Molecular Cloning of Wild Type and Fusion Genes Associated with the t(X;18) Translocation in Synovial Sarcoma

Chromosomal translocations have been well studied in haematopoietic tumours, but with respect to solid tumours, consistent translocations have only been observed in sarcomas. Synovial sarcoma is a soft tissue tumour occurring predominantly in the adolescent age group of 10-20. A key molecular event in the development of this disease is thought to be the t(X,:18) translocation involving the SS18 gene on chromosome 18 and the SX gene family members 1,2 and occasionally 4 on chromosome X. The aim of this project was to clone cDNA for the fusion genes produced, SS18/SSXI and SS18/SSX2, and also the full-length wild type genes SSX1, SSX2, and SS18. This bank of clones would then be used for functional studies into the role these genes play in normal and malignant environments. To achieve this a PCR based cloning strategy was employed. By amplification of the gene of interest using specific primers located in the 5’ and 3’ flanking regions of the gene, full-length cDNA could be generated for insertion into plasmid. Fully characterised clones were transfected into cell lines to study transcript and protein expression. During this project sequencing S18/SSX2 clone led to the discovery of an unidentified exon (exon 8) in the SS18 portion of the fusion gene. Analysis of wild type SS18 gene expression indicated mRNA splicing into at least two transcripts in human tissues and four in mice, with varying expression levels throughout the tissue types tested. In melanoma tumour samples over expression of the SS18 transcript lacking exon 8 was observed indicating a possible role for this particular transcript in the tumourigenesis process. The molecular function of the genes associated with synovial sarcoma is largely unknown, although recent publications point toward a role in transcription control. The clones produced in this study will provide a platform for in vitro studies into the unction of the wild type and fusion proteins

Mutation Status in Myeloid Malignancies Informs Clinical Insight

The last decade has seen significant development in understanding the genetic basis of myeloproliferative neoplasms (MPN), with the underlying somatic mutational landscape revealed by genome and targeted exome sequencing. The aim of this research was to conduct a series of original investigations to develop insight into the utility and clinical application of these disease markers. Initially, three recently identified disease-specific molecular markers (mutations in JAK2, CALR and CSF3R) were utilised to measure the burden of disease post allogeneic transplantation using methods based on quantitative PCR, capillary electrophoresis and Sanger sequencing. The measured variant allelic frequency of the disease was compared against percentage donor chimerism – a short tandem repeat PCR based method with high discriminatory power that enables a semi-quantitative measure of engraftment based on the calculation of donor and recipient DNA in the blood. All three mutation markers were shown to be stable and robust for monitoring purposes throughout the disease course, inversely correlating with percentage donor chimerism. The increased sensitivity of the JAK2 V617F qPCR used in the JAK2 study highlighted the potential of these disease specific markers to detect molecular and haematological relapse in patients several weeks before clinical manifestation and proved an advantageous adjunct in the clinical assessment of transplantation efficacy. In a second series of studies, the incidence and significance of recurrent MPN mutations in specific clinical scenarios and MPN disease cohorts was investigated. A baseline for the incidence of JAK2, CALR and MPL mutations in Irish patients referred for suspected MPN was established and shown to be comparable with that of other Western European cohorts. To extend this finding, the incidence and significance of these mutations was investigated in specific clinically relevant disease cohorts, revealing a distinct molecular IV pathogenesis in paediatric patients with essential thrombocythaemia (ET) when compared to adults, a low incidence of CALR mutations in splanchnic vein thrombosis patients that correlated with the aetiology of the disease and the presence of CALR driver mutations in familial MPN cases. Additionally, the presence of a JAK2 V617F mutation in utero in ET was established for the first time and the dynamic nature of MPN disease clones over the course of disease was revealed, thus informing insight into the clonal evolution and molecular heterogeneity of MPN patients. This information was collated with results from a number of audits based on clinical referral patterns to a routine molecular diagnostic facility to inform the development of a molecular diagnostic testing algorithm for patients with suspected MPN. In the third series of studies novel next generation sequencing (NGS) technology was applied to investigate somatic mutations in acute myeloid leukaemia (AML) related patient cohorts. A proprietary NGS panel for recurrently mutated genes possessing diagnostic and prognostic significance in AML was evaluated on the Ion Torrent (ThermoFisher) NGS platform across three molecular diagnostic centres in Ireland, Northern Ireland and Scotland. This verified the technique to be robust and reproducible for mutation detection in AML patients. The technology was subsequently deployed to profile mutation status in a clinically high-risk AML patient cohort undergoing transplantation therapy and in a familial AML disease setting to clarify the genotype, reveal the diverse clonal architecture of patients and identify additional driver mutations relevant for existing and emerging therapeutic interventions. Taken together, the studies in this thesis inform clinical understanding and decision making in MPN and AML related settings, provide novel insight into underlying mutational status and clonal behaviour in myeloid malignancies and have the potential to translate into optimised and improved treatment for these patient cohorts

Marketing as a means to transformative social conflict resolution: lessons from transitioning war economies and the Colombian coffee marketing system

Social conflicts are ubiquitous to the human condition and occur throughout markets, marketing processes, and marketing systems.When unchecked or unmitigated, social conflict can have devastating consequences for consumers, marketers, and societies, especially when conflict escalates to war. In this article, the authors offer a systemic analysis of the Colombian war economy, with its conflicted shadow and coping markets, to show how a growing network of fair-trade coffee actors has played a key role in transitioning the country’s war economy into a peace economy. They particularly draw attention to the sources of conflict in this market and highlight four transition mechanisms — i.e., empowerment, communication, community building and regulation — through which marketers can contribute to peacemaking and thus produce mutually beneficial outcomes for consumers and society. The article concludes with a discussion of implications for marketing theory, practice, and public policy

A review of Australian Government funding of parenting intervention research

Objectives: Parenting is central to children's optimal development and accounts for a substantial proportion of the variance in child outcomes, including up to 40% of child mental health. Parenting is also one of the most modifiable, proximal, and direct factors for preventing and treating a range of children's problems and enhancing wellbeing. To determine the effectiveness of new approaches to parenting intervention, and to evaluate how to optimise reach and uptake, sufficient funding must be allocated for high quality research. Method: We reviewed funding awarded by the National Health and Medical Research Council (NHMRC) and Australian Research Council (ARC) for parenting intervention research during 2011–2020. Results: Parenting intervention research received 0.25% of the NHMRC and ARC research budgets. Conclusions: There is a substantial mismatch between the funding of parenting intervention research and the impact of improved parenting on short‐ and long‐term child outcomes. To rectify this, it is critical that Australian Government funding schemes include parenting interventions as priority areas for funding. Implications for public health: Changes in allocation of funding to parenting research will support the establishment of evidence for the effective development, implementation and dissemination of parenting interventions to maximise health outcomes for children and their families

Assumption without representation: the unacknowledged abstraction from communities and social goods

We have not clearly acknowledged the abstraction from unpriceable “social goods” (derived from communities) which, different from private and public goods, simply disappear if it is attempted to market them. Separability from markets and economics has not been argued, much less established. Acknowledging communities would reinforce rather than undermine them, and thus facilitate the production of social goods. But it would also help economics by facilitating our understanding of – and response to – financial crises as well as environmental destruction and many social problems, and by reducing the alienation from economics often felt by students and the public

Monitoring Minimal Residual Disease in the Myeloproliferative Neoplasms: Current Applications and Emerging Approaches

The presence of acquired mutations within the JAK2, CALR, and MPL genes in the majority of patients with myeloproliferative neoplasms (MPN) affords the opportunity to utilise these mutations as markers of minimal residual disease (MRD). Reduction of the mutated allele burden has been reported in response to a number of therapeutic modalities including interferon, JAK inhibitors, and allogeneic stem cell transplantation; novel therapies in development will also require assessment of efficacy. Real-time quantitative PCR has been widely adopted for recurrent point mutations with assays demonstrating the specificity, sensitivity, and reproducibility required for clinical utility. More recently, approaches such as digital PCR have demonstrated comparable, if not improved, assay characteristics and are likely to play an increasing role in MRD monitoring. While next-generation sequencing is increasingly valuable as a tool for diagnosis of MPN, its role in the assessment of MRD requires further evaluation