A major goal of our laboratory is to confer resistence specifically to the Odontoglossum ringspot virus [ORSV; sometimes referred to as tobacco mosaic virus strain O (TMV-O)] in orchids. The chosen strategy may also provide cross-protection to other pathogens. The experimental design for the entire project is presented here along with the results obtained in several preliminary experiments performed in this research. Our approach involved RT-PCR amplification of the viral coat protein gene with gene-specific primers and digestion of the cDNAs into oligonucleotides. These fragments were cloned into the selectable vector pG35barB (which confers herbicide resistence) in both sense and antisense orientations. The cloned DNA was coated with tungsten beads and shot into orchid callus tissue using a makeshift biolistic gun. Tranformant callus cells were selected for by herbicide resistance. Unfortunately the potential transformants became contaminated with fungus and could nto be analyzed to determine which oligonucleotide was received and the effect each oligonucleotide had on pathogen resistance. Due to the uncertainty of the relatedness between ORSV and TMV-O, we also sequenced the coat protein gene of TMV-O and compared the amino acid sequence with those of several strains of ORSV: the Japanese strain had the highest percent amino acid similarity (99.4%), the Type strain the second highest (98.7%), and the Korean strain the lowest (96.9%). It was concluded that TMV-O is most likely one strain of ORSV, the Japanese strain. / Department of Biology

A photolabile terpolymer containing 6-nitroveratyloxycarbonyl (NVOC) protected amine, epoxy and trimethoxysilyl functionality in 1:3:2 monomer ratio was synthesized to template glass surfaces for specific site directed coupling of non-probe oligomers and probe oligonucleotides. Non-probe oligomers were introduced to the surface to control the environment of the probes by reducing probe-to-probe and probe-to-surface interactions. The trimethoxysilyl group served as the anchoring site for the terpolymer to be covalently bound to glass and silicon wafers. Amine terminated non-probe oligomers were coupled to the epoxy sites and thiolated 19-mer SMN1 probes were directed to the deprotected amine sites via the heterobifunctional linker, sulfosuccinimidyl-4-[maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC). Characterization of the terpolymer was done using 1H NMR, 13C NMR, MALDI-ToF and elemental analysis. NVOC deprotection was monitored by UV absorption, and surface characterization of the bound terpolymer on silicon wafers was investigated with XPS, ToF-SIMS, ellipsometry and static contact angle. Neutral polyethylene glycol (PEG), negatively charged methacrylic acid (MAA) oligomer and dC20 oligonucleotides were used as non-probe oligomers. The probe density on the surface was estimated to be 2.2 ± 0.3 x 10^12 molecules/cm2 and the presence of the oligomers on the surface did not significantly affect probe immobilization efficiency. The mixed films were functional for target hybridization and its selectivity towards partially-mismatched targets was investigated at different solution pH, ionic strength and temperature. It was demonstrated that pH can be tuned to ameliorate non-specific adsorption and ionic strength governed the selectivity of the surfaces. Improved selectivity was achieved at high salt concentration (1 M NaCl) on PEG and dC20 mixed films at room temperature. The MAA surface did not show significant improvements in selectivity. This indicated that charge of the oligomers does not dominate control of selectivity. The results suggested that the terpolymer construct played a role in depression of the melting temperature of the hybridized duplex to within 5 to 10 oC of room temperature. With the melting temperature shifted closer to room temperature, it is possible to improve selectivity for room temperature detections of single nucleotide polymorphism.

Bibliography: leaves 189-205. / xi, 205 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this PhD project is to screen feral goat rumen fluid for the presence of new organisms that may play a role in the detoxification of tannins and to investigate their mechanisms of action. An enrichment experiment is conducted to screen rumen fluid for anaerobic bacteria capable of growing in the presence of high levels of "Acacia" condensed tannin. Four morphologically-distinct bacteria are isolated, confirming that resistance is a property shared by more than one organism. One isolate is chosen at random for further characterisation and is identified as a strain of "Selenomonas ruminantium" subspecies "ruminantium". It is arbitrarily designated strain K2. "Selenomonas ruminantium" K2 is shown to be not only tannin-resistant but also able to grow on tannic acid. It is proposed that this bacterium obtained energy for growth from tannic acid. The thesis examines the molecular mechanisms controlling tannin resistance or tannin degradation in rumen microorganisms. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1997

Bibliography: leaves 189-205. / xi, 205 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this PhD project is to screen feral goat rumen fluid for the presence of new organisms that may play a role in the detoxification of tannins and to investigate their mechanisms of action. An enrichment experiment is conducted to screen rumen fluid for anaerobic bacteria capable of growing in the presence of high levels of "Acacia" condensed tannin. Four morphologically-distinct bacteria are isolated, confirming that resistance is a property shared by more than one organism. One isolate is chosen at random for further characterisation and is identified as a strain of "Selenomonas ruminantium" subspecies "ruminantium". It is arbitrarily designated strain K2. "Selenomonas ruminantium" K2 is shown to be not only tannin-resistant but also able to grow on tannic acid. It is proposed that this bacterium obtained energy for growth from tannic acid. The thesis examines the molecular mechanisms controlling tannin resistance or tannin degradation in rumen microorganisms. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1997

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 71-80).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 82-88).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 120-127).

Theses (Ph. D.)--Marshall University, 2004. / Title from document title page. Includes abstract. Document formatted into pages: contains xiv, 192 p. including illustrations. Bibliography: p. 157-192.