The work presented herein focuses on the study of novel metallodrugs and their interaction with various possible targets and off-targets in the form of biomolecules such as peptides, proteins, DNA, and small molecules via the use of ultra-high resolution Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry. Beyond traditional platinum(II) metallodrug such as cisplatin and oxaliplatin, new designs of metallodrugs are being conceived to attack and kill cancerous cells via new mechanisms of action in an effort to exceed the potency, selectivity, and effectiveness of metallodrug treatments. These new metallodrugs range from various activation strategies, to specific target binding, and even catalysis of cell-disrupting processes. As a result these novel drugs can have a wide array of effects on various biomolecule species the may encounter in the body. It is the aim of this thesis to show that mass spectrometry, specifically FT-ICR Mass Spectrometry, is uniquely suited to studying the wide array of metal-based drugs, their biomolecule targets/off-targets, and the numerous reaction products produced from their interactions. Though the study of metallodrug-modified biomolecules via mass spectrometry was shown to be challenging in many cases, mass spectrometry is currently the only analytical technique viable for studying the complex systems involved to a useful level of detail. The majority of the thesis focuses on study of isolated biomolecules interacting with novel metallodrugs and the MS and tandem-MS based study of the resulting observable components. A great range of metallodrugs and biomolecule interactions were observed. A photoactivated platinum(IV) metallodrug (trans, trans, trans[Pt(py)2(OH)2(N3)2]) was shown to produce a variety of platinum(II) based modifications to the peptides studied when activated with blue (463nm) visible light, with the ligand configurations varying depending on whether a histidine amino acid residue was present, allowing retention of both pyridine ligands, or not, allowing release of any of the bound ligands. Tandem MS studies using electron based dissociations showed the primary preference of binding to be at the Histidine residues, and when not available the complexes could bind to lysing functional groups, distinctly different behaviour to previously studied Pt(II) complexes. Oxidation of peptide species was also found to be a significant product of these reactions. Tandem MS studies located the oxidation sites to methionine and tryptophan residues, the latter of which provided insights into the oxidation mechanism. The oxidation process was found to be due to a hydroxyl radical process, not a singlet oxygen mechanism. UV/vis and EPR measurements were also undertaken and supported the results found. Studies into the interaction of a potent osmium(III) compound with isolated DNA strands showed that the metallodrug could bind to both guanine and cytosine sites along the biomolecule. Analysis of these species via CAD MS/MS proved challenging due to damage and eventual dissociation of the metal complex modifications. Whereas electron detachment dissociation enabled the elucidation of the two distinct binding locations. CAD MS/MS was found to be useful for studying hydrogen bonded/pi-stacking stabilised structures. Further investigations into DNA MS and MS/MS lead to the study of the platinum(IV) compound interactions with DNA stands, showing a vastly improved rate of reaction for the compound when compared to the previous peptide reactions. In addition uniform retention of pyridine ligands and no oxidation was observed, providing further evidence of the biomolecule playing a key role in the activation process of the Pt(IV) drugs. MS/MS studies of platinated DNA resulted in similar findings to the osmium-based metallodrugs. Experiments with native MS of DNA showed that observation and interrogation of duplex-DNA strands is possible, even with DNA strands which are not stabilised easily in ammonium acetate solutions, and without annealing. MS, MS/MS, and MS3 was achieved on duplex DNA strands using large concentrations of ammonium acetate and potassium chloride solutions, along with extremely carefully tuned MS source and transfer parameters. The effects of metals on tandem-MS techniques was uniquely apparent during the study of functionalised Iridium-based metallodrug modified peptides. The iridium complexes were shown to effectively quench many electrons used during ECD MS/MS. Though the species were still able to be studied using optimised ECD and CAD MS/MS parameters. The reactivity of metal centres was also shown to affect their own bound ligands, as observed herein with rhodium(II) compounds. The rhodium piano-stool complexes were shown to vastly accelerate and enable room temperature activation of C-H bonds within arene ligands towards hydrogen-deuterium exchange experiments, with some compounds achieving full exchange of available groups within just 1 hour. The process was studied using FT-ICR MS to track the exchange process and observe sequential exchange for 10 different rhodium compounds. An iridium analogue was also studied, which was ineffective, displaying the Rh metal centre’s unique chemistry for this reaction. A novel method for the enhancement of the electrospray ionisation process was conceived and developed in order to achieve improvement of analyte charging, so-called “supercharging”. Using a combination of standard electrospray ionisation and atmospheric chemical ionisation, CH5 +“superacid” ions were introduced into the ESI plume and enabled solution-additive free supercharging of analytes under a variety of conditions. The achievement of higher charge states/enhancement of charge is of uniform benefit to biomolecule characterisation and could help reduce the need for solution phase additives which can disrupt many chemical processes and biomolecule structure. The final section of the thesis is concerned with the escalation of metallodrug-biomolecule interaction from isolated biomolecule up to full-cell proteomes via FT-ICR MS. The great array of challenges faced in previous studies is addressed and strategies for accurate and reliable studies of large metallodrug-modified systems are outlined and tested. Two major strategies are proposed, one based on liquid-chromatography mass spectrometry with modified data processing techniques. The other using a niche MS/MS technique known as two-dimensional mass spectrometry, which would enable whole proteome characterisation without chromatographic separation. Preliminary result using both approaches and future outlook are presented.

In spite of the vast amount of evidence on the benefits of blood pressure (BP) lowering that has accumulated to date, hypertension (HTN) remains the leading risk factor for disease and disability worldwide. Since the first BP-lowering agents became available in the 1950s, their effects have been tested thoroughly by means of the best evidence-providing approach, namely, large randomised controlled trials (RCTs). In the same way, the pharmacogenomics of HTN have the potential to identify genetic biomarkers that predict the response of BP-lowering agents through genome-wide association studies (GWAS), which analyse quantitative traits at millions of markers across the genome to identify genetic variations that could contribute to HTN. For the most part, computational approaches and software tools have played a significant role in translating RCTs and GWAS findings. This thesis aims first to systematically review the BP responses of main BP-lowering agents, including angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), calcium channel blockers (CCBs), diuretics (DIs) and beta-blockers (BBs) in RCTs, and second to identify the single nucleotide polymorphisms (SNPs) associated with the BP-lowering responses of CCBs and BBs on Nordic Diltiazem (NORDIL) subjects using GWAS. Description of the research results: Following the Population Intervention Comparison Outcome Study (PICOS) design framework, a literature search of multiple sources resulted in the identification of 10,577 publications, with 5,568 unique records identified after duplicates were excluded. In total, 184 studies were identified as potentially eligible, of which 82 RCTs with a total of 197,684 participants were selected for quantitative synthesis. With regard to BP-lowering strategies, 13 studies with 41,886 participants focused on lowering BP intentionally, while the remaining 69 studies (155,798 participants) were classified as unintentional BP-lowering studies. Risk of bias in included studies: Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) framework, all included studies were described as RCTs; however, most studies did not address how treatment randomization occurred or how allocation of treatment was concealed. All included studies also stated that they were double-blind studies, but again, most did not describe how the double blinding was ensured throughout the studies. The risk of attrition bias was avoided as all randomized participants were included in the analysis. All of the studies had a low risk for reporting bias. BP-lowering agents were added to randomly allocated treatment to control high BP; consequently, one potentially unclear source of bias was present in 13 of the 82 studies. The overall quality was rated to be acceptable to high. In all, 48 studies were rated to be high-quality studies, and 34 studies were rated as acceptable quality. Effect of intervention: After a systematic search and selection process, 56 studies were included in the analysis of delta BP response, 37 studies were included in the analysis of single-measure BP response and 20 studies were included in the analysis of repeated measures. A number of BP-lowering agents showed a significantly (P<0.05) superior BP response in comparison with other agents included in the review; however, the level of BP response was still small. CCBs were superior to ACEIs in lowering both systolic BP (SBP) and diastolic BP (DBP). DIs were superior to ACEIs and CCBs in lowering SBP. ARBs were superior to BBs in lowering SBP. CCBs and DIs were significantly superior to placebos in lowering both SBP and DBP. Genome-wide study: Following NORDIL quality control standards, a final set of 3,850 samples and 500,905 SNPs was available for analysis. In total, 51 SNPs showed a significant (P<1X10-5) association with BP response. The top discordant signals identified in NORDIL included five SNPs for SBP on BB arm, seven SNPs for DBP on BB arm, 12 SNPs for SBP on CCB arm and nine SNPs for DBP on CCB arm. Discordant SNPs from the NORDIL were replicated, based on the interests of five collaborative RCTs; including 11 SNPs for SBP on BB arm, 22 SNPs for DBP on BB arm, 23 SNPs for SBP on CCB arm and 18 SNPs for DBP on CCB arm. However, no SNP achieved a genome-wide significance of (P<5x10-8). Future recommendations: Further systematic reviews of RCTs comparing different BP-lowering agents are required to provide evidence of the options for BP-lowering medication. Specifically, there is a need to study BP response as an outcome by itself, taking into account different BP-lowering agent combinations,including classes and sub-classes, along with co-morbidities such as type 2 diabetes mellitus, coronary heart disease and chronic renal failure. Regarding the genome-wide study, further studies are needed to clarify the potential contribution of plausible SNPs in relation to CCB and BB response in HTN. These studies should include comprehensive sequencing of the candidate interval, genotyping of variants in many population samples, testing for association, functional studies and investigation of interactions with other genes or environmental factors. Furthermore, genome-wide studies need to identify directionally discordant signals between SNP and BP response for BB and CCB and confirm the validity of a SNP BP response by analysing the SNP effect on mortality.

In this thesis, a multi-scale approach is provided to a pharmacokinetic and a pharmacodynamic problem. The first part of this research provides a realistic mathematical physiological model of the liver to predict drug drug interactions (DDIs). The model describes the geometry of a lobule (liver unit) and integrates the exchange processes, diffusion and active transport, between the hepatocytes and the blood and possible drug-drug interactions such as; reversible inhibition, mechanistic based inhibition (MBI) and enzyme induction. The liver model is subsequently integrated into a PBPK model with 7 compartments (artery blood, venous blood, gut, liver, kidney, lung, rest of the body). To assess the efficiency of the model to predict DDIs, 77 clinical DDI studies were compared to the model. These 77 clinical studies represent 5 victim drugs (midazolam, simvastatin, triazolam, cerivastatin and nifedipine) and 30 perpetrator drugs. The reversible inhibition, MBI and induction parameters for the majority of the perpetrators were estimated with in vitro experiments and adjusted for the human liver size. The PK parameters, such as clearance and absorption rate, and the physiological parameters were obtained from the literature. The DDIs were measured as the ratio of the AUC (Area Under the Curve of the blood concentration) or the ratio of the maximum concentration Cmax of the victim drug administered with and without the perpetrator drug. The predicted ratios were compared with the clinical observation by calculating the geometric fold error GMFE. The GMFE for AUCratio and Cmax,ratio were calculated to be 1.54 and 1.34, respectively. Moreover, the PBPK model excluding the gut compartment under-predicts both inhibition (lower AUCratio) and induction (higher AUCratio) which strongly suggests that the gut DDI component can not be neglected for accurate clinical prediction. However, the static combined model by Fahmi et al. [1, 2] without the gut component fortuitously predicts the clinical AUCratio better than inclusion with the gut component. To conclude, the model predicts DDIs relatively well as it is in the lower range of errors reported in the literature (1.47-2.00 [1, 2]). Moreover, the model is able to predict the pharmacokinetics of drugs and provides a dynamic description of the DDIs, such as the enzyme level and spatial distribution within a lobule. Furthermore, the perpetrator dose regimen can be changed or the error in the in vitro parameters can be integrated to observe their influences on the AUC ratio. The second part of this research explored the Warburg effect in a avascular tumour growth model incorporating a cell shedding term to account for tumour shrinkage. The tumour model was based on an extension of the Ward and King model [3], where two sub-populations; living cells and dead cells are considered. Three diffusion equations for glucose, lactate and the drug are considered and included into the model for growth rate, natural death rate and a death rate due to the drug. The simulation of the model without a drug shows similar behaviour to the original model by Ward and King despite the presence of the shedding term and predicts an extracellular pH of 6.8. However, when a drug treatment is added, the model is able to simulate the shrinkage of the tumour unlike the original model. Moreover, two scenarios with a basic, neutral and acidic drug were explored, assuming similar efficiency at physiological pH to assess the effect of changes in the extracellular pH. Acidic or weak base drugs seem to be more efficient in low pH environment as the fraction of neutral form is greater and therefore more drug is available to cross the cell membrane to reach its target.

There is increasing awareness of the prevalence of deliberate self-harm (DSH) although the phenomenon is still poorly understood. Those who self-harm often have a poor long-term prognosis, yet systematic focused treatment interventions are scarce. DSH appears to share fundamental characteristics with addictive behaviour, including; impulsive or compulsive urges to act in the presence of triggers, positive and negative reinforcing consequences and endorsement of the diagnostic criteria for clinical dependence. Given this fact, a behavioural mode of DSH may be appropriate. A range of events are anecdotally reported to trigger DSH. This thesis was designed to identify these cues, to develop an understanding of how those who self-harm respond to these cues and the processes by which these cues may operate to maintain DSH. An intervention based on the management of urges to self-harm in the presence of these cues was developed. Study I identified that triggers for DSH (interpersonal, intrapersonal and environmental) were similar to those that reliably predict addictive behaviour. Respondents endorsed the diagnostic criteria for dependency and reported that the act of DSH reduced negative emotions. The second two studies identified self-reported cue reactivity, and generalised hyperarousal to both DSH and neutral stimuli in those who self-harm but no evidence of psychophysiological cue reactivity. Study IV used ERP methodology to evaluate cue reactivity at the CNS level and to evaluate two mechanisms by which cues might operate to maintain DSH. There was some preliminary support for enhanced preconscious attentional bias towards emotional, but not environmental DSH cues, and no support for emotional interference. Study V identified that those who self-harm exhibited enhanced tolerance to physical and psychological stressors, and that priming with interpersonal distress did not impact on this tolerance. Finally, a single case intervention study identified a reduction in DSH, reduced psychophysiological arousal and urges to self-harm and improved clinical symptomatology. However, clinical improvements were not time-locked to targeted exposure intervention phases. The clinical and theoretical implications for these findings are discussed.

No description available.

Bulk solids technology plays a key part in manufacturing process when handled material is an assembly of solid particles in large quantities. Micro-dispensing technology for bulk solids can improve operation efficiency in dispensing relatively small amount of material. The demand for the technology covers a wide range of areas where materials have diverse flow behaviours and flow problems due to different physical properties. This Ph.D. project aims to investigate bulk solids flow behaviour and fluidizing mechanism in a hopper under the influence of vibration, and to develop a bulk solids micro-dispensing technique to demonstrate the dispensing process of active pharmaceutical ingredients (API), excipients and biomaterials. Experiment work in this project includes design of vibratory dispenser hopper and dispensing test with vibratory dispensers where mechanical vibration and ultrasonic vibration is utilized as the driving force to fluidize coarse granules and fine powders, respectively. The results suggest that the vibratory dispenser is capable of accurately and fast dispensing “dropwise” bulk solid in a small amount per drop. A doming controlled flow mechanism is identified in the vibratory dispenser. Bulk solids dome formed in the dispenser hopper plays as a “valve” of flow under the influence of vibration. The dispensing test results show that the design parameters of dispenser hopper, i.e. orifice size, hopper angle and hopper diameter, and vibration signal parameters, i.e. frequency and amplitude, affect the flow rate and dosage conformity in the vibratory bulk solids micro-dispensing technique. Additionally, a triboelectric charging phenomenon is investigated in the ultrasonic vibration dispenser and a solution to the charging issue is proposed with modifying surfaces of dispenser hopper by using platinum plating method. Pt-coated surface reduces the triboelectric charge generated in the dispensing process and improves the flowability of powders. The correlation between discharge rate and design parameters of ultrasonic vibration dispenser is derived. The derived equations are used to predict dosing results in the application of producing solid form oral drugs and biomaterial dry powder libraries for high-throughput screening (HTS) experiment.

Although traditional Cognitive Behaviour Therapy (CBT) has achieved many clinical successes, approximately 30-50% of patients are resistant to this form of treatment. This trans-diagnostic group of treatment resistant patients typically have chronic, co-morbid, and/or personality disordered symptoms and often engage in a range of maladaptive behaviours (e.g., substance abuse, deliberate self-harm). Acceptance and Commitment Therapy (ACT) is a relatively modern psychological treatment which proposes that the formally dissimilar symptoms this group present result from a common cause; namely, excessive entanglement with, and a need to escape from or avoid, unwanted private events such as thoughts, feelings, and memories (experiential avoidance). Preliminary evidence from clinical trials suggest that ACT may prove efficacious with treatment resistant patients. In this thesis, four studies were designed to examine the theoretical underpinnings and clinical utility of ACT. Studies 1 and 2 tested the ACT-derived prediction that diverse maladaptive behaviours serve a common experiential avoidance function. In support of this hypothesis, structural equation modelling showed that experiential avoidance predicted significant maladaptive behaviour covariance. Moreover, using the same method, a cross-sectional design showed that experiential avoidance partially mediated the effect of Negative Affect Intensity and Childhood Trauma on the tendency to engage in maladaptive behaviours. Studies 3 and 4 extended these theoretically-based investigations into the applied domain, pilot testing ACT for a sample of patients whose symptoms had been resistant to, or relapsed following, standard care. Study 3, a pre-post uncontrolled trial, revealed significant reductions in psychological distress with gains maintained at 6 and 12-month follow-up. Study 4, a randomised control trial comparing ACT to a CBT treatment as usual (CBT-TAU) condition, showed that ACT achieved more enduring effects than CBT-TAU. Furthermore, exploratory analyses suggested that, for the ACT group alone, reductions in experiential avoidance during treatment predicted follow-up outcomes. These findings support the use of ACT for treatment resistant patients.

It is just forty years since the identification of immunoglobulin E (IgE) as the reagin responsible for allergen induced immediate hypersensitivity reactions. IgE exerts its biological actions through the binding of its Fc fragment to specific Fc receptors on effector cells. There are two predominant Fc receptors for IgE – FcεRI, which has a very high affinity for IgE and FcεRII, which shows less avid binding. For much of the first two decades after the identification of IgE, it was thought that FcεRI expression was limited to mast cells and basophils and that IgE binding to other cell types such as Blymphocytes and antigen presenting cells (APCs) was mainly due to FcεRII. However with major advances in characterisation and functional knowledge of FcεRI, particularly in the last fifteen years, it has become apparent that FcεRI can be expressed on several more cell types that may be involved in initiation and maintenance of allergic inflammation – including APCs (monocytes and dendritic cells) and possibly eosinophils. The research described in the four papers forming this thesis was completed during this period and evaluated FcεRI expression on different cell types, their potential roles in allergen induced inflammatory responses and whether successful therapeutic strategies for allergic disorders may involve actions on FcεRI+ cells. The relative expression of FcεRI on peripheral blood basophils, monocytes and eosinophils from atopic and non-atopic subjects and any relationship with serum IgE concentrations was assessed in the first paper. The second study examined a potentially important role for basophils as a cellular source of rapidly releasable IL-4 which may help initiate allergen induced TH2 responses. The next study investigated the possible effects on allergen induced early and late asthmatic responses of the immunosuppressive drug cyclosporin A which had been shown both to inhibit mast cell and basophil degranulation and cytokine secretion (particularly by CD4+ T-cells). The final study evaluated FcεRI expression on these cell types as well humoral factors (e.g. seasonal changes in allergen specific IgG and IgE) in subjects who, after 3 to 4 years of grass pollen immunotherapy, had continued on either active or placebo immunotherapy for a further 3 years. A historical perspective explaining some of the reasons the studies were done is provided in the introductory chapter whilst the discussion chapter at the end reviews how many of the findings of the study have evolved in subsequent years right up to the present day and finishes off with a brief synopsis of how rapidly increasing knowledge of the regulatory functions of dendritic cells (expressing FcεRI and often monocyte derived) has resulted in better understanding of the mechanisms of allergen specific immunotherapy and is leading to more effective treatment modalities.

Chest physiotherapy is integral to the management of mechanically ventilated children and previous research has confirmed that chest wall vibrations are the manual techniques used most frequently by physiotherapists in this population. Chest wall vibrations involve the application of a compressive force to the chest during expiration, with the aim of removing accumulated secretions and improving lung aeration. However, these techniques are largely unquantified and may vary greatly between practitioners and clinical units, with any significance of such variability remaining unknown. In order to evaluate the effectiveness of any therapy it is important to have a means of quantifying the treatment. It is challenging to measure techniques which involve manual contact between the therapist and patient, and at the inception of this project no means existed of directly measuring the force applied through the hand during treatments. The effectiveness of chest physiotherapy in mechanically ventilated patients is likely to be influenced by the interactions between different treatment components, such as the magnitude and pattern of the chest wall vibrations and the accompanying lung inflations. It is therefore essential to assess both the forces applied during the vibrations with the simultaneous changes in air flow, recording the ventilatory pattern throughout the treatment. The study hypotheses were: 1. It is possible to create a technique to measure chest wall vibration forces during clinical treatments, and to relate such forces to simultaneous changes in respiratory flows, volumes and pressures 2. Maximum and mean force applied during chest wall vibrations increase with the size and age of the child 3. Manual lung hyperinflations with chest wall vibrations result in an increase in peak expiratory flow above that observed during baseline mechanical ventilation 4. After adjusting for inflation volume, application of chest wall vibrations result in an increase in peak expiratory flow above that obtained during manual lung inflations alone The primary objectives of this research were to: 1. Develop a method of quantifying chest wall vibration forces and a means of evaluating simultaneous changes in force with those of respiratory flow and pressure in ventilated infants and children of all ages 2. Estimate the variability over time, within and between individual physiotherapists when treating the same and different subjects with chest wall vibrations 3. Conduct a study to: i) Assess the feasibility of measuring force and respiration in a population of critically ill, mechanically ventilated children ii) Characterise the magnitude and pattern of forces applied during chest wall vibrations and evaluate the direct effects of these manoeuvres on flow and pressure changes in the lungs iii) Determine the relative contribution of manual lung inflations and chest wall vibrations to any observed increase in expiratory airflow A secondary objective was to explore the short term effects of chest physiotherapy, by recording changes in ventilation, respiratory system mechanics and blood gases following treatment. The thesis comprises four chapters: Chapter 1 contains a comprehensive literature review of published studies demonstrating the current knowledge base of the respiratory problems of mechanical ventilation in children, chest physiotherapy in intensive care and the relationship of chest physiotherapy to normal mechanisms of airway clearance. Chapter 2 describes the process of creating a dynamic force-sensing technique to characterise manual chest physiotherapy, detailing protocol and analysis refinement during pilot force and respiratory data collection. Assessment of the variability within and between physiotherapists is also assessed. Chapter 3 details a clinical study undertaken in intensive care units at Great Ormond Street Hospital for Children NHS Trust, London. The results are presented and interpreted. Chapter 4 discusses the findings of the thesis in relation to earlier research, highlights the strengths and limitations of the current study, interprets the clinical implications of the research and suggests future work.

At least one patient will experience a potentially serious intravenous (IV) drug error every day in an ’average’ hospital. IV drug errors have been estimated to be a third of all drug errors. Previous drug error research has focused on observation of nurses and errors they make but has not attempted to understand the decision-making processes used during the preparation and administration of IV drugs. The aim of this study was to explore the decision-making processes that novice and experienced IV nurses use during IV drug administration and how this influences risk taking and errors. A three-phased ethnographic study was carried out in a specialist cancer hospital, using focus groups, observation and interviews. Three focus groups with 14 registered nurses were used to develop culturally relevant definitions related to error and experience. Observation of the two wards took place over a week each. Twenty nurses were observed preparing and administering IV drugs, and then interviewed about their procedure. Data analysis was carried out using a five stage approach. Definitions of drug error, IV drug error, novice and experienced IV givers were developed from the focus groups. Four major themes were identified and represent findings from the direct observation and interview of the nurses: interruptions; lack of identification/knowing the patient; routinised behaviour; prevention of errors. One of the key findings was the lack of checking of patient identity prior to IV drug administration, which appeared to be based on the nurses feeling they knew the patient well enough although this was in contrast to the checking of drugs even if they were familiar with them. Implications for practice included: exploring new and effective methods of education based on behavioural theories; involving staff in updating and writing policies and procedures; and formal assessment of staff during IV preparation and administration.