No description available.

No description available.

Rhynchosporium commune is one of the most destructive pathogens of barley worldwide. It can cause crop yield losses of up to 40% in the UK and decrease in grain quality. Populations of R. commune can change rapidly, defeating new barley resistance (R) genes and fungicides after just a few seasons of their use. Fungicide use is one of the major modes of management of Rhynchosporium and is heavily relied on the agricultural industry. Fungicides that were effective in the past are no longer effective in controlling the disease and many are only effective when used in mixtures. Beyond the currently effective fungicides there is limited new chemistry available so there is a very real need for development in this area. In pathogenic fungi, the cell wall components play a key role in the establishment of pathogenesis. The cell wall forms the outer structure protecting the fungus from the host defence mechanisms. It is involved in initiating the direct contact with the host cells by adhering to their surface. The fungal cell wall also contains important antigens and other compounds modulating host immune responses. R. commune germinated conidia and interaction transcriptome sequencing generated a list of over 30 different cell wall proteins (CWPs) potentially involved in pathogenicity. R. commune genome and interaction transcriptome sequencing provided further information about the extent of CWP families as well as a subset of genes expressed during barley colonisation by R. commune. The use of bioinformatic techniques allowed for the analysis of gene sequences. Putative cell wall associated genes were compared to the sequences from the fungal database via sequence similarity, sequence alignments 15 and conserved domain searches to better understand their function. Phylogenetic analysis also allowed us to understand the evolutionary relationship between R. commune genes and related genes in other organisms. Transcription profiling of R. commune CWPs during the development of infection helped to prioritise them for functional characterisation. Targeted gene disruption unfortunately did not yield mutants but has furthered our understanding of this technique in R. commune for future attempts. Functional complementation was successful however and allowed the uncovering of the function of RSA9. The results show that R. commune RSA9 functions as an allantoicase, an enzyme which breaks down purines as a source of nitrogen when conditions are nitrogen limited. The use of chemical cell wall inhibitors allowed us to better understand the role of carbohydrate cell wall components in R. commune fitness and virulence. Inhibition of cellulose production by DCB showed reduced growth, germination and pathogenicity of R. commune. Similar results were observed when beta-glucan synthesis was impaired; as inhibitor concentration increased, growth and germination of the fungus decreased. The composition of R. commune cell wall was also uncovered during this research. Techniques such as HPLC and FTIR eluded the composition of monosaccharides and polysaccharides respectively. In addition the structure of R. commune cell wall was observed by microscopy, namely TEM. This project revealed some much needed information on the R. commune cell wall and the relation of its components to fitness and virulence during infection of barley.

Thesis (M. Ph.)--Chinese University of Hong Kong. / Bibliography: leaves 90-110.

Digitized by Kansas Correctional Industries

Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of glycolipids that possess significant immune modulating properties. To provide discrete synthetic compounds for biological assay, this thesis describes the syntheses of three PIM molecules, namely dipalmitoyl PIM2 (12), PIM4 (84), and PIM6 (108), and two PIM2 analogues designed for increased stability, PIM2ME (147) and PIM2MA (148). The synthesis of all of these molecules involved mannosylation of 1-O-allyl-3,4,5-tri-O-benzyl-D-myo-inositol (22), which was prepared from methyl α-D-glucopyranoside in 8% yield over 8 steps, using a Ferrier reaction strategy. A common intermediate, 3,4,5-tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-D-myo-inositol (9), was used for the syntheses of 12, 147, and 148. This compound was prepared by bis-mannosylation of the C-1 and C-6 hydroxyl groups of 22 with 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl trichloroacetimidate (63) to give, after protecting group manipulations, the α,α-pseudo-trisaccharide 9 in 37% over 4 steps. The selectivity of the desired α,α-product was found to be increased by the selection of Et₂O as the solvent for the glycosylation reaction. The C-1 hydroxyl group of 9 was coupled to benzyl (1,2-di-O-palmitoyl-sn-glycero)-diisopropylphosphoramidite (28) using 1H-tetrazole. Global debenzylation of the resulting product gave PIM2 (12) in 23% yield over 6 steps from 22. In a similar fashion 9 was coupled to 1-O-hexadeconyl-2-O-hexadecyl-sn-glycero-3-O-benzyl-(N,N-diisopropyl)-phosphoramidite (156), and subsequent deprotection gave PIM2ME (147) in 30% yield over 2 steps from 9. Coupling of 9 with 2-deoxy-1-O-hexadeconyl-2-O-hexadeconylamino-sn-glycero-3-O-benzyl-(N,N-diisopropyl)-phosphoramidite (172) and subsequent deprotection gave PIM2MA (148) in 47% yield over 2 steps from 9. A modified approach was required for the syntheses of PIM4 (84) and PIM6 (108). A selective glycosylation of the C-6 hydroxyl of 22 with an orthogonally protected mannose donor would allow extension of the manno-oligosaccharide in a 2+3 or 4+3 glycosylation strategy required to build the pseudo-pentasaccharide or pseudo-heptasaccharide core of 84 or 108 respectively. Sequential mannosylation of 22, firstly at the more reactive C-6 hydroxyl, with 2-O-acetyl-3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-α-D-mannopyranosyl trichloroacetimidate (85), was followed by mannosylation at the C-2 hydroxyl with 63. Removal of the silyl protecting group followed by a 2+3 coupling with the dimannoside donor, 2-O-acetyl-6-O-(2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-3,4-di-O-benzyl-α-D-mannopyranosyl trichloroacetimidate (95), gave a pseudo-pentasaccharide intermediate. Protecting group manipulations followed by coupling of the of the C-1 hydroxyl group of the inositol ring to phosphoramidite 28, and a global debenzylation, gave PIM4 (84) in 6% yield over 9 steps from 22. During the synthesis of PIM6 (108), thioglycosylation chemistry was explored and found to be comparable to reactions with trichloroacetimidate donors. Similar methodology was used for the synthesis of PIM6 (108) as had previously been carried out for the synthesis of PIM4 (84). Mannosylation at the more reactive C-6 hydroxyl of 22 with either phenyl 2-O-benzoyl-3,4-di-O-benzyl-6-O-triisopropylsilyl-1-thio-α-D-mannopyranoside (112) or 2-O-benzoyl-3,4-di-O-benzyl-6-O-triisopropylsilyl-α-D-mannopyranosyl trichloroacetimidate (113), was followed by mannosylation at the C-2 hydroxyl with 63. Removal of the silyl group followed by a 4+3 coupling with either of the tetramannoside donors, phenyl (2-O-benzoyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]2)-(3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]2)-(3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]6)-2-O-benzoyl-3,4-di-O-benzyl-1-thio-α-D-mannopyranoside (109) or (2-O-benzoyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]2)- (3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]2)-(3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1[to]6)-2-O-benzoyl-3,4-di-O-benzyl-α-D-marmopyranosyl trichloroacetimidate (131) gave a gave a pseudo-heptasaccharide intermediate. Protecting group manipulations followed by coupling of the of the C-1 hydroxyl group of the inositol ring to phosphoramidite 28, and a global debenzylation, gave PIM6 (108) in 9% yield over 9 steps from 22. To aid characterisation of 108, a sample was deacylated to afford dPIM6 (144) which gave the same spectral data as a sample from a natural source. The compounds PIM2 (12), PIM4 (84), PIM2ME (147), and PIM2MA (148) were assayed for adjuvant activity and were found to have comparable activity to fractions isolated from natural sources. The analogue PIM2ME (147) gave the best results and is currently undergoing further development.

No description available.

No description available.

No description available.

Typescript. / Bibliography: leaves [150]-173. / xiii, 173 l illus., table