The Mll gene was originally identified through its role in infant acute myeloid and lymphoid leukaemias. Mll also has a role in normal haematopoiesis as identified using mouse knockout models. Homozygous mutation of Mll is embryonic lethal, which has limited research into its role in haematopoiesis. To overcome this embryonic lethality, a conditional knockout mouse model of Mll was established. In this model, exons 9 and 10 of Mll were flanked by LoxP sites ('floxed'), which recombined to induce deletion of exons 9 and 10 in the presence of the Cre recombinase. Deletion of exons 9 and 10 lead to complete loss of the MLL protein as detected by immunoblotting. By breeding mice homozygous for the floxed Mll allele to mice that carried the Cre recombinase under the control of the Vav promoter, it was possible to delete Mll within the haematopoietic system. Analysis of foetal and adult haematopoiesis in the absence of Mll was carried out using the new model. In embryos lacking Mll, the foetal liver showed a marked reduction in the number of colony forming cells as well as both long and short term haematopoietic stem cells. When transplanted into lethally irradiated recipients with wild type competitors, Mll deficient foetal liver cells were unable to contribute to reconstitution of the haematopoietic system. In adult mice, removal of Mll had no apparent effect on the steady state haematopoietic system. Populations of myeloid, lymphoid and stem cells were unaffected. However, in competitive repopulation assays, Mll deficient bone marrow cells were unable to compete with wild type cells. This work suggests Mll is needed for the correct development of foetal liver haematopoiesis and also to maintain self-renewal potential in adult haematopoietic stem cells. However, it appears that Mll is not needed to maintain adult haematopoiesis under homeostatic conditions.

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Bowman's capsule parietal epithelial cell activation occurs in several human proliferative glomerulonephritides. The cellular composition of the resulting crescentic lesions is controversial, although a population of renal progenitor cells, which in adult healthy kidney contributes to the physiological cell turnover, has been proposed to be a major constituent. In this study we try to get light into the mediators involved in the aberrant progenitor cell proliferation and migration into the Bowman's space, which occurs in presence of an extended glomerular injury. To this aim, we studied 36 renal biopsies of patients with proliferative and non proliferative glomerulopathies. In parallel, we also analyzed the Munich Wistar Fromter rats with proliferative glomerulonephritis, characterizing for the first time a population of renal progenitor cells also in rodents. We demonstrated that dysregulated progenitor cells of the Bowman's capsule invade the glomerular tuft exclusively in proliferative disorders. In both humans and rats, up-regulation . of the CXCR4 chemokine receptor on progenitor cells was accompanied by high expression of its ligand, stromal-derived factor-1 (SDF-1) in podocytes. Parietal epithelial cell proliferation might be sustained by increased expression of the angiotensinII type1 (AT1) receptor. Treatment with the antihypertensive drug angiotensin-converting enzyme (ACE) inhibitor, reduces the number and the extension of crescents, limiting progenitor cell proliferation and migration. Moreover, ACE inhibitor normalized the expression of CXCR4, SDF-1 and ATl receptor on progenitor cells. These results suggest that glomerular crescents derive from the proliferation and migration of renal progenitors in response to injured podocytes. The SDF-1/CXCR4 axis, together with the AngII/ ATl receptor pathway, contributes to the dysregulated response of renal progenitors. Targeting the AngII/ AT1/ CXCR4 pathway may be beneficial in severe forms of glomerular proliferative disorders.

The focus of my thesis is to apply systems biology approaches to obtain a better understanding of complex cellular systems. In particular, my work concentrates on the function of haematological cells. The first part of the thesis applies a novel, predictive strategy to identify new regulators of a signalling pathway in human immune cells. Cdc42 is a membrane associated GTPase that is an important regulator of cytoskeleton rearrangements and is required for natural killer (NK) cell activation. Previous work had shown that Cdc42 activity oscillates during NK immune surveillance after an initial increase, suggesting that Cdc42 activity is regulated in NK cells by a number of signalling molecules. The aim of the project is to predict proteins required for Cdc42 activity in NK cells and test the predictions. Using a bioinformatics methodology, 13 different proteins were predicted to interact with Cdc42 and form feedback loops. To determine whether any of the predicted proteins were required for Cdc42 activity, NK cells were transfected with a Cdc42 biosensor, each of the predicted targets was downregulated with siRNA and Cdc42 activity was quantified by FRET/FLIM microscopy in the presence of target cells. The screen identified AKT1 and the p85a subunit of PI3K as novel regulators of Cdc42 activity. Depletion of each of these targets also results in an impaired cellular cytotoxic response. This proof of principle study demonstrates the power of a predictive approach in the NK immune surveillance context to identify novel regulators of Cdc42. The second part of my thesis is based on using a predictive methodology, called the ’Phenolog approach’ to predict novel proteins involved in cancer. To determine whether the predicated proteins are required to maintain genome stability, two of the predicted targets were tested using a human primary T cell system, in which cellular mechanisms are normal. Quiescent T cells were transfected with siRNA, the cells were stimulated to enter the cell cycle and chromosome integrity was analysed by interphase FISH. The two predicted targets tested were AND1, a DNA replication protein, and SEC13, a component of the nuclear pore complex. Depletion of each of the targets led to a number of chromosomal abnormalities,indicating that their normal expression during the G0—>G-i transition is required to maintain genome stability. The third part of my thesis focuses on the systematic analyses of the chromatin proteome of T cells during cell cycle progression and identifying changes in the proteome caused by depleting the DMA replication protein Mcm7. Reducing the induction of Mcm7 causes DMA damage, premature chromatid separation and genome instability (291). Initially, the chromatin proteome obtained by a native, non-crosslinked chromatin extraction method was compared with that obtained after formaldehyde crosslinking. In addition, the methodology was compared with the proteome obtained previously using the CSK extraction method (290). To analyse the effects of depleting Mcm7, quiescent primary T cells were transfected with Mcm7 siRNA and the cells were stimulated to enter the cell cycle. The proteins were crosslinked with formaldehyde, chromatin was isolated and the chromatin-bound proteome in Mcm7-depleted and control cells were analysed by LC-MS/MS. Changes in the nuclear proteome caused by depleting Mcm7 were quantified by a label-free spectral counting method. Network analyses (HumanNet) were used to identify protein interaction sub-networks. The analyses identified that downregulation of Mcm7 affects a number of processes, including DNA replication, DNA damage, transcription and ribosome biogenesis.

Introduction Haematopoietic progenitor cells (HPCs) may attenuate the response to acute vascular injury by maintaining endothelial integrity and function. The aim of this study was to determine whether circulating HPC number and function, and mobilising cytokines, reflect significant carotid disease or correlate with restenosis following carotid endarterectomy (CEA). Methods Initially the assay conditions to measure HPC number and function were optimized. HPC numbers were measured by flow cytometry (CD133+ve/CD34+ve) and early colony forming unit assay (eCFU). HPC function was measured by migration assay and by staining for senescence associated B-galactosidase (SA-Bgal). HPC number and function was then measured in 62 patients undergoing CEA pre-operatively, 1 day post operatively and 6 weeks post-operatively. Restenosis was assessed by duplex scanning at 3, 6 and 12 months. The circulating profile of GM-CSF, PIGF, SDF1 and VEGF was measured by multiplex ELISA. Results HPC numbers (P<0.001) and eCFU counts (P=0.001) fell rapidly 24hrs after CEA. The percentage post-operative fall in CD133+ve/CD34+ve cell numbers negatively correlated with degree of restenosis at the 6 month scan (r= -0.38, p=0.013). The percentage fall in eCFU number negatively correlated with degree of restenosis at the 6 (R=-0.42, P=0.008) and 12 month scans(R=-0.49, P=0.026). The migration rate of HPCs isolated from pre-operative blood also negatively correlated with restenosis at the 6 (R=-0.5, P=0.009) and 12 month scans(R=-0.53, P=0.05). Pre-operative SDF1 levels correlated with falls in CD133+ve/CD34+ve number (R=0.42, P=0.044) and eCFU counts (R=0.56, P=0.004), though not with restenosis. Conclusion HPC function appears to be linked to the development of carotid artery restenosis following endarterectomy. These data support the concept that HPCs have a role in regulating remodelling of the unstable and injured arterial wall.

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No description available.

The urinary tract (UT) is a sterile environment despite being constantly challenged by pathogens from its surrounding environment. Protection of the uroepithelium from microbial assault involves the innate immune system, but still, to date, little is known of the actual mechanisms that function in such defences. The focus of the research presented in this thesis, which used RT4 and VK-2 E6/E7 in vitro cell systems to model the bladder and vaginal epithelia respectively, was to interrogate the mechanisms by which these epithelia recognise and respond to microbial assault. The RNA of bladder RT4 cells challenged with motile and non-motile Escherichia coli strains was analysed via a TLR related gene array. The data supported upregulation of the NF-κB dependent cytokine IL-8 and thus NF-κB signalling was used to measure the uroepithelial response to microbial infection. Flagellin, recognised by TLR-5, and responsible for bacterial motility, induced a significant (p<0.001), 44.9±1.7 fold increase, in RT4 NF-κB signalling within 4h of challenge and with use of TLR-5 blocking antibody was identified as the key activating PAMP in the UT. Other bacterial cell wall components, namely LPS and peptidoglycan, failed to induce comparable NF-κB activation (maximum 10.7± 0.8 and 6.3±0.6 fold increase at 16 hours post challenge). These data led to the hypothesis that strains associated with urinary tract infection (UTI) have reduced or no motility. However, 12/24 clinical strains associated with UTI were motile and induced a NF-κB signalling response. Variability in the responses (3.4 0.3 to 40.3 1.8 fold at 8 hours post challenge), could not be explained by flagellin, suggesting that other factors, host and bacterial, may also contribute to the intensity of the response. Analysing UPEC strains isolated from the urine of patients carrying a C1174T SNP encoding a truncated TLR-5 revealed such strains to induce normal NF-κB signalling (39.5 0.3 fold) in RT4 cells. These data emphasised the importance of TLR-5 and bacterial motility in the innate response of the host to a UTI. Human (h) βD-2, a member of the Defensin family with microbial killing properties, is important in the innate defences of the uroepithelium. To identify agents that could be used therapeutically to enhance the innate defences of the UT, a reporter construct (phβD-2-Luc), containing 2 kbp of hβD- 2 5’UTR sequence, was transfected into RT4 and VK-2 E6/E7 vaginal cells. Reporter activity (fold increase) was significantly increased with calcitriol (VitD, 10 nM), or 17β-oestradiol (17β-oes, 4 nM), treatments when challenging VK-2 E6/E7 with either flagellin (68.1±8.5 vs. 196.3±28.3 for VitD, p<0.001, or vs. 199.5 37.7 for 17β-oes, p<0.01) or E. coli NCTC 10418(19.9±3.8 vs. 40.1±4.3 for VitD, p<0.01) or vs. 40.2±6.6 for 17β-oes, p<0.05). The hβD-2 reporter data also revealed Zymosan (50 μg/ml) enhancing reporter activity in both cell lines (RT4 34.8±5.0 and VK-2 E6/E7 27.2±2.9, both p<0.001) and had a synergistic effect to flagellin increasing reporter activity by more than 60 % (p<0.05). These data suggested that Zymosan, vitamin D as well as oestrogen regulate the hβD-2 gene. The expression of hβD-3, another member of the Defensin family with microbial killing properties, was identified in human bladder biopsies so its expression was further investigated in vitro. While no hβD-3 expression was measured in the RT4 cells, constitutive and inducible expression was identified in VK-2 E6/E7 cells. Human βD-3 expression was enhanced at 8 hours following challenge of the cells (PBS 70.5±13.4) with E. coli NCTC 10418 (1094.5±293.8, p<0.001), flagellin (518±139.2, p<0.01) and Zymosan (280.8±65.7, p<0.01). Induction was also observed with LPS but at 16 hours (27.0±8.1 (PBS), 612.2±260.8, p<0.001) but not with peptidoglycan (32.2±12.3, p>0.05). These data suggested that hβD-3 functions in the defence of urogenital tract and as its expression was inducible, a potential target for therapeutic strategies. The fungal cell wall β1,3-glucan, Zymosan, induced NF-κB signalling as well as hβD-2 in the RT4 and VK-2 E6/E7 cells, and hβD-3 expression and secretion in VK-2 E6/E7 cells. Molecular analysis of the cell RNA identified the expression of three isoforms (417, 330, 211 bp) of the Dectin-1 (CLEC7A) receptor and immunocytochemistry identified the receptor protein localised to the cell membrane. Following Zymosan challenge of VK-2 E6/E7 cells, receptor clustering and the phosphorylation of a Dectin-1 signalling protein, SYK was observed supporting the presence of functional receptors. Induction of NF-κB signalling by Zymosan was inhibited by blocking TLR-5 (4.2±0.4 vs 2.8±0.1, p<0.01) suggesting functional interactions between Dectin-1 and TLR-5. These data provide support for the presence of Dectin-1 receptor in the urogenital tract. In conclusion, the data presented in this thesis confirms the importance of TLR-5 in the defence of the urogenital tissues. Investigations of hβD-2 and hβD-3 expression and their enhancement in the UT have led to the discovery of a role for the Dectin-1 receptor. This receptor represents a target for future therapeutic strategies to enhance the innate response.

ATP activates P2X receptors in the apical surface of kidney epithelial cells. However, tubular fluid is unusual - with low sodium levels, high calcium levels and acidic in nature. This study investigated how P2X receptors operate in extreme ionic environments. Xenopus oocytes easily tolerated extreme ionic environments. Their membrane properties and pharmacological profile were examined under normal and extreme ionic conditions. It was confirmed that oocytes lack endogenous P2X receptors, and were unaffected by P2X receptor antagonists and allosteric modulators. Also, oocytes lacked muscarinic receptors. The pharmacological profile of P2X2 and P2X4 receptors expressed in oocytes were examined under normal and extreme ionic conditions. Each P2X subtype was activated by ATP and CTP and this activity was affected by extracellular ions. At rat P2X2, reduced extracellular sodium had an inhibitory effect on ATP signalling, which was reversed by acidifying of the medium or intensified by adding calcium to the medium. At rat P2X4, reduced extracellular sodium had a biphasic action (potentiation, then inhibition) on ATP signalling, which was insensitive to acidification but was intensified by adding calcium. A unique electrophysiological property of P2X2-expressing oocytes was used to investigate an ATP-releasing mechanism. An inward current (Ix) was identified and this current was blocked by the P2 receptor antagonist, suramin, and inhibited by the ATP-degrading enzyme, apyrase. The amplitude of IX was affected by the flow rate of the superfusate, confirming that ATP was released by a mechanosensory process. The present results show that ATP may activate P2X receptors present in the kidney, even in the extreme ionic environment of the distal nephron. Salt and water reclamation in the kidney may depend on flow rate, since a common mechanosensory mechanism can release enough ATP to activate those P2X receptors regulating ENaC and AQP2 channel function in the distal nephron.

No description available.