Applications of resonance Raman and Fourier transform infrared microspectroscopy in malaria research
thesisposted on 19.01.2017 by Webster, Grant Thomas
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This thesis investigates applications of Raman and FTIR microspectroscopy in malaria research. It was found that the use of biospectroscopy was able to generate significant new insights into discriminating lifecycle stages of the malaria parasite, detecting structural changes in malaria pigment and understanding the effects of chloroquine on supramolecular interactions in malaria pigment and its precursors. This dissertation is presented in the “thesis by publication” format, with the three results and discussion chapters (Chapters 3, 4 and 5) comprised of either published, accepted or submitted manuscripts. The first chapter provides a general introduction to malaria, with particular emphasis on the Plasmodium falciparum drug resistant strain of malaria parasite. This chapter also introduces the different models of quinoline drug binding that are likely to occur within the digestive vacuole (DV) of the parasite, as well as introducing the various macromolecules that are present within infected erythrocytes. The second chapter discusses relevant biospectroscopy theory and introduces the data analysis techniques used in the application of Raman and FTIR spectroscopy to malaria research. The third chapter describes FTIR spectroscopic investigations, focusing on discriminating the different intra-erythrocytic lifecycle stages (i.e. ring, trophozoite and schizont stage) of the malaria parasite by observing the specific lipid composition of each stage. In addition, haemozoin growth within the digestive vacuole (DV) of the parasite was observed using FTIR spectroscopy. As part of a larger study, the relevant section in the second manuscript of this chapter (II) builds upon the above findings by describing how the use of FTIR-ATR and Raman spectroscopy allows for direct observation of physical changes related to quinoline drug attachment in synthetic malaria pigment (β-haematin). The FTIR-ATR spectra show the effect of quinoline drug binding on β-haematin by revealing changes in the surface propionic acid groups (1744 cm-1). The fourth chapter describes investigations that used resonance Raman spectroscopy to detect haemozoin (malaria pigment) and the structural changes that occur in haemozoin in infected erythrocytes after chloroquine (CQ) treatment. Raman spectra show that CQ binds via π-π interactions between adjacent and oriented porphyrins, thereby disrupting the haemozoin aggregate and reducing excitonic interactions between adjacent Fe(III)PPIX units. The final chapter explains how resonance Raman in combination with UV-Visible spectroscopy can reveal the effects of varying concentrations of Fe(III)PPIX and CQ on excitonic interactions in solution and on the extent of supramolecular interactions in malaria pigment and other related haem aggregates. Resonance Raman spectra of haematin and haemin solutions are reported for 413 nm and 514 nm wavelengths. Enhancement of A1g modes (1569 cm-1 and 1370 cm-1) and B1g modes (1124 cm-1 and 755 cm-1) as a function of increased concentration are observed when irradiating with 514 nm laser excitation but not 413 nm. This wavelength dependent enhancement can be rationalised by considering an excitonic coupling mechanism. As the concentration of haematin increases there is an increased probability of supramolecular interactions occurring between Fe(III)PPIX units. This study provides new insight into the nature of excitonic coupling through supramolecular and other concerted interactions and may have important implications in understanding energy transfer processes in haem systems.