The increased need for macromolecular therapeutics, such as proteins and nucleotides, to reach intracellular targets asks for more effective delivery vectors and a higher level of understanding of their mechanism of action. Cell Penetrating Peptides (CPPs) have been shown to deliver a range of macromolecules into cells either through direct plasma membrane translocation or by endocytosis. All known endocytic pathways involve cellcortex remodelling, a process shown to be regulated by reorganisation of the actin cytoskeleton. Links between actin remodelling and CPP uptake has been shown but more information is required to determine the extent of this association and how it could influence further research into improving the delivery capacity of these entities. This project, by using the CPP octaarginine (R8) investigated how actin disorganisation influences the cellular entry of this peptide when attached to a model fluorophore Alexa 488 or Enhanced Green Fluorescent Protein (EGFP). A confocal microscopy technique was initially developed, allowing for high-resolution and spatial characterization of the actin cytoskeleton at different cell depths. Analysis using this developed method was used to highlight that serum starvation has a strong influence on the capacity of R8 to cause membrane blebs and possibly macropinocytosis. Using a range of direct or indirect actin inhibitors this work also highlighted how they can rapidly cause dramatic cellular deformities beyond the level of actin or more subtly affect actin organisation. Further confocal studies revealed that choice of cell line significantly affects the effect of actin disruption on CPP entry and that this is highly dependent on the nature of the probe. This was exemplified by results showing inhibition of EGFP-R8 uptake in HeLa cells treated with cytochalasin D, ! ! ii! latrunculin B and jasplakinolide but a dramatic increase in uptake in A431 cells when they were treated with these drugs. The regulation of actin dynamics involves various kinases including Rho–associated kinases ROCKs, and Src family kinases. The ROCK inhibitor Y27632 induced the formation of actin needles running perpendicular to the plasma membrane of A431 cells and increased EGFP-R8 internalisation. By contrast, Src inhibitor PP2 had little effect on both the actin cytoskeleton and EGFP-R8 uptake but completely abrogated the effects of Cyt D on cellular uptake. This demonstrates for the first time that pretreatment of actin with one inhibitor can negate the endocytic effects of another actin inhibitor working on a different target. Overall this study highlights the importance of analysing actin in detail to identify how CPPs and possibly other drug delivery vectors and formulations interact with cells to gain entry. Under defined experimental conditions R8 can modify the actin cytoskeleton and requires a functional or dysfunctional actin network to allow for maximal cellular entry.

Background Artificial nutrition and hydration (ANH) is the provision of nutrition and/or fluid via artificial means into the gut or into the blood. Decisions about ANH can be difficult and emotive. Dietitians assess patients to recommend whether ANH is indicated, and manage patients once ANH has begun. This research aimed to give voice to dietitians’ experiences of their involvement in decision-making about ANH, by exploring dietitians’ perceptions of their role and exploring how different contexts influenced dietitians’ experiences of decision-making about ANH. Methods Sixteen dietitians were interviewed twice for a qualitative phenomenological study which explored their experiences of decision-making related to ANH. The transcribed interviews were analysed using an interpretive phenomenological framework. Findings Three themes emerged from the data: ‘so much more than just deciding on a feed’; ‘wanting to be heard’; and ‘the emotional roller coaster’. Professional autonomy, being recognised as an expert, and wanting to be involved in decisions were important for all participants. Some participants were involved in decision-making and some implemented decisions made by others. When their professional expertise was not recognised, the emotional experiences were negative. Emotional labour and moral distress were displayed by some. Professionalism was shown by speaking out and acting as moral agents. Some believed that their emotions should not be shown. None of these experiences have been reported by UK dietitians before. Conclusions This study contributes rich and complex new knowledge to the understanding of dietitians’ experiences. These experiences included strong and complex emotional responses and correspondingly complex strategies to cope, which enabled dietitians to protect themselves, colleagues, and patients. Professional identity and challenges to their professionalism were also important aspects of their experiences. This research has expanded the concept of professionalism within dietetics. Implications for practice include the need to develop emotional intelligence and for dietitians to raise their profile by building strong collaborative partnerships with healthcare staff to facilitate their professionalism. Guidance on ethical decision-making for dietitians is required.

G protein-coupled receptors (GPCRs) are often described as ‘gate-keepers’ of the eukaryotic cell and their roles primarily involve translating extra-cellular stimuli via G protein coupling and intracellular signalling to govern various physiological responses. These functional parameters are wide ranging and include the modulation of visual sensory organs, to the control of vasoreactivity and heart rate. Importantly, their flexible architecture can facilitate small molecule interaction within a ‘binding-pocket’ and GPCR research often focuses on this relationship to identify and design novel and effective ligands to manipulate GPCR activation and signalling. GPR35 is a poorly characterised, Class A GPCR and despite reports of two potential endogenous activators; kynurenic acid (KYNA) and lysophosphatidic acid (LPA) in recent years, it is still widely acknowledged as an ‘orphaned’ GPCR. This is reflected by the propensity of its ligands, both endogenous and synthetic, to demonstrate extreme species orthologue-selective properties. Despite this, investigators have highlighted various roles for GPR35 relating to pain, inflammation, hypertension and heart failure, and investigations have suggested that activation of GPR35 leads to a selective coupling of Gα13, a G-protein understood to mediate Rho A and ROCK 1/2 signalling. However, a lack of functional ligand pairs has hampered further research to examine a role for GPR35 and subsequent Gα13 signalling in these disease models. In this study, we have characterised a series of highly-potent, synthetic ligands at human, rat and mouse orthologues of GPR35, revealing that GPR35 agonist, pamoic acid and antagonists, CID-2745687 and ML-145, are highly potent and selective for the human orthologue of GPR35. This has provided us with the opportunity to assess a functional role for GPR35 within a cardiovascular disease setting using functional ligand pairs in cells of a human origin, for the first time. Vascular smooth muscle cell (VSMC) migration and proliferation are central to neointima formation in vein graft failure. Despite a detailed understanding of VSMC migration and proliferation mechanisms, there are no pharmacological interventions which effectively prevent vein graft failure through intimal occlusion. In this study, we demonstrated that primary vascular cells expressed robustly detectable levels of GPR35, and these were comparable to those demonstrated in the colon, which has been previously reported to highly express GPR35. Human GPR35 is potently activated by the selective agonist pamoic acid and reference ligand zaprinast and blocked by antagonists CID-2745687 and ML-145. Following exposure of VSMC to pamoic acid or zaprinast, cell migration was enhanced and these effects were blocked by co-incubation with CID-2745687 or ML-145. Pamoic acid induced HSV SMC migration was also blocked in the presence of two distinct Rho A pathway inhibitors, Y-16 and Y-27632. Activation of this pathway was also reflected by remodelling of the cytoskeletal architecture in HSV SMCs to significantly elongate the cell and promote a contractile, migratory phenotype following pamoic acid stimulation and this effect was also blocked following co-administration with either antagonist. Additionally, we also demonstrated that following exposure to pamoic acid or zaprinast, human saphenous vein endothelial cell (HSV EC) integrity and proliferation were significantly improved and this was blocked following co-administration with either antagonist, suggesting a protective role for GPR35 in the vascular endothelium. Results from a previous study demonstrated that lack of GPR35 expression resulted in an elevation in systolic blood pressure (SBP) by up to 37.5mmHg in mice. The recent identification of mast-cell stabilising compounds as highly potent agonists at GPR35 provided the opportunity to pharmacologically target GPR35 within a rodent model of hypertension. Therefore, we also aimed to test if pharmacological manipulation of GPR35 via stimulation with the novel, rodent selective agonist amlexanox, modulated blood pressure and end-organ related damage in 6-12 weeks of age stroke prone spontaneously hypertensive rats (SHRSPs). Radiotelemetry acquisition of haemodynamic properties highlighted that pharmacological agonism of GPR35 exacerbated hypertension and end-organ damage in the SHRSP and this was evident following an elevation in SBP by 20mmHg throughout the trial. Moreover, quantification of heart mass and cardiomyocyte size revealed that GPR35 agonism induced cardiac hypertrophy. Collagen staining revealed enhanced renal fibrosis in both the interstitial and perivascular regions of the kidney from amlexanox treated animals, compared to vehicle controls. Additionally, large vessel myography highlighted that endothelium-dependent vasorelaxation was reduced by 20% in amlexanox treated SHRSPs. Fundamentally, these results suggest that GPR35 is involved in regulating vascular tone and we hypothesise that this may involve the Gα13-Rho A-ROCK1/2 signalling pathway demonstrated to mediate a contractile, migratory phenotype in human primary VSMCs. Taking these data together, the results suggest that GPR35 antagonists might be of clinical use to therapeutically target and inhibit activation of GPR35 in the setting of vascular remodelling during acute vascular injury, and hypertension and its related end organ damage.

Findings from randomised controlled trials (RCTs) on primary care treatments for non-specific low back pain (NSLBP) often show modest or non-significant differences in responses to treatments. The overall response to treatment within arms, however, is often large. This raises the question of the non-specific effects associated with using the treatments and whether the size of these non-specific effects is much larger than the size of effects associated with the specific components of treatments. Non-specific effects in clinical trials, defined in this thesis as the effects on the overall improvement of symptoms (i.e. response to treatment) that is not attributed to the treatment itself, contribute to the clinical course of symptoms and can be related to the patient, the symptoms, the healthcare practitioner, the communication between the patient and practitioner, the nature of treatment provided and the setting and environment of the clinical encounter. The objectives of this study were examining: 1) the pattern of within-arm overall responses to treatments in RCTs on non-specific low back pain; 2) sources of variation in responses to treatments by investigating the association of non-specific factors with overall responses to treatments; 3) the influence of patient characteristics on responses to treatments using individual patient data from RCTs; and 4) whether merely participating in RCTs adds to the size of response to treatments (the ‘trial effect’). The findings suggest that responses to treatments for NSLBP follow a pattern of an early large improvement in symptoms within 13-27 weeks of starting treatment followed by a smaller further improvement. This pattern was common to arms of RCTs regardless of the type of treatment. There was evidence that participants who had back pain episodes of less than 13 weeks showed larger responses to treatments than those with longer duration. There was weak inconclusive evidence for the association with age, gender, history of back pain, overall trial quality, adequacy of patient blinding and adequate compliance. There was no evidence that participating in RCTs led to larger improvement in back pain symptoms compared with participating in cohort studies. In conclusion, there is some evidence for the association of factors that are not related to the treatments with responses to treatments for NSLBP. Insufficient data hindered the assessment of other non-specific factors that were considered to be important, such as practitioner-patient communication.

Low back pain affects a large proportion of the general population. For some individuals, back pain becomes chronic, complex and difficult to treat effectively, with patients reporting continued pain and disability. A biopsychosocial framework has been adopted within research and clinical practice, as psychosocial factors have been recognised to be important in terms of pain management and recovery from back pain. Coping-related factors have been identified as particularly important, however a comprehensive examination of a wide range of coping factors is missing from available literature. A systematic review of the published literature identified important psychological factors that are predictive of low back pain outcome. Several factors emerged as potentially important, but fear avoidance beliefs appeared to be the most consistent predictor. Very few studies were found that investigated the role of behavioural coping, therefore a new measurement instrument was developed to aid further research. A detailed analysis of the Coping Strategies Questionnaire-24 was undertaken. Exploratory and confirmatory factor analyses were used and it was concluded that the measure was appropriate for use within this thesis. Data from a large cohort of primary care low back pain patients (n = 1,591) was used for analysis. Cross-sectional analyses revealed potential confounders of the relationship between coping and outcome at 12 months follow-up, which were controlled for within the longitudinal analyses. Only five coping variables were independently predictive of outcome – anxiety, depression, catastrophizing, self-efficacy and passive behavioural coping – along with pain duration and employment status. Change in coping over time predicted low back pain outcome, and it was found that coping worsening was particularly important. Coping worsening also partially mediated the relationship between pain duration and outcome. The major thesis findings were integrated into an overall conceptual model of coping, and key implications of this for clinical practice and research were discussed.

Human immune deficiency virus (HIV) currently affects around 34 million people worldwide, but due to recent advancements in antiretroviral treatments the morbidity and mortality of HIV has greatly improved (UNIAIDS, 2009). Tenofovir (TFV) is a nucleoside reverse transcriptase inhibitor (NRTI) that is currently widely used in most HIV regimens due to its favourable pharmacokinetic and pharmacodynamic properties, which allows for once daily dose (Gallent et al., 2004). TFV is used to treat HIV in both treatment experienced and treatment naive patients; it is also used as a treatment for hepatitis B and has recently been approved for use as a pre-exposure prophylaxis treatment. The preclinical trials for TFV showed that it had a good safety profile, however there has been an increasing number of patients presenting with TFV induced renal damage specifically proximal tubule dysfunction (Cooper et al., 2010). It was hypothesised that TFV was inducing this tubule damage via mitochondrial dysfunction, as this has been widely reported as an adverse reaction associated with NRTI’s (Kontorinis et al., 2003). Within these studies in vitro cell models were modified via culturing them in galactose media in order to probe for mitotoxins (Marroquin et al., 2007). The cytotoxicity and mechanisms of cell death induced via TFV were elucidated within these modified cell lines. Work presented has shown that TFV causes delayed mitochondrial cytotoxicity, which causes a cell cycle arrest within the G2/M phase leading to an accumulation of cell within the S phase. It was shown that the cell cycle arrest is likely due to the inhibition of mitochondrial polymerase γ leading to decreased mitochondrial DNA and ultimately cell death firstly via apoptosis (caused by release of caspase 3/ 7), and then necrosis when cellular ATP levels become too low to sustain apoptosis. Work presented also showed that autophagy induced by TFV, may play a protective role within cells at lower time period. A study within this thesis using patient urine samples was presented to look for a possible biomarker of TFV proximal tubule dysfunction. Kidney injury molecule-1 (KIM-1) was selected as previous studies have shown that it is specific to proximal tubule cells (Ichimura et al., 2008). Results demonstrated that TFV did not significantly alter the KIM-1 / creatinine ratio within patient urine samples. However the data from this pilot study are novel and should be used for future research into the possible use of KIM-1 as a biomarker for TFV induced proximal tubule dysfunction. The biomarker work presented within this thesis should be carried forward to power a future study using a larger and adequate patient cohort. The work presented within this thesis has now opened doors for further study into this subject area especially with regards to the study of KIM-1 as a biomarker, which is a pressing matter as TFV is now widely available for use as a PrEP treatment.

Community pharmacists are increasingly expected to improve disease management both by aiming to improve the effective use of medicines and by reducing the occurrence and severity of preventable adverse drug reactions (ADRs). The current PhD research began by assessing the challenges to the medicines reconciliation from patients' perspectives. A questionnaire based audit identified two important risk factors for reporting ADRs: being unaware of why medicines were prescribed and not recalling prior warnings about possible ADRs. These findings led to the evaluation of pharmacists' engagement with patients within pharmacy services such as the New Medicine Service (NMS). A questionnaire-based service evaluation of the NMS provided support for this service as an opportunity to improve identification and management of ADRs. This evaluation also highlighted the poor contribution of pharmacists towards reporting of ADRs. The findings led to the evaluation of ADR reporting by community pharmacists. This audit-based study identified lack of time and uncertainty about the seriousness of ADRs as the main barriers towards spontaneous reporting. The reporting of ADRs linked to high blood pressure medicines in this study indicated the need to assess the role of community pharmacists in the management of high blood pressure. A systematic review and meta-analysis suggested that pharmacist-led interventions made a significant impact on the management of systolic and diastolic blood pressure. Learning from two audits, a service evaluation and a systematic review led to the development of a randomised controlled trial that assessed the impact of written pharmacistled education on patients with high blood pressure. Interventions by pharmacists working in community pharmacies were associated with improvements in the control of hypertension, however, the mean difference in blood pressure between the intervention and control group was not statistically significant. Compared to participants in the control group, there was a significant improvement in the knowledge about hypertension and its treatment in the intervention group. The participants in this study gave a positive response about the involvement of pharmacists in the management of long-term medical conditions such as hypertension. The UK government wants to see a central role for pharmacists in patient care. The evidence presented in this research suggests that pharmacists have the potential to play a bigger role in patient care. Patients also recognize this potential and appear to be willing to seek pharmacists' advice on health-related issues. Pharmacists would need to identify their specific learning needs to help them deliver a more patient-centred care. They would also require the support and recognition from other stakeholders in particular the GPs.

Orally administered therapeutic agents need to cross the mucosal epithelial membrane in the intestine to reach the systemic circulation. This intestinal epithelial membrane is covered by a biopolymer barrier, namely, mucus which protects the underlying layer through trapping or degrading of foreign particles and macromolecules. Thus, mucus can restrict the systemic absorption of some therapeutic agents such as peptides by enzymatic degradation. Nanoparticles (NPs) could serve as a carrier for these peptides to protect them from environmental conditions in the mucus and to increase their bioavailability. However, these NPs can be trapped themselves by the mucus, hence, a proper nano-strategy should be selected to deliver these peptides orally. NPs delivery through intestinal mucus barrier has been studied extensively, where various in vitro tests and mucus models were investigated to mimic the in vivo test. In this thesis, two mucus models were assessed for their suitability as intestinal mucus barrier through which NPs diffusion can be studied. Also, multiple particle tracking (MPT) technique was exploited to study the diffusion and interaction of nanoparticles through pig intestinal mucus barrier. This technique (MPT) was used to understand the factors affecting the diffusion through mucus of NPs representing various nano-strategies such as PEGylated NPs and mucolytic NPs. Based on data obtained for the diffusion of NPs, we adopted a nano-strategy mimicking the capsid shell virus in which the NPs surfaces are densely covered with oppositely charged groups but with overall neutral charge. To do so, polyelectrolyte (PEC) NPs based on the self-assembly of (+) chitosan and (-) polyacrylic acid (PAA) were synthesized and the diffusion of these densely charged NPs was studied. After proving the concept, RAFT technique was used to synthesize zwitterionic densely charged NPs in which butyl methcrylate (BMA) was used as the lipophilic core and sulfobetaine as the shell of NPs. Native mucus prepared by our group was found to be a proper model to study NPs diffusion through it by the MPT technique. Study of diffusion of NPs representing various nano-strategies through mucus revealed the impact of various properties of these NPs on their diffusion. For example, particle size, zeta potential, type and molecular weight of the polymer, type and concentration of the diffusion enhancer and method of synthesis of NPs were detected to affect the diffusion of these NPs. For PEC NPs, the data obtained showed a relation between the zeta potential of NPs and their diffusivities through the mucus, where the highest diffusivity was obtained for the neutrally charged PEC NPs. Accordingly, sulfobetaine NPs were highly efficient NPs in term of their stability, charge density, particle size and importantly their diffusivities through the mucus barrier which was significantly higher as compared with all other tested NPs and related to the ratio of the sulfobetaine polymer in the NPs. This indicates that densely charged viral like NPs can be promising carriers to improve the mucus permeation of some therapeutic agents.

This thesis is concerned with the development of electrochemical approaches for the determination of carcinogenic genotoxic impurities in pharmaceuticals. Investigations focus on the genotoxic impurity hydrazine as it is a particularly challenging species for current methodologies used in the pharmaceutical industry. A metal nanoparticle functionalised polycrystalline boron-doped diamond macroelectrode is utilised for hydrazine determination in stationary solution, in the presence of two important electrochemically active, inner sphere, active pharmaceutical ingredients, namely acetaminophen and promazine. It is shown that this sensor can be tuned for the application by simply changing the chemical identity of the nanoparticles. A new design of flow cell for flow injection analysis is then developed, in order to exploit a hydrodynamic approach for the electrochemical determination of genotoxic impurities. The bespoke flow cell is combined with an inlaid boron doped diamond microband electrode and the flow injection analysis response is analysed numerically and against various models of dispersion to demonstrate the quality of the method. Finally, this flow injection analysis sensor is employed for the trace detection of hydrazine in a large excess of acetaminophen. Quantitation is demonstrated down to 0.274 parts per millions with respect to the amount of acetaminophen - surpassing the required safe guidelines set by the pharmaceutical industry for the quantitation of genotoxic impurities.

BACKGROUND: Hospital formularies are usually the gatekeepers for pharmaceutical drugs. Typical majority members of hospital formularies are physicians, although most of the time the formulary is chaired by a pharmacist. As German hospitals are struggling with a difficult economic environment the question arises: what kind of decision-making criteria are applied when pharmaceutical drugs should be added to the formulary list? Information regarding this topic is scarce due to the sensitive topic of decision-making. OBJECTIVES: Build a single decision-making framework which will be created to explain hospital drug funding decision-making and identify underlying mechanisms which explain processes and structures. The results can be used by hospitals to initiate knowledge sharing and provide a basis to analyse local formulary committee decision-making practice. Additionally, they can be used by the pharmaceutical industry to better adapt to the specific needs of the hospital decision-makers. METHODS: In this study, a mixed-methods approach has been used to confirm and further detail a preliminary hospital formulary decision-making framework derived from literature. An online survey was used to get insights on the structure of German hospital formularies and the relative importance of different decision-criteria. Additional semi-structured expert interviews were used to get in-depth information on the underlying mechanisms which influence decision-making on drug funding. RESULTS: Decisions for or against a pharmaceutical drug are influenced by a variety of perceived objective and specifically subjective criteria. Despite a consistency in a dominant, high impact role of pharmacists and lead physicians every hospital formulary member has different relative weighting of decision criteria. Drug funding decision-making in German hospital formularies is highly individual but usually starts with a quasi-rational preference influenced by a mixture of analytic and intuitive criteria. The decision to use more analytic or more intuitive criteria is influenced by a variety of factors. The two most important ones are uncertainty and power. The resulting individual preference is then challenged and adapted in a group decision-making process.