Hypoxia-inducible factor 1 alpha is required for the tumourigenic and aggressive phenotype associated with Rab25 expression in ovarian cancer

The small GTPase Rab25 has been functionally linked to tumour progression and aggressiveness in ovarian cancer and promotes invasion in three-dimensional environments. This type of migration has been shown to require the expression of the hypoxia-inducible factor 1 alpha (HIF-1α). In this report we demonstrate that Rab25 regulates HIF-1α protein expression in an oxygen independent manner in a panel of cancer cell lines. Regulation of HIF-1α protein expression by Rab25 did not require transcriptional upregulation, but was dependent on de novo protein synthesis through the Erbb2/ERK1/2 and p70S6K/mTOR pathways. Rab25 expression induced HIF-1 transcriptional activity, increased cisplatin resistance, and conferred intraperitoneal growth to the A2780 cell line in immunocompromised mice. Targeting HIF1 activity by silencing HIF-1β re-sensitised cells to cisplatin in vitro and reduced tumour formation of A2780-Rab25 expressing cells in vivo in a mouse ovarian peritoneal carcinomatosis model. Similar effects on cisplatin resistance in vitro and intraperitoneal tumourigenesis in vivo were obtained after HIF1b knockdown in the ovarian cancer cell line SKOV3, which expresses endogenous Rab25 and HIF-1α at atmospheric oxygen concentrations. Our results suggest that Rab25 tumourigenic potential and chemoresistance relies on HIF1 activity in aggressive and metastatic ovarian cancer. Targeting HIF-1 activity may potentially be effective either alone or in combination with standard chemotherapy for aggressive metastatic ovarian cancer

Studies on the AROM Pentafunctional Enzyme in Saccharomyces cerevisiae and Aspergillus nidulans

Aromatic amino acids are synthesised via the shikimate pathway. AROM is a pentafunctional enzyme which catalyses the five central steps of the shikimate pathway and is found in fungi and yeast. This thesis describes the purification and characterisation of AROM from overexpressing strains of Saccharomyces cerevisiae and Aspergillus nidulans. S. cerevisiae AROM was purified 30-fold with a 10% yield from a yeast overexpression strain which exploited the ubiquitin-fusion system of yeast. The purified protein was found to possess all five enzyme activities in a similar ratio to that observed in crude extract and had a subunit molecular weight of 175kDa. The main S. cerevisiae protein was shown to have several minor, lower molecular weight contaminants following SDS PAGE and three of these were found to cross-react with anti-AROM antibodies raised against Neurospora crassa AROM. The peak AROM fraction eluted from gel filtration chromatography was found to be composed of two proteins which were separable by native PAGE and both of which were shown to have shikimate DH activity. The poor recovery and multiple protein bands suggested that during the AROM preparation limited proteolysis was occurring despite a number of anti-proteinase measures. No means of eliminating limited proteolysis during S. cerevisiae AROM isolation were found and purification studies were carried out on the AROM of A. nidulans in the hope that proteolysis might not be as problematic in this species. A rapid procedure for the purification of A. nidulans AROM from the overexpresion strain A. nidulans 1314 has been developed which results in 13-fold purification and a 9% yield. The subunit molecular weight was estimated at 175kDa and the native molecular weight suggests that the protein is a dimer. The N-terminal DHQ synthase activity in the AROM protein was found to be severly deficient in both crude extract and the purified protein from A. nidulans 1314. This was independently attributed to the introduction of a missense muta tion in this region of the polypeptide. A preliminary limited proteolysis study was carried out on AROM purified from A. nidulans 1314 which suggested that the proteolysis pattern is complicated and which show that the DHQase and shikimate DH enzyme activities are least susceptible to limited proteolysis

Learning to Desist: Exploring the relationship between engagement in prison education and desistance from crime

University of Technology Sydney. Faculty of Arts and Social Sciences.This qualitative study explores the relationship between the engagement in prison education and desistance from crime. As a practitioner, I saw a disconnect between the growth learners appeared to experience over time in class and the dominant deficits-based policies, curriculum and pedagogical practices. Education was being understood as a criminological factor based on neoliberal ideas around increased employability leading to reduced reoffending. It felt such an understanding may not sufficiently capture the value of education for learners in prison nor its impact. This thesis applies the theoretical lenses of learning and desistance to adult male learners’ experience of intensive prison education to develop a more robust understanding of its impact on incarcerated learners and the value of quality education within prisons. Significantly, this thesis adopts atypical prison education research methodology, drawing on a strengths-based, more socially-just Appreciative Inquiry approach, together with ethnographic case study and thematic analysis to explore the self-identified best learning experiences of and impact on thirteen adult male learners in full-time basic skills education. The unique purpose-built Intensive Learning Centre in a medium security prison in New South Wales provided an exceptional opportunity to research the learning process when the conditions of learning space, program, staff relations and equal pay opportunities are optimal. Findings indicated that even the most reluctant learners seemed to experience attitudinal shifts towards their capacity to learn, capabilities, and both the desire and ability to desist from crime. Three key themes of Being, Becoming and Belonging were identified as significant to the learners, especially in relation to their experiences of place, culture, identity and basic skills acquisition which were bound by a sense that the educational space, programs and relationships were profoundly normalising and emancipatory. This thesis shows that engagement in high-quality prison education, even at the basic skills level, within fit-for-purpose learning spaces does much more than increase learners’ employability by raising their literacy and numeracy levels to a ‘functional’ standard. In addition, basic-skills education in prison can support the development of learners’ hope, capability, agency, empathy and an interest in civic engagement–characteristics mirrored by successful desisters. Accordingly, this has important implications for prison education policy, programs and pedagogy as well as staff professional development. This thesis suggests that understanding desistance as a learning process and learning as a capability-building process beyond employability may help us support our learners better, develop and deliver better prison education and, ultimately, better prisons in which the desistance process can be catalysed and assisted