Follicular T-helper (T_FH) cells are a subpopulation of CD4+ lymphocytes, which within germinal centres, determine differentiation of B-cells into memory cells and antibody-secreting plasmacytes. T_FH are therefore critical players in vaccine-induced immunity. Study of T_FH has been limited, as they are thought to be tissue resident cells, which do not normally re-circulate. While accessing blood is straightforward, access to lymph node tissue responding to vaccine is very limited. Therefore data on functions of tissue-resident human T_FH cells remains sparse. This thesis details establishment of an ethically approved peri-surgical window of opportunity study and development of novel tissue processing techniques and laboratory assays designed to overcome this hurdle. I randomized 42 consenting breast cancer patients due to undergo sentinel lymph node biopsy to be vaccinated with combined tetanus/diphtheria/polio vaccine ipsilateraly, contralateraly or not at all prior to surgery. A vaccine draining, non-sentinel node was studied in the context of vaccine-specific antibody and circulating lymphocyte response over the seven weeks following vaccination. Only lymph nodes draining the ipsilateral vaccine site were enriched for two CD4+derived populations; T_FH (CD45RO+CXCR5+ICOS+PD1+) and pre-T_FH (CD45RO+CXCR5+ICOS+PD1-). In blood, transient increases in absolute numbers of these same populations were observed one week following vaccination (mean-fold-increase: T_FH = 6.3; P = 0.002. Pre-T_FH = 4.0; P=0.002). In contrast a related population (CD45RO+CXCR5+ICOS-PD1+) showed no enrichment within vaccine-draining nodes or changes in circulating numbers post-vaccine. Total IgG, IgM and IgG1-4 isotype immunoglobulin vaccine response was assessed. Response correlated with predominant cell-type increase in blood: CD45RO+CXCR5+ICOS+PD1- were prevalent in slow-responders, correlating with increases in immunoglobulin-switched plasmablasts (r = 0.90; 95%CI 0.74-0.97. P < 0.0001), whereas CD45RO+CXCR5+ICOS+PD1+ were prominent in fast-responders, associated with increasing unswitched plasmablasts (r = 0.79; 95%CI 0.51-0.90. P < 0.0001) and plasma cells (r = 0.57; 95%CI 0.17-0.81. P = 0.007). Dichotomisation of response according T_FH sub-population tallies with measurable B-cell antibody and blast changes following vaccination. This possibly reflects memory state, suggesting different roles of T_FH and pre-T_FH in primary and secondary responses. Further study of the function of T_FH in lymphoid tissue-should focus on these two dynamic populations.

My thesis underlines the role and current evidence of minimally invasive surgical (MIS) technique for benign and malignant renal conditions. Evidence is presented for the use of laparoscopic and robotic partial nephrectomy along with expanding indications for ureteroscopy. Systematic reviews of ureteroscopy for large stones, obese patients, patients with bleeding diathesis and children is presented. Use of ureteroscopy for endoscopic diagnosis and management of upper urinary tract tumours is also presented. The work is comprised of 12 peer-reviewed published papers including 8 systematic reviews and 4 original research papers using retrospective or prospective case series on the subject. Based on the evidence, new insight into MIS for renal conditions will help clinicians and patients in informed decision-making. Minimally invasive surgery is a step in the right direction for management of various benign and malignant renal conditions. My work demonstrates evidence-based outcomes, which will ensure widespread adoption of these techniques in future.

Members of the Tumor Necrosis Factor superfamily of cytokines are an important source of costimulatory signalling required for the proper activation of T cells. One such receptor-ligand pair is Death receptor 3 (DR3) and its ligand TNF-like ligand 1A (TL1A). Genetic variants in TL1A are associated with inflammatory bowel disease and TL1A-DR3 interactions are required for full pathology in murine models of asthma, multiple sclerosis and colitis, with DR3 on T cells thought to play a major role in promoting immunopathology. Tumor Necrosis Factor superfamily cytokines, including TL1A, are synthesised as type II transmembrane proteins, some of which are subsequently cleaved to yield a soluble form in the serum. We have investigated the biological function of transmembrane versus soluble TL1A in mice using a mutant that prevents cleavage of TL1A from the plasma membrane. Overexpression of either full length or membrane restricted TL1A promoted an IL-13 driven small intestinal pathology which appeared earlier in mice where soluble TL1A was present. We hypothesise that this may be due to cell-type specific effects of the different forms of TL1A. In these same mice we identified increased total IgA in the serum, and went on to demonstrate that DR3 deficient mice have a defect in IgA in response to immunisation and TL1A can promote IgA class switching in vivo. Lastly we assessed the role of DR3 in lupus-like murine models. Here we found lack of DR3 was protective for the development of kidney disease. Overall we have shown that different forms of TL1A can have differing functions and that TL1A is important for components of both the humoral and cellular immune response.

Cancer survivors can experience a number of consequences of their disease, both physical and psychological. In this thesis, I aimed to clarify the psychosocial processes that reduce or increase post-traumatic stress after cancer. “Social support,” for instance, has often been linked to post-traumatic stress, but I describe several problems with this concept. Alternatively, inspired by the Social Identity Approach (SIA) to health, I proposed that group identification, or a sense of belonging and commonality with social groups, might predict levels of post-traumatic stress after cancer. In line with Social Cognitive Processing Theory (SCPT), I also proposed that cancer survivors’ perceptions that members of their social groups are closed, judgmental, and unreceptive in conversations about cancer (“group constraints”) might predict post-traumatic stress. I proposed that these constructs might act in tandem to predict levels of post-traumatic stress. To begin my programme of research, I conducted a meta-analysis that established factors contributing to post-traumatic stress disorder (PTSD). I also found an increased prevalence of PTSD among cancer survivors compared to controls without cancer. This meta-analysis demonstrated that cancer survivors can experience PTSD related to their illness. Subsequently, I undertook a series of quantitative questionnaire studies to establish psychosocial determinants of post-traumatic stress. My questionnaire included scales measuring post-traumatic stress, social support, group identification, and group constraints. In a pilot study, I used convenience sampling to test out the acceptability of the questionnaire and to provide a foundation for further hypothesising. Then, I distributed this piloted questionnaire to colorectal cancer survivors in Tayside. In initial “wave 1” cross-sectional analyses of this data, I found that social support was not an independent predictor of post-traumatic stress in multivariate analyses. However, group constraints consistently and independently predicted post-traumatic stress. Group identification exerted an effect on post-traumatic stress by reducing group constraints. I then distributed the same questionnaire a second time to the same respondents from wave 1 in order to collect longitudinal data. The results of the data from respondents at both waves 1 and 2 revealed that social support was, in fact, a likely causal predictor of post-traumatic stress. Constraints within the family also causally reduced post-traumatic stress. Family identification was a weak causal factor in reducing post-traumatic stress, but reciprocally, post-traumatic stress reduced group identifications. The longitudinal data also provided further, albeit not strong, support for the hypothesis that family identification reduces post-traumatic stress by reducing constraints on conversations within the family. Finally, in light of the consistent evidence from the quantitative studies above that conversational constraints are associated with increased post-traumatic stress, I conducted a qualitative interview study with a small number of cancer survivors aiming to characterise their experiences of conversations about cancer. The participants reported a broad range of experiences, beyond just constraints versus openness. These findings have broad implications for both theory and practice. For instance, in the final chapter, I discuss ways in which these results motivate further research on intersections between the Social Identity Approach and cancer-related post-traumatic stress. I also discuss implications for interventions going beyond traditional trauma exposure techniques.

There is much evidence in the literature supporting the hypothesis that immune cells affect the tumour cells. For example, Mattes et al, show that the tumour (i.e., B16-OVA melanoma) is eliminated by Th1 cells, but Th2 cells reduce the size of tu- mour (i.e., not eliminate the tumour cells). However, Xie et al show that the tumour B16 cells are eliminated by Th1 cells. Moreover, Kobayashi et al show that the growth of B16-F10 cells is associated with a large number of Th2 cells. Also, Chen et al show the growth of B16 melanoma cells is associated with a shift from anti-tumour M1 macrophages to pro-tumour M2 macrophages. Thus, there are contra- dictory experimental results on the interactions between the tumour and the immune system, and the biological mechanisms behind these results are still poorly understood. Furthermore, chemotherapy drugs affect not only cancer cells but also immune cells. In this thesis, we start with a simple model for the interactions between cancer cells and a chemotherapy drug (Cyclophosphamide), and use it to confirm (in combination with experimental data for tumour growth) that Cyclophosphamide has nonlinear drug decay. The second investigation presents a mathematical model to illustrate the dynamics of immune cells both in the absence and presence of cancer. We show that the tumour is eliminated in the presence of type-I or type-II or a mix of type-I and type-II cytokines, and the tumour grows in the presence of type-II cytokines. The third investigation involves a mathematical model of the drug effect on the tumour and immune interaction. We show that tumour growth is delayed when the drug is injected before the switch between the Th1 and Th2 cells that dominate the immune response. However, tumour grows faster when the drug is injected after this switch between the Th1 an Th2 cells. A more significant effect of the drug was observed when we assumed that drug injection occurred before the switch between M1 and M2 cells (i.e., before the switch between Th1 and Th2 cells) and this lead to a significant reduction in tumour size as a result of the decrease in the M2 cells. In our final investigation, we develop a PDE model for the spatial interactions between a chemotherapy drug and cancer cells, and calculate analytically the speed at which the tumour invades the domain (in a travelling wave manner). Moreover, we present a spatial model for the interaction between a chemotherapy drug, cancer cells and macrophages, and we investigate the speed at which the tumour invades the domain (in a travelling wave manner).

Polo like kinase 1 (PLK1) has many functions in the orchestration of cell division. It is an oncogene and its overexpression is associated with poor prognosis. PLK1 is a cell cycle-dependent protein; its levels are low in the early phases of the cell cycle and increase as cells go through the cycle, peaking in G2/M. In this thesis, our focus has been on two critical areas in PLK1 research. 1. Tumour suppressor p53 mediates transcriptional repression of PLK1. There have been several mechanisms suggested for this repression both by direct and indirect p53 involvement. In light of the recently proposed mechanism suggesting that p53-p21-DREAM-CDE/CHR is responsible for p53 mediated repression of G2/M proteins (including PLK1), we reassessed the event(s) by which p53 downregulates PLK1. While investigating the effect of different p53 stabilising agents on PLK1 levels, different agents were found to give rise to different cell cycle profiles. This could account for differences in the extent of PLK1 downregulation in response to different agents: i.e. that the levels of PLK1 after treatment reflect the phase in which the cycle is arrested. Further investigation showed that p53 mediated repression of PLK1 is partly p21 dependent, consistent with repression occurring (partly) through DREAM. Also, we found that different cells use different or overlapping mechanisms for this repression. Thus While in HCT116 cells, mutation of CDE/CHR elements (through which DREAM acts) abolished the PLK1 repression, U2OS cells were only partly dependent on CDE/CHR elements for this repression. Additionally, Serine 15 phosphorylation was found to be partly required for the repression and only wild type p53 (but not mutated forms of p53 that lack transactivation capacity) could repress PLK1 expression. These data are consistent with the idea that p53 repression is indirect, but do not rule out other mechanisms. 2. PLK1 has been extensively studied as a target for cancer therapeutics. PLK1 inhibition causes arrest in prometaphase and activation of the DNA damage response. Part of this thesis aimed at investigating the DNA damage response (DDR) induced in mitosis by PLK1 inhibitor and the consequences of such inhibition. Also, the results were compared with the results obtained/reported in response to microtubule poison, nocodazole. Our investigations showed some differences in the mitotic arrest-induced DDR observed by PLK1 inhibition and microtubule poisons. PLK1 inhibition resulted in both telomeric and non-telomeric γ-H2AX foci which were not caspase/CAD dependent, whereas nocodazole treatment resulted in caspase dependent DNA damage which was mostly on telomeres. DNA damage sensing protein kinases involved in DDR by PLK1 inhibition were found to be ATR and DNA-PK, whereas for nocodazole DNA-PK was the main kinase involved. Also, in clonogenic survival assays, more surviving colonies were observed in response to PLK1 inhibitor as compared with nocodazole treatment. Interestingly, PLK1 inhibition resulted in recruitment of the 53BP1 (one of the components of the DNA repair pathway) in mitosis which may not be beneficial and suggests further investigations on chromosomal abnormalities that PLK1 inhibition may bring about.

Context: There is little institutionalised palliative care in Greece; hence most patients with advanced cancer die in hospitals with unrecognised palliative care needs. There is little evidence on the care these patients receive. Patients’ perspectives and experiences of the illness and care, and their preferences on issues around death and dying have not been examined within the Greek context. Aim: To explore in-depth the care received by patients with advanced cancer in Greece, and how they and their families experience the illness and care throughout the last months of life. Methods: A prospective longitudinal, qualitative approach was adopted, employing an ethnographic methodological framework. Thirteen patients with advanced cancer were followed until death, from the point they came into contact with either one of two medical wards or a day clinic in an oncology hospital in Athens. The data were collected using participant observation, semi-structured interviews and discussions. The patients, 34 family members, and 32 health professionals involved in their care were interviewed (total 75 hours) and observed (total 830 hours). Data were analysed in both Greek and English, using thematic network analysis and case study analysis. Results: Six men and seven women (patients) took part in the study, age ranging 30-75 years. Primary diagnoses included breast, lung and colon cancer. They were followed for 40 days to six months prior to death. Twelve out of the 13 died in the hospital (one died at home); their last admission ranged 10-70 days. Care focused on cure and prolonging life until the late stages of the disease, allowing little opportunity for transitioning to a palliative phase. Decision-making was doctor and/or family focused. The patient’s voice was expressed through families. When patients chose to be actively involved, they did so through: a) planning care near the end of life, b) maintaining their role within the family, and c) assuring the space and time for their dying preparation. Dying trajectories were dependent on illness progression and doctors’ attitudes; decision-making of care; symptom control; preferences for care and priorities of life, which shifted over time for patients and families; and patients and families values and attitudes towards death and dying. Patients’ wishes for symptom relief increased as the illness progressed, whilst their focus shifted from the illness to the relationship with their loved ones, often resulting in changing preferences in support of their families’ wishes. Near death, patients actively negotiated space and time for preparing to die, and performed dying rituals. Families proved to have a central role in care provision. Members with a dominant role within the family most often adopted a decision-making role, whilst others retained the care-giving one. As the illness progressed, the focus of family’s decision-making changed from cure to care and the symptom burden increased. Also, in cases where the decision maker within the family changed during the illness trajectory, the decision-making shifted to the preferences of the new decision maker. All patients expressed a preference to die at home. Most families changed their preference from home to hospital death due to lack of health care support at home. Conclusions: The study showed the all importance of the interplay between decision making, illness progression, awareness of dying, family dynamics and their change over time in shaping patient trajectory at the last months of life. A framework of mindset orientation of goals and priorities is proposed whicch may provide a new prospect for approaching trajectories in palliative care. In Greece, the journey of advanced cancer patients over their last months of life is shaped by a combination of factors including doctors’ attitude towards cure rather than care; the lack of symptom control; the lack of availability of home care services; families’ resources to advocate for the patient and the role of the patient within the family. Preferences and choices over care changed over time for some patients and their families due to these factors. For palliative care to develop within Greece, the reasons behind these attitudes need to be further studied and challenged, and suitable training developed. Also, proper multi-professional teamwork, an integral component of palliative care, needs to be developed.

Tuberculosis (TB) caused approximately 1.5 million deaths worldwide in 2014 and is the second biggest killer due to a single infectious agent. Issues with current TB treatment such as interactions with antiretroviral therapy for HIV co-morbidities, poor compliance and the emergence of resistance to current antibiotics mean that there is an urgent need for new antitubercular drugs. InhA is an enoyl reductase that is the primary target for isoniazid, a first-line antitubercular drug. This enzyme plays a key role in the synthesis of mycolic acids which are key components of the TB cell wall. As such, InhA is an attractive target for future antitubercular drugs. Isoniazid is a pro-drug which is activated by the enzyme, katG, and resistance to isoniazid has been primarily attributed to mutations in katG. Thus, drug discovery for InhA to date has focused on direct inhibitors which do not require activation, mainly using virtual screening and structure-based drug design approaches. In recent years a structural mutation, S94A InhA, has been detected in clinical isolates conferring resistance to isoniazid in the absence of katG mutations, suggesting it is becoming clinically relevant in the context of isoniazid resistance. To date, no direct InhA inhibitors are available clinically, and only two S94A InhA inhibitors have been reported. In this thesis, two main approaches were used to evaluate and identify novel InhA inhibitors. Firstly, recently discovered wild-type (WT) InhA inhibitors at the University of Nottingham were further evaluated by determining their ability to inhibit the activity of the S94A InhA mutant, given the apparent increasing importance of this mutation in isoniazid resistance. In addition, their ability to inhibit the growth of Mtb, E. coil and S. aureus was determined in order to provide a more relevant inhibitory profile. Secondly, novel fragment-linking drug discovery tools were investigated as this may provide an alternative route for drug discovery and has not yet been reported for InhA. A series of WT InhA inhibitors previously discovered at the University of Nottingham were evaluated by determining their inhibitory activity for S94A InhA and determining their ability to inhibit the growth of Mtb, E. coli and S. aureus to provide a more relevant inhibitory profile as lead compounds. Two compounds were found to inhibit the activity of S94A InhA more strongly than WT InhA, which is of significant interest since only two S94A InhA inhibitors have been reported in the literature. Another compound was discovered that displayed an ability to inhibit the growth of Mtb and M. bovis at 25 ug.m1-1 whilst displaying no inhibitory activity for E. coli or S. aureus at the concentrations tested. To further explore the S94A InhA mutant, crystallization studies were then performed. The ultimate aim was to obtain structural information for the binding modes of the two inhibitors that displayed a more promising inhibitory activity for S94A InhA compared to WT InhA as this would aid future structure-based drug design. Successful crystallization conditions were determined for WT InhA-NADH and S94A InhA-NADH, which were similar to those reported in the literature. Novel fragment-linking drug discovery approaches were then explored for InhA. In order to explore the feasibility of this approach for InhA, molecular docking studies were conducted on a series of deconstructed InhA inhibitors to compare the binding poses of the fragments to the complete inhibitor. Findings suggested that the binding position of some of the fragments were preserved, when compared to the corresponding portion of the inhibitor. The structures of these fragments provide a starting point for further investigation and the possible de novo design of fragments for a fragment-linking approach. An experimental fragment-linking approach, in situ click chemistry, was also developed for InhA with the aim of identifying novel inhibitors. The method development consisted of using molecular docking studies to select azide and alkyne fragments, developing and optimizing an in situ method, and developing a suitable LC-MS/MS analytical method to detect the formation of 1,2,3-triazoles. A novel disubstituted 1,2,3-triazole was detected in a larger amount in the presence of InhA, by approximately twelve-fold, suggesting the enzyme was catalyzing the formation of this triazole. Attempts were made to validate the precise site of binding for the azide and alkyne fragments, but were unsuccessful and further work is required to determine the inhibitory activity of this compound. This in situ click chemistry tool will provide an exciting novel drug discovery approach in addition to the current reported methods for InhA.

No description available.

No description available.