A thesis presented to the Faculty of Medicine, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Medicine / Since changes occur in size, shape and haemoglobin content of red cells in disease a classification of the anaemias based on the morphology of the red cell has been widely used ( Wintrobe, 1956 ) . While such classification has a limited usefulness from the diagnostic and therapeutic approach it has two serious defects.. / IT2018

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree of Master of Science in Epidemiology (Epidemiology and Biostatistics). 2016 / Background Reducing severe acute respiratory illness (SARI)-associated mortality in African children remains a public health priority and an immense challenge. The pneumococcal conjugate vaccine (PCV) was introduced into the South African routine immunization programme in 2009. The objectives of this study were: I. To describe the demographic characteristics, clinical presentation, respiratory pathogens of children aged <5 years hospitalized with SARI in an urban (Chris Hani-Baragwanath Hospital, Soweto) and a rural (Matikwana and Mapuleng Hospitals, Mpumalanga) setting in South Africa from 2009-2013 and II. To compare the factors associated with mortality among children aged <5 years hospitalized with SARI in these two sites separately. Methods Hospitalized children with SARI were enrolled into an active, prospective sentinel surveillance program. Clinical and epidemiologic data were collected until discharge. Nasopharyngeal aspirates were tested for influenza (A and B) and eight other respiratory viruses. In-hospital case-fatality proportion (CFP) and risk factors for mortality were determined for each hospital site separately using unconditional logistic regression. Results The in-hospital CFP was significantly higher in the rural (6.9%, 103/1486) than the urban (1.3%, 51/3811) site (p<0.001). This was observed among both HIV-infected (urban: 6.6%, 17/257) vs. (rural: 12.9%, 30/233) (p=0.019) and HIV-uninfected children (urban: 0.6%, 13/2236) vs. (rural: 4.2% 36/857) (p<0.001). In the urban site the only factor that is independently associated with death on multivariate analysis was HIV infection (odds ratio (OR) 12.1, 95% confidence interval (CI) 5.8-25.2). In the rural site HIV infection (OR 3.5, 95% CI 1.7-6.9), age <1 year (OR 3.5, 95% CI 2.0-6.1) vs. 1-4 years, any respiratory virus detected (OR 0.4, 95% CI 0.2-0.6), pneumococcal infection(OR 4.5, 95% CI 1.8-10.8) and malnutrition (OR 12.8, 95%CI 1.2-134.6) were independently associated with mortality. Conclusion SARI mortality was higher in the rural setting. Even in the era of PCV availability pneumococcus is still associated with mortality in rural areas. Efforts to prevent and treat HIV infections in children and reduce malnutrition may reduce SARI deaths. / MT2017

INTRODUCTION: Despite best current therapy, acute lymphoblastic leukemia (ALL) still remains the most common cause of cancer-related death in children and young adults. Relapse is the main reason for treatment failure in ALL patients and occurs in 15-20% of these patients. Current risk stratification criteria have not been sufficient to predict relapse in ALL patients. The Philadelphia (Ph) chromosome is a chromosomal abnormality found in a subset of high risk ALL patients and is associated with a poor prognosis. Recent genome-wide studies have identified focal deletions of the Ikaros gene (IKZF1) in 70-80% of B-cell ALL patients that have the Philadelphia (Ph) chromosome. Subsequent studies have also found a strong correlation between IKZF1 deletions and ALL patients (Ph+ and Ph-) that relapse. IKZF1 is required for normal lymphoid development and loss of IKZF1 results in haploidinsufficiency or the overexpression of dominant negative IKZF1 isoforms, in particular Ik6 in high risk ALL patients. Most studies used DNA microarrays to detect IKZF1 deletions. Multiplex ligation probe dependent amplification (MLPA) is a low cost, rapid technique that can detect small DNA copy number changes of up to 50 targets in a single reaction and is not as technically challenging to analyse as arrays. MLPA has also been suggested to be used as an alternative to array based techniques in developing countries. METHODS: There were 31 ALL (paediatric and adult) patients that were tested using MLPA and 24 ALL patients tested using reverse transcriptase PCR (RT-PCR) to detect IKZF1 copy number changes and IKZF1 isoform expression pattern respectively. RT-PCR was validated with DNA sequencing and MLPA was validated with Fluorescent in situ hybridization (FISH). MLPA was also compared to cytogenetics in certain cases. RESULTS: MLPA detected 156 copy number changes (7.1 aberrations per sample) in 22 leukemic patients. IKZF1 deletions accounted for the majority of the aberrations (41%) and were detected in 53% of Ph+ ALL patients (n=15) by MLPA. IKZF1 deletions were detected at presentation and relapse in Ph+ and Ph- ALL patients. IKZF1 isoform Ik6 was detected in 70% of Ph+ and relapsed ALL patients after performing RT-PCR. IKZF1 deletions of exons 4-7 resulted in exclusive expression of Ik6. MLPA results were also correlated with certain aneuploidies detected with cytogenetics. CONCLUSION: This study showed that IKZF1 deletions could have assisted with prognosis in certain ALL cases and thus, newly diagnosed ALL patients should be screened for IKZF1 deletions. MLPA proved to be a reliable, rapid and cost effective technique to detect small copy number changes in multiple genes and should be implemented as a diagnostic test to detect IKZF1 deletions.

University of Macau / Institute of Chinese Medical Sciences

There is a huge unmet clinical need for a new, safe and effective minimally invasive treatment for Acute Ascending Aortic Dissection (AAAD) (1). In 2012 AAAD has a mortality rate of 1-2% per hour within the first 24 hours, and even with contemporary surgical techniques, advanced intensive and post operative care, the mortality from AAAD following surgery in most series remains in the unacceptable range of 10-30% at 30 days (2;3). 28% of patients presenting with AAAD are denied life saving surgery often because of age or co-morbidity - medical therapy alone associated with an in hospital mortality rate in excess of 50% (2;4-6). Currently available endovascular stent grafts used in the descending thoracic and abdominal aorta are not adequately designed to be utilised within the ascending aorta. They have a large stowed diameter 22-25 French (F), with a rigid covering of either Dacron or ePTFE, and a stiff inflexible delivery system unlikely to traverse the aortic arch without complication. While the contemporary results of elective surgery for ascending thoracic aortic aneurysm (ATAA) are good, with an elective mortality of <5%, surgical results for AAAD have improved little over the last 20 years, with a 30 day mortality rate between 10-30% (3;7). With the emerging role of endovascular stent grafts in the treatment of thoracic aneurysm and dissection, with shorter hospital stays and improved outcomes I believe now is the time for the development of a percutaneous solution for AAAD. Potential ascending thoracic aortic graft (ATAG) designs must take into account the very close proximity of intimal tear to both the coronary arteries and aortic valve, allowing a 4 good proximal graft seal without compromising coronary flow or aortic valve competence. ATAG should have a low profile, with a thin non porous covering and a flexible delivery sheath with accurate and precise deployment characteristics. Following a literature review and novel anatomical data collection from computerised tomography (CT) and magnetic resonance imaging (MRI) scans of AAAD and ATAA patient cohorts, it seems that 3 embodiments of ATAG should be designed and developed, all sharing advanced core technologies including a laser-cut nitinol stent frame, thin polyurethane (PU) material covering and accurate and precise deployment mechanisms: 1) The “supra-coronary tubular ATAG”, for treating AAAD with an intimal tear in the ascending aorta, no coronary or aortic valve involvement and adequate landing zones above the coronary arteries and before the right brachiocephalic trunk (RBCT). It is likely that this graft will be capable of treating at least a third of all patients with AAAD (8). 2) The “inverted t-shirt ATAG” to proactively protect coronary artery flow and achieve proximal seal within the sinuses in patients with an intimal tear in close association or involving the coronary arteries. 3) The “valved ATAG” to treat patients who have significant aortic regurgitation (AR), to achieve a proximal seal at the annulus when anatomy suggests it would be difficult to achieve with embodiment 1) or 2), and in those patients who have a hugely dilated aortic root, so that the ATAG can seal proximally at a relatively normal annulus size, and seal distally at a normal ascending aorta diameter 5 proximal to the RBCT. This could be the treatment option for the 25-35% of AAAD patients who currently require aortic valve repair or replacement (9). The most complex of the 3 devices above is embodiment 2), the “inverted t-shirt ATAG”, as it must ensure proximal aortic seal within an often dilated sinus, without compromise to aortic valve and proactively protect both coronary arteries with 2 coronary sleeves. Basic proof of concept (PoC) of this embodiment has been demonstrated in vitro within a normal sized aortic glass model, with some important study limitations, nevertheless it does demonstrate that tracking an ATAG branched graft with 2 coronary sleeves is possible over 3 guidewires and deploying accurately within the aortic root under both direct vision and fluoroscopy. Following successful PoC deployment I then specified and had manufactured a 2nd Generation ATAG (2G ATAG), with a laser-cut nitinol frame, longitudinal tie bars, and a novel thin PU graft covering material. The 2G ATAG has been shown to have adequate radial strength when compared to competitor devices, and can be stowed to 28 F for deployment. During ATAG development 2 patents have been filed, and I wrote with Professor Rothman a successful NIHR I4I grant for £743,000 to take ATAG from the current 28 F 2G device, towards the goal of an 18 F device with bench testing, in vitro flow rig and deployment analysis, and in collaboration with the Royal Veterinary College (RVC) into an animal model over the next 3 years (beyond the scope of this thesis). I hope that within this next development cycle ATAG can be iterated into a device that might be ready to embark on a first in man (FIM) trial to offer the AAAD population an effective and less invasive treatment strategy.

Acute myeloid leukaemia is a clonal disorder characterised by recurrent chromosomal translocations. One of the commonest, is the t(8;21) which results in part of the AML1 gene being juxtaposed to most of the ETO gene with the resultant chimeric protein, AML1-ETO, acting predominantly as a transcriptional repressor. Despite the extensive literature available, the exact mechanism by which the chimeric protein results in AML has not been fully elucidated. By using exon arrays and high throughput sequencing as tools it was hoped to gain further insights into the molecular basis of this disease. Gene expression profiling using the exon arrays highlighted molecular pathways and specific genes that play a key role in the pathogenesis in t(8;21). Exon arrays were also used to profile individual exon expression of the ETO gene. This demonstrated that the genomic breakpoint of ETO in the t(8;21) is variable between different patients. This technique also resulted in the discovery of a new exon in the ETO gene. This novel exon results in formation of alternative transcripts of AML1-ETO and was shown in mouse models to play a key role in leukaemogenesis. Chromatin immunoprecipitation followed by high throughput sequencing revealed novel aspects of AML1-ETO binding. A number of novel genes that bind AML1-ETO were recognized and in conjunction with the expression data, a number of hypothesis on how AML1-ETO binding effects gene expression are made.

Introduction: Newly qualified doctors are frequently first to the scene in managing acutely unwell in-patients. Failures in clinical assessment, basic management and early escalation of care lead to avoidable patient morbidity and mortality. Analyses of adverse events have highlighted the importance of non-technical skills training to improve patient safety. These are a combination of cognitive (such as decision making) and social skills (such as team working), which complement knowledge and technical ability, and contribute to safe and effective care. In order to train and assess junior doctors in these skills, we must first have an accurate understanding of what they involve. This research project was designed to identify the critical non-technical skills required by junior doctors to manage acutely unwell patients safely and effectively. It aimed to develop a tool to observe these skills that could be used in training, assessment and research. Method: A literature review was used to develop an initial framework to categorise the non-technical skills required in this domain. Twenty-nine in depth semi-structured interviews were then completed with junior doctors. A critical incident technique was utilised: Junior doctors were asked to recall a challenging case in which they managed an acute medical emergency. Interviews were transcribed and coded using template analysis. A panel of subject matter experts were then consulted in order to refine this framework and develop an assessment tool for observing these skills. This involved two focus groups and an iterative process, returning to the original data to verify any changes. Results: Four categories of critical non-technical skills were identified: Situation awareness, decision-making, task management and teamwork. Each of these had between three and four sub-categories. Descriptors, exemplar behaviours and an assessment scale were developed to allow these non-technical skills to be observed and rated using a behavioural marker system. During the development of this tool, exploration of the data revealed the influence of factors such as hierarchy and culture on the behaviour of junior doctors. Conclusions: The performance of newly qualified doctors in acute care is influenced by the complex clinical environments in which they work. This can have profound implications for patient outcomes. The framework developed by this research allows us to be explicit about the types of behaviours that are required to keep patients safe. If this tool can be integrated into clinical training, then it could be used by clinicians to support the development of safe and effective skills and reduce the current level of avoidable patient harm.

Acute respiratory infections (ARI) are a major global cause of morbidity and mortality. Vitamin D deficiency has been reported to associate with susceptibility to ARI and with greater severity and poorer control of asthma and chronic obstructive pulmonary disease (COPD). Clinical trials of vitamin D for the prevention of ARI have yielded heterogeneous results, with some showing protection and others not. This may reflect variation in the frequency of genetic variants influencing response to vitamin D supplementation in different populations. The impact that genetic variation in the vitamin D pathway has on vitamin D status, disease phenotype and response to vitamin D supplementation in prevention of ARI has not been comprehensively investigated. Methods: I conducted: 1. A systematic review and meta-analysis of clinical studies which have investigated vitamin D as a potential therapy for ARI; 2. Three cross-sectional studies (in n=297 adult asthma patients, n=278 COPD patients, and n=272 older adults) to investigate potential environmental determinants (lifestyle and anthropometric) and genetic determinants (35 single nucleotide polymorphisms [SNP] in 11 vitamin D related genes) of serum 25-hydroxyvitamin D concentration (25[OH]D) and clinical phenotype; 3. Three prospective studies investigating the influence of genetic variation in the vitamin D pathway on a) susceptibility to ARI (main effects analysis) and b) efficacy of vitamin D supplementation for the prevention of ARI (interaction analysis). Results: My systematic review identified consistent reports of an inverse association between vitamin D status and risk of ARI in observational studies, and heterogeneous reports from clinical trials. My cross-sectional studies identified a range of classical environmental factors which predict vitamin D status in the three study populations, but did not identify any genetic variants in the vitamin D pathway that associate with vitamin D status. I identified an association between vitamin D deficiency and decreased lung function in COPD patients, but no associations between vitamin D deficiency and asthma phenotype. Finally, my analysis identified a haplotype of 5 single nucleotide polymorphisms in the vitamin D receptor (VDR) gene which significantly modify the effect of vitamin D supplementation on risk of upper respiratory infection in COPD patients. Conclusions: I identified environmental determinants that predict 25(OH)D concentrations in all three study populations, but only found an association between vitamin D deficiency and disease severity in COPD patients. Furthermore, I identified a haplotype in VDR which modifies the effect of vitamin D supplementation in COPD patients to result in a significantly reduced risk of ARI.

Patients with Acute Myeloid Leukaemia (AML) present with the signs and symptoms of bone marrow failure. This finding spans the genetic and phenotypic diversity of the disease. The mechanism which underlies it is poorly understood. This thesis explores the effect of AML on the normal haematopoietic stem cell (HSC) population, using primary human diagnostic bone marrow samples. Previous work from our group suggested that AML induces a state of quiescence in HSCs, producing a differentiation block responsible for the observed cytopenias1. Reversal of this process might offer an alternative to the current treatment of patients with palliative transfusions. I have developed a flow cytometry-based technique to differentiate normal HSCs from leukaemia cells, selecting cells with the CD34+38-ALDHhighCLL1- expression signature. Validation of this technique by assessment of sorted cells by FISH and PCR, suggests it is successful in 73% of AML samples. In a further 25% of samples, it selects for a population significantly enriched for normal HSCs. We used this panel to investigate the concentration of HSCs at AML diagnosis, compared to controls. We show that there is no significant difference between HSC concentration at AML diagnosis (n=38, median [HSC] 2.5 cells/μl) and controls (n=24, median [HSC] 2.4 cells/μl). HSC concentration was not significantly affected by AML karyotype, patient age or gender. However, those patients presenting with a low HSC concentration at diagnosis (< 0.1 HSC/μl) were found to have a significantly worse outcome both in terms of overall and relapse-free survival, an effect apparently independent of age, gender and underlying karyotype. HSC concentration at diagnosis with AML may therefore represent a new independent prognostic marker. We then studied CD33 expression patterns on HSCs within Core Binding Factor mutated AML (n=37) at diagnosis, and found its expression to be significantly lower than on HSCs within controls (n=9) (17% versus 58%, p=0.005). CD33 expression on HSCs from AML samples rose significantly from diagnosis to remission (n=16) (17% to 58%, p=0.0001). This mirrors previous findings from our group using CD34low AML samples, and is, we believe, the first time that the antigenic signature of normal HSCs has been shown to be modified. 6 by the presence of AML. However, an in vitro assay to test the significance of these changes in terms of the cytotoxicity of GO towards normal HSCs did not demonstrate a significant difference between HSC subgroups. Finally, we attempted to investigate the mechanism by which AML might induce HSC quiescence by studying the comparative transcriptomes of HSCs from CD34low AML (n=6) and controls (n=6) by RNA-Seq, using direct cell to cDNA synthesis, followed by amplification. A first attempt resulted in poor quality data, with a significant proportion of reads mapping to non-coding DNA regions. A repeat approach, using utilising immediate RNA extraction post sorting resulted in significantly better quality data Bioinformatics analysis revealed differential expression of 6 genes between the 2 datasets (GNPDA1, ADGRG3, MIAT, WDR31, RP11-244H3.1 and RXFP1). GO enrichment studies using David highlighted a number of pathways including the TNF signalling pathway (p=0.003; after Benjamini-Hochberg correction p=0.51). Validation of these findings by independent qPCR, and functional exploration of enriched signalling pathways remains outstanding.

Phosphoinositides (PIs) are pivotal lipid molecules with both scaffolding and signalling functions regulating key aspects of cellular physiology. For example, phosphatidylinositol (3,4,5)-trisphosphate, generated by phosphoinositide 3-kinase (PI3K), is an essential mediator of the PI3K/AKT signalling pathway, which is crucial for cell proliferation, survival and apoptosis. Constitutive activation of this signalling cascade has been identified in acute myeloid leukaemia (AML), the most common haematopoietic malignancy in adults, and experimental deletion of the PI3K antagonists PTEN and SHIP cause leukaemia in mice. However, little is known regarding the role of other PI modulator proteins in AML. Thus, in this thesis, a lentivirally delivered small hairpin RNA (shRNA) library targeting 103 genes (345 pLKO knockdown constructs) with presumed or established roles in PI metabolism was utilised to screen for genes required for AML blast cell viability/proliferation and differentiation. First, knockdown constructs were tested for their impact on proliferation/viability in seven human AML cell lines by measuring fold change in fluorescence of the cell viability dye alamarBlue relative to controls (cells transduced with a non-targeting control hairpin) over three days. This identified 13 candidate genes selected with the criterion that two or more knockdown constructs per gene reduce cell viability/proliferation relative to control by greater than or equal to50 % across all cell lines. From these candidate genes, PIP4K2A, INPP5B and IMPAD1 were selected for downstream validation experiments, which reproduced the observation from the primary screen. For INPP5B and IMPAD1, knockdown constructs also reduced clonogenic potential of primary human AML samples but only showed a modest effect on normal CD34+ haematopoietic stem or progenitor cells (HSPCs) in a methylcellulose based assay. This could be recapitulated in a murine setting where knockdown constructs targeting both genes reduced clonogenic potential of murine MLL- AF9 AML cells with little effect on normal KIT+ HSPCs. In line with this, Inpp5b knockout KIT+ BM cells either failed to immortalise or weakly immortalised, following forced expression of the powerful MLL-AF9 oncogene. A further screen was performed to identify regulators of THP-1 blast cell differentiation, by seeding knockdown construct transduced cells into methylcellulose based semisolid media. After ten days of incubation the degree of macrophage differentiation was evaluated by light microscopy and an arbitrary differentiation score was given. With the criterion that greater than or equal to2 knockdown constructs per gene received the highest differentiation score, reflecting terminal macrophage differentiation of all seeded cells, SBF2 was identified as the top-scoring hit. Validation experiments have confirmed macrophage differentiation based on cytospin preparations of SBF2 knockdown THP-1 cells. Moreover, xenograft assays have shown that knockdown constructs targeting PIP4K2A and SBF2 delayed or abrogated in vivo leukaemogenesis. Thus this work has identified novel roles for PI modulator genes in human AML with possible therapeutic potential.