A quantitative, explorative, descriptive contextual study was conducted to determine to what extent the malaria control measures proposed by the Tanzanian government had been implemented by parents of children between the ages 0-5 years who lived in Bukumbi village. Structured interviews were conducted with 40 parents of children who had been admitted for malaria treatment during 2007, and the data analysed by computer. Although respondents had a basic knowledge of preventive measures they did not implement actions preventing the anopheles mosquitoes’ breeding in this tropical area. The vicious cycle of poverty, malaria episodes and lack of proper malaria health education hampered the implementation of control measures such as the spraying of houses with insecticides. Although the government of Tanzania subsidises insecticide treated bed nets the respondents did not maintain these nets and did not renew the insecticide treatment of these nets. The incidence of malaria is unlikely to decline in the Bukumbi village unless all identified factors are addressed. / Health Studies / M.A.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

In Tanzania laboratory services were observed to be not providing the quality of services required. It is assumed that the perceived discrepancy between malaria diagnosis and confirming laboratory result might be attributed to incompetence of health personnel. Objective The objective of this research was to explore the competence and extend to which health personnel in Muhimbli hospital comply with procedural norms in malaria diagnosis. Methodology A quantitative approach of explorative descriptive design was used. A survey was done using observation guidelines based on existing policies and norms. Actual practice of malaria diagnosis compared with the policies and procedural norms. Result The data revealed that health personnel are not competence in malaria diagnosis. Conclusion Competence of health personnel is important in malaria diagnosis, a special guideline should be developed and in-service training be implemented to minimize errors in reporting for malaria investigation. / Health Studies / M. A. (Public Health)

A quantitative, explorative, descriptive contextual study was conducted to determine to what extent the malaria control measures proposed by the Tanzanian government had been implemented by parents of children between the ages 0-5 years who lived in Bukumbi village. Structured interviews were conducted with 40 parents of children who had been admitted for malaria treatment during 2007, and the data analysed by computer. Although respondents had a basic knowledge of preventive measures they did not implement actions preventing the anopheles mosquitoes’ breeding in this tropical area. The vicious cycle of poverty, malaria episodes and lack of proper malaria health education hampered the implementation of control measures such as the spraying of houses with insecticides. Although the government of Tanzania subsidises insecticide treated bed nets the respondents did not maintain these nets and did not renew the insecticide treatment of these nets. The incidence of malaria is unlikely to decline in the Bukumbi village unless all identified factors are addressed. / Health Studies / M.A.

The aim of this study was to describe mothers’ knowledge, attitudes and practices regarding malaria in children under five years old. A descriptive cross-sectional study design was used. A structured questionnaire was administered by face-to-face interviews during data collection. The study population of this research composed of mothers of children under five years old suffering from malaria in Thyolo district. Economic-demographic characteristics that were significantly associated with mothers’ knowledge were age (p=0.018), formal education (p=0.001), income (p=0.005), and type of a house (p=0.002). Sources of malaria information that were significantly associated with mothers’ knowledge included television (p=0.004), radio (p=o.005), and posters (p=o.00019). Treatment-seeking behaviour was significantly associated with education (p=0.017). Treatment prior to hospitalisation was significantly associated with mothers’ education (p=0.0001), number of children that passed away (p=0.015), distance to the health facility (p=0.013), lack of money (p=0.019), and time taken at the hospital to get treatment (p=0.016). Recommendations were made to improve mothers’ malaria knowledge for further research / Health Studies / M.A. (Public Health)

Erythrocytes infected by mature forms of the Plasmodium falciparum parasite adhere to other components of the vascular space, a behavior considered critical to the pathogenesis of severe malaria. Adhesion is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP1), a highly variant antigen expressed by the parasite and subject to switching during the course of an infection. The host ligands of PfEMP1 include CD36, ICAM-1 and the ABO antigens. By employing a series of population- and family-based association studies from multiple African populations, we examined whether variation in the genes underlying these molecules affects susceptibility to severe malaria. Our results suggest that a common frameshift mutation in the ABO glycosyltransferase, responsible for blood group O, is associated with protection from severe malarial phenotypes (P=2x10⁻⁷), particularly severe malarial anaemia. However, we found no significant disease associations with variation in either the ICAM1 or CD36 genes. We focused on two particular functional polymorphisms, the missense ICAM-1Kilifi and the CD36 nonsense mutation T1264G. We genotyped both markers in around 10,000 individuals, but neither demonstrated an association with severe malarial phenotypes. Malaria has been a profound selection pressure shaping human genetic diversity. The last decade has seen the development of several haplotype-based methods to detect signatures of recent positive evolutionary selection. These techniques are potentially invaluable tools in our hunt for genetic variants that protect from life threatening malaria. We used simulations and empirical data from the International HapMap Project to demonstrate the validity of searching for long regions of haplotype homozygosity, as an approach to finding alleles undergoing selective sweeps. We analysed genetic data from a range of populations, particularly those utilized by HapMap, to investigate whether our candidate genes were associated with signals of recent positive selection. We characterized the distribution of a selection event associated with the CD36 1264G allele, focused in Central-West Africa, and demonstrated a novel signal of low population differentiation at the ABO gene, suggestive of longstanding balancing selection. Our work confirms that variation in the host ligands of PfEMP1 modulates severe malaria susceptibility, and highlights the value of using signals of selection, along with functional experiments and genetic association studies, to dissect the biology of severe malaria.

Malaria remains one of the world's major health problems, especially in developing countries. A better understanding of the pathology and pathophysiology of severe malaria is key to develop new treatments. Different approaches have been used in malaria research including the in vitro co-culture models with endothelial cells and both murine and simian animal models. However these are open to controversy due to disagreement on their representativeness of human disease. Using human post-mortem tissue in malaria research is another important approach but is practically challenging, limiting the availability of post mortem samples from malaria patients. The work in this thesis had two main themes. First I examined the role of the endothelial signalling Angiopoetin-Tie-2 receptor pathway in malaria. Ang-2 has been shown to be a significant biomarker of severe and fatal malaria. I examined the tissue specific expression of proteins from this pathway in post-mortem brain tissues from fatal malaria cases, but found no difference between cerebral malaria and non-cerebral malaria cases. Ang-2 correlated with the severity of malaria in these patients. An attempt to examine the interaction of hypoxia and the Ang-Tie-2 pathway in vitro using a co-culture model of human brain endothelial cells was unsuccessful due to contamination of the cell line. The second part of the thesis aimed to utilise molecular pathology techniques including miRNA and whole-genome microarrays. I have shown for the first time that these can be successfully applied to human post-mortem tissue in malaria. First I used archival tissues to examine the microRNA signature in the kidney of patients with malaria associated renal failure. Second I optimised a protocol to preserve post mortem tissue for molecular pathology, from an autopsy study in Mozambique. Using the subsequent total mRNA transcriptomic data and bioinformatics analysis this work has expanded our knowledge of differential gene expression and the families of genes which are dysregulated in the brain in response to malaria infection.

by Ng Wang Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 131-139). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / List of Tables --- p.ix / List of Figures --- p.x / List of Abbreviations --- p.xiii / Table of Contents --- p.xv / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter Chapter 2: --- Literature Review --- p.3 / Chapter 2.1 --- Malaria --- p.3 / Chapter 2.1.1 --- Global picture --- p.3 / Chapter 2.1.2 --- Malaria mechanics --- p.4 / Chapter 2.1.3 --- Life cycle of malaria parasite --- p.4 / Chapter 2.2 --- Treatment of malaria ´ؤ malaria drugs --- p.5 / Chapter 2.2.1 --- Antimalarial drugs --- p.5 / Chapter 2.2.2 --- Drug resistance --- p.6 / Chapter 2.3 --- Treatment of malaria - malarial vaccines --- p.7 / Chapter 2.3.1 --- Malarial vaccine developments --- p.7 / Chapter 2.3.2 --- Transmission blocking vaccines --- p.7 / Chapter 2.3.3 --- Pre-erythrocytic vaccines --- p.9 / Chapter 2.3.4 --- Blood stage vaccines --- p.10 / Chapter 2.4 --- The major merozoite protein - gpl95 --- p.11 / Chapter 2.5 --- Plants as bioreactors --- p.12 / Chapter 2.5.1 --- Products of transgenic plants --- p.13 / Chapter 2.6 --- Transgenic plants for production of subunit vaccines --- p.14 / Chapter 2.6.1 --- Norwalk virus capsid protein production --- p.15 / Chapter 2.6.2 --- Hepatitis B surface antigen production --- p.15 / Chapter 2.7 --- Tobacco and Arabidopsis as model plants --- p.16 / Chapter 2.7.1 --- Arabidopsis --- p.16 / Chapter 2.7.2 --- Tobacco --- p.17 / Chapter 2.8 --- Transformation methods --- p.17 / Chapter 2.8.1 --- Direct DNA uptake --- p.17 / Chapter 2.8.1.1 --- Plant protoplast transformation --- p.17 / Chapter 2.8.1.2 --- Biolistic transformation --- p.18 / Chapter 2.8.2 --- Agrobacterium-mediated transformation --- p.18 / Chapter 2.8.2.1 --- Leaf-disc technique --- p.18 / Chapter 2.8.2.2 --- In planta transformation --- p.19 / Chapter 2.9 --- Phaseolin --- p.20 / Chapter 2.10 --- Detection and purification of recombinant products - Histidine tag --- p.21 / Chapter 2.11 --- Aims of study and hypotheses --- p.22 / Chapter Chapter 3: --- Materials and Methods --- p.24 / Chapter 3.1 --- Introduction --- p.24 / Chapter 3.2 --- Chemicals --- p.24 / Chapter 3.3 --- Expression in tobacco system --- p.24 / Chapter 3.3.1 --- Plant materials --- p.24 / Chapter 3.3.2 --- Bacterial strains --- p.25 / Chapter 3.3.3 --- Chimeric gene construction for tobacco transformation --- p.25 / Chapter 3.3.3.1 --- The cloning of pTZPhasp/flgp42-His/Phast (F1) --- p.26 / Chapter 3.3.3.2 --- The cloning of pBKPhasp-sp/flgp42-His/Phast (P9) --- p.30 / Chapter 3.3.3.3 --- The cloning of pHM2Ubip/flgp42-His/Nost (C2) --- p.30 / Chapter 3.3.4 --- Confirmation of sequence fidelity of chimeric gene by DNA sequencing --- p.33 / Chapter 3.3.5 --- Cloning of chimeric gene into binary vector --- p.34 / Chapter 3.3.6 --- Triparental mating of Agrobacterium tumefaciens LBA4404/pAL4404 --- p.35 / Chapter 3.3.7 --- Tobacco transformation and regeneration --- p.36 / Chapter 3.3.8 --- GUS assay --- p.37 / Chapter 3.3.9 --- Genomic DNA isolation --- p.37 / Chapter 3.3.10 --- PCR amplification and detection of transgene --- p.38 / Chapter 3.3.11 --- Southern blot analysis --- p.38 / Chapter 3.3.12 --- Total seeds RNA isolation --- p.39 / Chapter 3.3.13 --- RT-PCR --- p.39 / Chapter 3.3.14 --- Northern blot analysis --- p.40 / Chapter 3.3.15 --- Protein extraction and SDS-PAGE --- p.40 / Chapter 3.3.16 --- Western blot analysis --- p.41 / Chapter 3.4 --- Expression in Arabidopsis system --- p.42 / Chapter 3.4.1 --- Plant materials --- p.42 / Chapter 3.4.2 --- Bacterial strains --- p.42 / Chapter 3.4.3 --- Chimeric gene construction --- p.42 / Chapter 3.4.3.1 --- The cloning of pBKPhasp-sp/His/EK/p42/Phast (DH) --- p.43 / Chapter 3.4.3.2 --- The cloning of pTZPhaSp/His/EK/p42/Phast (EH) --- p.45 / Chapter 3.4.3.3 --- The cloning of pBKPhasp-sp/His/EK/flgp42/Phast (DHF) and pTZPhasp/His/EK/flgp42/Phast (EHF) --- p.45 / Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.45 / Chapter 3.4.5 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.49 / Chapter 3.4.6 --- Transformation of Agrobacterium tumefaciens GV3101/pMP90 with chimeric gene constructs --- p.49 / Chapter 3.4.7 --- Arabidopsis Transformation --- p.49 / Chapter 3.4.8 --- Vacuum infiltration transformation --- p.50 / Chapter 3.4.9 --- Selection of successful transformants --- p.51 / Chapter 3.4.10 --- Selection for homozygous plants with single gene insertion --- p.51 / Chapter 3.4.11 --- GUS assay --- p.52 / Chapter 3.4.12 --- Genomic DNA isolation --- p.52 / Chapter 3.4.13 --- PCR amplification and detection of transgenes --- p.52 / Chapter 3.4.14 --- Southern Blot analysis --- p.52 / Chapter 3.4.15 --- Total siliques RNA isolation --- p.53 / Chapter 3.4.16 --- RT-PCR --- p.53 / Chapter 3.4.17 --- Northern blot analysis --- p.53 / Chapter 3.4.17 --- Protein extraction and SDS-PAGE --- p.54 / Chapter 3.4.18 --- Western blot analysis --- p.54 / Chapter 3.5 --- In vitro transcription and translation --- p.54 / Chapter 3.5.1 --- In vitro transcription --- p.54 / Chapter 3.5.2 --- In vitro translation --- p.55 / Chapter 3.6 --- Particle bombardment of GUS fusion gene --- p.56 / Chapter 3.6.1 --- Chimeric gene constructs --- p.56 / Chapter 3.6.2 --- Particle bombardment using snow bean cotyledon --- p.61 / Chapter Chapter 4: --- Results --- p.63 / Chapter 4.1 --- Tobacco system --- p.63 / Chapter 4.1.1 --- Chimeric gene constructs --- p.63 / Chapter 4.1.2 --- Tobacco transformation and regeneration --- p.65 / Chapter 4.1.3 --- GUS activity assay --- p.67 / Chapter 4.1.4 --- Molecular analysis of transgene integration --- p.68 / Chapter 4.1.4.1 --- Genomic DNA extraction and PCR --- p.68 / Chapter 4.1.4.2 --- Southern blot analysis --- p.70 / Chapter 4.1.5 --- Molecular analysis of transgene expression --- p.72 / Chapter 4.1.5.1 --- Total RNA isolation and RT-PCR --- p.72 / Chapter 4.1.5.2 --- Northern blot analysis --- p.75 / Chapter 4.1.6 --- Genomic PCR to confirm whole gene transfer --- p.76 / Chapter 4.1.7 --- Biochemical analysis of transgene expression --- p.78 / Chapter 4.1.7.1 --- Protein extraction and SDS-PAGE --- p.78 / Chapter 4.1.7.2 --- Western blot analysis --- p.78 / Chapter 4.2 --- Arabidopsis system --- p.83 / Chapter 4.2.1 --- Chimeric gene constructs --- p.83 / Chapter 4.2.2 --- Arabidopsis transformation and selection --- p.85 / Chapter 4.2.3 --- Selection of transgenic plants --- p.87 / Chapter 4.2.4 --- Assay of GUS activity --- p.91 / Chapter 4.2.5 --- Molecular analysis of transgene integration --- p.92 / Chapter 4.2.5.1 --- Genomic DNA extraction and PCR --- p.92 / Chapter 4.2.5.2 --- Southern blot analysis --- p.96 / Chapter 4.2.6 --- Molecular analysis of transgene expression --- p.99 / Chapter 4.2.6.1 --- Total RNA isolation and RT-PCR --- p.99 / Chapter 4.2.6.2 --- Northern blot analysis --- p.106 / Chapter 4.2.7 --- Genomic PCR for confirmation of whole gene transfer --- p.107 / Chapter 4.2.8 --- Biochemical analysis of transgene expression --- p.108 / Chapter 4.2.8.1 --- Protein extraction and SDS-PAGE --- p.108 / Chapter 4.2.8.2 --- Western blot analysis --- p.108 / Chapter 4.3 --- In vitro transcription and translation --- p.112 / Chapter 4.4 --- Particle bombardment of p42/ GUS fusion gene --- p.115 / Chapter Chapter 5: --- Discussion and Future perspectives --- p.117 / Chapter 5.1 --- Failure in detecting transgene expression --- p.117 / Chapter 5.2 --- Poor transgene expression --- p.120 / Chapter 5.2.1 --- Bacillus thuringiensis toxin and green fluorescent protein --- p.120 / Chapter 5.2.2 --- AT-richness --- p.121 / Chapter 5.2.3 --- Deleterious sequence - AUUUA --- p.123 / Chapter 5.2.4 --- Presence of AAUAAA or AAUAAA-like motifs --- p.125 / Chapter 5.2.5 --- Codon usage --- p.126 / Chapter 5.3 --- Future perspectives --- p.127 / Chapter Chapter 6: --- Conclusion --- p.129 / References --- p.131

Malaria, caused by the parasite Plasmodium, continues to exact high global morbidity and mortality rate next only to tuberculosis. It causes 300-500 million clinical infections out of which more than a million people succumb to death annually. Worst affected are the children below 5 years of age in sub-Saharan Africa. Plasmodium is a protozoan parasite classified under the phylum Apicomplexa that also includes parasites such as Toxoplasma, Lankestrella, Eimeria and Cryptosporidium. Of the four species of Plasmodium affecting man viz., P. falciparum, P. vivax, P. ovale and P. malariae, Plasmodium falciparum is the deadliest as it causes cerebral malaria. The situation has worsened recently with the emergence of drug resistance in the parasite. Therefore, deciphering new pathways in the parasite for developing lead antimalarial compounds is the need of the hour. The discovery of the type II fatty acid biosynthesis pathway in Plasmodium falciparum has opened up new avenues for the design of new antimalarials as this pathway is different from the one in human hosts. Although many biochemical pathways such as the purine, pyrimidine and carbohydrate metabolic pathways, and the phospholipid, folate and heme biosynthetic pathways operate in the malaria parasite and are being investigated for their amenability as antimalarial therapeutic targets, no antimalarial of commercial use based on the direct intervention of these biochemical pathways has emerged so far. This is due to the fact that the structure and function of the targets of these drugs overlaps with that of the human host. A description of the parasite, its metabolic pathways, efforts to use these pathways for antimalarial drug discovery, inhibitors targeting these pathways, introduction to fatty acid biosynthesis pathway, discovery of type II fatty acid biosynthesis pathway in Plasmodium falciparum and prospects of developing lead compounds towards antimalarial drug discovery is given in Chapter 1 of the thesis. In the exploration of newly discovered type II fatty acid biosynthesis pathway of P. falciparum as a drug target for antimalarial drug discovery, one of the enzymes; β-hydroxyacyl- acyl carrier protein dehydratase (PfFabZ) was cloned and being characterized in the lab. The atomic structure of PfFabZ was not known till that point of time. Chapter 2 describes the homology modeled structure of PfFabZ and docking of the discovered inhibitors with this structure to provide a rationale for their inhibitory activity. Despite low sequence identity of ~ 21% with the closest available atomic structure then, E. coli FabA, a good model of PfFabZ could be built. A comparison of the modeled structure with recently determined crystal structure of PfFabZ is provided and design of new potential inhibitors is described. This study provides insights to further improve the inhibition of this enzyme. Enoyl acyl carrier protein reductase (ENR) is the most important enzyme in the type II fatty acid biosynthesis pathway. It has been proved as an important target for antibacterial as well as antimalarial drug discovery. The most effective drug against tuberculosis – Isoniazid targets this enzyme in M. tuberculosis. The well known antibacterial compound – Triclosan, a diphenyl ether, also targets this enzyme in P. falciparum. I designed a number of novel diphenyl ether compounds. Some of these compounds could be synthesized in the laboratory. Chapter 3 describes the design, docking studies and inhibitory activity of these novel diphenyl ether compounds against PfENR and E. coli ENR. Some of these compounds inhibit PfENR in nanomolar concentrations and EcENR in low micromolar concentrations, and many of them inhibit the growth of parasites in culture also. The structure activity relationship of these compounds is discussed that provides important insights into the activity of this class of compounds which is a step towards developing this class of compounds into an antimalarial and antibacterial candidate drugs. Components of the green tea extract and polyphenols are well known for their medicinal properties since ages. Recently they have been shown to inhibit components of the bacterial fatty acid biosynthesis pathway. Some selected tea catechins and polyphenols were tested in the laboratory for their inhibitory activity against PfENR. I conducted docking studies to find their probable binding sites in PfENR. On kinetic analysis of their inhibition, these compounds were found to be competitive with respect to the cofactor NADH. This has an implication that they could potentiate inhibition of PfENR by Triclosan in a fashion similar to that of NADH. As a model case, one of the tea catechins; EGCG ((-) Epigalocatechin gallate) was tested for this property. Indeed, in the presence of EGCG, the inhibition of PfENR improved from nanomolar to picomolar concentration of Triclosan.conducted molecular modeling studies and propose a model for the formation of a ternary complex consisting of EGCG, Triclosan and PfENR. Docking studies of these inhibitors and a model for the ternary complex is described in Chapter 4. Docking simulations show that these compounds indeed occupy NADH binding site. This study provides insights for further improvements in the usage of diphenyl ethers in conjugation or combination with tea catechins as possible antimalarial therapeutics. In search for new lead compounds against deadly diseases, in silico virtual screening and high throughput screening strategies are being adopted worldwide. While virtual screening needs a large amount of computation time and hardware, high throughput screening proves to be quite expensive. I adopted an intermediate approach, a combination of both these strategies and discovered compounds with a 2-thioxothiazolidin-4-one core moiety, commonly known as rhodanines as a novel class of inhibitors of PfENR with antimalarial properties. Chapter 5 describes the discovery of this class of compounds as inhibitors of PfENR. A small but diverse set of 382 compounds from a library of ~2,00,000 compounds was chosen for high throughput screening. The best compound gave an IC50 of 6.0 µM with many more in the higher micromolar range. The compound library was searched again for the compounds similar in structure with this best compound, virtual screening was conducted and 32 new compounds with better binding energies compared to the first lead and reasonable binding modes were tested. As a result, a new compound with an IC50 of 240 nM was discovered. Many more compounds gave IC50 values in 3-15 µM range. The best inhibitor was tested in red blood cell cultures of Plasmodium, it was found to inhibit the growth of the malaria parasite at an IC50 value of 0.75 µM. This study provides a new scaffold and lead compounds for further exploration towards antimalarial drug discovery. The summary of the results and conclusions of studies described in various chapters is given in Chapter 6. This chapter concludes the work described in the thesis. Cloning, over-expression and purification of PanD from M. tuberculosis, FabA and FabZ from E. coli are described in the Appendix.

The aim of this study was to describe mothers’ knowledge, attitudes and practices regarding malaria in children under five years old. A descriptive cross-sectional study design was used. A structured questionnaire was administered by face-to-face interviews during data collection. The study population of this research composed of mothers of children under five years old suffering from malaria in Thyolo district. Economic-demographic characteristics that were significantly associated with mothers’ knowledge were age (p=0.018), formal education (p=0.001), income (p=0.005), and type of a house (p=0.002). Sources of malaria information that were significantly associated with mothers’ knowledge included television (p=0.004), radio (p=o.005), and posters (p=o.00019). Treatment-seeking behaviour was significantly associated with education (p=0.017). Treatment prior to hospitalisation was significantly associated with mothers’ education (p=0.0001), number of children that passed away (p=0.015), distance to the health facility (p=0.013), lack of money (p=0.019), and time taken at the hospital to get treatment (p=0.016). Recommendations were made to improve mothers’ malaria knowledge for further research / Health Studies / M.A. (Public Health)