CD55 is one of the complement regulatory/inhibitory proteins and is over-expressed on a wide range of solid tumours. CD55 is also known to be deposited within tumour stroma and is secreted in an active soluble form, mediated by matrix metalloproteinase-7. The complement cascade forms part of the innate immune system and culminates in cell lysis of targeted cells. As a complement regulatory protein, the primary function of CD55 is to accelerate the decay of complement components preventing formation of the membrane attack complex. CD55 is also known to be a ligand for the T cell early activation antigen CD97, and their interaction has been shown to inhibit the proliferation of activated T cells. This project aimed to develop anti-tumour immunotherapeutics aimed at exploiting CD55 as a tumour associated antigen. Initial strategies were to develop monoclonal antibodies, specific to identified epitopes from within the CD55 protein sequence, capable of binding, and neutralising CD55s decay accelerating activity. Developed antibodies would also have the potential to induce antibody dependent cell cytotoxicity, thus blocking CD55 protection of tumours and mediating an active anti-tumour response. Antibodies were raised specific to CD55 derived linear peptides, which have been used for the assessment of CD55 expression in breast tumour sections. Monoclonal antibodies failed to recognise natively expressed protein on viable tumour cells and alternate strategies were developed. An effective immunotherapy for the treatment of cancer would engage both cellular and humoral mediated responses for effective clearance of target cells. In order to achieve this, a DNA vaccine incorporating a human IgG Fc tail was developed expressing the active sites of CD55, containing HLA-A*201 restricted heteroclitic epitopes. The vaccines were used to immunise HLA-A*201 HHDII transgenic mice and CD55 specific responses were assessed. One of the vaccines analysed, elicited CD55 specific antibodies capable of recognising tumour cells in vitro and also generated epitope specific CD8+ T cell mediated lysis of epitope bearing cells. The frequency of CD55 specific T cells was obtained via antigen specific IFN gamma release ELISPOT assays and the cytokine profile of responses generated was assessed via luminex analysis. In conclusion, CD55 remains a viable target for immunotherapies aimed at CD55 bearing tumours. DNA vaccines encoding modified epitopes are capable or raising cellular and humoral responses to this antigen and further studies should be completed in order to determine anti-cancer effects in tumour bearing models.

Cytotoxic chemotherapy remains the mainstay of treatment for patients with cancer, however immunotherapy is starting to emerge as an additional modality of treatment. Evidence suggests that chemotherapy can synergise with immunotherapy to improve responses. Although CD8 T cells have been regarded as the main anti-tumour effector cell, the role of CD4 T cells in orchestrating CD8 and other anti-tumour responses is increasingly recognised. However, the CD4 T cell population contains effector and suppressive subsets with diverse and opposing functions. This thesis describes the establishment of a murine mesothelioma model with which to study the effects of different CD4 subsets on anti-tumour immune responses, and investigate their capacity to provide cognate help to tumour antigen specific CD8 T cells. Haemagluttin (HA) specific CD4 T cells from transgenic mice were polarised in vitro into Th1, Th2, Th17 and Treg subsets and adoptively transferred alongside HA specific CD8 T cells into mice bearing HA expressing tumours derived from a mesothelioma cell line. The effects of the different CD4 subtypes on tumour growth and their capacity to provide ‘help’ to CD8 T cells was investigated in a prophylactic treatment model and in the context of treatment with gemcitabine chemotherapy. Results showed that survival and behaviour of in vitro differentiated CD4 subtypes after adoptive transfer was highly variable and that only Th1s displayed anti-tumour activity when injected prophylactically, prior to tumour inoculation. Cytotoxic chemotherapy did not provide a favourable environment for adoptive transfer of in vitro differentiated CD4 cells. No antitumour activity was seen against established tumours, which may have been due to overriding tumour induced immunosuppressive mechanisms. Successful treatment of established tumours that had been treated with chemotherapy required both the provision of HA specific CD8 cells and the prior removal of an established, endogenous regulatory CD4 T cell population.

This thesis explores the views and experiences of people with lung cancer, and family members, of discussing preferences and wishes for end of life treatment and care. It presents an interpretive analysis based on the application of a constructivist grounded theory approach. Study participants included: eighteen men and seven women with lung cancer and nineteen family members. Participants were mainly from lower socio-economic groups living in the north of England. Single, joint and group interviews were used to gather data. Interview transcripts were analysed using a constant comparative method and conceptual models were drawn to aid the development of the theoretical interpretation. The study found that preferences and wishes for future care and treatment were not the main concern of people with cancer; rather, any concerns for the future were about the social aspects of death. Participants talked about their experiences of facing death whilst striving to live in the present. Planning for one’s own dying and eventual death was not something that people with lung cancer reported having discussed, except when, out of concern for their families, practical arrangements needed to be made following death. The disclosure of a poor prognosis had a huge emotional effect on participants, who ascribed a variety of meanings to this news. Participants’ reported that clinicians usually focused on their disease; they did not recall being offered any ‘options’ or ‘choices’ for future care. They commented that their preferences and wishes for future treatment and care were influenced by their clinician, spouse, other family members and their knowledge of others affected by cancer. The theory ‘maintaining integrity in the face of death’ is proposed. This theory purports that patients with advanced lung cancer and their families focus on acting and talking as ‘normal’ to help them balance living in the present whilst facing death. This thesis makes several contributions to knowledge. First, it provides the views of people from an underrepresented group of cancer patients from lower socioeconomic classes who are rarely included in research. Secondly, it shows how people facing the end of their life place little importance on choice. They focus instead on living in the present and carrying on as normal, which challenges current UK policy that seeks to promote individual patient choice at the end of life through advance care planning. The study findings suggest that policy makers and health and social care professionals need to develop ways of helping people prepare for a ‘social’ rather than a physical or ‘medicalised’ death: a focus on developing advance care planning that provides information to support people’s practical needs at the end of life, delivered as a family intervention, thereby helping people living with lung cancer to maintain their integrity in the face of death.

Medication errors are a significant global concern and can cause serious medical consequences in children. Double checking of medicines by two nurses is one strategy used by many children's hospitals to prevent errors from reaching paediatric patients. This thesis involves different studies that evaluated the effectiveness of the double checking process in reducing and preventing medication administration errors in a children's hospital. In addition, a systematic review was conducted of medication errors studies in the Middle East. A systematic review was also conducted of published studies of double checking. Six electronic databases were searched for articles that assessed the double checking process during the administration of medicines. Sixteen articles were identified. Only one of them was a randomised controlled clinical trial in a clinical setting. Only one study was conducted in a children's hospital. The review found that there is insufficient evidence to either support or refute the practice of double checking and more clinical trials are needed to evaluate the double checking process in children's hospitals. Based on the findings that were highlighted from the systematic review, a prospective observational study of paediatric nurses using the double checking process for medication administration was undertaken. The study aimed to evaluate how closely double checking policies are followed by nurses in different paediatric areas, and also to identify any. medication administration errors during the study period. 2,000 drug dose administration events were observed. There was variation between paediatric nurses adherence to double checking steps and different medication administration errors were identified. Based on the observational study, a semi-structured questionnaire study was developed. It was designed to explore the paediatric nurses' knowledge and opinions about the double checking process. The study showed that many nurses have insufficient knowledge on the double checking process and the hospital policy for medication administration. A simulation study was conducted to examine whether single or double checking is more effective in detecting and reducing medication errors in children. Each participant in this study was required to prepare and administer medicines in scenarios for two "dummy patients" either with another nurse (double checking) or alone (single checking). Different confounders were built into each scenario (prescribing and administration) for nurses to identify and address during the administration process. Errors in drug preparation, administration and failure to address confounders were observed and documented. The main findings from this study were that the double checking process is more likely to identify medication administration errors and contraindicated drugs than single checking. The time taken for drug administration was similar for both processes. Another systematic review was conducted to identify the published medication errors studies that have been undertaken in the Middle East. The review identified 45 studies from 10 Middle Eastern countries. Nine of the studies focused on medication errors in paediatric patients. Educational programmes on drug therapy for doctors and nurses are urgently needed in the Middle East. These studies have contributed to the field of medication safety by providing more information about double and single checking medication administration processes in paediatric hospitals. More educational and training programmes for nurses about the importance of double checking and improving their adherence rate to the double checking steps during medication administration are required to improve its effectiveness.

Prescribing errors affect the safety of patients of all ages, however, the risk of prescribing errors may be higher in paediatric patients. This is due to the complexity of calculating doses, age-specific pharmacokinetic and pharmacodynamic changes and the higher usage of unlicensed and off-label medications. This research explores the prevalence and nature of prescribing errors in children and the interventions used to reduce such errors. A multi-centre observational study was conducted to evaluate the prevalence and nature of prescribing errors in paediatric settings. Clinical pharmacists working in thirteen different UK hospitals were asked to identify prescribing errors in inpatient medication orders. The overall error rate was 8.7% of total orders which was similar to the rate of prescribing errors in adults (8.9%) in the EQUIP study. Dosing errors were found to be the most common and the majority of errors were classified as minor. Severity assessment of these errors was conducted by an expert panel to evaluate the severity of all errors which are reported in chapter 2 as data were missing from some of the original error reports. Although there were discrepancies between the panel’s and the original pharmacists’ assessment in 234 of 892 errors, minor errors were still the most common. A second observational study was conducted to evaluate the prevalence and nature of paediatric prescribing errors in electronic medication orders as data for electronic prescribing were only reported from one hospital. Pharmacists were shadowed in their routine clinical work by the researcher who documented all prescribing errors identified by the pharmacists. The overall rate of errors was 5.2% of total orders which was significantly lower than the rate of errors found in the written orders (8.7%) in the previous study conducted in the same hospital using the same methods and before the introduction of an electronic prescribing system. The most common types of errors identified were omission of regular medications on admission followed by under-doses, non-measurable doses and overdoses. Most errors were classified as significant followed by minor errors due to the increased numbers of omission of regular medications on admission. Education is frequently recommended as being key to reducing prescribing errors therefore a systematic review was conducted to identify and evaluate educational interventions which have been used to reduce prescribing errors in children. Only 25 studies met the inclusion criteria. These studies used various educational interventions such as lectures and feedback. Eighteen studies reported that the interventions were effective in reducing errors. Multi-faceted interventions seemed to be more effective than single-strategy interventions. It was difficult to compare the various strategies due to differences in study definitions, denominators and methods of data collection. The impact of interventions is often evaluated by testing the prescribing ability of the doctors involved. A systematic review was therefore conducted to examine the literature on the tests used to assess prescribing competency in doctors caring for children. Only 16 studies met the inclusion criteria. Six studies were designed specifically to evaluate doctors’ dose calculation skills, seven studies used assessments asking the participants to write prescriptions according to given clinical cases and three studies had general questions about prescribing in children. The validity and reliability of the identified tests were not evaluated in any of the studies however. This project has contributed to the field of prescribing errors in children by providing more information about the prevalence and nature of such errors including the most common types and serious errors. Further research is still needed to identify the best ways of avoiding these.

The complex process by which cancer cells invade local tissue and metastasise is responsible for approximately 90% of cancer related deaths. The cell biological events that underlie this transition to malignancy are driven by invariable alterations within the genome, however relatively little is known about the genetic determinants involved. If identified, novel genes which perturb the rate of tumour progression could become potential targets for future therapeutic intervention. Using a novel in vivo system, it is possible to characterise the behaviour of transformed cells during the early stages of tumour development and follow these cells in real time, thus improving our understanding of the critical events that initiate cell proliferation, tumour cell invasion and metastasis. Using Drosophila as a model organism it is possible to generate neoplastic tumours within the dorsal thorax whereby clones of transformed cells are homozygous mutant for a specific tumour suppressor gene. By specifically labelling these transformed cells with GFP, their behaviour can be observed in high temporal and spatial resolution within the living epithelium. RNAi technology can also be employed to simultaneously knock-down expression of an additional gene specifically within the mutant tissue. This forms the basis of a large-scale screen for novel genes that may promote tumour progression in this epithelium. The screen is now almost complete and so far we have screened through almost 500 genes, the majority of which have previously been implicated in cancer but remain uncharacterised. We have observed a wide range of phenotypes, with genes affecting cell proliferation, invasion, cell shape, actin organisation, junction integrity and epithelial multilayering. By setting ‘thresholds’ for particular phenotypes ‘hits’ have been identified which drastically enhance tumour progression, and these genes are in the process of being fully characterised to further our understanding of their role in tumour progression.

Germ cell tumours (GCTs) are a class of tumours classified histologically into two main types: seminoma and non-seminoma. Prior studies revealed that there is a significant difference in global DNA methylation between those two types, where non-seminomas represent more differentiated cells and exhibit a high level of methylation compared with seminomas that resemble the precursor cells of GCTs. A number of studies have reported that silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the silenced genes and the genomic targets that are methylated in GCTs have not yet been systematically identified. Furthermore, many methylation studies in GCTs do not include the level of gene expression in their investigation. We hypothesized that the methylation of genes might play an important role in gene silencing in GCTs, so the main focus of this thesis was studying the relationship between the gene methylation and gene expression in GCT cell lines representing seminoma and non-seminoma. We analysed genome methylation and gene expression of these cell lines using the Illumina infinium Human Methylome 450 bead chip system and Affymetrix Gene Chip Human Genome U133 Plus 2.0 arrays, respectively. We also compared our results with gene expression data from primary tumours in order to identify which events were shared in primary GCTs tumour. qPCR analysis was carried out after treatment of cells with the demethylation agent, 5-aza-2-deoxycytidine, to confirm that expression of identified genes was regulated by methylation. These analyses showed that differential methylation of CpG islands between seminoma and non-seminoma cell lines correlated well with differential gene expression and revealed that hypermethylation of CpG islands near the transcriptional start site was more strongly correlated with low gene expression than was methylation of other regions. Meanwhile, methylation analysis identified uniquely methylated genes and features for each cell line, which may imply an underlying mechanism of their development. One-hundred and forty-seven silenced genes which exhibited a difference in methylation and expression between seminoma and non-seminoma cell lines were identified, some of these genes were also differentially expressed in primary tumours. Re-expression of selected silenced genes in non-seminoma cells after treatment with 5-aza-2-deoxycytidine confirmed that methylation played a role in gene silencing. Some of the genes identified are closely associated with pluripotency and implicated in chemosensitivity (PRDM14, KLF4, TDRD12, DDX43, MNS1, and RBMXL2). Silencing of these genes could therefore account for the progression process from seminoma to non-seminoma. PRDM14 was given special attention as it plays an important role in germ cell development and maintenance of germ cell pluripotency. The role of PRDM14 in GCT biology was studied, revealing that high expression of PRDM14 in combination with 5-aza-2-deoxycytidine treatment increased the response of cells to chemotherapy compared with those that had low levels of PRDM14. In addition, this study supports a growing body of literature on PRDM14 suggesting that this gene plays a critical role in DNA demethylation.