Molecular analysis of leukaemia genomes during malignant transformation and leukaemia progression

This thesis details a tripartite approach to understanding genetic and epigenetic modifications in human leukaemia. First, an in vitro mouse model of precursor B-cell leukaemia was established by retroviral transduction of primary B-cell precursors with the oncogenes BCR-ABL1 or MYC. The model was used to study DNA damage incurred during the initial phase of oncogene activation i.e. oncogenic stress. Analysis of γH2AX (a mark of DNA damage) and the H3K27 acetylation histone modification (a mark of active enhancers and promoters) by chromatin immunoprecipitation sequencing (ChIP-seq) and mRNA expression (RNA-seq) were combined to establish that lineage specific transcription predisposes lineage specific genes to DNA damage. This was functionally validated with a lineage-switch model and technically validated using FISH. Second, the same cells were maintained during their recovery from the period of acute oncogenic stress until stably transformed. Re-analysis of the transformed populations using the same H3K27Ac ChIP-seq and RNA-seq techniques showed that the stable transformation of these cells is paralleled by activation of a subset of intergenic enhancers which induce an aberrant transcriptome enriched for cell activation and metabolic pathways. Finally, the techniques used in these in vitro experiments and the knowledge of oncogenic enhancers gained from them were then applied to the clinical setting. EVI1, a key haematopoietic transcription factor that regulates differentiation, is aberrantly expressed in a subset of myeloid leukaemia where it is universally associated with a poor prognosis. A patient sample biobank was established and suitable samples were filtered using a combination of RT-qPCR and FISH. Thus, H3K27AC ChIP-seq and newly optimised chromosome conformation capture sequencing (3C-seq or 4C) was used to understand the epigenetic interactions which result in aberrant expression. This has revealed several interactions between the EVI1 promoter with up- and down-stream intergenic regions which must be further investigated.Open Acces

Mapping and prediction of archaeological sites of habitation by modern humans using GIS and expert mapping on the south coast of South Africa

South Africa contains many archaeological resources including shell middens from the Middle Stone Age (MSA) and Later Stone Age (LSA). These shell middens give researchers insight into the behaviour of modern humans where the first fossil evidence appears in Africa around 200 000 years ago (Klein, 2008). Research into shell middens is therefore vital to understand the origin of human kind. This study investigates whether Geographical Information Systems (GIS) is a useful tool for predicting locations of unknown shell midden sites using the characteristics of known areas of modern human habitation. This was done using suitability analysis and expert mapping techniques. Ground truthing of the results of the desktop analysis revealed that GIS is not a useful tool for predicting sites of modern habitation as the characteristics that determine human habitation are too variable

The Effect of Continuous and Pulse Dose Ammonium Chloride Regimens on the Urine pH of Goats

Ammonium chloride (NH4Cl) has been the primary preventive modality for struvite urolithiasis in goats. This study investigated the effect of continuous and pulse dose NH4Cl therapeutic regimens on urine pH in ten goats. The initial regimen (feed additive) consisted of 0.007% NH4Cl as a feed additive. Following this week long regime, the two treatment regimens were designed as a standard ten goat cross-over design. The first treatment regimen (continuous) consisted of daily administration of a titrated dosage of NH4Cl for ten days, followed by four days without treatment. The third treatment regimen (pulse) used daily administration of a titrated dose of NH4Cl for three consecutive days followed by four days without treatment for three treatment periods. Ammonium chloride dosages were titrated to result in a urine pH of < 6.5 (target level) prior to commencing treatment regimens. Urine pH was evaluated once daily during feed additive regimen and twice daily during the treatment regimens. A Bayesian methodology was used to determine the daily odds ratios for production of target urine pH during treatment regimens. The odds ratios were also calculated between pulse dosages during the pulse regimen. The feed additive regimen did not result in target urine pH within 7 days. Treatment with the continuous regimen resulted in target pH, however, pH returned to >6.5 within five days, (odds ratio 0.23-1.56 at Treatment Time 10). The odds ratios for each pulse period of the pulse dose regimen were 2.20-7.45, 0.41-1.68 and 1.59-5.62 respectively. The results of this study indicate that variability in response to therapy warrants titrating individual dosages of NH4Cl, continuous therapy results in a loss of effectiveness, and pulse dosage is effective in repeatedly producing a urine pH of <6.5

Economic and Practical Factors in Diagnosing HNPCC Using Clinical Criteria, Immunohistochemistry and Microsatellite Instability Analysis

Aim: To determine a cost-efficient strategy for HNPCC molecular diagnostic testing. Methods: 138 families referred to a Regional Genetics Service had hMLH1 and hMSH2 mutation analysis. The sensitivity and specificity of clinical selection criteria with or without immunohistochemistry (IHC) and microsatellite instability (MSI) analysis to further refine case selection and the effect of these approaches on the cost of mutation analysis were examined. Results: Clearly deleterious mutations were identified in 49/138 (35.5%) of all families tested. The most sensitive criteria for identifying families with MMR mutations were the full Bethesda guidelines but these have poor specificity. IHC and MSI were useful pre-screening tools. Conclusion: A cost-efficient approach in laboratories where IHC and/or MSI analysis are available, is to use inclusive (non-specific) criteria to select cases, followed by IHC and then MSI. Where one or both results are abnormal, proceed to further mutation analysis. Where MSI or IHC or tumour blocks are not available, more restrictive clinical criteria may be more appropriate for cost-efficient case selection

Stopping rape: towards a comprehensive policy

The need to stop rape is pressing. Since it is the outcome of a wide range of practices and institutions in society, so too must be the policies to stop it. This important book offers a comprehensive guide to the international policies developed to stop rape, together with case study examples on how they work. The book engages with the law and criminal justice system, health services, specialised services for victim-survivors, and educational and cultural interventions, as well as how they can best be coordinated. It is informed by theory and evidence drawn from scholarship and practice from around the world. It will be of interest to a global readership of students, practitioners and policy makers as well as anyone who wants to know how rape can be stopped

Use of oxygen-loaded nanobubbles to improve tissue oxygenation: bone-relevant mechanisms of action and effects on osteoclast differentiation

Gas-loaded nanobubbles have potential as a method of oxygen delivery to increase tumour oxygenation and therapeutically alleviate tumour hypoxia. However, the mechanism(s) whereby oxygen-loaded nanobubbles increase tumour oxygenation are unknown; with their calculated oxygen-carrying capacity being insufficient to explain this effect. Intra-tumoural hypoxia is a prime therapeutic target, at least partly due to hypoxia-dependent stimulation of the formation and function of bone-resorbing osteoclasts which establish metastatic cells in bone. This study aims to investigate potential mechanism(s) of oxygen delivery and in particular the possible use of oxygen-loaded nanobubbles in preventing bone metastasis via effects on osteoclasts. Lecithin-based nanobubbles preferentially interacted with phagocytic cells (monocytes, osteoclasts) via a combination of lipid transfer, clathrin-dependent endocytosis and phagocytosis. This interaction caused general suppression of osteoclast differentiation via inhibition of cell fusion. Additionally, repeat exposure to oxygen-loaded nanobubbles inhibited osteoclast formation to a greater extent than nitrogen-loaded nanobubbles. This gas-dependent effect was driven by differential effects on the fusion of mononuclear precursor cells to form pre-osteoclasts, partly due to elevated potentiation of RANKL-induced ROS by nitrogen-loaded nanobubbles. Our findings suggest that oxygen-loaded nanobubbles could represent a promising therapeutic strategy for cancer therapy; reducing osteoclast formation and therefore bone metastasis via preferential interaction with monocytes/macrophages within the tumour and bone microenvironment, in addition to known effects of directly improving tumour oxygenation

Combined inhibition of p97 and the proteasome causes lethal disruption of the secretory apparatus in multiple myeloma cells.

Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma

Stellenbosch Media Forum 2008

Stellenbosch Media Forum is an annual publication written and produced by the BPhil (Journalism) class of that specific year in the Department of Journalism, Stellenbosch University.Earlier this year Koos Bekker, owner of Media24, said he would not buy the New York Times even though his company could afford it. According to Moneyweb.co.za Bekker said the days of print media are numbered and the New York Times is old news. Die afgelope klompie jare het die media 'n transformasie ondergaan. In Suid-Afrika spesifiek is talle beperkinge op die media in die post-94-era opgehef. Die media funksioneer in 'n vryemark-stelsel en die algemene persepsie is dat dit meer fokus op die kommersiele as tevore. Met die ekonomiese afplatting is daar boonop gerugte van personeelverminderings en word selfs meer verwag van die Gideonsbende wat in die nuuskantore oorbly. Exposure to a variety of TV channels and internet websites is increasing the visual stimulation of media audiences. Media products are being redesigned to satisfy specific needs, in specific niches. One big change in terms of design is that more - and bigger - visuals and less text are being used. Vanjaar het e.tv die eerste 24-uur-nuuskanaal in Suid-Afrika geloods en al hoe meer drukmediaprodukte fokus op hul aanlyn-teenwoordigheid. Die Mail & Guardian het in Junie sy webtuiste herontwerp en sy groepblog, Thoughtleader, het die prys vir die Beste Suid-Afrikaanse Blog in die 2008 Suid-Afrikaanse Blogtoekennnings gekry. The Times, die Sunday Times se interaktiewe dagblad, het vanjaar sy eerste verjaarsdag gevier en bewys (sover) dat die konsep van 'n koerant wat met 'n webblad geintegreer is, wel werk. We are living in exciting times as far as development in the media is concerned. And that is why this year's edition of SMF has as its theme "Change in the media". Die veranderende media is hoofsaaklik te danke aan die ontwikkeling van tegnologie, soos dat jy jou nuus op jou selfoon kan kry. Nuttig, veral in Suid-Afrika waar die toegang tot breebandinternet gebrekkig is. Die koms van blogs noop koerante nou om onmiddellikheid en interaksie na te volg. These developments also influence journalists, sources and their audiences. With the advent of democracy in South Africa, changes in the consumer demographics of certain media products have occurred. And, oh yes, women have also advanced in the media since 1994, both as producers of media, and how they are represented. And then there is the youth, who "owns" new media technologies. And the disabled, who can get access to a new world through media technology. But, there are still many people in South Africa who do not have access to media, because of socio-economic circumstances: too poor to own the latest technology; illiterate and forgotten by the media elites. Die rol van die media, om debat te stimuleer, as waghond op te tree en die stem van die stemloses te wees, word deur al hierdie veranderings uitgedaag. Toenemende kommersialisering kan mediavryheid van binne erodeer. En dan is daar steeds die moontlikheid van politieke inmenging, al is mediavryheid grondwetlik verskans. Tradisionele kunsvorme in die media, soos kortverhale, radiodramas en boekresensies word gemarginaliseer, maar tog is daar die moontlikheid dat hulle kan aanpas en bly voortbestaan. Another challenge is the media's coverage of environmental issues, which has to be in sync with the phenomenon of global warming. Sport reporting also has to adapt to new developments, with sport writers now needing to have a knowledge of economics and politics as well. Een ding is seker: maatskappye, mense, produkte en onderwerpe wat by die media betrokke is, sal soos 'n verkleurmannetjie moet aanpas om te kan oorleef