Plasmodium falciparum and Mycobacterium tuberculosis infections collectively cause as many as five million deaths world-wide each year. In the most afflicted populations, currently available drugs and vaccines appear inadequate. By offering insight into the pathophysiology of diseases, genetic studies provide options for new therapeutic approaches to major health problems. The results of case-control studies of genetic factors associated with disease outcomes in malaria and tuberculosis in an African setting are presented in this thesis. Glucose-6-Phosphate dehydrogenase (G6PD) deficiency, the commonest enzymopathy of humans, affects over 400 million people. The geographical correlation of its distribution with the historical endemicity of malaria suggests that this disorder has risen in frequency through natural selection by malaria. However, attempts to confirm that G6PD deficiency is protective in case-control studies of malaria have yielded conflicting results. Hence, for this X-linked disorder, it is unclear whether both male hemizygotes and female heterozygotes are protected or, as frequently suggested, only females. Furthermore, how much protection may be afforded is unknown. In two large case-control studies of over 2000 African children, I found that the common African form of G6PD deficiency (G6PD A-) is associated with a 46-58% reduction in risk of severe malaria for both female heterozygotes and male hemizygotes. A mathematical model incorporating the measured selective advantage against malaria suggests that a counterbalancing selective disadvantage, associated with this enzyme deficiency, has retarded its rise in frequency in malaria-endemic regions. There is some evidence that two T helper cell subsets, Thl and Th2, regulate the immune response and thus influence the course of infections in mammalian hosts. These T cell subsets are reciprocal and associated with distinct cytokine profiles. Th2 T cell differentiation is promoted mainly by interleukin-4. Analysis of an IL-4 promoter polymorphism indicates that homozygosity for a putatively upregulatory IL-4 promoter variant is associated with a signficantly increased risk for severe malaria whilst heterozygotes are protected against this condition. Epidemiological evidence implicates host genetic factors as major determinants of variable susceptibility to tuberculosis. Most attempts to define the genetic factor(s) have focused on the HLA genes but only one result, an association of HLA-DR2 with increased susceptibility to disease in Asian populations, has been reported with any consistency. The genetic component in tuberculosis is likely to be determined by multiple genes and, therefore, in this study, the role of both HLA and non-HLA candidate genes was investigated. No association was found with variants of the macrophage gene, NRAMP1, the homologue of which has been implicated in the regulation of genetic resistance in the mouse model. Examination of certain class I and class II HLA alleles as well as the -590 interleukin-4 promoter polymorphism also did not show any association with disease. However, heterozygotes for a promoter polymorphism at position -238 of the tumour necrosis factor gene and homozygotes for dysfunctional variants of the gene encoding the collectin, mannose binding protein, were both at increased risk of developing pulmonary tuberculosis.

No description available.

Plasmodium falciparum is a protozoan parasite and the causative agent of the most lethal form of malaria, a major disease in the tropical world. Chloroquine has been very effective in treatment of this disease, however the emergence of chloroquine-resistant strains in most geographical regions where malaria is endemic has made difficult the control of malaria. In addition, resistance to other antimalarials has been observed in these regions. The objective of this study was to determine the molecular mechanisms of multidrug resistance in P. falciparum. We have selected in vitro a P. falciparum strain resistant to actinomycin D from a parental drug sensitive clone, 3D7. Interestingly, we found that the actinomycin D resistant clone is less sensitive to chloroquine and mefloquine (antimalarial drugs) and rhodamine123. Comparison between parental 3D7 and resistant P. falciparum did not show differences in the copy number or level of expression of pfmdr1 previously implicated in chloroquine or mefloquine resistance. Furthermore, to identify if other P-glycoprotein homologues are involved in resistance, we used oligonucleotide primers to conserved sequences in ABC domains. An ABC protein, a homologue to the subunit 4, of the 26S proteasome complex has been cloned. To determine if this gene was involved in resistance to actinomycin D, a Northern blot was done. Surprisingly it was found a decreased in the expression of this gene in the resistant cell line, 3D7R/actD2, in comparison with its parental cell line, 3D7. Studies are in progress to determine the role of the PFS4 subunit in the resistance phenotype of 3D7R/actD2.

Data based abnormality detection is a growing research field focussed on extracting information from feature rich data. They are considered to be non-intrusive and non-destructive in nature which gives them a clear advantage over conventional methods. In this study, we explore different streams of data based anomalies detection. We propose extension and revisions to existing valve stiction detection algorithm supported with industrial case study. We also explored the area of image analysis and proposed a complete solution for Malaria diagnosis. The proposed method is tested over images provided by pathology laboratory at Alberta Health Service. We also address the robustness and practicality of the solution proposed. / Process Control

The life cycle of the malaria parasite is very complex and provides a number of potential targets for vaccination. In this thesis, data on two plasmodial asexual blood stage antigens (RAP-2 and Pf332) are presented. A partial aim of the work presented herein was to investigate the mechanisms responsible for the destruction of erythroid cells in anaemia, and more specifically to define the role of the rhoptry associated protein (RAP)-2 and other members of the RAP complex, RAP-1 and -3 in processes resulting in anaemia. Antibodies to the RAP complex were shown to have the potential to mediate the destruction of RAP-2-tagged erythroid cells by phagocytosis or by complement activation and lysis. In addition, antibodies to RAP-1 and RAP-2 could induce the apoptotic death of RAP-2- tagged erythroblasts. The frequency and functionality of naturally occurring RAP-2 antibodies in the sera of anaemic and non-anaemic Cameroonian children were also investigated. All sera tested contained RAP-2-reactive antibodies by both immunofluorescence and flow cytometry. The anaemic group of children had higher levels of IgG than the non-anaemic ones, while the levels of IgM were similar. With respect to IgG subclasses, higher levels of IgG3 were seen in the non-anaemic individuals as compared to anaemic subjects. The non-anaemic individuals recognised a greater proportion of RAP-2-tagged RBCs and activated complement to a greater extent than the anaemic ones. Earlier studies observed that humans continuously exposed to malaria, recognised Pf332 extensively. Further studies revealed that Pf332 antibodies were able to inhibit parasite growth and cytoadherence in vitro. Making use of Pf332-C231, a sub-fragment of Pf332, we studied the effects/mode of action of C231-specific antibodies on P. falciparum parasite growth and development in vitro. The antibodies appeared to act mainly on late stage parasites by two main mechanisms: 1) through the induction of abnormal/pyknotic parasites, and, 2) RBC lysis (disintegration of RBCs), thus limiting parasite growth and development. The antibody isotype in this context was IgG. Following the removal of immune pressure, parasites resumed growth, albeit at a much slower rate. The results suggest that during natural infections, antibodies to C231 could play a role in parasite control. In summary, these data suggest that antibodies to both antigens could be instrumental in immune responses leading to disease control, but could also mediate pathology. / At the time of the doctoral defense, the following publication was unpublished and had a status as follows: Paper 3: Manuscript.

Background: The number of children under the age of five who dies of malaria per thousand births is sixteen in Tanzania. Even though improvements have been made there are still many challenges in the care of malaria infected children. Aim: The aim of this study was to investigate the experiences that nurses and relatives to malaria infected children have regarding the pediatric malaria care at Kilimanjaro Christian Medical Centre, Tanzania. Method: Semi structured interviews were conducted among three nurses and three relatives to malaria infected children within a pediatric ward at Kilimanjaro Christian Medical Centre, Tanzania. The interviews were recorded, transcribed and then analysed. Result: All the nurses agreed that the major challenge in the care of malaria infected children is the lack of knowledge from the relatives about prevention of malaria. This results in a lack of adherence among the relatives concerning prevention. The relatives agreed about their lack of knowledge about malaria and they wished for more education. They also considered the workload to be an issue for the nurses at the ward. The availability for the child to get treatment depends whether they are from a rural area or city. Conclusion: To continue the fight against malaria among children it is of great importance to focus on the relatives lack of knowledge about malaria, the workload issue and the long distance to hospital.

There is an abundance of information available on the molecular mechanisms of antigenic variation in Plasmodium falciparum. The variant antigen PfEMP1, which mediates antigenic variation as well as cytoadherence and rosetting, has been extensively characterised. Genes coding for the antigen belong to the gene family var, and several var genes have been cloned and characterised. The rodent malaria parasite P. chabaudi is a widely studied in vivo model for P. falciparum. The P. c. chabaudi AS parasite strain has been shown to exhibit antigenic variation and the variant antigen has been detected by surface fluorescence. As with P. falciparum, there is a link between antigenic variation and cytoadherence, however genes coding for the variant antigen in P. chabaudi have not been cloned to date. Therefore, potentially useful in vivo experiments on antigenic variation are restricted. In this thesis it is shown for the first time that the P. c. adami DS parasite strain also exhibits antigenic variation. / Chapter 3 describes efforts to locate genes coding for variant antigens in P. c. adami DS. The main strategy involved a genome survey, by sequencing and analysing randomly selected clones from a P. c. adami DS genomic library. DNA sequences were compared to Plasmodium spp. sequence databases to look for similarity to var genes or other genes encoding variant antigens. Of the 297 clones analysed none had significant sequence similarity to genes coding for variant antigens. However, in a small proportion of sequences some similarity to var genes was noted. Several genes of potential interest were identified, most importantly the gene coding for the vaccine candidate rhoptry associated protein 1 (RAP1), which was subsequently cloned and characterised. Further attempts to locate var gene homologues in P. c. adami involved amplification of P. c. adami genomic DNA using degenerate oligonucleotide primers corresponding to conserved regions of var genes. This strategy proved to be unsuccessful, most likely due to lack of sequence similarity between P. falciparum and P. c. adami genes. In several vaccination studies with the apical membrane antigen 1 (AMA1) of P. c. adami DS, mice were significantly protected against homologous parasite challenge. However, some mice developed late, low-level breakthrough parasitaemias. In Chapter 4, the characterisation of two such breakthrough parasitaemias is described. The ama1 genes of the breakthrough parasites were found to be identical to the ama1 gene of the parental parasites. Similarly, no alteration in AMA1 expression was observed. However, the breakthrough parasites were found to be more resistant than the parental parasites to the effects of passive immunisation with rabbit antisera to AMA1, RAP1 and possibly also MSP119. P. chabaudi infections in mice have been previously shown to consist of a primary parasitaemia followed by a short period of subpatency, and a recrudescent parasitaemia. In surface immunofluorescence studies with P. c. chabaudi, parasites of the recrudescence were shown to be distinct from parasites of the primary parasitaemia, with respect to antigens expressed on the surface of late trophozoite- and schizont-infected erythrocytes. / Chapter 4 describes similar surface immunofluorescence assays carried out with P. c. adami infected erythrocytes, and quantitation of fluorescence by flow cytometry. As with P. c. chabaudi, the recrudescent parasites were found to be antigenically distinct from the primary parasitaemia, indicating that antigenic variation had taken place. Because breakthrough parasites from the AMA1 vaccination trial were similar to recrudescences in peak and duration, we hypothesised that breakthrough parasitaemias, like recrudescent parasitaemias, occur as a result of antigenic variation. In Chapter 4 it was shown by surface immunofluorescence and flow cytometry using hyperimmune sera raised against different parasite populations, that breakthrough parasites express antigens on the surface of late trophozoite- and schizont infected erythrocytes that differ from those expressed by the parental and recrudescent parasites. These results support the hypothesis that switching of the variant antigen on the infected erythrocyte surface enables parasites to evade protective antibody responses directed against merozoite antigens. / Chapter 5 describes the cloning and characterisation of P. c. adami RAP1 which was identified in the process of the genomic survey described in Chapter 3, as well as P. berghei RAP1. Both rodent parasite orthologues of RAP1 were found to have 30% sequence similarity to P. falciparum RAP1, and 6 of 8 cysteines were conserved in the rodent parasite orthologues. However the three polypeptides vary significantly in size. P. c. adami RAP1 and P. berghei RAP1 consist of 691 aa and 604 aa respectively, whereas P. falciparum RAP1 consists of 783 aa residues. These size differences reflect very different N-terminal sequences prior to the first cysteine, whereas the cysteine-rich C-terminal regions are more conserved. Both P. falciparum RAP1 and P. c. adami RAP1 contain N-terminal repeats, however they bear no sequence similarity to each other. P. berghei RAP1 lacks N-terminal sequence repeats that are characteristic of P. falciparum and P. c. adami RAP1. The large cysteine-rich C-terminal region P. c. adami RAP1 (PcRAP1 C3) was expressed in E. coli as a hexa-his fusion protein. Rabbit antiserum to recombinant PcRAP1 C3 was used to characterise the expression and sub-cellular localisation of the RAP1 antigen. P. c. adami RAP1 was found to have a Mr of approximately 80,000 and was shown by immunofluorescence to localise to the merozoite rhoptries. Passive immunisation of mice with rabbit anti-RAP1 serum was shown to protect against fulminant parasitaemia and mortality. In a mouse vaccination trial using the recombinant PcRAP1 C3 polypeptide partial protection was conferred against homologous parasite challenge.

Malaria has long affected the world both socially and economically. Annually, there are 1.5-2.7 million deaths and 300-500 million clinical infections (WHO, 1998). Several antimalarial agents (such as chloroquine, quinine, pyrimethamine, cycloguanil, sulphadoxine and others) have lost their effectiveness against this disease through drug resistance being developed by the malarial parasites (The- Wellcome-Trust, 1999). Although there is no hard-core evidence of drug resistance shown on the new antimalarial compounds (artemisinin and artesunate), induced resistant studies in animal models have demonstrated that the malarial parasites have capabilities to develop resistance to these compounds (Ittarat et al., 2003; Meshnick, 1998; Meshnick, 2002; Walker et al., 2000). Furthermore, a useful vaccine has yet to be developed due to the complicated life cycle of the malarial parasites (The- Wellcome-Trust, 1999). As such, the re-emergence of this deadly infectious disease has caused an urgent awareness to constantly look for novel targets and compounds. In this present study, Plasmodium falciparum (clone 3D7) was cultured in vitro in human red blood cells for extraction of total RNA which was later reverse transcribed into cDNA. The áI-, áII- and â-tubulin genes of the parasite were then successfully amplified and cloned into a bacterial protein expression vector, pGEX- 6P-1. The tubulin genes were then sequenced and analysed by comparison with previously published homologues. It was found that the sequenced gene of áItubulin was different at twelve bases, of which only six of these had resulted in changes in amino acid residues. áII- and â-tubulin genes demonstrated 100% sequence similarity with the published sequences of clone 3D7, but differences were observed between this clone and other strains (strains NF54 & 7G8) of â-tubulin. Nevertheless, the differences were minor in áI- and â-tubulins and there was greater than 99% homology. Subsequently, all three Plasmodium recombinant tubulin proteins were separately expressed and purified. Insoluble aggregates (inclusion bodies) of these recombinant tubulins were also refolded and have been tested positive for their structural characteristics in Western blot analysis. Both soluble and refolded recombinant tubulins of malaria were examined in a drugtubulin interaction study using sulfhydryl reactivity and fluorescence quenching techniques. Known tubulin inhibitors (colchicine, tubulozole-c and vinblastine) and novel synthetic compounds (CCWA-110, 239 and 443) were used as the drug compounds to determine the dynamics and kinetics of the interactions. In addition, mammalian tubulin was also used to determine the potential toxicity effects of these compounds. Similarities were observed with other published reports in the binding of colchicine with the recombinant tubulins, hence confirming proposed binding sites of this compound on the Plasmodium recombinant tubulins. Two synthetic compounds (CCWA-239 and 443) that have previously tested positive against P. falciparum in vitro were found to bind effectively with all three tubulin monomers, while displaying low binding interactions with the mammalian tubulin, thus indicating that these compounds have potential antimalarial activity. Therefore, this study has satisfied and fulfilled all the aims and hypotheses that have previously been stated.

Proefschrift Universiteit van Amsterdam.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 99-100).