The initial aim of this project was to characterise the integrative recombination mechanism of bacteriophage ÖAR29 , to provide a better understanding for development of the shuttle plasmid pBA as a site-specific Bacteroides integration vector. RT-PCR showed that the previously identified ÖAR29 recombination genes, integrase (Int) and excisionase (Xis), were transcribed from pBA in E. coli SCS110, B. thetaiotaomicron AR29 and B. uniformis AR20. In silico derived amino acid sequences from both genes showed only very low levels of similarity to other known Int and Xis in GenBank. To improve understanding of the phage recombination system, the ÖAR29 genome was sequenced. This revealed a 35,558 bp double-stranded DNA genome with GC content of 39.11%. Bioinformatic analysis identified 53 open reading frames (>30 codons) and gene promoters and terminators that allowed the genome arrangement to be compared with other phages. Comparison of deduced gene products with proteins from other phages identified 6 reading frames, allowed tentative identification of 7 others, but left 40 ORFs unidentified. Those with strong homology to known genes were: large terminase subunit (44.66 kDa), dnaC (27.94 kDa), helix-turn-helix (HTH) transcription regulator (14.69 kDa), cI repressor (26.48 kDa), amidase (18.42kDa) and a novel integrase (54.22 kDa). The integrase gene is located 162 base-pairs downstream of the phage attachment (attP) core site, rather than the previously suggested location upstream of the integration site. The ÖAR29 attP was shown to include a 16-bp att core region, 117 bp upstream of the previously suggested location. Integration of ÖAR29 was found to occur at the 3’end of an arg-tRNA gene on the AR29 genome (attB). Imperfect direct repeats with a consensus sequence (ANGTTGTGCAA) were found surrounding the attP core. A review of pBA sequence showed that only the 5’ end (435 bp) of the newly identified Int gene was cloned in pBA. Despite this, PCR analysis revealed integration of pBA into the AR29 genome. Serial subculturing of pBA transformed AR29 was able to cure AR29 of the ÖAR29 prophage, providing an improved host for integrative plasmids, and for detailed studies of AR29 physiology and ÖAR29 life cycles.

Thesis (Ph.D.)--Murdoch University, 2005. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 211-230.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Jaquelin P. Dudley. Vita. Includes bibliographical references. Also available from UMI.

Thesis supervisor: Kevin G. Rice. Includes bibliographical references (p. 170-181).

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-158). Also available in print.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [154]-164).

Thesis supervisor: Wendy J. Maury. Includes bibliographic references (p. 56-66).

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references.

Non-viral vectors have been used to deliver therapeutic genes to treat different diseases. There are a variety of non-viral vectors such as liposomes, cationic polymers and peptides. Among all, pH responsive peptides showed excellent DNA transfection efficiency in many types of cell. These peptides are capable of changing their structural conformation as pH decreases, adopting a disordered structure which can destabilize endosomal membrane and therefore enhancing the release of DNA from endosomes into cytosol. Traditional pH responsive histidine-rich peptides showed good DNA transfection efficiency and low toxicity to the cells when compared with other non-viral vectors. However, their low pKa value restricted these peptides to be protonated only at late endosomal stage, in which DNA is extremely susceptible to endosomal degradation. This hindered the DNA to be released to the cytosol efficiently and therefore reduced DNA transfection efficiency. In response to this, it is of great interest to probe into the insertion of either 2,3-diaminopropionic acid (Dap) or methylated-2,3-diaminopropionic acid Dap(Me) to the peptide as alternative pH sensitive components. The pKa values for both Dap and Dap(Me) peptides are higher than that of histidine. It is anticipated that the higher pKa value, the protonation of peptide could be happened at an earlier stage of endosomal maturation. Such protonation of peptide destabilizes the endosome membrane rapidly, causing the release of DNA to the cytosol effectively and hence improving DNA transfection efficiency. In this experiment, LADap(Me)4-L1 peptide was the optimal candidate within the series. It showed good DNA transfection efficiency and cell viability in A549 cells among all Dap and Dap(Me) peptides. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences