MSc (Chemistry) / Department of Chemistry / This dissertation focuses on the application of 1, 3, 5-triazine in a pharmaceutical point of view since it has wide range of uses as described in chapter 1. Malaria is one of the most prevalent and deadly infectious diseases worldwide though there are already many synthesized anti-malarial drugs which are in use presently, drug resistance seems to be one of the major problem and combination therapy seems to be the only solution for now. Hence in this study we used hybridization as a tool (Figure 9) to synthesize new antimalarial drugs using 1, 3, 5-triazine as an intermediate linker, linking known anti-malarial drug, different amine and chloroquine-like amines together using nucleophilic substitution reaction. As explained in chapter four of this dissertation, triazine is used to synthesize mono-, di- and tri-amino-1, 3, 5-triazine substituted products. Using THF as a solvent and K2CO3 as a base changing in temperatures, from 0 oC 25 oC or reflux. Some products were synthesized using microwave irradiation. The application of triazine as an intermediate linker in the above mentioned condition yielded five mono-amino substituted dichloro-1, 3, 5-triazine (21-25) in an average yield of 82%, three amino substitution chloro-1, 3, 5-triazine (26-28) in an average yield of 87%, two amino substituted-1, 3, 5-triazine (29, 30) in an average of 90%, nine chloroquine-like synthesized compounds (33-41) in 84 % average yields respectively. / NRF

MSc (Biochemistry) / Department of Biochemistry / In 2017 malaria cases were estimated at 219 million and of these 435 000 resulted in death. Malaria is transmitted by female Anopheles mosquitoes which thrive in tropical and sub-tropical areas. Malaria is caused by five species from the genus Plasmodium, namely P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. P. falciparum causes the most severe form of the disease. P. falciparum has a complex life cycle in the human and mosquito hosts exposing the parasite to environmental changes, resulting in upregulation of heat shock proteins (Hsps). These Hsps facilitate protein folding and protein disaggregation. Hsp70 is a molecular chaperone whose function is to facilitate protein folding. P. falciparum Hsp70-x is the only member of this family of proteins that is exported to the erythrocyte cytosol by the parasite. PfHsp70-x has been implicated in the development of malaria pathogenesis. This is largely due to its association with P. falciparum erythrocyte membrane protein 1 (PfEMP1), an important virulent factor that is exposed to the exterior of the infected erythrocyte. In tumour cells, cell surface- bound Hsp70 is known to sensitize the tumour cells to cytolytic attack that is mediated by NK cells. Cell surface bound Hsp70 is thought to recruit NK cells and Granzyme B (GrB) via its 14 amino acid sequence, TKDNNLLGRFELSG, known as the TKD motif. Both PfHsp70-x and human Hsp70 (hHsp70) contain the TKD motif. Thus, this study sought to investigate the role of Hsp70 in facilitating the selective targeting of malaria parasite-infected erythrocytes by GrB. To this end, recombinant hHsp70 and PfHsp70-x were successfully expressed in E. coli and purified. Using slot blot and ELISA, it was observed that both PfHsp70-x and hHsp70 directly interact with GrB. PfHsp70-x showed greater affinity for GrB than hHsp70. In addition, using parasites cultured at the erythrocyte stage it was noted that GrB exhibits potent antiplasmodial activity (IC50 of 0.5μM). In addition, the findings suggest that GrB interacts with both Hsp70s (of parasite and human origin) resident in the infected erythrocyte. This makes GrB a promising antimalarial agent. / NRF

Malaria is a devastating parasitic disease in humans caused by species in the genus Plasmodium. With over 100 million cases and at least 1.5 million fatalities each year, the disease accounts for 4-5% of all fatalities in the world. A recent increase in the number of malaria cases in South Africa has imposed severe costs on the economy and public health. Immunity to malaria is a multi-component system involving both B and T celllymphocytes. Pc96 is a 96 kDa antigen identified in the mouse malaria model Plasmodium chabaudi adami. It is known to be associated with the outer membrane of mouse erythrocytes infected with the parasite and has shown protective roles in mice challenged with P. chabaudi adami. A specific T cell clone has been identified that adoptively provides protection to athymic mice infected with P. chabaudi adami. Antibodies raised against Pc96 identified proteins that induced the proliferation of the protective T cell clones. At least four other antigens of different species of. malaria share at least one cross-reactive epitope. In an attempt to identify a Plasmodiumfalciparum homologue ofPc96, the amino-acid sequence was used in a BLAST search of the P. falciparum genome database, identifying a 403 kDa protein with a high degree of homology to Pc96. Sequence alignments indicated a region spanning 90 amino acids in Pf403 that overlaps the Pc96 amino acid sequence. A 178 kDa protein in P. yoelii yoelii (Pyy178) was shown to be highly similar to Pc96. Tvcell epitope prediction programs identified putative T cell epitopes in Pc96 which appear to be conserved in Pf403 and Pyy178. A casein kinase IT phosphorylation site was also identified in this region and is conserved in both sequences. PCR primers were designed to amplify regions of the MAL3P6.11 gene coding for Pf403 from P.falciparum genomic DNA. An 817 bp region in the MAL3P6.11 gene was amplified. This codes for the region ofPf403 that shows high homology to Pc96 and contains the conserved T cell epitopes and casein kinase phophorylation site. A BamHI site was incorporated into the forward primer to facilitate in-frame ligation with cloning vectors. The PCRproduct obtained was verified by restriction analysis using HindIII and EcoRI sites within the fragment. The 817 bp peR product was cloned into the pMOSBlue vector using a blunt-endedPCR cloning kit, and transformed into MOSBlue competent cells. Recombinants were identified using the uIV complementation system, and verified by PCR, plasmid DNA isolation, and restriction digestion analysis. The insertDNA in pMOSBlue was cut out with BamHI and sub-cloned into the BamHI site in the pMAL-C2x expression vector. Sequencing ofthe construct confirmed the identity of the cloned insert and showed the sequence to be in frame with the malE gene coding for maltose binding protein (MBP). The fusion protein, MBP-Pf32 .5, was induced and expressed as a 75 kDa protein comprising ofthe 32.5 kDa region ofPf403, and MBP (42.5 kDa) and was detected by anti-MBP antibodies, by western blotting. This recombinant protein has many applications for further studies involving the characterisation of the Pf403 protein, and the determination of possible roles that the protein may have in stimulating an immune response during human malaria infections. / Thesis (M.Sc.) - University of Natal, Pietermaritzburg, 2003.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-06-05T21:05:38Z No. of bitstreams: 1 Bruno de Bezerril Andrade - 2010.pdf: 74164438 bytes, checksum: a1cccf3d1f924ff7710fc4a73a190f0a (MD5) / Made available in DSpace on 2012-06-05T21:05:38Z (GMT). No. of bitstreams: 1 Bruno de Bezerril Andrade - 2010.pdf: 74164438 bytes, checksum: a1cccf3d1f924ff7710fc4a73a190f0a (MD5) / Universidade Federal da Bahia. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A malária é considerada uma das mais importantes doenças infecciosas do mxmdo. Esta doença é causada por diversas espécies do protozoário Plasmodium sp., principalmente o Plasmodium falciparum e o Plasmodium vivax, transmitido por mosquitos do gênero Anopheles. Apesar dos esforços governamentais e privados para o desenvolvimento de estratégias para o controle da doença, o panorama atual da malária está piorando, muito em razão do aparecimento de cepas de parasitas resistentes aos medicamentos. Os casos fatais são relatados principalmente na Áfiica e são causados pelo Plasmodium falciparum. Apesar de ser menos letal, a malária causada pelo Plasmodium vivax é mais amplamente distribuída e pode apresentar também alta morbidade e mortalidade. Na maioria das áreas endêmicas, estudos têm identificado vários fatores relacionados à imunidade clínica ou susceptibilidade aos parasitas. Assim, pelo menos quanto à malária causada pelo Plasmodium falciparum, idade, polimorfismos genéticos e exposição repetida ao parasita são considerados importantes determinantes da evolução da doença. Infelizmente, pouco tem sido feito na identificação de fatores preditores consistentes que poderiam ser usados para avaliação clínica. Este quadro é ainda pior para malária causada pelo Plasmodium vivax, provavelmente porque muitos pesquisadores consideram que é uma doença benigna. Além disso, como a maioria do conhecimento atual sobre a patogênese da malária não ajudou a reduzir a ocorrência da infecção e suas complicações, novas abordagens são necessárias para superar este cenário desfavorável. Esta Tese reúne um conjunto de seis manuscritos que visam identificar potenciais determinantes da gravidade da malária em uma área endêmica da Amazônia Ocidental Brasileira. Em primeiro lugar, um método preciso e eficaz para o diagnóstico da malária foi rastreado através da comparação de vários testes, incluindo um software baseado em redes neurais artificiais. O ensaio molecular mostrou-se o mais eficiente para o diagnóstico da malária sintomáticos e assintomáticos. Além disso, a utilização racional de um teste rápido para diagnóstico da malária pode ser promissora em áreas onde há dificuldade na formação continuada dos técnicos diagnósticos. A rede neural artificial indicou que o balanço de citocinas é um forte determinante do quadro clínico. Em outro estudo, uso de sorologia para mensuração de anticorpos IgG contra o sonicado de glândula salivar do vetor Anopheles darlingi mostrou-se útil para a avaliação da exposição ao Plasmodium vivax e também para estimar a imunidade clínica á malária. Em um terceiro estudo com foco na identificação de outros fatores relacionados à imunidade clínica, a exposição natural ao vírus da hepatite B mostrou-se associada à redução da gravidade clínica da malária causada tanto pelo Plasmodium vivax quanto pelo Plasmodium falciparum. No que diz respeito exclusivamente à malária vivax, os casos graves apresentaram uma intensa e desregulada resposta inflamatoria sistêmica. Nestes pacientes, a enzima antioxidante superóxido dismutase-1 surgiu como um excelente marcador da gravidade e mostrou-se envolvida na patogênese da doença grave, na qual há uma liberação de grandes quantidades de heme livre. Em conjunto, os manuscritos desta tese adicionam importantes informações no entendimento dos mecanismos determinantes da gravidade da malária, extremamente / Malaria is considered one of the most important infectious diseases that ever threaten the world. This disease is caused mainly by the infection with Plasmodium falciparum or Plasmodium vivax transmitted by Anopheles mosquitoes. Despite governmental and private efforts for the development of key strategies for the disease control, the actual panorama of the Plasmodium infection is getting worse due to the emergence of drug resistMt parasite strains. The lethal cases are reported mostly in Africa and are caused by Plasmodium falciparum. Albeit being less lethal, Plasmodium vivax infections are more widely distributed can cause high morbidity and eventually death. In most endemic areas, studies have indentified a number of factors related to clinical immunity or susceptibility to the parasites. Thus, at least regarding the falciparum malaria, age, genetic polymorphisms and repeated exposure to Plasmodium are considered most important determinants of the disease outcome. Unfortunately, little has been made in the screening of reliable predicting factors that could be ultimately used for clinical evaluations. This landscape is even worse for vivax malaria, probably because many researches consider it as a benign disease. Moreover, as most of the current knowledge about the malaria pathogenesis did not truly help to relieve the disease burden, new insights are necessary to overcome this unfavorable scenario. This thesis brings together a set of six manuscripts that aim to identify potential determinants of the disease severity linked to the immunopathogenesis in an endemic area from the western Brazilian Amazon. First, a precise and effective method for malaria diagnosis was screening by comparing multiple tests, including a software based of artificial neural networks. The molecular assay showed to be the most efficient for the diagnosis of symptomatic and asymptomatic malaria. In addition, the rational use of a rapid test for the diagnosis of malaria may be promising in areas where there is difficulty in continued training of technical human resources. The artificial neural network indicated that the cytokine balance is a strong determinant of the clinical presentation. In another study, the use of serology for measuring IgG antibodies against the sonicate salivary gland of Anopheles darlingi vector is a promising marker of exposure to Plasmodium vivax and can also estimate the clinical immunity. Intriguingly, the natural exposure to the hepatitis B virus appeared as an important factor associated with reduced clinical severity for both vivax and falciparum malaria. Concerning solely the vivax malaria, severe cases have an intense and unregulated inflammatory response. In these patients, the antioxidant enzyme superoxide dismutase-1 has emerged as an excellent marker of severity and was involved in the pathogenesis of the severe disease in which there is a release of large amounts of free heme. Together, the manuscripts of this thesis add important information in understanding the mechanisms that determine the severity of malaria.

Malaria is one of the three most deadly diseases in Africa. Although there are available treatments, their efficacy has been greatly reduced over the past two decades due to the development of resistance to currently available drugs. This has necessitated the search for new and effective antimalarial agents. This project approached the search for new antimalarial compounds in two ways: (i) by screening natural products isolated from marine algae against the Plasmodium parasite and (ii) by modification of selected isolated active compounds to target 1-deoxY-đ-xylulose 5-phosphate reductoisomerase (DXR), an enzyme found in the nonmevalonate isoprenoid biosynthetic pathway of Plasmodium Jalciparum. It was envisaged that such a compound would exhibit dual action on the Plasmodium parasite. Extracts obtained from 22 marine algae were prefractionated by solvent partitioning and were screened for anti plasmodial activity against the chloroquine sensitive (CQS) P. Jalciparum D 10 strain. Overall, 50% of the algae screened produced at least one crude fraction with activity against P. Jalciparum. Extracts of the algae Sargassum heterophyllum, Plocamium cornutum, Amphiroa ephedrea and Pterosiphonia cloiophylla gave the most promising results. Fractionation of S. heterophyllum afforded three tetraprenyltoluquinols (3.1, 3.2 and 3.5) and an all-trans-fucoxanthin (3.6). Three new compounds (4.5, 4.6 and 4.7) and two known halogenated monoterpenes (4.1 and 4.4) were isolated from P. cornutum. Each of the isolated compounds from both S. heterophyllum and P. cornutum showed antiplasmodial activity with IC₅₀ values ranging from 2.0 - 15.3 μM for S. heterophyllum and 13 - 230 μM for P. cornutum. Attempts to synthetically modify halogenated monoterpene 4.4 by dihydroxylation and phosphorylation in order to inhibit the DXR enzyme was unsuccessful. However, the hemiterpene analogue (5.42) of the halogenated monoterpenes was successfully phosphorylated and dihydroxylated to give compound 5.45 which showed promising activity against DXR. The result obtained indicated that the proposed phosphorylation and dihydroxylation of the halogenated monoterpene 4.4 would result in the synthesis of a potent DXR inhibitor and therefore a potential antimalarial agent with dual mode of action on the Plasmodium parasite.

This study investigated the implementation of the roll back malaria (RBM) programme at household and at health post levels and examined factors that negatively impact on malaria prevention and control activities. Quantitative, descriptive, analytic crosssectional research, guided by the conceptual framework of the Health Belief Model, was conducted. Structured interviews were conducted with 857 women (for the household survey in phase 1) and 53 health extension workers (HEWs) in phase 2 of the study, in nine malaria endemic districts of Sidama Zone, southern Ethiopia. Data were analysed using SPSS version 20. The study’s findings indicate that 53.3% (n=457) of the household respondents and 24.5% (n=13) of the HEWs had low levels of overall malaria-related knowledge. Household respondents aged 25-34 years, (p<0.01); regularly received malaria-related information, (p<0.001) and the less poor women (p<0.001) had good levels of knowledge. Of the households, 38.9% (n=333) reported poor RBM practices. Wealth, knowledge, perceived threat of malaria and perceived benefits of implementing malaria preventive measures were positively associated with good RBM practices. Indoor residual spraying (63.6%; 422 out of 664), consistent use of insecticide treated bed nets (51.6%; 368 out of 713), and environmental sanitation (38.6%; 331 out of 857) were the most commonly implemented malaria prevention strategies in the study area. Out of the 252 reported malaria cases, 53.6% (n=135) occurred among children under five years of age who also comprised 50.0% (n=16) of 32 reported malaria-related deaths. The RBM practices were poorly implemented in the study area despite malaria prevention and control efforts. Slow progress in behavioural changes among household members, lack of transportation services for referring malaria patients, lack of support given to HEWs and lack of feedback and supervision from higher level health care facilities were identified as potential challenges facing RBM implementation in the study area. Future efforts need to focus on effective behavioural changes based on intervention studies and regular monitoring of the RBM programme. The workloads of the HEWs should also be reconsidered and lay health educators should be used more effectively. Health posts should always have sufficient anti-malaria drugs and other resource such as rapid diagnostic kits. / Health Studies / D. Litt. et Phil. (Health Studies)

Controlling and eliminating malaria in Indonesia is a challenging endeavour. Evidence-based strategic plans should be critically formulated to overcome a complex mosaic of infection risk across the 5000-km-long archipelago of thousand islands and distinctive habitats. This project aimed to thoroughly explore the challenges and opportunities for controlling/eliminating malaria and present the application of malaria cartographic tools to allow malaria control agencies and their partners to comprehensively assess the prospects for reaching pre-elimination, monitor and evaluate the effectiveness of future strategies against the baseline generated. First, the historical context of malaria in Indonesia and important methods of control and their impact in the context of the political systems that supported them was comprehensively described. A series of distribution maps of twenty Anopheles malaria vector mosquitoes in Indonesia were also produced, supported by comprehensive reviews of each species’ bionomics and susceptibility to insecticides. Then, the application of malaria cartographic tools for malaria control/elimination in Indonesia was explored. The first high spatial resolution (1 x 1 km) baseline endemicity maps of Plasmodium falciparum and P. vivax malaria were generated, together with corresponding estimates of population at risk and clinical burdens of each species in 2010. Low malaria prevalences of these parasites were predicted in western areas, with high prevalences in eastern Indonesia. Over 132 million people in Indonesia lived at risk of P. falciparum transmission with 70% of them in areas of unstable transmission and 30% in stable transmission. There were an estimated 7.7 million P. falciparum clinical cases across the populations at risk. Meanwhile, nearly 130 million people lived at risk of P. vivax malaria with 79% living in unstable and 21% in stable transmission areas. This infection caused 1.5 million estimated clinical cases in 2010. Both estimates were 30-fold and 6-fold higher than routinely reported numbers, respectively. Finally, this project revealed the substantial multi-faced problems that impede current efforts towards the pre-elimination agenda. High rates of undiagnosed clinical cases, insufficient competence of malaria microscopy, inadequate primaquine dosing against P. vivax malaria infections, insufficient evidence of vector control interventions, wide diversities of vector mosquitoes and their bionomics, mosquito resistance against insecticides and inadequate malaria surveillance systems are challenging the task of controlling and eliminating malaria. A diverse range of strategies enabling locally-specific approaches must be implemented for controlling and eliminating malaria in Indonesia. Strategic recommendations are listed and future research priorities are proposed for further study.

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