The use of antiretroviral therapy (ART) during pregnancy and lactation has significantly reduced the rate of mother-to-child transmission (MTCT) of HIV. However, pregnancy is known to affect the pharmacokinetics of many drugs, including key antiretroviral (ARV) drugs. In addition, ARV use during lactation raises questions about unintended exposure of breastfed infants to maternal drugs through breast milk. For drugs with significant genetic contribution to observed pharmacokinetic variability, we hypothesised that polymorphisms in drug disposition genes may accentuate or attenuate pregnancy-induced changes and/or breastfed infants’ exposure. HIV positive pregnant women and nursing mothers taking efavirenz (EFV)- or nevirapine (NVP)-based ART were recruited from three hospitals in Benue State, Nigeria. A novel strategy involving a preliminary pharmacogenetic association study was used to investigate the magnitude of pregnancy-induced changes in EFV and NVP pharmacokinetics in women stratified by single nucleotide polymorphisms (SNPs) in disposition genes. EFV apparent clearance (CL/F) was higher and AUC0-24, Cmax and Cmin were significantly lower in pregnant compared with postpartum women. When stratified based on the SNP with the highest predictive power, pregnant women with CYP2B6 516GG genotype were especially at risk. In the NVP cohort, exposure was also significantly lower in pregnant compared with postpartum women. When stratified based on composite CYP2B6 516G > T and 983T > C genotypes, Cmin was below target in most patients with combined CYP2B6 516GG and 983TT during pregnancy and postpartum. Cmin was below target in at least 50% of pregnant women with one or two variant alleles, compared with 0% in postpartum women. The intensive pharmacokinetics of EFV and NVP in breast milk and pharmacogenetic predictors were described for the first time. Breast milk pharmacokinetic parameters of EFV in breast milk differed significantly between patient groups stratified by CYP2B6 516G > T. The median time averaged milk-to-plasma concentration (M/P) ratio was 1.10 (range: 0.57-1.71) and the paediatric dose weight-adjusted exposure index was 4.05% (1.08-13.8). The resulting infant plasma concentration was influenced by CYP2B6 516G > T, highest up to 8 days of age at 1590 ng/mL (190-4631) and decreased by about 90% in the age stratum 9 days to 3 months. NVP AUC0-12, Cmax and Cmin in breast milk were significantly lower in patients with composite CYP2B6 516GG/983TT than those with at least one variant allele. The M/P ratio was 0.88 (0.74-1.2) and paediatric dose weight-adjusted exposure index was 3.64% (1.99-9.88). Infant plasma concentration differed significantly based on CYP2B6 516G > T/983T > C and CYP3A4 20230G > A (*1G), highest in those exposed through both breast milk and post-exposure prophylaxis compared with either alone. A breastfeeding physiologically-based pharmacokinetic (PBPK) model to predict infant exposure to maternal drugs through breast milk was developed and validated, with over 90% of all individual observed data points within the predictive interval. This thesis presents details about five different studies where these findings were observed. Their clinical implications in the context of current knowledge and practice were also explored.

Introduction. HIV-associated neurocognitive disorders appear to remain common despite access to antiretroviral therapy (ART). Penetration of ART into the central nervous system (CNS) is highly variable between drugs and between individuals. Cerebrospinal fluid (CSF) concentrations of many antiretroviral medications fall below the minimum inhibitory concentration for wild type virus. HIV-1 RNA can be detected in the CSF at greater levels than in plasma (CSF/plasma discordance), however the clinical significance of this is unclear, and the degree of difference considered pathological varies. Whether the CNS can act as a sanctuary site leading to persistent HIV detection in plasma is not known. Methods. The PARTITION study recruited HIV positive adults from 13 UK clinical sites. Paired CSF and plasma was collected from patients undergoing LP for clinical indication (group A) and subjects with unexplained viraemia despite ART (group B). The study aimed to determine a) the prevalence of CSF/plasma discordance and factors associated with this occurrence, and b) the prevalence of HIV-1 RNA detection in CSF in those with HIV-1 RNA persistence in plasma. A sensitive assay detected HIV-1 RNA below 50 copies/ml in a subgroup and a cytometric bead array determined CSF biomarkers. A matrix of clinical features and CSF/plasma biomarkers was related to cognitive decline in subjects from the CHARTER study. Drug concentrations in CSF and plasma were measured by mass spectrometry assays and related to host genetic factors in subjects in the PARTITION study and a Vietnamese cohort with tuberculous meningitis. Results. CSF/plasma HIV discordance occurred in 13% of this cohort and was associated with nadir, but not current, CD4 cell count. CSF/plasma discordance occurred in 7 of 40 (18%) of subjects with ongoing viral detection in plasma vs. 0 of 39 of those without. Residual HIV-1 RNA detection below 50 copies/ml was also associated with CSF HIV-1 RNA detection. Resistance associated HIV mutations were detected in CSF of subjects with CSF/plasma discordance. CSF/plasma discordance above 0.5log10 was associated with raised profiles of inflammatory CSF proteomic biomarkers compared to those without discordance. In the CHARTER cohort, cognitive decline over 18 months was associated with lower concentrations of CSF TNFa and plasma IGF1/2. CSF concentrations of efavirenz were associated with the CYP2B6 c.516G>T single nucleotide polymorphism. Efavirenz metabolites were mainly glucuronidated in CSF, were present at neurotoxic levels, and were related to degree of blood brain barrier permeability. Host genetic factors were did not relate to CSF DRV concentrations. Conclusions. CSF/plasma discordance is a frequent occurrence, likely related to processes established during advanced immunosuppression not fully reversed by ART. It is associated with CSF HIV resistance and raised CSF biomarkers, even at levels 0.5-1log10 which have been considered non-significant in some studies. Potentially neurotoxic CSF concentrations of efavirenz relate to host genetic factors.

Chronic lymphocytic leukaemia (CLL) is a lymphoid malignancy which despite advances in the treatment options available is still incurable. Characterised by the gradual accumulation of CD5+ B cells, the paradigm that this is due to failed apoptosis has been challenged and a significant proliferative component has been identified. However, despite the crosstalk between pathways which regulate metabolism and proliferation the metabolic characteristics of these cells are not fully understood. Furthermore, there is a renewed interest in the field of cancer cell metabolism because of the Warburg effect, a hallmark of malignancy whereby cells preferentially switch to aerobic glycolysis and rapidly consume glucose. This has led to the development of new drugs such as AZD3965 an inhibitor of monocarboxylate transporter 1 (MCT1), which along with MCT4 mediates the export of lactate, a toxic bi-product of glycolysis, out of the cell. The aim of this project was to assess whether therapeutically targeting MCT -1 and -4 would be a viable approach for CLL. Chapter 2 of this thesis examines expression of MCT -1 and -4 as well as a specific chaperone protein needed for the surface expression of these proteins, CD147. This chapter confirms the presence of both MCT -1 and -4 and CD147 in normal B cells as well as demonstrating for the first time that these transporters are expressed in CLL cells using Western blotting and qRT-PCR to assess the MCTs and flow cytometry to measure CD147. The levels of both MCTs and CD147 are demonstrated to be significantly reduced in CLL cells in comparison normal B cells likely due to the adoption of a quiescent phenotype to aid cell survival. The following chapter investigates this further by assessing whether there are any changes in expression under the influence of microenvironmental stimuli, specifically CD40 ligand (CD40L). In this chapter it is demonstrated for the first time that MCT4 is upregulated in CLL cells in response to CD40L. Analysis of gene expression using a Fluidigm Biomark™ array suggests this is due to the induction of glycolysis and that CLL cells may promote fatty acid synthesis as well as instigating changes in the metabolism of the tumour stroma possibly to provide substrates. Finally, chapter 4 evaluates the sensitivity of CLL cell lines to AZD3965 using cell death and cell viability assays. Both MEC-1 and HG3 CLL cell lines are shown to be resistant to MCT1 inhibition using AZD3965 and silencing of MCT4 using siRNA cells also has no effect on the viability of MEC-1 cells. That MCT4 can compensate for MCT1 inhibition is shown by the transient expression of MCT4 in a Raji cell line where only MCT1 is expressed. Taken together, the data presented in this study indicates that while the inhibition of MCT1 is likely to be ineffective dual inhibition of both MCT -1 and -4 may be a viable strategy for the localised inhibition of CLL in the secondary tissues. Furthermore, MCT inhibition in this disease may have the potential to negate mechanisms of resistance and protection from oxidative stress mediated by CD40L.

Background: Problems with acceptability of healthcare interventions can undermine the validity of randomised evaluation studies. Hence, assessing acceptability is an important methodological issue. However, the research literature provides little guidance on how to define and assess acceptability. Acceptability of a healthcare intervention could be different, depending on the perspective taken: patients and healthcare professionals may have different views. Perceptions of acceptability may also change according to when acceptability is assessed, in relation to a person’s engagement with the intervention. A person can have perceptions about prospective acceptability (i.e. prior to taking part in the intervention); concurrent acceptability (i.e. whilst taking part in the intervention) and retrospective acceptability (after participating in the intervention). Objectives: The overall aim of this programme of research was to define acceptability in the context of healthcare interventions and to develop a Theoretical Framework of Acceptability (TFA) that can be applied to assess acceptability from two stakeholder perspectives: healthcare professionals and patients. The specific objectives were to: 1) Identify, from the published literature, how the acceptability of healthcare interventions has been defined, operationalised and theorised; 2) Theorise the concept of acceptability and develop a theoretical framework of acceptability (TFA) to guide assessment and develop preliminary assessment tools; 3) Use the tools to apply the TFA to assess intervention acceptability qualitatively, and 4) Apply pre-validation methods to develop preliminary versions of two TFA-based questionnaires. Methods: Six studies were conducted: 1. A systematic overview of reviews of published studies to investigate how the acceptability of healthcare interventions has been defined, theorised and assessed. The results of this study formed the basis for study 2. 2. Inductive and deductive methods of reasoning were applied to theorise acceptability and to develop the Theoretical Framework of Acceptability (TFA). 3. Semi-structured interviews with eligible participants who declined to participate in a Randomised Controlled Trial (RCT) comparing a new patient-led model of care with standard care, for managing blepharospasm and hemifacial spasm. The TFA was applied to identify whether participants’ reasons for refusal were associated with prospective acceptability of the intervention or with other factors. 4. Application of the TFA to analyse semi-structured interviews to assess healthcare professionals’ retrospective acceptability of two feedback interventions delivered in a research programme aimed at developing and evaluating audit and feedback interventions to increase evidence-based transfusion practice. 5. An extension of Study 3: semi-structured interviews with patients who agreed to participate in the RCT, at three-month follow-up, to assess patients’ concurrent acceptability of the standard model of care and the patient led model of care for managing blepharospasm and hemifacial spasm. 6. Pre-validation methods were applied to develop two TFA-based questionnaires applicable to the RCTs described in Studies 3, 4 and 5. Results: Study 1: acceptability had not been theorised and there was no standard definition used in the literature. Operational definitions of acceptability were often reported and often reflected measures of observed behaviour. Study 2: proposed definition: Acceptability is a multi-faceted construct that reflects the extent to which people delivering or receiving a healthcare intervention consider it to be appropriate, based on anticipated or experienced cognitive and emotional responses to the intervention. The TFA was proposed as a multi-component framework that can be applied to assess intervention acceptability across three temporal perspectives: prospective, concurrent and retrospective. The TFA consists of seven component constructs: Affective attitude, Burden, Ethicality, Intervention Coherence, Opportunity Costs, Perceived Effectiveness and Self-efficacy. Studies 3-5: It was feasible to apply the TFA in these empirical studies. Study 6: Two acceptability questionnaires were developed; the TFA informed the development of items reflecting the seven component constructs of the TFA. Conclusion: Despite frequent claims that the acceptability of healthcare interventions has been assessed, acceptability research could be more robust. Investigating acceptability as a multi-component construct resulted in richer information about the acceptability of each intervention, and suggestions for enhancing intervention acceptability across three temporal perspectives. The TFA offers the research community a systematic and theoretical approach to advance the science and practice of acceptability assessment for healthcare interventions.

Regulatory T cells (Treg) are enriched in tumour tissue relative to other compartments. Anti-tumour immunity is promoted through their depletion. It is hypothesised that Treg are recruited to human colorectal cancer (CRC) via a specific combination of chemokine receptors and integrins, blockade of which reduces tumour Treg recruitment, ameliorating the anti-tumour immune response. A systematic examination was conducted of receptors expressed by CRC-isolated Treg and the cognate ligands expressed by CRC. The effects of receptor inhibition were tested in murine models of colorectal cancer. Human CRC-infiltrating Treg exhibit a specific chemokine receptor signature, expressing significantly higher levels of CCR5 than conventional T cells. CRC expresses the ligands for CCR5 at significantly higher levels than distal tissue. Isolated Treg migrated towards CCR5 ligands in vitro and suppressed allogeneic T cell proliferation. CCR5 inhibition in murine models of CRC led to delayed tumour growth but had no effect on tumour Treg infiltration compared with vehicle control. CCR5 inhibition is unlikely to provide any significant reduction in the infiltration of Treg into human CRC. Given the effects CCR5 inhibition had on tumour growth, CCR5 antagonists command further investigation into their potential role as novel therapeutic agents in the treatment armoury against human CRC.

Dry powder inhaler (DPI) formulations are usually comprised of a mixture of micronised active pharmaceutical ingredient (API) with aerodynamic diameter 1-5 m to allow deposition in the lower airways, and a coarse (~70 micron) excipient, typically \(\alpha\)-lactose monohydrate, used to aid the handling, metering and dosing of the formulation. These components are usually combined in a secondary manufacturing process such as high shear blending (HSB), which is used primarily to distribute the cohesive drug particles throughout the bulk excipient to create a chemically homogeneous formulation. This thesis explores the use of HSB to produce mimic DPI formulations and assesses the effect of different blending and storage regimes on various physicochemical properties of such powders. A novel fluidised bed elutriation (FBE) technique was developed to test the ability of fine mimic drug particles to separate from coarse particles in such formulations, along with conventional air jet sieve (AJS) and Next Generation Impactor (NGI) studies. Results showed that, generally, blending regimes were seen to have little effect on the in vitro performance of these mimic formulations, with extended storage at high humidity having a more profound effect on the separation of fine particles. Tests on the performance of formulations using the FBE technique showed that fluidisation performance alone is insufficient to identify blending-induced changes; however, analysis of the fine elutriated fraction has shown subtle changes in the populations of particles due to HSB. Notable differences were observed between the mimic cohesive and adhesive drugs, indicating the ability of these tests to identify formulations with different adhesive properties. In addition, a study to determine the specific energy input (SEI) required to achieve chemical homogeneity suggested that extended high shear blending beyond a given energy input may only alter the size distribution of the formulation, and not improve homogeneity, thus having implications for the manufacture of such products.

Transarterial Chemoembolization (TACE) is a leading therapy in patients suffering from intermediate hepatocellular carcinoma (HCC). TACE is a transarterial therapy that involves injection of particles and chemotherapeutic agents to the tumour site. Once administrated, the particles block the blood flow to the tumour while also allowing targeted delivery. Several therapies have proven to increase survival in people suffering from HCC where drug eluting beads (DEBs) have become a frequently used method. Despite their success, DEBs are limited to drugs that interact with the embolizing material’s functional groups. Also, the embolizing beads must be calibrated to a patient’s blood vessel size where smaller sized beads will display faster release rates. In order to overcome these disadvantages, a Particle-in-Particle (PIP) system is proposed. Small microparticles (1 – 3 µm) are to be manufactured to suit a specific drug where they will function as a drug delivery component. These microparticles are then to be encapsulated into larger microparticles (100 – 1000 µm) which will act as an embolizing component of the PIP system. Polymers and particle production methods are to be investigated in order to produce a PIP system capable of targeted delivery of a wider class of drugs with identical release rates.

Adaptive radiotherapy (ART) aims to adapt the treatment plan to account for inter-fraction anatomical variations, based on online acquired images. However, ART workflows are not –yet– routinely used in clinical practice, primarily due to the dramatic increase of the workload required and the inadequate understanding of optimal methods to maximise clinical benefit. This thesis reports on investigations of procedures for the automation of the ART process and the identification of optimal adaptation methodologies. Investigated auto-segmentation algorithms were found insufficient for an automated workflow, while a hybrid deformable image registration (DIR), incorporating both intensity based and feature-based components, revealed the most accurate and robust performance. An evaluation method was proposed for interfraction treatment monitoring through dose accumulation following DIR. The robustness of several treatment methods to observable anatomical changes were investigated, highlighting cases whereby substantial dosimetric consequences may arise. Offline ART workflows were explored, specifically investigating the effects of treatment monitoring frequency, adaptation method (simple re-plan or re-optimisation addressing cumulative dose), and adaptation timing. Contrary to simple re-planning, re-optimisation demonstrated its ability to compensate for under-/over-dose, however, non-uniform dose distributions and hot-spots may be generated. Therefore established planning techniques are applicable for re-planning while advanced approaches are required for treatment re-optimisation accounting for radiobiological consequences.

The aim of the present research was to determine a range of principles for the development of virtual natural environments (VNEs), using low-cost commercial-off-the-shelf simulation technologies, for bedside and clinical healthcare applications. A series of studies have been conducted to systematically investigate different aspects of the VNEs on a wide variety of participants, ranging from undergraduate and postgraduate students, hospital patients and clinicians, to West Country villagers. The results of these studies suggest that naturalistic environmental spatial sounds can have a positive impact on user ratings of presence and stress levels. High visual fidelity and real-world-based VNEs can increase participants’ reported ratings of presence, quality and realism. The choice of input devices also has a significant impact on usability with these types of virtual environment (VE). Overall, the findings provide a strong set of principles supporting the future development of VNEs. Highly transferrable tools and techniques have also been developed in order to investigate the exploitation of new digital technology approaches in the generation of believable and engaging real-time, interactive virtual natural environments that can be modified and updated relatively easily, thereby delivering a system that can be regularly modified and updated to meet the needs of individual patients.

Cannabis remains the most commonly used illicit substance during adolescence, yet little is understood about the influences on changes in use patterns. There has been more focus on risk as compared to resilience in assessments of cannabis and psychopathology. This thesis aimed to assess self-reported factors influencing changes in patterns of cannabis use in adolescents, and to integrate resilience in the assessment of cannabis use, alcohol use, and psychopathology. A 6-month prospective design involving a sample of 288 adolescents recruited from schools and from Child and Adolescent Mental Health Services was utilised. Participants completed the Cannabis and Young People Questionnaire, Resilience Scale for Adolescents, Community Assessment of Psychic Experiences, and Depression Anxiety Stress Scales. Multiple factors influenced changes in cannabis use, with an overarching influence of peers. Cannabis use was not related to psychopathology, nor did it moderate the relationship between psychopathology and resilience. However personal competence emerged as a negative predictor of psychopathology. Level of social resources was the strongest negative predictor of alcohol use, and alcohol users had higher levels of depression. Therefore, there may be potential for utility of resilience factors, notably personal competence, and social resources in prevention and early intervention for mental health in adolescents.