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A urinary tract infection (UTI) is one of the leading reasons for treatment in primary healthcare. It is estimated that 50% of the female population in the UK will have least one occurrence of the infection in their lifetime. It is a debilitating condition and causes a variety of lower urinary tract symptoms (LUTS). The recommended practice for detecting a UTI is by analysing a urine specimen and culturing the sample for bacterial growth and antibiotic sensitivities. There are two main specimen collection methods: the midstream urine (MSU) and the catheter specimen of urine (CSU). The CSU is recognised as the gold standard, but requires an invasive procedure. The MSU which is the non-invasive clinical standard is regarded as insufficient because the method is frequently reported as contaminated with skin and vaginal flora, but the definitions of contamination in the literature varies. Drawing on the published body of knowledge, this study aimed to investigate and determine what constitutes contamination using microbiological culturing and the uroplakin-3 cell staining technique that detects the presence of cells that originate from the bladder. A two phase, single blind, cross over design study was conducted, comparing four different urine specimen collection methods. Experiment one tested the hypothesis that a MSU has equal merits to a CSU when capturing urothelial cells that are indicative of a UTI. A total of 60 patients and 30 controls were recruited into the study. The MSU specimens were compared with the CSU specimens to determine urinary cell origin using uroplakin-3 staining. The findings proved that the cells found in the MSU were not contaminants as commonly assumed, but were inflammatory markers of infection invading the lower urinary tract. Experiment two tested the hypothesis that if a MSU has equal merits to a CSU, then a directly voided urine specimen (natural urination) will be the optimal method when capturing the majority of urothelial cells that have been exfoliated from the bladder. A total of 31 patients were recruited and the MSU specimens were compared with the directly voided urine to determine the proportion of cells that originate from the bladder. The findings demonstrated that the directly voided urine was the optimal method and had the ability to capture predominant urothelial cells. A qualitative study of patient views and experiences of urine specimen collection was conducted. Thirty patients were interviewed and the data were analysed for recurrent themes. The study had shown an ideal urine specimen is that which is sensitive to the underlying pathology of a UTI. It is also a urine specimen that is easy to collect. The direct void is the recommended method of choice but should be accompanied with microscopy. Uroplakin staining should be initiated to further detect the positive presence of uroepithelial cells when distinguishing the difference between urinary contamination.

Approximately 40% of women will experience at least one urinary tract infection (UTI) in their lifetime and 25% of these women will go on to suffer recurrent UTIs. After the menopause, the risk of developing a recurrent UTI doubles, which is putatively linked to reduced estrogen levels. Topical vaginal estrogen treatment in postmenopausal women has been shown to reduce the incidence of UTIs. However, the mechanism by which vaginal estrogen helps to protect against UTIs is not well understood. Antimicrobial peptides (AMPs), secreted in response to infection and functioning via bacterial cell lysis, are an important component of the host innate immune response to infection. The aim of this project was to investigate the effects of estrogen treatment on vaginal epithelial AMP expression and synthesis. Immortalised vaginal epithelial cells (VK2 E6/E7), used to model the vaginal epithelium, were treated with 4nM 17β-estradiol for seven days and then challenged with 50ng/ml flagellin (isolated from Escherichia coli clinical UTI isolate) for 24 hours. Combined estrogen pretreatment plus flagellin resulted in a 2.3- and 2.1-fold increase in expression of the AMPs human β-defensin 2 (hBD2) and hBD3, respectively, above that observed with flagellin challenge alone (p-values < 0.001). Microarray analyses identified upregulation of the AMPs LCN2, RNase 7, S100A7, S100A12, SLPI, by estrogen pretreatment plus flagellin in addition to hBD2 and hBD3. Furthermore, several genes relating to keratinisation (for example keratin and SPRR genes) and inflammation (for example SERPINB4, S100A8 and S100A9) were also upregulated suggesting that estrogen pretreatment stimulated multiple protective responses in VK2 cells. A hBD2 luciferase reporter vector containing a 2032bp hBD2 promoter region was used to measure hBD2 expression in response to estrogen and flagellin treatments. Reporter activity in VK2 cells significantly increased 2.1-fold following seven day estrogen pretreatment (4nM) plus flagellin (50ng/ml) challenge compared to flagellin challenge without estrogen pretreatment (p=0.0367). Six estrogen response elements (EREs) were identified in the hBD2 promoter and were mutated by site-directed mutagenesis. Mutation of each of the EREs abolished hBD2 gene expression potentiation by estrogen pretreatment plus flagellin, suggesting that hBD2 is regulated through ER-α or ER-β binding to EREs in the hBD2 promoter. Pathway analysis of the microarray data identified IL-17A as a potential regulator of AMP expression. Thus, VK2 cells were challenged with exogenous IL-17A in concentrations ranging from 0.1ng/ml to 100ng/ml for 24 hours. The expression of hBD2, LCN2, RNase 7, and S100A7 was significantly increased between 2- and 21- fold in an IL-17A dose dependent manner (p-values < 0.001). In addition, estrogen pretreatment of VK2 cells prior to challenge with IL-17A (100ng/ml) plus flagellin (50ng/ml) significantly increased the expression of hBD2, hBD3 and S100A7 by between 1.4- and 1.6-fold compared to IL-17A plus flagellin without estrogen (p-values < 0.05). These data indicated an important role for estrogen in AMP expression even in the presence of proinflammatory cytokines, such as IL-17A. Altogether, these data indicated that estrogen is an important regulator of innate epithelial defences of the urogenital tract. These results suggest that estrogen protects against UTIs by augmenting the vaginal antimicrobial response to infection and by strengthening the epithelial barrier to infections. Hence, loss of estrogen following the menopause leaves postmenopausal women susceptible to repeated UTIs.

Background: A male cause is the main or a contributing factor in up to 50% of couples presenting with infertility. Azoospermia (lack of sperm in the semen) is present in approximately 15% of infertile men. Unlike psychosocial aspects of female infertility, male attitudes to their own infertility are poorly understood. Male infertility can potentially have a significant impact on psychological and social aspects of men’s lives, impacting negatively on self-image, relationships and causing psychological distress. Objective data in this area is lacking. Aim: The aim of this study was to assess the impact of the diagnosis of azoospermia on men’s psychological and social functioning. Method: This was a qualitative interview study, involving fifteen men with azoospermia attending a fertility clinic. All men were over twenty three years of age and suffering from primary infertility for more than a year. Participants gave their own account of how they perceived the experience of receiving the diagnosis, undergoing further investigations and having treatment. Data were collected between June 2013 and November 2013. The interviews were fully transcribed and analysed thematically using NVivo® software. Results: Major themes that emerged from the interviews included ‘reaction to the initial diagnosis’, ‘lack of cause and explanation’, ‘effect on interpersonal relationships’, ‘disclosure of the diagnosis’, ‘support seeking’ and ‘decisions regarding fertility treatment’. Key findings highlighted a feeling of shock and disbelief as a prominent part of men’s experience. Many men said that they never expected to be told of a ‘completely zero’ sperm count. Finding out was described as ‘heartbreaking’, ‘devastating’, ‘confusing’ and ‘sad’. The possibility of biological fatherhood was perceived as non-existent by some, with one commenting: ‘It felt as it was the end of the world’. One third of participants felt the diagnosis to be a threat to their masculinity, and to have a negative impact on their sense of self-confidence. Men found the lack of a precise aetiology frustrating and distressing. Most men were reluctant to share the diagnosis beyond close family members. The diagnosis brought partners closer together in most cases. Most men did not feel the need to seek external psychological support following the diagnosis and were satisfied with the support provided by clinic staff and their partners. A sperm retrieval operation was in most cases the only hope for establishing biological fatherhood. Decision- v making with regards to this and donor sperm treatment took into account multiple factors, including the risk of potential complications and side effects, their partner’s influence, and attitudes towards using donor sperm. Conclusion: Male infertility impacts substantially on men’s quality of life and healthcare professionals should be aware of this when investigating and treating patients with azoospermia. An improved understanding of men’s experiences is important for the provision of optimal clinical and psychosocial care. Better education and publicity about male factor infertility will reduce stigma and encourage men to seek help sooner. Men find the lack of specific aetiology frustrating and therefore further research is required into the aetiology of male infertility.

Urinary tract infections (UTIs) are the second most common cause of infection worldwide, making them a huge economic burden. People can carry diagnostic loads of bacteria within the bladder without experiencing any symptoms, this is asymptomatic bacteriuria (ABU). ABU prevalence increases with age, with around 19% of those over 60 being susceptible. ABU can reactivate sporadically, causing symptomatic episodes, known as recurrence. Current treatment is with antibiotics, either short courses or prophylaxis. Guidelines state to only treat symptomatic episodes. However, UTI symptoms are often diffuse and unclear, especially in older people. Thus, ABU is inappropriately treated in up to 52% of cases, encouraging antibiotic resistance. Better ways of discriminating between symptomatic and asymptomatic cases are needed. This would allow for more efficient treatment and patient management. To improve understanding of ABU, a longitudinal clinical pilot study was performed. For 6 months, every 2 weeks a urine sample and symptom questionnaire was collected from 30 patients over 65 who were clinically diagnosed with recurrent UTIs. The aim was to analyse potential changes in the host response and the colonising bacteria around periods of symptoms to see if distinct urinary profiles could be seen between symptomatic and asymptomatic states. Allowing for analysis into potential predictive biomarkers for symptoms. Uropathogenic Escherichia coli (UPEC) is known to cause the majority of UTIs, thus was the bacterial focus for this project. The study firstly allowed us to test the feasibility of such a demanding study design. It was possible to fully recruit to the study with all 30 patients completing its entirety, thus suggesting such a design could be repeated or scaled up in the future. The study produced a large database of bacterial isolates as well as urine samples. Urines were measured for a wide range of immune response proteins. Despite varying levels of immune activation, UPEC was able to evade host-defences and thrive in the bladder long-term. The study showed that ABU patients can carry significant bacterial loads of UPEC, even with antibiotics, questioning the advantages of prophylactic treatment of ABU patients. No distinct host or bacterial profile was identified between symptomatic states. No biomarkers could be seen to predict symptomatic episodes in these patients. However, what was observed was an unexpected level of dynamic variability within individuals and across the patient cohort.

End-stage kidney disease is increasing in prevalence and is associated with high levels of morbidity and mortality. At present, the only treatment options are dialysis or renal transplantation. However, dialysis is very costly and is associated with high levels of morbidity, whereas the problem with transplantation is that there is a shortage of organ donors. For these reasons, over recent years, there has been an increasing interest in developing novel therapies in the field of regenerative medicine including stem cell based therapies and tissue engineering. Stem cells could be used in a number of ways to develop new therapies for kidney disease. Firstly, they could be administered as cell therapies to patients with kidney disease, and secondly, they could be used to generate specific types of renal cells in vitro that could be used for understanding disease mechanisms and for drug discovery programmes. The barriers to the development of novel stem cell therapies include the difficulties in expanding kidney-derived stem cells in culture without altering their phenotype, and directing their differentiation to specific types of renal cells. These issues could be addressed by developing biomaterial substrates that provide an appropriate microenvironment for the successful culture and differentiation of stem cells. Within this study we interrogated a wide range of biomaterial substrates for their capability to direct the differentiation of kidney derived progenitor / stem cells. These materials were thoroughly characterised in terms of their physicochemical properties, such as surface chemistry, nanotopography and wettability by employing a wide range of analytic techniques, including X-Ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), colorimetry and contact angle measurements. We firstly investigated a range of polyacrylates. These substrates were novel in that, they were precisely designed to mimic cell binding motifs of the extracellular matrix stereochemically by using monomeric precursors that display particular chemical functional group chemistries, namely amine, hydroxyl, carboxyl groups or aliphatic spacer groups. We found that these materials differed strongly in the presence and distribution of surface functional group chemistries and topographical features, including the distribution of surface artefacts on a macroscale. Moreover, some of these materials were able to direct the differentiation into specialised renal cell lines. Two substrates, namely ESP 003 and ESP 004, directed the differentiation of kidney derived stem cells into podocytes and two further substrates, namely ESP 007 and BTL 15, directed differentiation into functional proximal tubule cells. These four substrates stimulated cell differentiation to an extent of about 40 to 50% after only 96 h in cell culture. We were moreover able to identify surface physicochemical cues, including surface micro- and nanoscale topography and surface functional group chemistries that are important to stimulate the differentiation process. In addition, we investigated a range of plasma polymer coatings composed of allylamine and octadiene that were provided as homo-or copolymers and in form of chemical gradients, the latter one differing in the amount of nitrogen functional group chemistries across the surfaces. We found that substrates with higher allylamine content displayed a greater amount of nitrogen functional groups and therefore increased in wettability. Moreover, those plasma polymer substrates with higher amine functionality directed kidney progenitor cell differentiation into podocytes, whereas substrates with higher octadiene concentration directed cell differentiation into functional proximal tubule cells, both to an extent of 35 to 45% after only 96 h in culture. To further study cell differentiation, we then incorporated gold nanoparticles underneath these plasma coatings, either in form of homogeneous coatings or in form of a nanoparticle density gradient. We found that surface topographic gradients increased cell differentiation into podocytes 3- to 4-fold, whereas differentiation into proximal tubule cells was only dependent on surface chemistry. Our studies on plasma polymer substrates highlighted not only the great potential of plasma polymers to modify surface functionality of a wide range of surfaces, but also emphasized the great capabilities of surface gradients, whether chemical or topographical in nature, to effect cellular fate. In summary, the results of this study include the identification of biomaterial substrates that have the potential to differentiate kidney-derived progenitor/stem cells in vitro and of the cues that are necessary to assist in the differentiation process. In the future, these biomaterials could be useful for directing the differentiation of pluripotent stem cell-derived renal progenitors to specific types of renal cells that could be used for applications in regenerative medicine and drug discovery programmes.

Mesothelial cells have been described to possess progenitor characteristics and contribute to regeneration through differentiation. However, it is not clear what the effects of prolonged culture have on the mesothelial cell properties and relative plasticity; as long-term cultures have not yet been established as a result of early senescence. Understanding the effects of time in culture is crucial for the development of novel therapies. In this thesis, we demonstrated that mesothelial cell cultures isolated from murine omentum could be cultured for more than 40 passages and showed relatively stable population doubling times. While initially, the cells did down-regulated the expression of mesothelial markers Wilm’s tumor protein 1 (Wt1) and mesothelin and epithelial genes; their mesenchymal profile was maintained. This along with the increased Snail2 expression suggested the cells were progressing through the mesothelial to mesenchymal (MMT) transdifferentiation programme. TGF-β and more recently EGF are known mediators of MMT. Targeting signalling through these receptors with small molecule inhibitors LY364947 and PD153035, slowed the rate of gap closure, in vitro and increased zonula occludens 1 accumulation at cell-cell contacts. Which seemed to be mediated through the MEK5/ERK5 pathway. Furthermore, siRNA-mediated transient knockdown of Zeb1 and Zeb2 transcription factors also achieved attenuated the rate of migration. Next, we moved onto to accessing the progenitor properties of the mesothelial cells with time in culture. The expression of stem cells markers Bmi1 (Proto-Oncogene, Polycomb Ring Finger), Sox9 ((Sex Determining Region Y)-Box 9) and CD34 were downregulated with repeated passaging. However, the low passage mesothelial cells exhibited clonogenicity and could differentiate into osteoblasts and adipocytes. Finally, in an embryonic kidney rudiment assay, we could show that the mesothelial cells co-existed with the embryonic kidney cells and allowed renal structures to form in their presence. Ultimately, we have demonstrated that the mesothelial cells from the omentum maintain some mesothelial characteristics with prolonged culturing. The cells showed clonogenic and multi-lineage potential and expressed a number of stem cell genes. The MMT programme is complex and could be partly reversed by targeting TGF-βR1 and EGFR tyrosine kinases, which along with transiently silencing the Zeb transcriptional factors seem likely key targets in ameliorating pathological fibrosis.

Avian Pathogenic Escherichia coli (APEC) is an important pathogen of the poultry industry, responsible for 43% of condemnations of broiler birds at slaughter and annual losses of between 1-8% of a laying flock. APEC is also a public health concern; consumption of poultry meat has been linked with urinary tract infections (UTI) in humans, and also as a reservoir of potential antimicrobial resistance genes. In this study of strain 3770, a reproductive tract associated isolate of APEC, the isolate was characterised for typical APEC virulence phenotypes, and also underwent full-genome sequencing in order to further advance our understanding of the pathogenomics of reproductive tract associated APEC infections. Additionally, a population study examined the virulence gene profiles within a population of reproductive tract associated E. coli, with a particular focus on virulence factors associated with infections in avian species and UTI in humans. It was found that 3770 exhibited characteristic extra-intestinal pathogenic E. coli (ExPEC) virulence phenotypes; serum resistant, adhesive, and able to persist in the short term within phagocytic cells. It was also able to induce reproductive tract infections via the aerosol route. The genome sequence was 5.02Mb in size with a high number of virulence genes. The most closely related E. coli was an adherent-invasive E. coli isolate associated with Crohn's Disease. The population study of reproductive tract infections revealed a high level of variance within the population, and a higher prevalence of ibeA and K1 capsule genes than seen in other populations. These results provide further evidence that there is no one single APEC pathotype. It is likely that virulence in APEC isolates is a complex relationship between the virulence profile of the bacterium and the health status of the host.

Aims: (1) Explore the physiological effect of renal denervation (RDN) (2) Explore the efficacy of distal denervation on blood pressure (BP) reduction in patients undergoing directly observed anti-hypertensive therapy (DOT) to minimise measurement bias (3) Evaluate the 6-month safety of distal denervation Methods: Patients with resistant hypertension were recruited and underwent assessment of drug compliance by assaying urinary drug levels. All subsequent measurements were recorded under DOT. Pre-denervation, office and ambulatory BP were measured, and patients underwent bilateral renal angiography and invasive measurement of aortic and renal arterial pressure and blood flow velocity. RDN was performed using the Symplicity Spyral catheter, denervating in the main renal arteries and each distal branch >3mm diameter. Invasive and non-invasive measurements were repeated 6-months post denervation under DOT. Results: 16 patients underwent denervation (age 63±12 years) with referral office SBP 180±18 mmHg. In total each patient received 22.6±5.0 ablations, 9.3±2.9 ablations in the main trunk and 13.3±4.8 ablations distally. At 6-months follow-up, overall unblinded 24-hour SBP reduction was -5.1±7.5 mmHg (p=0.020), with DBP reduction -3.4±4.9 mmHg (p=0.018). At 6-months follow-up an overall increase in renal blood flow velocity occurred at rest (1.91±3.51cm/s, p=0.04) and under identical sedation states (1.81±3.44 cm/s, p=0.05). Patients with the largest reduction in ambulatory SBP at 6-months had the largest increase in renal blood flow acutely after RDN (R 2=0.60, p < 0.001) and the largest decrease in renal resistance (R2=0.56, p < 0.001). Quantitative vessel angiography showed no significant change in any main or distal renal artery dimensions at 6-months. Conclusion: This unblinded study of distal RDN showed a significant reduction in ambulatory systolic and diastolic BP with no safety concerns at 6-months. These exploratory results suggest that acute changes in renal hemodynamics may be predictive of blood pressure response at 6-months follow-up.

Estimated to affect one in six couples in the UK, infertility is an issue of great public health importance. This thesis provides a critical overview of the methodological issues in defining and studying infertility, and investigates the epidemiology of infertility, particularly prevalence and early life and reproductive risk factors. An initial literature review critically evaluated different approaches to defining and measuring infertility, and provided an overview of current prevalence, trends, and existing literature on the determinants of infertility. Two datasets were analysed for the investigation of the epidemiology of infertility. The first was the Uppsala Birth Cohort Study Multigen, which describes the experiences of over 6000 Swedish women born between 1915 and 1929. Two indicators of fertility were used: general and age-specific fertility rates, and time to first live birth. These were analysed with respect to specific early life factors: gestation, birthweight, birthweight for gestational age, and ponderal index. The results provide no evidence to support the hypothes is that these markers of inutero growth are associated with fertility in adult women. The second dataset was the National Women's Health Study, a population-based survey conducted in 2001 which collected information on the reproductive histories of over 7000 UK women. These data were used to describe the epidemiology of infertility in the UK, providing rarely reported data on the prevalence of infertility, help-seeking for fertility problems, and the use of treatment for fertility problems. The second stage of this work investigated the relationship between prior adverse reproductive outcomes and secondary infertility. The results suggest that secondary infertility is associated with prior adverse pregnancy outcome including termination, miscarriage and ectopic pregnancy, although with the exception of prior ectopic pregnancy, associations were weak and often inconsistent. The implications of these findings, and recommendations for future studies on infertility, are discussed.