The discovery of new β-haematin inhibitors has become one focus for researches in response to the resistance of P. falciparum malaria parasites that emerged towards well-known antimalarials. While hundreds of new β-haematin inhibitors have been discovered using detergent mediated high-throughput screening methods, a crucial aspect is understanding exactly how these β-haematin inhibitors behave in the malaria parasite and inhibit the formation of haemozoin. What is known, is that well-known β-haematin inhibitors like chloroquine cause increased amounts of exchangeable haem in the parasite digestive vacuole and form a Fe(III)PPIX-inhibitor complex by accumulating at high concentrations which consequently inhibits parasite growth. Another important focus is on understanding the digestion of haemoglobin and its role in haemozoin formation. This research investigates the in vitro modelling of cellular haemozoin and inhibition by various β-haematin inhibitors across different scaffolds and the role of haemoglobin degradation in P. falciparum malaria parasites. The investigated β-haematin inhibitors resulted in micromolar IC50 (NF54) values and decreased parasite growth with increases in concentration. Using a pyridine-based parasite haem fractionation plate method, these β-haematin inhibitors were shown to target haemozoin formation by causing increased amounts of exchangeable haem that corresponded to decreasing amounts of haemozoin in chloroquine-sensitive parasites. The amounts of exchangeable haem were shown to be inversely proportional to the percentage of parasite growth in the presence of these β-haematin inhibitors. It was apparent that there was a tendency for parasite growth inhibition activity to decrease as the amount of exchangeable haem present in chloroquine sensitive parasites increased, although, the trend was not statistically significant. Moreover, it was observed that experimental cellular accumulation ratio values were low in comparison to chloroquine and amodiaquine. Based on the experimental cellular accumulation ratio values, it was deduced that the accumulation of these β-haematin inhibitors was not primarily due to pH trapping and more complex than previously proposed. Further investigations into the exchangeable haem amounts as a function of intracellular test compound amounts at the IC50 values of these β-haematin inhibitors highlighted that there was an apparent 1:1 relationship with the amount of intracellular exchangeable haem, indicative of complex formation. Transmission electron microscopy images were obtained for untreated parasites that showed intact parasites inside red blood cells with clearly visible haemozoin crystals dispersed throughout the parasite digestive vacuole, whilst, treated parasites showed less defined haemozoin crystals as a result of inhibition. Moreover, electron energy-loss spectroscopy revealed that untreated parasites exhibited a strong iron signal which was associated with haemozoin in the parasite digestive vacuole with a weaker signal attributed to the red blood cell cytoplasm. Similarly, a strong iron signal was shown in the digestive vacuole of treated parasites which was associated with less defined haemozoin crystals. A halo around these haemozoin crystals was observed and was suggested to be indicative of the build-up of exchangeable haem. Additionally, a strong bromine signal attributed to a bromine-containing β-haematin inhibitor, test compound 1, was also observed in the same region as the haemozoin crystals. Overlaid signal distribution maps for iron and bromine showed direct evidence of Fe(III)PPIX and test compound 1, suggesting complexation. High-quality Raman spectra were obtained for the Fe(III)PPIX species in red blood cells, chloroquine sensitive parasites and synthetically prepared samples for the Fe(III)PPIX porphyrin dominated spectral region of 1700-500 cm-1 at an excitation wavelength of 532 nm. From the spectra, a putative Fe(III)PPIX-test compound 1 complex was identified and shown to be similar to the synthetically prepared counterpart, haematin-test compound 1 mixture. It was highlighted that a unique peak at 1080 cm-1 indicated π- π interactions between the pyrrole-imidazole ring and thus confirming that the formation of this putative Fe(III)PPIX-inhibitor complex occurs. The confocal Raman true mapping technique proved to be efficient and reliable for imaging the signal distribution of haemozoin at the Raman peak of 754 cm-1 and 1080 cm-1 for the Fe(III)PPIX-test compound 1 complex which co-localized in the digestive vacuole of chloroquine sensitive parasites. Moreover, oxy- and deoxy-haemoglobin was observed to be localized to the red blood cell, where, deoxy-haemoglobin was located on the outer parts of the parasite. Principle component analysis, based on the Raman peak positions, exhibited significant differences in the spectra for Fe(III)PPIX species in red blood cells, chloroquine sensitive parasites and synthetic samples where clusters were observed to separate mainly along principle component 1. These data proved that the spectra of the Fe(III)PPIX-test compound 1 complex was the same as its synthetically prepared counterpart but different from the remaining Fe(III)PPIX species. In comparison to the Fe(III)PPIX-test compound 1 complex, the cluster separations were observed to be significant, where, no significant separation was observed for the Fe(III)PPIX-test compound 1 complex and the haematin-test compound 1 mixture. Based on this, it was evident that a Fe(III)PPIX-test compound 1 complex existed in the digestive vacuole of treated chloroquine sensitive parasites. To fully understand the inhibition of haemozoin, the development of a haem pathway model is necessary, but, requires certain prerequisites. Bioinformatics data from PAXdb and ExPASy revealed that chloroquine resistance (Dd2) parasites, containing 1337 previously identified proteins with an average abundance-weighted molecular weight of 40,483 ± 77 g/mol. With this, the protein mass per cell for red blood cells, chloroquine-sensitive and - resistant parasites were consistent across three protein quantification methods was measured and revealed that chloroquine resistant parasites had a significantly higher protein mass per cell than chloroquine sensitive parasites and in turn a higher total number of protein molecules per cell. Aspartic proteases are 4-fold higher in concentration than cysteine proteases with histo-aspartic protease having the highest concentration in chloroquine resistant parasites. Along with these data, a time point quantification for chloroquine sensitive parasites throughout the blood-stage showed that the amount of haemoglobin decreased in a sigmoidal manner and corresponded to a linear increase in the amount of haemozoin and relatively constant exchangeable haem amount. This was consistent with Giemsa smears that showed that for early time points, large initial decreases in the amount of haemoglobin were observed between the early trophozoite to late trophozoite stage.

Plasmodium falciparum (P. falciparum) infection modulates the response of innate immune cells. The aim of this work was to study the impact of P. falciparum infection and P. falciparum-derived molecules on the response of dendritic cells (DC) and monocytes. In paper I we investigated the effects of natural hemozoin (nHZ), a P. falciparum-derived molecule, on the phenotype and functionality of DC. We found that exposure to nHZ impaired the capacity of DC to mature. Paper II is a follow-up on paper I, where the underlying transcriptional events preceding the nHZ-induced impairment of DC maturation were investigated. More specifically, we examined the involvement of certain transcription factors, subunits of chromatin remodeling complexes and histone modifications in the regulation of DC maturation. Our findings suggest that nHZ-exposure of DC does not lead to recruitment or enrichment of molecules needed for transcriptional activation. In paper III we investigated P. falciparum effects in vivo in sympatric ethnic groups with differential susceptibility towards P. falciparum infection living in Burkina Faso. The aim of this study was to establish the transcriptional networks underlying the relatively better protection against P. falciparum infection observed in the Fulani ethnic group compared to other sympatric ethnic groups. Our findings reveal differential gene expression in monocytes of infected Fulani compared to uninfected Fulani and the difference concerned multiple classes of genes including signal transduction, immunological responses and chromatin remodelers. The results provide new aspects on molecules and regulatory mechanisms that are involved in the relatively more protective response against P. falciparum infection. Taken together, the work presented in this thesis leads to a deeper understanding of the P. falciparum-induced modulation of responses of innate immune cells and the underlying mechanisms possibly regulating those responses. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.</p>

Sardinia was an island with a history of a malarious environment until eradication efforts were conducted from 1946 to 1950. While historic documents suggest the disease was introduced from North Africa around 500 BC, no study has been conducted to test for the presence of malaria in prehistoric native populations, such as the Nuragic people of the Bronze Age. However, it has been suggested that aspects of the Nuragic culture, for example the stone structures found throughout the island, are adaptations to a malarious environment. The purpose of this dissertation is to test the hypothesis that malaria was present in prehistoric Sardinia. In addition, the value of applying anthropology, pertaining specifically to prehistoric investigations, to understand and combat malaria is supported. To test for the presence of malaria, multiple lines of evidence were used to analyze human skeletal remains from a Middle Bronze Age tomb. Because malaria does not result in a specific pattern of bony responses that can be identified through a gross analysis of the remains, additional lines of evidence were used. These included an osteological analysis for the possible presence of conditions related to malaria (e.g., inherited hemolytic anemias) and the collection of bone samples to test for ancient malaria DNA, Plasmodium falciparum histidine-rich protein II, and the malarial pigment hemozoin. In addition, a review of the literature pertaining to the ecology and history of Sardinia were used with archaeological data to evaluate if it was possible the malaria parasite was affecting humans on the island during prehistory. While it was interpreted that conditions were favorable for malaria to infect individuals during this time, and possible cultural adaptations were noted, no conclusive evidence was found by analyzing skeletal remains. More work is needed to diagnose malaria better in human remains and understand the health of populations in Sardinia during the Bronze Age. Considering the coevolution of malaria parasites, humans, and mosquitoes is a necessary step in developing methods to combat malaria as the parasite and disease vector become more resistant to medicine and insecticides. In particular, applying anthropological methods and theories shows promise for fighting this disease.

Malariapigment Hemozoin wird üblicherweise als nicht-toxische, hochmolekulare, parasitäre Speicherform des nicht abgebauten, toxischen Häms aus dem Wirtszell-Hämoglobins betrachtet. Unaufgereinigtes Pigment, wie wir es im infizierten Erythroyzten finden und wie es nach Schizontenruptur freigesetzt wird, kann man als die "natürliche Diät" bezeichnen, die Makrophagen in Malaria-infizierten Wirten aufnehmen. Nach Aufnahme in den Makrophagen persistiert Hemozoin in den Lysosomen und wird nicht abgebaut. Das Häm-abbauende Enzym, die Häm-Oxygenase wird nicht induziert. Hemozoin ist eine potente Quelle für Radikale, woraus Lipoperoxide und davon abgeleitete Hydroxyaldehyde,wie 4-Hydroxynonenal resultieren . 4- Hydroxynonenal in Konzentrationen, wie sie in Hemozoin-beladenen Monozyten nachgewiesen wurden, hemmen die Proteinkinase C. In immunopräzipitierter Proteinkinase C aus Hemozoin- haltigen Makrophagen wurden ProteinkinaseC-Hydroxynonenal-Komplexe nachgewiesen. Die Hydroxynonenal-bedingte Hemmung der Proteinkinase C (und anderer bisher nicht untersuchter Enzyme und Prozesse) könnte die Hemozoineffekte auf den oxydativen burst und die Phagozytose erklären. Der Phorbolester-induzierte oxydative burst ist irreversibel gehemmt in Monozyten, die entweder Hemozoin oder aber Hemozoin-haltige infizierte Erythrozyten phagozytiert haben. Die Hemmung der NADPH-Oxydase, das für den oxydativen burst verantwortliche Enzym, durch intrazelluläres Hemozoin, sollte beträchtlich zur burst -Hemmung beitragen. Monozyten phagozytieren Hemozoin-haltige, infizierte Erythrozyten oder isoliertes Hemozoin , sind danach jedoch unfähig, erneut zu phagozytieren, wie es Monozyten nach Phagozytose und Verdau von nicht-infizierten Erythrozyten physiologischerweise tun. Schließlich ist die Expression von Membranantigenen, die für die Immunantwort von Bedeutung sind, in Hemozoin-haltigen Monozyten vermindert. Die Induktion des für die Präsentation externer Antigene verantwortlichen Histokompatibilitätskoplexes (MHC) Klasse II durch Interferon-gamma ist in Hemozoin-beladenen Monozyten aufgehoben. Sowohl das Interzelluläre Adhäsionsprotein 1 (CD54) als auch p150,95 Integrin (CD11c) sind in Hemozoin-haltigen Monozyten vermindert Oberflächen-exprimiert. Trotz der verschiedenen funktionellen Einschränkungen sind Hemozoin-beladene Phagozyten vital. Bei Plasmodium-falciparum-Malaria enthält ein hoher Anteil von Gewebsmakrophagen und zirkulierender Monozyten und Leukozyten große Mengen an Hemozoin. Wichtige Funktionen wie oxydativer burst , Phagozytose und die Expression von MHC Klasse II sind in Hemozoin- beladenen Phagozyten gestört. Es scheint deshalb gerechtfertigt, die Hemozoin-Beladung als wichtigen Faktor in der gestörten Immunantwort bei der P.falciparum-Malaria zu betrachten. / Malaria pigment hemozoin is generally considered to be a non-toxic, high-molecular-weight, parasitic storage form of undigested,toxic, host-hemoglobin-heme.Crude pigment, as present in infected erythrocytes and shed after schizont rupture, may be considered the 'natural diet' ingested by macrophages in malaria-infected hosts. After ingestion by macrophages hemozoin persists in the lysosomes without being degraded. The heme-degrading enzyme, the heme-oxygenase, is not induced. Hemozoin is a powerfull source of radicals that generates lipoperoxides and derived, toxic hydroxyaldehydes such as 4-hydroxynonenal. High concentrations of 4-hydroxynonenal, which have been detected in hemozoin-fed macrophages, inhibit protein kinase C. Complexes between hydroxynonenal and protein kinase C have been detected in immunoprecipitated protein kinase C from hemozoin-fed macrophages. Hydroxynonenal-mediated inhibition of protein kinase C (and of other as yet unidentified enzymes and processes) may explain hemozoin-mediated effects on oxidative burst and phagocytosis. The phorbol ester-eliceted oxidative burst is irreversibly suppressed in monocytes fed with hemozoin or hemozoin-containing, infected erythrocytes. The inhibition of NADPH-oxidase, the enzyme responsible for oxidative burst, by ingested hemozoin should considerably contribute to burst inhibition. Monocytes avidly ingest infected hemozoin-containing erythrocytes or isolated hemozoin but are unable to repeat the phagocytic cycle as monocytes do after phagocytosis and digestion of non-infected erythrocytes. Finally , the expression of membrane antigens involved in the immune response is decreased in hemozoin-loaded monocytes. The induction of the major histocompatibility complex (MHC) class II by interferon-gamma, that is responsible for presentation of external antigens, is abrogated in hemozoin-loaded monocyte. The intercellular adhesion molecule 1 (CD54) as well as the p150,95 integrin (CD11c) are decreased on the surface of monocytes containing hemozoin. Despite multiple functional impairments, hemozoin-loaded phagocytes remain alive. In Plasmodium-falciparum malaria large portions of resident macrophages and circulating monocytes and leukocytes contain massive amounts of hemozoin. Important functions like oxidative burst, phagocytosis and the expression of MHC class II are severely impaired in hemozoin-fed phagocytes. It seems therefore likly that hemozoin loading may play an important role in the impairment of the immune response seen in P.falciparum malaria.

A dissertation submitted to the Faculty of Medicine University of the Witwatersrand, Johannesburg For the degree of Master of Science in Medicine / Chloroquine-resistant Plasmodium falciparum accumulate significantly less chloroquine than susceptical parasites, and this is thought to be the basis of their resistance ( Fitch, 1970 ). Martin et. al ( 1987 ), recently demonstrated that in the presence of verapamil, a calcium channel blocker, chloroquine-resistant P falciparum becomes chloroquine-sensitive, with an increase in the chloroquine accumulation.The mechanism of such reversal has yet to be elucidated / IT2018

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-19T13:19:42Z No. of bitstreams: 1 Clarissa Cunha Santana Avaliação... 2015.pdf: 1937824 bytes, checksum: 1d212794cc3bec0caa6b8414a186531b (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-19T13:19:53Z (GMT) No. of bitstreams: 1 Clarissa Cunha Santana Avaliação... 2015.pdf: 1937824 bytes, checksum: 1d212794cc3bec0caa6b8414a186531b (MD5) / Made available in DSpace on 2016-02-19T13:19:53Z (GMT). No. of bitstreams: 1 Clarissa Cunha Santana Avaliação... 2015.pdf: 1937824 bytes, checksum: 1d212794cc3bec0caa6b8414a186531b (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A malária é uma doença causada por cinco espécies de parasitos do gênero Plasmodium que causa anualmente a morte de milhares de pessoas, principalmente em países pobres da África. Muito antiga, uma diversidade de fármacos já foram empregados na tentativa de erradicação da doença, entretanto o aparecimento de cepas resistentes, bem como efeitos adversos gerados pelo tratamento impossibilitou tal ação. Os quinolínicos configuram uma grande parte destes tratamentos, apresentando uma notável atividade antimalárica. Neste trabalho nós avaliamos o potencial antimalárico de três novos derivados quinolínicos BS 260, BS 318 e BS 373 em culturas de Plasmodium falciparum, cepa w2, cloroquina resistente. BS 373 apresentou melhor atividade contra culturas de Plasmodium falciparum e, assim como o BS 318, foi capaz de inibir a biocristalização de hemozoína pelos parasitos. A microscopia eletrônica de transmissão revelou uma desorganização celular, diminuição do tamanho e quantidade de cristais de hemozoína no vacúolo digestivo, bem como vacuolizações citoplasmáticas e presença de estruturas membranares no vacúolo digestivo, o que indica a ocorrência de um processo autofágico nas células tratadas com 10 LM e 20 LM do BS 373. A presença de cristais citoplasmáticos indica a ocorrência de autólise pela ruptura da membrana do vacúolo digestivo. Por fim, o efeito dos tratamentos se mostrou irreversível nos parasitos com 24 horas de tratamento para BS 318 e BS 373, enquanto que para BS 260 essa irreversibilidade só foi observada após 48 horas. Nossos dados mostram que os derivados quinolínicos testados são efetivos contra culturas de P. falciparum, configurando bons candidatos à novas moléculas antimaláricas. / Malaria is a disease caused by five Plasmodium species that cause deaths of thousands of people annually, mostly in poor countries of Africa. Very anccient, a variety of drugs have been used in an attempt to eradicate the disease, however the emergence of resistant strains, as well as adverse effects caused by treatment prevented such action. The quinoline are a large part of these treatments, presenting a remarkable antimalarial activity. In this paper we evaluate the antimalarial potential of three new quinoline derivative BS 260, BS 318 and BS 373 in Plasmodium falciparum chloroquine resistant, w2 strain, cultures. BS 373 showed the best activity against Plasmodium falciparum cultures, while and analogously to BS 318 was able to inhibit the hemozoin formation by parasites. The transmission electron microscopy revealed a cell disorganization, decreased size and amount of hemozoin crystals in the digestive vacuole, cytoplasmic vacuolization and presence of membrane structures in the digestive vacuole, which indicates an autophagic process in cells treated with 10 LM and 20 LM BS 373. Cytoplasmic being crystals indicate parasite cell autolusis caused by digestive vacuole membrane disrupture. Finally, the effect of treatment proved irreversible on parasites at 24 hours of treatment for BS 318 and BS 373, whereas for BS 260 this irreversibility was only observed after 48 hours. Our data show that the quinoline derivatives tested are effective against P. falciparum cultures, setting good candidates for new antimalarial molecules.