Structured resuscitation training (SRT) programmes for healthcare professionals have been developed to try and achieve an optimum standard of resuscitation management amongst the participants and thus improve patient care. The effectiveness of these has not been systematically investigated. Aims 1. To systematically review the literature regarding the effectiveness of structured resuscitation programmes. 2. To investigate, in particular, aspects of the effectiveness of the Neonatal Life Support course (a SRT programme which takes place within the author's area of clinical practice). Methods A systematic review of the literature on structured adult, paediatric and neonatal resuscitation training was carried out using Best Evidence Medical Education (BEME) methodology. Over a 22 month period, candidates undertaking a one day Neonatal Life Support Course at the Liverpool Women's Hospital were recruited into a follow-up study whose aim was to assess their retention of resuscitation skills over time and their confidence at performing neonatal resuscitations. Candidates repeated the Neonatal Life Support 'airway' test at 3-5 months and, if successful, they were subsequently retested at 12-14 months. Prior to the test, candidates were asked to complete a confidence questionnaire and following the test, peer assessment review forms were distributed by their line manager to their peers as part of a multi-source feedback exercise.

Cryptococcal meningitis (CM) is a leading cause of death in HIV -infected patients in Africa. The thesis examines novel strategies for treatment and prevention of HIV - associated CM, and reports: i) A literature review of HIV -associated CM. ii) The results of a large combined cohort analysis identifying high baseline fungal burden and abnormal mental status as key determinants of mortality in patients with CM. iii) A description of the epidemiology, clinical features and outcomes of CM in Cape Town, based on a series of observational cohort studies, showing that CM is the commonest cause of adult meningitis in Cape Town, with an in- hospital mortality of ~30%, and that ART roll-out has yet to substantially reduce the number of cases or impact on in-hospital mortality. iv) A report of a randomized controlled trial demonstrating that the novel immunotherapeutic strategy of short course adjunctive IFN-y increases the rate of clearance of cryptococcal infection in HIV -associated CM. v) Results from flow cytometry and luminex analysis examining immune responses to cryptococcal infection in late stage HIV -infection, and assessing the effect of adjunctive IFN-y on the host immune response. vi) The results of a large trial demonstrating that screening for cryptococcal antigenaemia on entry into ART programmes allows the identification of patients at risk of developing CM, potentially enabling their prevention. The thesis is based on fieldwork carried out over a three-year period in Cape Town, South Africa, followed by additional laboratory work at the National Institutes of Health, Bethesda, USA and St. George's University of London.

The experiments in this thesis evaluated the proposal that GluR-1"7" mice display impairments in affective and motivational processes (Mead & Stephens, 2003a). The introductory experiments examined sensorimotor and affective aspects of behaviour in GluR-l"A and wild-type control mice (Chapter 2). These studies attempted to evaluate any performance-based behavioural impairment which may have interfered with learning. Chapter 3 assessed BLA-dependent learning on a Pavlovian fear conditioning paradigm (e.g., LeDoux, Sakaguchi & Reis, 1986). The simple nature of this learning task, and the large body of evidence implicating the amygdala in this form of learning provided an opportunity to examine the influence of the GluR-1 mutation on emotional learning (Maren, 2000a Cardinal et al., 2002). Chapters 4 and 5 made use of separate Pavlovian and instrumental preparations which characterised different affective and sensory-specific associatively activated outcome representations (Blundell, Hall & Killcross, 2001 Balleine, Dickinson & Killcross, 2003 Corbit & Balleine, 2005). The results are discussed in respect to a failure of GluR-1"7" mice to attribute affective and motivational incentive value to the sensory-specific properties of a US an account which furthers that proposed by Mead and Stephens.

No description available.

Novel osteochondral repair tissue engineering strategies are investigating the use of a single scaffold, with a portion for osteogenic and chondrogenic differentiation, and a single cell source, most notably the mesenchymal stem cell, to facilitate osteochondral differentiation and repair in a single construct. However, this approach requires robust differentiation protocols to ensure that the correct balance of each cell type is produced and maintained. Techniques used to analyse osteogenic and chondrogenic differentiation are well established, but many of the current methods described are qualitative, based on imaging stained cells or sections under a microscope. To facilitate higher throughput screening of chondrogenic differentiation in human MSCs, a novel culture technique using V shaped 96 well plates has been developed combining three robust and quantitative assays. Additionally, two reporter cell lines have been developed that express luciferase under the control of an osteogenic (osteocalcin) or chondrogenic (col2a1) promoter in order to streamline differentiation assays. The use of growth factors to elicit differentiation is well established; with BMP-2 used to enhance osteogenic differentiation and TGF-61 used to enhance chondrogenic differentiation. However, there are several limitations of using growth factors in regenerative medicine and consequently, the use of growth factor mimics was investigated. Two promising growth factor mimics were identified that could support both osteogenic and chondrogenic differentiation; LE135 and imperatorin. LE135, a retinoic acid receptor antagonist, significantly enhanced chondrogenesis with increased GAG production, col2a1 promoter activity and versican mRNA expression and had no significant effect on osteogenic differentiation. Imperatorin, a coumarin derivative, significantly enhanced early stage osteogenesis (alkaline phosphatase activity) and had no significant effect on late stage osteogenesis (mineralisation). Furthermore, chondrogenic differentiation was enhanced by imperatorin with significantly increased GAG production.

Stem cell derived endothelial cells have a growing number of applications in clinical medicine and biomedical research and will be critical in any organ regeneration programme. Endothelial cells can now be derived from a number of distinct stem cell populations including embryonic stem cells, blood progenitors and induced pluripotent stem cells. In order for stem cell derived endothelial cells to be used optimally it is important that they are fully assessed for the cardinal characteristics of endothelial cells on mature vessels. With this in mind, in my thesis I have assessed the ability of stem cell derived endothelial cells to display hallmarks of authentic endothelial cells from vessels including, alignment under shear stress, responses to pathogen stimuli and vasoactive hormone release. My group has previously shown that endothelial cells from human embryonic stem cells did not respond to agonists of toll-like receptor-4 (TLR4). TLR4 is important to allow endothelial cells to sense infection, but is also associated with cardiovascular disease. In my PhD, I showed that, whilst these cells have no TLR4 they have a fully functional NOD1 receptor pathway which allows the cells to sense Gram-negative bacteria. This may be relevant to their use therapeutically and allowed me to speculate that these cells are immune competent, but through lack of TLR4 might be protected from vascular inflammation. In order for stem cell derived endothelial cells to be useful in in vitro assays however, it is important that they express all the key hallmarks of endothelial cells from vessels. Endothelial cells grown from blood progenitors or from induced pluripotent stem cells did have TLR4 responses and released vasoactive hormones at levels comparable to endothelial cells from vessels. In the later part of my PhD, I applied endothelial cells derived from blood progenitor cells to key assays to study endothelial cell biology and pharmacology. This included an assay to detect cytokine storm reactions to drugs, which currently limits the development of biological drugs. This assay, employing cells derived from stem cells of individual patients also has applications in personalised medicine. In my final chapter, I was able to grow BOEC from a patient with a homozygous mutation that results in loss of function of the enzyme cPLA2, which is thought to be critical to prostaglandin and prostacyclin release, and used these cells to study this pathway. I showed that cPLA2α is the dominant isoform responsible for the release of prostacyclin from endothelial cells and provided a proof-of-concept that BOEC can be used to phenotype patients. In summary, my thesis includes characterisation of stem cell derived endothelial cells, and includes applications of adult progenitor derived cells in assays to study pharmacology and cell biology with a view to developing personalised medicines and cell therapies.

Mesenchymal stem cells/multipotent stromal cells (MSCs) have a variety of unique properties that have made them a popular cell type in the study of tissue engineering. Their ability to undergo osteogenic, adipogenic and chondrogenic differentiation has long been appreciated. In addition to their capacity for skeletogenic differentiation, there are suggestions that MSCs have additional roles in organising tissue vasculature through interactions with endothelial cells (ECs). However, suitable experimental models to test these unique MSC activities are lacking and the mechanisms are unclear. Here, we have developed a novel 3D in vitro co-culture spheroid model of MSCs and ECs. Development and further investigations into this 3D co-culture spheroid model has resulted in many novel discoveries. Using calcium depletion from cell culture media to quantify osteogenic differentiation of MSCs in both 2D and 3D represents a complimentary assessment method that is non-destructive. Co-culture of MSCs and ECs was also found to promote osteogenic differentiation whilst having no detrimental effects on cell viability during long-term culture. Further investigations into the 3D co-culture of MSCs and ECs demonstrated spontaneous endothelial organisation. Using this model it was possible to track endothelial restructuring and identify the signalling processes involved, ultimately focusing on platelet-derived growth factor and notch signalling. Using a combination of pre-differentiated MSCs and ECs within the 3D co-culture system, osteochondral spheroids were developed. These spheroids were analysed using a combination of novel and traditional imaging techniques; it was found that osteochondral spheroids self-organised into distinct bone and cartilage regions, similar to those observed at the osteochondral boundary and during early endochondral ossification. This in vitro multiple cell culture system represent a simplified tractable model of osteochondral tissue.

No description available.

Haemopoietic stem cells are capable of generating a variety of mature cells. One of the important steps in stem cell biology involves the commitment of a multipotent precursor to a single lineage. As yet the molecular mechanism for cell fate determination is unknown. This work was focused on commitment of the granulocyte/macrophage (GM) bi-potential precursor towards the macrophage lineage. Published studies have noted that the activity of tyrosine kinase, fyn, correlated with macrophage maturation and increased expression of the macrophage-colony stimulating factor receptor (M-CSFR) in the human myelomonocytic cell line, THP-1. Furthermore, it was also reported that treatment with fyn anti-sense reduced M-CSFR up-regulation and cell adhesion, indicative of macrophage commitment and/or maturation. To investigate whether fyn was involved in the transcriptional activation of c-fms and potentially in macrophage commitment in primary haemopoietic cells I used real-time RT-PCR to determine levels of fyn and c-fms. Examination of fyn RNA levels revealed that it was unlikely to regulate c-fms and suggested that fyn is most likely regulated by c-fms. Having ruled out fyn as a potential positive regulator of macrophage commitment, I subsequently set out to identify other potential regulators. To do so, I examined the expression profiles of single granulocyte/macrophage (CFU-GM), macrophage (CFU-Mac) and neutrophil (Pre-N) precursors using a DNA microarray approach. My single cell microarray data provided a "snap-shot" of the expression profiles of the CFU-GM, CFU-Mac and Pre-N progenitors and revealed the presence of groups of genes whose expression correlated with the developmental stage of the single cells. Furthermore, analysis of the expression profiles identified changes in gene expression due to cell handling and culture treatments. Real-time RT-PCR analysis was used to validate a set of array genes potentially involved in macrophage commitment. For genes expressed at levels greater than 1 copy in lO4 total cDNA molecules a strong correlation was revealed between microarray hybridisation and real-time PCR data. Further analysis of enriched bone marrow precursors and a multi-potent cell line revealed a set of eleven genes as potential regulators of macrophage commitment and five genes as potential early marker of commitment. My results provide a precedent for new experimental studies of the mechanisms underlying monocyte lineage determination and more general mechanisms of commitment that can be extended to different tissues and embryo development.

Pluripotent stem cells including induced pluripotent stem (iPS) cells and embryonic stem (ES) cells are known for their distinctive property of indefinite self-renewal in an undifferentiated status, with the potential to differentiate into all types of cells. Current protocols used in the differentiation of human iPS cells and human ES cells towards erythropoiesis utilize two main approaches: (1) embryoid body (EB) formation, which influences heterogeneity of the produced population, and/or (2) co-culture with mouse stromal cells, where obstacles of purification of the cells rise, which makes the xeno-free culture requirement difficult to achieve, in addition to the cytokine supplements. Moreover, these protocols reported low efficiency in number and functionality, especially with human iPS cells, and required long culture times. One of the major challenges in erythroid cell production from human ES/iPS cells is achieving full maturation and enucleation of erythroid cells in serum-free and feeder-free condition, in order to ensure a completely xeno-free culture condition suitable for clinical applications. In this study, we have designed a novel protocol for direct differentiation of human iPS cells towards erythroid cells under serum-free conditions bypassing the EB formation step without requiring co-culture. Our protocol involves three steps: (1) hematopoietic/erythropoietic induction, followed by (2) erythroid differentiation, and finally, (3) erythroid maturation and enucleation. Differentiated cells were separated into normoxia and hypoxia conditions. As early as day 7 of culture, an early hematopoietic marker, CD34, was observed, followed by a high expression of CD45, which is a pan leukocyte marker in parallel to less expression of an early erythroid marker, CD71. Over the culture period, an increase in the expression of the late erythroid marker, CD235a, was monitored, which reached high levels by the end of the 28-day culture protocol. Further studies on functional and morphological analysis using CFU assay showed that the cell population on day 14 were able to form erythroid progenitor colonies, i.e. BFUEs. Immunocytochemical staining showed the presence of heme-containing proteins, which was later confirmed by globin expressions by qPCR. Interestingly, staining with new methylene blue confirmed reticulocyte morphology, which indicated that partial maturation was achieved. Hypoxia condition is a key regulator for erythropoiesis and haemoglobin formation, as indicated by the BFU-Es formed under hypoxic conditions, together with formation of adult-type haemoglobin, as shown in qPCR. Further studies on maturation of those cells are required in order to achieve fully mature and functional erythroid cells phenotype. This thesis thus presents a direct differentiation protocol toward erythroid cells using human iPS cells in serum-free and feeder-free system, bypassing EB-stage and resulting on high efficiency of erythroid cells formation within 4 weeks of culture, which include partial maturation and formation of adult-type haemoglobin.