Induced pluripotent stem cells (iPS cells) have shown great promise in the newly-developing field . of regenerative medicine. Recently, however; there have been conflicting reports regarding their immunogenicity, even when derived in an autologous fashion, due to the ectopic expression of certain developmental antigens. Dendritic cells (DC) are professional antigen presenting cells and therefore serve as potential tools for modulating the immune response, particularly with respect to acceptance of stem cell-derived tissues. We hypothesised that DC differentiated from iPS cells, so-called iPS, may have the capacity to induce tolerance to tissues differentiated from the same parent iPS cell line. As a first step towards this goal, we have derived iPS cells from both embryonic and adult mouse fibroblasts, developed protocols for their differentiation along the DC lineage and characterised the resulting DC both phenotypically and functionally, using bone-malTow derived-DC (bmDC) for comparison. Furthermore, their potential for inducing tolerance as a function of regulatory T cell induction has also been explored. Our data suggest that, although ipDC are able to function in a similar manner to bmDC both in vitro and in vivo, they display important differences with respect to cytokine production and MHC class II expression which may reflect their early embryonic origin and render them pro-tolerogenic. One application for this novel population of DC might be the induction of tolerance to autologous iPS cell-derived tissues. In order to assess the need for tolerance under such circumstances, we have investigated the reported immunological rejection of iPS cells when transplanted syngeneically. Although we were unable to coroborate previous findings showing immunological rejection of iPS cells under such circumstances, we were able to show that the formation of teratomas from iPS cells is a stochastic and very variable process, unlike their generation from ES cells. Importantly, we were able to exclude the involvement of any antigen-specific component to the failure of teratoma survival by inducing blanket tolerance to iPS cell-derived tissues, using a cocktail of non-depleting monoclonal antibodies specific for CD4, CD8 and CD40L. This is the first repOlted study deriving dendritic cells from mouse iPS cells and demonstrating their extensive characterisation. Additionally, the work presented in this thesis adds to the growing body of evidence of the immunological propelties of iPS cells with our work demonstrating that they appear not to attract an adaptive immune response but, however, show significant heterogeneity with respect to their ability to engraft in vivo.

Introduction: Mortality from acute bacterial meningitis (ABM) in sub-Saharan adults is 50-60%, twice that in well-resourced settings. To date, interventions designed to impact on outcome have been ineffective in this setting. This thesis addressed the hypothesis that high mortality is due in part to delayed or inappropriate care, and that this may be improved by goal directed resuscitation. Methods: Clinical and laboratory surveillance data from a large central hospital in Malawi were analysed. Clinical predictors of poor outcome were synthesised into the Malawi Adult Meningitis Score (MAMS). To assess feasibility and outcome, patients with suspected ABM were recruited in the emergency department and observed in year one under routine clinical care (Phase 1), and then managed with a meningitis-specific EGDT clinical care bundle in year two (Phase 2). Laboratory data were tested for outcome associations. Results: A significant decline in the total number of CSF isolates over 12 years was noted, entirely in children under 5 years of age coinciding with Hib vaccination. Adult meningitis incidence remained unchanged, despite significant ART provision. Analysis of 715 historical meningitis episodes showed that the mortality rate was 54% at day 40; HIV seroprevalence was 87% and treatment delays were marked. Coma, seizures, tachycardia and anaemia but not HIV were significantly associated with mortality. The MAMS predicted outcome with good agreement, estimated sensitivity was 74%, specificity was 55%. EGDT was found to be feasible, more clinical targets were met using EGDT including reduced time to antibiotics, iv fluids, blood transfusion and airway placement. There was no significant difference in outcome at 40 days between the two cohorts. Neither high pneumococcal load nor the presence of EBV co-infection in the CSF were associated with poor outcome. CSF white cell counts were lower in non-survivors, other markers of inflammation were not associated with outcome. Conclusion: The incidence of bacterial meningitis is not falling in adults in Malawi. Important clinical predictors were identified and synthesised into a prediction tool. EGDT for meningitis is feasible in adult patients in Malawi; a larger trial is required to test the impact on outcome. Further work to improve pre-hospital care and identify novel adjunctive treatments is necessary.

Recreating the 3D spatial environment of the eNS allows neural cells in vitro to behave more like their counterparts in vivo, providing robust and controllable model systems that mimic key aspects of the cell biology of the nervous system. A simple, consistent and physiologically relevant model system, which uses a multi-well plate format and can potentially be used at a scale suitable for commercial R&D, has been developed. The model uses an engineered neural tissue which is prepared by a process of initial glial cell self-alignment within a tethered 3D collagen hydrogel and subsequent stabilisation of the gel. Stabilisation is achieved using RAFT technology which entails partial removal of interstitial fluid thereby increasing matrix and cell density. To establish viable production technology for the manufacture of eNS tissue models, the parameters that govern glial cell self-alignment were optimised via development of an assay system that requires a small number of cells. A CNS eo-culture system suitable for widespread adoption will require various combinations of cells to suit specific neuroscience research requirements. Both primary neuronal and glial cell types, relevant cell lines and stem cells were incorporated within engineered neural tissues and then assessed using a range of measures including neural cell survival, morphology, differentiation and sensitivity to stabilisation. In a bid to determine the limitations of these 3D eNS model, neuron-glial interactions, markers for myelination, electrophysiological responses and glial cell behaviour in response to injury and insult were also investigated. Initial studies reveal the new model system can be scaled down to facilitate increased throughput, be assembled quickly and reliably using various neural cell sources, and the eo-cultures exhibit characteristic behaviours that mimic in vivo scenarios.

This thesis investigates whether Oogonial Stem Cells (OSCs) exist and can be isolated from the adult mouse ovary. It also examines their ability to differentiate into oocyte-like cells in vitro. Transmembrane domains in the DEAD-box polypeptide 4 (DDX4) – the germline marker used in this antibody-based isolation – are examined using in silico protein modelling. The specificity of this antibody to DDX4 is tested in male and female germline cells. It is shown that the DDX4 antibody may have some specificity for DDX4, but the existence of a surface-bound DDX4 is unlikely. OSC-like cells can be isolated from the ovary using the DDX4 antibody by Fluorescence-Activated Cell Sorting (FACS). However, gene expression analysis and protein immunofluorescence show that these cells do not initially express DDX4 or possess germline identity. Despite this, they acquire some pre-meiotic and oocyte-specific markers in culture, including DDX4. Critically, the cells never express meiosis-specific markers even in the presence of meiotic enhancers BMP4 and retinoic acid. It is unlikely that these ovarian cells are being sorted by means of a cell surface DDX4 expression, because another antibody to a larger DDX4 epitope fails to detect DDX4 in isolated cells. These findings highlight that freshly isolated OSCs are not germ stem cells, and are not being isolated by their DDX4 expression, but instead may reflect an artefact of the antibody used or procedure adopted. Altogether this work offers support to the established dogma that the adult ovary is populated at birth by a fixed number of oocytes, and that adult de novo production is a rare or insignificant event.

Haematopoietic stem cell transplantation (HSCT) has become a central treatment modality in the management of various hematologic malignancies, but it is not without treatment sequelae. The major complication of HSCT is acute or chronic graft-versus host disease (GvHD). GvHD is an immunologically mediated disease that contributes substantially to transplant-related morbidity and mortality. One reason for the lack of progress in the treatment of acute GvHD (aGvHD) is the lack of reliable biomarkers. There is a need to develop diagnostic tools that can identify patients who are at higher risk of aGvHD progression following allogeneic HSCT and predict GvHD occurrence before clinical symptoms manifest. During the past decade, many reports have identified genetic variants such as single nucleotide polymorphisms (SNPs) that influence the risk of aGvHD after allogeneic HCT. In addition, since miRNAs are key regulators of gene expression, miRNA-related SNPs including SNPs in miRNA genes and target sites may function as regulatory SNPs through modifying miRNA regulation to affect the phenotypes and disease susceptibility. Firstly, this study investigated the impact of rs2910164 and rs2431697 in miR-146a, rs3027898 in IRAK1 and rs10511792 in MICA for their association with HSCT outcome and showed that there was a significant association between carrying the C variant in rs2910164 in miR-146a and an increased non relapse mortality (NRM) post-HSCT. For rs2431697 in miR-146a, the presence of the T allele was associated with a trend towards an increased NRM in patients post-HSCT. In the case of rs3027898 in IRAK1, the C allele was associated with a decreased risk of relapse in patients which was more apparent when patients were homozygous for the C allele. For rs1051792 in MICA, this study showed that the MICA-129 Met variant was significantly associated with low overall survival (OVS) post-HSCT, which was more apparent in the group of patients receiving non-TCD treatment. This study also revealed that the presence of the MICA-129 Met allele in patients was significantly associated with an increased risk of relapse and the presence of the MICA-129 Val variant in patients was significantly associated with an increased risk of developing aGVHD post-HSCT. Investigation of gene expression and the protein levels of MICA in the GI tract showed that there was a significant association between decreased expression of MICA and aGvHD which was observed again in the case of MICA protein levels, where high levels of MICA protein were observed in patients with no active GIGvHD. Assessment of the levels of iii soluble MICA in sera of patients post HSCT showed a significant association between high levels of soluble MICA and aGvHD post-HSCT. Alongside MICA, this study investigated the mRNA and protein levels of a panel of genes (C1QTNF7, LGALS7, ANP32A, HTRA1, PIK3AP1, PSTPIPI, MSR1 and CXCL9) in RNA from blood samples and patient sera at different time points pre and post-HSCT. This study showed that there was a significant downregulation in the expression levels of MSR1 and ANP32A in aGvHD patients post-HSCT while a significant upregulation in the expression levels of CXCL9 was observed in aGvHD patients. Investigation of the association between the levels of proteins and the incidence of aGvHD showed that there was a significant association between upregulated protein levels of LGALS7 and aGvHD. Finally, a microRNA profiling in GI samples taken from aGvHD patients was performed aiming for the identification of miRNAs associated with the incidence of aGvHD in the GI tract after HSCT. This study identified 4 miRs that were dysregulated in patients in association with aGvHD, and a validation study was carried out for hsa-miR-34a-5p which expression was shown to be significantly decreased in patients with aGvHD (1-4) compared those patients with no aGvHD.

Mouse embryonic stem cells (mESCs) are derived from the inner cell mass of the blastocyst. When cultured in 3-D suspension in the absence of feeder cells, they form aggregates called embryoid bodies (EBs) that spontaneously differentiate to generate an outer layer of extra-embryonic endoderm with underlying basement membrane, an inner layer of primitive ectoderm, as well as a central proamnioticlike cavity. Furthermore, EBs can undergo a process that resembles gastrulation and can generate derivatives of the three embryonic germ layers. EBs are therefore very useful as model systems for investigating the early stages of mammalian development, and also for generating specific lineages such as ectoderm, mesoderm and endoderm, which can be used to investigate the mechanisms regulating the differentiation of specific lineages, as well as for regenerative medicine research. However, when used as a source for generating specific cell types of interest, EBs can be problematic, because only a proportion of cells within each EB will differentiate to become the required cell type. For this reason, there has been much interest in developing more efficient 2-D culture systems for directing the differentiation of mESCs to specific cell types. However, it is not clear whether cell types differentiating in EBs are equivalent to the corresponding cell types generated in 2-D differentiation cultures and have the same differentiation potential. To address this question, this study has compared the properties of nascent mesoderm arising in EBs with nascent mesoderm arising in 2-D differentiation culture. In order to do this, a mESC reporter line was created where a gene encoding the far-red fluorescent protein E2-Crimson (E2C) was knocked into the Rosa26 locus of an E14-Bra-GFP mESC line. This line enabled GFP+ nascent mesoderm cells to be isolated using fluorescence activated cell sorting so that the expression of key genes could be analysed, and then the fate of the cells could be tracked in living mice in vivo or following incorporation into developing organs ex vivo due to the fact that they constitutively express E2C. After confirming that the novel reporter E14-Bra-GFP/Rosa26-E2C mESC line displayed typical mESC properties and behaved similarly to unmodified mESC lines, the effectiveness of the E2C reporter for tracking cells in vivo and in vitro was assessed. Although E2C expression was stable, the fluorescence signal was quite weak, which meant that while it was possible to detect E2C in cells in vitro and on histological sections, tracking them in living mice was not feasible. For this reason, the study focused on comparing the gene expression profile of mesoderm isolated from EBs and 2-D cultures using quantitative reverse transcription PCR (RT-qPCR), and then their differentiation potential was assessed by incorporating the mesodermal cells into mouse kidney rudiments ex vivo. The most striking result from the RT-PCR analysis was that the mesodermal cells isolated from the EBs expressed > 20-fold higher levels of the lateral plate mesoderm gene, Foxf1, compared to the mesoderm cells derived from the 2-D culture system. Surprisingly, neither the mesodermal cells isolated from the EBs, nor those isolated from the 2-D system integrated into developing nephrons within kidney rudiments cultured ex vivo. This result was unexpected because the kidney is derived from the mesodermal lineage, specifically the intermediate mesoderm, and so it was anticipated that nascent mesoderm cells would be able to contribute to the developing kidneys. However, further analysis showed that the EB-derived mesoderm cells differentiated into PECAM+ endothelial-like cells within the rudiments. It is known that the lateral plate mesoderm gives rise to the vasculature in the developing embryo, so the fate of the EB-derived nascent mesodermal cells accompanied with their high levels of Foxf1 suggested that they were likely to have been already specified as lateral plate mesoderm. In contrast, the mesodermal cells derived from the 2-D culture system showed a limited capacity to generate PECAM-1+ cells, and instead, appeared to integrate into the renal stroma. It can therefore be concluded that Bra+ mesodermal cells generated using different in vitro culture systems have different properties, and might already be specified to more differentiated mesodermal lineages, such as paraxial, intermediate or lateral plate mesoderm. To facilitate future progress, it would be useful to generate dual reporter mESC lines that enabled the expression of Bra and a marker of either paraxial, intermediate or lateral plate mesoderm to be monitored simultaneously in real-time during in vitro differentiation culture.

Background: Studies suggest that cancer and non-cancer patients have needs (e.g. physical, psychological, religious, spiritual needs and information needs) that are not being adequately met. The review undertaken has presented a strong argument in favour of the case for a comprehensive holistic assessment of supportive and palliative care needs (Ahmed, 2010; Ahmed et al., 2014). At present, there is no widely used systematic, evidence-based, holistic approach to screening patients for supportive and palliative care needs. There is evidence to indicate a lack of studies on the clinical utility of tools (Ahmed, 2010; Ahmed et al., 2014; Ahmed et al., 2015). The Sheffield Profile for Assessment and Referral for Care (SPARC) is a multidimensional screening tool which gives a profile of needs to identify patients who may benefit from additional supportive or palliative care, regardless of diagnosis or stage of disease. Despite rigorous psychometric development, preliminary field-testing, and validation, the clinical utility of SPARC has yet to be established (Ahmed, 2010; Ahmed et al., 2014; Ahmed et al., 2015). This doctoral study was conducted within the context of a pragmatic randomised controlled trial and nested within the MRC framework for evaluating complex interventions. From reviewing the literature, it became increasingly apparent of the importance of combining quantitative and qualitative research methods approaches in the development and evaluation of complex interventions in palliative care research. This study provides an opportunity to 'test-drive' SPARC with patients that have supportive and palliative care needs. The hypothesis was that the use of a validated multidimensional holistic screening tool for supportive and palliative care needs, namely; SPARC, would lead to improved recognition of supportive and palliative care needs, and improved health care outcomes for patients (Ahmed, 2010; Ahmed et al., 2014; Ahmed et al., 2015). Aims and Objectives: The primary objective was to design and undertake a pilot study to evaluate clinical outcomes associated with the use of SPARC. The trial itself focussed primarily on outcomes, not on the processes involved in implementing the intervention. An additional element of this doctoral study and a secondary objective was to undertake a process evaluation (comprising of retrospective case note reviews, semi-structured interviews with patients and health care professionals) (Ahmed et al., 2015). Methods: This was an open, pragmatic, randomised controlled trial. Patients (n=182) referred to the palliative care service were randomised to receive SPARC at baseline (n=87) or after a period of two weeks (waiting-list control n=95). Primary outcome measure is the difference in score between Measure Yourself Concerns and Wellbeing (MYCAW) patient-nominated Concern 1 on the patient self-scoring visual analogue scale at baseline and the two-week follow-up. Secondary outcomes include difference in scores in the MYCAW, EuroQoL (EQ- 5D), and Patient Enablement Instrument (PEI) scores at Weeks 2, 4, and 6. As part of a process evaluation, case notes were reviewed at week 8, and semi-structured interviews were undertaken with a sub-group of patients and health care professionals (Ahmed et al., 2015). Results: There was a significant association between change in MYCAW score and whether the patients were in the intervention or control group X² trend = 5.51; degrees of freedom = 1; P = 0.019). A higher proportion of patients in the control group had an improvement in MYCAW score from baseline to Week 2: control (34 of 70 [48.6%]) vs. intervention (19 of 66 [28.8%]). There were no significant differences (no detectable effect) between the control and intervention groups in the scores for EQ-5D and Patient Enablement Instrument at 2, 4, or 6-week follow-up (Ahmed et al., 2015). Most patients interviewed [30/33], found SPARC either quite easy to complete, fairly straightforward, simple or had no problems in completing it. Only a small number of participants found questions on SPARC 'too sensitive or upsetting'. A crucial finding in the context of the trial was the large proportion of patients interviewed [30/33] who did not experience or report any noticeable change, or beneficial effects after completing SPARC (EAPC abstract, 2015). Most health care professionals had something positive to say about SPARC and had previous experience of using SPARC, and most were considering using it at some point in the future. A number of barriers were identified to the relief of distress highlighted by SPARC. Only 5/164=3.0% patient notes made any direct reference to SPARC. Conclusion: This trial result identifies a potential negative effect of SPARC in specialist palliative care services, raising questions that standardised holistic needs assessment questionnaires may be counterproductive if not integrated with a clinical assessment that informs the care plan (Ahmed et al., 2015). This is supported by review of case notes, and the interview data from patients that indicate that most patients felt that no particular action or benefit followed from the completion of SPARC (Ahmed et al., 2015). Only a few patients who had no recent contact with palliative care service and scored high for some SPARC items were recalled by the service and reassessed. Overall, participants and health care professionals considered SPARC an acceptable and relevant tool for the clinical assessment of supportive and palliative care needs (EAPC abstract, 2015). The potential negative effect of SPARC in a specialist palliative care service could be due to the failure of health care professionals to act on identified needs in a timely manner, or related to the raising of patients' expectations that are not subsequently met. The qualitative study helps in the interpretation of the outcome results, and provides useful insights into how SPARC might be used in practice. Early identification of and monitoring of symptoms is only useful if effective treatment programs or systems are in place to address identified needs, and we must consider and evaluate new methods to achieve practice change. The effective integration of SPARC into routine care and standard operating systems requires further investigation (Ahmed, 2010; Ahmed et al., 2014; Ahmed et al., 2015).

Predictable levels of transgene expression will be essential for full exploitation of human ES cells (hES) in basic research and medicine. In practice however, transgene expression in mammalian cells is often found to be silenced, low or variegated presenting a serious drawback in this area of research. Unpredictability of transgene expression arises mostly as a consequence of variable transgene copy number and/or inhibitory effects of surrounding chromatin structures in which the transgene has randomly integrated. We have addressed this issue in hES cells, by combining gene targeting and site-specific recombination at the (3-casein and the hypoxanthine phosphoribosyltransferase {hprt) genes. A neomycin expression cassette flanked by heterospecific lox sites is first introduced by homologous recombination (HR). In a subsequent step, recombinase-mediated cassette exchange (RMCE), permits the exchange of the neomycin gene with a transgene containing a blasticidin-oct-4-GFP expression cassette flanked by the same set of heterospecific lox sites. Using a promoter trap strategy, site-specific integrants can be directly selected with blasticidin. While we failed to target the p-casein gene in hES cells, we obtained homologous-recombinants with targeting frequencies of up to 0.4 % at the hprt gene in the male hES cell line (HI). F/prMargeted HI cells were shown to maintain a normal karyotype; to exhibit common hES cell surface markers; and to differentiate down the osteogenic lineage pathway. Additionally, subsequent targeted correction of hprt-targeted HI mutant cells was achieved, restoring structure and function of the hprt gene. Following RMCE, site-specific integrant were recovered with 100 % efficiency using a promoter trap strategy at the hprt locus. However, site-specific integrants were characterised by a progressive down-regulation of GFP, possibly as a result of transcriptional interference from the BSD cassette or inhibitory elements present at the site of integration. Further work will be required to investigate the nature of this silencing. Targeting transgenes with reliable transgene expression at specific loci in human ES cells will be important for modifying specific ES cell-derived tissues for therapeutic applications in regenerative medicine. Engineering the hprt locus should prove very useful for addressing issues such as directed differentiation, immunotolerance as well as protection against tumorigenesis.

Background: Several studies have shown that newly qualified doctors often feel unprepared to provide acute care, and that such feelings are a source of anxiety and stress. The main aim of this thesis was to explore how newly qualified doctors perceive and negotiate the complex challenge of assessing and treating acutely unwell patients in the early days of their professional practice. The thesis begins by examining preparedness in a local context and proceeds to explore preparedness in acute care throughout the UK. A variety of qualitative research methods are then employed to explore behavioural influences and patterns of error within acute care contexts. Methods: In the first instance, a questionnaire study was undertaken at the University of Edinburgh, involving feedback on preparedness for practice over three consecutive years from 2007 to 2009, against 13 major programme outcomes, from graduates and their educational supervisors. In order to gain a more global perspective, a systematic literature review synthesising work examining the perceived preparedness of UK graduates in acute care versus other General Medical Council mandated outcomes was then undertaken using five databases archiving medical, educational, nursing and psychological literature. Preparedness ratings in relation to each outcome were mapped to a novel generic rating scale to allow comparisons between studies. Six focus groups involving 36 clinicians were conducted and analysed using a constructivist grounded theory approach. The developing theory and relationships between emergent themes were refined and validated by further interviews with participants. Subsequently, 38 newly qualified doctors participated in high-fidelity simulated acute care scenarios. Each scenario was immediately followed by a debriefing which encouraged articulation of cognitive processing. Errors were identified and coded where possible using Reason's generic error modelling system (GEMS). Remaining errors were coded inductively using a modified framework analysis to discern further patterns within the data. Results: University of Edinburgh graduates consistently felt well prepared in consultation and communication skills but less prepared in acute care and prescribing. Educational supervisors felt that graduates were least well prepared in acute care and practical procedures. The literature search recovered 256 articles, of which 10 satisfied the inclusion criteria. These articles suggested that graduates perceive themselves to be the least well prepared in acute care and prescribing, and senior doctors and other healthcare colleagues perceive newly qualified doctors to be less well prepared in acute care than any of the other outcomes. Three broad themes emerged from the focus group data: cognitive challenges, roles and responsibilities and environmental factors. Exploration of the relationships between the themes led to the development of a conceptual framework. Using evidence from the simulated scenarios, corresponding debriefs and field notes, 164 of the 243 simulated scenario errors could be classified according to the original version of GEMS. A further 26 errors were coded using two novel categories: compound error and submission error. Multidimensional analysis involving both the amplified GEMS classifications and iteratively-developed key subject areas revealed specific patterns of error such as the propensity for rule-based mistakes relating to hospital systems. Discussion and Conclusions: This thesis adds to existing work which emphasises the complex inter-relationships between emotion, affect, decision-making and behaviour. It is the responsibility of the medical education community to ensure that newly qualified doctors are aware of the roles that these factors play in errors and adverse events. Emotional skills training, particularly with reference to dynamic, high-stakes situations, should form an integral part of basic medical training. Medical training and assessment structures currently emphasise and reward personal knowledge and academic attainment above collaboration and emotional maturity. In the drive to improve patient safety, a key component is to nurture doctors who understand human fallibility and who feel empowered to seek help, safe in the knowledge that they will not be deemed to have failed.

Haematopoietic stem cells (HSC) are a rare population of cells that have the ability to self-renew and differentiate giving rise to various blood lineages, thereby reconstituting the whole haematopoietic system. This is an essential characteristic, exploited in bone marrow transplantation therapy in response to myeloablative treatment. Due to their rarity, the lack of sufficient HSC numbers for transplantation has proved to be a major clinical issue. Separately, in the development of leukaemia, acquired mutations in HSCs give rise to malignant cells. These cells, like HSCs, have the ability to self-renew and differentiate forming immature blasts and are termed cancer (leukaemic) stem cells. They are thought to remain in a quiescent state and are therefore not targeted by standard chemotherapy, inducing relapse in haematopoietic malignancies. In this study, a cross species stem cell based screen was conducted on a 12,000 small molecule library across a range of adult and embryonic tissue types with a view to identifying compounds that would (i) expand HSCs ex vivo and in vivo for transplantation and (ii) eradicate cancer stem cells in leukaemia. A number of small molecules were identified as lead compounds and were assessed in our investigation. We found that Yohimbine, an alpha-2 adrenergic receptor (adra-2) antagonist, and Oxa-22, cis-2-Methyl-5-trimethylammoniummethyl-1,3-oxathiolane iodide (M3 Muscarinic acetylcholine receptor agonist) elicited a 2- and 1.5- fold increase in HSC frequency (respectively) in vivo. Further competitive transplantation studies showed that Yohimbine and Oxa-22 treated cells also enhanced the reconstitution of B cells and T cells respectively. In parallel, we also assessed Oxa-22 and a third compound, Phthalylsulfathiazole- an antibacterial sulphonamide, in the leukaemic setting to ascertain whether compounds could target leukaemic stem cells (LSCs). We found that these compounds promoted proliferation in acute myeloid leukaemia (AML) cell lines. Furthermore, when Oxa-22 and Phthalylsulfathiazole were administered in vivo models of AML, they accelerated disease progression by increasing the number of LSCs. Collectively, these results show that using small molecules we can target neuronal related pathways to enhance HSC number and function. Further investigation is required to elucidate the exact mechanisms of the compounds however, these data may prove to be influential in directing new methods of stem cell expansion for transplantation therapies. Small molecules targeting neuronal or antibacterial related pathways were also found to target malignant LSCs and alter their behaviour. By driving LSCs out of their dormant state, these small molecules may pave the way for potential targeting of LSCs in conjuction with standard current chemotherapies that incorporate and kill proliferating cancer cells.