Bibliography: leaves 239-259. / 259 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Concentrations of minimal medium components and fermentation parameters were optimized for high level constituitive expression of recombinant methionyl porcine growth hormone in E. coli. Improved yields were obtained when the magnesium concentration was reduced, and the ammonium chloride concentration increased. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1995

Bibliography: leaves 239-259. / 259 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Concentrations of minimal medium components and fermentation parameters were optimized for high level constituitive expression of recombinant methionyl porcine growth hormone in E. coli. Improved yields were obtained when the magnesium concentration was reduced, and the ammonium chloride concentration increased. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1995

Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Science, Discipline of Biochemistry, 2004. / "June, 2004" Includes bibliographical references (leaves 137-156).

Picobirnavirus is an unclassified dsRNA virus, which is associated with viral gastroenteritis in humans and animals. Picobirnavirus dsRNA has been detected in many cases when diagnostic PAGE screening for rotavirus dsRNA is performed. During this routine diagnosis, picobirnavirus dsRNA has been detected in the faeces of patients with and without viral gastroenteritis. Despite the common occurrence of picobirnavirus infection in humans and animals, its direct involvement in causing gastroenteritis has not been established. No molecular studies have been done on picobirnavirus except sequencing and epidemiology studies. Like all RNA viruses, picobirnavirus encodes a RNA-dependent RNA polymerase. The picobirnavirus RNA-dependent RNA polymerase has only been identified on the basis of its amino acid sequence. The catalytic activity of the polymerase has not been studied to date. In this study, a porcine picobirnavirus was studied at a molecular level to establish the activity of the protein encoded by segment 2 of its genome. To determine the identity of this putative picobirnavirus RNA-dependent RNA polymerase, its open reading frame (ORF) was successfully amplified by PCR, cloned and sequenced. Subsequently the ORF was successfully sub-cloned into baculovirus and bacterial expression vectors. The protein encoded by picobirnavirus segment 2 was successfully expressed as a recombinant protein in a soluble form in both baculovirus and bacterial expression systems. In the baculovirus system, two recombinant baculoviruses were constructed. One recombinant baculovirus expressing a histidine tagged protein and another one expressing an untagged protein. In bacterial expression systems, a recombinant protein fused to a Glutathione-S-Transferase (GST) tag at the N terminal end was expressed. The GST tag allowed easy purification of the expressed GST fusion protein by affinity chromatography on immobilized glutathione. Subsequently the GST tag could be removed from the purified recombinant protein by proteolysis with thrombin. Both tagged and untagged putative picobirnavirus RNA-dependent RNA polymerase from the bacterial expression system were shown to have an affinity for heparin. This implies that the protein might have an affinity for nucleic acids. Picobirnavirus genome segment 2 ssRNA was generated from full length picobirnavirus genome segment 2 cDNA by in vitro transcription. The recombinant E.coli expressed proteins (tagged and untagged) was tested for ssRNA binding and RNA replicase activity. No RNA binding or replicase activity was observed with either tagged or untagged recombinant protein. This study reports the first evidence other than conserved polymerase motifs, that the protein encoded by segment 2 of picobirnavirus is most likely a RNA-dependent RNA polymerase. Copyright / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

Doctor of Philosophy / Department of Animal Sciences and Industry / John M. Gonzalez / This series of studies focuses on the impact of intra-litter variation on fetal myogenesis, and the ability of porcine progenitor cells to respond to growth promoting stimuli. In study 1, the smallest (SM), median (ME), and largest (LG) male fetuses from each litter were selected for muscle morphometric analysis from gilts at d-60 ± 2 and 95 ± 2 of gestation. On d-60 and 95 of gestation LG fetuses had greater whole muscle cross-sectional area (CSA) than ME and SM fetuses, and ME fetuses had greater whole muscle CSA than SM fetuses. Indicating that SM and ME fetuses are on a delayed trajectory for myogenesis compared to LG fetuses. At d-60 the advanced trajectory of LG compared to ME fetuses was due to increased development of secondary muscle fibers; whereas, the advanced myogenic development of LG and ME fetuses compared to SM fetuses was due to the presence of fewer primary and secondary muscle fibers. At d-95 of gestation the advanced myogenic development of LG and ME was due to increased hypertrophy of secondary muscle fibers. For study 2, porcine fetal myoblasts (PFM) were isolated from SM, ME, and LG fetuses from d-60 ± 2 of gestation fetuses and for study 3, porcine satellite cells (PSC) were isolated from the piglet nearest the average body weight of the litter. Both myogenic cell types were utilized to evaluate effects of porcine plasma on proliferation, differentiation, and indications of protein synthesis. For the proliferation assay, cells were exposed to one of three treatments: high serum which consisted high-glucose Dulbecco's Modified Eagle Medium supplemented with 10% (vol/vol) fetal bovine serum, 2% (vol/vol) porcine serum, 100 U penicillan/mL, 100 µg of strepmycin/mL, and 20 µg of gentamicin/mL (HS), low serum which consisted of HS without 10% FBS (LS), and LS supplemented with 10% (wt/vol) porcine plasma (PP). Treatments for the differentiation and protein synthesis assays consisted of either HS or LS media that either contained porcine plasma at 10% (wt/vol; PPP) or 0% (wt/vol; PPN). The HS-PFM had a greater proliferation rate compared to the LS and PP-PFM, and PP-PFM had a greater proliferation rate compared to LS-PFM. The LG fetuses’ PFM had a reduced proliferation rate compared to SM and ME fetuses’ PFM, which were similar. The PPP-PFM had a decreased myotube diameter compared to PPN-PFM. Small fetuses’ PFM had a greater myotube diameter compared to ME and LG fetuses’ PFM, and ME fetuses’ PFM had a greater myotube diameter compared to LG fetuses’ PFM. The proliferation rate of PP-PSC was decreased compared to the HS- and LS-PSC, and HS-PSC had a greater proliferation rate compared to LS-PSC. The PPP-PSC had greater differentiation capacity and myotube diameter than PPN-PSC. In conjunction these results indicate divergent myogenic development among different fetal sizes within a litter and suggest that porcine plasma supplementation stimulates myogenic progenitor cell activity in an age specific manner.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a global disease which takes a significant toll on the pork industry and the welfare of pigs. The causative agent – PRRS virus (PRRSV) – is a single-stranded, positive-sense RNA virus of the Nidovirales order. In the process of replication, PRRSV induces the rearrangement of cellular membranes to form double-membrane vesicles (DMVs). These structures are thought to have a role in a) concentrating viral structures to increase their chances of interacting with one another, and b) preventing elements of the cellular immune response from detecting viral structures. Previous work has suggested that the DMV originates from the autophagy pathway – a highly-conserved mechanism for cells to recycle extraneous organelles and proteins during times of stress. Other work suggests that the DMV may be a co-opted EDEMosome – a recently-discovered vesicle which is involved in regulating the level of endoplasmic reticulum-associated degradation (ERAD). This thesis explores these possibilities – using immunofluorescent imaging as well as examining the proteomic and ribonucleic acid composition of the DMV as isolated by flow cytometry or separated from other organelles by density gradient – calling both candidate pathways into question and suggesting other candidate structures such as exosomal vesicles.

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"June, 2004" / Includes bibliographical references (leaves 137-156) / xvi, 156 leaves : ill. (some col.), plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Science, Discipline of Biochemistry, 2004

Reproduction de : Thèse d'exercice : Médecine vétérinaire : Toulouse 3 : 2007. / Titre provenant de l'écran titre. Bibliogr. p. 117-121.