The mechanisms underpinning gene regulation in P. falciparum malaria remain largely elusive, though mounting evidence suggests a major role for epigenetic feedback. Interestingly, long non-(protein)-coding RNAs (lncRNAs) have been found to play a dominant role in initiating and guiding the transcriptional, epigenetic, and post-transcriptional status of specific loci across a broad range of organisms. LncRNAs are uniquely poised to act co-transcriptionally on neighboring loci, and/or to remain physically tethered at their site of origin, and through sequence-specific binding activities can impart temporal and spatial specificity to ubiquitously expressed nuclear protein complexes. Proteins, on the other hand, must be translated in the cytoplasm, and hence lose memory of their transcriptional origins. Encouraged by these features of lncRNAs, we set out to investigate the regulatory capacity of P. falciparum lncRNAs on a genome-wide scale. First, we surveyed transcriptional activity across approximately one quarter of the P. falciparum genome using a custom high-density DNA tiling array. We predicted a set of 60 developmentally regulated intergenic lncRNAs, and found that many of these novel loci neighbored genes involved in parasite survival or virulence pathways. Remarkably, upon further analysis of intergenic lncRNA properties, we discovered a family of twenty-two telomere-associated lncRNAs encoded in the telomere-associated repetitive element (TARE) region of P. falciparum chromosome ends. We found that each lncRNA-TARE was encoded adjacent and divergent to a subtelomeric var virulence gene. Moreover, we found that lncRNA-TARE expression was sharply induced between the parasite DNA replication and cell division cycles, that lncRNA-TARE loci contained numerous transcription factor binding sites only otherwise found in subtelomeric var promoter regions, and that the GC content and evolutionary sequence conservation of lncRNA-TAREs was similar to that of P. falciparum ribosomal RNA. Next, we set out to assemble P. falciparum intergenic lncRNA and antisense RNA transcript structures using state-of-the-art deep sequencing and computational tools. Towards this end, we harvested an unprecedented sample set that finely maps temporal changes across 56 hours of P. falciparum blood stage development, and developed and validated strand-specific, non-polyA-selected RNA sequencing methods. This enabled the annotation of over one thousand high-confidence, bona fide lncRNA transcript models, and their comprehensive global analysis. We discovered an enrichment of negatively correlated, tail-to-tail overlapping sense-antisense transcript pairs, suggesting a conserved role for antisense-mediated transcriptional interference in P. falciparum gene regulation. We also discovered a highly correlated spliced antisense counterpart to a gene required for sexual commitment, that the expression of an intriguing subset of antisense transcripts significantly dropped during parasite invasion, and that lncRNA-TARE and 'sterile' var virulence gene transcription was markedly up-regulated during parasite invasion. Lastly, we predicted over one thousand circular RNAs (circRNAs), and validated six circRNA transcript structures. Importantly, this thesis work represents the first focused investigation of lncRNAs in P. falciparum malaria, with the characterization of a compelling family of telomere-associated lncRNAs and numerous antisense RNAs. The data, methods, and results herein offer exceptional technological advancements coupled with compelling insights into the biology of the devastating human pathogen P. falciparum malaria. It is my hope that this work will facilitate future P. falciparum lncRNA functional studies and the strand-specific profiling of additional P. falciparum samples.

Malaria is a daunting epidemic killing millions of people annually and no region is harder hit than Sub-Saharan Africa (SSA). Each year there are more than 247 million malaria cases in SSA, resulting in more than 600,000 deaths. Despite a comprehensive understanding of the parasite and its transmission, worldwide eradication campaigns have failed to adequately control or eliminate the disease. This paper provides a meta-analysis of historical and current approaches to malaria eradication throughout SSA, highlighting past success and perceived failure to avoid repetitive progression down a path of narrowly focused eradication efforts. Through consideration of the economic costs associated with malaria, as well as a critique of current international elimination strategies, this analysis suggests sizeable and widespread returns to pursuing eradication measures. However, this paper finds that current methods are not sufficient to eradicate the malaria burden and multi-dimensional and all-encompassing approaches are essential to making malaria history.

Severe anemia is a major life-threatening complication of malaria. Inappropriately low reticulocytosis in malaria patients with anemia suggests insufficient erythropoiesis, of which the mechanisms and implications are not clear. The principle growth factor that promotes erythropoiesis is erythropoietin (Epo). Studies determining the serum level of Epo in malaria infected patients have been inconclusive. Furthermore, the role of Epo and the erythropoietic response to Epo stimulation during malaria have never been examined. The purpose of the experiments performed in this thesis was, thus, to investigate the role of Epo and erythropoiesis in relation to anemia during blood-stage malaria using the murine model of Plasmodium chabaudi AS. A murine Epo specific ELISA, which was determined to be less biased by the presence of other cytokines in the samples as compared to the conventional Epo bioassay, was first developed to facilitate the research. The kinetics of Epo production in the kidney and the levels in the serum were characterized. It was demonstrated that Epo production during blood-stage malaria is mainly regulated by the degree of anemia and that renal cytokines may have only a minor effect on this response. Next, the roles of Epo and erythropoiesis during blood-stage malaria were investigated by neutralization of endogenous Epo or by administration of exogenous Epo. Timely onset of Epo-induced reticulocytosis was shown to be important for the alleviation of malarial anemia and survival. However, reticulocytosis in response to Epo stimulation is severely suppressed by infection with malaria. Dissection of the upstream events of erythropoiesis demonstrated that blood-stage malaria compromises the generation of reticulocytes by suppressing the proliferation, differentiation, and maturation of erythroid-lineage cells at various stages of erythroid development. Taken together, our data provide important insights for understanding the patho

Effective case management of malaria is hampered by the spread of parasite resistance to nonartemisinin antimalarials. To counteract the impact of drug resistance, the World Health Organization (WHO) has endorsed artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated Plasmodium falciparum malaria. Currently recommended ACTs are artemether-lumefantrine, artesunate plus amodiaquine, artesunate plus mefloquine, artesunate plus sulfadoxine-pyrimethamine and dihydroartemisinin-piperaquine. This study sought to review evidence of the efficacy and safety of different non-artemisinin antimalarials in combination with artesunate, artemether or dihydroartemisinin for the treatment of uncomplicated P. falciparum malaria in non-pregnant adults and children. The search for randomized controlled trials (RCTs) was conducted in the Cochrane Central Register for Controlled Trials (CENTRAL), MEDLINE, EMBASE and in ClinicalTrials.gov in January 2009. The eligibility and the methodological quality of trials were assessed and data were extracted, using standard forms. Data were captured and analyzed in Review Manager Software, versions 4.2 and 5.0. The outcomes assessed were: treatment failure, fever and parasite clearance time, calculating the relative risk (RR) and a weighted mean difference (WMD) with a 95% confidence interval and p-values, indicating statistical significance at 0.05. Thirty-seven trials with 6862 participants were included. Artesunate combined with amodiaquine had a statistically significant lower risk of treatment failure compared to the combination of artesunate with sulfadoxine-pyrimethamine (RR=0.57, 95% CI [0.33, 0.97], p=0.04, seven trials, N=1341). In addition, treatment with artesunate plus mefloquine was significantly associated with a lower risk of treatment failure compared to artesunate plus azithromycin (RR=0.04, 95% CI [0.00, 0.64], p=0.02, one trial, N=54). There was no significant difference when either mefloquine or atovaquone-proguanil were combination partners with artesunate (RR=2.6, 95% CI [0.93; 7.24], p=0.07, one trial, N=1066). When artesunate was combined with chloroquine, primaquine or azithromycin and compared with artesunate monotherapy, there was no statistically significant difference in the risk of unadjusted treatment failure. Each of these comparisons had one trial each. Artesunate plus chloroquine was quicker at clearing fever compared to artesunate plus sulfadoxinepyrimethamine (WMD= -7.20, 95% CI [-12.53, -1.87], one trial, N=132). Few trials adequately reported adverse events. There was no significant difference observed in the risk of adverse events between artesunate plus amodiaquine compared with artesunate monotherapy, however, adverse events were significantly less in artesunate plus amodiaquine compared to artesunate plus methylene-blue. Artesunate plus amodiaquine on the other hand had significantly more adverse events reported compared to artesunate plus sulfadoxine-pyrimethamine. The findings of this study support the implementation of artemisinin-based combination therapy for the treatment of uncomplicated malaria. Most crucially, this review found a greater advantage of combining amodiaquine with artesunate compared to sulfadoxine-pyrimethamine. The efficacy of artesunate plus mefloquine was superior to that of artesunate plus azithromycin. Furthermore, the combination of artemisinins with chloroquine, primaquine and azithromycin has shown very low efficacy and these combination therapies should not be recommended. The reporting of efficacy was not standardized as many trials did not differentiate between re-infections and recrudescences. Adverse events were also not adequately reported. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Pyrimethamine efficacy was investigated in vitro on the blood asexual stages, the sexual stages and exflagellation in Plasmodium falciparum. Gametocytogenesis was stimulated following the standard methods on five isolates of Plasmodium falciparum. From these five isolates, RSA 2, 3 and 5 produced gametocytes which reached maturity within seven days and the gametocytes were able to exflagellate. Isolate MW2 produced young gametocytes which disappeared within ten days. NF54 produced mature gametocytes which lasted for 24 hours only. There were no statistically significant differences between the static and the synchronization methods of gametocyte stimulation for any of the isolates. The effect of pyrimethamine was investigated by adding a known concentration of the drug (For RSA 2, MW2 and NF54, l00nmol/ℓ; RSA 3 and 5, 3000nmol/ℓ pyrimethamine) to the culture medium for seven days during gametocyte stimulation. The results of this investigation show that there was gametocytocidal activity on the isolates that were used and pyrimethamine also had a schizontocidal action on NF54 and the young gametocytes of this isolate were destroyed by the drug. At concentrations which were inhibitory to asexual parasites, the drug had a sporontocidal effect on isolate RSA 2 but not on isolate RSA 5. The pyrimethamine MIC values for asexual parasites ranged from 300nmol/ℓ to > 3000nmol/ℓ (RSA 2 and 5 were not inhibited at 3000nmol/ℓ ). These results are consistent with those found in previous studies when pyrimethamine resistance was first detected in South Africa. The chloroquine MICs indicate a good correlation with the results obtained from previous drug sensitivity tests for all the isolates examined using both the 48-hour in vitro test and isotope incorporation for growth assessment. The isobolograms constructed to determine relationship between chloroquine and pyrimethamine indicated no synergism for isolates RSA 2 and 5, but the Σ relative IC[50]s indicated a weak synergism. Both the isobolograms and the Σ relative IC[50]s for the isolates RSA 6, 9 and 14 indicated an antagonistic action between chloroquine and pyrimethamine. The results obtained from this study have important implications for malaria control in South Africa. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

Anopheles melas is a brackish water mosquito found along the coast of West-Africa where it can be the dominant malaria vector locally. In order to facilitate genetic studies of this species and to examine the usefulness of microsatellite markers when used in a sibling species, 45 microsatellite loci originally developed for Anopheles gambiae were sequenced in An. melas. These loci were evaluated on their suitability as polymorphic markers based on repeat structure, length, and polymorphism in wild An. melas populations. Of the 45 loci, 18 were not considered promising markers in An. melas. A total of 48 out of 90 An. gambiae primers contained at least one mismatch with the An. melas annealing site. An. melas-specific primers were designed for 27 loci, and their variability was examined in two wild populations from Equatorial Guinea. Based on a low level of polymorphism, Hardy-Weinberg disequilibrium, or poor amplification, a further 12 loci were excluded. The remaining fifteen loci were screened in four additional wild populations from a wider geographic region including Equatorial Guinea, Cameroon, The Gambia, and Guinea Bissau. These loci showed an average heterozygosity ranging from 0.18 to 0.79, with 2.5 to 15 average alleles per locus, yielding 13 highly polymorphic markers and two loci with more limited variability in a wide geographic region. To examine the effects of cross species amplification, five of the original An. gambiae markers were also amplified in the An. melas populations. Null alleles were found for one of these An. gambiae markers. We discuss the pitfalls of using microsatellite loci even in a very closely related species, and conclude that in addition to the well-known problem of null alleles associated with this practice, many loci may prove to be of very limited use as polymorphic markers even when used in a sibling species. Fifteen An. melas-specific markers were subsequently amplified and analyzed in 11 wild An. melas populations from throughout the range of this species, including Bioko Island, Equatorial Guinea. We analyzed pair-wise population differentiation between all populations, and found that all but two comparisons were significant (p-val.<0.05), and populations clustered into three distinct groups representing Bioko Island, Central Africa, and West Africa populations. A Bayesian clustering analysis found little, if any, evidence for migration from mainland to Bioko Island populations, although there was evidence of migration from Bioko Island to the West population cluster, and from the Central to the West population cluster. Simulations of historical gene followed these same patterns and further support our predictions of unidirectional gene flow. Comparison of 1161 nucleotides amplified and sequenced from the ND4 and ND5 regions of the mtDNA showed that differentiation between An. melas population clusters is on par with levels of differentiation between member species of the An. gambiae complex, with low support for internal nodes in a maximum likelihood tree, which suggests that observed An. melas clusters are not monophyletic. From this we hypothesize that Bioko Island An. melas populations are derived from Tiko, Cameroon, and that these populations became isolated from one another when sea levels rose after the last glaciation period (?10,000-11,000 years ago), cutting off Bioko Island populations from the mainland and significantly reducing migration. Our conclusions have implications for vector control within the region, as Bioko Island is the subject of an intensive malaria control campaign, and the lack of migration from mainland West Africa to Bioko Island make it unlikely that eradicated populations of this malaria vector will be repopulated by mainland immigrants.

The two main causes of child mortality and morbidity in Africa are malaria and invasive bacterial diseases. In addition, co-infections in sub-Saharan Africa are the rule rather than the exception. However, not much is known about the host-pathogen interaction during a concomitant infection or how it affects the outcome of disease. In order to study the immunological responses during malaria and bacterial co-infections, we established a co-infection mouse model. In these studies we used two pathogenic bacteria found in malaria co-infected patients: Streptococcus pneumoniae and Relapsing fever Borrelia duttonii. Hosts co-infected with malaria and Borrelia showed greatly increased spirochetal growth but low parasite densities. In addition, the co-infected hosts presented symptoms of experimental-cerebral malaria, in an otherwise unsusceptible mouse model. This was found to be a consequence of a dysregulated immune response due to loss of timing and control over regulatory mechanisms in antigen presenting cells thus locking the host in an inflammatory response. This results in inflammation, severe anemia, internal organ damage and pathology of experimental cerebral malaria. On the other hand, in the malaria - S. pneumoniae co-infection model we found that co-infected hosts cleared the bacterium much more efficiently than the single infected counterpart. This efficiency of clearance showed to be neutrophil dependent. Furthermore, in vitro studies revealed that neutrophils isolated from malaria-infected hosts present an altered migratory effect together with a significantly increased capacity to kill S. pneumoniae. This suggests that a malaria infection primes neutrophils to kill S. pneumoniae more efficiently. Furthermore, a study was carried out on plasma samples from Rwandan children under the age of five, on which a full metabolomics profile was performed. We showed that these children could be divided in different disease categories based on their metabolomics profile and independent of clinical information. Additionally, the mild malaria group could further be divided in two sub-groups, in which one had a metabolomic profile resembling that of severe malaria infected patients. Based on this, metabolite profiling could be used as a diagnostic tool to determine the distinct phase, or severity of a malaria infection, identify risk patients and provide helpful and correct therapy.

Background Recent malaria epidemics in KwaZulu-Natal indicate that effective anti-malarial therapy is essential for malaria control. Although artemether-lumefantrine has been used as firstline treatment for uncomplicated Plasmodium falciparum malaria in northern KwaZulu- Natal since 2001, its efficacy has not been assessed since 2002. The objectives of this study were to quantify the proportion of patients treated for uncomplicated P. falciparum malaria with artemether-lumefantrine who failed treatment after 28 days, and to determine the prevalence of molecular markers associated with artemether-lumefantrine and chloroquine resistance. Methods An observational cohort of 49 symptomatic patients, diagnosed with uncomplicated P. falciparum malaria by rapid diagnostic test, had blood taken for malaria blood films and P. falciparum DNA polymerase chain reaction (PCR). Following diagnosis, patients were treated with artemether-lumefantrine (Coartem®) and invited to return to the health facility after 28 days for repeat blood film and PCR. All PCR P. falciparum positive samples were analysed for molecular markers of lumefantrine and chloroquine resistance. Results Of 49 patients recruited on the basis of a positive rapid diagnostic test, only 16 were confirmed to have P. falciparum by PCR. At follow-up, 14 were PCR-negative for malaria, one was lost to follow-up and one blood specimen had insufficient blood for a PCR analysis. All 16 with PCR-confirmed malaria carried a single copy of the multi-drug resistant (mdr1) gene, and the wild type asparagine allele mdr1 codon 86 (mdr1 86N). Ten of the 16 samples carried the wild type haplotype (CVMNK) at codons 72-76 of the chloroquine resistance transporter gene (pfcrt); three samples carried the resistant CVIET allele; one carried both the resistant and wild type, and in two samples the allele could not be analysed. ii Conclusions The absence of mdr1 gene copy number variation detected in this study suggests lumefantrine resistance has yet to emerge in KwaZulu-Natal. In addition, data from this investigation implies the possible re-emergence of chloroquine-sensitive parasites. Results from this study must be viewed with caution, given the extremely small sample size. Recommendations A larger study is needed to accurately determine therapeutic efficacy of artemetherlumefantrine and resistance marker prevalence. The high proportion of rapid diagnostic test false-positive results requires further investigation. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2013.

The main purpose of the project was to create an initial lab set-up for a dig-ital in-line holographic microscope and a reconstruction algorithm. Different parameters including: light source, pin-hole size and distances pinhole-object and object-camera had to be optimized. The lab set-up is to be developed further by a master student at the University of Nairobi and then be used for malaria detection in blood samples. To acquire good enough resolution for malaria detection it has been found necessary to purchase a gray scale camera with smaller pixel size. Two dierent approaches, in this report called the on-sensor approach and the object-magnication approach, were investigated. A reconstruction algorithm anda phase recovery algorithm was implemented as well as a super resolution algorithm to improve resolution of the holograms. The on-sensor approach proved easier and cheaper to use with approximately the same results as the object-magnication method. Necessary further research and development of experimental set-up was thoroughly discussed. / Projketet har gått ut på att bygga en billigare och enklare metod för att identifiera malaria i blodprover. Malaria är ett stort problem i en mängd områden i världen. Flera av dessa är fattiga och kan i nuläget inte tillhandahålla den här tjänsten till sin befolkning. Förutom att dyr apparatur krävs måste även utbildad personal lägga ner mycket tid för att kolla en stor mängd blodprover för att statistiskt säkerställa om en person har malaria eller inte. Vårt mål var att bygga en labbuppställning för "Digital in line holographic microscopy" och en rekonstruktionsalgoritm som en masterstudent vid Nairobi universitet ska fortsätta utveckla. Vi kom också fram till vilken upplösning som krävdes för att kunna urskilja malaria i blodproverna. Digital in line holographic microscopy går till så att man har en ljuskälla som riktas genom ett pinnhål, ljuset som går genom pinnhålet ljuser upp det prov, blodproverna i vårt fall, man vill undersöka och det resulterande ljuset fångas på en kamera. Med kunskap om fourieroptik går det att rekonstruera den digitala bilden man fångat på kameran, innan rekonstruktion är den ett hologram vilken är svårtydd. Labbuppställningen byggdes delvis med en 3D printer. För att förbättra resultaten implementerades flera algoritmer vilka lade ihop en mängd förskjutna bilder till en bättre bild, så kallad super resolution. Vi lyckades inte komma till den upplösning som krävdes för att urskilja malaria men gjorde en grundlig förstudie och en utförlig beskrivning av det arbete som väntar den student som fortsätter med projektet. Framför allt beskrevs värden på parametrar och vilken typ av kamera som ska användas för att optimera uppställningen.

The lead essay measures the long-term impact of famine severity during the 1959-1961 Great Chinese Famine on contemporary per capita GDP and rural household income in China. Empirical results present a consistently negative relationship between famine severity and per capita GDP in 2010 supported using an instrumental variable approach. The instrumental variable (IV) based on the sequence in which the Chinese Communist Party (CCP) took over continental China, exploiting the relationship between a local community's demonstration of loyalty to the new CCP regime, the radicalism of leadership during the Great Leap Forward social and agricultural reform starting in 1958, and the consequences of the Great Famine. The second essay utilizes the interaction of malaria prevention and the historical geographic distribution of malaria endemicity to estimate the average global impact mosquito-control has had on population growth. The differential benefit mosquito-control health campaigns may have had with respect to the initial malaria prevalence provides useful counterfactual groups for empirical analysis as well as possible evidence for the divergence in population development between the temperate and tropical regions of the world.