involved in the regulation of apoptosis. Pre-clinical data for XAC 1396-11, a small molecule inhibitor of X-linked inhibitor of apoptosis protein (XIAP), showed induction of apoptotic cell death in a concentration- and time-dependent manner in NSCLC cells.

Investigation of the function of the pro-migratory molecules 5T4 and N-cadherin in embryonic stem cells pluripotency and differentiation

Spencer, Helen Louise January 2008 (has links)
Similarities between tumours and embryonic trophoblast invasive properties led to the investigation of cell surface molecules whose function may overlap. Murine monoclonal antibodies directed against human placental syncytiotrophoblast previously identified a novel protein, ST4, which is a 72kDa cell surface glycoprotein. 5T4 protein expression on colorectal, gastric and ovarian carcinomas is associated with a poorer clinical outcome in these patients. It is hypothesised that 5T4 is required by cells undergoing major structural changes, with over-expression of 5T4 linked to a more motile cellular phenotype. Expression of cell surface 5T4 antigen has been shown to be an early marker of embryonic stem (ES) cell differentiation. Furthermore, cell surface ST4 antigen often displays reciprocal expression compared to the adhesion protein E-cadherin. Epithelial-mesenchymal transition (EMT) events occur during tumour cell metastasis and embryonic development and is associated with down-regulation of E-cadherin. Recent data has identified an EMT-like event during ES cell differentiation. This thesis aimed to investigate the function of 5T4, Ncadherin and E-cadherin in EMT-like events during ES cell differentiation.

Development and evaluation of a rapid molecular assay and a mask sampling method enabling the study of tuberculosis transmission

Cheah, Eddy Seong Guan January 2010 (has links)
Mycobacterium tuberculosis (Mtb) is transmitted in small aerosol droplets expectorated by individuals with active pulmonary tuberculosis (TB). There is still no established method for sampling these infectious aerosols and measuring the bacterial concentration in them. Little is known about the relationship between tuberculous aerosol output and infectiousness of TB cases; medical practice currently relies on the acid-fast smear status to gauge this and determine when it is safe to discharge TB patients. This study involved the development and evaluation of a rapid molecular assay and a mask sampling approach to increase our knowledge relating to TB transmission. A molecular assay targeting the mycobacterial 16S rDNA and differentiating Mtb complex from non-tuberculous mycobacteria was developed to facilitate recruitment of TB patients for the mask sampling study. Although the assay did not achieve this outcome during the study, we demonstrated its clinical utility on mycobacterial isolates. A second molecular assay targeting the Mtb global lineagedefining and locally prevalent RD750 polymorphism was also developed for preliminary genotyping of Mtb strains to assess for potential recent transmission events. Both the 16S and RD assays were performed in single thermocycler runs but in separate reaction tubes and utilised a combination of TaqMan and SYBR Green technologies to simultaneously differentiate two products. The 16S-RD assay shows promising potential for direct specimen analysis and detection of mixed infections. A novel method of mask aerosol sampling coupled to a mycobacteriophage amplification assay developed in this study was able to detect and to some extent quantify the amount of respiratory-borne Mtb. This approach could potentially be more reliable than smear microscopy in assessing TB infectivity. With further optimisation and improvement, both the 16S-RD assay and the mask sampling method have potential to facilitate better understanding of TB transmission and subsequently contribute to public and clinical management of this disease.

Molecular differences in sporadic breast cancer in young women (≤ 35-year old) : analysis of TGFBI, DDB2 and MCM5

Touma, Dona January 2011 (has links)
The aim of this study was to investigate mRNA and protein expression of three genes (Transforming Growth Factor Beta Induced (TGFBI), Damaged-Specific DNA Binding (DDB2) and Minichromosomal Maintenance-5 (MCM5)) in breast cancers. Q-RT-PCR (36 cancers, 8 normal/benign tissues and 9 organoid samples), western blotting (6 cell lines, 6 cancers and 4 normal/benign tissues) and immunohistochemistry (67 breast cancers) were performed, and for TGFBI functional assays (viability, apoptosis and invasion) were carried out in two cell lines (ZR-75-1 and MDA-MB-468) after transient transfection with recombinant TGFBI and vector controls. TGFBI showed reduced mRNA and protein expression in all cancer cell lines relative to HBL-100. The mRNA levels were also significantly lower in breast cancers compared to normal/benign tissues. Immunohistochemistry results showed that 46 / 67 breast cancers were negative or had <1% nuclear staining. There was a significant correlation between TGFBI mRNA levels and patient age; with lower levels expressed in younger women (p=0.04). Higher expression of DDB2 mRNA was observed in ER/PR positive (MCF-7, T47-D and ZR-75-1) compared to ER/PR negative (HBL-100, MDA-MB-231 and MDA-MB-468) cell lines. Higher DDB2 mRNA levels was significantly correlated with ER positive (p=0.04) and grade II (p=0.02) tumours; lower levels were associated with younger patient age (p=0.025). In addition, higher DDB2 protein expression was associated with ER (p=0.001) and PR (p=0.004). Elevated MCM5 mRNA and protein levels were observed in MCF-7 and MDA-MB-231. MCM5 immunoreactivity was significantly correlated with low grade (p=0.02) and ER/PR positive (ER p=0.04 and PR p=0.01) tumours. Transfection with TGFBI had no effect on viability, apoptosis and invasion of ZR-75-1 and MDA-MB-468 cells. In conclusion, the results fail to support the hypothesis of this study, namely that expression of TGFBI, DDB2 and MCM5 could contribute to the more aggressive features of sporadic breast cancers in younger women.

Age-associated changes in promoter CpG island methylation and their potential role in cancer development

Gautrey, Hannah Elizabeth January 2013 (has links)
Changes in DNA methylation patterns are a hallmark of both cancer and ageing, and may underlie susceptibility to developing age-related diseases such as cancer. To uncover the impact that such variation has on health and ageing, we assessed DNA methylation patterns in the peripheral blood leukocytes (PBL) of 480 participants in the Newcastle 85+ study to determine the levels, and degree of inter-individual variation, of DNA methylation in the promoter region of a panel of genes by Pyrosequencing. We found considerable inter-individual variation in promoter CpG methylation in several genes and a remarkable similarity to leukaemic patterns of aberrant methylation. This included specific methylation of the same sets of genes, strong correlations between methylation of the genes (in a pattern reminiscent of the CpG island methylator phenotype observed in cancer) and the presence of densely methylated alleles in highly methylated individuals, identical to patterns observed in cancer cells. This suggests that ageing and cancer related methylation may be closely linked. Further analysis of PBL DNA methylation levels in Newcastle 85+ study participants with a previous history of cancer (n=113) versus cancer-free individuals (n=113) found significantly higher methylation in those with a cancer history (10.71% vs. 10.21%, p=0.04). Further, a separate group of 72 individuals diagnosed with cancer within the 3 year duration of the study had similarly increased methylation levels (10.96% vs. 10.36%, p=0.03), suggesting that pre-existing methylation in normal cells may increase risk of cancer and may be evident prior to clinically detectable disease. In addition, individuals with increased DNA methylation were more likely to be categorised as frail (as defined by Fried) than those with lower DNA methylation measures, indicating that disrupted methylation patterns are associated with detrimental effects on healthy ageing. Subsequently, a GWAS analysis found that SNPs in two genes, DSCAM and DSCAML1, appeared to be associated with determining DNA methylation levels in the Newcastle 85+ study participants.

Pharmacogenetic study of Fc gamma receptor and HER2 genes in breast cancer

Cresti, Nicola January 2013 (has links)
Breast cancer is a complex set of diseases with different biological and clinical characteristics. An important contribution to this diversity is provided by germ-line genetic variations. The HER2-positive breast cancers have been extensively studied with particular regard to their biology and targeted treatments. However, the influence of pharmacogenetic (PG) factors on these aspects remains largely unexplored. This research focused on the possible effects of common single nucleotide polymorphisms (SNPs) on specific aspects of HER2-positive disease. Initially we analysed two coding SNPs in the HER2 gene (Ile655Val and Ala1170Pro) in breast cancer patients and evaluated their potential association with HER2 expression in tumour samples. The proline variant of the Ala1170Pro SNP was associated (odds ratio = 1.7, p = 0.01) with HER2 over-expression/amplification in over 360 breast cancer patients. In contrast, Ile655Val was not associated with HER2 over-expression/amplification. Bioinformatics tools predict that Ala1170Pro might affect the structure or function of the HER2 protein. The same variants were explored in the context of DNA extracted from the patients’ primary tumours in 241 patients. We hypothesized that the proline allele of Ala1170Pro could undergo allele-specific amplification during the development of HER2-positive tumours. This hypothesis, however, was not confirmed. Although the association of the proline allele of Ala1170Pro with HER2 positivity is intriguing, the role of the two SNPs in HER2 over-expression/amplification remains to be elucidated. Trastuzumab has radically changed the treatment of HER2-positive breast cancer. However, resistance to treatment and toxicity can limit its effectiveness. The second objective of this project was the analysis of PG, biomarker and pharmacokinetic (PK) parameters in trastuzumab-treated patients. Fc Gamma Receptors (FcgRs) are key proteins in the trastuzumab-induced Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and two coding SNPs in these genes (FCGR2A His131Arg and FCGR3A Phe158Val) were analysed. The measurement of trastuzumab in plasma was made possible by the development of a novel cell-based ELISA. Only 28 patients with advanced disease treated with trastuzumab were recruited. However, we observed a possible association of the valine allele of the FCGR3A Phe158Val SNP with a longer time to progression (p = 0.03). Cardiac toxicity was assessed in a group of 139 patients treated with adjuvant trastuzumab. Although a role of germ-line genetic variants could not be demonstrated, the analysis highlighted the challenges and limitations encountered in the conduct of an observational pharmacogenetic study. This project leaves a legacy archive composed of germ-line DNA samples, tumour DNA samples, plasma samples and tumour FFPE blocks from over 360 breast cancer patients. These samples and data are available for the exploration of further potential factors which might influence the biology of the disease and/or its response to treatment.

Investigating leukaemic propagation in childhood acute lymphoblastic leukaemia

Bomken, Simon Nicholas January 2013 (has links)
Childhood acute lymphoblastic leukaemia (ALL) does not possess a propagating cell hierarchy, at least as defined by B-cell precursor immunophenotype. Indeed, many, or even all, leukaemic blasts may have the potential to propagate the disease. This unusual characteristic mirrors the substantial capacity for clonal expansion demonstrated by fully differentiated normal lymphoid cells. This Fellowship aimed to investigate the genetic programmes underlying the propagation of acute lymphoblastic leukaemia. An initial candidate approach confirmed the expression of PIWIL2, a gene critical to the maintenance of germline stem cells, in both cell line and primary ALL. Knockdown of PIWIL2 resulted in reduced cellular proliferation and significant prolongation of doubling time in two ALL cell lines, SEM (MLL/AF4) and 697 (E2A/PBX1). Unexpectedly, PIWIL2 was also found to be expressed in peripheral lymphoid cells from healthy donors, but not terminally differentiated cells of myeloid origin, suggesting that PIWIL2 may have a previously unidentified function in both normal and malignant lymphoid cells. A second project has developed an in vitro genome-wide RNAi screen to identify candidate genes involved in the clonal propagation of ALL. This project has assessed a serial re-plating assay using feeder cell co-culture to provide a surrogate niche environment. Initial results have demonstrated the feasibility of such an approach. The benefit of using a co-culture re-plating assay, as compared to a standard suspension culture approach, remains under investigation. ii Finally, this Fellowship developed a protocol for the lentiviral transduction of patient-derived leukaemic blasts and cloned and validated a novel lentiviral vector capable of in vitro analysis, in vivo disease monitoring and RNAi. With these, it will now be possible to validate candidate leukaemic propagation genes in vivo, using primary leukaemic material. The results of these studies will provide candidates for the development of novel therapeutic agents for children with ALL.

BRCA1 N-terminal interacting partners

Pangon, Laurent Luc Gilles January 2008 (has links)
The product of the breast and ovarian cancer predisposition gene, BRCAI, is a large protein with two main recognised motifs; the N-terrninal RING and C-terrninal BRCT. The N-tenninus has been reported to interact with at least eight proteins (MSH2, ATFI, ERalpha, BAPI, p300, NPMlB23, UbcH5A and BARDI). In order to test the relevance of these interacting partners to the BRCAI tumour suppressor activity, we intended to test patient derived missense variants upon each interaction. We found that of the eight potential partners, only BARDI and the ubiquitin conjugating enzyme UbcH5A interacted with the BRCAI N-terrninal region, by yeast two-hybrid assays. The BRCAI/UbcH5A interaction was also confirmed in mammalian cells by FRET analysis. To determine }Vhether these interactions, or their abrogation, might fonn part of the BRCAI-mediated tumorigenesis pathway we undertook an extensive mutagenesis study. Our results show that all known pathogenic missense mutations and the majority ofBRCAI N-terrninal unclassified variants, found in populations enriched for family history ofdisease, weakened or abolished the BRCAI/UbcH5A interaction. Loss of ubiquitin ligase activity measured biochemically, and in cells, correlated with BRCAI/UbcH5A disruption. Finally, in silico prediction, based on evolutionary conservation and protein structural analysis supported and complemented our mutagenesis results. Overall, our data provide functional evidence that the BRCAI/UbcH5A interaction is highly sensitive to missense substitution and suggest that the ubiquitin pathway is likely to play an important role in BRCAI-mediated tumorigenesis. Even though we could not detect a direct interaction between BRCAI and MSH2, our study on MSH2-deficient primary human cells implicated MSH2 in the homologous recombination repair pathway.

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