This study describes developments and in vitro characterisation of lipid microparticles prepared using spray-drying for drug delivery to the lung via dry powder inhalers. Bioadhesive formulations such as prochitosome or chitosome powders have been introduced to overcome the drawbacks of liposome instability and potentially provide significant increase in the residence time of drug in the lung. Mannitol or lactose monohydrate (LMH) aqueous solutions were spray dried at inlet temperatures of 90, 130, 170 or 210ºC. Soy phosphatidylcholine and cholestrol (1:1 mole ratio) were used in all formulations. Cholesterol was added to increase vesicle membrane rigidity. Proliposomes containing salbutamol sulphate (SS) were prepared by incorporating various lipid:carrier (mannitol or LMH; 1:2, 1:4, 1:6, 1:8 and 1:10 w/w). Prochitosomes including SS or beclomethason dipropionate (BDP) were prepared by adding various chitosan glutamate:lipid ratios of 1:10, 2:10, 3:10 and 5:10 w/w. Chitosomes, including various cryoprotectants (mannitol, LMH, trehalose or sucrose), were prepared by including chitosan glutamate to liposomes generated from ethanol-based proliposomes in the ratio of 3:10 w/w chitosan to lipid. The spray-drying parameters for generation of dry powders were optimised by using an inlet temperature of 120ºC, outlet temperature of 73 ± 3°C, aspirator rate of 100%, suspension feed rate of 11%, and spray flow rate of 600 L/h using B-290 Buchi mini spray-dryer. The production yields were 48.1±2.84%, 69.73±2.05%, 61.33±2.51% and 58.0±3.0% for mannitol and 50.66±3.51%, 68.0±2.0%, 73.66±1.52%, 59.0±2.64% for LMH at 90, 130, 170 or 210ºC, respectively. The size of the particles were smaller than 5 µm for both carriers at of 90 and 130 ºC, whilst larger than 5 µm at inlet temperatures of 170 and 210 ºC. Particles had smooth, spherical and smaller size at 90 and 130 ºC than inlet temperatures of 170 and 210 ºC. Mannitol kept its crystalline properties after spray-drying, whilst LMH changed to amorphous at all drying temperatures. Mannitol-based proliposome particles were uniform, small and spherically shaped. In contrast, LMH-based proliposome particles were irregular and large. Entrapment efficiency of SS was higher for LMH-based proliposomes, however, fine particle fraction (FPF) was higher for proliposomes containing mannitol. Higher FPF was obtained for proliposome containing lipid to mannitol ratio of 1:6 (FF= 52.6%). Vesicles size decreased with increasing carrier ratio and the zeta potential was slightly negative for all formulations studied. Prochitosomes were small, porous and spherically shaped particles. Higher FPF was achieved for prochitosome powders containing chitosan to lipid ratio of 3:10 and 5:10 for both SS (FPF = 58.12±2.86% and 70.25±2.61% respectively) and BDP (FPF = 61.89±9.04% and 61.56±3.13% respectively). Zeta potential and the fraction of mucin adsorbed on the vesicles increased upon increasing chitosan concentration. Vesicle size decreased with increasing chitosan concentration. Entrapment efficiency (EE) of the formulations containing BDP was higher than that for SS. Moreover, the drug EE was higher using chitosomes compared to liposomes. LMH and trehalose-based liposome or chitosome particles were spherical with less tendency of agglomeration compared to mannitol and sucrose-based particles. Powders containing LMH, trehalose or sucrose were amorphous, whilst mannitol-based powder was crystalline. The FPF values were 14.39±1.81%, 32.29±0.15, 48.99±2.22% and 50.79±3.19% for mannitol, sucrose, LMH and trehalose-based liposome formulations, respectively. However, FPF% values were higher for chitosomes, being 23.48±3.38%, 33.89±0.66%, 54.88±1.85% and 55.9±2.74% for mannitol, sucrose, LMH and trehalose-based chitosomes, respectively. The EE of SS was increased upon coating liposome surface with chitosan regardless of cryoprotectant type. In conclusion, the findings of this study have demonstrated the potential of lipid microparticles in pulmonary drug delivery and that prochitosomes or chitosomes may offer great potential for enhancing drug resident time in the lung.

No description available.

Background: Prostate cancer affects over 40,000 men in the United Kingdom each year. The UK Government’s pledge to reduce inequalities within cancer care, relating to prostate cancer, has predominantly focused on the experiences of heterosexual men and overlooked the experiences of gay men. Hence, current recommendations relating to prostate cancer care may not reflect the unique needs of this marginalised group. Aims: This study aimed to explore the impact of prostate cancer on gay men to understand how the disease affected their life and, identify any specific psychosocial or support needs. Methods: This interpretive phenomenological study was guided by van Manen’s methodological and analytical approach. Twelve gay men were recruited from across the UK. Semi-structured interviews were conducted to explore participants’ experiences of prostate cancer. Analysis: Interpretative data analysis incorporating hermeneutic principles identified themes and sub-themes which were reflected through the lens of Merleau-Ponty’s four lifeworld existentials. Findings: The context in which prostate cancer is experienced is unique to gay men. Themes which convey the bodily impact of the disease (Corporeality) include ‘violation of identity’, ‘assault of the physical body’ and ‘the power of potency’. The changing sense of time (temporality) is revealed within themes of ‘threat to eternal youth’, ‘living in a state of flux’, ‘disrupted lives’ and ‘past, present and future horizons’. Relationships with others (relationality) are illuminated through the themes ‘the quest for mutual respect and equality’, ‘locating information’, ‘to tell or not to tell’, ‘changes and challenges’ ‘friendship’ and ‘in pursuit of peers’. Finally, the intimacy of space (spatiality) is uncovered within the themes ‘yearning for community’, ‘the power of proximity’ and ‘isolation’. Conclusions: This is the first UK study to explore the experiences of gay men with prostate cancer, and identified unmet needs unique to gay men diagnosed with prostate cancer which have implications for clinical practice, health policy and groups which provide support to men with prostate cancer.

Background: Patellofemoral pain (PFP) is a condition that has been described as 'the Loch Ness monster of the knee' and the 'black hole of orthopaedics' due to its indefinable and expansive nature when addressing the aetiology, management and treatment. Although significant and clinically important changes have been observed with the joint biomechanics, psychosocial factors, brain activity and neuromuscular activity a lack of understanding of PFP still remains. The overall aim of the study was to provide a unique and multifaceted investigation into the motor unit control, biomechanical, neuromuscular and psychosocial factors in understanding the movement control of PFP subjects and the response to a common clinical intervention. Method: Non-symptomatic (n=13) and symptomatic subjects (n=13) performed a single limb isometric squat in two conditions, no tape and with a medial glide tape application. Motor unit data from the Vastus Medialis (VM) and Vastus Lateralis (VL) was recorded using sEMG Decomposition. Muscle activity of the Gastrocnemius (GAS), Rectus Femoris (RF), Biceps Femoris (BF) and Gluteus Medius (GMed) were collected using sEMG. Kinematic and kinetic data from the lower limb were recorded. All systems were synchronised for simultaneous data collection. Measures of conscious motor processing were made using the Movement Specific Reinvestment Scale and pain levels recorded using the Numerical Rating Scale. Results: Grouped Tape Response: The mean motor unit firing exhibited an increase in the VM firing rate and a decrease in the VL firing rate within the tape condition, across both subject groups, suggesting a modification in the load bias across the Vasti muscles. The common drive, a physiological phenomenon describing common fluctuations in the motor unit firings and consequently a measure of the nervous systems signals to control pools of motor units, increased in the VM and decreased for the VL in response to the tape condition in symptomatic subjects showing that the tape may be providing enhanced feedback to the nervous system that responded by modifying the 'control' to the motor units. There were no changes however in the non-symptomatic subject group, perhaps suggesting their motor units were already controlled efficiently. Motor unit recruitment analyses, through regression analysis of the motor unit firing rate and knee joint moment, showed the VM motor units in the nonsymptomatic subjects were recruitment at a higher firing rate in the tape condition but interestingly no change in the VL and no change in the symptomatic subjects. Both non-symptomatic and symptomatic subjects demonstrated significant reductions in transverse plane knee joint range of moment, illustrating a more controlled rotational knee joint after the application of tape. There were no significant changes found in the coronal or sagittal joint mechanics. Symptomatic subjects demonstrated a propensity to consciously control their movements, suggestively disrupting automatic motor control tasks. Symptomatic subjects presented with pain scores of 4.2/10 on the numerical rating scale. Individual Tape Response: Exploration of individuals' response to tape, opposed to pooling data and treating as homogenous groups, exhibited a non-uniform response with variable increases, decreases and no changes across the different measurements taken. The exploration of the data with this method is in line with common clinical presentation of PFP subjects and presents rationale for new ways to view the data as to not mask the true physiological behaviours. Comparison of groups: Motor unit recruitment analyses comparing the subject groups demonstrated that symptomatic subjects had a significantly different motor unit recruitment strategy for the Vasti muscles, where the larger motor units were firing faster compared to the non-symptomatics larger motor units in both VM and VL for the same level of force. Symptomatic subjects demonstrated a lower common drive to the VM and higher common drive in the VL compared to non-symptomatic subjects, which after the application of tape became the same level as the non-symptomatics. Symptomatic subjects also exhibited significantly lower muscle activity in the GAS, BF, RF and GMed, thus suggestively increasing the muscle activity bias to the Vasti muscles. No changes were seen in the joint biomechanics or mean motor unit firing rate between the groups. Discussion These findings suggest that the nervous system offers a portfolio of solutions to control and distribute force, which can be manipulated through a common taping intervention. The results show that the motor unit firing rate in the VM increases and decreases in the VL, coupled with altered motor unit recruitment strategies thus inferring that there may be a re-distribution of force across the Vasti with the application of tape. This is interestingly complimented with an increase of common drive in the VM and decrease in the VL between subject groups. The presence and then change of common drive within the Vasti so that the motor units are firing in unison, more so with tape, and providing a tantalising prospect that the VM muscle is acting more efficiently and controlled with tape. The novel findings of the neuromuscular system and its modification were alongside the increase in torsional joint control. However, it is evident that the motor unit firing rate, common drive and motor unit recruitment present variable responses amongst individuals, offering different solutions to achieve the same goal; increasing the force and its control within the muscle. The underlying mechanism for the observed findings are unable to be expressed definitively, however it can be deduced that the application of tape presents proprioceptive feedback to the muscle that alters the motor unit pool; consequently adjusting the force and its control within and across muscles leading to an increase in knee stability. Conclusion: The key implications from this work is that the application of tape can offer clinically meaningful changes to the sensory-motor control system, through the manipulation and alteration of the motor unit pool, suggestively from an enhanced proprioceptive feedback mechanism. However, researchers and clinicians should consider the individualistic responses and the potential to mask true physiological findings by assuming homogeneity within patient populations with data analyses and clinical decision processes respectively. This work offers unique and novel insights into both the behaviour of patients with Patellofemoral Pain and also the effects of a taping intervention, thus providing additional clinical understanding and also tantalizing opportunities for future work exploring musculoskeletal or neurological disorders and insight into the sensory-motor control strategies.

The term agenda setting has been used variably across the healthcare literature, in particular in writings on doctor-patient communication, medical education, and behaviour change. No attempt has yet been made to integrate these different conceptualisations. This impacts both investigation and teaching of this communication skill. The studies in this thesis aim to clarify a conceptual foundation, and to develop a measure of agenda setting, for use in teaching clinicians. A context of long-term condition management was selected. In these clinical encounters clinician and patient agendas naturally intersect and may disagree, and patient participation is essential to effective management. Phase 1 of this thesis involved a structured literature review, and focus group study with clinicians in primary and secondary care to map components of agenda setting. These were refined through a consensus group study involving patients, clinicians, researchers and educators. An integrated model of agenda setting is proposed that adopts new terminology: agenda mapping involves establishing shared focus, and agenda navigation involves tracking natural shifts in focus throughout an interaction. Agenda mapping includes six core domains: (1) identifying patient talk topics, (2) identifying clinician talk topics, (3) agreeing shared priorities, (4) agreeing focus, (5) collaboration, and (6) engagement. Clarifying these domains established a foundation for measurement. Phase 2 of this thesis addresses measurement of agenda mapping. A review of measures confirmed that no existing measure includes all agenda mapping domains. The Evaluation of AGenda mapping skiL Instrument (EAGL-I) was developed, and tested in a study with third year medical students. EAGL-I scores were shown to represent reliable and valid assessment of agenda mapping. Conditions, under which reliable assessment may occur, are also discussed. Educators and researchers now have a tool for use in teaching agenda mapping to clinicians. Further investigation of agenda mapping in long-term condition management may also now progress.

Ryanodine receptors (RyRs) are the largest known ion channels composed of four identical subunits. Interactions between structural/functional domains have been proposed to regulate channel activity and play an important role in the pathogenesis of RyR-associated disorders. RyR2 mediates the release of calcium form sarcoplasmic reticulum of cardiac myocytes and its dysfunction is associated with life-threatening arrhythmias. The principal aim of this study was to characterise the self-association of the RyR2 N-terminus biochemically and evaluate its impact on channel function. Moreover, its role in channel dysfunction observed in arrhythmia-susceptible individuals was tested together with dantrolene’s ability to rescue the disease phenotype. RyR2 N-terminus self-association is mediated by multiple sites with two critical oligomerisation determinants located in the loops connecting strands β8-β9 and β20-β21, predicted to reside at the inter-subunit interface. N-terminus self-association is further stabilised by disulphide bonds most likely involving multiple cysteine residues with cysteine 361 contributing to this process. Normal N-terminal inter-subunit interactions within the full-length RyR2 appear to prevent spontaneous activation of the channel at diastolic calcium. Channel hypersensitivity is a common feature of the arrhythmia-associated phenotype suggesting that abnormal N-terminus self-interaction might be involved in RyR2 pathology. Indeed the presence of arrhythmia-linked mutations (L433P and R176Q) compromises the ability of the RyR2 N-terminus to oligomerise. Defective N-terminus self-association appears to underlie the functional impairment of RyR2L433P. The mutated channel displays compromised [3H]ryanodine binding and reduced stability of tetrameric assembly, both of which can be rescued by dantrolene at clinically relevant concentrations. Notably, dantrolene’s primary mode of action appears to involve stabilisation of the N-terminal inter-subunit interactions. In summary, the work presented here provides important insights into a novel domain-domain interaction and its role in the regulation of RyR2 function.

In wound healing and tissue repair, the presence of α-smooth muscle actin (α-SMA) containing myofibroblasts leads to wound closure and collagen-rich scar formation. This thesis investigated mechanisms of transforming growth factor-β1 (TGF-β1)-mediated differentiation and the dysfunctions involved in age-associated loss of differentiation. The loss of epidermal growth factor receptor (EGFR) and hyaluronan (HA) production, and diminished interaction of HA with its receptor CD44 (compromising its function) were the principal contributors to aged fibroblast resistance to differentiation. In response to TGF-β1, CD44 relocated to EGFR held in cholesterol-rich membrane-bound lipid rafts. This was HA-dependent, as hyaluronidase or 4-methylumbelliferone treatments restricted CD44 motility and prevented CD44-EGFR co-localisation. Additionally the intracellular signalling cascade was found to be a sequential phosphorylation of extracellular signal regulated kinase 1 & 2 followed by Ca2+/calmodulin kinase II. The activation of both proteins was required for differentiation. Elevated microRNA-7 (miR-7) expression was found in aged fibroblasts. Overexpressing miR-7 in young fibroblasts attenuated the expression of EGFR and inhibited differentiation. When miR-7 was inhibited, EGFR and hyaluronan synthase 2 expression, CD44 membrane motility, and TGF-β1-mediated differentiation in aged fibroblasts were restored. Activation of EGFR drove miR-7 promoter activity and expression in a JAK/STAT1 dependent manner. Treatments of aged fibroblasts with 17β-estradiol (E2) resulted in decreased miR-7 expression and, when TGF-β1 was added, restored the α-SMA-positive phenotype. E2 treatments had no impact on STAT1 phosphorylation; leading to the hypothesis that E2 regulation of inflammatory mediators may be involved. The data demonstrated different points of intervention for the promotion or prevention of TGF-β1-regulated myofibroblast differentiation. The interactions between HA-CD44 and EGFR were crucial elements in the differentiation process and the importance of miR-7 was apparent. The mechanisms shown here may have direct implications for modifying the wound healing response, particularly for developing therapeutic strategies to improve healing in the elderly.

Hereditary haemophilia A (HA), an X-linked bleeding disorder, is caused by mutations in the coagulation factor VIII gene (FVIII abbreviates protein, gene symbol F8). The aim of this study was (1) to characterise F8 mutations in HA cohort from Pakistan, (2) to investigate whether in vitro thrombin generation (TG) differs according to mutation type, and (3) to compare haemophilia joint health score (HJHS) and Gilbert score with severity, age, TG and underlying mutation in HA cohort which had minimal access to haemostatic replacement therapy. Methods: One hundred HA individuals and 100 healthy controls were recruited; clinical details were recorded. Results: Phenotypic measurements were re-evaulated in Cardiff; the essential regions of F8 were screened. Ninty two individuals were diagnosed with HA, 7 with haemophilia B and 1 was normal. The F8 defects were characterised and comprised point mutations, inversions and frameshifts. Thirty novel variants were identified. No significant difference was observed in vitro TG between severes with null mutation and those with a missense change. HJHS was strongly correlated with Gilbert score (r =0.98), both were siginificantly higher in severe compared with nonsevere before the age of 12 years (P≤0.01) but not thereafter. According to developmental age (<12 years, 12-16 years and > 16 years), both scores were significantly lower in the youngest group (P≤0.001). In severes there was no correlation between in vitro TG and joint score, no significant difference was observed for either joint score according to the underlying mutation type. Whereas in nonseveres, negative correlation between in vitro TG and joint score was observed. Conclusions: F8 defects in Pakistan is heterogenous; in severe HA in in vitro TG are not influenced by underlying mutation.

The ryanodine receptor (RyR) is a large (563 kDa) tetrameric calcium channel that controls the efflux of calcium ions from the large internal stores within myofibrils called the sarcoplasmic reticulum. The cytoplasmic domain is connected to the pore via an "interacting-domain" (I domain) which plays an important role in the mechanism of channel regulation. Within the I domain is a calcium binding region which resembles a lobe of calmodulin and is thus called the calmodulin-like-domain (CaMLD). CaMLD has a binding partner region, the calmodulin-binding-domain (CaMBD) located in the cytoplasmic domain. Mutations in the cardiac isoform of RyR have been shown to result in the clinical presentation of a lethal disease condition called catecholaminergic polymorphic ventricular tachycardia (CPVT). Thus, mutations proximal to the CaMLD and CaMBD region are of particular interest, since if these mutations affect the binding of calcium or result in any conformational change of the protein, then the role of the mutation could be explained. Computer simulation methods were employed to investigate the role of CaMLD and CaMBD in the overall biology of RyR. The individual domains and also the entire section of RyR containing both domains were modelled. This latter model underwent a simulated folding process to generate a tool to further investigate the role of CPVT mutations within this region in the context of the structural changes brought about by the changes in amino acid residues of the protein. CaMLD and CaMBD were produced as recombinant proteins and selected CaMLD CPVT mutants generated. The calcium dependent interaction of these two domains was examined through a range of functional techniques, in particular circular dichroism. All the studied mutants displayed a calcium induced conformational change associated with the functional protein. However, one particular mutation was identified which appeared to significantly weaken the structural stability and have reduced calcium sensitivity when compared to the wild type recombinant protein.

This thesis presents the development and testing of an online lovingkindness meditation (LKM) intervention. Previous studies were systematically reviewed, showing complex but encouraging evidence that LKM can enhance the wellbeing of individuals and communities by promoting pleasant emotions and empathic attitudes. However, previous randomised controlled trials (RCTs) are small and have methodological limitations. An online RCT was conducted, which recruited 809 adult volunteers to test whether an LKM intervention offered to the general population improves wellbeing through pleasant emotions, psychological resources, empathy and altruism. LKM was compared to a light physical exercise course (LE). Participants followed prepiloted videobased instruction, wrote about their experiences in online diaries and interactive fora, and completed questionnaires and an objective measure of helping behaviour. The data were analysed using a mixed methods approach. Both courses led to greater wellbeing. LKM participants were significantly less anxious and more likely to donate money to charity than LE participants. Differences in other outcomes were not significant. Attrition was high but generally unrelated to the interventions’ content. The pathways to wellbeing differed. LKM was an emotionally intense experience, generating deep reflections and an increased connectedness with self and others. LE led to increases in relaxation and physical wellbeing which generated a sense of achievement. Some participants had early difficulties with LKM, in which personal factors played an important role. The study provides suggestive evidence that both LKM and LE enhance pleasant emotions, psychological resources and wellbeing, and that LKM specifically stimulates empathy and altruism. The LKM training platform used in this study is available for immediate largescale implementation as an inexpensive public health intervention. However, future research is needed to confirm present findings and devise LKM interventions that reduce the negative impact of initial training. Completion rates might be improved by nesting online RCTs within cohort studies.