Thesis (Ph. D.)--Oregon State University, 1991. / Includes mounted photographs. Typescript (photocopy). Includes bibliographical references (leaves 112-119). Also available on the World Wide Web.

Thesis (Ph. D.)--Illinois State University, 1993. / Title from title page screen, viewed February 13, 2006. Dissertation Committee: Brian J. Wilkinson, Radheshyam K. Jayaswal (co-chairs), Anthony E. Liberta, Herman E. Brockman, Hou Tak Cheung. Includes bibliographical references (leaves 115-124) and abstract. Also available in print.

Thesis (Ph. D.)--Illinois State University, 1995. / Title from title page screen, viewed May 2, 2006. Dissertation Committee: Radheshyam K. Jayaswal (chair), Brian J. Wilkinson, Anthony J. Otsuka, Herman E. Brockman, Hou T. Cheung. Includes bibliographical references (leaves 131-144) and abstract. Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (M.Sc.)--York University, 2005. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 56-62). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss &rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR11827

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Title on signature page has the Greek symbol for tau. Typescript. Vita. Includes bibliographical references (leaves 167-196). Also available on the Internet.

Naegleria fowleri is a free-living amoeba that causes primary amoebic meningoencephalitis (PAM). In the United States, there are between 0-8 cases of PAM per year, with approximately 98% of cases resulting in death. High case fatality and limited treatment options highlight the need for better understanding of this organism in terms of its biology and pathogenicity. Transfection is a useful tool that allows for the study of gene function, but at present no transfection systems have been established for N. fowleri. This study attempts to establish a transfection system for N. fowleri using the piggyBac vector, with the hope of eventually using the piggyBac transposon system to identify novel genes related to pathogenicity in N. fowleri. To accomplish this, 5’ and 3’ regulatory regions for genes in the N. fowleri genome were amplified and inserted into a piggyBac vector with a GFP reporter gene via molecular cloning, and vectors introduced to the amoeba via electroporation. Although no GFP was visualized after transfection, there are several routes for optimization of the transfection system that could be explored. Development of a transfection system could allow for the study of pathogenicity in vivo, by either utilizing the transposon system of piggyBac or the expression of reporter genes for visualization of amoeba during the course of infection. Further elucidating N. fowleri pathogenicity factors could reveal new drug targets, give new information about the organism’s biology, and help better define an effective treatment regimen to combat PAM.

In addition to substrate-associated cellulases, Cellulomonas fimi secretes endoglucanases ( endo-1, 4-β-D-glucan glucanohydrolases, EC 3.2.1.4. ) which are recovered from the cellulose-free culture supernatant of cells grown on microcrystalline cellulose. Two such enzymes, C3.1 and C3.2 with Mrs of 130'000 and 120'000, respectively, were purified to homogeneity. The two endoglucanases were shown to share the same N-terminal amino acid sequence and to hydrolyze carboxymethylcellulose ( CMC ) with similar efficiencies ( 236u/mg protein for C3.1 and 367u/mg protein for C3.2 ). The recombinant lambda vector L47.1-169 was identified from a C.fimi DNA-lambda library on the basis of hybridization with C3.1/2-specific oligonucleotide probes. The subclone pTZ18R-8 only moderately expressed CMCase activity. The 5'-terminus of cenC ( the gene coding for C3.1/2 ) was localized in the insert by Southern transfer experiments and nucleotide sequence analysis. Results from total C.fimi RNA-DNA hybrid protection analyses defined the boundaries of cenC in pTZ18R-8 and led to the tentative identification of -10 and -35 promoter sequences. To improve the expression of cenC, its entire coding sequence, except for the start codon GTG, was fused in frame to the ATG codon of a synthetic ribosomal binding site ( PTIS ) and placed under the transcriptional control of the lac p/o system. Induction of the resulting clone ( JM101[pTZP-cenC] ) led to impaired growth in liquid cultures because overproduction of CenC inhibited cell division'" and eventually led to cell death. Analysis of cell fractions by SDS-PAGE revealed a dominant ( >10% of total cell extract proteins ), clone-specific protein with a Mr of approximately 140'000 which was found exclusively in the cytoplasmic fraction. Conversely, 60% of the total CMC-hydrolyzing activity was localized in the periplasmic fraction indicating that the export of CenC is required for maximal expression of endoglucanase activity. Isolation of the cellulolytic activities from an osmotic shockate led to the purification to homogeneity of two recombinant cellulases, CenC1 and CenC2, with Mr of 130'000 and 120'000, respectively. Both enzymes hydrolyzed CMC with similar efficiencies ( 278u/mg protein for CenC1 and 390u/mg protein for CenC2 ). In addition, amino acid sequence analyses showed the two enzymes to have the same N-termini as the native enzymes and proved furthermore that the CenC leader peptide was functional in Escherichia coli. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The polyhomeotic (ph) locus of Drosophila melanogaster has been characterized genetically. Early studies showed that ph is a member of the Polycomb (Pc) group. These genes have similar phenotypes and are required for normal segment determination. Recent analyses of amorphic ph mutations show that the ph locus is complex, has a strong maternal effect and plays a role in cuticular development. To test the function of ph at the molecular level, the cloning of the ph locus was undertaken. One strain had been shown to contain a P element insertion near ph. A genomic library was prepared from this strain and a recombinant phage containing this P element insertion was isolated by transposon tagging. The DNA flanking the insertion was used as a starting point for a chromosomal walk. A series of overlapping phage spanning 170 kilobases was isolated. Southern blot analysis was used to determine the locations of important deficiency breakpoints within the region covered by the walk. A distance of approximately 35 kb was shown to separate the two deficiency breakpoints which include ph. This interval was found to contain rearrangements in four of the seven ph alleles which were examined by Southern blot analysis. The interval also contains a repeated sequence. The relationship between the genetic and molecular structure of ph is discussed. / Science, Faculty of / Zoology, Department of / Graduate

M.Sc. / As part of an investigation into differential gene expression in response to abiotic and chemical inducers of acquired resistance in tobacco, a PCR fragment of 660bp was repeatedly found in RNA preparations from treated cell suspensions by differential display analysis. The fragment (D1B) was isolated, purified, cloned and sequenced. The nucleotide sequence of the fragment was compared with sequences in the BLAST sequence database and was found to be homologous to the mitochondrial orfx genes from Arabidopsis thaliana, Beta vulgaris, Oenothera berteriana, Oryza sativa and Marchantia polymorpha. In order to obtain the full sequence of the gene specific primers were designed using the Arabidopsis sequence as template. The primers were designed to complete the 5’-end of the gene and were designed to overlap the D1B fragment previously found. A fragment (C3Y) of 460bp was isolated, purified, cloned and sequenced. The complete sequence (D1B and C3Y combined) was 851bp long and showed 96% homology with the Arabidopsis orfx gene on the nucleotide level and 87% homology on the translated amino acid level. The sequence was submitted to the Basic Local Alignment Search Tool (BLAST) database as accession gi: 24209907. In plant genomes, the orfx gene is closely linked to important structural genes such as the nad subunits of complex I (NADH: ubiquinone oxidoreductase). Orfx codes for a hypothetical protein that shows homology to the mttB (membrane targeting and translocation) gene found in E. coli. In bacteria the gene is essential because if deleted, the organism was no longer viable. Functional analysis of the bacterial gene revealed a novel pathway specific for membrane targeting and secretion of cofactor containing proteins, such as iron-sulphur (Fe-S) clusters, of which the mttB gene encodes one subunit. It is thought that a similar pathway might be responsible for the correct localisation and assembly of such Fe-S containing protein complexes in the inner mitochondrial membrane of higher plants. The differential display result may be indicative of a general up-regulation of mitochondrial gene expression in response to the triggering of plant defences or a possible specific effect on the expression of the orfx gene. A hypothesis was formulated that chemical inducers of plant defences affect the mitochondria of treated plant cells to result in increased production of reactive oxygen intermediates (ROI), similar to the oxidative microbursts proposed to be involved in systemic required resistance. Using a dichlorodihidrofluorescein (H2DCFDA) assay, it was found that salicylic acid (SA), benzo (1,2,3) thiadiazole-7-carbothioc acid S-methyl ester (BTH) and isonitrosoacetophenone (INAP) increased ROI production within cells in a dose dependant manner. The biochemical basis of this effect could possibly be related to the inhibition of the NADH:ubiquinone oxidoreductase activity of complex I of the mitochondrial electron transport chain by SA, BTH and INAP. / Prof. I.A. Dubery