Improving the repopulation potential of human umbilical cord blood (UCB) haemopoietic stem cells (HSCs) remains a paramount goal in HSC transplantation (HSCT) therapy. This implies enhancing the homing and engraftment potential of UCB-CD34+CD133+ cells to the bone marrow (BM). Although an array of molecules continues to be identified as ‘key’ homing molecules, the molecular mechanisms controlling HSC homing are still not fully understood. The regulatory implications of hypoxia in the BM, with the concomitant stabilisation of hypoxia inducible transcription factor-1α (HIF-1α), are becoming more apparent, yet at the commencement of this thesis no study had explored whether hypoxia induced signalling can be adopted to regulate the homing and engraftment of transplanted HSCs. The aim of this DPhil project was thus to investigate whether hypoxic conditions as detected in the BM influence the adhesion of UBC-CD133+ cells to osteoblasts, BM stromal cells and BM endothelial cells-60 (BMEC-60), as well as their transmigration towards chemokine SDF-1α across BMEC-60. Increasing the exposure of UCB-CD133+ cells to 1.5% O2 doubled the percentage of transmigrating cells (p<0.05), and while hypoxia stimulated UCB-CD133+ cells preferentially adhered to IL-1β stimulated BMEC-60, their adhesion to non-stimulated (BMEC-60) was significantly improved (p<0.001). To help unravel the underlying molecular mechanisms, we attempted to examine the potential involvement of hypoxia regulated scaffolding protein HEF-1/NEDD9/Cas-L (HEF-1) in the increased percentage of migrating UCB-CD133+ cells after hypoxia pre-conditioning. The role of HEF-1 in HSCs is unexplored, and its multifunctional contribution in a variety of processes including cell migration, attachment and invasion make HEF-1 a prime candidate as a contributing homing molecule. After identifying a suitable short-hairpin RNA (shRNA) sequence to knockdown HEF-1, generating lentiviral (LV)-particles in house and optimising transduction protocols, HEF-1 knockdown was achieved in haemopoietic model cell lines KG-1 and KG-1A (KG-1/KG-1A–HEF1). Significantly decreased KG-1A–HEF1 cell adhesion to non-stimulated BMEC-60 was detected. Together, these studies provide a promising platform to further explore the role of HEF-1 in hypoxia induced UCB-CD133+ cell transmigration towards the key homing molecule SDF-1α.

GATA1 and KLF1 are transcription factors that regulate genes which are important for the development of erythroid cells. The GATA1 transcriptional co-factor FOG1 has been shown to be essential in a wide range of GATA1 dependent cellular functions. Here we tried to understand the diverse mechanisms by which GATA1 and KLF1 recognize their binding sites, how the GATA1 recognition mechanisms are affected by complexation with either FOG1 or KLF1 and how the GATA1 recognition mechanisms affect the transcriptional regulation of the erythroid differentiation. We profiled the DNA binding specificities/affinities of a GATA1 fragment (mGATA1NC), that contains only the two DNA binding domains (N-terminal and C-terminal Zn finger), and the DNA binding specificities/affinities of a KLF1 fragment (mKLF1257-358), that contains the three DNA binding domains, using a novel methodology that combines EMSA with high throughput sequencing (EMSA-seq (Wong et al., 2011a)). We also profiled the DNA binding specificities of the C-terminal Zn finger of GATA1 alone (mGATA1C), the wt-mGATA1, the wt-mGATA1/wt-mFOG1 complex and the mGATA1NC/mKLF1257-358 complex. At first, we confirmed that the N-terminal Zn finger of GATA1 has a strong preference for the “GATC” motif, whereas the C-terminal Zn finger of GATA1 has a strong preference for the “GATA” motif. Next, we found that in the mGATA1NC, both DNA binding domains can bind simultaneously a wide range of different positional combinations of the co-occurring “GATA” and “GATC” motifs, on the same DNA sequence. The wt-mGATA1 did not show the ability to bind in the same co-occurring motifs implying an effect of the non-DNA binding domains of the protein in the regulation of its DNA binding specificities. On the contrary, complexation of wt-mGATA1 with the wt-mFOG1 partially restored its ability to bind in a now limited range of different positional combinations of the co-occurring “GATA” and “GATC” motifs, on the same DNA sequence. Similar observations were made for other pairs of GATA1 N-terminal and C-terminal Zn finger specific motifs. We then projected the GATA1 DNA binding specificities/affinities in vivo and we classified the GATA1 ChIP-seq peaks in low, medium or high affinity based on the number of the GATA1 motifs. We noticed that high affinity GATA1 ChIP-seq peaks tend to appear in regions with low nucleosome occupancy. We also noticed that GATA1 ChIP-seq peaks in the enhancer regions are usually high affinity whereas GATA1 ChIP-seq peaks in the proximal promoter regions are usually low affinity. Additionally, we observed that high affinity GATA1 ChIP-seq peaks are usually found in regions with increased levels of H3K4me2 and are associated with a higher decrease in the H3K4me3 levels on the TSS of the nearby genes. None of these GATA1 related in vivo observations were found for the KLF1 ChIP-seq positions. These findings significantly advance our understanding of the DNA binding properties of GATA1, KLF1 and their complexes and give an insight on the importance of the GATA1 DNA binding affinities in the regulation of the erythroid transcriptional program. Overall the work establishes an experimental and analytical framework to investigate how transcriptional co-factors can change the DNA binding specificities of specific transcription factors and how integration of the transcription factor DNA binding affinities with in vivo data can give novel insights into the transcriptional regulation.

Against the background of a dilemma experienced by researchers during a genomics study at an established biomedical research centre in Kenya, the broad aims of this thesis are to develop appropriate responses to important ethical questions on sharing information on a common and serious genetic condition, sickle cell disorder, and assess the responsibilities of researchers in this regard. Using an empirical approach to normative reflection across two phases of qualitative research, I explore the nature of important moral concerns related to sharing sickle cell disease information from researchers’ and community members’ points of view; and develop a bottom-up normative analysis around the questions generated. This analysis interweaves community experiences, processes of community reasoning and ex situ normative reflection; placing community views and values centrally while referencing these to wider ethical debates, commentaries and guidelines in the literature. Two main outputs of this thesis are to provide recommendations for information sharing on SCD findings in the genomics study in Kilifi; and to propose a set of key issues to consider for this type of information in other studies and geographic settings. I conclude that researchers have a strong responsibility to share SCD information on affected children with families as a form of ancillary service (validating tests, counselling and care); but less responsibility to actively share carrier information. Concurrent responsibilities are working collaboratively with the Ministry of Health/District General Hospital to plan and implement services for SCD; ensuring counselling services support family stability as far as reasonably possible; and to build forms of community engagement and informed consent that counter risks of diagnostic interpretations of research.

Over a third of the world’s population lives at risk of potentially severe Plasmodium vivax malaria. Unique aspects of this parasite’s biology and interactions with its human host make it harder to control and eliminate than the better studied Plasmodium falciparum parasite. Spatial mapping of two human genetic polymorphisms were developed to support evidence-based targeting of control interventions and therapies. First, to enumerate and map the population at risk of P. vivax infection (PvPAR), the prevalence of this parasite’s human blood cell receptor – the Duffy antigen – was mapped globally. Duffy negative individuals are resistant to infection, and this map provided the means to objectively model the low endemicity of P. vivax across Africa. The Duffy maps helped resolve that only 3% of the global PvPAR was from Africa. The second major research focus was to map the spatial distribution of glucose-6-phosphate dehydrogenase enzyme deficiency (G6PDd), the genetic condition which predisposes individuals to potentially life-threatening haemolysis from primaquine therapy. Despite this drug’s vital role in being the only treatment of relapsing P. vivax parasites, risks of G6PDd-associated haemolysis result in significant under-use of primaquine. G6PDd was found to be widespread, with an estimated frequency of 8.0% (50% CI: 7.4-8.8%) across malarious regions. Third, it was important to represent more detailed descriptions of the genetic diversity underpinning this enzyme disorder, which ranges in phenotype from expressing mild to life-threatening primaquine-induced haemolysis. These variants’ spatial distributions were mapped globally and showed strikingly conspicuous distributions, with widespread A- dominance across Africa, predominance of the Mediterranean variant from the Middle East across to India, and east of India diversifying into a different and diverse array of variants, showing heterogeneity both at regional and community levels. Fourth, the G6PDd prevalence and severity maps were synthesised into a framework assessing the spatial variability of overall risk from G6PDd to primaquine therapy. This found that risks from G6PDd were too widespread and potentially severe to sanction primaquine treatment without prior G6PDd screening, particularly across Asia where the majority of the population are Duffy positive and G6PDd was common and severe. Finally, the conclusions from these studies were discussed and recommendations made for essential further research needed to support current efforts into P. vivax control.

The presence of TP53 mutations has been associated with inferior outcome in diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). In DLBCL, the impact of the TP53 codon 72 polymorphism and MDM2 SNP309 has not been clearly elucidated, whereas MDM2 SNP309 was suggested as a poor-prognostic marker in CLL. In addition, p16INK4a promoter hypermethylation has been implicated as a negative prognostic factor in DLBCL. The aim of this thesis was to further evaluate these molecular markers in well-characterised materials of DLBCL and CLL. In paper I, we investigated the prognostic role of TP53 mutation, codon 72 polymorphism and MDM2 SNP309 in DLBCL (n=102). The presence of TP53 mutations (12.7%) correlated with a poor lymphoma-specific and progression-free survival, and a particularly pronounced effect was observed in the germinal center subtype. Neither the MDM2 SNP309 nor the TP53 codon 72 polymorphism had an impact on age of onset or survival. In paper II, we applied pyrosequencing to measure the level of p16INK4a methylation in DLBCL (n=113). Thirty-seven percent of cases displayed p16INK4a methylation; however, no clear association could be observed between degree of methylation and clinical characteristics or lymphoma-specific survival. In papers III–IV, we investigated the prognostic role of MDM2 SNP309 (n=418) and TP53 mutation (n=268) in CLL. No correlation was observed between any particular MDM2 SNP309 genotype and time to treatment and overall survival. Furthermore, no association was found between the different MDM2 SNP309 genotypes and established CLL prognostic markers. TP53 mutations were detected in 3.7% of CLL patients; where the majority showed a concomitant 17p-deletion and only three carried TP53 mutations without 17p-deletion. We confirmed a significantly shorter overall survival and time to treatment in patients with both TP53 mutation and 17p-deletion. Altogether, our studies could confirm the negative prognostic impact of TP53 mutations in DLBCL, whereas MDM2 SNP309 and TP53 codon 72 polymorphisms appear to lack clinical relevance. We also question the role of p16INKa methylation as a poor-prognostic factor in DLBCL. Finally, the presence of TP53 mutation in CLL appears to be rare at disease onset and instead arise during disease progression.

Chronic lymphocytic leukemia (CLL) is a heterogeneous malignancy displaying varying clinical outcome, where molecular markers today can divide patients into prognostic subgroups. Despite the introduction of new agents for treatment, remissions are usually not sustained in CLL and resistance towards treatment can partly be explained by aberrant apoptosis. The aim of this thesis was to find new drugs for CLL patients resistant to conventional therapy and to analyze genes involved in apoptosis within different prognostic subgroups. In paper I-II, the in vitro activity of substances was investigated using the fluorometric microculture cytotoxicity assay (FMCA). When evaluating rapamycin (paper I), an inhibitor of mTOR, in 97 tumor samples from different entities, CLL was found to be one of the most sensitive tumor types. Combination experiments on patient CLL cells indicated that rapamycin acted synergistically with the CLL drugs vincristine and chlorambucil. An investigation of 20 anti-cancer agents in cells from 40 CLL patients (paper II) revealed that prednisolone and rolipram displayed high activity in poor-prognostic patients, in particular IGHV unmutated CLL. Furthermore, when used in combination these agents were found to produce a synergistic effect. In paper III, the anti-apoptotic BCL2 family member BFL1 was evaluated in 37 CLL cases. Levels of BFL1 were higher in fludarabine-resistant patients compared to fludarabine-sensitive patients. In addition, the high expression of BFL1 inversely correlated to fludarabine-induced apoptosis in CLL cells. A single nucleotide polymorphism in the anti-apoptotic BCL2 gene (-938C&gt;A) has been suggested as a novel poor-prognostic marker in CLL. In paper IV, we investigated this BCL2 polymorphism in 268 CLL patients and correlated genotypes to clinical data. However, no association could be confirmed between this polymorphism and clinical outcome or established prognostic markers. In conclusion, this thesis has shown that rapamycin is a potential drug for treatment in CLL. Furthermore, prednisolone and rolipram were identified as interesting candidates for treatment of poor-prognostic patients. Finally, the anti-apoptotic protein BFL1 may contribute to chemoresistance and hence represents a potential therapeutic target in CLL, whereas from our data, the BCL2 -938C&gt;A polymorphism does not appear to have any prognostic significance.

The clinical course of chronic lymphocytic leukemia (CLL) is highly heterogeneous, which has prompted the search for biomarkers that can predict prognosis in this disease. The IGHV gene mutation status and certain genomic aberrations have been identified as reliable prognostic markers of clinical outcome for this disorder. However, the search for more feasible prognostic markers in CLL is still being pursued. Recently, certain single nucleotide polymorphisms (SNPs) in the GNAS1, BCL2 and MDM2 genes and the RNA expression levels of the LPL, ZAP70, TCL1, CLLU1 and MCL1 genes were suggested as novel prognostic markers in CLL. In papers I-III, we performed genotyping analyses of the GNAS1 T393C, BCL2 -938C&gt;A and MDM2 SNP309 polymorphisms in 268-418 CLL patients and related the genotypes with clinical data. Association studies between the polymorphisms and established prognostic markers (i.e. IGHV mutation status, genomic aberrations, CD38 expression) were also performed. Our studies did not find any significant relationship between these SNPs with either clinical outcome or other known prognostic markers in CLL. In paper IV, we measured the RNA expression levels of LPL, ZAP70, TCL1, CLLU1 and MCL1 in 252 CLL cases and correlated these levels with clinical outcome. Here, we verified that high expression of all these RNA-based markers, except MCL1, were associated with an unfavourable prognosis. We also confirmed a close relationship between IGHV mutation status and the RNA-based markers, especially for LPL and CLLU1 expression. Among the RNA-based markers, multivariate analysis revealed LPL expression as the strongest independent prognostic marker for overall survival and time to treatment. Furthermore, the RNA-based markers could add further prognostic information to established markers in subgroups of patients, with LPL expression status giving the most significant results. In summary, data from papers I-III could not verify the GNAS1 T393C, BCL2 -938C&gt;A and MDM2 SNP309 polymorphisms as prognostic markers in CLL. Future SNP markers must hence be confirmed in large, independent cohorts before being proposed as prognostic marker in CLL. In paper IV, we conclude that LPL expression appears to be the strongest among the RNA-based markers for CLL prognostication. Further efforts to standardize LPL quantification are required before it can be applied in the clinical laboratory to predict clinical outcome in this disease.

Investigation of the relationship between iron deficiency, iron supplementation and susceptibility to infection, was suggested by the author's initial observations of an association of anaemia with serious bacterial infections in infancy in Papua New Guinea. The bulk of previous longitudinal clinical intervention studies in infancy showed beneficial effects of iron supplementation. However, defects of control and design and recording in these studies and contradictory anecdotal reports left the question unresolved. A prospective, placebo-controlled, randomised, double-blind trial of iron prophylaxis (3ml intramuscular iron dextran = 150mg Fe) to two month old infants was carried out on the North Coast of Papua New Guinea where there is high transmission of malaria. A literature review, pilot studies, protocol, demography, geography and laboratory methods developed are described. Findings indicate that the placebo control group became relatively iron deficient over the first year of life and that the iron dextran group had adequate, although not excessive iron stores and a higher mean haemoglobin; however, the prevalence and effects of malaria recorded in the field were higher in the iron dextran group. Analysis of field and hospital infectious morbidity in the trial indicated a deleterious association with iron dextran for all causes including respiratory infections (the main single reason for admission). Total duration of hospitalisation was significantly increased in the iron dextran group. Analysis of other factors showed (1) a higher admission rate associated with low weight-for-height recorded at the start of the trial; (2) a significant positive correlation between birth haemoglobin and hospital morbidity rates; (3) increased malaria rates in primiparous mothers of the cohort infants who received iron infusion during pregnancy; (4) lower relative risk of malaria associated with iron prophylaxis in individuals with alpha thalassaemia, which was found to be highly prevalent in this region. In conclusion, it is suggested that policies of iron supplementation, total dose iron injection and routine presumptive iron therapy for anaemia which are widely in practice in malaria endemic areas should be closely reviewed.

The avian immune system is a complex system of defence mechanisms that protect bird hosts against threats from ubiquitous pathogens. According to the co-evolutionary models, variability in immune traits of hosts is the key component providing ability to adapt and enhance their defence mechanisms in presence of constant selective pathogen pressure. Domestic chicken (Gallus gallus f. domestica) is used as a model organism in avian biology and also is one of the most important food-producing animals, not only for their meat but also for the egg production. Unfortunately, in research usually only inbred chicken lines are used and modern poultry husbandry is tight with unilateral breeding towards highly productive breeds. Those approaches decrease intra-population polymorphism in chickens. However, especially in case of farm animals, searching and extending the pool of immune variability and enhancing pathogen resistance is crucial for sustaining healthy and biologically secure populations and their products. Morphologically highly distinct traditional chicken breeds, which have evolved for hundreds years under different selective pressures, may represent this desirable immunological variability. In my thesis I described variability in chosen immunological traits, haematological parameters and proteomic...

v III. Abstract (EN) According to the Disposable soma theory, senescence is mainly influenced by adaptive allocation of resources among the physical growth, reproduction and self-maintaining processes, which depend on expected survival of an individual. The amount of allocated resources may be influenced by external environmental factors such as heavy metal pollution or by inner factor e.g. reproduction. The aim of my diploma thesis was to clarify the relationships between senescence, selected haematological traits, reproduction and efficiency of innate immunity mechanism. Moreover, we are interested in the role of heavy metal pollution in ageing and haematology. The heterophiles to lymphocytes ratio (H/L) was used as a marker of long-term stress caused by these physiological expenses. This study summarizes partial data collected within the long-term research of great tits (Parus major) in Prague, Czech Republic, I participated in. We have more than 300 samples of repeatedly captured individuals at our disposal. The structure of this unique dataset allows us to observe changes in the traits of our interest during an avian lifespan. Our results show that during aging, males experience a noticeable increase in the H/L ratio compared to females. Although we have not found the effect of age on the brood size,...