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Based on previous studies of the novel Lactobacillus reuteri DPC16 strain, an in vitro investigation on the supernatant antimicrobial activity and the culture safety against normal gastrointestinal microflora and gastric mucus was done in this thesis. DPC16 cell-free supernatants (fresh and freeze-dried, designated as MRSc and FZMRSc) from anaerobic incubations in pre-reduced MRS broth, have shown significant inhibitory effects against selected pathogens, including Salmonella Typhimurium, E. coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes. These effects were mainly due to the acid production during incubation as evidenced by the negation of such activity from their pH-neutral counterparts, and this acidic effect was shown to reduce the pathogen growth rate and decrease the total number of pathogen cells. By incubation of concentrated (11 g/L) resting cells in glycerol-supplemented MRS broth, another DPC16 cell-free supernatant (designated as MRSg) has shown very strong antimicrobial effect against all target pathogens. As indicated by a kinetic profile, this activity developed in a sigmoidal fashion as incubation proceeded, reaching to maximum activity after 6-8h and maintained at the same level thereafter. Further study has shown that the antimicrobial activity of this supernatant was pH-independent, effective across a pH range of 4.6 to 6.5, and acted on both Gram-negative and Gram-positive pathogens. Using the minimum effective dose, a time course investigation has provided evidence that this supernatant affected the growth of the target pathogens by elongating the lag phase and lowering the total cell number at the end of the incubation. Lastly, it was found that the strong antimicrobial effect of MRSg was bactericidal at high concentrations and bacteriostatic at low concentrations. However, it also found that the viability of DPC16 cells also decreased as incubation prolonged, which suggests that this glycerol-derived supernatant had a lethal effect to its own cells. Nevertheless, this lethal effect was exerted to a much lesser extent compared with that to the pathogens. The last DPC16 cell-free supernatant was designated as SGF, which was produced from secondary fermentation of the same resting cells in glycerol-water. SGF did not show a significant antimicrobial activity, which suggests that this specific strain is not capable of utilising glycerol in the absence of fermentable carbohydrates. The antimicrobial activity found in MRSg matched with previously published characteristics of reuterin, which is a unique antimicrobial substance synthesised by L. reuteri when incubated with glycerol. Therefore, a study on the production kinetics of reuterin by DPC16 was carried out. Supernatants of both MRSg and SGF were studied. Results showed that glycerol utilisation occurred in both MRSg and SGF, whereas the bioconversion of glycerol into reuterin was different. In MRSg, glycerol was constantly utilised by DPC16 resting cells, and by the end of an 18h incubation 85.8 mM of glycerol was utilised, where 72.8% was transformed into reuterin. The formation of reuterin initiated with an inclining production and reached the maximum rate of 10.9 mM/h after 6h of incubation, with the total production of 64 mM of reuterin at the end of the 18h incubation. This reuterin production in MRSg followed a similar pattern to that of its antimicrobial activity, which suggests a certain correlation between reuterin formation and the increase of antimicrobial activity in MRSg. Therefore, the major antimicrobial component in MRSg that accounted for its potent antimicrobial activity was very much likely to be reuterin. In SGF, however, detectable reuterin was negligible even though some glycerol may have been absorbed into the highly concentrated DPC16 resting cells. This has responded to the antimicrobial activity assay in that due to the lack of essential carbohydrate nutrient for normal cell metabolism, there was no glycerol utilisation and hence no reuterin synthesis. Having studied the antimicrobial activity of L. reuteri DPC16 and the production of antimicrobial-competent reuterin, two safety issues (the impact on growth on other normal commensal probiotics and mucin degradation activity) of this strain were assessed to further evaluate its probiotic potential. By using similar in vitro assays as in the antimicrobial test, the same set of DPC16 supernatants have demonstrated no adverse effect on the growth of either Lactobacillus acidophilus, Lactobacillus plantarum, Pediococcus acidilactici, or Bifidobacterium lactis DR10. No stimulatory effect was found either. By incorporating purified porcine gastric mucin into classic mucin-degradation assays in both liquid and agar media, DPC16 has not exhibited the same mucinolytic activity as that of the faecal flora cultures. Thus, it can be concluded that L. reuteri DPC16 is as safe to the host as normal commensal microflora.

A new variant of human apolipoprotein B has been identified in a subject with decreased low density lipoprotein cholesterol and apolipoprotein B concentrations. Although no clinical signs of fat malabsorption were observed the subject was diagnosed, on the basis of his low cholesterol and apolipoprotein B level, as having hypobetalipoproteinaemia. The variant of apolipoprotein B was first identified by Western blot analysis. The analysis revealed an abnormal low molecular weight form of apolipoprotein B as well as normal apolipoprotein B-100 indicating that the subject was heterozygous for a truncated form of apolipoprotein B. The new variant (apo B-32) was a result of a C→T transition at nucleotide 4548 in exon 26 of the apolipoprotein B gene. This mutation changes a CAG codon which codes for glutamine into a TAG stop codon resulting in translation of a truncated apolipoprotein B protein approximately 32% the length of normal apolipoprotein B-100. Although only 32% of the length of apolipoprotein B-100, apo B-32 was still capable of forming lipoprotein particles as indicated by its presence in both the low density and high density lipoprotein fractions. This density distribution is unique since apo B-32 is the shortest known truncated apolipoprotein B to be found in the low density lipoprotein fraction. This finding clearly indicates that the region of apo B-32 is important in the lipid binding characteristics of apolipoprotein B-100. The binding of apo B-32 to heparin confirmed three heparin binding sites previously predicted to be in the amino-terminal 30% of apolipoprotein B-100. Isolated lipoproteins formed from apo B-32 appeared to be similar to high density lipoproteins in size and composition. However, unlike high density lipoproteins, the apo B-32 lipoproteins in plasma were partially precipitated by polyanionlcation reagents normally used to precipitate very low density and low density lipoproteins. The presence of both apolipoproteins Al and E on the apo B-32 lipoproteins suggested that apolipoprotein Al or E may mediate the metabolism of apo B-32 since apo B-32 does not posses the receptor binding region for the low density lipoprotein receptor. Four further subjects were identified as having reduced low density lipoprotein and apolipoprotein B concentrations. However a lack of any truncated apolipoprotein B in their plasma made it difficult to link their hypobetalipoproteinaemia with the apolipoprotein B gene. The cause of the hypobetalipoproteinaemia in these subjects remains uncharacterised although future linkage analysis studies in these individuals and family members will at least establish whether their hypobetalipoproteinaemia is related to the apo B gene.

People experience the irritating tearing and burning sensation of lachrymatory factor (LF, propanthial S-oxide) when cutting or chopping onion bulbs. LF is produced by lachrymatory factor synthase (LFS) specifically from 1-propenyl sulfenic acid, a breakdown product of trans-1-propenyl-L-cysteine sulfoxide (1-PRENCSO) by alliinase. This thesis describes strategies to produce a tearless onion by using RNA interference (RNAi) silencing. To determine whether a gene silencing cassette can silence lfs gene transcripts from onion (Allium cepa L.), a crop recalcitrant to genetic transformation, a gene silencing assessment system was developed by using a model plant as a host for the gene of interest. Tobacco (Nicotiana tabacum) plants transgenic for LFS enzyme activity from onion were first produced by introducing a CaMV 35S-onion-lfs gene construct. These plants were then subjected to a second transformation with an RNAi construct directed against the lfs gene sequence. LFS enzyme activity assay showed that the transgenic plants, containing both the lfs gene and the RNAi construct, had significantly reduced LFS activity. This observation was supported by Western analysis for the LFS protein and further validated by quantitative RT-PCR analysis that demonstrated a significant reduction in the lfs transcript level in the dual transformants. This work demonstrated that the RNAi construct is a suitable candidate for the development of a tearless onion. This model plant RNAi system has wide reaching applications for assessment and targeting of plant secondary pathway genes, from poorly studied or recalcitrant plant species, that are important in pharmacological, food and process industries. The functional RNAi vector identified in the model system was transformed into onion. Endogenous lfs transcript levels were successfully reduced by up to 43-fold in six transgenic lines. In consequence, LFS enzyme activity was decreased by up to 1573-fold and this observation was supported by Western analysis for the LFS protein. Furthermore, the production of the deterrent LF upon tissue disruption was reduced up to 67-fold. Subjective olfactory assessment of silenced lines indicated that the pungent odour given off by the leaf and bulb material was much reduced compared with that of non-transgenic counterparts, and that this was replaced by a sweeter milder onion odour. A novel colorimetric assay demonstrated that this silencing had shifted the 1-PRENCSO breakdown pathway so that by reducing LFS protein, more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. A consequence of the raised thiosulfinates levels was a marked increase in the downstream production of a non-enzymatically produced zwiebelane isomer that has never previously been identified, and other volatile compounds, di-1-propenyl disulfides and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophenes, which had previously been reported either in small amounts or had not been detected in onions. These raised volatile sulfur compounds provide an explanation for the unique flavour notes of the LF reduced onion and are predicted to have health benefits akin to those found in garlic. These results demonstrated that silencing of LFS enzyme activity by introducing an RNAi construct directed against the lfs gene sequence simultaneously reduced levels of the deterrent LF and increased the desirable thiosulfinates in onions.

Microplitis croceipes, one of the nectar feeding parasitoid wasps, has been found to associatively learn chemical cues through feeding. The experiments on M. croceipes are performed and recorded by a Sony camcorder in the USDA-ARS Biological Control Laboratory in Tifton, GA, USA. The experimental videos have shown that M. croceipes can respond to Coffee odour in this study. Their detection capabilities and the behaviour of M. croceipes with different levels of coffee odours were studied. First, the data that are related to trained M. croceipes behaviour was extracted from the experimental videos and stored in a Microsoft Excel database. The extracted data represent the behaviour of M. croceipes trained to 0.02g and then exposed to 0.001g, 0.005g, 0.01g, 0.02g and 0.04g of coffee. Secondly, indices were developed to uniquely characterise the behaviour of trained M. croceipes under different coffee concentrations. Thirdly, a preliminary model and its parameters were developed to classify the response of trained wasps when exposed to these five different coffee odours. In summary, the success of this thesis demonstrates the usefulness of data analysis for interpreting experimental data, developing indices, as well as understanding the design principles of a simple model based on trained wasps.

Introgression of genes from crops into ruderal populations is a multi-step process requiring sympatry, synchronous flowering, chromosomal compatibility, successful pollination and development of the zygote, germination, establishment and reproduction of hybrid progeny. The goal of this thesis was to generate data on as many steps in this process as possible and integrate them into a predictive statistical model to estimate the likelihood of successful introgression under a range of scenarios. Rape (Brassica napus) and wild turnip (B. rapa var. oleifera) were used as a model system. A homozygous dominant mutation in the rape genome conferring herbicide resistance provided a convenient marker for the study of introgression. Potential differences between wild turnip populations from a wide range of geographic locations in New Zealand were examined. Hand pollination established the genetic compatibility of rape and wild turnip and a high potential for gene introgression from rape to wild turnip. Interspecific hybrids were easily generated using wild turnip as the maternal plant, with some minor differences between wild turnip populations. The frequency of successful hybridisation between the two species was higher on the lower raceme. However, the upper raceme produced more dormant interspecific hybrid seed. Field trials, designed to imitate rare rape crop escapes into the ruderal environment, examined the ability of rare rape plants to pollinate wild turnip plants over four summers. At a ratio of 1 rape plant for every 400 wild turnip plants, the incidence of interspecific hybridisation was consistently low (<0.1 to 2.1 % of total seed on wild turnip plants). There was a significant year effect with the first season producing significantly more seed and a greater frequency of interspecific hybrid progeny than the other years. The frequency of interspecific hybrid progeny increases when the ratio of rape: wild turnip plant numbers increases. The relative importance of anemophily and entomophily in the production of interspecific hybrids was examined. Wild turnip plants produced twice as many seeds with bee pollination relative to wind pollination. However, the frequency of interspecific hybrids under wind pollination was nearly twice that for bee pollination. Light reflectance patterns under UV light revealed a marked difference between wild turnip and rape flowers compared to near identical appearance under visible light. The data indicates that bees are able to distinguish between rape and wild turnip flowers and exhibit floral constancy when foraging among populations with these two species. Hybrid survival in the seed bank, germination and seedling establishment in the field are important components of fitness. Seed banks established in the soil after the field trials described above germinated in subsequent spring seasons. The predominantly brassica weed populations were screened for herbicide resistance and the numbers of interspecific hybrids germinating compared to the original frequency in the field trial results. Frequency of interspecific hybrids was reduced in the populations compared to the original seed deposit. Seed with a known frequency of interspecific hybrid seed was sown in a separate trial, and the frequency of interspecific hybrids compared at 0, 4, 6, and 8 weeks after sowing. Poor germination resulted limited competition between seedlings, however the frequency of interspecific hybrids declined over time indicating low plant fitness. There were no significant population effects on any parameters tested. Interspecific hybrids grown in a glasshouse were backcrossed to the parental species and selfed within the plant and within populations. Pollen from the interspecific hybrids was found to have markedly reduced fertility. Interspecific hybrid plants had low female fertility, with the majority (88%) of the pollinated flowers aborting the siliques. Of the remaining siliques, most (98%) had only one to three seeds per silique. Inheritance of the herbicide resistance gene was regular in backcrosses but highly skewed following self pollination with an excess of herbicide-sensitive progeny. Production of a stochastic predictive model integrated the information acquired over the practical work phase of this thesis and utilised the capabilities of @risk, a new application of a risk analysis tool. The three outputs examined were the number of flowering plants resulting from backcrosses to rape and wild turnip and self pollination of the interspecific hybrid progeny. Five scenarios were modelled and all demonstrated the high likelihood of introgression failure in this system. In all scenarios, >75% of simulations resulted in no interspecific hybrid progeny surviving to flowering in the third generation. In all scenarios, and for all three outputs, the seed set on the interspecific hybrids of the second generation was the major factor that limited the number interspecific hybrid progeny surviving to flowering in the third generation.