Thesis (Ph. D.)--Illinois State University, 1992. / Title from title page screen, viewed January 27, 2006. Dissertation Committee: H. Tak Cheung (chair), Anthony Otsuka, Alan Katz, Brian Wilkinson, David Weber. Includes bibliographical references (leaves 108-120) and abstract. Also available in print.

Thesis (Ph. D.)--Illinois State University, 1989. / Title from title page screen, viewed October 12, 2005. Dissertation Committee: H. Tak Cheung (chair), David W. Borst, Herman E. Brockman, Arlan G. Richardson, Brian J. Wilkinson. Includes bibliographical references (leaves 145-156) and abstract. Also available in print.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 1996. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 1996. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

All developing embryos contain an extracellular matrix (ECM) consisting of proteins, glycoproteins, and proteoglycans. These components are important for morphogenetic processes such as cell migration, cell differentiation and cell death. The ECM of the starfish, Pisaster ochraceus, consists of three major components: A hyaline layer which coats the external surface of the embryo; a basal lamina which lines the basal surfaces of the epithelia; and a blastocoelic component which fills the embryonic cavity or blastocoel. Observations of chemically fixed asteroid embryos have revealed the hyaline layer to contain five sub-layers of fibrous strands encrusted with amorphous material. Strands of a similar nature form a meshwork within the fluid-filled blastocoel. Recent studies of the living embryo, however, have suggested that the ECM within the blastocoel of echinoderms, including the asteroid, is a gel-like substance and not a fluid with extracellular fibres. Since artefacts imposed by chemicals such as aldehydes and osmium are well documented, a method of preservation, which does not involve the use of these chemicals, may resolve the apparent conflict over the nature of the ECM of the asteroid embryo. Freeze substitution, an expensive cryofixation technique which has proven successful in fixing vertebrate tissue, does not require the use of aldehydes and osmium. The initial objective of this study was to devise an inexpensive, easily employable freeze substitution technique which would allow good preservation of cellular and extracellular elements of the embryonic starfish, Pisaster ochraceus. A plunge freezing apparatus was constructed which consisted of a Dewer flask filled with liquid nitrogen, a small cup was filled with cryogen and inserted into the nitrogen, and a motor which constantly stirred the cryogen. Embryos were isolated on copper freeze-fracture grids and plunged into the cryogen. After considering four different cryogens and four separate cryoprotectants, cryoprotecting asteroid embryos with propylene glycol and plunging them into supercooled propane was found to provide optimal preservation. Frozen embryos were freeze substituted in anhydrous ethanol at -90 °C, osmicated, and embedded for ultrastructural and histochemical analysis. Following freeze substitution, the blastocoel appears to contain a gel-like substance, rich in sulfated GAG's, with extracellular fibres and not a fluid with fibres. In addition, the hyaline layer was found to consist of at least six sub-layers of greater thickness than was seen in chemically fixed embryos. Histochemical studies demonstrated that both sulfated and unsulfated GAG's were present in these layers. The morphological differences among the sub-layers suggest that some sub-layers may have unique functions while others may have functions shared by other sub-layers. Freeze substitution also revealed the presence of microvillus associated bodies, structures which may represent major attachment points of the hyaline layer to the epithelium. Although the fixation of asteroid embryos by freeze substitution is a lengthy process, taking four to five days, the resulting preservation, particular!ly of the ECM components, justifies its use over chemical fixations. Material preserved by freeze substitution can be used for histochemical studies and, since aldehydes and heavy metals are not necessary for successful preservation, may also prove useful for immunocytochemical studies. / Medicine, Faculty of / Graduate

abstract / Biochemistry / Doctoral / Doctor of Philosophy

The ectodomain of influenza matrix protein 2 (M2e) may be an ideal candidate in the development of influenza universal vaccine due to its highly conserved property among different subtypes/strains of influenza virus. M2e based vaccines have been extensively studied and potent cross-subtype/strain protections have been reported. However, more and more M2e mutants of influenza virus have been identified in recent years. It is still unclear whether M2e based vaccines are effective against these M2e mutants of influenza virus. This study first evaluated cross-protection of an M2e tetrameric peptide vaccine based on H5N1 virus strain A/Vietnam/1194/04 (VN/1194-M2e) against lethal challenges of M2e mutants of H5N1 virus strain A/Hong Kong/156/97 (HK/156) and a novel H7N9 virus, because there are 3 or 5 amino acid differences between VN/1194-M2e and HK/156-M2e or VN/1194-M2e and H7N9-M2e. The results showed that the vaccination of VN/1194-M2e did not induce high level of cross-reactive antibodies against HK/156-M2e and just provided poor cross-protection against lethal challenge of HK/156 virus. In contrast, VN/1194-M2e vaccination induced high level of cross-reactive antibodies against H7N9-M2e. Consistently, the vaccination provided good cross-protection against lethal challenge of H7N9 virus. These results strongly suggested that some mutations in M2e, such as mutations at positions 10, 14 and 16 which found in HK/156 M2e, might affect the M2e vaccine efficacy, but some others, such as five mutations found in H7N9-M2e, might not be critical for the M2e immunogenicity. This study then investigated the relationship between the M2e immunogenicity and amino acid mutations of the M2e. Beside VN/1194-M2e (P0), we synthesized additional 10 M2e mutant peptides which contain different single or multiple mutations. The 3D structures of these M2e peptides were predicted and analyzed. The prediction results showed that group 1 peptides (P0, P10, P14, P16, P18, P20 and P18-20) exhibited either irregular structures or loose hairpin structures which might associate with well exposure of antigenic epitope, whereas group 2 peptides (P10-14, P10-16, P14-16 and P10-14-16) formed tight hairpin structures in which antigenic epitope might bury inside their own secondary structure. Vaccination efficacies of these M2e peptides were evaluated in mice for antibody responses and cross-protection against lethal challenge of VN/1194 and HK/156 viruses. Our results showed that vaccinations of group 1 peptides induced high levels of cross-reactive antibodies against VN/1194-M2e and good cross-protection against lethal challenge of VN/1194 virus. However, vaccinations of group 2 peptides vaccinations induced significantly lower VN/1194-M2e antibody responses and poor cross-protection against lethal challenge of VN/119 virus. Furthermore, both group 1 and group 2 peptides could just induce low levels of cross-reactive antibodies against HK/156-M2e and poor protection against lethal challenge of HK/156 virus. Although H5N1-M2e tetrameric peptide has been previously shown to protect mice from lethal challenges by different subtypes/strains of influenza virus, this study has shown that certain amino acid variations in M2e could weaken M2e immunogenicity but some others might not. The different secondary structures of M2es may probably associate with their immunogenicity. Our findings have provided valuable information for the development of M2e based universal vaccines. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The studies submitted for this thesis encompass two broad areas of interest. The first is the role of extracellular matrix during folliculogenesis, including ovulation and corpus luteum formation. The observations made were extended in a second series of studies investigating matrix and other parameters of morphologically distinct follicles. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1274421 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2007