This study then focused on protein transduction domains (PTD), such as the HIV-Tat and octaarginine, which have shown great promise as vectors for drug delivery and have demonstrated abilities to bypass drug transporters thereby increasing drug efficacy. However their mechanism of entry and eventual cellular fate is much debated in the literature. KG1a and K562 cells were found to be good models for studying the cellular dynamics of fluorescently conjugated PTD as their suspension status minimised background fluorescence resulting from non-specific binding of fluorescent peptides to tissue culture plastic. Immunofluorescence microscopy and flow cytometry implicates a predominantly endocytic mechanism of uptake for these peptides and their final cellular distribution is indicative of late endosomes and lysosomes. Their cellular dynamics suggests they may be able to bypass conventional MDR processes, making them ideal for the circumvention of both transporter mediated drug exclusion and drug sequestration.

Background: This thesis presents a study of the safety of medicines with respect to drug counterfeiting in developing countries (East Africa and the Middle East). Counterfeit medicines are also present in industrialised countries, but not on the same scale as in developing countries. The aim of the study was to establish the responsiveness of health care professionals at the practice level concerning the counterfeiting of medicinal products in developing countries focusing on six countries in the East African region and seven countries located in the Middle East. Method: The method of data acquisition used was by survey questionnaires issued in 13 developing countries (6 in East Africa and 7 in the Middle East). The questionnaires were delivered to the respondents either personally or by e-mail and the questionnaire, responses were returned by the same means. Respondents returned their questionnaire forms direct to the author either on the same day or later by e-mail. The data were analysed with regard to the specific questions. Results: The study findings suggested that the poorer the country, the higher the degree of counterfeiting. All the respondents (n: 2180) agreed that there was a fake or counterfeit medicine problem in their own country (71% of respondents in Africa and 63% of respondents in the Middle East considered this a major problem). Both branded and generic drugs were counterfeited and the extent of the problem and several other factors concerning counterfeited drugs differed significantly between industrialised and developing countries. The difference depended on drug regulation control and enforcement and also on the quality and the prices in the legal supply chain. In most industrialised countries like the USA, Japan or the members of the EU, the level v of drug counterfeiting is <1% of the total medicines market value. An exception is the former Soviet Union where up to 20% of the market is occupied by counterfeit drugs. In contrast, within regions of Africa, Asia and parts of Latin America, between 10-30% of the available medicines are fakes (WHO 2006) Conclusions: The study showed that healthcare workers were aware of the prevalence of counterfeit medicines and quite a number of them had encountered them in their supply role. There is an indication that the respondents tried to assure themselves of the quality of the drugs they purchased by using several methods. However, no rigorous effort was taken to confirm as well as report suspected counterfeit drugs to regulatory authorities. In the industrialised world, medicines regulatory authorities have developed strict standards and controls to ensure the safety and effectiveness of drugs. However, as this study has found, in less developed countries a lack of human and financial resources within the health sector as a whole restricted the activity of regulatory agencies, resulting in a sub-optimally regulated environment in which substandard drug production persisted without detection.

Background: Outcomes in the management of early rheumatoid arthritis (RA) have been significantly improved through the use of composite disease activity measures (such as the DAS28) and aggressive DMARD escalation until a lower disease activity target has been achieved. Imaging studies suggest that the DAS28 may be insensitive to low levels of subclinical active disease that is associated with an increased risk of flare and progressive joint damage. Further, in some cases, elevations of the DAS28 may not necessarily be related to on going active synovitis. In both instances, relying upon the DAS28 assessment alone may lead to patients being considered for an inappropriate treatment decision since, patients with active subclinical disease may not be considered for further DMARD escalation whilst patients with non-inflammatory causes of DAS28 elevation may be offered additional DMARD therapy that is either ineffective or potentially toxic. There is emerging evidence that musculoskeletal ultrasound (MSUS), gene expression profiles and inflammatory protein microarrays might provide useful additional disease activity information that allows clinicians to reach a treatment decision that is targeted at an individual patient’s specific needs Objectives: 1. To determine whether using MSUS assessment of global disease activity in addition to the DAS28 produces significantly better short-medium term clinical and radiological outcomes 2. To determine whether grouping early RA patients by either RA phenotype or disease activity level is associated with evidence of differential gene expression between the comparator groups 3. To determine the degree of correlation and agreement between the Multi-Biomarker Disease Activity (MBDA) test, the DAS28 and a MSUS disease activity assessment Methods 111 patients with either clinical diagnoses of early RA (symptom duration < 1 year) or anti-CCP antibody positive inflammatory arthritis were recruited to the Targeting Synovitis in Early Rheumatoid Arthritis (TaSER) study. Clinical consultations occurred monthly for 18 months and all participants were treated using the same step-up DMARD-biologic escalation protocol. Participants were randomised to either a DAS28 or MSUS assessment group. In the DAS28 group, DMARD therapy was escalated until DAS28 low disease activity (LDAS – DAS28 <3.2) had been achieved. In the MSUS group, MSUS assessment was indicated for instances of DAS28 LDAS or DAS28 moderate disease activity (3.2≤ DAS28 <5.1) with minimal clinical synovitis (28SJC ≤1). During MSUS assessment, the bilateral radiocarpal, index and middle MCP, index and middle PIP and 2nd and 5th MTP joints were examined for the presence of gray scale synovial hypertrophy and Power Doppler (PD) signal. Active disease was defined as the presence of grade 1 or higher PD signal in 2 or more joints. DMARD therapy was not changed if there had been significant escalation within the preceding 3 months. Intra-articular and intra-muscular corticosteroid injections were administered generously during periods of active disease. Blinded clinical outcomes were collected at baseline and every 3 months until study completion. Plain x-rays of hands and feet and MRI of the dominant wrist and hand were performed at baseline and study completion and will be graded by 2 independent radiologists who are blinded to participant’s randomisation group. Primary outcomes comprised: 1. mean change in DAS44 from baseline and 18 months, 2. mean change in MRI RAMRIS erosion score between baseline and 18 months. Secondary outcome measures included: between group comparisons of the DAS44 and ACR-EULAR remission rates, EULAR response criteria, HAQ, EURO-QoL 5D, CRP, ESR, 10cm pain visual analogue score, mean change in plain x-ray Sharp score (van der Heijde modification) and mean change in MRI RAMRIS synovitis and bone marrow oedema scores. 79 Participants donated additional blood samples for nested biomarker analysis at baseline, follow-up months 3 and 18. Baseline and 3 month PAXgene RNA samples were analysed with the assistance of the Systems Biology Group, Institute of Cardiovascular and Medical Sciences, University of Glasgow using an Illumina HumanHT-12v4 Beadchip microarray. Baseline, 3 month and 18 month serum samples were analysed by Crescendo Biosciences using their in house MBDA microarray. Additional whole blood, serum and plasma samples remain available for future polyomic analyses. For the gene expression analysis, participants were segregated into comparator groups based upon baseline and 3 month RA phenotypic and disease activity data. Comparator groups were intended to represent common clinical scenarios. Between group comparisons of gene expression were conducted in the R software package using the Linear Models for Microarray Data (Limma) plug-in. An adjusted p value <0.05 was considered to represent evidence of differential gene expression. For the MBDA analysis, the degree of correlation (Spearman’s rank correlation) between DAS28 and MBDA score was calculated at each time point and for all time points pooled together. The percentage agreement between MBDA, DAS28 and MSUS disease activity state categorisations was also calculated. Results 111 participants were recruited and 101 (91%) completed follow-up. 95 (86%) participants fulfilled 1987 ACR RA classification criteria and 107 (96%) fulfilled 2010 ACR-EULAR RA classification criteria. The presenting features appeared typical of an early RA cohort and, excepting gender, there were no statistical differences in baseline characteristics between the groups 414 MSUS assessments were performed, 369 MSUS assessments coincided with DAS28 LDAS, of which 92 (25%) identified active synovitis. 271 MSUS assessments coincided with DAS28 remission, of which 66 (24%) identified active synovitis. 45 MSUS assessments coincided with DAS28 moderate disease activity of which 15 (33%) identified active synovitis. Overall 71% of paired DAS28 and MSUS assessments agreed on the disease activity state MSUS-driven DMARD escalation was not associated with significant improvements in clinical outcomes. Both groups experienced a similar mean change in DAS44 between baseline and 18 months (DAS28 -2.51 vs MSUS -2.76, p 0.39). There were no statistically significant between group differences in the ACR core set variables at any of the time points, nor their mean change from baseline. Over the follow-up period, the MSUS assessment group demonstrated incremental increases in the proportion of participants with EULAR good responses and DAS44 remission and a significantly higher rate of DAS44 remission at study completion (DAS28 44% vs MSUS 65%, p=0.045). The impact of MSUS-driven DMARD escalation on radiological outcomes, medium-long term outcomes and adverse event rates remains to be determined. At baseline, gender (61 genes), RhF status (5 genes) and current smoking (1 gene) were associated with evidence of differential gene expression. The expression patterns of 19 genes changed following commencement of DMARD monotherapy. However, it was not possible to demonstrate evidence of differential gene expression in relation to disease activity level or phenotypic extremes at either time point. Up-regulation of 3 genes at baseline was associated with requiring DMARD escalation at 3 months. Otherwise, baseline gene expression was not predictive of 3 month disease activity state nor disease course over 12 months. Mean baseline interferon response gene score was not predictive of response to step-up DMARD therapy The MBDA test score correlated positively with DAS28 at a single time point (rs=0.58, p<0.0001) and the change correlated positively with corresponding changes in DAS28 (rs=0.56, p<0.0001).

The experiments presented in this thesis describe the roles of the post-junctional α1, α2-adrenoceptors (AR) and P2X purinoceptors in response to sympathetic nerve stimulation in mouse mesenteric and tail arteries. Such roles were determined by combining wire myography techniques and nerve stimulation alongside various selective antagonists. The influence of each receptor on the response to nerve stimulation was first defined in wild type (WT) mice before analysis of α1-AR knock out (KO) mice. Therefore the effect that genetic removal of the α1-ARs had on vascular response could be investigated. The response is the force generated in milligrams/tension when the nerves are stimulated and correspond to activation of post-junctional receptors and subsequent smooth muscle cell contraction. Systolic Blood Pressure and the α1-ARs The aim of the first study (Chapter Three) was to determine whether systolic blood pressure (BP) was different in the KO mice compared to WT controls. Tail cuff measurements of systolic BP were recorded in WT and AR KO mice. It was found that BP was not affected by loss of the α1A- and α1D¬-AR subtypes (ADKO) or by loss of all three α1-AR subtypes (α1-null). Therefore, the α1-AR role in maintaining BP may not be as crucial as previously understood or, the remaining receptors may have compensated for the loss. Calcitonin Gene Related Peptide (CGRP) in Mouse mesenteric and tail arteries The second study (Chapter Four) examined whether, under the stimulation parameters used, the potent vasodilator CGRP masked the vasoconstrictor response to nerve stimulation in mouse mesenteric and tail arteries. The potent neurotoxin capsaicin depletes the sensory nerves of CGRP. Responses prior to capsaicin incubation were compared with those following capsaicin treatment in WT, ADKO and α1-null mice. In mesenteric artery from each mouse strain, capsaicin incubation had no significant effect on the peak response to nerve stimulation. Furthermore, in the tail artery, capsaicin treatment had no significant effects on the responses in WT and α1-null mice. However, in the ADKO mouse tail artery preparations, capsaicin treatment significantly increased the peak response at low frequency stimulation. These findings may indicate an interaction between the α1-AR subtypes and the release of CGRP whereby the presence of all or none of the subtypes (WT and α1-null) has no effect on CGRP release but loss of the α1A- and α1D-AR subtypes alters the balance and reveals a CGRP induced effect. Response to perivascular nerve stimulation in Mouse mesenteric and tail arteries from Wild Type mice The aim of Chapter Five was to determine which receptors were involved in the response to nerve stimulation in mouse mesenteric and tail arteries. These results would then act as a comparison with the ADKO and α1-null responses in the later chapters. A contractile response to nerve stimulation was recorded in each of the studied vessels and abolished by combined blocked with antagonists for the α1-, α2¬-ARs and P2X receptors. Individual receptor blockade and component analysis then revealed the roles of the individual receptors. In the mesenteric arteries, the α1-ARs were the main contributors to the response followed by the P2X receptors and finally the α2-ARs which displayed both pre- and post-junctional effects. No interactions were discovered between the α1-ARs and the other receptors. The P2X receptors initiated the contraction and prolonged the response at the low frequency and contributed to the contractile response at the high frequency. In the tail artery, the α2-ARs were the dominant receptors but required the presence of the α1-ARs, and to some extent the P2X receptors in order to produce a full contractile response upon activation. The P2X receptors alone initiated the response at low frequency stimulation with both α1-ARs and P2X receptors involved in initiation of the contraction at the high frequency. Response to perivascular nerve stimulation in mesenteric and tail arteries from mice lacking α1A- and α1D-AR subtypes (ADKO) Chapter Six determined the role of the α1B-ARs in the response to nerve stimulation and therefore examined whether loss of the α1A- and α1D-AR subtypes (ADKO) affected the response. In the mesenteric arteries, at the low frequency, there was a potential interaction between the receptors. However, no single receptor was responsible for the contraction although there was a trend for the P2X receptors to be active at the beginning of the response and the α2-ARs to be active at the latter stage of the response. This was also true at the higher frequency. The ADKO vessels displayed no evidence of pre-junctional α2-ARs. In the mesenteric arteries, there is little role for the α1B-ARs. The overall contribution in the tail artery was reduced in the ADKO, particularly at the higher frequency. Similar to the WT mice, the α2-ARs were the main contributor to the response. The P2X receptors initiated the contraction but required the α2-ARs to be active at the low frequency. The α1B-ARs also required an interaction with the other receptors at low frequency stimulation in order to contribute to the initiation of the response. The α1B-ARs also contributed to the initiation of the response at the high frequency without requiring the presence of the other receptors. Response to perivascular nerve stimulation in mesenteric and tail arteries from mice lacking α1A-, α1B- and α1D-AR subtypes (α1-null) In the final study (Chapter Seven), α1-null mice were utilised in order to determine whether loss of the α1-ARs had a significant effect on the response to nerve stimulation in mouse mesenteric and tail arteries. Furthermore, the influence of the α2-ARs and P2X receptors were compared with the response in the WT vessels to determine whether they compensate for the loss of the α1-ARs. The removal of the α1-ARs dramatically reduced the response to nerve stimulation in the mesenteric arteries, with no apparent compensation from the α2-ARs or P2X receptors. There was no evidence of pre-junctional α2-ARs. The small response recorded at the high frequency was initiated by the P2X receptors and maintained by the α2-ARs and P2X receptors. The tail artery response was smaller in the α1-null mice compared with WT, particularly at the higher frequency. However, the response was still mediated largely by the α2-ARs with an interaction with the P2X receptors likely. A potential compensatory mechanism was recorded at the lower frequency as the response to P2X receptor antagonism was greater in the α1-null than in the WT mice. As was shown in the WT, activation of the P2X receptors initiated the contraction, particularly at the high frequency. Findings and Results Collectively, the findings of the studies presented in this thesis demonstrate that the α1-, α2-ARs and P2X receptors are involved in the response to nerve stimulation in the mouse mesenteric and tail arteries. Genetic removal of the α1-ARs in the ADKO and α1-null is most effective in the mesenteric arteries with little evidence of any compensatory mechanisms. In the mouse tail artery, the α2-ARs are the main contributors to the response and so loss of the α1-ARs has less of an effect. Interactions between the receptors were most clearly shown in the tail artery with little interaction between the α1-ARs and the other receptors demonstrated in the mesentery. Furthermore, it has been demonstrated here that systolic BP is unaffected in the KO mice and there is little input from the CGRP nerves using the parameters tested. From these results, the importance of α1-AR activation in nerve mediated responses was determined. The absence of a compensatory mechanism to match the response lost in the ADKO and α1-null mice, and the presentation of a normal BP alongside the survival of these transgenic mice challenges the importance of the α1-ARs being crucial in the maintenance of vascular tone. This therefore complements the knowledge that treatment of primary hypertension with α1-AR antagonists is largely unsuccessful. The contractile response mediated by α2-ARs and P2X receptors may be used as potential therapeutic targets in controlling hypertension.

The traditional paradigm relying on drug discovery to treat and heal the body is changing. Medicine for the 21st century is moving towards using the body’s internal language of DNA and RNA to cure disease and repair injuries to the body. We now appreciate the complexity of signalling through the genome and its transcribed RNA. The role of micro RNAs and short interfering RNAs are gaining much interest as potential therapeutics. This interest has been sparked by the discovery that the dysregulation of micro RNAs is the origin for a spectrum of diseases from cancer through to osteoporosis. Small regulatory RNAs have been shown to influence stem cell maintenance, proliferation and differentiation, offering the potential to produce new tissue by manipulating RNA levels. However delivery of these molecules is fraught with difficulties. Without protection these molecules are quickly degraded in vivo and in vitro before reaching their intended target. With this in mind, this thesis aims to investigate the potential role for gold nanoparticles to deliver small regulatory RNAs and in turn produce a non-toxic and physiologically significant effect upon the cells. Initial investigations revealed the importance of PEG density and AuNP concentration; with lower PEG densities, allowing attached therapeutic siRNA against C-Myc to reduce C-Myc protein levels and cell proliferation. Subsequently we determined that modulating the expression of osteo-suppressive miRNA, with a nucleic antagonist sequence was able to influence osteogenesis in two cell models (MG63s and hMSCs). This thesis has shown that AuNPs can be used to effectively deliver therapeutically active small molecules to cells in vitro.

Background: From personal experience, plus a brief overview of literature, the researcher surmised that the potentially traumatic impact on patients of losing their analyst to terminal illness/sudden death had received scant attention from the psychoanalytic community. Listening directly to patients (as opposed to analysts) had been particularly overlooked. Literature Review: This initial hypothesis was confirmed/refined by this Review: it was felt that focusing on the account that patients gave of their experience would be the most appropriate means of eliciting information. Knowledge that would assist the psychoanalytic community to most effectively ensure the wellbeing of future patients. Methodology: Interpretative Phenomenological Analysis (IPA) seemed to most appropriately meet the aims of this research. 14 semi-structured interviews were conducted, allowing the flexibility in questioning that is an inherent characteristic of IPA. For consistency collecting, transcribing and analysing of data were undertaken by the researcher. Accepted ethical procedures were followed. Findings and Discussion: Five Superordinate Themes emerged based on 21 Major Themes – Changes in Analysis before Termination; Aftercare in Relation to Outcomes; The Inherent Nature of Analysis inc. Requirements of Analytic Training; Emotional/Psychic Effects; Experience Utilised. Within these 5 Superordinate Themes issues/dilemmas were identified that were detrimentally affecting, in some cases seriously, patients’ wellbeing. The majority of these issues had been identified, in some form, over the past 50 years but not adequately acknowledged/acted upon. Some new issues emerged, including problems that are occurring in the interface between formal executors and sick analysts. Suggestions were given that might be helpful for the profession to take forward. Conclusions: The researcher has separated out the variables that appear to affect the outcome for patients into those that are largely ‘fixed’ as opposed to those that are ‘more malleable’ and urges the psychoanalytic community to act speedily on the latter.

Cell survival is modulated by a cocktail of ion channels engaging cell life and death decisions through controlling key cellular messages such as apoptosis and proliferation. Unnatural regulation of these processes results in various disorders, for example neurodegenerative diseases, as well as the cancers. Nowadays, these pathologies are affecting millions of people per year in the world. Potassium (K+) ion channels appear to play a potent role in such illnesses since they can control many cellular gates in cell physiology such as ionic homeostasis and signalling cascades. Amongst the K+ channels, small (SK1-3) and intermediate (SK4) conductance Ca2+-activated potassium ion channels have recently been shown to save cells, thereby protecting mitochondrial function which serves as a cell survival platform. In the case of other ion channels, for instance transient receptor potential melastatin 7 (TRPM7), it is also repeatedly stated that such membrane channels shows an impressive and differential role in excitable and non-excitable cell survival. This channel also modulates ionic homeostasis of crucial ions in cellular physiology such as Ca2+. This study reveals that central nervous system (CNS) and breast cancer cells differentially express SK1-4 ion channel subtypes, and their functional presence is pharmacologically confirmed, however, in most cases these results were further clarified through small interference RNA (siRNA) method. Similarly, functional TRPM7 channel expression in CNS cells is also confirmed. In the CNS, SK1-4 channel activation rescues neurons from oxidative stress, whereas, TRPM7 channel inhibition protects CNS cells from this hydrogen peroxide (H2O2) harmful effect, as well as hypoxia and apoptosis, so improving cell survival. Excitingly, SK1-4 channels differentially exist between wild-type and Huntington’s affected mouse striatal cells, where diseased cells lack SK1-3 channels, key players in action potential activity. Interestingly, SK2 or SK3 channel subtypes are also functionally expressed in breast cancer cells with various phenotypes. This study established that these ion channels are powerful agents in a survival role, in fact controlling growth through cross-talk with an apoptotic avenue “intrinsic pathway”. SK2 or SK3 channel activation enhances cell viability, while its inhibition dampens cell growth. It is very noteworthy that SK2 and SK3 channels are not expressed in non-tumorigenic breast cells. In brief, SK1-4 and TRPM7 molecules are clearly implicated in the survival of diverse cell types through an apoptotic route, indicating that these ionic regulators are promising targets in channelopathies related to cellular degeneration and growth.

Adhesion as a scientific phenomenon has been researched for the past 70 years, as the notion of two entities contacting effects a huge expanse of daily activities, from writing to sophisticated cellular and bacterial interactions essential for growth and survival. Inherently, a robust and adequate model of adhesion was acquired, one in which biological aspects were considered. Initially, the methodology required was optimised using the atomic force microscope (AFM) by testing a model bone substrate against ultra-high molecular weight polyethylene (UHMWPE), a material commonly found in the articulating acetabular cup. Once a force mapping technique was established experimentation continued to bacterial adhesion against model bone samples of various roughness, establishing that the adhesion phenomena occurs at a scale dependency due to the alterations in the topography of the surface at the micro to nano level. Aseptic loosening and osteolysis are major causes of failures in implanted biomedical devices at the hip. These issues are governed by the deterioration of the moving components, producing particles known as wear debris associated with the metals, bone cement, and UHMWPE materials initiating an immune response which is detrimental to the surrounding cells and tissues adjacent to the implant. The notion of mechanical aspects altering the health of mammalian cells has been ignored throughout the research of implantations and their effect on the cells by foreign bodies; the only concept studied to date is the viability and functionality post exposure. Therefore, this thesis aims at observing ii mesenchymal and osteoblast (both rodent and human) cells associated to wear debris (metal and polymeric particles of various sizes and compositions) exposure and the effect this has on cell nanomechanical and adhesive properties using the AFM techniques. The data obtained indicated that Cobalt nanoparticles were more damaging on all cell types than Titanium and polymeric particles.

Skeletal muscle atrophy is characterised by a loss of muscle weight and volume involving a reduction in muscle fibre diameter in the absence of degenerative changes and/or a reduction in the actual number of fibres. In humans there are a wide range of stimuli for skeletal muscle atrophy including bed rest, extreme training, malnutrition and cancer. The underlying cellular and molecular mechanisms governing the process of atrophy are beginning to be elucidated. However, there remains much to be learnt to allow the development of rational therapeutic interventions. A miniature neuromuscular stimulator was used to impose artificial levels of activity on the rat Tibialis anterior muscle in vivo. Continuous electrical stimulation at a frequency of 20 Hz for 7 days resulted in a 12% (+/- 2%) decrease in muscle weight. Foxo1 is known to play a central role in skeletal muscle atrophy. Therefore transcriptional changes in 12 Foxo1 target genes were measured following artificial in vivo stimulation and compared with changes in the same genes in published in vitro models of muscle atrophy. The standard in-vitro models used were treatment of C2C12 myotubes for 24 hours with glucose free media, giving a 56% (+/- 2%) decrease in myotube diameter, or 24 hours treatment with 1μM dexamethasone (Dex), which we found produced no significant change in myotube diameter. Marked transcriptional differences were observed between in vivo and in vitro atrophy models. These differences highlighted the need to develop a model based on muscle inactivity that more clearly reflected muscle behaviour in vivo. A tetrodotoxin (TTX) nerve cuff was used to block all efferent impulse activity in the common peroneal nerve, and therefore to induce progressive disuse atrophy in the dorsiflexors over a period of 14 days. This resulted in a maximal 51% (+/-1%) loss in mass of the treated Tibialis anterior muscle compared to that in the untreated control limb. This atrophy was achieved in the absence of any histological signs of damage or degeneration of the muscle fibres. It was therefore possible to block muscle activity for 14 days, and then reverse the blockade, allowing the same muscle fibres to recover without detriment over the subsequent 7 days. Microarray analysis was used to compare genome-wide transcript changes following 3, 7 and 14 days of nerve blockade, 14 days of nerve block with 7 days of recovery, or 7 days electrical stimulation at 20Hz. Following quality control and normalisation of data(n=4), systematic bioinformatics analysis including GO-term enrichment revealed key signalling pathways involved in the process of disuse atrophy. A working hypothesis was developed which centres on the transcriptional control of myogenin (Myog) through a sensory role of the neuromuscular junction. This involves both class II histone deacetylases (Hdac4/5) and the janus kinase and signal transducer and activator of transcription (Jak/Stat) pathway, and influences potential downstream Myog targets including the ubiquitin E3 ligases Trim63 and Fbxo32, as well as genes involved in neuromuscular junction formation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to confirm the temporal profiles for expression of key genes central to this model. These included a 33-fold increase in Myog mRNA after 3 days nerve block, and a 180-fold increase in transcript for the alpha subunit of the nicotinic acetylcholine receptor after 14 days of nerve blockade. To establish whether our findings were transferable to atrophy induced by other mechanisms, and to examine potential chemical interventions, we returned to the C2C12 in vitro model. The transcript levels of the same key genes were measured in myotubes following treatment with glucose-free media as well as media containing chemical inhibitors of the signalling pathways central to our hypothesis: the class II Hdac inhibitor MC1568, the Stat3 inhibitor S3I-201, and in addition to the Class I Hdac inhibitor MGCD0103. Differences were again observed in the patterns of change in transcript levels between in vivo and in vitro treatments as well as between in vitro treatment groups. The atrophy associated with glucose starvation was a 56% (+/- 2.%) reduction in myotube diameter relative to cells grown in normal media. While MC1568 and S3I-201 treatment reduced this in vitro atrophy by a small amount (to 42% +/- 4% and 21% +/- 6%, respectively, relative to cells grown in normal media), surprisingly the class I Hdac inhibitor MGCD0103 markedly blunted starvation atrophy with a decrease in myotube diameter of only 3% (+/- 7%). The discovery of therapeutic targets that could alleviate skeletal muscle atrophy is an exciting and potentially productive avenue of research. Skeletal muscle atrophy has wide ranging causes and debilitating consequences for patients. This project represents a starting point in the systematic development of drugs that could benefit these patients by improving quality of life and decreasing morbidity.

Adverse drug reactions remain a major health issue with delayed type hypersensitivity reactions developing in a high number of individuals. The cellular immunological processes that underlie drug-specific responses in hypersensitive patients have been previously described; however the involvement of the humoral immune system has not been studied in great detail. Consequently, this thesis explores the nature of the piperacillin-specific B cell response in hypersensitive patients and compares the cellular and humoral immune response that develops in patients with cystic fibrosis (CF) exposed to repeated courses of the drug. Initial studies involved characterization of B cell proliferation, B cell phenotype and the nature of total and drug-specific IgG antibody secretions using peripheral blood mononuclear cells (PBMC) from hypersensitive patients. For comparison PBMCs from 2 groups of individuals were assessed: piperacillin naïve healthy volunteers and piperacillin tolerant patients with CF. ELISA, and ELISpot were used to detect piperacillin-specific B cells responses and IgG secretion. T lymphocyte proliferation was assessed with the lymphocyte transformation test (LTT). T lymphocytes from hypersensitive patients, but not tolerant patients or naïve donors were stimulated to proliferate in the presence of the drug. The peak concentration for T cell activation was 1 mM. Phenotypic assessment of hypersensitive patients B-cells revealed an increase in CD19+CD27+ expression in response to piperacillin treatment in vitro. IgG secreting immortalized B-cell lines also expressed a pure CD19+CD27+ phenotype. Piperacillin stimulation of hypersensitive patient PBMC also led to an increase in the secretion of IgG. In contrast, IgG secretion was not detectable following piperacillin stimulation of PBMC from tolerant patients and healthy controls. Western blotting and mass spectrometric methods were applied to characterize -lactam-protein covalent binding. Bovine serum albumin (BSA) binding was time- and concentration-dependent with hapten densities (i.e., the extent of selective lysine residue modification) and anti-piperacillin antibody binding affinity increasing with increasing molar ratios. Lysine residues in BSA at positions 4, 12, 131, 132, 136, 211, 431, 524, and 537 were modified by piperacillin. Epitope profiles also showed similar lysine residues were modified with amoxicillin, benzylpenicillin and flucloxacillin though the extent of ionisation at each site of modification was drug-dependent. A hapten inhibition ELISA used to assess the specificity of the antidrug antibodies revealed the total antibody binding to aztreonam, amoxycillin, benzylpenicillin and penicillin V BSA adducts. This indicates a lack of cross-reactivity with piperacillin-specific IgG antibodies. Subsequently, LTT and ELISA were employed to screen the piperacillin-specific T cell response and IgG antibodies during piperacillin therapy. It was established that piperacillin-specific T cells were detectable on and following clinical diagnosis of hypersensitivity. Moreover, piperacillin-specific T cell responses were detected in a small number of patients currently classified as drug tolerant. A significant difference in piperacillin-specific IgG was observed when plasma form LTT positive and negative blood samples were compared. LTT positivity was associated with higher levels of piperacillin-specific IgG. Furthermore, a significant decrease in piperacillin-specific IgG was seen 24 h post-desensitisation (graded drug challenge). Piperacillin-specific T cell clones isolated from hypersensitive patients were used to explore the effect of plasma bearing anti-piperacillin IgG on the T cell response. Eleven piperacillin-specific CD4+ and CD8+ T-cell clones were generated from 2 hypersensitive patients. All clones were stimulated to proliferate with piperacillin in a concentration-dependent manner. IFN-γ and IL-5 secretion was seen to predominate following piperacillin stimulation. There were no differences in piperacillin-specific T-cell proliferation when piperacillin-specific antibody bearing plasma and plasma from naive volunteers were compared. However, attenuation in IFN-γ secretion was observed with plasma bearing anti-piperacillin antibodies alone. Collectively, the data presented in this thesis begins to describe the different components of the drug-specific humoral and cellular immune response that develops in piperacillin hypersensitive patients with CF.