CYP24A1 (25-hydroxyvitamin D-24-hydroxylase) is a useful enzyme target for a range of medical conditions including cancer, cardiovascular and autoimmune disease, which show elevated CYP24A1 levels and corresponding reduction of calcitriol (the biologically active form of vitamin D). Calcitriol has antiproliferative and pro-differentiating properties, however use of calcitriol as a therapeutic drug is limited by hypercalcaemia. An alternative approach is the use of CYP24A1 inhibitors to prevent the metabolism of calcitriol. The aim of this research is to design and synthesise novel inhibitors of CYP24A1 to enhance the endogenous levels of circulating calcitriol. Furthermore, it is important to develop compounds that are selective for CYP24A1 over CYP27B1 so that the generation of calcitriol itself is not blocked. In order to understand the requirements of inhibitor binding to the enzyme-active site, it would be useful to have a 3D structure of both human CYP24A1 and CYP27B1. However, to date, no human crystal structures are available for either of these enzymes. Therefore, a homology model for CYP24A1 has been developed and published. A CYP27B1 homology model was developed, using a combination of homology modelling, molecular dynamics simulations, and molecular docking to understand the satisfactory explanation of the binding selectivity of the CYP27B1 model with the natural substrate and with selective inhibitor complexes. Docking results for CYP27B1 showed amino acids Arg107, Asn387 and Asp320 have an important role in binding interactions to form hydrogen bonds with inhibitors. The development of potent and selective inhibitors from three azole series was investigated. Development of series one using pyridine, imidazole and triazole as the haem binding group was synthesised successfully. The compounds exhibited weak potency and IC50 ranging between 10.2 to 28.4 μM against CYP24A1. Owing to the low CYP24A1 inhibitory activity the compounds were not evaluated against CYP27B1. The series two bis(3-methyl-1-phenyl-1H-pyrazol-5ol) was synthesised successfully. Two compounds were the moderate CYP24A1 inhibitors and so were further evaluated against CYP27B1. However, these compounds showed enzymatic inhibition (IC50 = 0.57 μM and 0.41 μM) against CYP27B1, that is they were more selective for CYP27B1 which could be rationalised from docking experiments. A series of (E)-N-(2-(1H-imidazol-1- yl)-2-(phenylethyl)-3/4-styrylbenzamides have been synthesised using an efficient synthetic route and shown to be potent inhibitors of CYP24A1 (IC50 0.11 - 0.35 μM) compared with the standard ketoconazole. Molecular modelling using our CYP24A1 homology model showed the inhibitors to fill the hydrophobic binding site, forming key transition metal interaction between the imidazole nitrogen and the haem Fe3+ and multiple interactions with the active site amino acid residues.

The drive to increase interprofessional teamwork in the healthcare environment has gained significant traction in recent years. This has partly been as a consequence of UK inquiries that have cited breakdowns in communication and teamwork as contributory factors leading to poor patient outcomes. One method to prepare practitioners for interprofessional teamworking is interprofessional education (IPE). The General Pharmaceutical Council specifies that IPE must be embedded within UK Master of Pharmacy (MPharm) programmes. However, there is a paucity of literature examining IPE related to pharmacy and limited knowledge of pharmacists' interprofessional interactions with healthcare professionals (HCPs). This makes it challenging for pharmacy educators to design IPE sessions that are reflective of practice. To address this, a mapping process was undertaken to identify IPE sessions that are delivered in UK MPharm programmes (17/29 schools responded). This identified significant variation in IPE sessions delivered in terms of learning outcomes addressed, topics covered, and the range of student HCPs involved. A mixed method study was then undertaken to explore pharmacists' interprofessional interactions in practice. A questionnaire was disseminated to pharmacists in Wales via community pharmacies (61.9% response) and hospital pharmacy departments (estimated 59.1% response). Analysis of returned questionnaires identified that although the extent of interprofessional collaboration varied pharmacists in both sectors most frequently interact with doctors and nurses. Semi-structured interviews were undertaken with pharmacists from both the community (n=14) and hospital (n=15) sectors to explore the nature of interactions. Using deductive and inductive thematic analysis, the nature of pharmacists' interactions with HCPs was elucidated, facilitators and barriers to interactions were determined and suggestions for IPE developed. Findings from these studies resulted in a series of recommendations for pharmacy educators and policy makers to facilitate pharmacists' interprofessional collaboration in practice and aid the development of relevant IPE that is of value to learners.

Birds face many challenges during a breeding attempt including predation, food availability, pathogens and parasite load. Cyanistes caeruleus (blue tit) prefer human placed nest boxes, however the stable microclimate presented within the nest box exacerbates the challenges posed by ectoparasites and potentially pathogenic bacteria, with reductions in breeding success reported. The addition of green plant material to help control these deleterious effects has been reported within Mediterranean climes but comparable studies have not been undertaken in temperate regions. This study introduces novel molecular approaches including next generation sequencing methods to assess the nest microbiome. This approach avoids the culturing bias of previous work in this area. Terminal-Restriction Fragment length Polymorphism (T-RFLP) analysis was used to assess bacterial richness progression through the breeding attempt with more traditional methods used to assess bacterial load. DNA barcoding was performed to identify bacteria, ectoparasites and vascular plant fragments present within the nest with vegetation surveys conducted around a subset of nests to assess if any active selection of plant material was occurring. Bacterial species richness and load were relatively stable between nest build and clutch completion with a significant increase in both post fledging, following the introduction of nestling faeces in the nest and reduced time for nest sanitation. DNA barcoding provided marked increases in the taxonomic knowledge of nest dwelling biota with 169 bacterial taxa, thirteen species of ectoparasite and 154 vascular plant taxa identified. Although ectoparasites and pathogenic bacteria were detected within the nest no effect was seen upon hatching or fledging success. It is more likely that a reduction in fitness would be observed post fledging. A high proportion of plant material containing volatile compounds was recorded within the nest, however active selection could not be confirmed.

Antiepileptic drugs (AEDs) are prescription only medications which were firstly introduced in the 1880s to treat epilepsy. However, the rapid growth in the drug discovery market led to a new generation of AEDs with multiple mechanisms of action. These new drugs represent a promising treatment for many diseases in addition to epilepsy such as neurological disorders, psychological disorders and substance and alcohol abuse treatment as substitutes for benzodiazepines and methadone. However, their multiple roles triggered their misuse potential and concern on their abuse potential was raised in the literature, the media, and by many addiction organizations. Hence, this research highlights some of the AEDs which have raised concern and discusses their therapeutic effects, mechanism of action as well as their overdose and abuse probability from a forensic toxicology point of view. Some AEDs have a narrow therapeutic index and require therapeutic drug monitoring in order to attain the optimum response. The majority of published analytical methods focuses on their analysis in serum and plasma within therapeutic ranges and includes a maximum of 11 AEDs in one analytical step. Therefore, a robust and accurate method was developed for the simultaneous analysis of 15 common AEDs and two of their major metabolites in whole blood using LC/MS/MS. The method was validated according to the standard practices for method validation in forensic toxicology (SWGTOX, May 2013) over a wide concentration range to include AED therapeutic and toxic concentrations which make it suitable for both clinical and forensic analysis. Stability studies are of great importance in forensic cases where it takes up to a few weeks for autopsy, sampling, drug screening and finally confirmation analysis. However, reports specifically addressing the stability of antiepileptic drugs in whole blood are relatively scarce compared with those for drugs of abuse. Thus, using the previous method, the stability of AEDs in whole blood was investigated under different storage conditions. The LC/MS/MS method developed for AEDs analysis in whole blood was successfully transferred to another laboratory and extended to include 18 AEDs and 4 metabolites. It was revalidated for AEDs analysis in serum and plasma in addition to whole blood. Before any new method can be adapted to routine forensic analysis, it has to be validated using authentic samples. A total of 467 previously processed samples were reanalysed using the transferred method. The results were compared to the reference laboratory's values and these showed a very good correlation. The prevalence of AED abuse, namely gabapentin and pregabalin, was investigated among prisoners. 904 urine samples were collected from 8 prisons in Scotland over a one month period. Firstly, a simple and accurate method was developed and qualitatively validated for 21 AEDs in urine to screen the urine samples. Secondly, the method was quantitatively validated for the positive AEDs. Drug analysis in hair has multiple applications in clinical laboratories and forensic toxicology. However, only a few papers have considered conventional AEDs analysis in hair for therapeutic drug monitoring purposes. As part of this research, AED extraction from hair samples was investigated. Six different digestion methods and 4 clean-up procedures were compared for 16 AEDs. An LC/MS/MS method was qualitatively validated using the extraction procedure that attained the highest recovery with all AEDs. Subsequently, two authentic hair samples were tested and the method was quantitatively validated for the positive AEDs in these samples.

Each cell must replicate and segregate its DNA for the process of cell division. During the replication of the circular chromosome present in Escherichia coli the unwinding of the two parental strands leads to the formation of positive supercoils ahead of the replication fork. If the replication fork is free to rotate this might turn into precatenanes, intertwining of the two newly replicated chromosomes, behind the fork. If not resolved, supercoiling ahead of the fork can stall replication, while precatenation will turn into catenation at the end of replication, and will prevent chromosome segregation. Bacteria have mechanisms for the resolution of these undesired topologies which are performed by type II topoisomerases: Topoisomerase IV (Topo IV) and DNA gyrase. Topo IV is known to resolve precatenanes and catenanes, and gyrase can remove positive supercoiling. Both enzymes act by transporting one double stranded DNA helix through a gap made in a second double stranded segment of DNA. Interestingly, there is no direct evidence for precatenane formation on chromosomal DNA in living cells. Therefore, the aim of this work was to develop a method to detect precatenation in the bacterial chromosome. We tried to answer the following questions: Does precatenation occur on the bacterial chromosome in vivo? Is it Topo IV or DNA gyrase that decatenates in vivo? Are there mechanisms other than Topo IV or DNA gyrase responsible for precatenane and catenane unlinking in E. coli? The method consisted of using site-specific recombination between two recombination sites recognized by ΦC31 integrase. Once the integrase binds to the sites and recombines, the segment between the two sites is excised as a circle. After DNA replication, two circles will be produced, one from each sister chromosome. If the sisters were precatenated, the two circles might be catenated. Therefore, detection of catenanes after recombination occurred would indicate the presence of precatenation on the chromosome. Catenanes were detected as products of site-specific recombination on plasmid DNA in vivo when both type II topoisomerases were inhibited, suggesting that both Topo IV and DNA gyrase can decatenate DNA in vivo. Site-specific recombination was then used to detect precatenation on a plasmid replication intermediate model and on the chromosome. Interestingly, the products of recombination on the replication intermediate model and on the bacterial chromosome were the monomer of the 2.5 kb circle and its dimer. Formation of the dimer suggested that the two sister chromosomes co-localize after replication fork has passed and that might be due to precatenation. However, precatenation was not directly detected. Optimization of the method is required to obtain direct evidence of precatenation. In additional work, the level of supercoiling (Lk-Lk0) of a 398 bp circle produced by Xer recombination between closely spaced psi sites was determined to be -1. This together with previous results allowed the total linkage change of Xer recombination at psi to be determined as +4. Thus, the Xer reaction is driven by loss of four negative supercoils. This result is fully consistent with the Holliday junction model for strand exchange by the Xer recombinases and all other tyrosine recombinases.

Experimenting with novel algorithms and configurations for the automatic management of Cloud Computing infrastructures is expensive and time consuming on real systems. Cloud computing delivers the benefits of using virtualisation techniques to data centers instead of physical servers for customers. However, it is still complex for researchers to test and run their experiments on data center due to the cost for repeating the experiments. To address this, various tools are available to enable simulators, emulators, mathematical models, statistical models and benchmarking. Despite this, there are different methods used by researchers to avoid the difficulty of conducting Cloud Computing research on actual large data centre infrastructure. However, it is still difficult to chose the best tool to evaluate the proposed research. This research focuses on investigating the level of accuracy of existing known simulators in the field of cloud computing. Simulation tools are generally developed for particular experiments, so there is little assurance that using them with different workloads will be reliable. Moreover, a predictive model based on a data set from a realistic data center is delivered as an alternative model of simulators as there is a lack of their sufficient accuracy. So, this work addresses the problem of investigating the accuracy of different modelling tools by developing and validating a procedure based on the performance of a target micro data centre. Key insights and contributions are: Involving three alternative models for Cloud Computing real infrastructure showing the level of accuracy of selected simulation tools. Developing and validating a predictive model based on a Raspberry Pi small scale data centre. The use of predictive model based on Linear Regression and Artificial Neural Net- works models based on training data set drawn from a Raspberry Pi Cloud infrastructure provides better accuracy.

The metastatic spread of cancer cells is a major contributor to cancer patient deaths. In order to disseminate from one part of the body to another, invasive tumour cells must perform a complex cascade of steps. Common to several stages of the metastatic process is the ability of tumour cells to squeeze through narrow spaces. One adaptation that allows cancer cells to adjust to confined environments is a change in the cell mechanical properties, which results in diminished cell stiffness. Such an altered cellular physical property is thought to contribute to the invasive and metastatic properties of cancer cells. There is little currently known about the factors or signalling pathways that modulate cell stiffness; therefore, the identification of factors that modify tumour cell plasticity could identify potential drug targets for anti-metastasis chemotherapy. I hypothesized that within the distributions of parameters in tumour cell line populations, it would be possible to select for cells with an augmented ability to squeeze through narrow gaps, thus highlighting factors that contributed to cell deformability. To identify factors that help cancer cells migrate through confined spaces, MDA MB 231 human breast cancer cells and MDA MB 435 melanoma cells underwent 3 rounds of selection, using tissue culture inserts with 3 μm pores. This selection approached allowed me to isolate cell populations with enhanced abilities to pass through psychical constrictions, as well as augmented invasive abilities in vitro and in vivo. Additionally, populations of small volume cells were selected from parental MD MB 231 cells by flow cytometry. By obtaining the matching cell size control, I found that increased pore invasion was not solely dependent on small cell or nuclei size. In fact, properties unique to the pore-selected invasive cells were: decreased actin cytoskeleton anisotropy and cell rigidity (Young’s modulus) as determined by Atomic Force Microscopy (AFM). To identify signalling pathways that were associated with observed cytoskeleton and elasticity changes, RNA sequencing was performed on parental, pore-selected and flow-selected MDA MB 231 cells, and parental and pore-selected MDA MB 435 cells and a stringent comparison was performed. Such an approach allowed identification of common genes, which were used for further analysis by applying GSEA. The most common gene signature was found to associate with 3 increased signalling through the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) pathway. Blocking the activity of the MAPK/ERK pathway with two pharmacologically distinct MEK inhibitors resulted in actin stress fibre restoration, increased cell stiffness and restrained cell invasion through collagen matrices. I was able to identify, for the first time that changes in cytoskeletal organisation mediated by increased signalling through the MAPK/ERK pathway resulted in cell plasticity alterations. Therefore, drugs that block Ras-MAPK signalling would likely provide clinical benefit by reversing the effect of this signalling pathway on tumour cell plasticity and subsequently restraining migratory and invasive capabilities.

The genome of the eukaryotic parasite Trypanosoma brucei is both dynamic and unconventional in several aspects. In comparison with other eukaryotic genomes, where the majority of protein coding genes are associated with their own transcriptional promoters, T. brucei transcribes almost all protein-coding genes polycistronically. Transcription initiates from broad regions that lack defined promoter sequences and RNA Polymerase II then traverses up to hundreds of genes, generating a pre-mRNA that then requires trans-splicing and polyadenylation to generate mature mRNAs. Termination of transcription, via virtually unknown processes, occurs where two multigene transcription units converges or, in some cases, adjacent to a downstream transcription initiation site. RNA Polymerase II transcribes the majority of protein-coding genes in this manner, negating any differential gene expression via transcriptional control. A further unusual aspect of the genome is the dedication of as much as a third of the coding capacity to elements of antigenic variation. When infecting the mammalian host, parasites express a dense protein coat of variant surface glycoprotein (VSG). In order to evade host immune elements, T. brucei switches expression to antigenically distinct VSGs, employing a repertoire of ~2,000 genes. Both transcriptional and recombination-based strategies enable the parasite to either switch transcription between ~15 expression sites, each housing a distinct VSG, or relocate VSG sequence from silent gene arrays into an active VSG expression site. Although multiple factors have been found to regulate these processes, the events which trigger a VSG switch by either pathway are unclear. R-loops are three stranded structures containing an RNA-DNA hybrid and displaced single-stranded DNA. Although potentially deleterious to genome integrity, R-loops have been linked to transcription initiation and termination, DNA replication and recombination events. In this study, the potential for R-loop involvement in these fundamental genome functions of T. brucei was investigated. Firstly, Ribonuclease (RNase) H enzymes, which resolve the RNA-DNA hybrid portion of R-loops, were characterised, revealing T. brucei expresses potentially three distinct catalytic enzymes, two functioning in the nuclear genome and one in the kinetoplast(mitochondrial) genome. Nuclear RNase H activity was depleted by null mutation or RNAi mediated knockdown of the nuclear RNase H enzymes, showing that while one RNase H, TbRH1, is non-essential, loss of the other, TbRH2, caused several growth and genome integrity defects. As it was hypothesised to increased levels of RNA- DNA hybrids of the genome, RNA-DNA hybrids were mapped in wild type parasites and those lacking RNases H using a specific antiserum, S9.6. This mapping identified the conserved formation of R-loops at centromeres, retrotransposon-associated genes, rRNA and tRNA genes. R-loop enrichment was also uncovered at RNA Polymerase II transcription start sites, as documented in mammalian genomes. DNA damage was specifically increased at these sites after TbRH2 depletion, indicating efficient resolution of these transcription initiation-associated R-loops is critical for genome maintenance. In contrast, R-loops were not associated with DNA replication or transcription termination suggesting RNA-DNA hybrids are not involved in these processes in T. brucei. The most abundant sites of R-loop enrichment were found to be at the nucleosome depleted regions located between the coding regions of polycistronically transcribed genes and are associated with polyadenylation and trans-splicing, highlighting a novel correlation of R-loops with pre-mRNA processing. Lastly, R-loops were mapped to VSG expression sites where their abundance increased after ablation of RNase H activity, an effect that was associated with both increased DNA damage and VSG switching, uncovering an R-loop-driven mechanism of antigenic variation.

Site-specific recombinases of the Xer family are essential in most bacteria with circular chromosomes for the resolution of chromosome dimers arising after genome replication. In Helicobacter pylori, a gastric pathogen implicated in peptic ulcer disease and gastric cancer, the chromosome dimers are resolved by a single Xer recombinase, XerH. Interestingly, many H. pylori strains carry a second Xer recombinase, XerT, usually encoded on a large conjugative transposon TnPZ. Remarkably, XerT is not involved in chromosome dimer resolution, but was shown to be required for the mobilization of TnPZ in vivo. In this thesis, I investigated the molecular mechanisms of XerH- and XerT-mediated recombination by combining X-ray crystallography with protein biochemistry and microbiology. I solved the crystal structure of the XerH tetramer in a post-cleavage synaptic complex with its substrate DNA site, difH. To our knowledge, this is the first structure of an Xer recombinase bound to DNA. The structure and additional biochemical data provided key insights into the ordering and regulation of difH binding and first strand cleavage by XerH. Moreover, I investigated the regulation of XerH recombination by FtsK – a host factor usually required for Xer recombination – and found that XerH can resolve plasmids in the absence of FtsK in E. coli, but additional factors might be required for recombination of chromosome-borne difH sites. In the second part of this work, I studied the mechanism of XerT-mediated TnPZ transposition. XerT’s binding and cleavage sites on transposon ends were mapped and XerT activity was reconstituted in vitro by trapping cleavage and strand exchange products. In addition, the complete TnPZ excision has been reconstituted in vivo in E. coli, indicating that XerT is sufficient to catalyze TnPZ mobilization. Based on the results, a testable model for TnPZ excision and integration was proposed. In summary, this work provides valuable insights into the mechanisms of the two Xer recombinases of H. pylori and enhances our understanding of Xer recombination (a process essential for bacterial survival) and conjugative transposition (important in the spread of antibiotic resistance among bacteria), which in the future could help develop new therapeutic agents against deadly pathogens such as H. pylori or help control the spread of antibiotic resistance.

Epidemiological research has identified thigh musculature and knee ligament injuries are of most concern in professional soccer. With strength identified as a modifiable risk factor, isokinetic dynamometry has become a popular means of injury screening. However, isokinetic assessments are criticised based on their lack of functional relevance to injury aetiology. Study one describes isokinetic screening of the thigh musculature in an elite cohort of male soccer players assessed at 60, 180 and 270º·s-1 across four knee joint angles (70-40°). This study identified strength characteristics were velocity and angle dependent, advocating the use of novel metrics and a range of angular velocities for future isokinetic screening. Study two identified these procedures of assessment were modifiable during late stage rehabilitation of an elite soccer player. When considering the influence of gender and age on lower limb injury incidence, studies three and four identified the proposed isokinetic procedures were sensitive to differences in playing age between male and female youth and adult soccer players. In addition to isokinetic assessments, kinematic analysis of functional movements were completed. Study five comprised kinematic (sagittal and frontal planes) assessment of single leg hop and change of direction tasks performed using sidestep and crossover techniques with female soccer players. When considering previous criticisms of isokinetic testing not being functionally relevant, the knee angles and angular velocities identified in study five were used to predict knee flexor and extensor torque expressed by knee joint angle and velocity (study six). Study seven compared the predicted strength values to the knee joint moments exhibited during the hopping tasks. Strength assessments should use joint angle and velocity to profile musculature strength with increased functional relevance, and may identify additional training needs. These studies also allow strength profiling during the completion of functional tasks that may inform increasingly specific interventions.