http://hdl.handle.net/10468/67 Mon, 20 May 2013 00:10:19 GMT 2013-05-20T00:10:19Z http://hdl.handle.net/10468/962 FLT3-driven redox signalling in acute myeloid leukaemia Corcoran, Aoife Acute myeloid leukaemia refers to cancer of the blood and bone marrow characterised by the rapid expansion of immature blasts of the myeloid lineage. The aberrant proliferation of these blasts interferes with normal haematopoiesis, resulting in symptoms such as anaemia, poor coagulation and infections. The molecular mechanisms underpinning acute myeloid leukaemia are multi-faceted and complex, with a range of diverse genetic and cytogenetic abnormalities giving rise to the acute myeloid leukaemia phenotype. Amongst the most common causative factors are mutations of the FLT3 gene, which codes for a growth factor receptor tyrosine kinase required by developing haematopoietic cells. Disruptions to this gene can result in constitutively active FLT3, driving the de-regulated proliferation of undifferentiated precursor blasts. FLT3-targeted drugs provide the opportunity to inhibit this oncogenic receptor, but over time can give rise to resistance within the blast population. The identification of targetable components of the FLT3 signalling pathway may allow for combination therapies to be used to impede the emergence of resistance. However, the intracellular signal transduction pathway of FLT3 is relatively obscure. The objective of this study is to further elucidate this pathway, with particular focus on the redox signalling element which is thought to be involved. Signalling via reactive oxygen species is becoming increasingly recognised as a crucial aspect of physiological and pathological processes within the cell. The first part of this study examined the effects of NADPH oxidase-derived reactive oxygen species on the tyrosine phosphorylation levels of acute myeloid leukaemia cell lines. Using two-dimensional phosphotyrosine immunoblotting, a range of proteins were identified as undergoing tyrosine phosphorylation in response to NADPH oxidase activity. Ezrin, a cytoskeletal regulatory protein and substrate of Src kinase, was selected for further study. The next part of this study established that NADPH oxidase is subject to regulation by FLT3. Both wild type and oncogenic FLT3 signalling were shown to affect the expression of a key NADPH oxidase subunit, p22phox, and FLT3 was also demonstrated to drive intracellular reactive oxygen species production. The NADPH oxidase target protein, Ezrin, undergoes phosphorylation on two tyrosine residues downstream of FLT3 signalling, an effect which was shown to be p22phox-dependent and which was attributed to the redox regulation of Src. The cytoskeletal associations of Ezrin and its established role in metastasis prompted the investigation of the effects of FLT3 and NADPH oxidase activity on the migration of acute myeloid leukaemia cell lines. It was found that inhibition of either FLT3 or NADPH oxidase negatively impacted on the motility of acute myeloid leukaemia cells. The final part of this study focused on the relationship between FLT3 signalling and phosphatase activity. It was determined, using phosphatase expression profiling and real-time PCR, that several phosphatases are subject to regulation at the levels of transcription and post-translational modification downstream of oncogenic FLT3 activity. In summary, this study demonstrates that FLT3 signal transduction utilises a NADPH oxidase-dependent redox element, which affects Src kinase, and modulates leukaemic cell migration through Ezrin. Furthermore, the expression and activity of several phosphatases is tightly linked to FLT3 signalling. This work reveals novel components of the FLT3 signalling cascade and indicates a range of potential therapeutic targets. Tue, 01 Jan 2013 00:00:00 GMT http://hdl.handle.net/10468/962 2013-01-01T00:00:00Z http://hdl.handle.net/10468/1099 Administration of a live culture of Lactococcus lactis DPC 3147 into the bovine mammary gland stimulates the local host immune response, particularly IL-1beta and IL-8 gene expression Beecher, Christine; Daly, Mairead; Berry, Donagh P.; Klostermann, Katja; Flynn, James; Meaney, William; Hill, Colin; McCarthy, Tommie V.; Ross, R. Paul; Giblin, Linda Mastitis is one of the most costly diseases to the dairy farming industry. Conventional antibiotic therapy is often unsatisfactory for successful treatment of mastitis and alternative treatments are continually under investigation. We have previously demonstrated, in two separate field trials, that a probiotic culture, Lactococcus lactis DPC 3147, was comparable to antibiotic therapy to treat bovine mastitis. To understand the mode of action of this therapeutic, we looked at the detailed immune response of the host to delivery of this live strain directly into the mammary gland of six healthy dairy cows. All animals elicited signs of udder inflammation 7 h post infusion. At this time, clots were visible in the milk of all animals in the investigation. The most pronounced increase in immune gene expression was observed in Interleukin (IL)-1β and IL-8, with highest expression corresponding to peaks in somatic cell count. Infusion with a live culture of a Lc. lactis leads to a rapid and considerable innate immune response. Sun, 01 Feb 2009 00:00:00 GMT http://hdl.handle.net/10468/1099 2009-02-01T00:00:00Z http://hdl.handle.net/10468/69 Effects of apigenin, lycopene and astaxanthin on 7β-hydroxycholesterol-induced apoptosis and Akt phosphorylation in U937 cells Lordan, Sinéad; O'Neill, Cora; O'Brien, Nora M. Oxysterols arise from the enzymic or non-enzymic oxidation of cholesterol and have been shown to be cytotoxic to certain cell lines. In particular, apoptosis induced by the oxysterol 7β-hydroxycholesterol (7β-OH) has been associated with the generation of oxidative stress, cytochrome c release and caspase activation. Due to the fundamental importance of apoptosis in pathological processes, the identification of substances capable of modulating this form of cell death is now actively researched. The objective of the present study was to investigate if apigenin, lycopene and astaxanthin could inhibit 7β-OH-induced apoptosis in U937 cells. Pretreatment with 0.1 µM-astaxanthin protected against apoptosis, while lycopene did not oppose the adverse effects of 7β-OH. At low concentrations, apigenin did not protect against oxysterol-induced apoptosis; however, at higher concentrations it intensified cell death. Additionally, we investigated the effect of 7β-OH, apigenin and astaxanthin on the activation of the serine threonine kinase Akt (phosphorylated Akt:Akt ratio) to determine whether the effect on cell viability and growth was linked to the Akt signalling pathway. Akt activation was decreased in the oxysterol-treated cells compared with control cells; however, this did not attain significance. Interestingly, activation of Akt was significantly reduced compared with control cells following incubation with apigenin and astaxanthin both in the absence and in the presence of 7β-OH. Our data suggest that apigenin, lycopene and astaxanthin failed to protect against 7β-OH-induced apoptosis, and the decrease in cell viability and the increase in apoptotic nuclei induced by the antioxidants appear to be associated with down regulation of Akt activity. Fri, 01 Aug 2008 00:00:00 GMT http://hdl.handle.net/10468/69 2008-08-01T00:00:00Z http://hdl.handle.net/10468/804 The identification and characterisation of Rab4 interacting proteins Lindsay, Andrew J. Rab4 is a member of the Rab superfamily of small GTPases. It is localized to the early sorting endosome and plays a role in regulating the transport from this compartment to the recycling and degradative pathways. In order to further our understanding of the role Rab4 plays in endocytosis, a yeast two-hybrid screen was performed to identify putative Rab4 effectors. A constitutively active mutant of Rab4, Rab4Q67L, when used as bait to screen a HeLa cDNA library, identified a novel 80kDa protein that interacted with Rab4-GTP. This protein was called Rab Coupling Protein (RCP). RCP interacts preferentially with the GTP-bound form of Rab4. Subsequent work demonstrated that RCP also interacts with Rab11, and that this interaction is not nucleotide-depenedent. RCP is predominantly membrane-bound and localised to the perinuclear recycling compartment. Expression of a truncation mutant of RCP, that contains the Rab binding domain, in HeLa cells, results in the formation of an extensive tubular network that can be labelled with transferrin. These tubules are derived from the recycling compartment since they are inaccessible to transferrin when the ligand is internalised at 18oC. The truncation mutant-induced morphology can be rescued by overexpression of active Rab11, but not active Rab4. This suggests that RCP functions between Rab4 and Rab11 in the receptor recycling pathway, and may act as a ‘molecular bridge’ between these two sequentially acting small GTPases. Quantitative assays demonstrated that overexpression of the truncation mutant results in a dramatic inhibition in the rate of receptor recycling. Database analysis revealed that RCP belongs to a family of Rab interacting proteins, each characterised by a carboxy-terminal coiled-coil domain and an amino-terminal phospholipid-binding domain. KIAA0941, an RCP homologue, interacts with Rab11, but not with Rab4. Overexpression of its Rab binding domain also results in a tubular network, however, this tubulation cannot be rescued by active Rab11. Sat, 01 Dec 2001 00:00:00 GMT http://hdl.handle.net/10468/804 2001-12-01T00:00:00Z