Melbourne Dental School - Theses
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ItemCharacterization of casein phosphopeptides( 1995)Casein phosphopeptides (CPP), containing the cluster sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, have been shown to stabilize amorphous calcium phosphate at neutral and alkaline pH and to be anticariogenic in various in vitro, animal and human experiments. Anticariogenic casein phosphopeptides (ACPP) therefore have commercial potential as toothpaste, mouthwash and food additives for the prevention of dental caries. The aim of this project was to comprehensively characterize the CPP produced under industrially-relevant conditions using the commercially available enzymes Novo trypsin PTN 3.0 S, Novo Alcalase® 2.4L and Pancreatin 4NF. To facilitate studies on the identification and characterization of CPP a simple and efficient purification procedure involving selective precipitation of Ca2+/ethanol-induced aggregates of the phosphopeptides from enzymic digests was developed. The individual peptides of the precipitates were purified using anion exchange FPLC and reversed-phase HPLC and then identified by solid-phase sequence analysis and amino acid composition analysis after vapour-phase hydrolysis. Prior to sequence analysis the phosphopeptides were covalently coupled to arylamine membranes and the phosphoseryl residues converted to S-ethylcysteinyl residues by calcium-ion-catalysed β-elimination in the presence of ethanethiol. Alternatively, in the event of low coupling efficiency to arylamine membranes, phosphoseryl residues were converted to S-ethylcysteinyl or S-propylcysteinyl residues and sequenced from polybrene-treated glass fibre discs. A method for the separation and identification of CPP using high performance capillary electrophoresis (HPCE) was also developed. The ability of HPCE to rapidly resolve phosphopeptides exhibiting varying degrees of phosphorylation, truncation and deamidation make it ideal for the product quality control of CPP. HPCE was also used to develop relationships between absolute electrophoretic mobility (µ) and peptide charge and size for CPP containing 2-5 phosphoseryl residues, with µ found to be proportional to q/M2/3 where q is the net negative charge and M is molecular mass of the peptide. The results showed that CPP can be produced using Novo trypsin and pancreatin with only minor modifications, relative to CPP produced using analytical-grade trypsin, such as slight truncation, deamidation and methionine oxidation. Deamidation and methionine oxidation most likely resulted from an elevated hydrolysis temperature and/or conditions employed during commercial casein (CN) production. Studies on the hydrolysis of casein at different enzyme:substrate (E/S) ratios by these enzymes suggest that for CPP production using Novo trypsin, a minimum degree of hydrolysis (DH) of 17.3% is required for maximal release of ACPP whilst for pancreatin, a DH of 19.0% is required. For pancreatin, it is recommended that this value is not exceeded so as to minimize the release of the truncated β-CN-4P(f7-24). CPP produced using alcalase were severely truncated relative to those prepared using Novo trypsin and pancreatin, resulting in the loss of residues suggested to be necessary for full anticariogenic activity. Decreasing E/S ratio slightly lowered the degree of truncation caused by alcalase, however, low E/S ratios also resulted in a reduction in CPP yield. It is likely that ACPP produced using alcalase would have much lower specific anticariogenic activity than those produced using Novo trypsin or pancreatin. In conclusion, it has been demonstrated that Novo trypsin PTN 3.0 S and Pancreatin 4NF are suitable enzymes for the production of CPP on a commercial scale and that Alcalase® 2.4L is unsuitable. For the production of CPP using Novo trypsin, a minimum DH value of 17.3% is required for maximal ACPP release whilst for pancreatin, a DH value of 19.0% is required. HPCE has been demonstrated to be an ideal method to monitor CPP quality.
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ItemAscorbic acid deficiency and Harvey-ras proto-oncogene expression in experimental oral mucosal carcinogenesis( 1996)The aims of the present project were to examine the role of L-ascorbic acid (L-AA) deficiency and expression of the proto-oncogene, Harvey-ras (H-ras), in in vivo rodent oral mucosal carcinogenesis. The inbred, mutant Osteogenic Disorder Shionogi (ODS) rats used in the present study are unable to synthesise L-AA because they lack the liver microsomal enzyme, L-gulono-y-lactone oxidase (GLO), needed in L-AA biosynthesis. Foot length measurements and random analyses of microsomal GLO, by using rabbit anti-GLO rat-antiserum, were undertaken to confirm the breeding of ODS rats. The minimal L-AA requirement for survival of ODS rats under experimental conditions were determined. Two levels of L-AA deficiency were examined in ODS rats, while outbred Wistar rats acted as controls in the carcinogenesis experiment. 0.5% m/v 4 nitroquinoline-1-oxide (4NQO) in propane-1, 2-diol was used topically to induce oral mucosal squamous cell carcinoma (SCC) in ODS (n=72) and outbred Wistar (n=48) rats with vehicle and untreated controls (n=168). Harvested palatal tissues were analysed qualitatively, by using a modified dysplasia index, and histometrically. Total RNA extracted from rat palatal SCC (n=22) were analysed for mutations in codons 12, 13, 59 and 61 of H-ras by using reverse transcription (RT)-polymerase chain reaction (PCR) and direct DNA sequencing. H-ras mRNA expression was examined in 4NQO-induced rat palatal SCC and untreated palatal samples using semi-quantitative RT-PCR. Foot length measurements differed significantly (p«0.01) between ODS and outbred Wistar rats, confirming the genotype of the former. Likewise, rat GLO was detected in the positive outbred Wistar control, but not in the six random ODS samples. ODS rats were shown to require 10 mg L-AA/day for prolonged survival under experimental conditions. Plasma L-AA levels in ODS rats were adjusted to one-eighth of one-half that of the outbred animals (48±2 µM). The carcinogenicity of 4NQO was confirmed in ODS and outbred rats, with palatal SCC detected after 20 weeks and by the 40th week, respectively. Dysplasia scores for 4NQO-treated ODS rats were significantly greater than for 4NQO-treated outbred Wistar rats at all time periods over the 24 weeks (p<0.05), but were not significantly different between ODS rats with plasma L-AA levels of 7µM and 26 µM (p=0.94). The mean palatal epithelial thickness measurements increased significantly with time in all 4NQO-treated animals (p=0.04) and were significantly different between varying levels of epithelial dysplasia (p<0.05), but the relative proportions of individual epithelial compartments were maintained. No H-ras mutation was detected in the specified codons in any 4NQ-induced palatal SCC samples compared to untreated palatal mucosal samples. It was concluded that L-AA deficiency, in the model of chemical carcinogenesis used, did not influence the process of experimental carcinogenesis. The absence of H-ras mutations at specified sites and the observed H-ras mRNA levels in 4NQO-induced rat palatal SCC suggested that the H-ras proto-oncogene was unlikely to have contributed to malignant transformation in the present study.
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ItemVertical root fracture and fracture-related properties of dentine( 1999)The prognosis of vertical root fracture (VRF) is unfavourable and endodontic procedures, especially lateral condensation, have been suggested as a cause of VRF. In addition, the mechanislTI of VRF resulting in a typical buccolinguaI fracture has not been investigated. The possibility of lateral condensation as a cause of VRF was investigated by comparing loads and strains during obturation with those at fracture. The mechanism of VRF was also investigated, in association with stress and strain distribution. Finally, selected tensile properties of dentine were examined in relation to patterns of fracture. Lateral condensation alone should not be a cause of VRF because loads and strains on the outer root surface generated during lateral condensation were much lower than at fracture in each tooth type. However, to avoid VRF, finger spreaders should be used to obturate root canals, because the results showed that loads and strains generated during lateral condensation using finger spreaders were significantly lower than when a hand spreader (D 11 T) was used. The mechanism of VRF was investigated using finite element analysis (FEA), and the results were validated by experimentally measuring outer root surface strains using strain gauges. The strain measurement results correlated well with predicted circumferential strain distribution from FEA models. FEA results showed that stress distribution was not uniform, and the highest tensile stresses were found on root canal surfaces in a buccolingual direction, concentrated in areas of greatest curvature of the root canal. The results suggested that the dentine thickness, root morphology and root canal shape, including root canal irregularities, all contributed to this pattern of non-uniform stress distribution. In addition, a root canal shape predisposing to stress concentration areas is a more influential factor than root morphology, and a severe oval root shape is more susceptible to VRF than less oval or circular root shape. The root with a combination of these factors is the most susceptible to VRF. The SEM photographs of fracture surfaces suggested that the microstructure of dentine may affect the tensile strength of dentine. Dentine was shown to behave as an anisotropic material with regard to ultimate tensile strength (UTS), with the highest UTS when the tensiIe force was perpendicular to tubule orientation. Thus, buccolingual fractures, which are the predominant pattern of VRF in all teeth, occur in a direction requiring the greatest energy to fracture. This suggested that the mechanisms which create local high tensile stress concentration or non-uniform stress distribution play a stronger role in VRF rather than dentine structure. UTS of dentine was shown to depend on the tubule orientation and location (distance from pulp cavity) of dentine. These suggested that both orientation and microstructure (hydroxyapatite and collagen fibrils) of intertubular and peri tubular dentine contributed to the anisotropy of dentine. Therefore, UTS of dentine should be described in relation to location (distance from pulp cavity) as well as tubule orientation. However, because of the complexity of dentine components and the small size of specimens, developing an improved testing system is necessary to achieve more information to understand fracture behaviour and other mechanical properties of dentine.