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Chapter one is an overall introduction reviewing the applications of compounds possessing rotational symmetry, in particular their role as asymmetric reagents/catalysts in various reactions. The attempted syntheses of two different types of C₃-symmetric homochiral triols are described in chapter two. Unfunctionalised and functionalised triamides were synthesised from the corresponding amine and 1,3,5-benzenetricarbonyl trichloride. Unfunctionalised N-substituted 1,3,5-triazacychlohexanes (triazines) were synthesised from the corresponding amine and formaldehyde, however reactions between chiral amino alcohols and formaldehyde failed to give the desired trihydroxy triazines. Chapter three describes the syntheses of a range of unfunctionalised chiral C₃-symmetric compounds, as well as a review of C₃-symmetric systems as host molecules. The successful synthesis of a range of unfunctionalised triethrs was carried out by reacting 1,3,5-tris(bromomehyl)mesitylene with some selected terpenes and other alcohols. Their role as host molecules was investigated, however they failed to bind with any of the various metals that were used. Their asymmetric induction capabilities were also tested when one of the triethers was used as a catalyst in the Et₂Zn/aldehyde reaction. The desired alcohol was indeed isolated, however it was found by ¹H nmr of the Mosher's ester to be a reacemic mixture. In chapter four the synthesis of the unfunctionalised triethers that was described in chapter three was extended to the synthesis of C₃-symmetric triethers containing functional groups. A range of tridentate ligands were synthesised containing either amino, ester, keto or hydroxyl terminal groups. In addition to these, trihydroxy ligands containing amino linkages in place of ethereal linkages were also synthesised. From these compounds three of the trihydroxy ligands were used in complexation reactions with Sn, Ti, Si, Ni, and Cu. The reactions using Si, Ni and Cu failed to give the desired complexes. However, strong evidence was obtained for the existence of the desired Sn and Ti complexes. Once again using the Et₂Zn/aldehyde reaction preliminary asymmetric induction studies were carried out using one of the Ti complexes. The reaction showed some selectivity in favour of the R enantiomer. Chapter four also describes the syntheses of some hexadentate ligands, in particular the synthesis of a chiral C₃-symmetric hexol, a C₃-symmetric ligand containing three β keto-ester groups, and also a 1,3-diketo derivative.

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No description available.

Successful bacterial pathogens manipulate crucial intracellular host processes as a virulence strategy. One particular potent mechanism utilized by bacterial phytopathogens is to inject virulence factors (effectors) directly into the host cell. While many effectors have been identified and shown to suppress plant immune responses, very few have well-characterized enzymatic activities or host targets. To overcome the challenges of functional analysis of effectors, I designed two heterologous screens to characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae. Specifically, my objective was to identify those P. syringae effectors that target evolutionarily conserved host proteins or processes and to subsequently elucidate the molecular mechanisms of these effectors. The first heterologous screen that I performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human cells to identify potential interacting host proteins. The second heterologous screen iii utilized a high-throughput genomics approach in yeast, known as the pathogenic genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous approach, I have identified HopZ1a as the first bacterial phytopathogen effector that binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin. In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and a conserved lysine residue (K289). Using the second heterologous screen in yeast, I have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK) signaling cascades. Together, my work has identified novel eukaryotic targets and elucidated the virulence functions of HopZ1a.

We studied the host specificity, interactions with plant immune systems, and virulence factors of the phytopathogenic Type III secretion system-carrying bacterium Pseudomonas syringae. In studying host specificity, we ran growth and pod assays using seventeen pathovars of P. syringae on kidney bean hosts. We tracked bacterial growth numbers over six days and compared pathovar growth patterns. To study immune interactions with host plants, we performed effector-triggered immunity induction and suppression assays with individual effectors in Arabidopsis thaliana to determine whether effector evolutionary age was related to resultant plant immune responses. No correlations were observed. To generate candidate virulence effectors, we sequenced mRNA from seven P. syringae pathovars grown in inducing media and pulled out hits to virulence-related genes.

We studied the host specificity, interactions with plant immune systems, and virulence factors of the phytopathogenic Type III secretion system-carrying bacterium Pseudomonas syringae. In studying host specificity, we ran growth and pod assays using seventeen pathovars of P. syringae on kidney bean hosts. We tracked bacterial growth numbers over six days and compared pathovar growth patterns. To study immune interactions with host plants, we performed effector-triggered immunity induction and suppression assays with individual effectors in Arabidopsis thaliana to determine whether effector evolutionary age was related to resultant plant immune responses. No correlations were observed. To generate candidate virulence effectors, we sequenced mRNA from seven P. syringae pathovars grown in inducing media and pulled out hits to virulence-related genes.

Successful bacterial pathogens manipulate crucial intracellular host processes as a virulence strategy. One particular potent mechanism utilized by bacterial phytopathogens is to inject virulence factors (effectors) directly into the host cell. While many effectors have been identified and shown to suppress plant immune responses, very few have well-characterized enzymatic activities or host targets. To overcome the challenges of functional analysis of effectors, I designed two heterologous screens to characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae. Specifically, my objective was to identify those P. syringae effectors that target evolutionarily conserved host proteins or processes and to subsequently elucidate the molecular mechanisms of these effectors. The first heterologous screen that I performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human cells to identify potential interacting host proteins. The second heterologous screen iii utilized a high-throughput genomics approach in yeast, known as the pathogenic genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous approach, I have identified HopZ1a as the first bacterial phytopathogen effector that binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin. In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and a conserved lysine residue (K289). Using the second heterologous screen in yeast, I have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK) signaling cascades. Together, my work has identified novel eukaryotic targets and elucidated the virulence functions of HopZ1a.

Toxoplasma gondii is the causative agent of Toxoplasmosis in human and animals. T. gondii isolates are highly diverse. Hundreds of genotypes have been identified, but only three clonal lineages, namely Type I, II and III are prevalent worldwide. In mouse model, T. gondii strains can be divided into three groups based on their virulence, including the virulent (LD100=1), the intermediately virulent (LD50 = 103-104) and the non virulent (LD50 > 105). The clonal Type I, II and III T. gondii strains belong to these three groups, respectively. Epidemiologic studies suggest the difference of virulence in mice may relate to the severity of toxoplasmosis in human infection. Therefore, it is necessary to understand biological differences in genetically different T. gondii strains and their effect on the host responses. To date, the majority of data published on this aspect has been limited to in vitro assays. Here, we used in vivo assays to investigate host responses upon infection of distinct Toxoplasma strains. Our studies examined host response to infection of the three widespread clonal lineages of T. gondii using a mouse model. The following results were revealed: (i) increased tissue burden in mice is the indicator of virulence of T. gondii. Quantification of parasite burden in the spleen of mice showed significantly more parasites for Type I strain than that of Type II and III strains, with the latter two having comparable parasite burdens. Given that the Type II strains are more virulent than the Type III strains in mice; this result suggests that difference in host response is the result of specific parasite-host interaction, which is not simply due to the difference of parasite tissue load. (ii) gene expression in the host is strongly influenced by parasite genetic background. Transcriptional profiles of mice infected with the above three types of T. gondii strains showed that the overall gene expression patterns are similar between Type I and Type II infected mice and both stimulated stronger and more polarized change comparing to Type III strain. These results emphasize the importance of studying T. gondii pathogenesis in the host with the consideration of parasite genetic diversity. Such research could possibly aid in select appropriate regimes to treat toxoplasmosis caused by diverse T. gondii strains.

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.