Since host cell-derived thymidine is not incorporated into Chlamydia trachomatis DNA, we hypothesized that chlamydiae must synthesize dTMF de novo for DNA replication. The only known enzyme performing de novo dTMP synthesis is thymidylate synthase (TS). The goals of this thesis were to provide biochemical evidence for the existence of TS in chlamydiae, to investigate the mechanism by which the parasite obtains folate, a necessary cofactor for TS, and to provisionally characterize chlamydial TS. Results of a series of in situ experiments using a mutant cell line as chlamydial host which is incapable of de novo dTMP synthesis suggest that C. trachomatis converts dUMP into dTXP. In vitro experiments conclusively establish these findings by the demonstration of TS activity in extracts prepared from host-free chlamydial reticulate bodies. Furthermore it was found that both sulfa-sensitive and sulfa-resistant chlamydial strains can synthesize folates de novo; however strains vary significantly in their ability to transport preformed folates from the host cell. A C. trachomatis gene which is capable of complementing thymidine auxotrophy in Escherichia coli deficient in TS was cloned. Auxotrophic E. coli containing the complementing chlamydial DNA sequence converts dUMP to dTMP, using methylene tetrahydrofolate as the cofactor. The complementing DNA fragment contains an open reading frame of 1587 bp. Surprisingly this open reading frame shows absence of sequence homology to known TS. Unique in vitro characteristics shared by the enzyme activities from both chlamydial extract and recombinant E. coli extract suggest that C. trachomatis might encode a novel TS.

No description available.

Trichogramma pretiosum Riley is a generalist egg parasitoid that is mass released in biological control programmes to control various insect pests on different plant species. The parasitism rates achieved by T. pretiosum vary across host species and across plant species, which suggests that the species of host and its host plant could both influence the host searching performance of this parasitoid. A comparative behavioural study across the eggs of two moth species, Helicoverpa armigera Hübner and Spodoptera litura Fabricius, was conducted because these two host eggs have different characteristics. Helicoverpa armigera eggs are laid singly, whereas S. litura eggs are laid in clusters and are covered with fine scales from the moth abdomen. I tested how these and other differences in host species affect the behaviour of T. pretiosum females towards their host species. The ovipositional responses of T. pretiosum females, from different natal hosts and of different ages since emergence, to eggs of H. armigera and S. litura were studied in choice and no choice tests. Helicoverpa armigera eggs were consistently preferred over S. litura eggs, regardless of the natal host and adult age. When only S. litura eggs were available as hosts, they were parasitized at statistically similar rates to H. armigera eggs. The adult lifespan and lifetime fecundity of T. pretiosum were variable but were significantly affected by natal host species and/or host species to which they were exposed. The ovigeny index (OI) was significantly lower in the parasitoids exposed to H. armigera eggs than in those exposed to S. litura eggs, regardless of the natal host, indicating that H. armigera eggs sustain the adult parasitoids better, through host feeding, than S. litura eggs. Trichogramma females, like other egg parasitoids, use chemical cues that originate not only from their host plants and host eggs, but also from the adult insects that deposit the hosts. These last include the scales left by the ovipositing moths. These host-associated cues are used by parasitoids over long and short distances to find their hosts. Olfactometer tests demonstrated that the odours from moth scales and host plants attracted T. pretiosum females significantly, and thus function as long range cues. By contrast, the odour from host eggs did not attract them in this way. The strength of attraction was similar across the scales from H. armigera and S. litura moths. The female parasitoids responded most strongly to odours from tomato and cabbage, whereas odour from corn was not such a strong attractant. A non-host plant tested did not attract the parasitoids at all. The odours of moth scales from both H. armigera and S. litura were more attractive to T. pretiosum females than was the odour of the host plants tested. When parasitoids have followed these long range cues to the nearby vicinity of their hosts, they respond to short range cues to localise the hosts. Moth scales did not attract female parasitoids over a short distance, but they did arrest their movement so that they remained longer in the area with scales. Trichogramma pretiosum females took significantly more time to localise S. litura eggs than it took for them to locate H. armigera eggs. Time to locate hosts did not increase significantly when the eggs had been washed with hexane, which suggests that cues other than chemicals are associated with the eggs and play a more significant role in host finding over short distances. On their emergence, parasitoids are confronted by several options, including mating, searching for hosts, and seeking food. Season influenced the daily pattern of T. pretiosum emergence, with cooler winter temperatures extending emergence over two days. In summer they emerged earlier in the day and all parasitoids in the tests emerged during the first day. Parasitoids left their emergence sites by walking before their wings expanded fully. Adults of T. pretiosum visit flowers, presumably for nectar. Olfactometer tests showed that both males and females of T. pretiosum were attracted to the odor of alyssum flowers. The attraction to flower odour was significant for males when they had had no access to nutrients for 24h, but was less significant when they had just emerged. On the contrary, the attraction to food was more significant in newly emerged females. For 1 day starved females, cues associated with host plants (tomato leaves) and food (alyssum flowers) were equally important. Nutritional sources influenced the adult lifespan and reproductive performance of the parasitoids. Honey was the best source of nutrition for adults of T. pretiosum, resulting in the longest lifespan and the highest reproductive output, followed by alyssum flowers, tomato leaves and water. The results are discussed in relation to (i) understanding the entire mechanism of host searching behaviour of egg parasitoids, (ii) interpreting the behavioural interaction between generalist parasitoids and their different host insects and plants, and (iii) the better use of egg parasitoids in biological control. Throughout the discussion suggestions are made for further research.

Includes bibliography. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Entomology, 1989

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.

Bibliography: leaves 113-144. / ix, 144 leaves, [56] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the functions of the maternal, protein secretions of the endoparasitoid wasps Venturia canescens Gravenhorst regarding their role in providing protection against the host's immune system. Also investigates mucinous protein secretions on the egg surface and coagulation reactions and their role in protecting eggs against host cellular attacks. / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied & Molecular Ecology, 2002

Thesis (Ph. D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1984. / Includes bibliographical references (leaves 113-128).

Thesis (M.S.) in Ecology and Environmental Science--University of Maine, 2006. / Includes vita. Includes bibliographical references (leaves 71-75).

Thesis (Ph.D) -- University of Adelaide, Dept. of Entomology, 1989. / Includes bibliography.

Thesis (Ph.D.)--University of Adelaide, School of Earth and Environmental Sciences, Discipline of Environmental Biology, 2004. / Includes bibliographical references (leaves 219-236).