The hematopoietic stem cell (HSC) is characterized by its ability to self-renew and produce all mature blood cells throughout the life of an organism. This is tightly regulated to maintain a balance between survival, proliferation, and differentiation. The HSCs are located in specialized niches in the bone marrow thought to be low in oxygen, which is suggested to be involved in the regulation of HSC maintenance, proliferation, and migration. However, the importance of hypoxia in the stem cell niche and the molecular mechanisms involved remain fairly undefined. Another important regulator of human HSCs maintenance is the tyrosine kinase receptor FLT3, which triggers survival of HSCs and progenitor cells. Mutations in FLT3 cause constitutively active signaling. This leads to uncontrolled survival and proliferation, which can result in development of acute myeloid leukemia (AML). One of the purposes with this thesis is to investigate how survival, proliferation and self-renewal in normal HSCs are affected by hypoxia. To study this, we used both in vitro and in vivo models with isolated Lineage-Sca-1+Kit+ (LSK) and CD34-Flt3-LSK cells from mouse bone marrow. We found that hypoxia maintained an immature phenotype. In addition, hypoxia decreased proliferation and induced cell cycle arrest, which is the signature of HSCs with long term multipotential capacity. A dormant state of HSCs is suggested to be critical for protecting and preventing depletion of the stem cell pool. Furthermore, we observed that hypoxia rescues HSCs from oxidative stress-induced cell death, implicating that hypoxia is important in the bone marrow niche to limit reactive oxidative species (ROS) production and give life-long protection of HSCs. Another focus in this thesis is to investigate downstream pathways involved in tyrosine kinase inhibitor-induced cell death of primary AML cells and cell lines expressing mutated FLT3. Our results demonstrate an important role of the PI3K/AKT pathway to mediate survival signals from FLT3. We found FoxO3a and its target gene Bim to be key players of apoptosis in cells carrying oncogenic FLT3 after treatment with tyrosine kinase inhibitors. In conclusion, this thesis highlights hypoxic-mediated regulation of normal HSCs maintenance and critical effectors of apoptosis in leukemic cells expressing mutated FLT3. / <p>On the day of the defence date the title of article II was "Hypoxia, via hypoxia-inducible factor (HIF)-1, mediates low cell cycle activity and preserves the engraftment potential of mouse hematopoietic stem cells" and one of the authors is no longer included in the article.</p>

<p>Background. Platelets (thrombocytes) are the smallest cells in the blood. Platelet fulfils functions as formation of blood clots when bleeding. Low levels leads to bleeding while high levels increase the risk of thrombosis (obstruction of the circulatory flow system). Platelet transfusions may be required for patients with systemic bleeding and for patients at higher risk of bleeding because of coagulation defects, sepsis (presence of bacteria in the bloodstream), or platelet dysfunction related to medication or disease. A pathogen-reduction system for platelet components would be a useful method since it reduces the risk of bacterial, protozoa, viral and white blood cell contamination. The Intercept Blood System method (IBS) for platelets, destroys DNA and RNA and was validated against the routine method in order to reduce pathogen transmission risk during transfusion. The validation of IBS, the trombocyte count for100 buffy coat concentrates from 2007 were compared to values for 100 buffy coat concentrates from 2006 that had been treated with gamma-radiation. Akademiska sjukhuset in Uppsala has a requirement that 75% of the platelet concentrates contain at least 300*10 9 platelets per unit. IBS fulfilled to 94% compared to 98% for the routine method.</p><p>Thus, the IBS-method was well above the required value and is now used at</p><p>Akademiska sjukhuset in Uppsala.</p>

Background. Platelets (thrombocytes) are the smallest cells in the blood. Platelet fulfils functions as formation of blood clots when bleeding. Low levels leads to bleeding while high levels increase the risk of thrombosis (obstruction of the circulatory flow system). Platelet transfusions may be required for patients with systemic bleeding and for patients at higher risk of bleeding because of coagulation defects, sepsis (presence of bacteria in the bloodstream), or platelet dysfunction related to medication or disease. A pathogen-reduction system for platelet components would be a useful method since it reduces the risk of bacterial, protozoa, viral and white blood cell contamination. The Intercept Blood System method (IBS) for platelets, destroys DNA and RNA and was validated against the routine method in order to reduce pathogen transmission risk during transfusion. The validation of IBS, the trombocyte count for100 buffy coat concentrates from 2007 were compared to values for 100 buffy coat concentrates from 2006 that had been treated with gamma-radiation. Akademiska sjukhuset in Uppsala has a requirement that 75% of the platelet concentrates contain at least 300*10 9 platelets per unit. IBS fulfilled to 94% compared to 98% for the routine method. Thus, the IBS-method was well above the required value and is now used at Akademiska sjukhuset in Uppsala.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease where a significant proportion of patients will develop an aggressive disease. Today, the mutational status of the immunoglobulin heavy variable (IGHV) genes is one of the strongest prognostic markers in CLL, where unmutated IGHV genes correlate with poor outcome. In addition, IGHV3-21 gene usage is associated with poor prognosis independent of mutational status. Recently, several genes were shown to be differently expressed between IGHV mutated and unmutated CLL and were suggested as prognostic markers. The aim of this thesis was to examine the applicability of these RNA-based prognostic markers in CLL. In papers I and II, the prognostic significance of LPL and TCL1A mRNA expression in CLL was investigated in 140 and 144 patients, respectively. High expression was found to be associated with inferior clinical outcome for both markers. However, CLL cases with mutated IGHV3-21 genes displayed low levels of LPL expression, indicating that LPL cannot identify this poor-risk patient group. In contrast, high TCL1A expression was detected in all IGHV3-21 cases. To elucidate the functionality of LPL in CLL, LPL lipase activity was measured in 33 cases. The lipase activity was found to be invariably low, implying an alternative function for LPL in CLL. In paper III, a comprehensive analysis of five RNA-based markers (LPL, TCL1A, ZAP70, CLLU1 and MCL1) was performed in 252 CLL patients. All RNA-based markers except MCL1 predicted clinical outcome, with LPL being the strongest. Moreover, LPL expression independently predicted overall survival when adjusted for established markers. All of the RNA-based markers added additional prognostic information to established markers, e.g. high LPL expression predicted an inferior outcome in patients with mutated IGHV genes or good-risk cytogenetics. For clinical application, over time stability of prognostic markers is crucial. In paper IV, the expression of LPL, TCL1A, ZAP70 and MCL1 was investigated in samples taken at diagnosis and at a follow-up of seven years in 104 CLL patients. LPL was found to be the most stable marker, displaying high correlation between the sequential samples, whereas ZAP70 and MCL1 varied significantly. TCL1A expression increased at follow-up, which may indicate disease progression as TCL1A promotes cell survival. In summary, this thesis highlights the applicability of RNA-based markers in CLL prognostication, both as single markers or in combination with established markers. In particular, LPL was shown to be the strongest RNA-based marker in terms of prognostic strength and stability.

This study was undertaken to investigate the relationship between Natural Killer cells and HLA-G at the placental barrier in HIV-I infected pregnant women and to establish the significance, if any, to in utero infection. Fifty-five HIV -I infected pregnant women were recruited into the study after consent was obtained. Blood samples were collected from both mothers and babies for viral loads and CD4+ cell counts. Placental samples were obtained from pregnancies at delivery and examined by immunoperoxidase immunohistochemistry methods using monoclonal antibodies to p24 antigens and Natural Killer (CD56+) cells. HLA-G expression was quantified using real-time polymerase chain reaction. Analysis of viral loads and CD4+ cell counts were undertaken in categories. No significant association was observed between the viral load of mothers and their CD4+ cell counts. Eighteen percent of the women in this study population had 5 log viral loads with a transmission rate of 0.27(95% Cl, 0.15 - O. 39). Maternal viraemia was significantly associated with transmission of infection to babies (p = 0.047). The odds ratio indicated that for every 1 log increase in maternal viral load the babies were 3.1 times more likely to acquire the infection (Exp (B) = 3.137 (95%CI, 1.015-9.696). Furthermore, the study found that a higher number of female babies were infected than males. Although not statistically significant the odds ratio indicated that female babies were 3.1 times more likely to become infected than males (Exp (B) = 3.110 (95%CI, 0.819-11.808). We report here the results of immunohistochemistry for p24 antigens and NK (CD56+) cells and compare them to the immunological responses of both mothers and babies at birth. HIV-1 antigens were detected in 94.5% of all placentas by immunohistochemistry. Infiltration of CD56+ was found in 98% of placental tissue. The analysis revealed that the presence of p24 antigens in placental tissue was not influenced by maternal viral load or CD4+ cell counts. Lower median NK cell values were observed in placentas of mothers with infected babies as compared with the uninfected cluster. Although not statistically significant, the risk of vertical transmission was increased 3.4 times more in placentas which had lower NK cell values. According to the odds ratio, babies CD4+ counts were affected by every 1 log increase in mother's viral load. Overall, maternal viral load emerged as a strong predictor for risk of infection from infected mothers to their infants. Our analysis indicated that female babies were 3.7 times more likely to acquire the infection than males. Using data obtained from real-time PCR we investigated the relationship between maternal viral load and the quantity of HLA-G expression (p = 0.045; 95%CI 1.029- 11.499). Logistic regression models revealed that mother's viral load was the strongest risk factor for vertical transmission. No statistically significant correlation was noted with HLA-G and viral transmission. However, the odds ratio indicated that the risk of infection increased by 1.3 with every 1 fold increase in HLA-G expression. An analysis of mother-to-child transmission rates by gender revealed that the odds ratio for transmission was 3.4 times more in female babies than in males. We then investigated the relationship between maternal viraemia and HLA-G expression. A positive correlation between maternal viral load and placental HLA-G was observed (p = 0.038). When gender susceptibility to HLA-G expression was explored a statistically significant association was observed in placental tissue of mothers with infected and uninfected male babies and HLA-G expression (p = 0.013). To conclude, the analysis found that HLA-G was up regulated 3.95 times more in placental tissue of mothers with infected babies than in mothers with uninfected babies. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2007.

No description available.

Le lymphome est un cancer des ganglions lymphatiques, lieu de prolifération et différenciation des cellules immunitaires en particulier des lymphocytes B qui sont des cellules productrices d'anticorps. Les lymphomes résultent de l’accumulation de mutations génétiques dans le génome d’une cellule B normale contribuant à la transformation en cellule B maligne. Cette cellule B dite transformée prolifère alors pour engendrer un clone de cellules B malignes qui s’accumulent au niveau des ganglions lymphatiques, formant alors une tumeur appelée ‘lymphome B’ (pour cancer lymphoïde issu de la transformation maligne des lymphocytes B). Le ganglion lymphatique normal a une structure histologique qui se décompose ainsi, du centre à la périphérie : le centre germinatif, la zone du manteau et la zone marginale. Les lymphomes B sont classés en différents sous-types histologiques en fonction de leur origine topographique au niveau du ganglion lymphatique et de leurs caractéristiques bio cliniques spécifiques. Parmi ces sous-types, une forme particulièrement agressive peut être distinguée : le lymphome à cellules du manteau. Ce sous-type de lymphome est caractérisé par des rechutes successives et une survie qui est généralement courte (médiane de survie de 4 à 5 ans) même si certains patients, avec des formes plus indolentes de lymphomes à cellules du manteau, présentent des survies prolongées (médiane de survie de 7 à 10 ans). Des biomarqueurs prédictifs de la courte survie manquent aujourd’hui, ce qui rend difficile la prise en charge optimisée des patients. Ce projet s’intéresse à cette question. Plus précisément, nous nous proposons de rechercher des mutations génétiques associées à la résistance thérapeutique. Notre approche sera basée sur le séquençage ciblé à haut débit du génome de cellules B tumorales issus de patients présentant des cas classiques du lymphomes à cellules du manteau mais aussi des cas plus particuliers comme ceux présentant des résistances thérapeutiques précoces, par exemple. Par cette approche dite de ‘cartographie’ à l’échelle du génome, nous espérons identifier des nouveaux prédicteurs moléculaires de la survie chez ces patients atteints de lymphome à cellules du manteau et également apporter de nouvelles connaissances dans l’interconnexion entre la génétique et l’épigénétique dans cette maladie. / Lymphoma is a cancer of the lymph nodes which are organs in which immune cells, particularly the antibody producing B cells, proliferate and differentiate before circulating in the blood and tissues to fight infection. B cell lymphoid cancers – ‘B cell lymphoma’ arise as a consequence of the occurrence of gene mutations in B cells. By affecting the functions of key B cell genes, these mutations drive the malignant transformation of the affected B cells which then begin to divide abnormally eventually destroying normal lymph node organization and function. The lymph node is divided into distinct micro-anatomical compartments or zones which are called (from the inner to outer most compartment – germinal centre, mantle zone, and marginal zone). B cell lymphoma classification follows this general organization and classifies tumours depending on the compartment of origin of the particular tumour B cell population. This classification thus defines lymphoma according to a ‘histological subtype’ with defined clinic-biological features. Among these subtypes, mantle cell lymphoma (MCL) is a particularly aggressive form of B lymphoid cancer. This type of lymphoma is characterised by successive relapses and short survival (median is 4 to 5 years), although some patients can show long survival. Predictive biomarkers of this clinical behavior are lacking. This project aims to address this question. More specifically we propose to perform whole ‘exome’ sequencing – i.e. sequencing of all protein coding sections of all known protein coding genes in the genome – of the tumour B cell DNA from patients who show refractory or early relapsing disease compared to patients who show relatively long survival. By doing this genome scale study we hope to identify new gene mutations that can serve as molecular predictors of survival and bring new knowledges in the understanding between genetics and epigenetics in MCL.

Commercial capture-based aquaculture of the Atlantic bluefin tuna (ABT), Thunnus thynnus (L.), has been prominent in the Mediterranean for over a decade. Owing to several limitations encountered in working with the species, including its high commercial value, there has been little research carried out relating to this species. The objective of this study was to examine several health parameters of captive ABT. These included an examination of coronary artery lesions, haematology, plasma biochemistry, assessment of immune function and changes in fatty acid (FA) flesh content through the on-growing period. Arteriosclerosis in fish is a pathologic condition of uncertain etiology and involves the main coronary artery in teleosts. Apart from reports of their widespread occurrence in salmonids, they have been described from a restricted number of wild ABT specimens but have not received further attention. This investigation analysed the effect of size and period of net-pen rearing on the prevalence and severity of arteriosclerotic lesions in ABT. Coronary arteries from wild and captive fish were investigated and prevalence was 100 %, but increasing structural degradation was observed with increasing fish size, suggesting that lesions progress throughout the life of the fish. Due to the limited availability of wild specimens, the effect of captivity on arteriosclerosis in ABT could not be adequately quantified, although observations suggest that the farming process has no major effect on arteriosclerotic lesions in ABT. Studies on the haematology, plasma biochemistry and immunology of ABT are limited. Haematological and plasma biochemical indices are useful in animal health assessment but use of these requires the establishment of species-specific ranges. Blood was collected from captive ABT specimens of varying weight (61-361 kg) and the major haematological (n = 45), plasma biochemical (n = 30) and immunological parameters (n = 45) were quantified. Size-based differences were found in haematological indices between experimental sub-groups including increased erythrocyte number and haemoglobin level in smaller ABT. No differences were found in immunological parameters except for total IgM levels, which were higher in the smaller individuals. Preliminary investigations indicated that disease prevalence in captive ABT is very low. Epidermal mucus is an important interface between fish and their environment and comprises immunological components which act as a first barrier against pathogen entry or colonisation. Mucus was collected from captive ABT and analysed for innate immune components. The presence of IgM was detected in the mucus of ABT by an enzyme-linked immunosorbent assay and several different enzymes were detected with an API-ZYM kit assay. Zymography experiments confirmed the presence of protease-like enzymes in the mucus, while enzyme assays quantified alkaline phosphatase, protease, esterase and cathepsin B activities. Lysozyme levels were high. The mucus agglutinated sheep erythrocytes but did not demonstrate complement or bacteriolytic activity. There is restricted information on the fatty acid composition of farmed ABT or how this is influenced when the fish are held under commercial aquaculture conditions. This study investigated the FA composition of farmed ABT, its variation by dorsal muscle region and the correlation between dietary FA composition with that of the fish. Analysis of flesh samples retrieved from farmed ABT did not reveal significant differences in the FA composition of experimental sub-groups irrespective of size, time held in captivity or diet. These results indicate that FA metabolism in ABT is substrate-selective. Gene expression measurements from several organs of ABT showed that expression of Δfad5 and elovl5, genes involved in FA metabolism, were highest in the brain followed by the liver but no expression of these genes was detected in the spleen. The findings of this research address aspects of health evaluation and nutritional status in farmed ABT and are discussed in terms of farming practice. Conclusions from some of these studies suggest that the practice of holding wild-captured stock in cages for periods of up to 18 months does not result in significant impact on ABT.

Thesis (PhD (Curriculum Studies))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The Faculty of Health Sciences at Stellenbosch University adopted a new set of guidelines for curriculum design in 1997, emphasising an orientation towards the requirements of the public sector general practice, a holistic approach and exposure to community lifestyle and disease patterns specific to various communities. In order to ensure the anchoring in the realities of the general practice, a family medical practitioner, appointed by the Academy of Family Practice, was included in the curriculum control structure of the faculty. It was further recommended that a family medical practitioner should be included in the curriculum committee of each discipline, where appropriate. The present research, starting from the assumption that the opinion of a single family practitioner is insufficient to determine the adequacy of the curriculum for general medical practice, aimed to conduct a comprehensive needs analysis of all stakeholders in the undergraduate haematology training programme at the Faculty of Health, Stellenbosch University, and to compare the findings with the existing curriculum. To this purpose, the opinions of five adult medicine haematologists, ten paediatric haematologists, four laboratory haematologists, ten interns, fourteen students and twenty general practitioners were surveyed. An open-ended questionnaire on the usefulness of the haematology module for hospital and independent general practice was analysed, using the “coding technique” method. On this basis, a list of subjects was drawn and, using a Delphi method, the participants in the study were asked to rate their importance for practice. The answers to the open-ended questionnaires revealed a few overarching concepts, the most important being the need to structure the material taught in the form of “approaches”, supporting the differential diagnosis, which is the most frequent task of a general practitioner. Among the outcomes identified in the panellists‟ answers, the need to adequately detect and assess the “red flag” signs for haematological cancers was proposed for consideration as an outcome in the next curriculum. The Delphi survey indicated a group of subjects which were rated as most important for practice and another group designated as devoid of utility. The remaining subjects, rated as of moderate importance, could be further classified as diseases usually managed by the general practitioner and pathology which would be referred to a specialist for management. These iv findings were compared with the existing curriculum and the discrepancies were analysed, resulting in a set of proposals towards a framework for a new undergraduate haematology curriculum. For the first time in the literature, as far as can be determined, this research presents outcomes and content for an undergraduate haematology course which were defined and rated for importance by consensus of the curriculum developers, specialists in the field and graduates of the course. The methods tested in this process and some of the trends revealed might be useful for curriculum development in other medical disciplines. / AFRIKAANSE OPSOMMING: Die Fakulteit van Gesondheidswetenskappe by die Universiteit Stellenbosch het in 1997 nuwe riglyne vir kurrikulumontwerp aanvaar. Hierdie riglyne beklemtoon `n bewustheid van die behoeftes van algemene praktyk in die openbare sektor, `n omvattende benadering tot en blootstelling aan die gemeenskapslewenstyl, asook aan siektepatrone eie aan verskillende gemeenskappe. Om te verseker dat die kurrikulum in die werklikhede van algemene praktyk geanker bly, is `n algemene praktisyn, aangestel deur die Akademie van Huisartskunde, ingesluit in die kurrikulum beheerstruktuur van die fakulteit. Dit is verder ook aanbeveel dat, waar van toepassing, `n huisarts in die kurrikulumkomitee van elke dissipline ingesluit moet word. Hierdie navorsing, wat van die veronderstelling gespruit het dat die opinie van `n enkele huisarts onvoldoende is om die toepaslikheid van `n kurrikulum vir algemene praktyk te verseker, het ten doel gestel om `n omvattende analise van behoeftes van alle belanghebbendes in die voorgraadse hematologie-opleidingsprogram by die Fakulteit van Gesondheidswetenskappe, Universiteit van Stellenbosch, te doen en om die bevindings met die bestaande kurrikulum te vergelyk. Die menings van vyf volwasse medisyne hematoloë, tien pediatriese hematoloë, vier laboratorium hematoloë, tien huisdokters, veertien studente en twintig algemene praktisyns is verkry. `n Oop-einde vraelys oor die bruikbaarheid van die hematologie-module vir hospitaal- en onafhanklike algemene praktyk is m.b.v die gekodeerde tegniek ontleed. Op grond hiervan is `n lys onderwerpe gekies en studiedeelnemers is deur van die Delphi-metode gebruik te maak, gevra om die graad van belangrikheid van elkeen aan te dui. Die antwoorde op die oop-einde vraelys het `n paar oorkoepelende konsepte uitgelig. Die belangrikste hiervan was om die materiaal wat gedoseer word te struktureer in die vorm van „benaderings‟, wat die vorming van `n differensiële diagnose ondersteun. Lg. is die algemeenste taak van die algemene praktisyn. Een van die uitkomste wat deur die studiedeelnemers geïdentifiseer is, nl. die vermoë om die `rooi vlag` tekens van hematologiese kankers korrek te bespeur en te assesseer, is voorgestel vir oorweging vir insluiting as `n uitkoms in die volgende kurrikulum.

No description available.