Medical Biology - Theses

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End date: 2023 × Start date: 2023 × Type: PhD thesis × Subject: Malaria ×

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    Identification of molecular pathways associated with susceptibility and immunity to severe dengue and malaria
    Studniberg, Stephanie Irene ( 2023-12)
    In this era of increasing globalisation, urbanisation, and worsening climate change, the geographical range of transmission-competent mosquito vectors is shifting. Mosquito-borne diseases such as malaria and dengue are gradually emerging in previously unaffected areas, and re-emerging in areas where they had once previously subsided. With alarming increases in dengue case incidence, and for the first time, a chapter addressing the influence of climate change on malaria transmission in the World Malaria Report, it is clear that these two important vector-borne diseases are of utmost global relevance. As per the World Health Organization (WHO) guidelines, individuals presenting with warning signs signifying progression to severe dengue are required to remain under hospital observation. However, these warning signs appear late in the disease course and are non-specific. Consequentially, hospitals become overwhelmed with patients admitted for in-patient observation, many of whom do not progress to severe dengue. Biomarkers to detect progression to severe dengue upon hospital presentation are much needed to improve patient triage and resource allocation. In malaria, despite the great achievement of the recommendation by the WHO for the use of the RTS,S and R21/Matrix-M vaccines in children living in endemic areas, reductions in malaria case incidence remain at a prolonged stall. It is clear that efficacious vaccines approved for children to adults are required to reduce the global malaria burden. Further elucidation of the molecular mechanisms underlying the immune response to dengue and malaria is imperative if these outcomes are to be achieved. To address these outstanding concerns, an integrative systems immunology approach was utilised to identify molecular pathways associated with susceptibility and immunity to severe dengue and malaria. The studies within this thesis have integrated data from single-cell mass cytometry, serology, and transcriptional profiling of peripheral blood mononuclear cells from individuals progressing to either dengue fever (DF) or dengue haemorrhagic fever (DHF), as well as individuals living in a malaria-endemic regions of Indonesia with either symptomatic or asymptomatic Plasmodium falciparum and Plasmodium vivax malaria. Integrative data analysis identified frequencies and transcriptional profiles of effector CD4+ and CD8+ T cells as important components of dengue immunity in individuals progressing to DF. Furthermore, high frequencies of defined populations of CD4+ non-classical monocytes were associated with increased odds of developing DHF. Our approach discovered a strong transcriptional phenotype of immunosuppression underlying asymptomatic P. falciparum malaria, suggesting that the carriage of these infections could preclude complete parasite clearance. Lastly, unlike symptomatic P. falciparum malaria that induced a highly inflammatory response, clinical P. vivax infection featured the upregulation of anti-inflammatory pathways and checkpoint receptors, providing a feedback loop to ameliorate symptomatic infection. Furthermore, gene set enrichment analysis revealed profound dysfunction of the blood monocyte compartment in both symptomatic and asymptomatic P. vivax malaria. Together, the findings in this thesis have critical implications for the deployment and efficacy of malaria vaccines, and for the development of diagnostic tools to predict disease outcomes for dengue patients at point-of-care.
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    Studies of Plasmodium-Iron Interactions
    Clucas, Danielle Bridget ( 2023-09)
    Background Iron deficiency anaemia and malaria co-exist across sub-Saharan Africa where they disproportionately affect young children and pregnant women. Iron supplementation is recommended to treat anaemia, but there are concerns regarding its safety and the potential protection afforded by iron deficiency. An interaction between host iron status and malaria risk has been hypothesised for decades, but confounding factors mean that conclusions are difficult to draw from field studies alone. In this thesis the complex interaction between Plasmodium and iron is investigated using clinical, pre-clinical and in vitro studies. Methods The effect of host iron deficiency on the risk of Plasmodium falciparum parasitaemia by PCR was assessed in a cohort of 711 anaemic Malawian pregnant women, and the risk of intravenous iron supplementation on subsequent risk of parasitaemia explored. These findings were dissected using Plasmodium berghei infection in C57Bl/6 mouse models of iron deficiency (Tmprss6 knockout (Tmprss6-KO)) and iron overload (inducible hepcidin knockout (iHamp-KO)). Tmprss6 knockdown (Tmprss6-KD) was used to assess the phenotype in Tmprss6-KO mice and to explore Tmprss6 as a druggable target; achieved through treatment of wild-type C57Bl/6 mice with siTMP, a GalNAc conjugate targeting Tmprss6. The effect of iron restriction on the parasite was investigated through iron chelation of in vitro P. falciparum cultures, followed by transcriptomic and proteomic analysis. A role of post-transcriptional regulation in the response to iron chelation was further explored. In the setting of known post transcriptional regulation of cellular iron in other organisms via the iron responsive element (IRE)/Iron regulatory protein (IRP) system, and with an IRP-like protein described in P. falciparum, the role of PfIRP was explored through the generation and characterisation of PfIRP-KO parasites. Results Among anaemic Malawian pregnant women iron deficiency was associated with a 53% reduced risk of P. falciparum parasitaemia (Adjusted risk ratio 0.47, 95% confidence interval (0.34, 0.60), p<0.0001), with this finding robust to varied definitions of iron deficiency. Intravenous iron did not increase the subsequent risk of P. falciparum parasitaemia. These findings were supported by the mouse models. Tmprss6-KO mice had improved survival when infected with P. berghei. Conversely, iHamp-KO mice exhibited decreased liver stage infection but an unaltered course in the blood stage of infection. Tmprss6-KD did not replicate the KO phenotype; the disease course was not changed in siTMP treated mice. In vitro, iron chelation inhibited parasite growth and induced substantial changes in the transcriptome and proteome of P. falciparum. Differential expression of genes and proteins involved in key processes in the parasite’s lifecycle, and with plausible links to iron were identified, as was a possible role for post-transcriptional regulation. Investigating this showed PfIRP-KO parasites were more susceptible to iron chelation. Transcriptomic and proteomic analysis identified proteins that might be regulated in an IRE/IRP-like manner. Conclusions This work adds to the current understanding of the complex interaction between Plasmodium and iron. Taken together, these data support iron deficiency being protective against P. falciparum infection. In vitro studies highlighted genes of potential further interest in P. falciparum iron homeostasis and support an iron related role for PfIRP.