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Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes (IEs) is a common property of field isolates associated with severe disease (Pain, Ferguson et al. 2001). Platelet receptors CD36 (Pain, Ferguson et al. 2001), P-Selectin (Wassmer, Taylor et al. 2008) and gC1qR (Biswas, Hafiz et al. 2007) mediate clumping. To characterize the molecular specificities of the clumping phenotype, I cloned clumping parasite line IT/C10 by limiting dilution. I characterized var gene expression in the IT/C10 clones using generic primers for the DBL tag region (Bull, Berriman et al. 2005). Clumping assays were conducted in the presence of specific reagents to delineate host factors hypothesized to contribute to development of the clumping phenotype. Finally, I conducted a clinical study with isolates from children with malaria in Kilifi, Kenya. This study shows that in parasite line IT/C10, platelet-mediated clumping is associated with Itvar30 suggesting a prominent role for the PfEMP-1 encoded by this var gene in development of platelet-mediated clumping. For IT/C10 parasites, platelet activation appears to be involved in platelet-mediated clumping. Platelet P-Selectin appears to mediate clumping using lectin-dependent interactions. To further elucidate the mechanisms that mediate clumping by host platelets, I have used a panel of platelet antagonists to delineate specific platelet activation pathways. Our results show that platelet activation plays an important role in platelet-mediated clumping. Finally, in this study, platelet-mediated clumping was associated with parasitaemia, but not with disease severity.

Typhoid disease, caused by infection with S. Typhi, is a significant cause of mortality and morbidity in resource–poor countries. Efforts have been made to generate a new generation of vaccines that are efficacious and can be given to infants, but have been hindered by a poor understanding of the protective immune response to S. Typhi infection, and in particular by the absence of a correlate of protection. Controlled human infection studies (‘challenge studies’) provide a model for investigating infectious diseases and appraising novel vaccines, including in typhoid disease. This DPhil described the development of a human challenge model of typhoid fever using S. Typhi Quailes strain administered to healthy adults in a sodium bicarbonate buffer. The careful characterisation and manufactured of the strain is described. Following ingestion of 103 CFU of S. Typhi 55% of participants developed typhoid disease, whilst ingestion of 104 CFU gave a higher attack rate of 65%. At this attack rate vaccine efficacy against human challenge should be demonstrable with a modest sample size. Validity of the model in the appraisal of vaccines was demonstrated using Ty21a, a live, oral, attenuated vaccine. Protective efficacy of Ty21a compared to placebo against challenge was 35%, comparable to that observed in some endemic settings, and the estimated protection in the first year after vaccination in Cochrane meta-analysis. Clinical, microbiological and humoral immune responses were investigated in participants challenged during model development. Typhoid disease was associated with a high fever in most, but not all participants, and a range of symptoms. Severity of disease was variable, and included asymptomatic bacteraemia, as well as fever and symptoms in participants in whom bacteraemia could not be demonstrated. Typhoid disease was associated with a strong humoral immune response to the flagellin and lipopolysaccharide antigens of S. Typhi but not the Vi polysaccharide capsule. Humoral immune responses were not demonstrated in participants without typhoid fever. There was a dose-response relationship to the clinical, microbiological and humoral responses with participants challenged with 104 CFU having more marked responses than those challenged with 103 CFU. Future success of challenge studies relies on the willing participation of healthy adult volunteers. The motivations for participation, and experiences of participants, were appraised by questionnaire. Whilst financial compensation was an important motivator, it was not the sole motivator. Participants were positive about their experiences, and most would participate again.

ATRX is a chromatin remodeling protein associated with X-linked Alpha-Thalassemia Mental Retardation syndrome and cancers that use the Alternative Lengthening of Telomere pathway. In the absence of ATRX there is a DNA damage response associated with telomeres and the expression of certain genes are perturbed. Recent findings (Law et al, 2010 Cell) have shown that ATRX is preferentially enriched at GC-rich tandem repeats in the genome. The mechanism for this localisation is unknown but may be related to the potential for these GC-rich tandem repeats to adopt non-B form DNA structures; ATRX has been shown to bind such structures (G4) in vitro. This study aims to understand the specific factors of the repeats that signal ATRX targeting. To address the research questions, an experimental system was developed, in which known targets, the ψζ VNTR and telomere repeats, were inserted into an inducible ectopic gene in the 293T-Rex cell line by site-directed recombination. ATRX was found to be enriched at the ectopic repeats compared to an endogenous negative control suggesting that it is recruited by the repeats independent of its original context. Furthermore, ATRX enrichment increased upon transcription of the ectopic gene, and this was dependent on the orientation of the repeat with the non-template strand being G-rich. Interestingly, when the repeat was transcribed, the distribution of ATRX across the repeats was asymmetrical with most ATRX binding downstream of the repeat. Moreover, there was a direct correlation between the repeat size and level of ATRX bound: the longer the repeat the higher the increase in ATRX enrichment. To determine the signal for ATRX binding, assays were performed to look for features which reflected the distribution of ATRX including H3K9me3, RNA polII, G4, R loops and DNA supercoiling. R loops look to be a strong candidate for the signaling of ATRX binding.

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.

Improving the repopulation potential of human umbilical cord blood (UCB) haemopoietic stem cells (HSCs) remains a paramount goal in HSC transplantation (HSCT) therapy. This implies enhancing the homing and engraftment potential of UCB-CD34+CD133+ cells to the bone marrow (BM). Although an array of molecules continues to be identified as ‘key’ homing molecules, the molecular mechanisms controlling HSC homing are still not fully understood. The regulatory implications of hypoxia in the BM, with the concomitant stabilisation of hypoxia inducible transcription factor-1α (HIF-1α), are becoming more apparent, yet at the commencement of this thesis no study had explored whether hypoxia induced signalling can be adopted to regulate the homing and engraftment of transplanted HSCs. The aim of this DPhil project was thus to investigate whether hypoxic conditions as detected in the BM influence the adhesion of UBC-CD133+ cells to osteoblasts, BM stromal cells and BM endothelial cells-60 (BMEC-60), as well as their transmigration towards chemokine SDF-1α across BMEC-60. Increasing the exposure of UCB-CD133+ cells to 1.5% O2 doubled the percentage of transmigrating cells (p<0.05), and while hypoxia stimulated UCB-CD133+ cells preferentially adhered to IL-1β stimulated BMEC-60, their adhesion to non-stimulated (BMEC-60) was significantly improved (p<0.001). To help unravel the underlying molecular mechanisms, we attempted to examine the potential involvement of hypoxia regulated scaffolding protein HEF-1/NEDD9/Cas-L (HEF-1) in the increased percentage of migrating UCB-CD133+ cells after hypoxia pre-conditioning. The role of HEF-1 in HSCs is unexplored, and its multifunctional contribution in a variety of processes including cell migration, attachment and invasion make HEF-1 a prime candidate as a contributing homing molecule. After identifying a suitable short-hairpin RNA (shRNA) sequence to knockdown HEF-1, generating lentiviral (LV)-particles in house and optimising transduction protocols, HEF-1 knockdown was achieved in haemopoietic model cell lines KG-1 and KG-1A (KG-1/KG-1A–HEF1). Significantly decreased KG-1A–HEF1 cell adhesion to non-stimulated BMEC-60 was detected. Together, these studies provide a promising platform to further explore the role of HEF-1 in hypoxia induced UCB-CD133+ cell transmigration towards the key homing molecule SDF-1α.

As more patients with large body surface area burns are surviving and requiring reconstructive surgery, there is a necessity for advances in the provision of bioengineered alternatives to autologous skin cover. The aims of this Thesis are to identify feasible source tissues of Endothelial Colony Forming Cells and Mesenchymal Stem/Stromal Cells for microvascular network formation in vitro with three-dimensional dermal substitute scaffolds. The working hypothesis is that pre-vascularised dermal scaffolds will result in better quality scarring when used with split thickness skin grafts. Human umbilical cord blood, peripheral blood and adipose tissue were collected and processed with ethical approval and informed consent. Samples were cultured to form endothelial outgrowth colonies and confluent Mesenchymal Stem/Stromal Cells, which were characterised using flow cytometry and expanded in vitro. Mesenchymal Stem/Stromal Cell multipotency was confirmed with tri-lineage mesenchymal differentiation. Primary cells were tested in a two-dimensional tubule formation co-culture assay and differences assessed using a proangiogenic antibody array. Tubule formation was tested in four different acellular dermal substitute scaffolds; Integra® Dermal Regeneration Template, Matriderm®, Neuskin-F® and De-cellularised Human Cadaveric Dermis. Umbilical cord blood was the most reliable source of Endothelial Colony Forming Cells, the yield of which could be predicted from placental weight. Microvasculature dissected free from adipose tissue was a reliable source of Mesenchymal Stem/Stromal Cells which supported significantly more tubule formation than Mesenchymal Stem/Stromal Cells from whole adipose tissue. Microvasculature Mesenchymal Stem/Stromal Cells secreted significantly higher levels of the proangiogenic hormone leptin, and addition of exogenous leptin to the tubule formation assay resulted in significantly increased tubule formation. Microvasculature was cultured in all four of the scaffolds tested, but depth of penetration was limited to 100µm. The artificial oxygen carrier perfluorocarbon was shown to increase two-dimensional tubule formation and may be useful in further three-dimensional scaffolds studies to improve microvascular penetration.

Multipotent haematopoietic stem cells (HSCs) supply the organism with mature blood cells of all lineages throughout adult life. These cells first originate in the dorsal aorta (DA) of the vertebrate embryo, and a multitude of signalling pathways regulate their specification in the embryo. The emergence of HSCs is dependent on appropriate arterial specification and vessel maturation, processes which are heavily dependent on Notch signalling. This arterial involvement of Notch obscures its later roles in HSC specification. The Notch ligand dll4 is crucially involved in arterial development in the mammalian embryo, while zebrafish embryos deficient for dll4 activity only exhibit minor arterial defects at the time of HSC emergence. Here, the zebrafish model has been exploited to reveal the first specific evidence for a role of dll4 in HSC specification. Dll4 is required for the expression of runx1, a transcription factor (TF) required for HSC specification, prior to any observed effects on vascular development. HSCs and all their derivatives are depleted in dll4 morphants. To disentangle the genetic requlatory cascade downstream of dll4 and upstream of runx1, RNA-seq was employed to discover downstream effectors of this signalling. Expression and functional screening of best candidate genes revealed seven genes with novel roles in HSC development. Foxc1b is a dll4 target predominantly mirroring the dll4 phenotype, and is thus likely to be the downstream effector of dll4, upstream of runx1. Interestingly, foxc1b also has a later dll4-independent role in HSC development, remarkably similar to that of cmyb. Taken together I show here for the first time a requirement of dll4 upstream of runx1 in HSC specification, mediated by foxc1b, followed by a later dll4-independent phase in HSC development.

Various diseases, including cancer, stroke and heart attack, are associated with disruption of the vascular system. However, lack of a profound understanding of the transcription regulation during vascular development hinders the formation of effective molecular intervention strategies targeting angiogenesis. Here we describe an enhancer of Neuropilin1 (Nrp1) from the second intron of the gene that directs arterial and coronary endothelial cell-specific expression. Mice transgenic for either human or mouse sequences of the Nrp1in2 enhancers drove expression of the LacZ reporter gene specifically in the endothelial cells within the arterial compartment from early in development, while no expression was detected in veins. In addition, the hNrp1in2 enhancer directed expression to the endothelial cells in the developing coronary vasculature, with the initial expansion from around the sinus venosus at E11.5, and eventually contributed to the capillary, venous and arterial compartments of the coronary vessels but not the endocardium. This expression pattern is consistent with that reported in the Apelin-nlacZ line (Red-Horse et al., 2010), making the Nrp1 enhancer the first identified mammalian regulating enhancer of the coronary endothelial cell. Phylogenetic footprinting, and a tissue culture reporter assay suggested that this enhancer contains a 184bp minimal core region hNrp1in2peakA2 that recapitulates the expression profile of the full length enhancer. hNrp1in2peakA2 has conserved and in vitro validated recognition sites for Gata, Ets, and Fox. The validated Fox and Ets sites form a functional FOX:ETS motif, and the FOX:ETS motif is responsible for synergistic activation ofthe enhancer by FoxC2 and Etv2 in reporter assays. Mutation introduction to the functional Ets sites or compound ablation of the Gata and Fox site in hNrp1in2peakA2 result in total loss of vascular expression, in terms of both arterial and coronary expression. The Fox, Ets and Gata recognition sites may be sufficient to achieve arterial- and coronary- specific expression of the hNrp1in2peakA2.

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.

DNA methylation is an epigenetic modification that plays an important role in development and differentiation. The patterns of DNA methylation are largely established in early embryogenesis and maintained during development. Abnormal DNA methylation patterns have been associated with many human diseases, including cancer. Despite its importance, little is currently known about the mechanisms that determine DNA methylation patterns throughout the genome. To shed light on the molecular mechanisms that regulate DNA methylation, this study investigates whether DNA methylation patterns are established and maintained normally when human DNA is placed into a heterologous murine environment as opposed to its natural, endogenous chromosomal environment. Here, a previously generated transgenic mouse model, containing 117 kb of human DNA bearing the human α-globin cluster and all of its known regulatory elements, was analysed. The pattern of DNA methylation of the endogenous human α-globin cluster was compared with that of the transgenic cluster in the background of mouse embryonic stem cells (ESCs) and tissues. It was found that, although the normal human DNA methylation pattern was largely established and maintained in a mouse background, the region immediately around the human α-globin genes themselves is generally less methylated in mouse compared to human ESCs. It was found that regions adjacent and up to 2kb from the CpG islands (CGIs), so-called CGI shores, were unusually hypomethylated: this seems to be the result of an extension of CGIs in humanised mouse (hm) ESCs compared to human (h) ESCs. Furthermore, this hypomethylation appeared to increase during development in both erythroid and non-erythoid cells. To identify any cis-regulatory sequences responsible for the hypomethylated state of human CGI shores in the mouse, 2-4 kb human test sequences containing the CGI associated with the human α-globin 2 (α2) gene and its adjacent hypomethylated shore were re-integrated into the mouse α-globin locus via recombination-mediated cassette exchange (RMCE). Human CGI shores became hypomethylated in the context of the re-integrated test sequences, indicating that the appearance of hypomethylation is determined by the underlying human DNA sequence in the test fragments. In summary, the data presented here reveal that human CGIs become extended when placed in a mouse background leading to hypomethylation of human CGI shores in the mouse compared to the pattern of methylation at the normal endogenous human locus. These findings suggest that species-specific factors determine DNA methylation near CGIs. The transgenic mouse model provides an excellent system to dissect out species-specific regulation of CGI shore methylation. Furthermore, this study lays the foundation for future experiments addressing the role of DNA methylation in regulating human gene expression in the murine context, and examining the validity of transgenic mouse models for the study of human gene regulation.

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.

The engraftment of haemopoietic stem/progenitor cells (HSPCs) from umbilical cord blood (UCB) into adult recipients, although advantageous in terms of sourcing units, the decreased need to match donor and recipient and reduced risk of graft versus host disease (GvHD), is delayed compared to grafts using HSPCs from mobilised peripheral blood (MPB) or bone marrow (BM). One reason for this is the limited number of HSPCs (CD34+/CD133+ cells) in a unit of UCB compared to MPB or BM. The CXCR4-CXCL12 axis is widely recognised as a key player in the bone marrow homing, retention, and engraftment of HSPCs. The aim of this thesis was to investigate whether the engraftment of HSPCs from UCB into the bone marrow could be improved. Firstly, a novel in vitro 3D time-lapse chemotaxis assay to assess the homing capacity of human UCB CD133+ HSPCs, towards the chemokine CXCL12 was developed. One advantage of this assay was that it distinguished cell chemotaxis from chemokinesis and allowed these parameters to be quantified. Human UCB CD133+ HSPC chemotaxis towards CXCL12 was inhibited by the CXCR4 antagonist, AMD3100. Importantly, the presence of CXCL12 or AMD3100 had no affect on cell chemokinesis. To complement the in vitro chemotaxis assay, a short term in vivo homing assay in NSG mice was successfully established. The effect of siRNA silencing of the CXCR4 co-receptor, CD164, which is also expressed on CD133+ HSPCs, on cell migratory and homing ability was investigated. CD164 knock-down using siRNA in human UCB CD133+ HSPCs did not demonstrate an effect on homing to NSG bone marrow in vivo or chemotaxis to CXCL12 in vitro. However, homing to NSG mouse spleen was significantly reduced in cells silenced for CD164. Following this, an 8 day HSPC expansion system using nanofibre scaffolds (Nanex) and differing cytokines was investigated. These serum and feeder free conditions yielded a significant expansion of cells that retained CD133+CD34+ expression and their in vitro chemotactic ability to CXCL12. Time constraints did not permit the engrafting ability of these cells to be analysed in an in vivo HSC reconstitution assay that was initiated. However these studies will provide the basis to support future related research in this laboratory.

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.

The Lon protease is a mitochondrial matrix quality-control protease belonging to the family of AAA+ proteins (ATPases associated with many cellular activities). We had previously found Lon to be upregulated in lung tumours with a non-angiogenic phenotype in a microarray study comparing these to conventional angiogenic tumours. In this project I set out to investigate whether Lon had any role in modulating the hypoxic response of tumour cells. Using a novel monoclonal antibody against Lon, I found that upregulation of Lon was present in breast and lung tumours and that higher levels of Lon are correlated with shorter overall survival in breast cancer patients. Targeting Lon with siRNA and shRNA in tumour cell lines reduced the normoxic and hypoxic stabilisation of HIF-α subunits. This is mediated through a mechanism independent of the activity of HIF-prolyl hydroxylases and independent of any changes in mitochondrial transcription. I found that the pre-imported form of Lon could bind and chaperone VHL in the cytoplasm potentially modulating VHL activity. In cell lines and human tumours, I observed that the proline-hydroxylated form of HIF-1α is induced by hypoxia and the hydroxylated form of HIF-1α is associated with shorter overall survival in breast cancer patients. This observation supports the notion that higher levels of Lon is associated with poor survival by downregulating VHL leading to higher levels of hydroxylated HIF. Finally I show that targeting Lon in cell lines is able to inhibit growth in a cell-line dependent fashion and partially reverses the Warburg effect, increasing oxygen consumption and reducing lactate production. In conclusion, I have demonstrated the broad therapeutic potential of targeting the Lon protease in tumours and highlighted a mechanism of post-hydroxylation HIF-regulation that has not been previously recognised in VHL competent tumours.

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.

Umbilical cord blood (UCB) is used as a source of haematopoietic stem cells (HSCs) for transplantation but shows defective homing to the bone marrow niche and delayed haematological reconstitution. Following transplantation, HSCs will home to the bone marrow in response to the CXCL12 chemokine, adhere to the bone marrow sinusoidal endothelial cells and then migrate into and lodge in bone marrow niches. In addition to CXCR4, a variety of molecules have been described as being important in these processes. In this laboratory, junctional adhesion molecule-A (JAM-A) was shown to be expressed on human UCB CD133⁺/CD34⁺ cells and regulated by hypoxia. In this thesis, further phenotypic studies show that this molecule is most highly expressed on human CD41a⁺ megakaryocytes and CD14⁺ monocytes/macrophages in UCB. JAM-A was also found to be expressed on all human UCB CD133⁺ cells, which have been shown by others to encompass the HSCs and early myeloid-lymphoid precursors and on the majority of CD34⁺ haematopoietic progenitor cells (HPCs). While it is also present on bone marrow sinusoidal endothelium (BMEC), JAM-A is not detected on cultured bone marrow mesenchymal stromal cells (MSCs). JAM-A blockade, silencing and overexpression experiments showed that JAM-A contributes to, but is not solely responsible for, the adhesion of CD34⁺ haematopoietic progenitor cells to IL-1β activated BMEC-60 cells and fibronectin. Lack of significance in cell migration suggested that JAM-A is more likely to act as an adhesion molecule or a regulator of adhesion rather than as a migratory molecule in such cells. Further functional studies using the proximity ligation assay highlight a potential association of JAM-A with CXCR4 and the adhesion molecules, tetraspanin CD82 and integrin β1. Mechanistic studies were commenced to establish if JAMA could modulate CXCR4 signalling following CXCL12 stimulation, but time constraints prevented these from being completed. These preliminary experiments which were carried out first in the Jurkat cell line lacking JAM-A or transduced to express JAM-A, however, suggest that JAM-A may modulate CXCL12-induced Rap1 phosphorylation and ERK1/2 phosphorylation. The former pathway is important for integrin function and the latter pathway is important in cell adhesion. The results described here, although requiring finalisation, support the hypothesis that JAM-A acts as an adhesion molecule and also may fine tune CXCR4 and integrin mediated functions on human CD34⁺ cells, thereby potentially regulating engraftment of these cells to the bone marrow niche.

Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies against fetal platelet antigens inherited from the father and which are absent from maternal platelets. In Caucasians, antibodies against the Leu33 (HPA-1a) polymorphism of integrin β3 (part of the platelet αIIbβ3 complex) account for >70% of cases. Antenatal screening for these antibodies does not currently take place in the UK, partly because of the absence of sensitive, predictive tests. We hypothesized that the poor sensitivity and predictive abilities of current assays are due to the use of β3 in an inappropriate conformation, resulting in sub-optimal binding of HPA-1a antibodies. We hypothesized firstly that in vitro induced changes to αIIbβ3 might alter accessibility of the HPA-1a epitopes to alloantibodies, thus reducing assay sensitivity. Secondly, we hypothesized that HPA-1a antibodies are stimulated by, and preferentially recognise, β3 in association with αv, a molecule present on placental syncytiotrophoblasts, and that reactivity against platelet αIIbβ3 reflects only cross-reactivity with αvβ3. Our first hypothesis was proven by demonstrating that use of the cation chelating compound EDTA, used by many diagnostic laboratories as a component of assay reagents or present in blood samples as anticoagulant, resulted in significantly reduced assay sensitivity. These findings were confirmed in an international workshop. Support for our second hypothesis was provided by demonstrating enhanced reactivity of a small panel of examples of anti-HPA-1a against αvβ3 compared to αIIbβ3 and by molecular modelling data. We also showed that HPA-1a antibodies can inhibit platelet function by using a novel application of the ROTEM® delta thromboelastograph and an immunofluorescence assay in which we demonstrated blocking of platelet function using a monoclonal antibody, PAC-1, that binds only to activated αIIbβ3. These studies provide possible explanations for the poor sensitivity and predictive abilities of current assays and suggest further areas for research.

Acute Myeloid leukemia (AML) is an aggressive haematological malignancy that mainly affects the elderly. Relapse is common and is thought to be due to the presence of chemotherapy resistant leukemic stem cells (LSC). Within the CD34+ disease (>5% of the blast cells expressing CD34) , two subtypes have been identified; an LMPP/GMPlike expanded type and a MPP/CMP-like expanded type, the former is the most common, accounting for around 80% of CD34+ AML. Both the GMP-like and LMPPlike expanded populations show LSC activity. To improve our understanding of the disease and gain better insight in to how to develop treatments, the molecular basis of the disease needs to be investigated. I investigated miRNAs in the GMP/LMPP-like expanded AML. miRNAs are small non-coding RNAs involved in the regulation of mRNA. In recent years miRNAs have been shown to be implicated in many different diseases. To investigate the role miRNAs play in AML, miRNA expression was profiled in leukemic and normal bone marrow. Bioinformatic analysis was then used to examine the different miRNA expression profiles between normal and leukemic marrow. Our study showed that miRNAs are dysregulated in AML. miRNAs from the miR-17-92 and its paralogous cluster miR-106b-92 were amongst the miRNAs to be found down regulated in AML As had been seen previously at an mRNA level, on an miRNA level the LSC populations more closely resembled more mature progenitor populations than HSC and MPP populations, however the LSC populations did display an aberrant stem cell-like miRNA signature.

The central problem for researchers of HIV-1 evolution is explaining the apparent design of the virus for causing pandemic infection in humans: understanding how HIV-1 spreads is key to halting the pandemic. Current knowledge of how HIV-1 spreads from host to host is based upon experimental observation and indirect inferences informed by theory. The hypothesis of this thesis is that diversity of HIV-1 around the time of transmission is important for viral adaptation to a new human host, rather than intrinsic superiority of particular strains found in infectious fluids from human donor hosts, and that studying recombination is important for understanding this behaviour. To demonstrate the apparent randomness of transmission, I test the null-hypothesis that hard selection accounts for between-host viral divergence in a rare case study of contemporaneous infection. I explain how the experimental data that I have generated and the analyses I have carried out address certain basic assumptions and predictions about HIV-1 transmission and may inform current strategies for vaccine design. Specifically, my approach contributes to the current literature on HIV-1, by investigating an alternative hypothesis to the single virion theory of sexual transmission and by characterizing the role of recombination in a pseudodiploid virus following multiple-infection.

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.

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.

It has been demonstrated that retinal photoreception among mammals extends beyond rods and cones to include a small number of intrinsically photosensitive retinal ganglion cells (pRGCs), which are capable of responding to light due to expression of the melanopsin (OPN4) photopigment. OPN4 may have therapeutic potential if ectopically expressed in the degenerate retina in cases where photoreceptors are lost, but the other molecules involved in this light induced transduction cascade are less well characterized. Therefore I sought to probe further the mechanism of OPN4 mediated light sensitivity by siRNA mediated knock down of specific molecules in two mice models in which complete loss of rods and cones renders them almost exclusively dependent on the OPN4 pathway for light sensitivity. I generated siRNA probes against the long transcript variant of murine Opn4 mRNA and first tested these probes on the murine Neuro2A (N2a) cell line, before assessing effects in C3H/HeN rd and rodless/coneless rd/rd cl mice. siRNA was injected intravitreally into one eye and pupillometry was assessed, combined with molecular analyses at various timepoints. Reverse transcription polymerase chain reaction (RT-PCR) analysis in N2a cells confirmed Opn4 knockdown and immunolabelling techniques identified >85% silencing with siRNA. Pupil responses in the rd and rd/rd cl mice were inhibited by the siRNA injections in vivo which confirmed the functional effect of Opn4 silencing detected by molecular analysis. I therefore present a novel reproducible in vivo model in which siRNA induced silencing of the melanopsin pathway can be assessed by pupillometry and compared to levels of mRNA and protein at specific timepoints. Probes against other putative candidate genes, such as TRPC3, may unravel the molecular interactions of this pathway. This may help in future to induce light sensitivity in other retinal neurons in patients who are completely blind from photoreceptor loss.

Melanoma is the most aggressive form of skin cancer and its incidence has risen more than any other cancer in the United Kingdom. Surgery is the mainstay of treatment for localised and regional disease. Hitherto prognosis for metastatic melanoma has been poor, with a median life expectancy of less than 12 months. However, in the last decade there have been considerable molecular advances leading to greater understanding of tumour biology and molecular characterisation of tumours. These developments have highlighted the heterogeneous nature of melanoma and provided a basis for personalised therapy. This thesis presents the clinical use of surgical (sentinel lymph node biopsy) or molecular (genetic mutation screening) techniques in Oxford to stratify melanoma patients into subgroups. The use of sentinel lymph node biopsy for patients with thick (greater than 4mm) primary tumours predicted overall and recurrence free survival. Screening for BRAF and NRAS mutation in patients with metastatic melanoma demonstrated associations between clinical and genetic characteristics, and differences in clinical outcomes. Identifying subgroups through genetic screening enables targeted therapy. This thesis further investigated the role of phosphatidylinositol-3-kinase (PI3K) inhibitors in melanoma cell lines as pro-senescence therapy. Inhibition of the PI3K axis showed an increased in DNA damage; a marker for senescence. The PI3K pathway was shown to regulate the anti-senescent factors TBX2 and TBX3. Evaluation of transcriptional activity indicated cooperation between the T-box and Forkhead factors, raising the prospect of combination therapy in metastatic melanoma. The management of melanoma is evolving. Following substantial molecular advances, a new generation of targeted therapies have emerged, demonstrating clinical responses in patients that would have been inconceivable a decade ago with conventional therapies. In this progressive era of targeted therapies, it should not be assumed that all melanomas respond or progress in a similar manner. Therefore personalised investigations and therapies should be offered to melanoma patients in order to improve clinical outcomes.

The alternative road map describes the separation of lympho-myeloid and myeloid-megakaryocyte-erythroid (myeloid-Mk-E) lineages as the earliest haematopoietic commitment event. However, a number of aspects of this lineage restriction process remain poorly understood. Herein this work identified a lympho-myeloid restricted progenitor in the embryo, which resembles the adult LMPP, and demonstrated that lymphoid lineage restriction is initiated prior to definitive haematopoiesis, much earlier than previously appreciated. In vivo fate mapping showed that lympho-myeloid progenitors significantly contribute to steady state myelopoiesis in the embryo. The early thymic progenitor (ETP) as most primitive cell in the thymus was characterised and demonstrated to sustain B, T and myeloid but not Mk potentials at the single cell level. The ETP therefore largely resembles the cellular properties of lympho-myeloid progenitors in bone marrow and foetal liver, which points to these cells as candidate thymus seeding progenitors (TSP). Furthermore the existence of a putative Mk progenitor was explored within the LSKCD150+CD48+Gata1pos compartment of a Gata1 reporter mouse providing the basis for a future prospective characterisation. Finally, this work evaluated the earliest lineage restriction of von Willebrand factor (Vwf)-EGFP+ and EGFP- haematopoietic stem cells (HSCs) through in vitro paired daughter fate mapping. Single Vwf+ HSCs showed heterogeneous Mk priming and more frequently sustained Mk potential after cell division. Moreover, analysis of lineage priming between daughter cells revealed the asymmetric expression of key lineage determinants and stem cell regulators, which might be employed as reporters for future fate mapping studies.

During embryonic development, precursor cells commit to specific cell fates in response to environmental cues through the establishment of lineage-specific gene expression programmes. Transcription factors are important downstream effectors of signalling pathways that initiate and maintain cell fate decisions. The haematopoietic transcription factor SCL (TAL-1) is an essential regulator of embryonic blood development. However, the exact stage at which SCL is required, its mechanisms of action, and its genomic targets are poorly understood. Characterising, jiow SCL functions - , during haematopoietic development will provide insights into how stern cells are specified. Using the embryonic stem cell/embryoid body (ES/EB) system to model early mouse development, we describe a critical role for SCL in mesoderm patterning. SCL is first expressed in PDGFRa+ FLK1+ mesoderm populations which contain lateral, paraxial and cardiac precursors. Through loss- and gain-of-function studies, we show that SCL drives lateral mesoderm specification and activates the haematopoietic programme in a direct DNA-binding independent manner, while actively repressing alternative mesodermal fates, specifically cardiac development, in a DNA-binding dependent manner. At a molecular level, we have identified direct genomic targets of SCL in Flk-1 + mesoderm populations. These include haematopoietic and cardiac transcription factors, cardiac-specific structural proteins, signalling proteins and general transcriptional repressors; thereby strengthening the dual function of SCL in mesoderm patterning. Finally, we have shown that the cardiac transcription factor GATA4 acts in a reciprocal manner, specifying cardiac precursors while repressing a lateral mesoderm fate. Collectively, this implicates SCL as a critical transcriptional regulator of cell fate decisions in early mesodermal precursors, employing distinct molecular mechanisms to impose a blood programme. Moreover, and extending earlier reports, we document the existence of an antagonistic cross-talk between haematopoietic and cardiac lineages during mesoderm patterning. In conclusion, this work offers a cellular and molecular platform to begin to dissect the network of genetic interactions involved in these developmental processes.

The role of Notch in cell fate determination and lineage restriction in the bone marrow (BM) is controversial in the field. Recent studies have convincingly shown that Notch is dispensable for haematopoietic stem cell (HSC) regulation in adult haematopoiesis (Maillard et al., 2008). In contrast, Notch signaling has been proposed to be of importance in the regulation of BM megakaryocyte progenitor differentiation, based on dominant negative genetic approaches, identifying a potentially distinct role for Notch in adult BM haematopoiesis (Mercher et al., 2008). Here, I found that by selectively ablating the gene coding the transcription factor recombination signal-binding protein J kappa (RBP-Jk), to which all canonical Notch signaling converges, canonical Notch signaling does not mediate HSC maintenance, neither in steady state nor in conditions of stress. Furthermore, I propose, in contrast with previous studies (Mercher et al., 2008), that canonical Notch signaling plays no role in myeloerythropoiesis cell lineage commitment in the BM. My data also show that key Notch target genes are suppressed by RBP-Jk, as their expression is unaffected in Notch1-deficient BM progenitors, while target genes are upregulated in Rbp-Jk-deleted megakaryocyte and erythroid progenitors. This establishes for the first time in mammalian cells in vivo, that Notch target genes are kept in a suppressed state by RBP-Jk, potentially restricting T cell commitment to the thymus and not to the BM, at the expense of myeloerythropoiesis. Notch signaling and GATA3 are two master regulators in T cell commitment (Han et al., 2002; Ho et al., 2009; Pui et al., 1999; Radtke et al., 1999; Zhu et al., 2004). However, although very well established as being involved in the thymic stages of T cell restriction, there is little evidence of Notch and GATA3 being involved in the migration of a thymus settling progenitor (TSP) from the BM to the thymus or in the establishment of the earliest thymic progenitor (ETP) in the thymus. From this thesis work, I conclude that Notch signaling is essential for the emergence of ETPs in the thymus in a NOTCH1-independent manner. Moreover, I demonstrate, as supported by a very recent published study (Hosoya et al., 2009), that GATA3 is important for the development of the earliest T cell progenitor. GATA1 and GATA2 mediate haematopoietic stem cell maintenance in the BM. GATA1 is required for erythropoiesis, megakaryocytes and eosinophils while GATA2 is important for the proliferation and survival of HSCs. In contrast, a role for GATA3 in the BM has never been established. By using a Gata3-conditional knockout mouse model, I demonstrate that GATA3 is dispensable for HSC maintenance in steady state and following active haematopoietic regeneration as well as for HSC self-renewal in the BM.

Replication-deficient adenovirus (Ad) and modified vaccinia virus Ankara (MVA) vectors expressing single Plasmodium falciparum antigens can induce potent T cell and antibody responses and have entered clinical testing using a heterologous prime-boost immunisation approach (Ad_MVA). This thesis describes a number of pre-clinical approaches aimed at enhancing the efficacy of these viral vectored vaccines targeting the blood-stage of malaria. First, the development of a highly efficacious malaria vaccine is likely to require a multi-antigen and/or multi-stage subunit vaccine. The utility of an Ad_MVA immunisation regime combining vaccines expressing the 42kDa C-terminus of the blood- stage antigen merozoite surface protein 1 (MSP142) and the pre-erythrocytic antigen circumsporozoite protein (CSP) in the P. yoelii mouse model was investigated. It was found that vaccine co- administration leads to maintained antibody responses and efficacy against blood-stage infection, but reduced secondary CD8+ T cell responses and efficacy against liver-stage infection. CD8+ T cell interference can be minimised by co-administering the MVA vaccines at separate sites, resulting in enhanced liver-stage efficacy. The mechanisms of CD8+ T cell interference were explored. Second, Ad_MVA regimes expressing blood-stage antigens that can protect against P. chabaudi and P. yoelii blood-stage infection were tested against P. berghei, but did not confer protection. Similarly, IgG from rabbits immunised against P. falciparum MSP1 (PfMSP1) could not protect mice from a chimeric P. berghei parasite expressing PfMSP1. Third, two molecular adjuvants, the C4bp α-chain oligomerisation domain (IMX108/313) and the Fc fragment of murine IgG2a, were tested for their ability to enhance immunogenicity of recombinant adenoviruses when fused at the C-terminus of a blood-stage antigen. IMX108/313 was found to adjuvant T cell responses of small (< 80kDa) antigens and this was associated with antigen oligomerisation. However, the Fc fragment did not adjuvant responses. Finally, it was found that using a strong early promoter to drive antigen expression enhances the immunogenicity of single administration MVA vaccines, but that this did not enhance post-boost immunogenicity in an Ad_MVA regime.

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.

Drug resistance is a significant challenge in the fight against malaria. Importantly, reduced efficacy has been reported against artemether (ATM)/Lumefantrine (LM) (LM-ATM), amodiaquine (AQ)/artesunate (AS) (AQ-AS), two important combination treatment regimens in Africa, and against piperaquine (PQ), a drug which has been evaluated as a potential alternative in Africa, in combination with dihydroarteminisin (DHA). Chloroquine (CQ) resistance in P.falciparum is associated with two main transporters PfCRT and PfMDR1. I investigated the mechanisms of resistance to PQ, LM and AQ, with the overall goal of identifying molecular markers that can be used to track resistance. I used CQ as a reference. The key antimalarial drugs were highly active against clinical isolates from Kilifi, Kenya with median inhibitory concentrations (IC50s) of <5nM for DHA and <55 nM for CQ, AQ, PQ, LM and DEAQ (desethylamodiaquine, the active metabolite of AQ). pfcrt-76 and pfmdr1-86 mutations were associated with AQ, DEAQ and LM but not DHA or PQ activity. Interestingly, > 20% of analysed isolates had decreased susceptibility to LM (IC50 >100nM); these isolates were the most susceptible to CQ and carried wild type genotypes at pfcrt-76 and pfmdr1-86. I observed that CQ resistance had been declining in Kilifi since 1993 (prior to CQ withdrawal) to 2006 (7 years after its withdrawal), similar to observations in Malawi. My results support the hypothesis that susceptibility to antimalarial drugs returns when drug pressure is removed, and suggest that the use of LM-ATM may hasten the return of CQ susceptibility. Continued monitoring of drug susceptibility is crucial. pfcrt-76 and pfmdr1-86 may be useful molecular markers of LM-ATM efficacy in Kilifi and other African sites. Using a microarray approach, I identified additional genes (including various transporters) that may contribute to LM resistance. I recommend further studies to clarify the exact roles of the identified genes.

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 Adark 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.

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.