<p>Malaria remains the most important parasitic disease worldwide. It is estimated that over 500 million infections and more that 2.7 million deaths arising from malaria occur each year. Most (90%) of the infections occur in Africa with the most affected groups being children of less than five years of age and women. this dire situation is exacerbated by the emrggence of drug resistant strains of Plasmodium falciparum. The work reported in this thesis focuses on improving the purification of PfHPRT by investigating the characteristics of anion exchange DE-52 chromatography (the first stage of purification), developing an HPLC gel filtration method for examining the quaternary structure of the protein and possible end stage purification, and initialcrystalization trials. a homology model of the open, unligaded PfHPRT is constructed using the atoomic structures of human, T.ccruz and STryphimurium HPRT as templates.</p>

The primary vector of malaria in the eastern United States, Anopheles quadrimaculatus (Say), was recently discovered to be a complex of five different cryptic species: A - An. quadrimaculatus, B - An. smaragdinus, C1 - An. diluvialis, C2 - An. Inundatus, D - An. maverlius (Reinert et al. 1997). In this research project, the goals were to determine which species were found in Texas, establish overall distribution patterns of those species, and observe the dates in which each specimens were collected so that any seasonal changes in species could be observed. Both An. quadrimaculatus (A) and An. smaragdinus (B) were identified from collections made throughout Texas from September 2002 through January 2005. Anopheles smaragdinus only made up 3% of the total specimens collected and neither An. inundatus nor An. maverlius were collected in Texas, even though they have both been collected in neighboring Parishes in Louisiana. Anopheles. quadrimaculatus' habitat and geographic range was found to be more extensive than An. smaragdinus. While An. smaragdinus was found only in the easternhalf of Texas with no collection south of Fort Bend County, An. quadrimaculatus was found throughout the eastern half of Texas, many of the southern Golf coast counties, and a few counties in far west Texas. The most common land cover where An. quadrimaculatus specimens were collected was on pasture/hay fields. This is very different from An. smaragdinus specimens in that pasture/hay was one of the least common land covers and the dominant land cover was woody wetlands. Overall, An. smaragdinus was usually associated with land covers that could provide shelter, while An. quadrimaculatus could be found among habitat that was more open and urban. There was no observed change in the species composition over time in this study. In fact, when An. smaragdinus was collected, An. quadrimaculatus was usually collected at the same time. Both An. quadrimaculatus and An. smaragdinus were collected throughout late spring, summer and early fall. Of course, the collection times of these species could have been an artifact of when most of the collectors were looking for An. quadrimaculatus (Say) specimens.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 338-352). Also available online.

Thesis (Ph. D.)--Case Western Reserve University, 2009. / [School of Medicine] Department of Genetics. Includes bibliographical references.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (M.A.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: secondary metabolites; hairy roots; tetraploid; artemisinin. Includes bibliographical references (p. 83-106).

Malaria is an infectious disease caused by eukaryotic protists in the genus Plasmodium. Approximately half of the world's population is at risk of malaria. The burden of Plasmodium falciparum malaria has increased in recent years due to the emergence of resistant strains, which have even been documented in regions previously reported as malaria-free. Although malaria vaccine research has been conducted and has showed recent positive results, there still remains no effective vaccine to prevent malaria in clinical practice. According to the World Health Organization, prompt confirmation of malaria infection by microscopy and/or rapid diagnostic test (RDT) is critical to control the spreading of malaria and to prevent the evolution of drug resistant Plasmodia strains. However, malaria diagnosis remains a significant challenge as many malaria endemic regions have inadequate access to microscopy, and antibody-based RDTs are restricted by their stability under tropical temperatures and by their cost. The objective of this study was to develop a new approach to malaria diagnosis using DNA aptamers to recognise proteins encoded by Plasmodium. The research is divided into two parts. Firstly, DNA aptamers against the diagnostic markers, P. falciparum histidine-rich protein 2 (HRP2) and P. falciparum lactate dehydrogenase (PfLDH), were selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Secondly, a selected PfLDH aptamer was incorporated into a gold nanoparticle detection system to develop an aptamer-nanoparticle conjugate as a new approach towards malaria diagnosis. The identified HRP2 and PfLDH aptamers were characterised by isothermal titration calorimetry (ITC) for their affinity to targets and were observed to bind with nanomolar affinity. As PfLDH aptamers were observed to have a higher affinity to their target, PfLDH, their specificities were further characterised by ITC using human lactate dehydrogenases, hLDHA1 and hLDHB. The PfLDH aptamers were shown to be highly specific to PfLDH with no observed affinity to human LDHs. After further characterisation, PfLDH aptamer 2008s was chosen for the next stage of the research to be combined with a nanoparticle as a route towards diagnostic application. In the second part of this study, PfLDH aptamer 2008s was conjugated to gold nanoparticles (AuNPs) to create aptamer-AuNP conjugates (2008s-AuNP). The aptamer-AuNP conjugates were characterised by their tolerance in different pH and salt concentration and in their sensitivity to PfLDH. This new approach of malaria diagnosis was further validated by incubating the aptamer-AuNP conjugates with various proteins and colour changes were observed specifically upon incubation with PfLDH but not with other proteins. Hence, a Plasmodium specific aptamer-AuNP conjugate to the malaria diagnostic marker, pLDH, has been developed in this research. This work lays the foundation for further development of novel rapid diagnostic tests based on nucleic acid aptamers and nanotechnology for robust and cost-effective malaria diagnosis with potential benefit not only for malaria but in a plethora of diagnostic applications. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

Drug resistance is one of the major impediments to control Plasmodium falciparum malaria worldwide. Heat shock protein 90 (Hsp90) is an essential component of the buffering capacity of eukaryotic cells as a part of the stress response. P. falciparum is no different and requires Hsp90 to chaperone proteins essential for cell cycle progression and drug resistance. Inhibition of P. falciparum Hsp90 (PfHsp90) may be able to not only cripple the parasite but also serve as an adjunctive antimalarial by circumventing drug resistance. The results presented in this thesis identify novel Hsp90 inhibitors that synergize with conventional antimicrobials, such as chloroquine (CQ), when used in combination. The objectives were to identify specific malaria Hsp90 inhibitors, the mechanism of the synergistic phenotype, and whether the strategy translates in vivo. To this end, the antimalarial activity of the purine analog PU-H71, and novel PfHsp90 inhibitors was tested. PU-H71 and the novel inhibitors APPA, harmine, and acrisorcin exhibited antimalarial activity in the nanomolar range and displayed synergistic activity with CQ. PU-H71 was able to reverse CQ resistance in a cell-based assay using the CQ-resistant strain W2. PU-H71 caused ring-stage arrest during the intra-erythrocytic cycle. Co-immunoprecipitation studies revealed that PfHsp90 interacts directly with the CQ resistance transporter (PfCRT). In the P. berghei mouse model of malaria, PU-H71 and harmine were able to reduce parasitemia and synergize with CQ. The interaction of PfHsp90 with PfCRT may underlie the synergistic phenotype. We conclude that PU-H71 and harmine are effective adjunctive antimalarial drugs that may be useful in combination therapies.