Evaluating legume species as alternative trap crops to chickpea for management of Helicoverpa spp. (Lepidoptera: Noctuidae) in central Queensland cotton cropping systems

Mounting levels of insecticide resistance within Australian Helicoverpa spp. populations have resulted in the adoption of non-chemical IPM control practices such as trap cropping with chickpea, Cicer arietinum (L.). However, a new leaf blight disease affecting chickpea in Australia has the potential to limit its use as a trap crop. Therefore this paper evaluates the potential of a variety of winter-active legume crops for use as an alternative spring trap crop to chickpea as part of an effort to improve the area-wide management strategy for Helicoverpa spp. in central Queensland’s cotton production region. The densities of Helicoverpa eggs and larvae were compared over three seasons on replicated plantings of chickpea, Cicer arietinum (L.), field pea Pisum sativum (L), vetch, Vicia sativa (L.) and faba bean, Vicia faba (L.). Of these treatments, field pea was found to harbour the highest densities of eggs. A partial life table study of the fate of eggs oviposited on field pea and chickpea suggested that large proportions of the eggs laid on field pea suffered mortality due to dislodgment from the plants after oviposition. Plantings of field pea as a replacement trap crop for chickpea under commercial conditions confirmed the high level of attractiveness of this crop to ovipositing moths. The use of field pea as a trap crop as part of an areawide management programme for Helicoverpa spp. is discussed.

Predicting survival of western rock lobsters Panulirus cygnus using discriminant analysis of hemolymph parameters taken immediately following simulated handling treatments

Instances of morbidity amongst rock lobsters (Panulirus cygnus) arriving at factories in Western Australia (WA) have been attributed to stress during post-harvest handling. This study used discriminant analysis to determine whether physiological correlates of stress following a period of simulated post-harvest handling had any validity as predictors of future rejection or morbidity of western rock lobsters. Groups of 230 western rock lobsters were stored for 6 h in five environments (submerged/flowing sea water, submerged/re-circulating sea water, humid air, flowing sea water spray, and re-circulated sea water spray). The experiment was conducted in late spring (ambient sea water 22°C), and repeated again in early autumn (ambient sea water 26°C). After 6 h treatment, each lobster was graded for acceptability for live export, numbered, and its hemolymph was sampled. The samples were analysed for a number of physiological and health status parameters. The lobsters were then stored for a week in tanks in the live lobster factory to record mortality. The mortality of lobsters in the factory was associated with earlier deviations in hemolymph parameters as they emerged from the storage treatments. Discriminant analysis (DA) of the hemolymph assays enabled the fate of 80-90% of the lobsters to be correctly categorised within each experiment. However, functions derived from one experiment were less accurate at predicting mortality when applied to the other experiments. One of the reasons for this was the higher mortality and the more severe patho-physiological changes observed in lobsters stored in humid air or sprays at the higher temperature. The analysis identified lactate accumulation during emersion and associated physiological and hemocyte-related effects as a major correlate of mortality. Reducing these deviations, for example by submerged transport, is expected to ensure high levels of survival. None of the indicators tested predicted mortality with total accuracy. The simplest and most accurate means of comparing emersed treatments was to count the mortality afterwards.

Metabolism of 1,8-Cineole in Rat: Its Effects on Liver and Lung Microsomal Cytochrome P-450 Systems

The monoterpene cyclic ether, cineole (l,8-cineole, I) also known as eucalyptol, is a component of many essential oils and is widely distributed in nature. It is extensively used in pharmaceutical preparations for external application and also as a nasal spray. It was reported earlier that cineole when administered to sheep may be largely oxidized in the system (Scheline 1978). However the mode of metabolism of cineole is not known. Hence the present study was undertaken to investigate the metabolic fate of this ubiquitous terpenoid following its administration to rats by gastric intubation.

Standard yet unusual mechanisms of long-distance dispersal: Seed dispersal of Corymbia torelliana by bees

Mellitochory, seed dispersal by bees, has been implicated in long-distance dispersal of the tropical rain forest tree, Corymbia torelliana (Myrtaceae). We examined natural and introduced populations of C. torelliana for 4 years to determine the species of bees that disperse seeds, and the extent and distance of seed dispersal. The mechanism of seed dispersal by bees was also investigated, including fruit traits that promote dispersal, foraging behaviour of bees at fruits, and the fate of seeds. The fruit structure of C. torelliana, with seed presented in a resin reward, is a unique trait that promotes seed dispersal by bees and often results in long-distance dispersal. We discovered that a guild of four species of stingless bees, Trigona carbonaria, T. clypearis, T. sapiens, and T. hockingsi, dispersed seeds of C. torelliana in its natural range. More than half of the nests found within 250 m of fruiting trees had evidence of seed transport. Seeds were transported minimum distances of 20-220 m by bees. Approximately 88% of seeds were dispersed by gravity but almost all fruits retained one or two seeds embedded in resin for bee dispersal. Bee foraging for resin peaked immediately after fruit opening and corresponded to a peak of seed dispersal at the hive. There were strong correlations between numbers of seeds brought in and taken out of each hive by bees (r = 0.753-0.992, P < 0.05), and germination rates were 95 ± 5%. These results showed that bee-transported seeds were effectively dispersed outside of the hive soon after release from fruits. Seed dispersal by bees is a non-standard dispersal mechanism for C. torelliana, as most seeds are dispersed by gravity before bees can enter fruits. However, many C. torelliana seeds are dispersed by bees, since seeds are retained in almost all fruits, and all of these are dispersed by bees.

The mortality of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) neonate larvae in relation to drop-off and soil surface temperature: The dangers of bungy jumping

The first larval instar has been identified as a critical stage for population mortality in Lepidoptera, yet due to the body size of these larvae, the factors that contribute to mortality under field conditions are still not clear. Dispersal behaviour has been suggested as a significant, but ignored factor contributing to mortality in first-instar lepidopteran larvae. The impact that leaving the host plant has on the mortality rate of Helicoverpa armigera neonates was examined in field crops and laboratory trials. In this study the following are examined: (1) the effects of soil surface temperature, and the level of shade within the crop, on the mortality of neonates on the soil after dropping off from the host plant; (2) the percentage of neonates that dropped off from a host plant and landed on the soil; and (3) the effects of exposure to different soil surface temperatures on the development and mortality of neonates. The findings of this study showed that: (1) on the soil, surface temperatures above 43°C were lethal for neonates, and exposure to these temperatures contributed greatly to the overall mortality rate observed; however, the fate of neonates on the soil varied significantly depending on canopy closure within the crop; (2) at least 15% of neonates dropped off from the host plant and landed on the soil, meaning that the proportion of neonates exposed to these condition is not trivial; and (3) 30 min exposure to soil surface temperatures approaching the lethal level (>43°C) has no significant negative effects on the development and mortality of larvae through to the second instar. Overall leaving the plant through drop-off contributes to first-instar mortality in crops with open canopies; however, survival of neonates that have lost contact with a host plant is possible, and becomes more likely later in the crop growing season.

Effect of changes in hook pattern and size on catch rate, hooking location, injury and bleeding for a number of tropical reef fish species

The Queensland Great Barrier Reef line fishery in Australia is regulated via a range of input and output controls including minimum size limits, daily catch limits and commercial catch quotas. As a result of these measures a substantial proportion of the catch is released or discarded. The fate of these released fish is uncertain, but hook-related mortality can potentially be decreased by using hooks that reduce the rates of injury, bleeding and deep hooking. There is also the potential to reduce the capture of non-target species though gear selectivity. A total of 1053 individual fish representing five target species and three non-target species were caught using six hook types including three hook patterns (non-offset circle, J and offset circle), each in two sizes (small 4/0 or 5/0 and large 8/0). Catch rates for each of the hook patterns and sizes varied between species with no consistent results for target or non-target species. When data for all of the fish species were aggregated there was a trend for larger hooks, J hooks and offset circle hooks to cause a greater number of injuries. Using larger hooks was more likely to result in bleeding, although this trend was not statistically significant. Larger hooks were also more likely to foul-hook fish or hook fish in the eye. There was a reduction in the rates of injuries and bleeding for both target and non-target species when using the smaller hook sizes. For a number of species included in our study the incidence of deep hooking decreased when using non-offset circle hooks, however, these results were not consistent for all species. Our results highlight the variability in hook performance across a range of tropical demersal finfish species. The most obvious conservation benefits for both target and non-target species arise from using smaller sized hooks and non-offset circle hooks. Fishers should be encouraged to use these hook configurations to reduce the potential for post-release mortality of released fish.

Utilisation of local ingredients in commercial pig feeds

The long-term competitiveness of the both the Vietnamese feed and pig production industries are constrained and under pressure whilst the industry is dependent on the use of imported feed ingredients in diets for animal production. These cost pressures are a result of import taxes, transport costs, currency fluctuations and feed supply limitations. By undertaking studies on available resources which are currently under-utilised and with potential as local feeds, we can prove their suitability for use as feedstuffs in pig diets and as replacements for imported feed ingredients. In undertaking this process we can lower feeding costs for pig production in Vietnam by the use of local feeds which are cheaper, generate new industries in Vietnam harvesting or processing these feeds and increase the incomes of Vietnamese workers who are involved in producing these by-products. Our project has shown that rubber seed, when processed correctly to lower the hydrogen cyanide content, is a safe and suitable protein meal feedstuff for use in pig diets with the potential to replace significant quantities of imported soybean and fishmeal in Vietnamese pig diets as long as diets are balanced for any amino acid shortfalls. Our peanut studies have shown that use of binders can help alleviate pig production problems with aflatoxin content in peanut meals. Further work is needed to characterise the fate of the bound aflatoxin to see if there is any meat residue risk. Cassava residue is a resultant by-product from starch extraction in both large and small cassava processing factories. Sub-samples from these two mill types were collected and evaluated for residue HCN. Analyses has shown that the processing and sun drying results in a product with relatively consistent low HCN content. Chemical analyses also reveal that significant residual starch also remains in this by-product. Digestibility studies and pig performance feeding studies have shown that cassava residue can be included in diets at 30% with no adverse effect, although the higher fibre content of this product means that strategically, cassava residue is more suitably used in finisher and sow diets. Research has examined the digestible energy content of a number of sunflower meal types available in Australia and identified major differences in their energy value based on processing, additionally, amino acid analysis has shown a significantly lower lysine content than previous reported. We also examined the digestible energy content of a number of Australian stylo forage legume harvest batches and identified the differences in their energy value based on age/harvest time of the forage legume. Analysis results of various stylo cuts showed that the early cut stylo has a higher starch content and lower fibre fraction content than observed in late and recut stylo which were allowed to grow longer. As a result the faecal digestible energy content was higher for the early cut stylo than for the subsequent cut stylo material which had been allowed to become woody. The results have shown that feeding of stylo meal does provide some nutritive value to the pig with increased energy and nitrogen supply, with a portion of the nitrogen presented which the pig is able to retain. Based on nutrient and fibre content stylo could have a useful role in sow feeding and satiety under non-stall housing situations. With increasing Vietnamese investment in rubber production seen with larger areas under plantations the amounts of rubber seed available for animal feeding will grow significantly over the next 15 years and the importance of the by-product ie rubber seed meal as a protein source in diets for Vietnamese pigs.

Latent TGF-beta Binding Proteins : Adhesive functions and matrix association of LTBP-2 and potential functions of LTBP-1 and LTBP-3 in mesothelioma

Latent transforming growth factor-beta (TGF-beta) binding proteins (LTBPs) -1, -3 and -4 are ECM components whose major function is to augment the secretion and matrix targeting of TGF-beta, a multipotent cytokine. LTBP-2 does not bind small latent TGF-beta but has suggested functions as a structural protein in ECM microfibrils. In the current work we focused on analyzing possible adhesive functions of LTBP-2 as well as on characterizing the kinetics and regulation of LTBP-2 secretion and ECM deposition. We also explored the role of TGF-beta binding LTBPs in endothelial cells activated to mimic angiogenesis as well as in malignant mesothelioma. We found that, unlike most adherent cells, several melanoma cell lines efficiently adhered to purified recombinant LTBP-2. Further characterization revealed that the adhesion was mediated by alpha3beta1 and alpha6beta1 integrins. Heparin also inhibited the melanoma cell adhesion suggesting a role for heparan sulphate proteoglycans. LTBP-2 was also identified as a haptotactic substrate for melanoma cell migration. We used cultured human embryonic lung fibroblasts to analyze the temporal and spatial association of LTBP-2 into ECM. By We found that LTBP-2 was efficiently assembled to the ECM only in confluent cultures following the deposition of fibronectin (FN) and fibrillin-1. In early, subconfluent cultures it remained primarily in soluble form after secretion. LTBP-2 colocalized transiently with FN and fibrillin-1. Silencing of fibrillin-1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP-2 indicating that the ECM association of LTBP-2 depends on a pre-formed fibrillin-1 network. Considering the established role of TGF-beta as a regulator of angiogenesis we induced morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) and followed the fate of LTBP-1 in the endothelial ECM. This resulted in profound proteolytic processing of LTBP-1 and release of latent TGF-beta complexes from the ECM. The processing was coupled with increased activation of MT-MMPs and specific upregulation of MT1-MMP. The major role of MT1-MMP in the proteolysis of LTBP-1 was confirmed by suppressing the expression with lentivirally induced short-hairpin RNAs as well as by various metalloproteinases inhibitors. TGF-beta can promote tumorigenesis of malignant mesothelioma (MM), which is an aggressive tumor of the pleura with poor prognosis. TGF-beta activity was analyzed in a panel of MM tumors by immunohistochemical staining of phosphorylated Smad-2 (P-Smad2). The tumor cells were strongly positive for P-Smad2 whereas LTBP-1 immunoreactivity was abundant in the stroma, and there was a negative correlation between LTBP-1 and P-Smad2 staining. In addition, the high P-Smad2 immunoreactivity correlated with shorter survival of patients. mRNA analysis revealed that TGF-beta1 was the most highly expressed isoform in both normal human pleura and MM tissue. LTBP-1 and LTBP-3 were both abundantly expressed. LTBP-1 was the predominant isoform in established MM cell lines whereas the expression of LTBP-3 was high in control cells. Suppression of LTBP-3 expression by siRNAs resulted in increased TGF-beta activity in MM cell lines accompanied by decreased proliferation. Our results suggest that decreased expression of LTBP-3 in MM could alter the targeting of TGF-beta to the ECM and lead to its increased activation. The current work emphasizes the coordinated process of the assembly and appropriate targeting of LTBPs with distinct adhesive or cytokine harboring properties into the ECM. The hierarchical assembly may have implications in the modulation of signaling events during morphogenesis and tissue remodeling.

The right path : The autobiography of a survivor /

Account of the German occupation of Kecskemet; fate of Jews of Kecskemet; liberation; immediate postwar experiences in Kecskemet; memories of childhood in Kotaj and Kecskemet; move to Budapest; training as soccer player in Budapest; return to Kecskemet and work in printing shop; fate of family members during the holocaust; early years of World War II in Kecskemet; entry into forced labor; life in labor camp; escape and hiding; liberation by Red Army; return to Kecskemet under Soviet Ukrainian occupation; return to printing business in Kecskemet; courtship and marriage in April 1945; reuinion with two sisters; birth of daugher; move to Budapest in 1949; work as printer in Budapest; life in Budapest under Communist domination; anti-Semitism; uprising of 1956 in Budapest; flight to Vienna; life in Vienna; emigration to USA; life in New York; move to Los Angeles; started business in food preparation; coached soccer team.

Characterization of small GTPase Cdc42 from the ectomycorrhizal fungus Suillus bovinus and Agrobacterium tumefaciens-mediated transformation of fungi

Ectomycorrhizal formation between the host tree, Pinus sylvestris and fungal symbiont, Suillus bovinus was investigated at the molecular level by isolating genes regulating the organization of the actin cytoskeleton in the fungal partner S. bovinus. An Agrobacterium tumefaciens mediated transformation (ATMT) system was developed for the ectomycorrhizal fungi in order to assign specific functions to the cloned molecules. The developed ATMT system was also used to transform a plant pathogenic fungus, Helminthosporium turcicum, to hygromycin B resistance. Small GTPases Cdc42 and Rac1, the regulators of actin cytoskeleton in eukaryotes were isolated from S. bovinus. Sbcdc42 and Sbrac1, are both expressed in vegetative and in the symbiotic hyphae of S. bovinus . Using IIF microscopy, Cdc42 and actin were co-localized at the tips of vegetative hyphae and were visualized in association with the plasma membrane in swollen cells typical to the symbiotic hyphae. These results suggest that the small GTPases Cdc42 may play a significant role in the polarized growth of S. bovinus hyphae and regulate fungal morphogenesis during ectomycorrhiza formation through reorganization of the actin cytoskeleton. The functional equality of Cdc42 was tested in yeast complementation experiments using a Saccharomyces cerevisiae temperature sensitive mutant, cdc42-1ts. The genomic clone of CDC42 was isolated from S. bovinus genomic DNA via specific primers for Cdc42. The analogous S. cerevisiae cdc42 mutations, dominant active G12V and dominant negative D118A, were generated in the Sbcdc42 gene by in-vitro mutagenesis. The ectomycorrhizal fungi, S. bovinus, P. involutus and H. cylindroporum were transformed using ATMT and phleomycin as a selectable marker. PCR screeing suggested that the T-DNA was inserted in all the three fungal genomes but the fate of integration could not be proved by Southern blot analysis. An alternative Agrobacterium strain, AGL-1 and selection marker, hygromycin was used to transform our model fungus S. bovinus. PCR and Southern analysis suggested an improved efficiency of transformation. All the transformed fungal colonies selected for hygromycin gave positives in PCR and the Southerns showed multiple or single copy T-DNA integrations into the S. bovinus genome. Using the same Agrobacterium strain and the selectable marker, a maize pathogen, H. turcicum was also subjected to ATMT. The H. turcicum transformation data suggested the single copy T-DNA integrations into the genome of the screened transformants that further confirms wider applicability of the ATMT. The plasmids carrying the wild-type (pHGCDC42) and the mutated Sbcdc42 alleles (pHGGV; pHGDA) under Agaricus bisporus gpd promoter were constructed in an A. tumefaciens vector. ATMT was used to transform S. bovinus with the plasmids carrying the wild-type and mutated Sbcdc42 alleles. The isolation of Sbcdc42 and Sbrac1 genes and some other functionally related genes from ectomycorrhizal fungus, S. bovinus will form the basis of future work to resolve the signalling pathway leading to ectomycorrhizal symbiosis. The development of ATMT system will be a valuable tool in analysing the exact function of signalling pathway components in ectomycorrhizal symbiosis or in plant pathogenic interactions. The transformation frequency and broad applicability along with the simplicity of T-DNA integration make Agrobacterium a valuable, new and a powerfull tool for targeted and insertional mutagenesis in these plant associated fungi. The developed ATMT systems should therefore make it possible to generate large number of transformants with tagged genes which could then be screened for their specific roles in symbiosis and pathogenecity, respectively.

Annual nitrogen dynamics and urea fertilizer recoveries from a dairy pasture using 15N; effect of nitrification inhibitor DMPP and reduced application rates

Direct nitrogen (N) losses from pastures contribute to the poor nitrogen use efficiency of the dairy industry, though the exact fate of applied N and the processes involved are largely unknown. Nitrification inhibitors such as DMPP can potentially increase fertilizer N use efficiency (NUE), though few studies globally have examined the effectiveness of DMPP coated urea in pastures. This study quantified the NUE of DMPP combined with reduced application rates, and the effect on N dynamics and plant–soil interactions over an annual ryegrass/kikuyu rotation in Queensland, Australia. Labeled 15N urea and DMPP was applied over 7 winter applications at standard farmer (45 kg N ha−1) and half (23 kg N ha−1) rates. Fertilizer recoveries and NUE were calculated over 13 harvests, and the contribution of fertilizer and soil N estimated. Up to 85% of the annual N harvested was from soil organic matter. DMPP at the lower rate increased annual yields by 31% compared to the equivalent urea treatment with no difference to the high N rates. Almost 40% of the N added at the conventional fertilizer application rate as urea was lost to the environment; 80 kg N ha−1 higher than the low DMPP. Combining the nitrification inhibitor DMPP with reduced fertilizer application rates shows substantial potential to reduce N losses to the environment while sustaining productivity in subtropical dairy pastures.

Spatial ecology of a specialist insect herbivore : the leaf-mining moth Tischeria ekebladella on the pedunculate oak Quercus robur

Herbivorous insects comprise a major part of terrestrial biodiversity, and their interactions with their host plants and natural enemies are of vast ecological importance. A large body of research demonstrates that the ecology and evolution of these insects may be affected by trophic interactions, by abiotic influences, and by intraspecific processes, but so far research on these individual aspects has rarely been combined. This thesis uses the leaf-mining moth Tischeria ekebladella and the pedunculate oak (Quercus robur) as a case study to assess how spatial variation in trophic interactions and the physical distribution of host trees jointly affect the distribution, dynamics and evolution of a host-specific herbivore. With respect to habitat quality, Tischeria ekebladella experiences abundant variation at several spatial scales. Most of this variation occurs at small scales notably among leaves and shoots within individual trees. While hypothetically this could cause moths to evolve an ability to select leaves and shoots of high quality, I did not find any coupling between female preference and offspring performance. Based on my studies on temporal variation in resource quality I therefore propose that unpredictable temporal changes in the relative rankings of individual resource units may render it difficult for females to predict the fate of their developing offspring. With respect to intraspecific processes, my results suggest that limited moth dispersal in relation to the spatial distribution of oak trees plays a key role in determining the regional distribution of Tischeria ekebladella. The distribution of the moth is aggregated at the landscape level, where local leaf miner populations are less likely to be present where oaks are scarce. A modelling exercise based on empirical dispersal estimates revealed that the moth population on Wattkast an island in south-western Finland is spatially structured overall, but that the relative importance of local and regional processes on tree-specific moth dynamics varies drastically across the landscape. To conclude, my work in the oak-Tischeria ekebladella system demonstrates that the local abundance and regional distribution of a herbivore may be more strongly influenced by the spatial location of host trees than by their relative quality. Hence, it reveals the importance of considering spatial context in the study of herbivorous insects, and forms a bridge between the classical fields of plant-insect interactions and spatial ecology.

Microbial diversity in the municipal composting process and development of detection methods

Composting refers to aerobic degradation of organic material and is one of the main waste treatment methods used in Finland for treating separated organic waste. The composting process allows converting organic waste to a humus-like end product which can be used to increase the organic matter in agricultural soils, in gardening, or in landscaping. Microbes play a key role as degraders during the composting-process, and the microbiology of composting has been studied for decades, but there are still open questions regarding the microbiota in industrial composting processes. It is known that with the traditional, culturing-based methods only a small fraction, below 1%, of the species in a sample is normally detected. In recent years an immense diversity of bacteria, fungi and archaea has been found to occupy many different environments. Therefore the methods of characterising microbes constantly need to be developed further. In this thesis the presence of fungi and bacteria in full-scale and pilot-scale composting processes was characterised with cloning and sequencing. Several clone libraries were constructed and altogether nearly 6000 clones were sequenced. The microbial communities detected in this study were found to differ from the compost microbes observed in previous research with cultivation based methods or with molecular methods from processes of smaller scale, although there were similarities as well. The bacterial diversity was high. Based on the non-parametric coverage estimations, the number of bacterial operational taxonomic units (OTU) in certain stages of composting was over 500. Sequences similar to Lactobacillus and Acetobacteria were frequently detected in the early stages of drum composting. In tunnel stages of composting the bacterial community comprised of Bacillus, Thermoactinomyces, Actinobacteria and Lactobacillus. The fungal diversity was found to be high and phylotypes similar to yeasts were abundantly found in the full-scale drum and tunnel processes. In addition to phylotypes similar to Candida, Pichia and Geotrichum moulds from genus Thermomyces and Penicillium were observed in tunnel stages of composting. Zygomycetes were detected in the pilot-scale composting processes and in the compost piles. In some of the samples there were a few abundant phylotypes present in the clone libraries that masked the rare ones. The rare phylotypes were of interest and a method for collecting them from clone libraries for sequencing was developed. With negative selection of the abundant phylotyps the rare ones were picked from the clone libraries. Thus 41% of the clones in the studied clone libraries were sequenced. Since microbes play a central role in composting and in many other biotechnological processes, rapid methods for characterization of microbial diversity would be of value, both scientifically and commercially. Current methods, however, lack sensitivity and specificity and are therefore under development. Microarrays have been used in microbial ecology for a decade to study the presence or absence of certain microbes of interest in a multiplex manner. The sequence database collected in this thesis was used as basis for probe design and microarray development. The enzyme assisted detection method, ligation-detection-reaction (LDR) based microarray, was adapted for species-level detection of microbes characteristic of each stage of the composting process. With the use of a specially designed control probe it was established that a species specific probe can detect target DNA representing as little as 0.04% of total DNA in a sample. The developed microarray can be used to monitor composting processes or the hygienisation of the compost end product. A large compost microbe sequence dataset was collected and analysed in this thesis. The results provide valuable information on microbial community composition during industrial scale composting processes. The microarray method was developed based on the sequence database collected in this study. The method can be utilised in following the fate of interesting microbes during composting process in an extremely sensitive and specific manner. The platform for the microarray is universal and the method can easily be adapted for studying microbes from environments other than compost.

Intracellular activities of Salmonella enterica in murine dendritic cells.

Dendritic cells (DC) efficiently phagocytose invading bacteria, but fail to kill intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium). We analysed the intracellular fate of Salmonella in murine bone marrow-derived DC (BM-DC). The intracellular proliferation and subcellular localization were investigated for wild-type S. Typhimurium and mutants deficient in Salmonella pathogenicity island 2 (SPI2), a complex virulence factor that is essential for systemic infections in the murine model and intracellular survival and replication in macrophages. Using a segregative plasmid to monitor intracellular cell division, we observed that, in BM-DC, S. Typhimurium represents a static, non-dividing population. In BM-DC, S. Typhimurium resides in a membrane-bound compartment that has acquired late endosomal markers. However, these bacteria respond to intracellular stimuli, because induction of SPI2 genes was observed. S. Typhimurium within DC are also able to translocate a virulence protein into their host cells. SPI2 function was not required for intracellular survival in DC, but we observed that the maturation of the Salmonella-containing vesicle is different in DC infected with wild-type bacteria and a strain deficient in SPI2. Our observations indicate that S. Typhimurium in DC are able to modify normal processes of their host cells.

Synthesis of neoglycopeptides and analyses of their biodistributionin vivo to identify tissue specific uptake and novel putative membrane lectins

Complex typeN-linked oligosaccharides derived from fetuin, fibrinogen and thyroglobulin were coupled to acetyltyrosine affording a series of neoglycopeptides with retention of terminal structures and the beta-anomeric configuration of their reducing endN-acetylglycosamine residue. The neoglycopeptides thus synthesized could be labelled to high specific activities with125I in the aromatic side chain of tyrosine. Analysis of the fate of these neoglycopeptides in conjunction with inhibition with asialofetuin and oligosaccharides of defined structure in micein vivo revealed the uptake of galactosylated biantennary compound by kidneys, in addition to the known itinerary of triantennary galactosylated complex oligosaccharide from fetuin to liver and the galactosylated biantennary chain with fucosylation in the core to bone marrows. On the other hand, the agalacto, aglucosamino biantennary chains with and without fucosylation in the core region are taken up by submaxillary glands while the conserved trimannosyl core with fucose is primarily concentrated in stomach tissue. These studies thus define new routes for the uptake of complexN-linked glycans and also subserve to identify lectins presumably involved in their recognition.