Medicine (RMH) - Theses
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ItemCharacterisation of novel epigenetic pathways in the malaria parasite Plasmodium falciparum( 2014)The protozoan parasite Plasmodium falciparum is the main agent responsible for the severe and potentially fatal form of human malaria, which is predominantly prevalent in tropical and sub-tropical countries. Malaria is transmitted through the bite of an infected female Anopheles mosquito and the parasites eventually invade and multiply asexually in red blood cells. Malaria pathogenesis is associated with the expression of clonally variant proteins on the surface of the infected erythrocyte which mediate cytoadhesion of the infected red blood cell to the endothelium of blood vessels. Thereby the infected erythrocytes sequester in the vasculature and avoid clearance by the spleen. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the main variant antigen expressed on the surface of the infected erythrocyte that is responsible for cytoadhesion and immune evasion. PfEMP1 is encoded by the var multi-gene family and var genes are expressed in a mutually exclusive manner. To escape the human immune system, the parasite switches expression of the single expressed var gene. These pathogenic processes of invasion and cytoadhesion are tightly regulated by epigenetic mechanisms. Epigenetics refers to heritable changes in gene activity that are caused by changes in chromatin structure rather than changes in the DNA sequence. Nucleosomes are the basic repeat elements that make up chromatin and are formed by DNA wrapping around a histone octamer. Chromatin can assume two principle states; the open and transcriptionally competent euchromatin or the tightly packed and silent heterochromatin. The majority of the parasite’s genome exists in a euchromatic state. Genes in the euchromatin compartment are not necessarily active; those that are not are maintained in a readily inducible state and can be transcribed later in the parasite’s life cycle. Some of the gene families involved in pathogenic processes such as invasion and cytoadhesion are largely restricted to the subtelomeric and central clusters of heterochromatin and are transcriptionally silent but single genes can assume a euchromatic structure and be activated. One of the epigenetic mechanisms of altering this chromatin state is through the exchange of core histones with variant histones. P. falciparum expresses the histone variants H2A.Z, a conserved histone variant present in the promoters of genes in many eukaryotes, and H2B.Z, a unique apicomplexan specific histone variant. Pf H2A.Z is enriched at promoters of euchromatic genes in an expression independent manner. In var genes however, Pf H2A.Z is enriched at promoters of only active var genes and not silent var genes. Here I explore the interaction between Pf H2A.Z and Pf H2B.Z and their correlation with gene expression. Through co-immunoprecipitation assays on isolated mononucleosomes I found that in the malaria parasite Pf H2A.Z and Pf H2B.Z are present in the same nucleosomes. These double variant nucleosomes also harbour the active euchromatin marks H3K4me3, H3K9ac and H4K12ac but not the repressive mark H4K20me3. To further investigate at which sequences in the genome Pf H2B.Z is present, I performed chromatin immunoprecipitation. I found that Pf H2A.Z and Pf H2B.Z are significantly enriched at the transcriptional start sites of most genes, but this enrichment does not correlate with gene activity. However, in var genes, Pf H2B.Z together with Pf H2A.Z is enriched near the TSS of only the transcriptionally active var gene, suggesting that nucleosomes containing Pf H2A.Z and Pf H2B.Z may have a distinct function in var gene regulation. One mechanism by which Pf H2A.Z and Pf H2B.Z may play a role in gene expression is through the acetylation of their N-terminal tails, and H2A.Z acetylation correlates with transcriptional activity in yeast and humans. I tested for the presence of N-terminally acetylated Pf H2A.Z and Pf H2B.Z in active and inactive genes, but variant histone acetylation did not correlate with gene activity in the set of genes examined. Instead, acetylated Pf H2A.Z and Pf H2B.Z correlated well with total Pf H2A.Z and Pf H2B.Z occupancy, indicating that variant histone acetylation at the residues investigated may have a role that is distinct from gene regulation in P. falciparum. Epigenetic marks have been studied widely in the asexual stages of the parasite but have not yet been investigated in detail in the sexual stage gametocytes. Cellular differentiation in eukaryotes involves changes in gene expression that are regulated by modifications in chromatin composition, and recent evidence suggests that this might also be important in P. falciparum gametocytogenesis. Consistent with a role of epigenetic mechanisms in sexual differentiation, I found that the total levels of some active and inactive chromatin marks slightly fluctuate during gametocyte development. Chromatin immunoprecipitation in mid stage female gametocytes revealed that similar to the asexual stages, Pf H2A.Z is generally enriched at the TSS of active and inactive genes, however the levels of enrichment did not correlate between asexual and sexual stages revealing a novel stage specific plasticity in double-variant nucleosome occupancy in gene regulatory regions. In yeast the multi-subunit chromatin remodelling SWR1 complex exchanges the H2A.Z/H2B dimer for the H2A/H2B dimer in a replication independent manner. The Swr1 protein is the catalytic subunit of this complex, so to determine if a similar histone deposition mechanism occurs in P. falciparum, I characterised PfSwr1. Cellular fractionation showed that throughout the asexual life cycle PfSwr1 is predominantly found in the nucleus. To investigate if PfSwr1 is involved in the Pf H2A.Z/Pf H2B.Z deposition, co-immunoprecipitation assays were carried out. I found that PfSwr1 associates with both core and variant histones consistent with a multistep process for depositing the Pf H2A.Z/Pf H2B.Z dimer into nucleosomes. These studies give key insight into the epigenetic mechanisms and their possible role in gene expression in the malaria parasite.
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ItemHLA-G and malaria in pregnancy( 2011)HLA-G is an atypical class I MHC molecule that promotes tolerance and attenuates the immune response. It is highly expressed on the placental cells that invade the maternal tissues, and is one of the ways in which the immune system is modified in pregnancy to allow maternal tolerance of the foetus. The modification of the maternal immune response may also inadvertently promote tolerance of infections in pregnancy, and HLA-G may contribute to this tolerance of infection. Malaria is an infectious disease with high rates of morbidity and mortality globally. The most severe form is caused by the parasite Plasmodium falciparum. In endemic areas it is primarily a disease of children. Immunity is gradually acquired and disease amongst adults is uncommon. However during pregnancy, the high rates of infection return. Malaria in pregnancy is associated with morbidity and mortality for the mother and the foetus. This study is based on the hypothesis that the immune changes in pregnancy that allow maternal tolerance of the foetus may also contribute to the increased rate of malaria in pregnancy. It contributes to the understanding of the pathology of malaria in pregnancy, but also to the knowledge of the immunological response to malaria, of the role of HLA-G in response to infection and how the changes in placental immunology may contribute to other infections in the placenta. This study shows there is an association between HLA-G genotype and Plasmodium falciparum infection in the placenta, that genotype only corresponds with mRNA for a specific pattern of malaria infection in the placenta, and that HLA-G expression correlates with an important consequence of malaria in pregnancy, low birth weight. These findings suggest the hypothesis that placentae that can quickly reduce their HLA-G expression are able to mount a more efficient immune response to the pathogen. Those who have a persistently elevated HLA-G are unable to mount an efficient immune response, and develop a chronic infection. The study also demonstrates a reduction in cytokine response to Plasmodium falciparum in the presence of placental cells, although this is not mediated by HLA-G.
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ItemEpidemiological and immunological studies of treatment for pregnancy associated malaria( 2010)Pregnant women are highly susceptible to malaria, and malaria in pregnancy causes a number of adverse outcomes such as maternal anaemia and delivery of low birth weight babies. Thus pregnant women are specifically targeted in malaria prevention efforts with control measures including IPTp and bed nets. Pregnant women are uniquely susceptible to malaria because Plasmodium falciparum infected erythrocytes can adhere to the placenta. This is mediated by the variant surface antigen (VSA) family Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Accumulation of infected erythrocytes in the placenta may subsequently result in acquisition of immunity targeting this protein especially among multigravid women. Longitudinal data collected over 9 years from Malawian pregnant women demonstrated decreased prevalence of peripheral and placental malaria, maternal anaemia and LBW. In the same time frame coverage with IPTp SP and bed nets increased. SP IPT doses were associated with protection against placental parasitaemia, maternal anaemia and LBW from 1997-2001, but not from 2002-2006. Bed net use was associated with protection from peripheral or placental parasitemia and LBW throughout the study, but not with anaemia. These results indicated decreased maternal malaria infection correlated with improved pregnancy outcomes. Increased bed net coverage explains more of this change than SP use. SP resistance may be compromising its effectiveness. In chapter 4 of this thesis, I explored the protective effect of immunity against pregnant associated malaria variant surface antigens (VSA-PAM) using sera from a group of Malawian pregnant women who undertook anti-malarial treatment. My results showed that the level of immunity against VSA-PAM was associated with improved anti-malarial treatment outcomes and decreased maternal anaemia at delivery. The finding presented in this thesis validated further studies investigating the role of antibodies against VSA-PAM in protecting against placental malaria infection. The decreased protective effect of SP-IPTp also urged the importance of replacing SP from the first line anti-malarial treatment drugs.