Functional study for the characterisation and validation of IFNAI as a tumour suppressor gene in melanoma pathogenesis

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel UniversityThe complexity of melanoma is pronounced at many levels, whereby both environmental influences and genetic predisposition are involved and interact. Embedded within this complexity is heterogeneity, a defining characteristic of this malignancy. The rearrangement of genomic material on chromosomes 1p, 6q, 9p or 10q, 11q and 17q has been frequently reported during the development and progression of cutaneous malignant melanoma (CMM), suggesting several putative tumour suppressor genes and oncogenes in these regions. The genomic complexity of chromosome 9p21 in melanoma development is well documented. This region encodes a potent cyclin-dependent kinase inhibitor CDKN2/INK4A/p16 as a tumour suppressor gene (TSG) that is frequently inactivated in melanomas. Functional evidence suggested the presence of additional TSG loci in the 9p21-22 chromosome region (Parris et al., 1999). In pursuit of identifying novel TSG(s), our previous group’s collaborative research provided experimental evidence that suggests IFNA1 as a candidate TSG for melanoma development. Therefore, the aim of this work was to provide a further functional validation of such tumour-suppressive activity in CMM. Firstly, I have successfully subcloned IFNA1 cDNA into pcDNA3 expression vector and established a panel of stably IFNA1-expressing clones. Subsequently, I have assessed their tumourigenicity in soft agar by measuring the colony-forming ability of each transfected clone. Expression analyses of IFNA1, at both post-transcriptional and translational levels, were also carried out. I have also demonstrated a strong correlation between anchorage-independent growth in soft agar and IFNA1 expression in qRT-PCR. The antiproliferative and pro-apoptotic effects of IFNα have been widely documented, however, the precise mechanisms that trigger and potentiate this behaviour are not completely known. Based on previous findings, I have investigated whether IFNA1 exerts its antitumoural activity through apoptosis. I was able to demonstrate a moderate relationship between anchorage-independent growth in soft agar and the apoptotic levels in the transfected clones. Although unpersuasive and inconclusive, the results seemed encouraging since this study was carried out using only the highly tumourigenic malignant melanoma UACC903 cell line

SCSA based MATLAB pre-processing toolbox for 1H MR spectroscopic water suppression and denoising

In vivo 1H Magnetic Resonance Spectroscopy (MRS) is a useful tool in assessing neurological and metabolic disease, and to improve tumor treatment. Different pre-processing pipelines have been developed to obtain optimal results from the acquired data with sophisticated data fitting, peak suppression, and denoising protocols. We introduce a Semi-Classical Signal Analysis (SCSA) based Spectroscopy pre-processing toolbox for water suppression and data denoising, which allows researchers to perform water suppression using SCSA with phase correction and apodization filters and denoising of MRS data, and data fitting has been included as an additional feature, but it is not the main aim of the work. The fitting module can be passed on to other software. The toolbox is easy to install and to use: 1) import water unsuppressed MRS data acquired in Siemens, Philips and .mat file format and allow visualization of spectroscopy data, 2) allow pre-processing of single voxel and multi-voxel spectra, 3) perform water suppression and denoising using SCSA, 4) incorporate iterative nonlinear least squares fitting as an extra feature. This article provides information about how the above features have been included, along with details of the graphical user interface using these features in MATLAB

New generation of electrochemical immunoassay based on polymeric nanoparticles for early detection of breast cancer

Screening and early diagnosis are the key factors for the reduction of mortality rate and treatment cost of cancer. Therefore, sensitive and selective methods that can reveal the low abundance of cancer biomarkers in a biological sample are always desired. Here, we report the development of a novel electrochemical biosensor for early detection of breast cancer by using bioconjugated self-assembled pH-responsive polymeric micelles. The micelles were loaded with ferrocene molecules as "tracers" to specifically target cell surface-associated epithelial mucin (MUC1), a biomarker for breast and other solid carcinoma. The synthesis of target-specific, ferrocene-loaded polymeric micelles was confirmed, and the resulting sensor was capable of detecting the presence of MUC1 in a sample containing about 10 cells/mL. Such a high sensitivity was achieved by maximizing the loading capacity of ferrocene inside the polymeric micelles. Every single event of binding between the antibody and antigen was represented by the signal of hundreds of thousands of ferrocene molecules that were released from the polymeric micelles. This resulted in a significant increase in the intensity of the ferrocene signal detected by cyclic voltammetry

Multispectral imaging flow cytometry reveals distinct frequencies of γ-H2AX foci induction in DNA double strand break repair defective human cell lines

Copyright @ 2012 International Society for Advancement of Cytometry. The article can be accessed from the links below.This article has been made available through the Brunel Open Access Publishing Fund.The measurement of γ-H2AX foci induction in cells provides a sensitive and reliable method for the quantitation of DNA damage responses in a variety of cell types. Accurate and rapid methods to conduct such observations are desirable. In this study we have employed the novel technique of multispectral imaging flow cytometry to compare the induction and repair of γ-H2AX foci in three human cell types with different capacities for the repair of DNA double strand breaks (DSB). A repair normal fibroblast cell line MRC5-SV1, a DSB repair defective ataxia telangiectasia (AT5BIVA) cell line, and a DNA-PKcs deficient cell line XP14BRneo17 were exposed to 2 Gy gamma radiation from a 60Cobalt source. Thirty minutes following exposure we observed a dramatic induction of foci in the nuclei of these cells. After 24 hrs there was a predictable reduction on the number of foci in the MRC5-SV1 cells, consistent with the repair of DNA DSB. In the AT5BIVA cells, persistence of the foci over a 24 hour period was due to the failure in the repair of DNA DSB. However, in the DNA-PKcs defective cells (XP14BRneo17) we observed an intermediate retention of foci in the nuclei indicative of partial repair of DNA DSB. In summary, the application of imaging flow cytometry has permitted an evaluation of foci in a large number of cells (20,000) for each cell line at each time point. This provides a novel method to determine differences in repair kinetics between different cell types. We propose that imaging flow cytometry provides an alternative platform for accurate automated high through-put analysis of foci induction in a variety of cell types.This article is made available through the Brunel Open Access Publishing Fund

Reduction of Acquisition time by Partition of the signal Decay in Spectroscopic Imaging (RAPID-SI) technique : preliminary In-vivo results and comparison with CSI

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