The alternative pathway (AP) of complement activation is implicated in several renal pathologies. These include membranoproliferative glomerulonephritis (MPGN) type II and atypical haemolytic uraemic syndrome (aHUS). Factor H (FH), comprised of 20 short consensus repeats (SCRs), is the primary regulator of the AP at the glomerular basement membrane (GBM) as evidenced by the fact that impaired factor H function is linked to both diseases. The current treatment options for MPGN type II and aHUS are limited and patients ultimately develop renal failure. There is a clear need for kidney targeted therapeutics to control AP dysregulation. I sought to generate novel recombinant FH fusion proteins to replace dysfunctional FH and to deliver the complement regulatory domain of factor H (SCRs 1-5) to the GBM. A protein containing the regulatory SCRs (FH1-5) and one containing the regulatory and cell-surface binding SCRs (FH1-5/18-20) was generated and expressed in a eukaryotic expression system. C3b binding and full complement regulatory function of FH1-5 and FH1- 5/18-20 was confirmed by in vitro assays. To target the GBM a B cell hybridoma (mAb3) was obtained which secretes an antibody that binds to the NC1 domains of collagen type IV alpha-3 chain, expressed predominately in the GBM. The mAb3 variable regions (Fv) were successfully cloned after elimination of an abundantly expressed endogenous light chain transcript. A mAb3 single chain FV (scFv) was cloned downstream of FH1-5 creating a FH1-5/mAb3 scFv fusion protein. As an alternative a fusion protein incorporating the scFv of the 1G6 monoclonal antibody (FH1-5/1G6 scFv) was generated. Low expression levels of the FH1-5/scFv proteins limited the functional analysis. Binding of both proteins to C3b and binding of FH1-5/1G6 scFv but not FH1-5/mAb3 scFv to bovine and murine NC1 domains was demonstrated using cell culture supernatants. These reagents remain in the early stage of development but have potential to be attractive alternatives for the treatment of aHUS and MPGN type II. In addition to MPGN type II, FH1-5/scFv may find application in the treatment or prevention of anti-GBM nephritis and renal ischaemia-reperfusion injury.

Atypical haemolytic uraemic syndrome (aHUS) is renal disease associated with mutations and/or polymorphisms in genes encoding complement proteins, including complement factor H (CFH), factor I (CFI) and membrane cofactor protein (CD46). Recently, deficiency of CFH-related (CFHR) proteins 1 and 3 (via loss of the CFHR3/CFHR1 gene block) was linked to the generation of autoantibodies to CFH. Around 10% of aHUS patients develop CFH autoantibodies, adding aHUS to a growing list of kidney diseases with a defined autoimmune component. IgA nephropathy (IgAN) is another such renal disease, where autoantibodies target an aberrantly glycosylated IgA1. To investigate the role of CFH and CFHR copy number variation in the control of complement activation in aHUS and IgAN, I have first generated a full panel of recombinant CFHR proteins in mammalian cell culture. These were then used to generate unique monoclonal antibodies (mAbs) and ELISA protocols to screen for autoantibodies. Using carefully optimised immunisation protocols, I used my recombinant CFHR proteins to produce several highly-specific CFHR mAbs. One of which targets CFHR1 (R1/1037) and three target CFHR4 (R4/244, R4/277 and R4/123). The generation of these antibodies have allowed putative ELISA screens to be developed to measure the concentration of CFHR4 in healthy individuals and aHUS patients. My full panel of CFHR proteins also enabled screening of both aHUS and IgAN patients for the presence of autoantibodies to CFH and the CFHR proteins. Screening of aHUS plasma did not indicate the presence of any novel CFHR autoantibodies. However, IgA autoantibodies against CFHR5 (~9%) and CFH (~32%) were detected in IgAN patients. Interestingly, 64% of IgAN patients show reactivity with bovine CFH. During this PhD, I have generated a panel of unique reagents for the study of CFHR proteins in health and disease. These have allowed me to demonstrate for the first time, the presence of CFH and CFHR5 autoantibodies in a preliminary cohort of IgAN patients.

In this thesis I present an investigation of a novel steroid hormone receptor cochaperone, FKBPL, and its role in male infertility. I present evidence of proposed functional mutations in the FKBPL coding sequence within a subset of the infet1ile male population, namely, those presenting with non-obstructive azoospermia, that were not present within fertile control populations. FUl1her, I show that FKBPL exhibits a cell specific pattern of expression in human testis consistent with a role in androgen receptor signalling. I then demonstrate that increased expression of FKBPL can potentiate androgen responsive gene expression in vitro. The androgen receptor and its co-factors are important also in the development and progression of prostate cancer. I present an examination of the histone modifying enzyme and androgen receptor co-activator, LSD 1, and report that it is of limited clinical utility as a prostate cancer biomarker and is not a critical factor in the maintenance of aberrant DNA methylation at the GSTP 1 locus in prostate cancer cell lines. I present further examination of the epigenetic landscape of prostate cancer using a panel of prostate cell lines representing progression from normal to late stage androgen-insensitive prostate cancer. I have demonstrated that a loss of epigenetic control at a specific set of gene loci correlates with malignant transformation while epigenetic control at other gene classes is maintained. Transcriptional analysis of candidate genes, coupled with analysis of associated promoter CpG density, reveals some genes that are repot1ed to be hypermethylated and silenced in prostate cancer to possess low density CpG promoters whose methylation is likely to be inconsequential to transcriptional regulation. This suggests that a reevaluation of purported candidate biomarkers in light of recent developments in our understanding of CpG promoter regulation might be necessary. FUl1her, I find that coordinate changes in the expression of in silica identified targets of methylation that are also androgen responsive is not characteristic of disease progression in prostate cancer, though some genes identified in this screen were able to discriminate normal from disease cells based on their transcriptional profile. I also show that epigenetic aberrations may be pharmacologically reversed, but that normal control of a subset of genes is lost as a consequence. This work collectively highlights the potential diagnostic and therapeutic translational applications of our growing understanding of the human epigenome in prostate cancer, and highlights some important considerations in the interpretation of epigenetic data in the prostate cancer field.

The most common cause of encrustation and blockage of long term urinary catheters is colonisation of the urinary tract by Proteus spp. However, the degree of encrustation experienced by those with Proteus colonisation differs markedly between individuals. This study assessed the range of problems experienced by those colonised by Proteus and the factors which may differentiate severe from mild encrustation in this group. 21 long term catheter users found to have Proteus on urine screening were followed for approximately 3 months with weekly microbiological and chemical urine analysis and examination of their catheters. There was considerable variation in catheter lifespan within and between individuals. Some with persistent Proteus colonisation had no encrustation problems, others experienced frequent catheter blockage. Proteus was usually a stable component of urinary and catheter flora. Rapid encrustation was associated with a lowered nucleation pH (pHn). There was no clear difference in voided pH (pHv) between rapid and slow encrusters, but rapid blockers had a lower mean safety margin (pHn - pHv). pHv was not a useful predictor of catheter blockage. pHn was variable within and between individuals, but was higher in those who encrusted slowly. It was dependent on the calcium concentration and, to a lesser extent, the magnesium concentration of urine, with rapid encrusters having higher urinary calcium. Proteus isolates were assessed for urease activity. Strains with higher urease activities produced more alkaline urine, but this did not clearly result in shorter catheter lifespans. Proteus isolates from catheter users were found to have greater antibiotic resistance, particularly to trimethoprim and amoxicillin, than other local urinary tract isolates. Courses of antibiotic appeared ineffective at altering the urinary flora. The source of Proteus urinary colonisation, examined using Dienes typing and pulsed field gel electrophoresis of bacterial DNA, was commonly found to be the subject's own intestinal flora.

In this doctoral thesis I am proposing a paradigm shift in the management of men with localised prostate cancer involving destroying areas of prostate cancer alone – so-called focal therapy. By doing so, I propose that side-effects of whole-gland therapies will be reduced whilst maintaining cancer control. I have carried out a phased and structured programme of work to test this hypothesis. First, I review the literature to define the current problem faced by men with localised prostate cancer – namely over-diagnosis and over-treatment with treatment-related side-effects. I demonstrate why multifocality of prostate cancer seems the key impediment to tissue preservation (focal therapy) and why our thinking on multifocality should change. Second, I then tackle how we can accurately localised disease. I evaluate and determine that that using template mapping biopsies is more accurate at localising clinically significant lesions compared to transrectal ultrasound-guided biopsies. Further, I show that histological features of poor prognosis predominantly reside with the index lesion in the presence of multifocal disease. I then evaluate the role of multi-parametric MRI in detecting and ruling-out clinically significant disease using two key and unique datasets of men. I show that the negative predictive value of multi-parametric MRI – the key attribute when proposing to leave parts of the prostate untreated - ranges from 90% to 95% for clinically significant cancer. Third, I evaluate the side-effects and early disease control outcomes of focal therapy within two prospective development studies which are research ethics committee and National Cancer Research Network approved. The Hemi-HIFU study demonstrated that approximately 90% of 20 men treated with hemiablation for unilateral cancer, achieved the trifecta status of pad-free, leak-free continence, erections sufficient for intercourse and cancer control at 12 months. The Focal-HIFU study demonstrated that focal therapy targeted individual lesions rather than one-half of a prostate leads to trifecta outcomes in 84% of 41 men. These results are extraordinary when one considers that trifecta rates after radical therapy are in the order of 50%. My data support the contention that focal therapy has a role in decreasing many of the harms associated with standard whole-gland therapies. This programme of work has now led to a number of multicentre trials testing the reproducibility of these findings with longer follow-up. It has also led to a change in standard practice across a number of international centres. Ultimately, comparative effectiveness research will be necessary to determine the true role of focal therapy in treating localised prostate cancer.

Two neurotransmitters, ACh and ATP, initiate nerve-mediated contractions in detrusor smooth muscle from guinea-pigs and humans with bladder over-activity whereas only ACh has a role in muscle from stable human bladders. Both Acetylcholine and ATP are rapidly broken down, by acetylcholinesterases and ectonucleotidases respectively. The activity of ectonucleotidase has been shown to be reduced in the unstable human detrusor compared to stable. The reduced activity may contribute to the development of the purinergic component of the unstable human detrusor contraction. In vitro muscle strip experiments showed that apyrase significantly reduced the force of EFS contractions in guinea pig and over active human detrusor. Tension in guinea-pig was reduced to 81.5 1 4.5 % of control and by 87.5 7.5 % in overactive human. Apyrase had no effect in stable human detrusor. In guinea-pig detrusor the ectonucleotidase inhibitor ARL 67156 significantly increased the force of EFS contractions. The ectonucleotidase activity was investigated by measuring the degradation of a number of concentrations of ATP by detrusor samples. The ATP degradation rate in guinea pig and human detrusor was significantly reduced by the presence of ARL 67156. The ARL 67156 sensitive fraction which represented the ectonucleotidase activity solely was not significantly different between the stable and overactive detrusor groups. RNA extracted from human detrusor samples was used in gene expression assays to investigate the presence of four members of the ectonuleotidase family. ENTPDase 1, 2, 3, and 5 was present in all 18 human detrusor samples. There was significantly less ENTPDase 1 and 3 in over active detrusor samples compared to those that were stable. There was no difference in the levels of ENTPDase 2 and 5 between the two detrusor groups. Novel ATP sensitive biosensors were used to measure the real time release of ATP from guinea pig detrusor over the frequency range 1-20 Hz. The ATP signal reached a maximal response at a lower frequency compared to tension. The ATP signal kl/2, 4.41 3.02 HZ, was significantly lower than that of the tension generated, 12.0 2.59 Hz. In the presence of 1 iM atropine the frequency dependence of the ATP signal and tension is comparable. There was no significant difference between the kl/2 of the ATP signal, 7.29 3.5 Hz, and that of tension, 3.12 1.6 Hz. The apyrase experiments show a correlation between the effect of apyrase on nerve-mediated contractions in human and guinea-pig detrusor and the proportion of atropine resistance. This is consistent with the hypothesis that ATP contributes to nerve-mediated contractions in guinea-pig and over-active human bladders by incomplete breakdown prior to its activation of detrusor smooth muscle. This was corroborated by an enhancement of contraction strength in guinea-pig tissue by ARL 67156, which would have reduced further ATP breakdown. The reduced ATP breakdown may be linked to the difference in the levels of ENTPDase 1 and 3 between stable and overactive detrusor. The use of ATP sensitive biosensors show it is possible to measure the real time release of ATP from detrusor. The ATP release is frequency dependent and there is evidence to suggest that there is preferential release at lower stimulation frequencies.

Aim: Embryo freezing is a standard practice in most fertility units. According to the latest Human Fertilisation Embryology Authority data, 2,032 babies were born in 2010 from 10,548 cycles using frozen-thawed embryos in the UK. However, the practical benefit of embryo freezing in the National Health Service (NHS) context, and the psychological impact of this practice are unknown, and need to be reviewed in the light of increasing demand for NHS support for assisted conception. Therefore, this thesis investigates the answer to the question, “Should we be freezing embryos?” from two aspects: the influence on in vitro fertilization (IVF) success rates from embryo freezing and the decision-making process by which couples decide whether or not to freeze any surplus embryos. Methods: Analysis of the cumulative pregnancy rate (CPR) following three cycles of IVF treatment including embryo freezing was performed using life table analysis. A qualitative interview study involving IVF couples was performed aiming to explore the personal and social factors that couples consider when deciding about embryo freezing. Results: It was found that embryo freezing imparts a modest benefit of about 4% increase in the overall CPR. The qualitative study showed that regardless of the practical benefits of freezing embryos and the ethical and other reservations that couples have about it, the vast majority of IVF couples wish to avail themselves of the opportunity to freeze any surplus embryos, and use every additional opportunity to maximize their chances to have a baby. The decision-making process was complex and nuanced, and was fully appreciated only on reflection. Conclusion: Findings from this study will inform couples who face the difficult decisions about embryo freezing. Although this study indicates that more detailed information may not have influenced their decision, it provides the basis for further study comparing the influence of more targeted information on freezing decisions.

A third of all men experience unpleasant lower urinary tract symptoms (LUTS) such as a poor stream and being unable to postpone urination, usually later in life. Two important investigations for these men are: a one-o clinic-based measurement of urine ow rate, and the patient's hand written record of volumes passed over the course of several days. Well acknowledged deficiencies in these tests have spurred research into home-based alternatives. `Home urodiagnostic' devices have been developed that obtain multiple measurements of flow rate and an electronic voiding diary. However, little conclusive evidence exists as to their clinical utility. The aim of this thesis is to investigate the value of home urodiagnostics in the assessment of men with LUTS. First, the improvement in clinical performance of an average rather than single flow rate measurement is calculated based upon the theory of combining variance, predicting benefit for thousands of men per year. Next, finding existing devices deficient, the characteristics and technical performance of a novel device are presented. Despite its low cost, it is found to meet the required standard. In a study of conventional versus home urodiagnostics in men with LUTS, the latter is better tolerated, less likely to fail and gave more reliable measurement of flow rate. A study in which home urodiagnostics was performed before and after prostate surgery reveals large variation in the response of flow rate to surgery. Subtle changes within an individual are demonstrable. Finally, home urodiagnostics is piloted within primary care, where the resulting data suggests benefit from a change in the management strategy of over a third of patients studied. In conclusion, home urodiagnostics shows promise for improving the assessment of men with LUTS. The next step is to evaluate the effect on patient reported outcomes in a large scale trial.

Lower urinary tract symptoms (LUTS) affect a considerable number of people worldwide, inflicting significant impact on their daily lives. Adequate tools that accurately assess LUTS are therefore vital to aid diagnosis and monitor treatment. Urinary diaries provide an inexpensive, non-invasive method and are frequently used to evaluate LUTS in clinical practice and research trials. Despite their common use, a validated diary does not exist. Diaries appear to have evolved in an ad hoc manner, adapted to reflect different patients or conditions under investigation, resulting in numerous designs. The need for a standardised validated urinary diary was therefore evident. Qualitative and quantitative methodologies were employed to develop the urinary diary. In phase 1, patient and clinician opinion was sought on diary content, format and duration using interviews and questionnaires. Including both opinions guaranteed that the resultant diary would be applicable and understandable to those asked to complete it, whilst ensuring the diary remained clinically relevant. Key themes were identified and incorporated into four draft diaries for further assessment. Applying an iterative process, four rounds of content validation using patient-completed diaries or interviews, and one round of clinician opinion were undertaken before a consensus was achieved. The final draft diary developed in phase 1 was subsequently subjected to further psychometric validation in phase 2. Construct validity, criterion validity and reliability of the diary were proven. Analysis of diary duration demonstrated that a reduction in diary length was feasible without significantly compromising reliability. Pilot analysis of diary responsiveness has shown that the diary is sensitive to change and further analysis will be the focus of future research. Ultimately, a valid and reliable three day diary has been produced and is recommended for the assessment of all patients with LUTS to provide a quantitative, objective measure of lower urinary tract function.

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