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Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest solid tumours, with 5-year survival of less than 5%. The aggressive nature of the tumour, the tumour-supportive environment (stroma), delayed diagnosis and poor chemotherapy response lead to poor patient prognosis. The tumour-stroma is a dynamic network of immune, mesenchymal, and endothelial stromal cells surrounded by an extracellular matrix rich in matricellular and architectural proteins. Periostin (POSTN), a matricellular protein primarily secreted by stromal cells, is often associated with cell proliferation and migration. POSTN overexpression has been observed in many tumour types, however downregulation in bladder carcinoma rises some questions. POSTN is an alternatively sliced gene, that encodes multiple different isoforms, whose function in PDAC is still unknown. The aim of this study is to isolate and clone these isoforms in order to produce recombinant proteins for further investigation. Methods: The transcript variants were extracted from PS1 cells (a pancreatic stromal cell line), converted to cDNA and amplified by RT-PCR. The synthesised DNA was ligated with plasmid vectors and cloned by traditional cloning into E.coli cells. Single colonies were sequenced and analyses, determining the transcript variant being cloned. The plasmids ligated with the variants of interest were isolated and transfected into PS1 cells. The recombinant proteins synthesised by the PS1 cells were then purified. Results: Multiple isoforms of POSTN were cloned in E.coli. Sequencing analysis revealed the exons present in each sample of the cloned colonies and the isoforms were identified. The plasmids of interest were successfully extracted from the colonies by using purification technique appropriate for subsequent mammalian cell transfection. Substantial progress was made in producing recombinant POSTN proteins. Conclusion: In this study the isolation of three cloned POSTN transcript variants were achieved. These variants are POSTN-202, 209 and 201-b.

Acute myeloid leukaemia (AML), also known as acute myelogenous leukaemia is a cancer of the myeloid line of blood cells, characterized by the rapid proliferation of abnormal myeloid cells accumulating in the bone marrow that interfere with normal haematopoiesis. Although AML is a relatively rare disease, which accounts for almost 1% of cancer deaths in the United Kingdom and 1.2% of cancer deaths in the United States, it is the most common acute leukaemia; primarily affecting adults, and its incidence is known to increase with age. To date, there is still very little understood about the risk factors for the development of AML beyond the onset of old age. However, some key pre-existing haematological disorders such as myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN) are now known to have a relatively high incidence of developing AML (15-52% and 7% respectively). In recent years, there have been great strides made towards understanding some of the major genetic changes that occur in AML. Normal haematopoiesis is strictly regulated by the HOX family of transcription factors which have been shown to play a key role in the development of cancer when elevated in the normal peripheral blood mononuclear cells (PBMCs) of the elderly. Together with its known role as an oncogene in AML, and a predictor of survival in this disease, it is suggested that HOXA9 expression in PBMCs could be a predictive marker of non-therapy related AML. This study suggests that elevated HOXA9 expression is indeed a predictive marker for AML development and attempts to shine a light on the progression of AML from pre-leukemic diseases such as MDS and MPN as well as offering a possible solution which may be used as a therapeutic drug either alone or in combination with current commonly used therapeutics.

Haemopoietic activity in Aplastic anemia (AA) is significantly reduced and is generally attributed to failure of Haemopoietic stem cells (HSC) within the bone marrow. The extent to which Mesenchymal stromal cells (MSC) are involved in the functional restriction of HSC is largely unknown. To understand this, the physical and functional properties of AA MSC were studied in vitro. MSC were characterised by their phenotype and ability to form adherent stromal layers. The functional properties of AA MSC were assessed through proliferative, clonogenic and cross¬over culture assays. Results show several alterations in the physiological and functional status of MSC in AA. Although AA MSC presented typical morphology and distinctive mesenchymal markers, stromal formation was significantly reduced; furthermore, their proliferative and clonogenic capacity was markedly decreased and ability to sustain haemopoiesis was also reduced as assessed by total cell proliferation and clonogenic ability of HSC. It was concluded that the biological characteristics of AA MSC are different from those of control MSC and their in vitro haemopoiesis-supporting ability significantly weaker. The second aim of this study was to identify serum proteins that change expression in untreated AA patients compared to Anti-thymocyte globulin treated patients and healthy controls that might be useful in understanding and monitoring of AA. Serum was profiled using SELDI¬TOFIMS, detecting a number of differentially expressed protein peaks. A combined strategy of chromatographic fractionation, ID gel electrophoresis, passive elution, trypsin digestion and MALDI-TOFIMS or LC-MS/MS analysis, formally identified six discriminatory peaks, further validated with Western blot and ELISA. They include acute phase response proteins: Haptoglobin, Transthyretin, Alpha-l-aeid glycoprotein and Apolipoprotein Al as well as Apolipoprotein Cl and C3 and represent novel disease- and therapy- associated AA biomarkers. Inflammatory biomarkers in AA serum are consistent with immune-mediated pathology of AA and suppressive action of Anti-thymocyte globulin, and can potentially be used for disease detection, diagnosis and monitoring.