No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

Parasites of the phylum Apicomplexa, including Toxoplasma gondii and Plasmodium falciparum, are major pathogens of man and domestic animals. Aspartic proteases play fundamental roles in eukaryotic biology, and have been validated as drug targets in several pathogens. The identification of the complete repertoire of seven putative aspartic protease genes in T gondii, called TgASP 1- TgASP7, was therefore undertaken, with the aim of determining the function of these proteases. The bioinformatic and phylogenetic analysis of these genes, as well as that of the putative aspartic proteases identified in the genomes of other sequenced Apicomplexa, was performed. Three of the TgASPs were selected for further study: TgASP 1, TgASP3, and TgASP5. These genes were cloned, and expressed in T gondii and in heterologous systems. The expression profiles, subcellular localizations, and post-translational modifications of these proteases are presented. The function ofTgASPl and TgASP3 was examined using traditional and conditional knock-out strategies. TgASPl localizes to punctate apical compartments in resting cells, but relocalizes dramatically to the nascent inner membrane complex of dividing cells. This protease is proposed to function in parasite replication. TgASP3 and TgASP5 localize to the trans- and cis-Golgi respectively, where they are postulated to act as maturases. Two as yet uncharacterized homologues ofTgASP3 were identified in Plasmodiumfalciparum (PfPMIX and PfPMX) and Plasmodium berghei (PbPMIX and PbPMX). These genes were cloned and expressed in different Plasmodium species. PfPMIX and PfPMX were tentatively localized to the Maurer's clefts, where they are postulated to play a role in merozoite egress from the infected red blood cell. The apparently essential roles played by several of these aspartic proteases make them attractive targets for chemotherapy.

Toxoplasma gondii is an important pathogen of humans and domestic livestock and is a major cause of abortion. It is widely distributed amongst mammals and birds and the epidemiology of human disease is complex due to the interactions between human and animal hosts. Cats are the definitive host for the parasite and rodents are believed to be involved in propagation of the parasite in cat populations. The aims of this study were to use molecular biological methods to investigate parasite transmission in wild rodents and humans.

No description available.

The aim of this thesis was to further our knowledge about the contribution of silent alleles on megabase chromosomes to the late stages of trypanosome infection and test the hypothesis that this contribution takes shape in a hierarchy of expression due to differences between alleles in terms both of flanking regions and coding sequence. This was achieved through a combination of bioinformatics and molecular studies. The initial approach was to undertake an extensive manual curation of the available VSG archive; this endeavour resulted in establishment of a fertile collaboration with the Trypanosoma brucei genome sequencing project, and in creation, with the aid of P. Ward and S. Menon, of a dedicated web-based tool to handle and query curated VSG genes. Out of an updated estimate of ~1600 VSG genes, 940 (between half and three quarters) were annotated and shown to be arranged in subtelomeric arrays and to be largely present as pseudogenes (~90%). By considering separately the hypervariable N-terminal domain (three types, A, B and C) and the more conserved C-terminal domain (types 1 to 4, with two additional types identified in this study), it appeared that most of the degeneracy lay in the C-terminal domain. This suggested that N-terminal domains (one third of them being intact) would be utilised by a process of segmental gene conversion yielding hybrid genes, by recombination with functional C-terminal ends resident at the expression site. Under the assumption that “order” within the genome (the presence of patterns within the VSG archive) helps inform “order” in VSG expression (a hierarchy based on different activation probabilities), it was somewhat surprising to detect little evidence of clear substructuring within the archive: no “classes” of VSGs could be identified, based on coding sequence and flanking sequence features. In keeping with the observed high level of divergence within the VSG archive, clear orthologue groups (here defined as alleles sharing >60% amino acid sequence identity) were found not to include more than three to four members and to be scattered at random across the arrays. Putative functional genes could not be separated into groups based on expected differences in activation probabilities, such as a different number of upstream 70-bp repeats, shown to be involved in copying silent alleles to the expression site.