It is widely believed that the sperm-specific protein phospholipase C zeta (PLCζ) is released into the oocyte upon fusion and initiates calcium (Ca²⁺) oscillations which regulate oocyte activation. Reduced amounts, abnormal localisation patterns, and aberrant forms of PLCζ underlie certain types of male-factor infertility, prompting significant interest in the use of this fundamental protein as a clinical therapeutic agent or diagnostic marker. This thesis systematically addressed a range of outstanding questions relating to the expression, localisation, and therapeutic/diagnostic application of PLCζ. Using immunohistochemical and quantitative immunofluorescence techniques, the current study investigated the effect of paternal age and epididymal maturation upon PLCζ expression and localisation in mouse sperm. Neither age nor epididymal maturity exhibited significant effect upon the total level of PLCζ in mouse sperm, although there was a significant trend towards post-acrosomal localisation as sperm matured during transit in the epididymis. Immunohistochemical analysis of mouse testis showed that the expression of PLCζ first appeared at the roundspermatid stage of spermatogenesis. Paternal age did not influence total levels of PLCζ expression or localisation pattern in human sperm samples (n=29), with PLCζ being predominantly localised to the post-acrosomal region. A significant reduction (34%) was detected in the proportion of sperm exhibiting PLCζ from an oocyte-activation deficient patient compared with fertile controls. Novel immunohistochemical analyses suggested, for the first time, that PLCζ is first expressed from the round spermatid stage in humans, and that the analysis of PLCζ expression in human testicular biopsies (n=29) may provide credible indication of the oocyte activation ability of surgically-retrieved sperm samples from infertile patients. Initial data also suggest that flow cytometry may serve as a useful prognostic/diagnostic tool for the quantitative analyses of PLCζ expression in human sperm samples. Mammalian and bacterial expression systems were utilised to generate recombinant human PLCζ protein as a therapeutic agent and for the generation of a novel monoclonal anti-PLCζ antibody respectively. Expression studies using HEK293T cells demonstrated that human PLCζ appeared to localise to the endoplasmic reticulum and that the EF hand domain appeared to be involved in regulating this pattern of localisation. Collectively, these studies extend our fundamental knowledge of how PLCζ is expressed in developing sperm and assist in the translation of PLCζ as a clinical therapeutic and diagnostic biomarker for oocyte activation ability.

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Urinary incontinence (UI) is a major health care problem which has significant implications for quality of life within an ageing population. Urinary continence can however, be restored by simple rehabilitative strategies and therefore nurses need to be knowledgeable and proficient in providing quality care for patients with UI. However, there is evidence that there is very limited content related to continence promotion within the current pre-registration nursing curricula and there are no specific modules dedicated to this area of clinical practice. Student nurses are also still expected to learn through doing i.e. delivering hands on care for patients with UI and are supported in practice by a qualified mentor. There is evidence however, that suggests that qualified staff have negative attitudes towards UI but what is not known is if these negative attitudes influence student nurses’ experiences of dealing with UI in clinical practice in their first year of study. In order to generate a deeper understanding of the lived experiences of adult branch student nurses a longitudinal phenomenological study was adopted to explore their experiences in their first year of study. Thirteen students volunteered to participate and were recruited from 2 United Kingdom (UK) Universities. The students were interviewed on two occasions; once at the beginning of their first year and again at the end of their first year. Data was analysed using phenomenological and hermeneutical approaches including iterative reading and interpretation to identify the themes that emerged from the data. The findings revealed 3 major themes; Being There, Being Understood and the Influence of Others. Being There, showed that student nurses are perplexed and confused about why loss of bladder function, is not viewed as something that requires assessment and investigation and is predominantly the remit of Healthcare Assistants (HCAs). Being Understood, captured the students’ problems in ‘finding a voice’ to express these concerns regarding this approach to UI care delivery in their desire not to ‘rock the boat’ and to ‘fit in’ with their nursing colleagues who were predominantly the HCAs in this study. The Influence of Others, encapsulates the role of the HCA in influencing the student nurses’ experiences of UI in their first year of study. Student nurses should spend up to 40% of their time in clinical placement supported by a qualified member of staff however, there is considerable evidence in this study that this support is infrequent. Student nurses’ experiences of UI therefore do not appear to be negatively influenced by qualified staff as they are infrequently their role models for this aspect of care in their first year of study. They are however, influenced by HCAs who have no formal training in this area of clinical practice or in supporting students in practice. The findings from this study have implications both for the education of student nurses and for HCAs.

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This thesis explores the experience of dialysis in young adults across three papers: the first paper, a systematic literature review, evaluates the evidence for direct and indirect relationships between social support and adherence to treatment regimens - including prescription, medication, dietary restriction, and fluid intake. It reviews the quantitative evidence, finding no consistent relationship between these two variables. The second paper presents findings from an empirical study, qualitatively exploring the lived experience of dialysis in patients aged 18-35 years. This cross-sectional study was undertaken according to the principals of interpretative phenomenological analysis (IPA), with semi-structured interviews undertaken with four male patients. Two interconnected aspects of experience were identified, forming two broad categories of themes: biographical disruption and biographical repair. Biographical disruption described the immediate and ongoing negative impact that dialysis had on patients' lives, including failure to complete developmental tasks and difficulty maintaining a place within social networks. Patients also perceived multiple barriers to initiating and sustaining intimate relationships (e.g. sexual dysfunction and body-image disturbance). Biographical repair revealed a process of adjustment and adaptation, with patients finding new meaning in life on dialysis through efforts to reconnect with lost peers and seek alternative interests. This study suggested that age - and developmental life-stages - are important determinants of illness experience and outcome. The third paper discusses implications for theory and clinical practice emerging from the first two papers. It emphasises the importance of considering the intersection between illness and age in both research and clinical contexts. The difficulties that young people have in maintaining a place within social networks is discussed in relation to social support structures, whilst the difficulties faced in establishing and maintaining intimate relationships are also considered within a developmental framework. This paper also contains personal reflections on the research process and outcomes.

Background. Exercise is a natural medicine that has been prescribed for the prevention and management of chronic diseases, to enhance quality of life, improve health status and promote longevity. Current efforts to implement exercise as routine practice in the conventional renal replacement therapy population have been hampered by a lack of randomised controlled data. The aim of this thesis was to investigate the effect of exercise as an adjunctive therapy to enhance outcomes pertaining to renal transplantation, vascular access, haemodialysis adequacy and muscle wasting in Stage 4 and 5 Chronic Kidney Disease patients. It was hypothesised that randomised controlled trials employing gold standard outcome measures would reveal significant beneficial effects of exercise that are strongly associated with quality of life, hospitalisation and survival in this patient population. Reports. The first report presents a systematic literature review of exercise in the kidney transplant population. The largest positive effects were noted on intermediate outcomes such as aerobic fitness and muscle strength. Presumably these adaptations contributed to the trends observed for improvement in quality of life. Whether exercise impacts on outcomes associated with longevity of life requires further study. The rest of the thesis focused on patients receiving the more popular form of renal replacement therapy, haemodialysis. The first empirical study of the thesis, appertaining to vascular access, investigated the feasibility of implementing a post-operative forearm exercise intervention for arteriovenous fistula maturation. Exercise had no effect on primary outcomes measures of arterial diameter (95% Cl, -0.24 [-1.12; 0.51] mm) and venous diameter (95% Cl, 0.16 [-1.84; 1.24] mm). It was concluded that future randomized controlled trials should investigate a similar protocol implemented before arteriovenous fistula creation to enhance surgery success and maturation. The second randomised controlled trial explored the effect of intradialytic exercise, in comparison to the traditional prescription of increased dialysis time, to enhance dialysis adequacy and solute removal. Increased haemodialysis time, but not exercise, increased equilibrated Kt/V urea compared to control trials (Extra time vs. control: 95% Cl, 0.15 [0.05; 0.26], P < 0.05; exercise vs. control: 95% Cl, 0.03 [-0.05; 0.12], P > 0.05). Exercise, but not increased time, increased phosphate reduction ratio (exercise vs. control: 95% Cl, 8.6 [0.5; 16.7] %,p < 0.05; extra time vs. control: 95% Cl, 5.0 [-1.0; 11.1] %, p > 0.05). Thus intradialytic exercise cannot replace the traditional prescription of increased haemodialysis time for improving dialysis efficacy, but may be a useful adjunctive therapy for serum phosphate control. The third study implemented a randomised controlled trial of intradialytic progressive resistance training for treating muscle wasting. The primary outcome measure of thigh muscle volume, as measured by magnetic resonance imaging, significantly increased following 12 weeks of training compared to a sham exercise control (95% Cl, 193 [63; 324] cm3). Intradialytic resistance exercise elicited an anabolic and strength response in haemodialysis patients. However, a surprising lack of a change in functional capacity despite increased muscle mass warrants further investigation. Conclusion. The findings suggested that exercise had a beneficial effect on factors relating to outcomes in Stages 4 and 5 Chronic Kidney Disease patients. However, to ensure effectiveness of interventions and to maximize programme efficiency, careful consideration of basic exercise and physiological principles is required. Nevertheless, the observed benefits of exercising outweighed its risks, thus supporting the initiative for exercise prescription as an adjunctive therapy for the management of this disease state.

This PhD sought to exploit the acute effects of exercise upon the kidneys to make tenable links to pathological states such as acute kidney injury (AKI) and chronic kidney disease (CKD). It is surprising that such associations with their potential clinical implications have received limited attention so far despite the ever-increasing number of healthy individuals participating in vigorous and physiologically challenging activities. The work herein has shown how experimental in-vivo exercise models may be used to simulate a “stressed” kidney with features that resemble diseased states. Summarising the key findings briefly, the first study (chapter 2) demonstrated that maximal-intensity exercise in the form of an 800 metre sprint resulted in increased urinary concentrations of an AKI biomarker (neutrophil gelatinase-associated lipocalin / NGAL), suggesting mild kidney stress or a concentrating effect. However, plasma NGAL concentrations decreased and urinary rises were independent of post-exercise proteinuria. There was also an inverse relationship between urinary volume and urinary NGAL concentrations – an observation that is also seen in oliguric AKI. The systematic review in the second study (chapter 3) found promise in post-exercise proteinuria as a predictor for CKD progression. Five studies (N = 351) that met inclusion criteria, examined prospective cohorts of Type I diabetics who were at risk of CKD. Through combining the results of the primary outcome in four studies (N = 318), the presence of post-exercise proteinuria was highly associated with elevated resting proteinuria at follow-up (χ2 test, P < 0.0001) and significant odds ratios (developing CKD following a positive exercise test vs. not developing CKD) were noted in each of these four studies (OR 2.3-52.0). However, there was great variability and questionable validity in the interventions that did not permit meta-analysis. It was evident that exercise interventions need to be refined and standardised before applying to other at-risk CKD populations. In the third study (chapter 4), it was demonstrated that a prior bout of muscle-damaging exercise established a pro-inflammatory state with elevated plasma interleukin-6 (IL-6) concentrations, and that with subsequent endurance-based exercise in the heat there was increased kidney stress as measured by increased urinary NGAL and plasma creatinine concentrations. The latter elevations met clincial criteria for AKI. Also, plasma IL-6 and plasma NGAL concentrations were positively correlated. Lastly, the final study (chapter 5) extended the findings of chapter 4 by isolating the role of pro-inflammatory IL-6 in AKI. Through infusion of recombinant IL-6 in healthy males to concentrations above 100 pg/ml, elevations in plasma NGAL concentrations were shown but not to AKI ranges. In addition, there were no changes to plasma concentrations of other AKI biomarkers such as creatinine or cystatin C. Overall, this suggests that IL-6 is able to modulate NGAL but is not responsible per se for AKI or kidney dysfunction. Thus, it is likely that additional physiological aberrations are needed.

In the kidney, unbound amino acids are freely filtered into the lumen of the nephron. For reabsorption to occur, they must be transported across the phospholipid bilayers of the tubular epithelium by selective transport systems. Mutations in these transport systems can lead to disease though a conferred lack of amino acid re-absorption. One such disease is cystinuria, caused by mutations in SLC3A1 and SLC7A9, which encode the two protein subunits of System b0,+, rBAT and b0,+AT, respectively. In healthy individuals System b0,+ mediates Na+- independent reabsorption of dibasic amino acids, and the cysteine dimer, cystine, in exchange for neutral amino acids. In cystinuric patients, these amino acids are not sufficiently reabsorbed causing a dibasic aminoaciduria and the precipitation of cystine crystals, leading to the formation of renal calculi. A cohort of cystinuric patients was recruited to the study, and both genes were screened for causal variants. A range of techniques was employed to enable the detection of small point mutations and large genomic rearrangements. Four novel missense variants were detected in SLC3A1. These were M465K, N254T, L416P and Y579D. In silico homology modeling of rBAT against the crystal structure of B. cereus oligo-1,6-glucosidase (PDB code 1UOK), predicted the location of these mutations in the extracellular domain of the protein. When rBAT cRNA was injected into Xenopus oocytes, uptake of the prototypical System b0,+ substrate [3H]arginine was observed, following the association of human rBAT with an endogenous oocyte light chain. A series of techniques was optimised to allow the characterisation of FLAG-tagged rBAT function and expression in oocytes, 1-6 days postinjection of cRNA. Mutations in rBAT lead to a mis-folding of the protein and its early degradation in the ER, preventing successful trafficking of the System b0,+ heterodimer to the renal epithelial membrane. This aberrant trafficking leads to reduced rBAT expression and System b0,+ activity in oocytes. Functional characterisation of the novel mutant proteins led to a decrease in the Vmax of [3H]arginine transport. Over-expression of rBAT in oocytes apparently overcomes the defect and leads to a recovery of function over time. However, [3H]arginine uptake in M465Kexpressing oocytes was still lower than that observed with wild-type rBAT even at 6 days postinjection. These data were supported by immunofluorescent detection of rBAT and the mutant proteins at the plasma membrane of oocytes. Western blotting of total membrane proteins from oocytes expressing mutated rBAT showed decreased total protein, suggestive of an increased rate of degradation associated with the pathogenic variants. An increased understanding of the effect of these mutations on the biogenesis of rBAT will contribute to the identification of novel therapeutic targets in the treatment of cystinuria.