Haematological malignancies and their treatment lead to prolonged periods of severe thrombocytopenia (platelet count ≤ 50 x 10⁹/l). Despite the use of prophylactic platelet transfusions, haemorrhage remains an important complication during this thrombocytopenic period. Within a 30 day period up to 70% of patients have clinically significant haemorrhage (World Health Organization (WHO) grade 2 or above bleeding) and up to 10% have severe or life-threatening haemorrhage (WHO grade 3 or 4 bleeding). Hence our current management of these patients to prevent haemorrhage is sub-optimal. The aim of this thesis was to identify clinical and laboratory factors that may predict the risk of haemorrhage in patients with haematological malignancies and severe thrombocytopenia. This was achieved via several different study designs and assessed the effect of clinical and laboratory factors on any or clinically significant haemorrhage and their effect on intracranial haemorrhage. This thesis has demonstrated that there is no consensus on how bleeding is assessed and graded in this patient group. Also it showed that the absolute immature platelet number may be a better alternative to the total platelet count to guide administration of platelet transfusions. Female sex, a previous history of a fungal infection, a high C-reactive protein, a high white cell count, a low platelet count, anaemia, impaired renal function, and recent clinically significant haemorrhage were all found to be independent risk factors for haemorrhage. Patients who were in complete remission from their haematological malignancy had a much lower risk of bleeding.

As our understanding of cancer biology increases and novel therapies are developed, an increasing number of predictive biomarkers are becoming clinically available. Aberrant acetylation has been strongly linked to tumourigenesis and the modulation of acetylation through targeting histone deacetylase (HDAC) has led to the introduction of many HDAC inhibitors. To date, two have had regulatory approval for the treatment of cutaneous T cell lymphoma (CTCL). Modifications in chromatin control underpin the mechanism of action of HDAC inhibitors. A genome wide loss-of-function screen identified HR23B as a gene that governs sensitivity to HDAC inhibitors. HR23B shuttles ubiquitinated cargo proteins to the proteasome and elevated levels may contribute to cell death mediated by this pathway. It also governs cell sensitivity to drugs that act directly on the proteasome. HDAC inhibitors influence proteasome activity and there may be a synergistic interaction with proteasome inhibitors. HR23B and HDAC6 interact and HDAC6 may be a negative regulator of apoptosis and a positive regulator of autophagy and through its ability to down-regulate HR23B, may impact on the cellular outcome of HDAC inhibitor treatment. Expression of HR23B has been correlated with clinical response to HDAC inhibitors in a retrospective analysis of CTCL patients. The tissue expression of HR23B and the autophagy marker LC3 has been investigated and there may be a reciprocal relationship in their expression in some tumours which may provide prognostic information and patients with low HR23B expression but high levels of autophagy appear to have a particularly poor prognosis. Well designed, biomarker-driven prospective clinical trials are needed to clarify the predictive and prognostic roles of HR23B.

Angiogenesis, the growth of new blood vessels, is vital to tumour growth. Prevailing dogma has been that tumours cannot grow without angiogenesis. Based on this premise, anti-angiogenic drugs are used clinically. However, the principle of angiogenesis as an absolute requirement for tumour growth has been challenged with reports that many tumours are entirely or partially non-angiogenic. This study describes and quantifies characteristics of non-angiogenic non-small cell lung tumours, demonstrates non-angiogenic growth in small-cell/neuroendocrine lung tumours and investigates the underlying pathogenetic processes by comparison with angiogenic lung tumours. Hypoxia is an important stimulus for angiogenesis. Differences in response to hypoxia may determine whether a tumour produces new vessels. In order to test this, levels of. necrosis, often considered a surrogate marker of hypoxic stress, were quantified but no difference in quantity of necrosis was found Moreover, immunohistochemical investigation of hypoxia and angiogenesis factors provided no unambiguous explanation for the differences in angiogenesis. Significant differences were seen, however, in fibrosis and inflammation, which were both greater in angiogenic tumours. Differences were greater for lymphocytes rather than cells of the ‘innate’ immune system. This provided an alternative hypothesis: angiogenesis occurs during wound healing and in the growth of granulation tissue, so it is possible that tumour angiogenesis is a response to factors produced by immune cells rather than the tumour itself. A tumour’s angiogenic status may, therefore, be determined by the response it provokes from the immune system. Further work to test this theory would compare levels of immunogenic factors such as Tumour Necrosis Factor and tumour cell surface antigens such as the HLA class I molecules. The study concludes with an investigation into the molecular basis of non-angiogenic growth using the technique of comparative genomic hybridisation (CGH) which allows amplifications and deletions of areas of DNA to be calculated. High-resolution array CGH was evaluated against conventional CGH, and the results compared with previous RNA studies from our laboratory. These revealed a set of genes with consistent changes in both RNA and DNA, several of which form part of known angiogenic and inflammatory pathways.

This research programme investigated the expression of prolyl hydroxylase (PHD) proteins in rodent tissues. The importance of PHD enzymes lies in their ability to render oxygen sensitivity to Hypoxia inducible factor (HIF), the principal mediator of intracellular oxygen homeostasis. The first part of this study focused on developing and validating anti-sera capable of detecting PHD proteins in rodent tissues. With these reagents, it was possible to assess the relative expression of each PHD protein in a number of different rat tissues. PHD2 was the most abundant isoform in all tissues studied. In contrast, an abundance of PHD1 was observed only in testis and skeletal muscle. A number of different tissue species of PHD3 were identified and their abundance was found to vary between different tissues. These observations provide further evidence of the principal role of PHD2 in regulating HIF in vivo, but also point towards additional roles for PHD1 and PHD3 in selected tissues. They highlight the potential for there being a complex interplay between different PHD enzymes which could, in the future, prove potential targets for therapeutic manipulation. This study also provides additional insights into the mechanisms underlying the phenotypes observed in PHD deletional mouse models which appear, in many cases, to be directly related to the abundance of a given PHD isoform. The emerging role of PHD3 as a promoter of sympathetic lineage apoptosis prompted further study of PHD3 expression in rat neuronal tissues. An abundance of PHD3 was demonstrated throughout the rat sympathetic nervous system, a finding which appeared at odds with its known role as a promoter of neuronal apoptosis and resulted in a series of collaborative studies which demonstrated a sympatho-adrenal phenotype in wild type compared to PHD3-/- mice. Further collaborative studies utilising wild type mice and those deleted of specific PHD isoforms, were carried out to assess the significance of the abundance of PHD3 and PHD1 noted here in rat hippocampus and testis respectively. While neither study demonstrated statistically significant phenotypes, these observations remain of interest and areas for future research.

TRAP1 is a Heat Shock Protein (HSP) chaperone to retinoblastoma but also associated to the tumor necrosis factor receptor. HSPs are primarily up regulated in cancer. Work in our lab noted a down regulation of TRAP1 in some non-small cell lung cancers compared to normal lung. The first aim of this project was to evaluate the effect of the loss of TRAP1 on cell proliferation using a spheroid model. The presence of TRAP1 in spheroids promoted cell proliferation and a faster onset of hypoxia. This suggests an oncogenic role for TRAP1 since rapid hypoxia development equates to poor prognosis. Micro array analysis showed that TRAP1’s loss was associated with increased transcrpition of the Junctional Mediating and Regulatory protein (JMY). JMY possesses an oncogenic property due to its ability to facilitate cell motility. Additionally it has tumor suppressor activity in promoting p53 activation. The second aim of this project was to produce an anti-JMY antibody and use it to characterize JMY and additionally verify the association between TRAP1 and JMY. JMY was found to be widely expressed in normal tissues and in many types of tumors. In neoplastic tissues, comparing primary versus metastatic tumors, JMY was found to have significantly higher expression in the metastatic compared with the primary tumors. A pilot study showed that nuclear co-expression of JMY and P53 was associated with shorter overall survival suggesting that a possible tumorigenesis mechanism could be via a deregulation/mutation of JMY/p53 or both. Finally, using 3 dimensional constructions, I demonstrated the distinct morphological difference between an angiogenic tumor and a non-angiogenic tumor. Additionally, I showed a characteristic cytoplasmic p53 sequestration in the non-angiogenic phenotype that is absent in the angiogenic phenotype. This could be the mechanism that the non-angiogenic tumor uses to adapt to hypoxia. This would imply that there is a potential for cancers to escape therapy by switching between these 2 phenotypes.

Human T lymphotropic virus type -1 (HTLV-1) infections are important causes of mortality and morbidity in endemic areas worldwide. There is neither a vaccine specific for the virus nor satisfactory treatment for the associated malignancy or inflammatory syndromes. HTLV-1 associated infective dermatitis (IDH) is a chronic dermatitis that has been observed in a variable proportion of HTLV-1 infected children. IDH may serve as an early clinical marker for HTLV-1 and an indicator of increased risk for developing other HTLV-1 associated conditions such as adult T cell leukaemia/lymphoma (ATLL) and HTLV-1-associated myelopathy or transient spastic paraparesis (HAM/TSP). However the mechanisms underlying IDH and the relationships with HAM/TSP and ATLL are poorly understood. We undertook skin biopsies from 14 cases with IDH, and controls which included five asymptomatic carriers (ACs) and 18 healthy uninfected individuals from South Africa. We conducted clinical assessments, proviral load, allergen-specific IgE, peripheral blood and cutaneous T cell and virological analyses. We obtained relevant clinical history and examined all cases and controls based on a pre-formed questionnaire. Despite the partial clinical similarities with atopic dermatitis, the individuals with IDH did not have an increased incidence of atopic disease including asthma or rhinitis. Furthermore house dust mite-specific IgE levels were not elevated in the cases compared to the controls, suggesting that atopy is not a predisposing factor for the development of IDH in HTLV-1 infected individuals. Circulating proviral load was significantly higher in those with IDH compared to asymptomatic carriers and skin biopsy revealed acanthosis, and lymphocytic epidermotropism associated with a superficial perivascular and periadnexal lymphocytic infiltration of CD8+, and CD4+ T cells. Furthermore IDH associated with an infiltrate of epidermal and dermal FoxP3+ T cells and lesional down-regulation of filaggrin expression compared to non-lesional skin. We did not observe an elevation of pro-inflammatory cytokines in the sera of individuals with IDH compared to the controls. We investigated integration patterns in the skin and blood of 10 cases with IDH, and two asymptomatic carrier (AC) individuals from South Africa. We first showed that the virus is present in the skin at high levels (total mean levels of 47.09 proviral copies per 1000 cells) as comparable to that which has been observed in blood (total mean levels 137 proviral copies per 1000 cells). Using a high throughput Illumina sequencing system in collaboration with Professor Bangham, we mapped and quantified the relationship between oligoclonal proliferation of HTLV-1 infected T cells in the skin and blood of IDH patients. It was found that in IDH, a selective outgrowth of certain clones is favoured, supporting the possibility of skin-specific factors exerting positive selection on proliferation. In IDH, there was not a preferential integration of the provirus in transcriptionally active regions of the gene sites, as had been observed in other HTLV-1 associated conditions. These observations imply that the selection forces that favour oligoclonal proliferation of HTLV-1+ T cells differ fundamentally between simple HTLV-1 infection and other events associated with the dermatitis. In conclusion, these data show that HTLV-1 is not associated with an atopic diathesis. Given the lack of elevated pro-inflammatory cytokines and presence of a cutaneous infiltrate of FoxP3+ T cells, the findings suggest that high levels of HTLV-1 replication promotes a regulatory environment leading to filaggrin down-regulation, cutaneous susceptibility to infection, and secondary inflammatory skin disease. Viral integration patterns would support the presence of skin-specific positive selection, perhaps eventually leading to expansion of particular clones with the potential to develop towards ATLL. It remains to be explained whether the high viral load in IDH changes over time, more specifically in the steps leading to ATLL.

Pathogenic gain-of-function mutations associated with increased paternal age, albeit harmful to embryonic development, are paradoxically enriched in the normal testis. Evidence from previous studies indicates that these so-called paternal age-effect mutations confer a proliferative advantage to the spermatogonia in which they arise, leading to clonal expansion within the normal testis over time. Recently, spermatocytic seminoma (SS; a rare testicular germ cell tumour that occurs mainly in older men) has emerged as a key link between the processes of somatic and germline mutation (Goriely et al, Nat Genet. 41:1247-52, 2009), suggesting that the proposed clonal expansion events can in some cases lead to testicular tumourigenesis. In this thesis, I have used immunohistochemistry to seek evidence for putative clones of cells in the normal adult testis. To address this, a screening approach was developed using markers chosen from analysis of normal testicular tissues and SS. The ontogeny of OCT2 and SSX expression in human testis, from embryonic development to adulthood, identified distinct subpopulations of spermatogonia at different maturation stages. Together, these data reveal the potential of OCT2 as a novel marker of A_dark spermatogonia (human reserve spermatogonial stem cells). In parallel with these observations, two distinct types of SS characterised by differential OCT2 and SSX immunoexpression were identified, providing new evidence for heterogeneity of this tumour. This work provided the backdrop to the detailed immunohistochemical study of normal adult testis by characterising in serial sections the expression of five spermatogonial markers, MAGEA4, SSX, FGFR3, OCT2 and SAGE1, and a proliferation marker, Ki67. Independent sections were screened with predetermined criteria set to identify unusual positively-stained cellular clusters within the seminiferous tubules. Several antigenic combinations previously described in SS were observed in a subset of these clones, suggesting differing genetic origins and a possible link with early events of testicular tumourigenesis. The size (minimum number of cells) of each clonal event was estimated and its correlation with the staining pattern of the molecular markers was investigated. In summary, the data presented in this thesis convincingly identify for the first time the previously hypothesised clonal events in the testis using immunohistochemical markers. My research will pave the way for future work involving genetic analysis of microdissected cells from these putative clones, aimed at identifying the underlying mutational events thought to be present.

Leukocyte extravasation is mediated in part by tissue specific chemotactic cytokines (chemokines) and specific chemokine receptors expressed on the surface of circulating cells. C-C chemokine ligand CCL25 is expressed exclusively in the intestine and thymus and mediates chemotaxis by cells expressing receptor CCR9. This chemokine and receptor pair may be relevant in the pathogenesis of intestinal inflammation, in diseases such as Crohn’s disease (CD) and coeliac disease. In this thesis I investigated CCR9 expression in situ, in tissues affected by intestinal inflammation, and also examined the effects of CCR9 antagonist treatment in patients. In vitro I investigated CCR9 function using human peripheral blood T cells enriched for CCR9 by cell sorting or all-trans retinoic acid treatment. Using tissues collected as part of a clinical trial in CD testing CCR9 antagonist, CCX282-B, I investigated ways of measuring if treatment reduced the number of CCR9 expressing cells in the intestinal mucosa. However, in situ staining for CCR9 by immunohistochemistry was unsuccessful, and in this thesis, I explored reasons why this might be the case. Treatment with CCX282-B did however, show a tendency to reduce T cell density in the intestinal mucosa, although results were highly variable between individuals. In an examination of human CCR9 function in vitro I demonstrate for the first time that CCL25 stimulates CCR9 surface internalization. These data clarify the observation that CCR9 staining by IHC produces poor results in tissues where ligand is abundant, such as the intestine and thymus. I describe a novel technique for measuring calcium flux in two populations simultaneously by flow cytometry, which confirmed that in a heterogeneous population of cells, only CCR9 expressing cells respond to CCL25 by calcium flux. Variability in clinical trials is partly created by the use of concomitant medications, and in CD, corticosteroids are widely used. For the first time I show that glucocorticoids (GC) impair CCR9 mediated chemotaxis, calcium flux and intracellular signalling without changes to CCR9 mRNA and surface protein expression. Reduced CCR9 mediated signalling was accompanied by an enhanced expression and function of co-expressed CXCR4, demonstrating that the effects of GC were receptor-specific and not mediated by non-specific toxicity or inhibition of cell signalling. In a second study CCX282-B was tested in patients with coeliac disease, and in this trial, there was no reported concomitant use of GCs. It was confirmed that dietary gluten stimulates significant T cell recruitment to the intestinal mucosa with a pronounced accumulation of intraepithelial lymphocytes (IEL) and a rise in the frequency of FoxP3 expressing cells. Patients on CCX282-B had lower IEL counts, and an equivalent proportion of FoxP3 expressing T cells, suggesting that CCR9 blockade restricted the recruitment of effector T cell subsets. This thesis confirms that the accumulation of T cells is central to inflammation in the intestine and that modulating chemokine receptor function may affect this. Furthermore, this thesis demonstrates that the function of CCR9 is suppressed by GCs, which are widely used therapeutically and therefore could identify a novel mechanistic basis for their activity in CD.

Treatment of lower respiratory tract infection (pneumonia and pleural infection) relies on the use of empirical broad spectrum antibiotics, primarily because reliable pathogen identification occurs infrequently. Another consequence of poor rates of pathogen identification is that our understanding of the microbiology of these infections is incomplete. This thesis addresses some of these issues by combining the acquisition of high quality lower respiratory tract samples, free from nasooropharyngeal contamination, with novel molecular microbiological techniques in an attempt to increase rates of pathogen identification. Four main areas are examined: (i) The role of so-called ‘atypical pneumonia’ bacteria in causing pleural infection. These pathogens have been previously identified in the pleural space infrequently and routine culture usually fails to isolate such bacteria. High sensitivity nested polymerase chain reaction (PCR) is a culture-independent technique which is used to undertake a systematic evaluation for these pathogens in pleural infection samples. (ii) The role of Pneumocystis jirovecii in pleural infection, either as a co-infecting pathogen or in monomicrobial infection. This fungus causes severe pneumonia, particularly in the immunosuppressed, but is increasingly recognised as a co-pathogen in community-acquired pneumonia, and is frequently isolated in the upper and lower respiratory tract in health. A high sensitivity real-time PCR assay is used to examine for this fungus. (iii) Ultra-deep sequencing of the 16S rRNA gene is used to perform a comprehensive microbial survey in samples taken from the multi-centre MIST2 study of pleural infection. The techniques employed allow analysis of polymicrobial samples and give very high taxonomic resolution, whilst incorporating methods to control for potential contamination. Further, these techniques provide confirmation of the results from the ‘atypical’ bacteria nested PCR study. (iv) Bedside ultrasound-guided percutaneous transthoracic needle aspiration (TNA) of consolidated lung is undertaken in patients with pneumonia, as part of the PIPAP study. An evaluation is undertaken of the efficacy and acceptability of TNA. Aspirate samples acquired are also processed using ultra-deep sequencing of the 16S rRNA gene. Other samples obtained as part of the PIPAP study, such as ‘control’ lung aspirates and ‘control’ pleural fluid samples, are similarly processed to enable calculation of sensitivity and specificity of the sequencing methodology.

Enteric fever caused by Salmonella enterica serovars Typhi and Paratyphi A are a major public health concern in Kathmandu. The aim of this thesis was to identify and assess the population most at risk by investigating epidemiologic trends of enteric fever within a subset population of Kathmandu. Therefore,the burden and incidence of enteric fever within the study population and the seasonal and gender distribution of enteric fever was assessed. Considerable burden of enteric fever, unrelated to population density, correlating with the seasonal fluctuations in rainfall was observed. This thesis also aimed to improve the understanding of enteric fever transmission by identifying probable transmission routes,hence various water and food samples were analysed and the extent of faecal contamination in them was determined. S. Typhi isolates were sequenced and genotyped and combined with GPS data to longitudinally study the local distribution and infer transmission of this human restricted bacterial pathogen. Extensive clustering of typhoid independent of population size and density and existence of an extensive range of genotypes within typhoid clusters including individual households with multiple cases was observed. These observations predict that indirect transmission had an overwhelming contribution for disease persistence, potentially through contaminated water. Consistent with this hypothesis, S. Typhi and S. Paratyphi A were detected in water supplies and it was observed that typhoid was spatially associated with public water sources and low elevation. A concurrent case-control study was also conducted which allowed for the determination of risk factors in the population at risk. These studies imply that resources should be allocated toward controlling the most important vectors of enteric fever, including food sold by vendors, chlorination of drinking water, construction of proper water distribution and sewage networks,vaccination campaigns and hygiene education.