Malaria is a deadly parasitic disease of humans spread through the bite of Anopheles spp. mosquitoes. Current control methods for the disease are broadly effective, but the spread of insecticide resistance in the principle Anopheline vectors of the disease raises the possibility of an increase in disease burden in the future. Transgenesis offers a novel alternative approach to vector control but requires the mass-release of virile, competitive, genetically altered male mosquitoes, thus the success of future transgenic release programmes depends greatly on how capable the transgenic strain is of surviving in field conditions and successfully introgressing with wild mosquito populations. Using a combination of laboratory and field-based ecological experiments, along with molecular biological and genomic approaches, we assessed both the fitness of two transgenic strains of Anopheles gambiae s.s. and the genetic and environmental factors determining survival, mating success and assortative mating behaviour in lab and field derived samples of non-transgenic Anopheles gambiae s.s. We found that imposed a fitness cost in both a transgenic strain carrying a phenotypic marker, and a second strain carrying a putative anti-malarial peptide sequence. Overt fitness costs were confined to larval development in both strains, although there was some evidence of a difference in egg production and morphology between strains. The anti-malarial peptide-carrying strain was significantly less fit, and suffered a fitness burden in hemizygote individuals as well as homozygotes. The possible sources of fitness differences are discussed. In semi-field-based crosses, we observed a significant interaction between the genetic and environmental background in the survival and mating success of non-transgenic strains; furthermore, the combination of laboratory rearing and a laboratory genetic background was sufficient to abolish the assortative mating behaviour normally observed between M- and S-molecular form An. gambiae populations.

During the second half of the twentieth century, the Antarctic Peninsula exhibited a rapid increase in air temperatures. This was accompanied by a reduction in sea ice extent, increased precipitation and a dramatic retreat of glaciers associated with an increase in heat flux from deep ocean water masses. Isotopic tracers have been used previously to investigate the relative importance of the different freshwater sources to the adjacent Bellingshausen Sea, but the data coverage is strongly biased toward summer and unambiguous determination of the different meteoric water contributions remains challenging. Here a high-resolution model is used to investigate the ocean's response to the observed changes in its different freshwater inputs (sea ice melt/freeze, precipitation/evaporation, iceberg melt, ice shelf melt and glacier melt). By developing the code to enable tracing of the sources and pathways of the individual components of the freshwater budget, it is shown that sea ice dominates the seasonal changes in freshwater content, but all sources contribute approximately equally to annual-mean freshwater fluxes and interannual freshwater flux anomalies. Ice shelf melting is shown to be the largest contributor to freshwater content on the annual mean. Decadal trends in the salinity and stratification of the ocean are investigated, and a 20-year surface freshening trend is found to be predominantly driven by decreasing autumn sea ice growth. By partitioning the freshwater in this way, insight is gained into the long-term freshwater balance and variability, and therefore the potential effects of a changing climate.

Successful treatment of prostate cancer (PCa) depends on early diagnosis and screening, which currently relies on the measurement of serum prostate specific antigen (PSA) levels. The overarching aim of the project was to generate molecularly imprinted polymers for PCa biomarkers, with subsequent integration with a sensing platform to allow for rapid, point of care detection and monitoring. The initial work involved the use of simple PSA epitopes for epitope imprinting using conventional imprinting techniques. A four amino acid sequence from the Cterminus of PSA was imprinted with MAA, Aam and Urea monomers to obtain bulk imprinted polymers. Apparent Kd of 102 μM, 154 μM, 194 μM was obtained for MAA, AAm, Urea based bulk mini-MIPs respectively. Epitope imprinting was further developed using a surface imprinting approach, via electropolymersiation of dopamine to detect an epitopic sequence from pro-PSA. An improvement in Kd from bulk-imprinted polymers, with an apparent Kd of 2.9 μM was obtained with the surface electrochemical MIP sensor. However, both epitope imprinting technique lacked sensitivity to measure clinical relevant concentrations of PSA (nM range). As a consequence, a more sophisticated technique called hybrid imprinting was developed to build an electrochemical MIP sensor. Hybrid MIP imprinting utilised an aptamer with established affinity towards PSA to trap the aptamer-PSA complex into a surface grown electropolymer (polydopamine). The resulting aptamer lined polymer pockets exhibited high selectivity and affinity towards PSA (apparent Kd 0.3 nM). The apta-MIP sensor was also able to discriminate from a homologous protein (human Kallikrein 2) and was resilient to fouling from serum proteins. The apta-MIP sensor was further translated to a MOSFET device whereby successful detection of PSA at clinically relevant concentration was obtained in human plasma. Although good sensitivity and selectivity was obtained with the hybrid-MIP sensors, further research is required to understand the binding mechanism of the template to the MIP.

CD40 is a member of the TNF receptor (TNFR) superfamily and its expression by a variety of cell types including tumour cells has suggested a possible role for CD40 in epithelial homeostasis and potentially in the pathogenesis of cancer. CD40 ligation by membrane-presented CD40L (mCD40L), but not soluble agonists, causes extensive apoptosis in malignant epithelial cells, including bladder and colorectal cancer cells, while sparing their normal counterparts. However, the role of CD40 in renal cell carcinoma (RCC) is relatively unknown and the effect of CD40 ligation in RCC cells has not been studied previously. This thesis aimed to investigate the effect of CD40 ligation in RCC cells, compare this to their normal counterparts (HRPT cells), and identify the mechanisms of CD40-mediated effects. The experimental work described in this thesis involved optimisation of assays for the detection of cell death (based on loss of plasma membrane integrity, DNA fragmentation and caspase activation) and for the detection of pro-inflammatory cytokine secretion. Immunoblotting techniques were adapted for a co-culture system to deliver mCD40L for detection of key intracellular CD40 signalling-associated mediators. Optimisation was also carried out for functional experiments using pharmacological inhibitors of intracellular mediators and caspases and for the detection of reactive oxygen species (ROS). Expression of CD40 by RCC cells was detected in RCC lines and in their normal counterparts HRPT cells and treatment with IFN-ɣ up-regulated CD40 expression in RCC cells. Cytotoxicity assays showed for the first time that mCD40L induced massive apoptosis in human RCC cells which further increased in the presence of IFN-ɣ, whereas it caused no cytotoxic effect in their normal counterparts (HRPT cells). By contrast, the G28-5 mAb did not cause death in RCC cells, and combination of IFN-ɣ with cross-linked G28-5 antibody did not render the G28-5 antibody significantly pro-apoptotic. Moreover, induction of cell death by mCD40L was accompanied by caspase-3/7 activation and DNA fragmentation in RCC cells, while mCD40L did not induce detectable DNA fragmentation in normal HRPT cells indicating that mCD40L triggered “apoptotic” cell death in RCC cells and in a tumour cell-specific fashion. ELISA assays showed that mCD40L induced marked secretion of IL-8 and IL-6 in RCC cells, which was stronger than that triggered by soluble agonist. More importantly, mCD40L induced GM-CSF secretion in RCC cells, but soluble agonist caused little GM-CSF release. In normal HRPT cells mCD40L caused secretion of IL-8 and IL-6 and a more pronounced secretion of GM-CSF, when it was compared to agonistic G28-5 mAb, confirming that CD40 on HRPT cells was functional despite being non-apoptotic. mCD40L triggered rapid induction of TRAFs 1, 2, 3 and 6 as early as 1.5h post CD40 ligation in RCC cells. By contrast, despite up-regulation of TRAF1 at 6h post CD40 ligation, in normal HRPT cells mCD40L down-regulated TRAF2 expression and caused no induction in TRAF3 expression. In addition, CD40-mCD40L interactions in RCC cells triggered MKK4/7 activation and downstream phosphorylation of both JNK and p38. Functional inhibition experiments demonstrated that JNK and p38 phosphorylation was essential in CD40-mediated apoptosis in RCC cells, and suggested that activation of p38 may be dependent on JNK activity. By contrast, inhibition of MEK1/2 and NF-кB did not alter CD40-mediated apoptosis in RCC cells, whilst inhibition of AP-1 caused moderate (not complete) reduction in apoptosis. This study demonstrated that induction of Bak and Bax occurred by 6h post CD40 ligation in RCC cells. Inhibition of caspase-9 significantly attenuated CD40-mediated apoptosis in RCC cells, while caspase-8 and 10 inhibition caused non-significant effects whilst no induction of death ligands was detectable, suggesting that CD40-induced apoptosis in RCC cells occurs via a direct, intrinsic apoptotic pathway. mCD40L triggered ROS production in RCC cells within 1h post CD40 ligation and ROS production were critical in the induction of death, as apoptosis was inhibited by the antioxidant NAC. Moreover, mCD40L triggered phosphorylation of the NOX subunit p40phox and the NOX inhibitor DPI attenuated apoptosis suggesting that a ROS-dependent NOX-triggered pathway may occur in RCC cells. By contrast, non-apoptotic CD40 agonist (G28-5 mAb) did not induce ROS production in RCC cells. Equally importantly, mCD40L caused rapid ASK-1 phosphorylation and down-regulated Trx-1 expression in all RCC lines. Collectively, this study has for the first time reported that mCD40L induced extensive apoptosis in RCC cells while sparing their normal cell counterparts. However, agonistic anti-CD40 antibody G28-5 did not cause cell death in RCC cells. Although additional functional experiments would be necessary to fully elucidate the functional mechanisms of apoptosis, it appears that CD40-mediated killing in RCC cells occurs via a TRAF3-p40phox-ASK-1-MKK4/7-p38/JNK pathway leading to caspase-9 and effector caspase-3/7 activation and intrinsic apoptosis. Importantly, whilst increasing ROS levels in RCC cells, mCD40L actively down-regulated Trx-1 expression. These findings have provided novel observations on the role of CD40 in regulating human RCC cell fate, and have also reinforced the importance of the quality of CD40 signal in determining functional outcome. Equally importantly, the findings have also assisted in the formulation of novel therapeutic avenues that may exploit CD40 for anticancer therapy and specifically for renal cell carcinoma.

New drug entities (NDE) are constantly being developed with most of them intended for oral administration. For this reason, there is a need to estimate their absorption in order to save time and money that would be lost if the drug enters the clinical stage and is then found to exhibit poor absorption. For many years, the use of animals was the most abundant method for studying pharmacokinetics to predict parameters such as intestinal absorption. However, these methods are time consuming, and expensive as well as being ethically unfavourable. As a result, developing other methods to evaluate a drug’s pharmacokinetics is crucial. The aim of this work was to develop in vitro methods for estimation of human intestinal absorption (%HIA) to replace the use of the aforementioned, less favourable methods involving the use of animals. Among the developed methods in this thesis is a unique type of chromatography known as micellar liquid chromatography (MLC) using biosurfactants such as bile salts as a mobile phase. Furthermore, studies investigated the effect of a change in the stationary phase in addition to investigating the effect of the change in temperature on the elution of the analysed compounds. It was found that R2PRED for the developed MLC methods was in the range of 43.3 % - 91.12 %. Another developed method was a spectrophotometric method based on the use of the solubilising effects of bile salts, as well as their binding to compounds. Therefore, two spectrophotometric methods were developed, a solubilisation method and a double reciprocal method, and used in the prediction of %HIA. It was found that the solubilisation method had a better predictability for %HIA than that of the double reciprocal method where R2PRED was found to be 82.32 % and 61.90 % respectively. Finally, a permeation method was developed using the ability of NaDC to form a hydrogel under specific conditions and applying the investigated drugs in an infinite dose to the prepared hydrogels. This facilitated the determination of permeability coefficients (Kp) that were then used in the prediction of %HIA using the obtained model. The two developed permeation methods were found to have close values of R2PRED for % HIA where R2PRED of the permeation method using flow through cells was found to be 79.8 % while that of the permeation method using Franz cells was found to be 79.67 %. In summary, this work reports several unique models for the in vitro prediction of human intestinal absorption, potentially removing the need for animal testing to predict %HIA.

The training regimes associated with competitive sport place athletes under both physical and psychological stress. Moreover, there are many further unique stressors associated with competitive sport (e.g., injury risk). This environment can leave athletes susceptible to a number of negative outcomes. Two important outcomes associated with the psychosocial and physiological consequences of sport are burnout syndrome and overtraining syndrome. Burnout and overtraining can have a number of cognitive, affective, motivational, and behavioral consequences (Goodger, Gorely, Lavallee, & Harwood, 2007; Meesuen et al., 2013). Subsequently, sport and exercise psychologists have sought to identify factors that may predispose athletes to these syndromes with a view to reducing their deleterious effects. One factor that has shown particular promise is perfectionism (see Hill & Curran, 2016). Although the existing literature has provided evidence for a relationship between perfectionism and burnout, an over-reliance on cross-sectional correlational designs has meant that conclusions regarding temporality and/or causality have been limited (Taris, 2000). Moreover, the only extant longitudinal study has several methodological limitations (Chen, Kee, & Tsai, 2009). As such, the first three studies of this thesis investigate the longitudinal direct, reciprocal, and mediational effects between perfectionism and athlete burnout. Finally, no study has investigated if perfectionism may predict changes in the susceptibility to overtraining syndrome over time. Using training distress as a marker of overtraining syndrome, the final study of this thesis sought to determine whether perfectionists are predisposed to experience training distress. Study one investigated perfectionism and burnout in junior athletes over a period of three months and the findings showed that perfectionistic strivings predicted decreases in burnout, whereas perfectionistic strivings predicted increases. Replicating and extending study one, study two investigated whether the two dimensions of perfectionism showed interaction effects in predicting changes in burnout in adult athletes over three months. The results of study two showed that the two dimensions of perfectionism interacted, with perfectionistic strivings buffering the incremental effect of perfectionistic concerns. Study three sought to examine whether the quality of motivation would mediate the longitudinal relationship between perfectionism and burnout in junior athletes over a period of six months. The findings of study three showed that autonomous motivation mediated the longitudinal negative relationship between perfectionistic strivings and burnout at both the between- and within-person level, whereas controlled motivation mediated the longitudinal positive relationship between perfectionistic concerns and burnout at the between-person level only. Study four investigated whether the two dimensions of perfectionism also showed divergent relationships with training distress in junior athletes over a period of three months and the findings showed that whereas perfectionistic strivings had a negative cross-sectional association with training distress, perfectionistic concerns had a positive association. Moreover, perfectionistic concerns predicted increases in training distress over the three month period, whereas perfectionistic strivings did not. Taken together, perfectionistic concerns appear to be a factor predisposing athletes to a higher risk of experiencing burnout and training distress, whereas perfectionistic strivings may be a protective factor. These divergent relationships may be explained by autonomous and controlled motivation. With this, the findings of the present thesis provide further evidence for the important role that perfectionism plays in sport.

The research work undertaken focused on the preparation and characterization of novel low molecular weight (LMW) hydrogels as functional biomaterials for tissue culture applications. To achieve this objective, new LMW compounds (as potential hydrogelators) were synthesized bearing a galactosamine or glucosamine moiety. The incorporation of carbohydrates was anticipated to confer molecular recognition of certain biomolecules upon the formed supramolecular gels and therefore act as potential anchor sites for cell-binding. The synthesis was based on short synthetic routes and low-cost starting materials were used as supplied. The target compounds were not confined only to those containing carbohydrates. A cinnamoyl-protected diphenylalanine hydrogelator was prepared and the properties of its corresponding hydrogel were investigated. Understanding the self-assembly mechanisms of supramolecular hydrogels is fundamental for the preparation and application of these novel materials. Therefore, a variety of techniques were employed for assessing and characterisation of gelation and to determine the configurational alignment of the formed fibres within the three-dimensional network of the gels. Specifically, the preparation and handling of hydrogels were optimized leading to robust gelation protocols. TEM and SEM microscopy revealed the size, shape and perplexing patterns of the fibres. XRD measurements verified polymorphism whereas rheology studies confirmed the viscoelastic properties of the gels. Non-covalent intermolecular interactions are the driving forces of the molecular packing, leading to higher order architectures. The combined spectroscopic analysis of the prepared hydrogels (by NMR, IR, UV-vis, CD) was advantageous to explore the nature of such interactions and allow the identification of key functional groups which actively participated in the self-assembly process. As a result of the CD work undertaken, utilisation of a synchrotron facility led to the establishment of a protocol for the evaluation of LMW hydrogels by SRCD spectroscopy, which was recently published. Finally, a preliminary biocompatibility study was undertaken to assess the toxicity of the hydrogels upon brain cancer cells. This project therefore required an interdisciplinary approach which involved the synthesis of a number of LMW compounds where some were found to be hydrogelators. This led to the preparation of their corresponding hydrogels and the study of their microscopic/macroscopic properties for the development of novel biocompatible materials suitable for tissue culture applications.

Introduction: Participation in ultra-endurance events has increased exponentially in recent years. Despite this, performance in such demanding events has been stagnant. Numerous studies have observed that ultra-endurance athletes consistently fail to meet the extensive energy demands and the current carbohydrate (CHO) recommendations, which may in part explain this plateau in performance. To date, little is known about the causes of suboptimal energy and CHO intake or the most effective strategies to address these inadequacies. Therefore, the aims of this thesis were to (i) explore the challenges to optimal nutritional intake, (ii) establish whether a gut-training programme could enable ultra-endurance athletes to meet the CHO recommendations and (iii) determine whether a short term high fat, low CHO diet (HFLC) or a low fat, high CHO (LFHC) diet prior to competition is more effective for ultra-endurance performance, when fuel availability is likely to be compromised. Methods: One hundred and seventy participants took part in the studies involved in this thesis, including 118 ultra-endurance athletes. The remaining participants consisted of three population groups with different levels of nutrition knowledge and experienced distance athletes, who were involved in the developmental phase of the first two studies. Both studies employed a two-phase approach to (i) adapt and evaluate a questionnaire for use with ultra-endurance athletes; and (ii) subsequently implement the questionnaire with these athletes (n = 101). The first questionnaire was completed alongside a series of 24 hr food diaries to explore the relationship between nutrition knowledge and food intake. The second explored the main factors that influence food choices during training and competition. The penultimate study required ultra-runners (n = 17) to follow a multicomponent dietary intervention (gut training + HFLC compared with gut training + LFHC diet), which was designed to overcome identified challenges to optimal nutritional intake and to optimise fuel iii availability in preparation for a 56 km ultra-endurance foot race. The final study explored the experiences of a subsample of ultra-endurance runners (n = 14) as they made their food choices during the race, using a series of face-to-face interviews. Results and discussion: The nutrition knowledge of ultra-endurance athletes was superior to the general population, however there was no relationship between knowledge and the adequacy of the ultra-endurance athletes diet. The most important factors that influenced the food choices of these ultra-endurance athletes were the avoidance of gastrointestinal symptoms (GIS) and the provision of adequate energy. These factors were followed closely by the desire for nutritious products and those that were easy to consume during training and competition. The multicomponent intervention successfully manipulated the CHO and fat composition of the 17 ultra-endurance athletes, however this did not affect their race performance. Furthermore, despite a period of gut training designed to improve the ultra-runners tolerance of high volumes of CHO, ultra-runners failed to meet the recommended rate of CHO intake and the severity of their GIS did not improve. Subsequent analysis of the interviews indicated that the processes involved in making food choices during the race were complex and dynamic. All ultra-runners altered their food choices during the race in response to triggers, such as hunger and taste fatigue. This resulted in the consumption of lower CHO density products, which may partially explain the suboptimal CHO intake. Conclusion: Advances in ultra-endurance performance appear to be restricted in part by the adequacy of the athlete’s nutritional intake. At present, strategies to address the multiple challenges to optimal nutritional intake have had limited success. However, practicing competition nutrition during training is likely to simplify iv the decision-making process during events, allowing ultra-endurance athletes to focus on their performance.

Background Regular engagement in physical activity (PA) provides children and young people with numerous physical, psychological and social health benefits. PA levels decline during adolescence and girls are less active than boys. Schools have been suggested as a promising location to target adolescent girls’ PA behaviours. Recently, numerous researchers have incorporated peer-led approaches into PA intervention designs. However, little is known about the feasibility, acceptability and effectiveness of these approaches. The overarching aims of the research programme were to investigate the effectiveness of school-based PA interventions for adolescent girls and assess the feasibility, acceptability and effectiveness of a novel school-based peer-led PA intervention to improve the PA levels and reduce the sedentary time (ST) of adolescent girls. Methods This programme of work included four studies. Study 1 was a systematic review and meta-analysis of the literature to assess the effectiveness of previous school-based interventions for adolescent girls. Study 2 was an exploration study, which assessed girls’ thoughts and perceptions of current school PA practices. Study 3 incorporated the design of a novel three-tier peer-led school PA intervention, with a mentoring component, as part of the Girls’ Peer Activity (G-PACT) project. University students (Mentors) delivered a series of leadership and PA educational sessions to a group of adolescent PA Leaders who disseminated this information to their Peers and encouraged them to engage in more PA. The G-PACT intervention was underpinned 7 by Social Cognitive Theory and Self-Determination Theory. This study evaluated the effectiveness of the G-PACT intervention on adolescent girls’ PA levels and ST. Study 4 was a feasibility and acceptability assessment of the G-PACT intervention in the secondary school setting utilising a qualitative approach. Results Study 1 established that school-based interventions for adolescent girls have a small but positive impact on girls’ PA levels. Interventions underpinned by theory and multi-component in nature were more effective. Study 2 provided an insight into girls’ school PA experiences, highlighting their enjoyment of PA with friends and the importance of choice over activities. Study 3 demonstrated that the G-PACT intervention with a fitness class-based after-school club was effective increasing girls’ PA levels. Study 4 indicated that the link between Mentors and Leaders in the G-PACT intervention was feasible and acceptable. However, the link between Leaders and their Peers requires refinement to improve the communication processes. Conclusion Schools are a promising setting to promote PA among this population, but past interventions only have a small positive impact on girls’ PA levels. Engaging with adolescent girls and listening to their needs is crucial to inform the development of complex interventions in a school setting. Novel interventions such as the G-PACT intervention should be encouraged as they show promise in increasing adolescent girls’ PA levels, and this innovative intervention approach warrants piloting before consideration on a larger scale.

This research explored the optimisation of three novel antimalarial series based on the MMV compound leads shown below. Extensive research began to optimise these potent hits through exploration of multiple functional group substitutions on two or more sites of the core structure (shown above). The highest activity tetrahydro-β-carboline analogue discovered during this research was LVT30 (IC50=1μM) which possesses an absent C3 interaction and a p-trifluoromethyl R2 substitution. Meanwhile, of the spirocyclic subset of derivatives, a high nanomolar activity compound was generated LVT86 (IC50=960 nM), possessing a butyl R1 chain and a m-trifluoromethyl R2 group and future work will include optimisation of this analogues pharmacokinetic properties. The most fruitful subseries of this project was a catalogue of 2-iminobenzimidazole compounds. In an attempt to optimise activity and increase potency, exploration involved variation of all three R positions and as a result, generated the most potent derivative over all three series. Compound LVTa95 possesses an inhibitory concentration of 33.4 nM and exhibits: a pentane R1 group, 2,4-dichloro R2 substitution and a hydroxy R3 group. As this compound existed as enantiomers, they were subsequently separated, and it was found both stereoisomers had similar activity.