No description available.

Clays and clay minerals (phyllosilicates) have been used for millennia to treat a range of human maladies, such as infected wounds and diseases of the skin. The unique chemistry of phyllosilicates allows them to support the wound environment and encourage healing. Their physicochemical properties can also be utilised to develop modified drug-release formulations and also enables their incorporation into polymer matrices for the development of advanced wound care materials. By developing novel antibacterial phyllosilicate-polymer composite materials it should be possible to support wound healing, whilst simultaneously treating infections locally to avoid systemic adverse effects and prevent the development of antimicrobial resistance. In this research project the clay minerals kaolin (KN), refined montmorillonite (rMMT), montmorilonite K10 (MMTK10), Laponite® RD (LRD), and Laponite® XL21 (LXL21) were investigated for their differing structure and physicochemical properties. Their ability to adsorb and desorb the antibacterial agents tetracycline (TC), doxycycline (DC) and ciprofloxacin (CIP) was determined through a series of adsorption kinetics and isotherm studies. LRD and LXL21 were shown to have the highest drug-carrying capacity and were also able to relinquish this drug-load to inhibit the proliferation of key wound pathogens; Staphylococcus epidermidis, Propionibacterium acnes, and Pseudomonas aeruginosa. XRD and FTIR analyses demonstrated that these drug molecules could be adsorbed into the interlayer space and edge groups of the Laponite® particles. LXL21-CIP composites were successfully incorporated into alginate polymer matrices through interaction of the exposed edge-groups on LXL21 and the hydroxyl groups of the alginate to produce novel nanocomposite film and foam materials. Selection of candidate materials was initially undertaken qualitatively with the support of a tissue viability nurse at the Royal Liverpool and Broadgreen University Hospitals NHS Trust. Important properties for wound-dressings such as adsorptive capacity, water vapour transmission rate, and keratinocyte compatibility were measured quantitatively and compared to materials already used for wound care in the UK. Both the film and foam materials were shown to have properties that would be beneficial for wound healing and were also able to release CIP in a controlled manner with notable activity against S. epidermidis, P. acnes, and P. aeruginosa. The nanocomposite film formulation developed in this research project showed promise for future clinical applications and future work should be undertaken to further optimise their manufacture and fully characterise their ability to support the healing of infected wounds. Although the nanocomposite foams require further research, the work presented in this thesis suggests they could also be promising materials for wound care applications.

No description available.

Synthetic cannabinoid receptor agonists or more commonly known as synthetic cannabinoids (SCs) were originally created to obtain the medicinal value of THC but they are an emerging social problem. SCs are mostly produced coated on herbal materials or in powder form and marketed under a variety of brand names, e.g. “Spice”, “K2”. Despite many SCs becoming controlled under drug legislation, many of them remain legal in some countries around the world. In Scotland, SCs are controlled under the Misuse of Drugs Act 1971 and Psychoactive Substances Act 2016 that only cover a few early SCs. In Saudi Arabia, even fewer are controlled. The picture of the SCs-problem in Scotland is vague due to insufficient prevalence data, particularly that using biological samples. Whilst there is evidence of increasing use of SCs throughout the world, in Saudi Arabia, there is currently no data regarding the use of products containing SCs among Saudi people. Several studies indicate that SCs may cause serious toxicity and impairment to health therefore it is important to understand the scale of use within society. A simple and sensitive method was developed for the simultaneous analysis of 10 parent SCs (JWH-018, JWH-073, JWH-250, JWH-200, AM-1248, UR-144, A-796260, AB-FUBINACA, 5F-AKB-48 and 5F-PB-22) in whole blood and 8 corresponding metabolites (JWH-018 4-OH pentyl, JWH-073 3-OH butyl, JWH-250 4-OH pentyl, AM-2201 4-OH pentyl, JWH-122 5-OH pentyl, JWH-210 5-OH pentyl, 5F-AKB-48 (N-4 OH pentyl), 5F-PB-22 3-carboxyindole)in urine using LLE and LC-MS/MS. The method was validated according to the standard practices for method validation in forensic toxicology (SWGTOX, May 2013). All analytes gave acceptable precision, linearity and recovery for analysing blood and urine samples. The method was applied to 1,496 biological samples, a mixture of whole blood and urine. Blood and/or urine samples were analysed from 114 patients presenting at Accident and Emergency in Glasgow Royal Infirmary, in spring 2014 and JuneDecember 2015. 5F-AKB-48, 5F-PB-22 and MDMB-CHMICA were detected in 9, 7 and 9 cases respectively. 904 urine samples from individuals admitted to/liberated from Scottish prisons over November 2013 were tested for the presence of SCs. 5F-AKB-48 (N-4 OH pentyl) was detected in 10 cases and 5F-PB-22 3-carboxyindole in 3 cases. Blood and urine samples from two post-mortem cases in Scotland with suspected ingestion of SCs were analysed. Both cases were confirmed positive for 5F-AKB-48. A total of 463 urine samples were collected from personnel who presented to the Security Forces Hospital in Ryiadh for workplace drug testing as a requirement for their job during July 2014. The results of the analysis found 2 samples to be positive for 5F-PB-22 3carboxyindole. A further study in Saudi Arabia using a questionnaire was carried out among 3 subpopulations: medical professionals, members of the public in and around smoking cafes and known drug users. With regards to general awareness of Spice products, 16%, 11% and 22% of those participants of medical professionals, members of the public in and around smoking cafes and known drug users, respectively, were aware of the existence of SCs or Spice products. The respondents had an overall average of 4.5% who had a friend who used these Spice products. It is clear from the results obtained in both blood and urine testing and surveys that SCs are being used in both Scotland and Saudi Arabia. The extent of their use is not clear and the data presented here is an initial look into their prevalence. Blood and urine findings suggest changing trends in SC use, moving away from JWH and AM SCs to the newer 5F-AKB-48, 5-F-PB-22 and MDMBCHMICA compounds worldwide. In both countries 5F-PB-22 was detected. These findings clarify how the SCs phenomenon is a worldwide problem and how the information of every country regarding what SCs are seized can help and is not specific for that country. The analytes included in the method were selected due to their apparent availability in both countries, however it is possible that some newer analytes have been used and these would not have been detected. For this reason it is important that methods for testing SCs are updated regularly and evolve with the ever-changing availability of these drugs worldwide. In addition, there is little published literature regarding the concentrations of these drugs found in blood and urine samples and this work goes some way towards understanding these.

Hypertension is a major risk factor for cardiovascular disease and mortality, and a growing global public health concern, with up to one-third of the world’s population affected. Despite the vast amount of evidence for the benefits of blood pressure (BP) lowering accumulated to date, elevated BP is still the leading risk factor for disease and disability worldwide. It is well established that hypertension and BP are common complex traits, where multiple genetic and environmental factors contribute to BP variation. Furthermore, family and twin studies confirmed the genetic component of BP, with a heritability estimate in the range of 30-50%. Contemporary genomic tools enabling the genotyping of millions of genetic variants across the human genome in an efficient, reliable, and cost-effective manner, has transformed hypertension genetics research. This is accompanied by the presence of international consortia that have offered unprecedentedly large sample sizes for genome-wide association studies (GWASs). While GWAS for hypertension and BP have identified more than 60 loci, variants in these loci are associated with modest effects on BP and in aggregate can explain less than 3% of the variance in BP. The aims of this thesis are to study the genetic and environmental factors that influence BP and hypertension traits in the Scottish population, by performing several genetic epidemiological analyses. In the first part of this thesis, it aims to study the burden of hypertension in the Scottish population, along with assessing the familial aggregation and heritialbity of BP and hypertension traits. In the second part, it aims to validate the association of common SNPs reported in the large GWAS and to estimate the variance explained by these variants. In this thesis, comprehensive genetic epidemiology analyses were performed on Generation Scotland: Scottish Family Health Study (GS:SFHS), one of the largest population-based family design studies. The availability of clinical, biological samples, self-reported information, and medical records for study participants has allowed several assessments to be performed to evaluate factors that influence BP variation in the Scottish population. Of the 20,753 subjects genotyped in the study, a total of 18,470 individuals (grouped into 7,025 extended families) passed the stringent quality control (QC) criteria and were available for all subsequent analysis. Based on the BP-lowering treatment exposure sources, subjects were further classified into two groups. First, subjects with both a self-reported medications (SRMs) history and electronic-prescription records (EPRs; n =12,347); second, all the subjects with at least one medication history source (n =18,470). In the first group, the analysis showed a good concordance between SRMs and EPRs (kappa =71%), indicating that SRMs can be used as a surrogate to assess the exposure to BP-lowering medication in GS:SFHS participants. Although both sources suffer from some limitations, SRMs can be considered the best available source to estimate the drug exposure history in those without EPRs. The prevalence of hypertension was 40.8% with higher prevalence in men (46.3%) compared to women (35.8%). The prevalence of awareness, treatment and controlled hypertension as defined by the study definition were 25.3%, 31.2%, and 54.3%, respectively. These findings are lower than similar reported studies in other populations, with the exception of controlled hypertension prevalence, which can be considered better than other populations. Odds of hypertension were higher in men, obese or overweight individuals, people with a parental history of hypertension, and those living in the most deprived area of Scotland. On the other hand, deprivation was associated with higher odds of treatment, awareness and controlled hypertension, suggesting that people living in the most deprived area may have been receiving better quality of care, or have higher comorbidity levels requiring greater engagement with doctors. These findings highlight the need for further work to improve hypertension management in Scotland. The family design of GS:SFHS has allowed family-based analysis to be performed to assess the familial aggregation and heritability of BP and hypertension traits. The familial correlation of BP traits ranged from 0.07 to 0.20, and from 0.18 to 0.34 for parent-offspring pairs and sibling pairs, respectively. A higher correlation of BP traits was observed among first-degree relatives than other types of relative pairs. A variance-component model that was adjusted for sex, body mass index (BMI), age, and age-squared was used to estimate heritability of BP traits, which ranged from 24% to 32% with pulse pressure (PP) having the lowest estimates. The genetic correlation between BP traits showed a high correlation between systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP) (G: 81% to 94%), but lower correlations with PP (G: 22% to 78%). The sibling recurrence risk ratio (λS) for hypertension and treatment were calculated as 1.60 and 2.04 respectively. These findings confirm the genetic components of BP traits in GS:SFHS, and justify further work to investigate genetic determinants of BP. Genetic variants reported in the recent large GWAS of BP traits were selected for genotyping in GS:SFHS using a custom designed TaqMan® OpenArray®. The genotyping plate included 44 single nucleotide polymorphisms (SNPs) that have been previously reported to be associated with BP or hypertension at genome-wide significance level. A linear mixed model that is adjusted for age, age-squared, sex, and BMI was used to test for the association between the genetic variants and BP traits. Of the 43 variants that passed the QC, 11 variants showed statistically significant association with at least one BP trait. The phenotypic variance explained by these variant for the four BP traits were 1.4%, 1.5%, 1.6%, and 0.8% for SBP, DBP, MAP, and PP, respectively. The association of genetic risk score (GRS) that were constructed from selected variants has showed a positive association with BP level and hypertension prevalence, with an average effect of one mmHg increase with each 0.80 unit increases in the GRS across the different BP traits. The impact of BP-lowering medication on the genetic association study for BP traits has been established, with typical practice of adding a fixed value (i.e. 15/10 mmHg) to the measured BP values to adjust for BP treatment. Using the subset of participants with the two treatment exposure sources (i.e. SRMs and EPRs), the influence of using either source to justify the addition of fixed values in SNP association signal was analysed. BP phenotypes derived from EPRs were considered the true phenotypes, and those derived from SRMs were considered less accurate, with some phenotypic noise. Comparing SNPs association signals between the four BP traits in the two model derived from the different adjustments showed that MAP was the least impacted by the phenotypic noise. This was suggested by identifying the same overlapped significant SNPs for the two models in the case of MAP, while other BP traits had some discrepancy between the two sources.

C-X-C motif receptor 2 (CXCR2) is a G-protein coupled receptor normally expressed on granulocytes, in particular CD11b +, Gr1+, Ly6G+ bone marrow derived suppressor cells (BMDCs) and once differentiated neutrophils.

Nitric oxide (NO) is a critical mediator of a variety of biological functions, including vascular and muscle relaxation, platelet aggregation, neuronal-cell function, microbicidal and tumoricidal activity, and a range of immunopathologies. NO is derived from L-arginine and molecular oxygen in a reaction catalysed by NO synthase (NOS). Three isoforms of NOS have been identified; neuronal constitutive NOS (ncNOS), endothelial constitutive NOS (ecNOS), and inducible NOS (iNOS). ncNOS and ecNOS are calcium-dependent, present constitutively in a variety of tissues and produce physiological concentrations of NO. However, large amounts NO are produced by iNOS which are expressed in cells such as macrophage after stimulation with a number of cytokines, including interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), and bacterial lipopolysaccharide (LPS). Findings of iNOS biological functions are based mainly on experiments using L-arginine analogues such as L-NG monomethyl arginine (L-NMMA) which competitively inhibits NO Synthase. These inhibitors are not NOS isoform selective and have differential bioavailability, and hence often render interpretation of results difficult. To directly define the iNOS biological functions, I constructed a strain of iNOS gene targeted mice. The first step of a gene targeting experiment is typically cDNA cloning and sequencing. A cDNA library was constructed in the vector λ ZAPII using mRNA isolated from J774 macrophages activated with IFN-? and LPS. Six independent positive colonies were found in the screen with both 5' and 3' specific probes and were subcloned in pBluescript. A full-length iNOS cDNA was constructed using the clones isolated from the library. Double strand cDNA sequence analysis showed that the J774 iNOS clone was identical to that of the Raw 264.7 macrophages iNOS cDNA sequence. A replacement-type targeting construct was prepared from 129/sv genomic DNA. A single targeted clone was identified amongst 636 screened after two independent electroporations of the CGR8 embryonic stem cell line. Gene replacement was detected by both 5' and 3' specific external probes. Five of ten chimeras generated gave germ line transmission. No homozygous mice were found by Southern blot analysis with 5' and 3' external probe in the offspring from heterozygous mice (FI) breedings. The iNOS gene had been altered by replacement with gene targeting construct and 5' iNOS gene translocation which could be detected by internal probe Southern blot analysis. Using the internal probe, mutant homozygous, heterozygous and wild-type mice were found in the offspring from heterozygous breedings. The ratio of these three genotypes was 21:47:25. Northern blot analysis with 5' iNOS cDNA probe showed that a large messenger appeared in the macrophages of homozygous mice when the cells were activated with IFN-γ and LPS in vitro. However there was no detectable iNOS protein translation by western blot analysis using monoclonal or polyclonal anti-iNOS antibodies.

It is well documented that the perinatal period (the time from pregnancy until the first year of a child’s life) is a crucial window of opportunity for foetal and infant development. Evidence suggests that parental psychopathology can have harmful consequences for the parent-infant relationship, parents’ relationship, infant’s development and later mental-health into adulthood. The focus of this thesis will be on exploring mindfulness as one possible non-pharmacological intervention that could benefit parents’ emotional well-being during the perinatal period. Chapter one contains a mixed methods systematic review of 14 published studies of Mindfulness and Acceptance Based Interventions (MABIs) during the perinatal period, for mothers experiencing anxiety and stress. The synthesis of quantitative and qualitative results were integrated and demonstrated tentative findings that MABIs can reduce anxiety, pregnancy anxiety and stress for mothers, whilst increasing self-efficacy and mindfulness. Qualitative themes discussed propose possible explanations for these findings. Methodological limitations are discussed in line with implications for clinical practice and recommendations for future research. Chapter two explores mothers’ and fathers’ experiences of mindfulness during parenting, one year following their attendance on the Mindfulness-Based Childbirth and Parenting programme (MBCP). Ten semi-structured interviews gathered rich, in-depth, idiosyncratic accounts which were subject to Interpretative Phenomenological Analysis (IPA). Two superordinate themes emerged from the data, ‘Getting closer to really living’ and ‘Greater connectedness and attunement’. Finally, Chapter 3 provides a reflective account of the author’s lived experience of the parallels between her own research journey and personal experience of becoming a mother. It addresses the author’s epistemological position and the researcher’s influence on the research process.

Telomeres are DNA-protein complexes which cap the ends of eukaryotic linear chromosomes. In normal somatic cells telomeres shorten and become dysfunctional during ageing due to the DNA end replication problem. This leads to activation of signalling pathways that lead to cellular senescence and apoptosis. However, cancer cells typically bypass this barrier to immortalisation in order to proliferate indefinitely. Therefore enhancing our understanding of telomere dysfunction and pathways involved in regulation of the process is essential. However, the pathways involved are highly complex and involve interaction between a wide range of biological processes. Therefore understanding how telomerase dysfunction is regulated is a challenging task and requires a systems biology approach. In this study I have developed a novel methodology for visualisation and analysis of gene lists focusing on the network level rather than individual or small lists of genes. Application of this methodology to an expression data set and a gene methylation data set allowed me to enhance my understanding of the biology underlying a senescence inducing drug and the process of immortalisation respectively. I then used the methodology to compare the effect of genetic background on induction of telomere uncapping. Telomere uncapping was induced in HCT116 WT, p21-/- and p53-/- cells using a viral vector expressing a mutant variant of hTR, the telomerase RNA template. p21-/- cells showed enhanced sensitivity to telomere uncapping. Analysis of a candidate pathway, Mismatch Repair, revealed a role for the process in response to telomere uncapping and that induction of the pathway was p21 dependent. The methodology was then applied to analysis of the telomerase inhibitor GRN163L and synergistic effects of hypoglycaemia with this drug. HCT116 cells were resistant to GRN163L treatment. However, under hypoglycaemic conditions the dose required for ablation of telomerase activity was reduced significantly and telomere shortening was enhanced. Overall this new methodology has allowed our group and collaborators to identify new biology and improve our understanding of processes regulating telomere dysfunction.

Muscarinic acetylcholine receptors (M1-M5) belong to the class A family of transmembrane G protein coupled receptors (GPCRs) and mediate various signalling processes. M1, M3 and M5 predominantly couple to Gq and promote intracellular calcium ion release from the endoplasmic reticulum. M2 and M4 preferentially couple Gi inhibiting adenylyl cyclase activity to thus decrease cAMP production and acting to regulate various ion channels. There is growing evidence that many GPCRs can exist as dimers or higher-order oligomers (Milligan, 2013) and muscarinic receptors are no exception (Alvarez-Curto et al., 2010). Herein, combinations of homomers and heteromers of co-expressed human M2 (hM2WT) and a RASSL (Receptor Activated Solely by Synthetic Ligand) form of the human M3 receptor (hM3RASSL) (Alvarez-Curto et al., 2011) were demonstrated to occur using N-terminal SNAP and CLIP tags in combination with homogeneous time resolved FRET (htrFRET). Stable Flp-In™ T-REx™ 293 cell lines able to inducibly express each of these receptor forms upon addition of doxycycline, and a cell line able to express both the hM3RASSL constitutively and hM2WT in a doxycycline inducible manner were generated. In these cells both hM3RASSL and hM2WT were detected after treatment with different concentrations of doxycycline via Western blots using tag-specific antibodies. Radioligand binding using [3H]-QNB indicated that similar amounts of hM2WT and hM3RASSL were expressed following induction with 5 ng.ml-1 doxycycline in the cells co-expressing the two receptors. Expression of the receptors was observed at the surface of live cells following labelling of the expressed receptors with SNAP and CLIP-specific cell impermeant substrates. Following induction with doxycycline each of hM2WT and hM3RASSL homomers and hM2WT-hM3RASSL heteromers were identified. Detection of oligomers was achieved following co-labelling with htrFRET-compatible substrates. Occupancy of hM2WT-hM3RASSL heteromers with the hM2WT agonist carbachol resulted in a marked, time and concentration-dependent decrease in detected heteromers and a concomitant, concentration-dependent increase in hM2WT homomers. The dynamics of interchange between heteromers and homomers was investigated by using a multiplex labelling approach and htrFRET. This method involved labelling with one energy donor and two energy acceptors capable of emitting at distinct wavelengths. Results confirmed the hM2WT-hM3RASSL heteromer to hM2WT homomer transition upon selective carbachol-mediated activation of hM2WT. A small increase in the hM3RASSL homomer was detected upon activation of the hM3RASSL with the selective agonist clozapine-N-oxide, but this was only observed in the absence of heteromers. Despite the presence of hM2WT-hM3RASSL heteromers the functional pharmacology of hM2WT and hM3RASSL receptor specific agonists was largely unaltered.