Dense deposit disease (DDD) is a rare, progressive and incurable kidney disease characterized by complement C3 accumulation along the glomerular basement membrane (GBM). It is the prototypical form of C3 glomerulopathy, which comprises renal disorders due to excessive C3 activation via the alternative pathway (AP). Human and murine studies indicate that renal inflammation in DDD is initiated by the specific C3 activation product iC3b. I have assessed the role of iC3b in DDD using the uniquely informative experimental model of C3 glomerulopathy in factor H (FH)-deficient mice. I demonstrate that coexisting deficiency of CD11b, the specific leukocyte receptor for iC3b, exacerbates the spontaneous renal phenotype in FH-deficient mice. This suggests that the iC3b-CD11b interaction may mediate partial renal protection in DDD. I also show that CD11b deficiency produces hypersensitivity to experimentally triggered glomerular injury in both FH-deficient and FH-sufficient mice. My second experimental approach in vivo and in vitro was to assess whether C3 activation in the circulation or on the GBM surface is the predominant cause of iC3b accumulation in DDD. This included experiments in which administration of a novel engineered human FH protein fragment reduced glomerular C3 staining in FH-deficient mice.

Brachytherapy using low dose rate (LDR) permanent seed implant or high dose rate (HDR) temporary implant is a well established treatment for prostate cancer. This study investigates the use of advanced dose calculation and imaging techniques to improve clinical prostate brachytherapy treatments. Monte Carlo (MC) simulations are used to assess the impact of source interactions and tissue composition effects that are ignored by the TG-43U1 dose calculation algorithm used in clinical practice. MC simulation results are validated using experimental phantom measurements. The development of prostate cancer may be driven by a dominant intra-prostatic lesion (DIL) but standard brachytherapy treatments prescribe the same dose level to the whole prostate. This study assesses the feasibility of multi-parametric (mp-MRI) guided focal boost treatments that escalate dose to the DIL to improve tumour control and of focal treatments that target the DIL to reduce treatment related side effects. Source interactions and tissue effects are shown to reduce the dose that is delivered to patients in LDR treatments, particularly for patients with calcifications, however the dosimetric impact is small compared to other uncertainties in LDR seed implant brachytherapy. For HDR treatments attenuation by steel catheters has only a small impact on dose distributions. Feasibility of mp-MRI guided focal boost HDR prostate brachytherapy is demonstrated in terms of tumour delineation and the ability to dose escalate the DIL without increased dose to normal tissues. The dosimetric feasibility of LDR and HDR focal therapy treatments is demonstrated. Focal therapy treatments are shown to be more sensitive to source position errors than whole gland treatments. MC simulations of focal therapy treatments show that there are no additional concerns in terms of dosimetric accuracy compared to standard whole gland treatments. Advanced dose calculation and imaging techniques can improve clinical prostate brachytherapy treatments.

Introduction Performance in healthcare systems is under constant scrutiny, with particular focus on safety, quality and efficiency. The management of many long-term diseases involves complex patterns of care processes. However, the mechanisms by which this works are poorly understood, with care pathways reflecting pre-determined, linear processes. Renal anaemia is a significant complication of chronic kidney disease that is expensive to treat and is associated with reduced quality of life and increased morbidity and mortality. There is wide variation between the clinical performance measures of centres for renal anaemia management, which researchers and practitioners have suggested is due to differences in organisation. Aims and objectives This research explores the features of high and low performing renal anaemia management services to build evidence for their improvement. Methods A multiple case studies approach examining anaemia management at eight renal centres with high and low performance was used. Data were collected using observation, interview and document sampling. The Unified Modelling Language was employed to define an abstract framework of the renal anaemia management service. A thematic analysis explored factors considered influential in the process and performance of renal anaemia management. Results System latency and reliability, regular communication and trust between decision makers all appear significant in producing high performance. The group of activities and coordination mechanisms used to manage renal anaemia in haemodialysis patients in high performing centres reflect these features. The current tax regime encourages practice that has neither quality nor efficiency at its core. Discussion The findings provide an important platform for performance improvement in renal anaemia management. In addition, the process modelling represents a departure from the major body of such work in healthcare, using a richer language to represent the complex and adaptive nature of healthcare systems. Therefore, the approach presented here may be a useful template for future work in this field.

No description available.

The ability of the urothelium to act as a urinary barrier is afforded by two key features: 1) It has the tightest barrier function of any epithelium, generated by tight junctions which assemble upon differentiation; 2) In response to damage, the quiescent urothelium can rapidly switch to a regenerative phenotype to enable regeneration and restitution of barrier function. Central to repair is the ability for the urothelium to sense damage; a process hypothesised to involve cell-cell communication. Direct cell-cell communication occurs through gap junctions, channels comprising connexin (Cx) proteins which allow for the passage of signalling molecules. Cxs have been linked to wound healing, as well as to maintenance of polarity and homeostasis in other epithelia, by both communication-dependent and independent mechanisms. A systematic characterisation of Cx expression has not been performed in urothelium and the significance of specific Cxs to urothelial physiology is not understood. This study aimed to investigate whether specific Cx proteins contribute to the regulation of barrier maintenance and restitution in human urothelium. Analysis of Cx transcripts identified Cx32, which was expressed in situ and highly induced upon in vitro differentiation of normal human urothelial (NHU) cells using two independent methods. In differentiated NHU cells, Cx32 proteins assembled into functional gap junction channels at cell borders, co-localising with the barrier defining tight junction proteins occludin and ZO-2. shRNA studies demonstrated that normal tight junction development and barrier function were dependent on the presence of intercellular Cx32, but not gap junction communication. In wound-healing experiments, intercellular communication through Cx32 channels inhibited urothelial cell migration and proliferation, in a process which involved suppression of activated SMAD3. Together the evidence presented here supports an unanticipated central role for Cx32 in orchestrating the homeostasis between barrier function and repair in human urothelium.

No description available.

Sacral nerve stimulation, or neuromodulation, has been shown to restore voiding in women with a specific type of urinary retention that is attributed to urethral sphincter overactivity. The therapy has gained popularity in this and other voiding dysfunctions, but its mechanism of action remains unexplained. This thesis explores the effects of neuromodulation on women with urinary retention. It incorporates a urodynamic study of the effect of neuromodulation on bladder and urethral (peripheral) function, a functional brain imaging PET (Positron Emission Tomography) study of cerebral (central) effects, and a review of the long-term efficacy of the technique. The urodynamics (including urethral pressure profilometry, cystometry, and sphincter electromyography) showed evidence of persistent urethral overactivity despite successful restoration of micturition. Together with the cystometric findings, this suggests that neuromodulation may facilitate voiding in this group by increasing detrusor contractility rather than by urethral relaxation. Review of the sacral nerve implants performed at this centre over several years reveals that approximately 75% continue to void at up to 5 years after surgery, while considering reasons for the loss of efficacy in other patients. The cerebral perception of bladder fullness was examined using PET scanning in a group of healthy female controls as well as women with retention treated by neuromodulation. The findings show that the brainstem activity which is present in healthy controls is not seen in retention patients until the neuromodulation is activated. The discussion addresses the respective roles of brainstem and cortical brain regions in the control of voiding function, and whether neuromodulation may 'normalize' cerebral activity. In conclusion, this thesis provides evidence, for the first time, of changes in brain activity following sacral neuromodulation in urinary retention, confirming that its effects may well be mediated by afferent innervation.

No description available.

Factor Inhibiting Hypoxia-inducible Factor (FIH) is an asparaginyl hydroxylasewhich regulates the transcription factor Hypoxia-Inducible Factor (HIF), viahydroxylation of a conserved asparagine residue of the HIF-alpha subunits (twoisoforms of which are well established, HIF-1 and HIF-2alpha - HIF-3alpha isless well characterized currently). Other targets of FIH have also been reported. Little is known about both FIH expression and function. This thesis willinvestigate the expression of FIH in rodents, in cell lines and in renal cancertissue samples. In addition, RNAi technology was used to study FIH function.Clear cell renal cell carcinoma (CCRCC) is commonly associated withinactivation of tumour suppressor von-Hippel Lindau protein (VHL) andconstitutive activation of HIF. The main question I have addressed in this thesisis whether FIH decreases HIF activation in this setting. To address this I inhibitedFIH using several approaches. Specifically, using hypoxia, dimethyloxalylglycine(DMOG) and RNA interference (RNAi). Each of these increased the expressionof HIF target genes in two different CCRCC cell lines, RCC10 and RCC4. Investigating three different CCRCC cell lines, FIH inhibition decreased numbers of RCC4 and RCC10 cells growing in culture, which is likely to be due to an increase in expression of pro-apoptotic, FIH-regulated HIF target genes. Interestingly, 786-O cells exclusively express the HIF-2alpha isoform. Attenuation of FIH in this setting did not affect expression of HIF target genes, nor affect growth in culture. To determine if this was due to lack of FIH or may be due tolack of HIF-1alpha, I introduced HIF-1alpha via a viral vector. Following this, sensitivity to FIH was clearly demonstrated. This implies either specific 'protection' of HIF-2alpha in 786-O or more general FIH selectivity for the HIF-1alpha isoform. My findings contrast with two reports suggesting that FIH expression is suppressed in CCRCC. My findings give insight into how FIH asparagine hydroxylation regulates HIF, in particular in renal cancer and makes this enzyme a potential target for therapeutic inhibition, in the majority of renal cancers.

Overactive bladder (OAB) syndrome is associated with increased spontaneous contractions of the bladder wall, potentially mediated by the release of chemical agents from the urothelium. Reduction of these spontaneous contractions offers a means to alleviate the symptoms of OAB. I tested two novel approaches using pig bladder in vitro or ex vivo preparations; bladder wall heating or injection of inert bulking agents. In vitro, intact (mucosa + detrusor muscle) preparations were heated to 42, 46, or 50oC by a heating coil. Preparations of only detrusor muscle or mucosa were heated by changing superfusate temperature. Experiments were done to examine the role of heat-sensitive TRPV1 channels during heating by using TRPV1 antagonists. The effect of heating on urothelium ATP release was also measured. Possible changes to tissue structure were histologically assessed with haematoxylin & eosin or van Gieson staining. Inert bulking agents (Tyrode’s, polyethylene glycol or coaptite) were injected into the sub-mucosal space and their effect on spontaneous contractions also measured. Ex vivo experiments with perfused pig bladders recorded spontaneous pressure variations when perfusate temperature was increased to 42°C. Spontaneous contractions in intact preparations were reversibly reduced when heated to 42, 46, or 50oC; TRPV1 antagonists had no effect. Heating to 42oC did not affect mucosa or detrusor-only preparations, but at 50°C contractions were abolished. Similar effects were seen in ex vivo experiments when heated to 42°C. No changes to tissue integrity were observed at 42 or 46°C. At all temperatures urothelial ATP release was increased. Spontaneous contractions were reduced by all bulking agents, coaptite was the most effective. These novel findings suggest possible clinical approaches to treat the symptoms of OAB by reducing spontaneous contractions.