Severe malaria caused by Plasmodium falciparum (P. falciparum) remains a leading cause of death in many low and middle income countries. The intraerythrocytic reproduction cycle of the parasite is responsible for all the symptoms and mortality of malaria. The merozoite, first invade a red blood cell (RBC) in the circulation, then grows, develops and multiplies within it by clonal division. Merozoite invasion is a complex process involving dynamic interactions between ligands in the merozoite coat and receptors on the red blood cell membrane. Therefore, filming the complete malaria invasion processes may shed the light on its mechanism. The rationale of this work is that learning how the various ligand-receptor interactions affect invasion phenotypes will lead us to a better understanding of the key biological and biophysical aspects of parasite growth in the blood. The work described has firstly involved the development of an optimised imaging platform for recording egress-invasion sequences. I used live cell microscopy to understand this stage of malarial infection better, by monitoring egress-invasion sequences in live cultures under controlled conditions and addressing the morphology and kinetics of erythrocyte invasion by P. falciparum. In addition, the erythrocyte invasion phenotypes of the various P. falciparum strains were systematically investigated for the first time by live cell microscopy. Furthermore, to better understand genetic recombination affecting erythrocyte invasion phenotypes, progeny from the 7G8 x GB4 cross was compared to their parents. In order to investigate specific receptor-ligand interactions and their distinct functional characterisations at each distinct stage, the enzymes that cleave receptors on the erythrocytes and antibodies targeting ligands on the merozoites were studied and their effects observed using the live-imaging platform. In the results, the functions of ligands on the merozoites demonstrated for the first time distinct and sequential functions of proteins during erythrocyte invasion, which could potentially guide the design of more effective malaria vaccines. In addition, I have designed microfluidic devices for studying blood stage malaria. Polydimethylsiloxane (PDMS) microfluidic devices are optically transparent, non-toxic and have biocompatible features. Building on previous work, I made specific microfluidic devices for achieving a high throughput of egress-invasion observations. Infected red blood cells were delivered into a microfluidic device channel containing cage-like "nests". The nests were designed to selectively trap these stiff, egress-ready cells, in order to obtain streams of merozoites on maturation. Uninfected RBCs were delivered from another input into a long serpentine channel co-flowing with the egressed merozoites. The results indicated that, during P. falciparum erythrocyte invasion under flow conditions, the morphological effect on erythrocytes and the kinetic properties show significant differences to those in static conditions. In addition, with optimised flow rates, it is possible to reach higher throughput of egress-invasion observations than static conditions. Both the static and flow experiments carried out in this study highlight important mechanisms and processes of malaria invasion, and represent new ways of studying blood stage malaria. Precise and high throughout recording of single-event host-pathogen interaction events will allow us to address a new area of fundamental biological questions in future work.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:744517 |
| Date | January 2018 |
| Creators | Lin, Yen-Chun |
| Contributors | Cicuta, Pietro |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/271829 |
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