Environmental determinants underlying commitment to asexual or sexual development

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Defining the global malaria burden and eradication strategies

Malaria impacts global health and economy and resulted in 216 million cases and 445000 mortalities in 2016 (1). The disease has been targeted for eradication through the concerted efforts of the World Health Organization (WHO) (1) and the continuation of flagship programs including the Malaria Eradication Agenda (malERA) (2) of the Roll Back Malaria campaign (3). Together, these endeavors emphasize an integrative, transdisciplinary approach that aims to improve available strategies targeting the parasite that causes malaria, the Anopheles mosquito vector transmitting the parasite and characterizing the transmission reservoir formed by these organisms (2). The current elimination strategies are based on lessons learnt from both successful and unsuccessful previous attempts at malaria elimination.

The Global Malaria Eradication Program (GMEP, 1955–1969) focused almost completely on indoor residual spraying (4), as the information on the feeding habits of the diverse Anopheles mosquito species was not available at that time (5,6). Subsequently, research into vector dynamics has identified important health and efficacy concerns in using “gold-standard” vector control methods. This resulted in adoption of integrated vector management as proposed by the WHO (7,8), which combines a number of interventions including long lasting insecticide treated nets and indoor residual spraying (7,9). Current vector control strategies are seen as the main factor that has led to dramatic decreases in malaria case numbers (1). However, confounding factors for sustained success with vector control include development of insecticide resistance (10) and changing biting behavior and migration dynamics of several of the Anopheline species that carry malaria parasites (5,6).

The apicomplexan Plasmodium parasites that transmit malaria contribute their own sets of challenges and problems to malaria eradication. In 2015, the results of the phase III clinical trial of the first malaria vaccine candidate, the RTS,S/AS01 vaccine, were published (11). The vaccine contained a region of recombinant P. falciparum circumsporozoite protein, which is essential for establishing a malaria infection in humans (12,13). Disappointingly, although the vaccine was able to reduce the symptomatic 2 malaria burden, it is far from completely effective and only offers transient protection (14). In addition, no effective vaccine candidate that also prevents transmission of Plasmodium parasites has entered clinical trials (1). The most effective interventions against the parasite to date have been the successes of the antimalarial drugs treating the symptoms of malaria infection (15).

The chemical interventions currently recommended by the WHO are mostly derivatives from natural products that had been used to treat malaria for centuries (16,17). The initial natural products, quinine and artemisinin, gave rise to subsequent generations of antimalarials, the 4- and 8-aminoquinolones; 4-methanolquinolines or artemisinin derivatives, respectively (15). These derivatives form the bulk of the current recommended treatment regime as artemisinin combination therapies (ACTs) (1), combining the rapid activity of artemisinin derivatives with the longer-lasting activity of, for instance, the quinoline compounds (18). The potent activity of artemisinin and its derivatives is believed to stem from Fe3+ (released by hemoglobin digestion) activating the drug in vivo (19), before alkylation of multiple parasite proteins result in the pleiotropic toxicity characteristic of this compound class (20). For the quinine-derivatives, the main mode-of-action is believed to be interference with the parasite’s ability to effectively sequester toxic heme moieties formed by hemoglobin digestion (18). Alternatively, the use of an artemisinin derivative is also recommended in conjunction with compounds targeting enzymes in the folate biosynthesis pathway of the parasite, dihydrofolate reductase (targeted by pyrimethamine) or dihydropteroate synthase (targeted by sulphadoxine), although widespread resistance to the antifolates preclude their use in most clinical settings (21).

The emergence of artemisinin resistance in Southeast Asia (22) and a case of reduced parasite clearance following treatment in Africa (23) resulting in decreased efficacy of the drug, raises serious concerns surrounding the continued use of ACTs as front-line treatment of malaria. These issues with the current interventions against the parasite have encouraged research into novel chemical scaffolds, resulting in promising drug candidates entering clinical development, most notably MMV048, OZ439, KAE609, KAF156 and DSM265 (15). The infectious, virulent nature of the parasite, however, necessitates the continual population of the antimalarial drug and vaccine candidate development pipeline, as proposed by the Medicines for Malaria Venture (MMV, www.mmv.org) if the goal of global malaria eradication is to be met (24).

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To this point, descriptions of effective lead chemical compounds (target candidate profiles, TCP) and potential medicines (target product profiles, TPP) were clearly defined and is used as a guided system for desirable attributes of the next generation of antimalarial medicines (25). For TCPs, multi-stage and -species activity, effective in targeting the spread of malaria as well as the symptomatic treatment of the disease, are emphasized (25). It is expected that identification of essential genetic moieties in the parasite (26) as well as identification of the druggable genome of the parasite (27) would overcome many of the previous obstacles in understanding drug activity that made it difficult to achieve the TCP and TPP guidelines. However, understanding the impact of antimalarials on the parasite is still incomplete without a solid knowledge base concerning the biology of the parasite.

Table of Contents :

  • List of figures
  • List of abbreviations
    • Chapter
    • Literature review
    • 1.1 Defining the global malaria burden and eradication strategies
    • 1.2 Causative agent of malaria and its complex life cycle
    • 1.3 A bifurcation in cellular fate defines intraerythrocytic P. falciparum parasite development: proliferate or differentiate?
    • 1.3.1 Resultant metabolic profile of the respective proliferative or differentiating forms of the
    • parasite
    • 1.3.2 Molecular reprogramming: Cell cycle progression in proliferating Plasmodium parasites
    • 1.4 Hard-wired regulation? The extrinsic and intrinsic regulatory signals that shape the bifurcation in parasite biology
    • 1.4.1 Environmental determinants underlying commitment to asexual or sexual development
    • 1.4.2 Controlled gene expression underlying differentiation or proliferation in the parasite
      • 1.4.2.1 Transcriptional control
      • 1.4.2.2 Post-transcriptional control
      • 1.4.2.3 Post-translational control
    • 1.5 Specific transcriptional regulators define asexual proliferation and sexual differentiation
    • 1.5.1 Epigenetic control of transcriptional landscape enabling proliferation or differentiation
    • 1.5.2 Transcription factors play decisive roles in proliferation or differentiation decision
    • 1.6 Hypothesis
    • 1.7 Aim
    • 1.8 Objectives
    • 1.9 Outputs related to the thesis
    • Chapter
    • Evaluating cell cycle progression of asexual P. falciparum parasites
    • 2.1 INTRODUCTION
    • 2.2 METHODS
    • 2.2.1 In vitro cultivation of intraerythrocytic P. falciparum parasites
    • 2.2.2 Perturbation of intraerythrocytic P. falciparum parasites with DL-α- difluoromethylornithine (DFMO)
      • 2.2.3 Measurement of nucleic content
      • 2.2.4 Oligonucleotide DNA microarray and analysis
      • 2.2.6 Microarray data analyses
      • 2.2.7 Gene association network filtering and coexpression regulation network inference
      • 2.2.8 Data availability
    • 2.3 RESULTS
    • 2.3.1 DFMO is an effective tool for arresting P. falciparum parasites in their life cycle
    • 2.3.2 The life cycle arrest induced by DFMO has physiological indications of a biologically relevant cell cycle arrest
    • 2.3.3 Cell cycle phase analysis of P. falciparum parasites undergoing cell cycle arrest and reversal
    • 2.3.4 Molecular characteristics of a quiescence-proliferation decision point
    • 2.3.5 Characterization of the cell cycle phases of P. falciparum parasites
    • 2.3.6 Molecular cues govern entry into the proliferative state of P. falciparum parasites
    • 2.4 DISCUSSION
    • Chapter
    • Intricate hierarchical transcriptional control regulates Plasmodium falciparum sexual differentiation
    • 3.1 INTRODUCTION
    • 3.2 METHODS
      • 3.2.1 Parasite culturing and sampling
      • 3.2.2 RNA isolation, cDNA synthesis and dye labeling
      • 3.2.3 Array hybridization and scanning
      • 3.2.4 Data analysis
      • 3.2.5 Data availability
    • 3.3 RESULTS
    • 3.3.1 High-resolution transcriptome displays clear distinction between sexual and asexual stages of development
    • 3.3.2 The gametocyte-specific transcriptional program reflects the molecular landscape of gametocyte development
    • 3.3.3 Timed regulation of gametocytogenesis enables sex-specific development and life cycle switching
    • 3.3.3.1 Both activation and repression events characterize the initial switch to differentiation
    • 3.3.3.2 Post-commitment, development and maturation is underpinned by separate transcriptional induction events of essential genes
    • 3.3.4 Epigenetic control contributes to timed gene expression during gametocytogenesis
    • 3.3.5 ApiAP2 transcription factors express at and regulate genes distinct to specific intervals during gametocytogenesis
    • 3.4 DISCUSSION
    • Chapter
    • The phenotypic consequences of proliferation/differentiation decisions of the malaria parasite
    • 4.1 INTRODUCTION
    • 4.2 METHODS
    • 4.2.1 Cell culture and preparation
    • 4.2.2 Optimization of Biolog Phenotype MicroarrayTM assay conditions
    • 4.2.3 Phenotype microarray analysis
    • 4.2.4 Data analysis
    • 4.3 RESULTS
    • 4.3.1 Suitability of the Biolog Phenotype microarray system for use with intraerythrocytic P falciparum parasites
    • 4.3.2 Translating signal produced from the Biolog Phenotype microarray system to informative parameters describing the metabolism of intraerythrocytic P. falciparum parasites
    • 4.3.3 Assessing total parasite metabolism of carbon and nitrogen substrates using phenotypic microarrays
    • 4.3.4 Rate and magnitude of carbon and nitrogen substrate metabolism in P. falciparum
    • parasites
    • 4.3 DISCUSSION
    • Chapter
    • Concluding discussion
    • 6. References

Philosophiae Doctor Biochemistry

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