Tubulointerstitial inflammation and fibrosis is frequently seen in patients with progressive renal failure, irrespective of the aetiology, and may represent a common pathway to renal failure. However, the role and function of tubulointerstitial lymphocytes in the pathogenesis of renal fibrosis is unknown. Using the wellcharacterised mouse model of unilateral ureteric obstruction (UUO) I determined the phenotype of infiltrating lymphocytes and by sequencing the complementaritydetermining region 3 (CDR3) of the variable β-chain examined whether there were clonal populations of T cells within UUO and normal kidney. There was a strong correlation between lymphocyte infiltration and tubulointerstitial expansion, α-SMA staining and collagen I deposition. A population of these infiltrating CD4+ and CD8+ lymphocytes also displayed the surface makers CD69 and CD44, and the nuclear proliferation marker Ki67. This suggests activation and proliferation of T cells in response to self-antigen recognition and the loss of immunological tolerance. Infiltrating T cells within UUO kidney demonstrated over expression of certain TRVβ gene segments in particular TRVβ3, suggesting a clonal population of lymphocytes. In normal and sham operated kidney over expression of T cells with the TRVβ13.2, 29 and 1 gene segments suggested populations of resident NKT cells expressing an invariant T cell receptor. On sequence analysis of the CDR3 region of infiltrating lymphocytes, large clonal populations of T cells were seen in individual UUO kidneys at 7 and 14 days after obstruction but not 28 days. These clonal populations in UUO kidney that were also in normal kidney had an identical amino acid motif within the CDR3 region. This suggests a population of T cells in normal kidney proliferate in response to injury. These lymphocytes could be a potential target for therapeutic interventions to prevent fibrosis in renal diseases that are not characteristically believed to be immune mediated.

This thesis examines Beach's hypothesis that the sexual behaviour of male rats is under multisensory control and is resistant to the loss of tactile information from the vibrissae. The loss of perioral sensation was found to profoundly disrupt sexual behaviour in sexually inexperienced males tested with females showing five ascending levels of precopulatory behaviour. Males did not copulate with females displaying presenting, presenting and hopping, hopping and darting levels of proceptive behaviour. However, males paired with darting females displaying the highest level level of proceptive behaviour did copulate in the presence of perioral anapsis caused by intramystacial injection of lidocaine. Injection of lidocaine intraperitoneally and intramasseterically showed that this effect was not due to systemic toxicity. Prior sexual experience was found to attenuate the effect of perioral anapsis and males given three prior copulatory trials copulated with presenting females in the presence of perioral perioral anapsis. Males given one prior copulatory trial also copulated with presenting females in the presence of perioral anapsis. These results complement recent research which has called for a revision of the multisensory hyopothesis and indicate that trigeminally mediated tactile information from the vibrissae plays an important role in the initiation of sexual behaviour in the naive male rat.

Urinary stones are a common medical condition affecting over 10% of adults in the developed world. Whilst many different materials are found in kidney stones, the majority are dominated by deposits of calcium oxalate (CaOx). In addition to the mineral phases, urinary calculi also contain macromolecules. It has been shown that many of these, particularly the proteins, can inhibit CaOx crystallisation and are therefore widely assumed to be relevant to stone pathogenesis. More recently, it has been proposed that certain urinary proteins facilitate the prevention of stone formation through their selective inclusion into the growing crystal and subsequent structural disruption through protease activities. Given that the intracrystalline location of proteins would be structurally challenging, their selective occlusion may support a functional role. This thesis explores these concepts. The crystallisation of CaOx in the urine of stone formers and healthy controls was compared. CaOx crystals grown in human urine and inorganic media were examined using a range of complementary techniques, e.g., electron microscopy, X-ray diffraction and thermogravimatric analysis. Proteins associated with the surface of different mineral phases and/or occluded within the crystal structures were isolated and identified using immunoblotting. The recovered proteins were quantified in terms of their crystal volume occupation, and the proportion of proteins taken up from the urine were investigated. Finally, six common methods used to study CaOx/protein interactions in vitro were compared. Significant differences were noted in the crystallisation parameters which define the urine samples from stone-formers and controls. These could not be attributed to electrolyte balance from which it is inferred that the influence of other urinary constituents such as proteins must be significant. Only a small proportion of the urinary proteins associated with the CaOx crystals and they contributed to less than 5% of the total crystal volume, suggesting that arguments for a preventative role in stone formation cannot be supported. Kinetic experiments demonstrated that two proteins, often proposed as inhibitors of stone formation had little effect on CaOx nucleation and growth rates. Different proteins were found to be surface-associated when compared to those that were occluded within the crystal structures. However, the same proteins all associated with both CaO monohydrate and dehydrate, which suggests unique template-mediated interactions are unlikely. The growth of CaOx in urine using six methods showed that the stabilisation of different hydromorphs and induction of some morphological changes in CaOx were protocol dependant and not, as previously suggested, attributable to protein interactions. The method used did not influence the type of protein associating with CaOx. These results indicate that whilst urinary proteins may be capable of modifying CaOx crystal growth they appear to do so through non-specific interactions. There is little evidence that proteins are present in urine as teleological inhibitors of stone formation.

Prostate cancer kills over 10,000 men every year in the UK, mainly as a consequence of the spread of the disease to other sites in the body. It is important to understand what controls the spread of prostate cancer cells, as this may lead to treatments that either slow down or prevent metastasis, and perhaps result in the cure of some of the advanced prostate cancers. Semaphorins act as chemotactic cues for axon guidance and cell movement, via their transmembrane receptors, plexins. It has been found that semaphorins and their receptors are overexpressed in some human prostate cancer cell lines. These cell lines have been screened for mutations in genes in the semaphorin signalling pathway. 13 somatic missense mutations in the cytoplasmic domain of the Plexin B1 gene have been identified in clinical samples of primary and metastatic prostate cancers. One of the mutations C5662T was predicted to result in a substitution of a Proline to a Serine. The aim of this research is to determine if the mutation C5662T in the Plexin B1 gene alters cell behaviour and contributes to prostate cancer progression. This thesis describes the method of constructing an expression vector containing the mutation and transfecting the mutation into COS7 cells to produce stable and transient clones. The effect of the mutation on cell collapse was examined. It was possible to show that Plexin B1 is involved in cell collapse and that Rnd1 is required for this process and this effect is independent of its ligand Semaphorin 4D (Sema4D). Although no functional difference between the wild type Plexin B1 and the mutant (C5662T) was observed, the rate of cell collapse may be important and more work will be needed to define whether this is a mutation that alters prostate cancer cell behaviour.

Introduction: Struvite stones (magnesium ammonium phosphate) account for 10-15% of renal stones and can grow rapidly forming staghorn calculi. With limited medical therapies available and surgery the mainstay of treatment, an understanding of the physico-chemical conditions causing struvite crystallization allows development of strategies to prevent their formation. At present, very little is known about the physico-chemical conditions that result in struvite crystallisation. This lack of understanding has two bases: i) the particular ionised concentrations of the reactants (e.g. Mg2+, NH4+, and phosphate) in the urine are unknown; ii) the prevailing chemical conditions that would modulate crystallisation are also unclear (e.g. pH, osmolality, other urinary constituents).Recent advances in the development of ion-selective electrodes allows accurate determination of urinary constituents in small (<1ml) undiluted samples. Methods: We have furthered developed an NH4+-ion selective electrode and magnesium ion-selective electrode to measure the urinary concentrations of each to aid our understanding of struvite deposition. [NH4+] and [Mg2+] were measured using plastic dip cast ion-selective electrodes dispersed in suitable plasticisers, using nonactin for NH4+ and several different neutral ligands for Mg2+. Data were validated against standardised and developing methodology, including colour spectrophotometry and computational algorithms. Urine samples were also subjected to analysis using clinical biochemical techniques. Results: We characterised an NH4+-ion selective electrode and made the first measurements of [NH4+] in undiluted urine samples, in normal subjects and those with stone disease. This technique was validated using colour spectrophotometry and then the technique was used to validate a computational algorithm. We have developed and characterised the Mg2+-ion-selective electrode but Ca2+ caused significant interference. Conclusions: Direct measurement of the urinary [NH4+] has been achieved for the first time with a value of about 25mM. This exciting new technique may now provide clinicians with an important point-of-care investigative tool in diagnosing and monitoring struvite calculogenesis.

Congenital abnormalities of the lower urinary tract of children adversely affect bladder function, both immediately and throughout development, and can result in renal failure. Such patients with pathological bladder states often require reconstructive surgery in an attempt to restore lower urinary tract function. However, it is unknown if there are functional defects to the musculature of the bladder - the detrusor smooth muscle. The aim was to examine the functional properties of human paediatric detrusor in normal patients and those with pathological bladder conditions. Full thickness bladder specimens were taken from pathological bladder conditions such as posterior urethral valves, neuropathic bladder, and bladder exstrophy. Normal tissue was obtained from those with urachal anomalies or from those undergoing ureteric reimplantation procedures. Ethical committee approval and patient consent were obtained at the time of surgery at Great Ormond Street Hospital. The bladder contractile properties were characterised by in vitro myopathy and electrical field stimulation in all patient groups. The histological properties were also examined in these groups. Additionally, with bladder exstrophy samples the viscoelastic properties of the tissue was measured by passive stretch studies to determine their contribution to the overall mechanical properties. Finally, intracellular Ca2+ responses in single detrusor muscle cells to agonist stimulation were measured by epifluorecence microscopy. Nerve and agonist-mediated contraction amplitude was significantly less in samples from patients with pathological bladders compared to those with normal bladder. Histological evaluation revealed an increase in the connective tissue to smooth muscle ratio in pathological bladders. In bladder exstrophy the tissue was mechanically stiffer but the single cell responses were similar to the normal detrusor responses following agonist application. The data shows that detrusor from paediatric patients with pathological bladders exhibited reduced contractility, whether elicited by excitatory nerves or agonist application. Functional innervation was reduced in pathological bladders and the pattern of excitatory neurotransmitter release was altered.

Primary keratinocytes form 3 types of colony with different morphologies termed holoclones, meroclones and paraclones, thought to be derived from stem, early and late stage precursor cells respectively (Barrandon and Green, 1987b, Rochat et al., 1994). Cancer cell lines produce colonies with morphologies analogous to those of holoclones, meroclones and paraclones, and consequently holoclone morphology is used as a surrogate marker for stem cell colonies. The aim of this study was to elucidate the relationship between clonogenicity, colony morphology and stem cells. Colonies formed by primary prostate epithelial cells and prostate cancer cell lines (DU145, PC3, LNCaP) were characterised. The proportions of colonies were not altered significantly by modification of culture conditions. In contrast to cancer cells, primary prostate epithelial cells form only two types of colony, termed types 1 and 2, which are analogous to holoclones and paraclones. Only type 1 colonies were highly proliferative, able to self-renew and express putative stem cell markers. Paradoxically, cells from DU145 meroclones formed holoclones and had self-renewal capacity (by serial cloning and xenografting). It is concluded that the major difference between holoclone and meroclone colonies from the cancer cell line DU145 is the proportion of stem cells within each colony, not the presence or absence of stem cells. Phage display was used to look for targets on the surface of cells in Type 1 colonies. Various experimental protocols were tested, but no targets were identified.

This thesis describes two groups of experiments, both relating to the condition of distal renal tubular acidosis (dRTA). In the first, an alternative diagnostic test of dRTA to the ‘gold standard’ short ammonium chloride (NH4Cl) test was assessed. This was achieved by the simultaneous oral administration of the diuretic furosemide and the mineralocorticoid fludrocortisone to increase distal sodium delivery and a-intercalated cell proton secretion. I evaluated 11 control subjects and 10 patients with known dRTA by giving oral NH4Cl or furosemide/fludrocortisone in random order on separate days. 3 subjects were unable to complete the study due to vomiting after the NH4Cl, however there were no adverse effects with furosemide/fludrocortisone administration. The urine pH decreased to less than 5.3 in the controls with both tests, whereas no patients were able to lower their urine pH below 5.3 with either test. The simultaneous administration of furosemide/fludrocortisone proved to be an easy, effective and well-tolerated alternative to the standard NH4Cl test for the diagnosis of dRTA. The second group were laboratory-based molecular physiology experiments. Anion exchanger 1 (AE1) mediates electroneutral anion exchange across cell membranes. It is the most abundant protein in the red cell membrane, but is also found in the basolateral membrane of renal a-intercalated cells, where it is required for urinary acidification. Point mutations have been described that convert the red cell AE1 into a cation conductance. AE1 mutations can also cause hereditary dRTA. I investigated the properties of four dRTA associated AE1 mutations (R586H, G609R, S613F and G701D) by heterologous expression in Xenopus Laevis oocytes. These mutants proved to be functional anion exchangers, unlike the red cell mutants, but also demonstrated a cation ‘leak’. I found a very large leak property in the G701D mutant, which is prevalent in SE Asia. I hypothesised that this property might confer a survival advantage. I characterised three other AR dRTA-associated AE1 mutants found in SE Asia, S773P, \Delta850 and A858D via similar transport experiments in AE1-expressing Xenopus oocytes. These three SE Asian mutants also had cation leaks of similar magnitude to that seen in G701D, a property that distinguishes them as a discrete group. The clustering of these cation-leaky AE1 mutations to malarious areas of SE Asia suggests that they may confer malaria resistance.

Acute urinary retention (AUR) is characterised by the sudden and painful inability to pass urine and is common in older men. It causes significant morbidity, frequently results in emergency hospital admission and often requires surgery. The aim of this thesis was to investigate ways of improving the management of men with AUR. The Hospital Episode Statistics (HES) database of the Department of Health was used to investigate mortality after AUR. In 100,067 men with spontaneous AUR, the one year mortality was 4.1% in men aged 45-54 and 32.8% in those aged 85 and over. In men with spontaneous AUR aged 75-84, the most prevalent age group, the one year mortality was 12.5% in men without comorbidity and 28.8% in men with comorbidity. The importance of comorbidity on mortality of men with AUR prompted the development of The Royal College of Surgeons of England (RCS) Charlson Score to improve comorbidity identification in HES. The RCS Charlson Score uses an explicit coding philosophy that is simple to use, more accurate than existing adaptations of the Charlson Score, reflects the current understanding of the prognostic impact of comorbidity and allows international comparisons. Given that many men with AUR are elderly, have significant comorbidity and therefore have a high risk of death, minimally invasive treatment alternatives to surgery for AUR were evaluated. Systematic literature review defined the role of prostatic stents as an effective treatment for frail and elderly men. Although only observational data were available a specifically developed checklist to assess methodological quality gave context to the findings. This thesis demonstrates that the management of AUR must focus not only on the prostate but also on the patient’s overall health status. The urologist should adopt a holistic approach when assessing and treating men with AUR to ensure the best possible outcomes.

The aim was to investigate whether chemokine ligand-receptor interactions are involved in the chemotaxis of prostate cancer to favoured metastatic sites. Initially, chemokine receptor mRNA expression, CXCR and CCR groups, was determined using conventional RT-PCR in cell lines derived from prostate cancer metastases, DU145, LNCaP and PC3, the primary prostate cancer cell line 1542 CPT3X and the normal prostate epithelial/ stromal cell lines 1542 NPTX, Pre 2.8 and S2.13. It was observed that in the cell lines derived from prostate cancer metastases, CXCR4 mRNA expression was relatively high. Using real-time quantitative PCR it was subsequently established that in DU145, LNCaP and PC3 cells, CXCR4 mRNA expression was 1287, 407 and 21 times respectively that of 1542 CPT3X. 1542 NPTX and 1542 CPT3X had similar levels of CXCR4 mRNA (the former had only twice that of the latter) and Pre 2.8 had no detectable CXCR4 mRNA expression. In laser microdissected patient primary tumour samples and patient benign tissue specimens CXCR4 mRNA expression was higher than that of the metastatic cell lines. Flow cytometry analysis showed that significantly higher levels of the CXCR4 protein were present on the cell membrane of the three metastatic cell lines. Cell migration assays revealed that chemotaxis of the metastatic cell lines PC3 and DU145 was enhanced by CXCL12 ligand and inhibited anti-CXCR4 antibody. We have demonstrated that human prostate cell lines derived from metastases express functional CXCR4 receptor and that CXCL12 ligand enhances their migratory capabilities. Also, primary patient tumours and patient benign tissue specimens express CXCR4 mRNA at high levels (it is suggested that in vivo post-transcriptional modification and/ or regulation of CXCR4 receptor at the protein stage may significantly affect cellular protein levels). These results suggest that the CXCL12-CXCR4 axis may be involved in the metastasis of prostate cancer to preferred organs.