An overview of the existing body of knowledge on the most widely researched areas of childhood leukaemia was presented and it was argued that a conceptual shift is required to achieve a more comprehensive understanding of the problem. This conceptual shift encompassed an ecosystemic approach. This study was conducted within a holistic systemic epistemology. A qualitative approach employing a case study method to provide rich descriptions of the context in which two leukaemia sufferer's symptoms were embedded. / Psychology / M.A. (Psyhcoloy)

An overview of the existing body of knowledge on the most widely researched areas of childhood leukaemia was presented and it was argued that a conceptual shift is required to achieve a more comprehensive understanding of the problem. This conceptual shift encompassed an ecosystemic approach. This study was conducted within a holistic systemic epistemology. A qualitative approach employing a case study method to provide rich descriptions of the context in which two leukaemia sufferer's symptoms were embedded. / Psychology / M.A. (Psyhcoloy)

Basic in vitro cell experiments were conducted on the P388 mouse leukaemia cell line to determine whether a supra-additive cell killing effect from combining hyperthermia with ionising radiation exists in the case of leukaemia. Methods were established to measure the cell kill, using a Coulter counter, from hyperthermia alone, radiation alone and several combined regimes. The cell kill from hyperthermia, in the range of 38-50 degrees for 30 minutes, 1 hour, 2 hours and 3 hours, and radiation, for 1, 3, 5, 9, 11 and 15 Gy was investigated. The approach used had various limitations, such as the underestimation of cell kill. Consistent trends, however, were found for the hyperthermia and radiation data, in accordance with the literature, which killed cells in a predictable manner. Subsequently, after other preliminary combined experiments were completed, the cell kill from both 5 and 11 Gy combined with hyperthermia at 43, 45 and 47 degrees for 2 hours were investigated. 5 Gy in combination with all levels of hyperthermia resulted in a direct additive cell killing effect. This, however, was not observed for 11 Gy in which a diminished effect was found. The overall level of cell kill from 5 Gy combined with hyperthermia was found to be equal, in the case of 43 degrees, or higher, as for 45 and 47 degrees, to that of those combined with 11 Gy. A supra-additive effect was not observed.

During stress conditions, glucocorticoids are secreted and exert most of their physiological responses by binding to and modulating the transcriptional activity of the glucocorticoid receptor (GR). Once activated, GR can regulate numerous cellular processes including inflammation, development, growth, metabolism and apoptosis. Although glucocorticoids have been used in the treatment of leukaemia for over 50 years, with the molecular mechanisms by which steroids exert their pro-apoptotic effect, the pathways responsible for the development of resistance to glucocorticoid treatment, as well as their role in the programmed cell death in other tissue types have not been precisely defined. Research has demonstrated that glucocorticoid-induced apoptosis requires a transcriptionally active form of GR and is executed by the induction of the intrinsic pathway of apoptosis. In addition, GR is regulated by diverse types of cytotoxic stress; including UV irradiation and hypoxia, which alter the receptor’s transcriptional activity through multiple mechanisms. These include post-translational modifications, subcellular localisation and interaction of the receptor with co-regulator proteins. The aims of this study are to identify novel members of the Bcl-2 family that are regulated at the transcriptional level by GR in both leukaemia and other tissue types where glucocorticoids promote cell survival. In addition, the molecular crosstalk between signalling pathways activated by cytotoxic stress conditions and the mechanisms by which they differentially modulate the apoptotic response will be investigated. Results obtained in this study have identified putative glucocorticoid response elements in the promoters of the BH3-only pro-apoptotic gene NOXA and the anti-apoptotic gene Mcl-1 and confirmed that both NOXA and Mcl-1 are direct GR transcriptional targets. The glucocorticoid-mediated expression of NOXA and Mcl-1 alters their protein-protein interaction pattern, leading to the subsequent destabilisation of Mcl-1 in cell lines that undergo glucocorticoid-induced apoptosis. Investigation into the effects that other cytotoxic stress pathways have on GR function have revealed that serine 226 phosphorylation of GR by JNK occurs in a rapid and transient manner. Phosphorylation has inhibitory effects on the transcription of GR targets in a gene-specific manner, including the differential regulation of NOXA gene expression. During hypoxia, glucocorticoids differentially regulate the GR and HIF-1 target genes, NOXA and Mcl-1, altering the apoptotic response. This study has provided additional insight into the molecular mechanisms that govern glucocorticoid-induced programmed cell death and revealed mechanisms by which glucocorticoids and cytotoxic stress pathways crosstalk, regulating apoptosis.

The promyelocytic leukaemia protein (PML) is a tumour suppressor initially identified in acute promyelocytic leukaemia (APL). In APL, PML and the retinoic acid receptor alpha (RARalpha) genes are fused as a consequence of the translocation t(15;17). The product of the chimeric gene is the oncogenic PML-RARalpha protein. The PML gene encodes multiple nuclear and cytoplasmic isoforms. PML nuclear isoforms (nPML) are the main components of the PML nuclear bodies (PML-NBs), sub-nuclear structures involved in the modulation of essential cellular players including the tumour suppressor p53. Nuclear PML has been intensively studied, while, the role of cytoplasmic PML remains poorly understood. Increasing evidence indicates that PML could bear cytoplasmic functions in both physiological and pathological settings. This study aims to gain more insights into the function of PML and PML-RARalpha cytoplasmic pool of proteins. Recently, two missense mutations resulting in truncated PML cytoplasmic protein (Mut PML) have been identified in aggressive APL cases. We found that Mut PML alters the structure and the function of the PML-NB mainly through the cytoplasmic relocation of nPML. Remarkably, Mut PML inhibits p53 transcriptional, growth suppressive and apoptotic functions. In the cytoplasm, Mut PML interacts and stabilizes PML-RARalpha, thus potentiating its block of RA-induced transcription and differentiation. A mutant of PML-RARalpha (Delta2) accumulating in the cytoplasm is able to inhibit RA-dependent transcription and differentiation, suggesting that cytoplasmic localization of PML-RARalpha may contribute to transformation. Finally, we found that Delta2 expression blocks G-CSF-dependent myeloid differentiation and causes partial transformation of primary haematopoietic progenitor cells.

Many haematological malignancies are associated with widespread alterations of the transcriptional and epigenetic programmes. Changes in DNA methylation provide the clearest example of epigenetic changes, but the mechanism(s) underlying such changes is unknown. To investigate this I studied DNA methylation across an ~80kb segment of the genome which is not known to be mutated in haematological malignancies. Methylation was perturbed in 35-100% of samples of DNA from individuals with a wide range of haematological malignancies but not in non-malignant haematological disorders. DNA methylation was comprehensively assessed by Southern blot analysis, classical bisulphite sequencing and using a newly developed capture bisulphite sequencing protocol. The results were also compared with analysis by MeDIP, an immunoprecipitation-based technique. These analyses provide methylation status at various levels including individual CpG resolution. This showed both gain and loss of methylation at CpG dinucleotides. Of interest, hypomethylation was most frequently seen in intergenic regions corresponding to transcription factor binding sites and areas of increased chromosome accessibility. These observations suggested that hypomethylation of the genome in haematological malignancies could arise from aberrantly expressed DNA binding proteins which, recruited to sequences in regions of open chromatin, would protect the underlying CpG dinucleotides from the methylation machinery. This, in turn, could lead to passive demethylation accumulating with increasing cell divisions. This hypothesis was tested with electrophoretic mobility shift assays using oligonucleotides representing the DNA underlying one such region. This showed that, compared to nuclear extracts from the lymphocytes of normal individuals, those from patients with CLL were enriched for a protein which binds to oligonucleotides containing the underlying sequence. Using a mass spectrometry approach, I identified a variety of proteins that may bind such regions and account for their passive demethylation in haematological malignancies.

Background: Few studies have investigated the relationship between physician and patient-assessed performance status (PS) in blood cancers. Methods: Retrospective analysis among 1418 patients with haematologic malignancies seen at Dana-Farber Cancer Institute between 2007 and 2014. We analysed physician-patient agreement of Eastern Cooperative Oncology Group PS using weighted kappa-statistics and survival analysis. Results: Mean age was 58.6 years and average follow-up was 38 months. Agreement in PS was fair/moderate (weighted kappa = 0.41, 95% CI 0.37-0.44). Physicians assigned a better functional status (lower score) than patients (mean 0.60 vs 0.81), particularly when patients were young and the disease was aggressive. Both scores independently predicted survival, but physician scores were more accurate. Disagreements in score were associated with poorer survival when physicians rated PS better than patients, and were modified by age, sex and severity of disease. Conclusions: Physician-patient disagreements in PS score are common and have prognostic significance.

Today, there is a substantial number of software and research groups that focus on the development of image processing software to extract useful information from medical images, in order to assist and improve patient diagnosis. The work presented in this thesis is centred on processing of images of blood and bone marrow smears of patients suffering from leukaemia, a common type of cancer. In general, cancer is due to aberrant gene expression, which is caused by either mutations or epigenetic changes in DNA. Poor diet and unhealthy lifestyle may trigger or contribute to these changes, although the underlying mechanism is often unknown. Importantly, many cancer types including leukaemia are curable and patient survival and treatment can be improved, subject to prompt diagnosis. In particular, this study focuses on Acute Myeloid Leukaemia (AML), which can be of eight distinct types (M0 to M7), with the main objective to develop a methodology to automatically detect and classify leukaemia cells into one of the above types. The data was collected from the Department of Haematology, Universiti Sains Malaysia, in Malaysia. Three main methods, namely Cellular Automata, Heuristic Search and classification using Neural Networks are facilitated. In the case of Cellular Automata, an improved method based on the 8-neighbourhood and rules were developed to remove noise from images and estimate the radius of the potential blast cells contained in them. The proposed methodology selects the starting points, corresponding to potential blast cells, for the subsequent seeded heuristic search. The Seeded Heuristic employs a new fitness function for blast cell detection. Furthermore, the WEKA software is utilised for classification of blast cells and hence images, into AML subtypes. As a result accuracy of 97.22% was achieved in the classification of blasts into M3 and other AML subtypes. Finally, these algorithms are integrated into an automated system for image processing. In brief, the research presented in this thesis involves the use of advanced computational techniques for processing and classification of medical images, that is, images of blood samples from patients suffering from leukaemia.

Student Number : 9000554P - PhD thesis - School of Molecular Medicine and Haematology - Faculty of Science / Dynamic cellular activity is fundamental to all life. Virtually all life processes, are modulated by the reversible phosphorylation of proteins, mediated by protein kinases and phosphoprotein phosphatases, respectively. This thesis focuses on three enzymes, namely: phosphoprotein phosphatase 1, phosphoprotein phosphatase 2A and protein tyrosine phosphatase-1B. Temporal variations in the expression of the enzyme proteins were examined in the human acute promyelocytic leukaemic cell line, HL-60. The cells were induced to differentiate along the macrophage pathway using phorbol-12-myristate- 13-acetate and along the granulocytic pathway using dimethyl sulfoxide, all-trans retinoic acid and 9-cis retinoic acid. Modulation of the rhythmic patterns of protein and messenger RNA was monitored in the absence and presence of inducing agents. Expression of protein in cell extracts prepared at various time intervals was determined by western immunoblotting, while mRNA expression was assessed by northern blotting and RT-PCR. The probe used for northern blotting was generated during the RT-PCR procedure. In addition, PTP-1B mRNA was cloned into an expression vector to produce recombinant protein. Results indicate that the expression of phosphoprotein phosphatase 1, phosphoprotein phosphatase 2A and protein tyrosine phosphatase-1B protein is dynamically regulated in proliferating HL-60 cells and modulated after being induced to differentiate along either the macrophage or granulocytic pathway. Similar changes were also noted with PTP-1B mRNA when using northern blot analysis. Using molecular cloning techniques, PTP-1B mRNA was successfully cloned into pGex-4T-1 expression vector to produce recombinant PTP-1B protein, which was checked by sequence and western blot analysis.

In Western Europe and North America, acute myeloid leukaemia (AML) is predominantly a disease of the elderly, with a median age at the time of presentation in excess of 60 years. However, many clinical trials in AML fail to recruit elderly adults due to a combination of strict entry criteria, or physician or patient bias. Thus, clinical outcome data from many trials may not be readily applicable to older patients with the disease. Furthermore, because the clinical outcome for many older patients with AML is frequently poor, elderly patients who receive intensive chemotherapy with curative intent are frequently selected for treatment on clinical criteria rather than on objective prognostic criteria that may define clinical outcome. The karyotype at the time of presentation may be considered one of the most important prognostic factors in adult AML. Therefore, the aim of this thesis were firstly to analyse the clinical outcome data from a cohort of elderly patients managed at a single centre in order to document the cytogenetic features of AML in an elderly population, to define the prognostic importance of presentation karyotype in the elderly, and to identify other prognostic factors. Retrospective analysis clearly demonstrated improved clinical outcome for older patients with AML over time, primarily as a consequence of improved supportive care and the delivery of more intensive chemotherapy. In addition, 'unfavourable' presentation karyotype, increasing age and raised serum LDH were found to correlate with poor clinical outcome Molecular cytogenetic techniques based upon fluorescence in-situ hybridisation technology offer the chance to detect and analyse cytogenetic aberrations at a higher resolution than can be achieved with conventional techniques. The cytogenetic data provided by comparative genomic hybridisation and mulitplex fluorescence in-situ hybridisation when used in the analysis of elderly patients with AML were found to correlate well with results obtained by conventional methods. Importantly, additive cytogenetic data were more likely to be provided if multiplex-fluorescence in-situ hybridisation was used in the analysis of cases with marker chromosomes or in cases with complex karyotype, although the technique was limited by an inability to reliably detect telomeric translocations. In addition, although both techniques can be used to complement conventional G-banding analysis, conventional FISH methods are often required to confirm the results.