Obesity results from complex interactions between biological, behavioural, social and environmental factors. The focus of this thesis, both the collection of papers presented and the extended abstract, is on community-based approaches to the prevention and treatment of obesity across five important key life stages. The importance of early life influences, including the intrauterine environment, and the subsequent impact on body fatness across the life-course is discussed. Published research included considers the impact of interventions which aim to limit gestational weight gain on baby’s’ birthweights and the wider influence on sustained breastfeeding. However research presented also suggests that pregnant women receive limited information from healthcare professionals and whilst pregnancy should provide an ideal opportunity for public health intervention, the postnatal period may be more acceptable as a time to provide support and advice. The prevalence of childhood obesity has continued to increase. The most significant predictor of childhood obesity is parental obesity. A paper is presented which demonstrates that engaging adults in lifestyle programmes can have a positive impact on both the diet and activity habits of their family. A systematic review considers how TV viewing during meal-times may influence the foods and drinks consumed with the findings suggesting that the practice of eating whilst watching TV has a negative impact on the diet quality. It is recognised that sugar-sweetened beverages (SSBs) contribute significantly to the free sugar dietary intake, particularly the %total energy intake of children and adolescents. A further published review asks the question, which interventions that aim to reduce the intake of SSBs lead to reduced body fatness in children. There is a need for both primary prevention activities to prevent unhealthy weight gain and interventions to treat overweight and obese children/adolescents. Published data provides evidence for an intervention which does lead to weight loss in obese adolescents, leading to enhanced self-esteem which helps to improve their lifestyles behaviours. Young adulthood represents a period of change with increasing independence. There may be few occasions to discuss weight management with young adults but a presented paper explores the opportunities in primary care. Obesity is associated with a number of chronic diseases, one example being type 2 diabetes which is now presenting in adolescents and young adults. Losing weight can have a significant clinical impact on the glycaemic control of people with diabetes and thus reduce the health burden associated with the condition. Type 2 diabetes is now reversible if significant weight loss is achieved. A paper is presented which evaluates weight management in people with diabetes and the associated impact on glycaemic control. Prevalence data suggests that 25 million adults in the UK are either overweight or obese. Given the health burden to both the individual and society, scalable solutions are required which are delivered in a community setting. Through collaboration with a commercial weight management organisation (CWMO), a series of papers are presented which explore the feasibility and effectiveness of referral from primary care to CWMOs. CWMOs, using behavioural strategies, are well placed to support the large numbers of people who need weight management guidance and a referral scheme may address the health inequalities seen in obesity prevalence. One example of a well-established behavioural strategy, target setting, is considered in more detail with evidence of the importance of target setting presented. As with all research, weight management research is confounded by methodological issues. Community- based programmes are often more complex than clinical trials and contamination may be an issue. The extended abstract explores the methodological issues related to the presented papers in more detail. Whilst we can continue to improve our methodology it is important that we advance our knowledge as to what works to reduce the obesity epidemic. Sadly there will probably not be one solution which is effective at an individual level given the psychosocial complexities of obesity. Ideally we need to promote a nutritionally balanced diet and adequate levels of daily activity which enable children and adults to maintain a healthy weight across the life-course starting at the very beginning of conception.

Introduction and Aims Despite the availability of evidence-based guidelines, blood transfusion, one of the commonest procedures in health care, manifests significant variation across different countries that appear to have comparable health systems. This is corroborated by extensive data on blood usage in coronary artery bypass graft surgery, a sentinel procedure, which also suggests that the variation may be related to institutional practice. Regional use of red cells within the same country, data for which had not yet been collated to date, was elicited, and it confirmed the same pattern of variation where the highest-using regions tend to transfuse double the red cells transfused in the lowest-using ones. Methods Since the reasons for it are largely unknown, analyses of available data from Australia, New Zealand, Canada, the USA, and 24 countries in Europe, were performed using linear correlation and regression analysis, to identify potential predictors and possible effectors of this variation. Results Apart from confirming a known demographic predictor, proportion of the population over 65 years (p = 0.01), another 4 predictors were identified including clinical activity as represented by coronary artery bypass graft surgery (p = 0.001) and health funding (p = 0.007), which together explained 70% of the variation in red cell usage. Similar regression models for platelets and plasma showed an R square value of 0.31 and 0.247 respectively. The variation in blood product use did not correlate at all with health service outcomes and performance, but correlated quite tightly with markers of supply sensitive care making a strong case for unwarranted variation in blood transfusion. Red cell use, for example, correlated with the availability of diagnostic technology as represented, amongst others, by the number of MRI units (r = 0.665, p = 0.001). Moreover, it was shown that countries that use more of one product use more of the others. A relationship was also found between blood product usage and cultural constructs capturing the notions of professional uncertainty and difficulty with accepting new evidence. Conclusions Irrational and evidence-denying variation in blood transfusion practice exists and can be measured relatively easily. Comparison of clinical use of blood in discrete geographical regions may be useful as a general measure of the effectiveness of the implementation of different tools to improve practice, not just within the context of blood transfusion but in clinical practice in general.

Chapter 1 outlines the potential of coordination and organometallic complexes in antimicrobial chemotherapy. Various historical examples are reviewed and their advantages and disadvantages discussed. Recent developments are then described, which have allowed the design of complexes with well-defined 3-dimensional structures, but in which the metal acts as a structural template rather than a reaction centre. Finally, consideration is given to the prospects for such compounds making a substantial contribution to the currently rather uncertain future of antimicrobial chemotherapy. Chapter 2 describes the discovery, synthesis and characterisation of new Class Ia (exo-pyridine) flexicates with varying ligand functionality, as well as some of the underlying challenges. The helicating effect of the linker connecting these bimetallic structures is subsequently explored, by investigating their structure, physical properties and aqueous stability. Chapter 3 focuses on the screening of water-soluble Class Ia flexicates for antimicrobial activity against various bacteria, allowing the establishment of lead compounds against Gram-negative bacteria. Various assays are then used in order to make further preclinical evaluations of the lead compounds and/or better understand the nature of the observed antimicrobial activity. For example, eukaryote toxicity and effect upon membrane integrity are assessed. Chapter 4 explores the use of tandem omics techniques (genomics, transcriptomics and proteomics) as means to develop credible hypotheses regarding the mechanism behind the potent activity of the lead compound against a pathogenic E. coli strain. The initial study describes the selection and characterisation of E. coli mutants tolerant the lead compound. Responses of the cell at the RNA and protein level respectively are then measured in response to the addition of the lead compound, at a sub-lethal dose. Chapter 5 Infers general conclusions from across this thesis and conveys suggestions for future work that may further expand this field. Chapter 6 details the experimental procedures used to carry out the work in this thesis. Appendix contains tables of crystallographic data.

Risk of cardiac conduction slowing (QRS/PR interval prolongations in monitored electrocardiograms) is assessed in nonclinical studies, where the current AstraZeneca strategy involves ensuring high margins to in vitro effects and statistical tests to identify in vivo effects. This thesis aims to improve QRS/PR risk assessment using pharmacokinetic-pharmacodynamic modelling for describing QRS/PR effects and evaluating translation to human effects. Data for six compounds were collected from the literature and previously performed in vitro (sodium/calcium channel), in vivo (guinea pig/dog) and clinical AstraZeneca studies. Mathematical models were developed and evaluated to describe and compare effects across compounds and species. Key results were that proportional drug effect models often suffice for small QRS/PR changes (up to 20%), while larger effects require nonlinear models. Heartrate correction and circadian rhythm models reduced residuals primarily for describing baseline PR intervals, with highest impact in humans followed by dogs and guinea pigs. Meaningful (10%) human QRS/PR changes correlated to low levels of sodium channel block (3-7%) and calcium channel binding (13-21%) and to small effects in guinea pigs and dogs (QRS 2.3-4.6% and PR 2.3-10%). This suggests that worst case human effects can be predicted by assuming four times greater effects at the same concentration from dog/guinea pig. Small changes in vitro and in vivo consistently translate to meaningful PR/QRS changes in humans across compounds. Accurate characterisation of concentration-effect relationships therefore require a model-based approach. Although the presented work is limited by the small number of investigated compounds, it provides a starting point for predicting human risk using routine QRS/PR data to improve the safety of new drugs.

The traditional approach to pharmacodynamic modelling relies on knowledge about the pharmacokinetics. A prerequisite for obtaining kinetic information is reliable exposure data. However, in several therapeutic areas, exposure data are unavailable including when the drug response precedes the systemic exposure (for example pulmonary drug administration) and when the drug is locally administered (for example ophthalmics). Dose-response-time (DRT) data analysis provides an alternative to exposure-driven pharmacodynamic modelling when exposure data are sparse or lacking. In DRT modelling, the response data are assumed to contain enough information about the drug kinetics, whereby a biophase model can be developed and act as the driver of the pharmacological response. The following work presents the fundamental principles of DRT modelling. This include the entire procedure of identifying a DRT model, encompassing the assessment of the biophase function and the pharmacodynamic model, extensions to cover population variations, identifiability analysis, parameter estimation, and model validation. To demonstrate the utility of the technique, two extensive pre-clinical DRT studies of the interaction between nicotinic acid (NiAc) and free fatty acids (FFA) are presented. The first study covered the response behaviour following intravenous and oral NiAc dosing in both normal (lean) and diseased (obese) rats. The second study extended the models of the first study to incorporate insulin as a driver of the FFA response. Moreover, data from chronic trials were analysed with the aim to quantitatively understand the adaptive behaviours associated with long-term NiAc treatments. The aim of this work is to answer the questions of when and how to use DRT data analysis, and what the limitations of the method are. The DRT models of the first study were successfully fitted to all response-time courses in lean rats, with high precision in the parameter estimates (relative standard errors (RSE) < 25%), visual predictive check (VPC) and individual plots that captured the population and subject trends, and "-shrinkages of less than 10%. The model for the obese rats were less precise, with specific parameters being practically non-identifiable (with, for example, RSE 250%). The results for both lean and obese rats were generally consistent with those of an exposure-driven reference model, albeit with less precision and accuracy in the parameter estimates. Finally, the model was able to describe non-linear biophase kinetics, present at high oral dosages of NiAc. The DRT models of the second study were able to capture the response-time courses for insulin and FFA on a population and individual level, and for both lean and obese rats. However, many parameters were uncertain (with RSE of, for example, 30-50%) and some were practically non-identifiable (with RSE of > 100%). The estimates were generally less precise and more inaccurate than those obtained in an exposure-driven reference model. Yet, most parameter estimates of the DRT models were within one standard deviation from those of the exposure-driven model. The final model was used to predict steady-state FFA exposures following repeated NiAc dosing for a range of different infusion protocols. The optimal dosing regimens consisted of infusions and wash-out periods were the wash-outs were 2h longer than the infusions. These predictions were consistent with those made by the exposure-driven model. Albeit, the DRT model predicted a slightly lower optimal reduction of FFA exposure. It is important to recognise that DRT analyses introduce bias and variability in the parameter estimates. To obtain reliable results, it is advisable to have rich pharmacodynamic data, covering drug administration at different routes, rates, and schedules. With these issues taken into account, the technique still performed well in the two extensive studies presented in this work. In conclusion, DRT data analysis is a modelling technique used in situations when exposure data are unavailable. The method is versatile and can describe a range of different pharmacological behaviours. Precision and accuracy is lost when comparing to an exposure driven pharmacodynamic modelling approach. Thus, DRT modelling is not to be considered as a replacement of the gold-standard pharmacokinetic-pharmacodynamic framework, but rather as a compliment when exposure data are unavailable.

In mathematical modelling for drug discovery, nonparametric methods are an alternative to the more commonly used parametric methods, and have the advantage of requiring fewer modelling assumptions. This thesis considers nonparametric methods for performing input estimation (deconvolution) -- inferring the input to a dynamical system based on measurements of the system’s state. A typical application is to determine the absorption profile of an orally administered drug. Commonly used input-estimation methods are restricted to system models that are linear. This thesis aims to develop and evaluate methods which can be applied to nonlinear systems, and which are additionally able to provide uncertainty estimates. An input-estimation method is considered to be a particular choice of 1) prior, 2) function parameterisation, 3) desired statistical quantity, and 4) estimation algorithm. Two classes of methods have been selected and implemented: direct optimal-control methods and Markov chain Monte Carlo (MCMC) methods. These have been evaluated on two pharmacokinetic and two body-weight modelling applications, using simulated as well as real data. Evaluation was based on several criteria, including accuracy, computational speed, and usability. The results show that the methods can achieve good accuracy, provided that data are relatively densely sampled. Properly applied, optimal-control methods can achieve very high speed, approximately 0.1s for typical problems, at the expense of not providing uncertainty estimates. For MCMC methods, the performance is highly dependent on the method settings as well as on the problem. In many cases, MCMC running times can be significantly reduced by a suitable choice of function parameterisation and sampling method. In all cases, estimation is based on clearly stated, quantifiable assumptions.

Background Bacterial wound infections (especially those involving biofilms) represent a major challenge to healthcare, and are responsible for significant morbidity and mortality. Owing to the rise in antimicrobial resistance, there is renewed interest in alternative antimicrobial agents for treatment of wound infections, where prevention of colonisation largely relies on topically applied biocides. Objectives The aim was to investigate the antibacterial activity of acetic acid (AA), following on from preliminary testing, and small-scale use on burns patients. This led on to the testing of additional products (SurgihoneyRO (SH1) and blue light (BL)), for which no prior evaluation (against biofilms) had been performed. Methods In vitro experiments were performed to test the antimicrobial activity of the agents against bacteria growing planktonically and as biofilms. Comparisons were also made to a range of commercially-available antimicrobial dressings (AMDs) and medical honeys. Results were assessed through measurement of biofilm biomass, and biofilm seeding using a crystal violet assay. Results All agents were effective against biofilms of a large panel of clinically important nosocomial wound pathogens. AA could prevent biofilm formation at concentrations of ≤0.31%, and eradication of mature biofilms was observed after 3 hours of exposure. SH1 prevented biofilm formation of 16 bacterial isolates at dilutions (from neat) of 1:2 to 1:128. Mature biofilms were highly susceptible to BL, with significant reduction in seeding observed for all isolates. Conclusions All of the test antimicrobial agents have shown promise in vitro for the treatment and eradication of biofilm infections caused by a range of important wound pathogens. However, there are still some unanswered questions. Clinical trials are planned, and it remains to be seen whether the in vitro findings will translate to the in vivo setting, where there is a complex interplay between host and pathogen, and many other factors that influence biofilm presence and persistence.

Chronic obstruction pulmonary disease (COPD) is a major cause of morbidity and mortality across the world. COPD is currently the fourth leading cause of death in the world and is predicted to become the third leading cause of chronic illness and death worldwide by 2030. There are several therapeutic strategies to reduce COPD symptoms and complications such as; bronchodilator medications, antibiotics, inhaled corticosteroids and rehabilitation. However, none of the available pharmacological or non-pharmacological treatments for COPD have been shown to delay or correct long-term defects in lung function. Small nucleic acids such as non-coding RNA (ncRNA) and interference microRNA (miRNA) have recently gained attention as a new class of therapeutics for various genetic diseases. Modulation of miRNA expression and function represents a promising strategy for therapeutic intervention in disorders such as inflammatory lung disease including COPD. In this study the aim was to design, formulate and characterise polymeric nanoparticles (NPs) containing miR-146a. This was followed by spray-dying using L-leucine and mannitol to prepare dry powder nanocomposite microparticles (NCMPs) for pulmonary delivery. Anionic and cationic poly (glycerol adipate-co- ω-pentadecalactone), (PGA-co-PDL), NPs were produced using poly (vinyl alcohol) and dioleoyltrimethylammoniumpropane (DOTAP) respectively. The particle size of the anionic NPs was 266.10±20.80 nm and the incorporation of DOTAP resulted in NPs of 244.80±4.40 nm at 15 % DOTAP concentration. The zeta potential (ZP) of 15 % DOTAP NPs was +14.8±0.26 mV. Fluorescently labelled synthetic miRNA (miR-146a) was adsorbed onto the surface of the optimum 15 % DOTAP NPs. The cell viability studies indicated that over 65 % of A549 cells remained viable after 24 h exposure to cationic NPs at a concentration of 1.25 mg/ml. The spray drying process was optimised to produce NCMPs with recovered NPs of 409.7±10.05 nm, yield of 86.05±15.01 % and low moisture content 2.02±0.03 %. The NCMPs produced had a spherical shape and corrugated surface. The in vitro aerosolisation analysis showed a mass mean aerodynamic diameters (MMAD) of less than 6 μm indicating the NCMPs would be deposited in the middle to deep lung region and a fine particle fraction (FPF) of 51.33±2.90 %. Internalisation of miR-146a loaded cationic NPs was observed in A549 cell lines using both fluorescence and confocal microscopy. The miR146a delivered to A549 cells as miR-146a-NPs and miR146a-NCMPs had a dose dependent reduction on target gene repression; interleukin 1 receptor-associated kinase (IRAK1) expression to 40 % and TNF receptor-associated factor (TRAF6) expression to over 20 %. Moreover, the miR-146a biological activity was maintained after spray drying. These findings demonstrate the promise of miR-146a-NPs/NCMPs as a dry powder pulmonary for the treatment of COPD, protecting miR-146a from degradation and enzymatic activity in the lung airways.

Women develop pulmonary arterial hypertension (PAH) more frequently than men suggesting that female sex and/or female sex hormones i.e. estrogens play a role in disease pathogenesis. Building evidence also implicates a role for microRNAs (miRNAs) in PAH. Little is known surrounding the interplay between sex/estrogens and miRNAs in PAH. Examining the sexual dymorphism in miRNAs with regards to PAH disease may provide insight into the sex bias observed in PAH. Loss-of-function BMPR-II mutations underlie heritable PAH. Here, we showed that in pulmonary artery smooth muscle cells (PASMCs) explanted from a pulmonary hypertensive mouse model with a knock-in BMPR-II mutation (BMPR-IIR899X+/-) there were differences in miRNA expression between sexes. Among the 20 miRNAs examined, 9 miRNAs exhibited significant change in expression within female BMPR-IIR899X+/- compared to female wild-type (WT) PASMCs but remained unchanged in male PASMCs. Of interest miRNA-96 demonstrated significant down-regulation in female BMPR-IIR899X+/- but remained unchanged in male BMPR-IIR899X+/-. In silico prediction software demonstrates the 5-HT1B receptor as a putative target of miRNA-96. The 5-HT1B receptor has previously been implicated in PAH development as it is thought to play a role in pulmonary artery vasoconstriction and pulmonary artery remodelling, two major hallmarks of PAH. To verify if 5-HT1B was a true target of miRNA-96 we carried out a 3’UTR luciferase reporter assay. Indeed we found over-expression of miRNA-96 could down-regulate the luciferase output of the luciferase reporter construct containing the 3’UTR of the 5-HT1B receptor. The down-regulation of miRNA-96 within the female BMPR-IIR899X+/- mouse PASMCs was associated with a concomitant increase in 5-HT1B mRNA and protein. This expression pattern was re-iterated in PASMCs explanted from female PAH patients. Here, we found female PAH patients had a decrease in miRNA-96 expression and an increase in 5-HT1B mRNA and protein but again expression remained unchanged in male patients compared to non-PAH controls. Interestingly we also found only female PAH patient PASMCs were proliferative to the mitogen serotonin (5-HT). This could be explained by the expression pattern of 5-HT1B observed. Next we examined the effect of miRNA-96 over-expression in human PASMCs. Here, we demonstrated that over-expressing miRNA-96 had the ability to reduce 5-HT1B protein expression; however, mRNA expression remained unchanged. In addition, we found that over-expressing miRNA-96 prevented serotonin-induced proliferation in PASMCs from female PAH patients. We have previously shown a relationship between estrogen and 5-HT1B as 17β-estradiol, the main pre-menopausal circulating estrogen, increases the protein expression of 5-HT1B in human PASMCs. Here, we examined the effect of 17β-estradiol on miRNA-96 expression. We found that 17β-estradiol decreased miRNA-96 expression suggesting 17β-estradiol’s effect on 5-HT1B expression could be mediated through a decrease in miRNA-96. To determine if endogenous 17β-estradiol also influences miRNA-96 expression we assessed the expression of miRNA-96 and 5-HT1B expression within whole lung homogenates from female and male mice that had been dosed with an aromatase (estrogen synthesising enzyme) inhibitor anastrozole. These mice have depleted circulating and local lung synthesis of estrogen, and elevated BMPR-II signalling. MiRNA-96 was elevated in the lungs from the estrogen-depleted female mice and this was accompanied by a decrease in 5-HT1B mRNA expression. No changes in miRNA-96 and 5-HT1B mRNA expression were observed within male lung tissue. These results further implicated a role for estrogen in regulating miRNA-96 and subsequently 5-HT1B. As over-expression of miRNA-96 in cell culture prevented a proliferative phenotype in human PASMCs we sought to assess the effect of over-expressing miRNA-96 in vivo. We utilised both the BMPR-IIR899X+/- and hypoxic mouse model of pulmonary hypertension (PH) to examine whether a miRNA-96 mimic could both reverse and prevent a PH phenotype. The miRNA-96 mimic was administered intravenously via the tail vein once a week for 2 weeks using the MaxSuppressor™ In Vivo RNA-LANCEr II delivery method. We first confirmed that in both of these models miRNA-96 was significantly depleted in the lungs of diseased mice. Secondly we showed that intravenous injection delivered miRNA-96 mimic to the pulmonary arteries. Our in vivo results demonstrated that dosing with miRNA-96 mimic reduced the right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and % of remodelled vessels in hypoxic and BMPR-IIR899X+/- female mice. Mice dosed with a negative control mimic showed no effect on PAH indices. Interestingly we also showed that hypoxic and BMPR-IIR899X+/- mice dosed with miRNA-96 mimic had a reduction in 5-HT1B protein expression compared to those dosed with negative control mimic. This is the first study to observe sexual dimorphism in miRNA expression with regards to PAH. We have provided novel data demonstrating how miRNA-96, under the potential influence of estrogen, plays a role in the development of PH in a sex-dependent manner, by regulating 5-HT1B expression and serotonin-induced proliferation. Restoring depleted miRNA-96 levels may present a novel therapeutic approach in PAH.

The kinesins form a superfamily of molecular motors that use energy from ATP turnover to generate force. They carry out a diverse set of cellular functions by transducing this force to transport cargos along microtubule (MTs) tracks. Kinesins are distributed among 14 families with different structural organisations all sharing a conserved motor domain that is comprised of a catalytic site for ATP hydrolysis and a MT binding site. Kinesins perform different functions with numerous key roles in mitosis. The kinesin-5 family member, Eg5, has been shown to play a key role in cross-linking and separating anti-parallel MTs. Inhibition of Eg5 leads to the formation of characteristic monoastral spindles, cell cycle arrest and may also lead to apoptotic cell death. This has led to Eg5 being identified as a potential target for drug development in cancer chemotherapy. To understand the mode of interaction between the protein and the potent drug candidate ispinesib, an extensive study using biochemical and biophysical techniques together with crystallography is presented in this thesis. Sustained chemotherapy imposes selection pressure on cancer cells, and can generate resistant mutants against chemotherapeutic agents. We have employed crystallographic, biochemical and biophysical approaches to understand the underlying molecular mechanism of resistance to Eg5 inhibition. These approaches establish that the point mutations in the inhibitor-binding pocket decrease the potency of SB743921, a potent Eg5 inhibitor binding at a site near loop L5, by more than 1000-fold. By determining crystal structures of the mutant Eg5 motor domains in the presence and absence of SB743921 and combining the results with calorimetric and molecular dynamics studies, we show that the development of resistance is due to changes in the global flexibility of the protein. In a second approach to address drug resistance towards allosteric inhibitors binding at the loop L5 site, we have also characterised inhibitor binding to a novel and distinct allosteric pocket in Eg5. This is the first experimental characterisation of protein-ligand interactions in this new site. These studies also revealed an unexpected second site. Extensive biophysical characterisation was used to determine the importance of each site to the observed biochemical activity. Finally, I have investigated kinesin-6 members as new targets for therapeutic intervention. Among kinesins involved in the mitotic phase, the members of kinesin-6 family are of particular interest as potential drug targets because of their important roles during the anaphase to telophase transition until completion of cell division by cytokinesis. This family consists of three members in humans; MKLP-1, MKLP-2 and MPP1 with a unique structural feature of a long insertion of around 100 amino acids in the loop L6 region. This unique feature makes them interesting not only as a drug target but also to understand their role in protein function. The motor domain of the kinesin-6 family members, MKLP-2 and MPP1 were cloned, expressed and purified and crystallisation trials were carried out. Moderately potent inhibitors were identified by biochemical screening of freely available compound collections. Selectivity analysis was also carried out against other kinesin members to provide specificity data about the inhibitor. In collaboration with Christophe Labiere from Dr. Catherine Guillou's laboratory at the CNRS, Gif-sur Yvette, France, a SAR investigation of the MKLP-2 inhibitor paprotrain was carried out using 135 analogues. This required extensive biochemical screening against MKLP-2 to identify more potent analogues of paprotrain. The first objective of this research is to investigate the mechanism by which existing Eg5 inhibitors block catalytic activity and the mechanism through which Eg5 develops resistance to these inhibitors. A second objective was to lay foundations for structural and biochemical characterisation of kinesin-6 family members. The results of this thesis provide a detailed understanding of Eg5 inhibition by ispinesib and describe an unexpected resistance mechanism towards allosteric Eg5 inhibitors dependent on unfavourable entropic effects in the mutant. The experimental characterisation of a new allosteric inhibitor binding site presented here, together with the crystal structure of an inhibitor binding at this site, provides a foundation for structure based design approaches. The work also provides an extensive study on MPP1 and MKLP-2 cloning, protein expression and purification together with screening and characterisation of their inhibitors.