Inflammation and oxidative stress are increasingly recognised as important independent mediators of preterm brain injury and have been implicated in the pathogenesis of cerebral palsy and cognitive impairment. Such exposures are common for the premature infant in whom infection and inflammatory morbidities occur in around 60%. Furthermore, many preterm infants require oxygen therapy and respiratory support due to lung immaturity. Epidemiological and experimental studies indicate that in addition to the independent effects of inflammation and extreme hyperoxia on the developing brain, inflammation preconditions the developing brain resulting in variable injury when exposed to subsequent hypoxia-ischaemia. However experimental studies employing exposure to more modest oxygen fluctuations are lacking. This thesis characterises a clinically relevant model of prematurity where the developing brain is exposed to low grade inflammation and oxygen fluctuation around a hyperoxic mean. We hypothesise that antenatal inflammation and postnatal oxygen fluctuation, both alone and in combination, have detrimental effects on developing white matter. Pregnant dams received intraperitoneal lipopolysaccharide (LPS) or saline on G18 and G19. Dams and their pups were then reared in room air or fluctuating hyperoxia (circa 10kPa) for seven days. We measured longitudinal brain and body growth in different experimental groups to 12 weeks. Whole brains were examined for mRNA expression of inflammatory cytokines (TNFα, IL-1β, IL-6 and IL-10) and markers of oxidative injury (iNOS, SOD2). To determine the effect of perinatal insults on developing white matter, we analysed the expression of myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in the internal and external capsule. We also examined white matter tracts for differences in microglia (CD68), oligodendrocyte progenitor cells (NG2), oligodendroglial cells (Olig2) and cell death (cleaved caspase3). Behavioural studies (Morris Watermaze Test, Elevated Plus Test and Open Field Test) were undertaken at 12 weeks of age to detect any long-term functional difference between the groups. Antenatal inflammation reduces both brain and body growth at P7. This normalises by P14 unless this inflammatory insult has been followed by postnatal oxygen fluctuation, where brain and body growth restriction persists until P14. We defined our inflammatory response at P1 following antenatal inflammation and did not observe elevation of mRNA at P1. We demonstrated increased SOD2 at this time point, indicating a reparative process. At P7 we observed a significant reduction in the oxidative response following combined exposure to antenatal inflammation and postnatal oxygen fluctuation, indicating a potential limit to, or suppression of, the reparative process. In terms of white matter injury, antenatal inflammation reduces myelination at P7. There is no synergistic effect of inflammation and oxygen fluctuation on MBP immunohistochemistry at P7. However, MBP mRNA expression is increased in pups exposed to both insults compared to those exposed to inflammation alone suggesting that the oxygen fluctuation may stimulate MBP production in response to oxidative injury. MBP mRNA levels and protein expression have all normalised by P14. We observed a reduction in total cell number in the external capsule and corpus callosum in the dual insult group, without an increase in caspase. In keeping with other studies we detected no effect of our perinatal insults on NG2+ve oligodendrocytes. Olig2+ve cell numbers were also consistent between experimental groups. In further characterisation of the cellular response, antenatal inflammation followed by postnatal oxygen fluctuation resulted in a decrease in GFAP mRNA at P7, an effect which was reversed and significantly increased by P14 suggesting delayed activation of the innate immune system. No difference was observed in microglial numbers between experimental groups. There was however, increased microglial cell death (CD68 + caspase) in the group exposed to antenatal inflammation. When this insult was combined with postnatal oxygen fluctuation there was a comparative decrease in microglial cell death, which may reflect an earlier peak of microglial cell death, due to an increased and sustained inflammatory stimulus. Morris Watermaze testing demonstrated that pups exposed to both insults took longer than controls to locate the hidden platform on day 1, which is a measure of spatial learning. The Elevated Plus Test and Open Field Test demonstrated that pups exposed to both insults were less anxious and took more risks than pups exposed to single insults. In conclusion, within a clinically relevant preterm model, antenatal inflammation transiently disrupts both brain and body growth and myelination of the motor tracts of the developing brain. Moreover, when combined with postnatal oxygen fluctuation, detrimental effects on growth are amplified and sustained. Decreased cell numbers are also observed within white matter tracts. In terms of long term functionality, these pups display disinhibition of behaviour as young adults. Collectively, this thesis demonstrates that synergistic actions of common low-grade perinatal insults may alter normal neurodevelopment, and that this may carry a risk of neurodevelopmental sequelae for preterm infants.

Constitutive activation of the ATM dependent DNA damage response and repair pathways have been reported in pre-malignant and malignant human tissues and may undermine the efficacy of genotoxic cancer therapies. Therefore, ATM inhibitors may overcome resistance to current cytotoxics and potentiate the effects of radiotherapy. A colorectal cancer model was investigated to develop a framework for the rational use of ATM inhibitors. HCT116 p21-/- cells display constitutive activation of the ATM DNA damage response but display a defect in the ionising radiation induced S-phase checkpoint, termed radioresistant DNA synthesis. This radioresistant phenotype is associated with increased basal levels of Cdc25A protein, deficient DNA damage-induced degradation of Cdc25A and Chk2 mis-localisation. HCT116 p21-/- and SW620 cells, which exhibit basal Chk2 threonine-68 phosphorylation, were unable to abrogate the S-phase checkpoint when treated with an ATM inhibitor, suggesting that the ATM– Chk2 arm is non-functional in these cells: inhibition of ATM did not potentiate the efficacy of ionising irradiation. To assess activation of the pathway a tumour microarray was created using 179 treatment naïve sporadic colorectal cancers; 152 were of the microsatellite stable phenotype. Phosphorylated Chk2 threonine-68 was present in 22 % of microsatellite-stable colorectal tumours and 33 % of tumours with the microsatellite instability phenotype. In a colorectal cancer cell line model constitutive activation of the ATM DDR pathway reflected an attenuated ATM-Chk2 axis and inhibition of ATM in these circumstances was unable to potentiate the efficacy of ionising irradiation. Basal Chk2 threonine-68 phosphorylation may reflect a deregulated ATM DNA damage response pathway and/or checkpoint adaption and therefore use of an ATM inhibitor in this background may have limited efficacy. A predictive model is proposed that integrates functionality of the ATM-Chk2 axis, p53 mutation status and defects in DNA repair pathways when considering ATM inhibitor therapy. Ultimately, molecular phenotyping and functional analysis of processes deregulated in cancer will permit individualisation of current treatment modalities, improving their efficacy and limiting patient toxicity.

CHEK1 inhibitors are currently in clinical trials for their ability to abrogate chemotherapy-induced CHEK1 activation and S phase arrest resulting in cancer cell apoptosis. No studies have yet identified ovarian cancers that could benefit from CHEK1-targeting therapy. I hypothesised that knowledge of CHEK1 and CHEK2 signalling in the DNA damage response can assist in identifying potential biomarkers for platinum responsiveness and CHEK-targeting therapy in ovarian cancer. In vitro studies investigated the CHEK1/2 inhibitor AZD7762 (AZD) and cisplatin (CP) in same patient-derived platinum-sensitive/resistant high-grade serous ovarian cancer cell lines (PEO1/PEO4 and PEO14/PEO23). Cytotoxicity assays confirmed higher CP IC50’s for PEO4 and PEO23 relative to PEO1 and PEO14 cell lines, respectively. AZD was more toxic to PEO1 cells and an additive effect of AZD with CP relative to CP alone was seen. A nontoxic AZD treatment to PEO4 cells sensitised the cells to CP when applied in combination. PEO14 and PEO23 cells had similar cytotoxicity profiles for combination treatments. BRDU DNA synthesis assays and cell cycle analysis revealed increased BRDU incorporation and accumulation in S phase when all cell lines were treated with CP. AZD treatment had a similar effect in PEO14 and PEO23 cells and increased the sub-G1 population, a marker of apoptotic DNA fragmentation, relative to control. Drug combination had no major effect on cell cycle distributions of both PEO14 and PEO23 cells relative to single agents but resulted in BRDU incorporation levels below CP and control levels for PEO14 cells. In PEO1 and PEO4 cells, AZD did not affect the cell cycle or DNA synthesis levels relative to control. Drug combination did not alter the cell cycle relative to CP treatment for PEO1 cells but decreased S phase and increased G2/M and sub-G1 populations in PEO4 cells. This was coupled with a decrease of CP-induced BRDU levels in PEO4 control levels. Apoptotic PARP cleavage/total PARP occurred early in CP treated PEO1 and PEO14 cells. A surrogate CHEK1/2 activity marker, p-CDC2 (Y15), decreased in all lines treated with AZD relative to control. Within PEO1 and PEO4 cells, greatest PARP cleavage was observed with combination treatment and coincided with high p-H2AX (S139), a DNA damage marker. p-CHEK1 (S317) and p-CHEK2 (T68), both ATR and ATM phosphorylation sites during DNA damage, increased for lone drug treatment and, to a greater extent, the combination drug treatments. PARP cleavage occurs across all treatments in PEO1 cells while it only occurs in the combination treatment for PEO4 cells. The latter coincides with a decrease in p-CHEK1 (S296) a CHEK1 autophosphorylation site, p-TP53 (S15), and p-BRCA1 (S1524), a homologous recombination marker, relative to the CP treated sample. In PEO14 and PEO23 cells, lone AZD and combination treatments had similar cleaved PARP/total PARP levels compared to the PEO14 CP treated cells. This was coupled with increased p-H2AX (S139), decreased CHEK1, and decreased CHEK2 autophosphorylation p-CHEK2 (S516). A human ovarian cancer xenograft model identified increases in p-H2AX (S139), CHEK1, p-CHEK1 (S317), p-CHEK2 (T68), and p-BRCA1 (S1524) in the carboplatin responsive cancers. In the paired pre- and post-chemotherapy human ovarian cancer samples, p-CHEK1 (S317) was elevated in post-chemotherapy responsive samples. In the first cohort, high p-CHEK1 (S317) was an independent poor overall survival biomarker and correlated with high p-H2AX (S139), MYC, p-CHEK1 (S296), p-CHEK2 (T68), p-CHEK2 (S516), and p-TP53 (S15). p-CHEK1 (S317) was associated with poor overall survival in serous ovarian cancers within the second pre-treatment ovarian cancer cohort. In conclusion, AZD can induce apoptosis in CP resistant cancer cells by synergising with CP to abrogate the S phase checkpoint, increase DNA damage, and inhibit CHEK1, and BRCA1 function. As a single agent, AZD can induce apoptosis by decreasing CHEK1 levels and CHEK2 activity. p- CHEK1 (S317) is a platinum responsive / poor prognostic biomarker.

RNF4 belongs to the family of SUMO-targeted ubiquitin E3 ligases (STUbLs). The role of STUbLs in maintaining genomic stability was first discovered in yeast. Theyeast STUbL mutants displayed genomic instability, elevated mutation rates, sensitivity to DNA damaging agents and also demonstrated synthetic lethality with other DNA repair genes. Although the role of vertebrate RNF4 in the DNA damage response was not yet established, it could rescue the Schizosaccaromyces pombe STUbL mutant phenotypes, showing that RNF4 is a functional homologue of the yeast STUbL proteins,and that it might be implicated in the vertebrate DNA damage response.A homozygous knockout of RNF4 in the DT40 chicken lymphocyte cell line was generated to investigate the involvement of vertebrate RNF4 in protecting cells against DNA damage. Although the complete loss of RNF4 did not affect cell proliferation or cell cycle distribution, the RNF4 -/- cells exhibited a selective hypersensitivity to some S-phase specific DNA damaging agents. This hypersensitivity could be rescued by introducing an ortholog of RNF4 from another vertebrate species, and this was dependent on a functional ubiquitin E3 ligase activity of RNF4.To explore the physiological function of RNF4 in the context of a wholeorganism, Danio rerio was chosen as an in vivo model. Danio rerio RNF4 sharedsimilar in vitro biochemical characteristics as RNF4 from other vertebrates – it was able to autoubiquitylate itself and also ubiquitylate SUMO2 chains. In Danio rerio, RNF4 is a maternally provided gene and is highly expressed in the adult gonads. In the ovaries, RNF4 expression was restricted to the early stage oocytes, suggesting a possible role in oocyte development. Loss-of-function studies in Danio rerio were performed using morpholino knockdown and zinc-finger knockout technologies, and the depletion of RNF4 in zebrafish did not affect early embryonic development or viability of the animal.The results presented in this thesis suggests that while vertebrate RNF4 is notlikely to be an essential gene in some vertebrates, it plays a role in the DNA damage response and might be implicated in gonad development in Danio rerio. The zinc-finger knockout model has just been established and a more in-depth analysis is necessary to shed more light on the in vivo functions of RNF4.

Vibration-based damage detection methods are used in structural applications to identify the global dynamic response of the system. The purpose of the work presented is to exhibit a vibration-based damage detection algorithm that localizes the sensor arrangements such that irregularities within the structural system can be detected, located, and quantified. Damage can occur in a structure either within the material or at a connection between segments; therefore two different types of specimens, a plate specimen and a connection specimen, were analyzed with the algorithm. Numerical and experimental analyses were completed for each of the specimen types, and the results prove that damage can be detected, located and quantified in each scenario. It is noted that the quantification of the damage is based on a supervised learning method (original and damaged states are known) and that the accuracy in which the damage is quantified within the scope of this work might have difficulty in unsupervised learning methods (only current state is known). This work will extend to be applied on a highway bridge as a basis for a structural health monitoring system, as preliminary results suggest that further refinement is needed.

Vibration-based damage detection (VBDD) methods are used to detect damage in structural members non-evasively. This investigation began with two objectives: to prove a VBDD method could detect damage using strain gauges both analytically and experimentally, and to then use that method to determine the distance from a damaged area that strain gauges could be effective. Work began simultaneously using finite element software and physical experiments. It was determined that a VBDD method could detect damage with strain gauges in both settings. A parametric study was then completed that used probabilistic methods to identify an effective range for strain gauges over the length of the structural member.

In a recent investigation conducted by the United States Air Force, the mechanical failure of the aileron lever, manufactured from 2014-T6 aluminum, caused the fatal mishap of a T-38 trainer aircraft. In general the locations of cracks are unknown and must be determined by simulation. In this study we propose to use a continuum damage modeling approach to determine the degradation and damage in a material as the number of cycles of loading increases. This approach successfully predicts the location of crack initiation, propagation path, and propagation rate. A stress-based model in conjunction with the successive initiation technique is utilized. Successive initiation is based on the idea that damage will accrue in a material. Each element inside a new material will have a value of 0 damage assigned to it. Over time, the damage that occurs due to stresses on individual elements will add until the damage reaches a value of 1. At that point, failure of the element will occur. A code was developed in ANSYS that can draw, mesh, and apply appropriate forces on the aileron lever for successive runs. By using the S-N curve for the 2014-T6 aluminum material, the material damage constants are found. This stress-based damage model is then used to determine the state of damage in each element. Each time the elements are stressed, a particular amount of damage will occur. When an element reaches a specific amount of damage, ANSYS will "kill" the element, resulting in the element no longer adding to the stiffness matrix of the material. Variability is a common occurrence in all aspects of engineering such as manufacturing, testing, and loading. A Monte Carlo simulation is used to determine the sensitivity of the results to variability of input parameters by ± 15%. Input parameters include loads, material properties and damage model constants. The Monte Carlo simulation indicates the only significant input in the initiation life of the material is the exponential value in the stress-based fatigue life equation. Material properties and load variations in the ± range will not significantly change the life prediction results.

Flood is one of the major natural disasters in Australia. It breaks down transportation and communication systems, disrupts businesses and causes damage to properties, loss of stock, crops and also loss of human lives. Better understanding of the impacts resulting from floods and other natural hazards can help to reduce the damages or losses. Flood damage assessment procedures estimate the impact of flood in monetary terms to help decision makers develop new policies, programs and development plans. This study examines different techniques, procedures and underpinning philosophies that have been used in some major emergency management and insurance organizations in the world such as RAM, ANUFLOOD, and HAZUS and NHRC. From this examination the study finds that none of them are suitable for mitigation and emergency purposes in producing economic flood damage estimation at fine resolution with high accuracy. From this perspective the study develops a rigorous procedure for property level economic flood damage assessment. The assessment procedures are embedded within GIS (Geographic Information System) technology which can model and analyse the multidimensional phenomenon of flood and damage characteristics of residential buildings. The damage estimation procedures developed in this study include flood modelling, collection and organisation of building inventory data; adopting a set of stage-damage curves; and measuring damage at property level. The quality of damage estimates derived from the procedures is highly influenced by quality of input data. The study also includes the implementation of the damage assessment procedure on the study area (a segment of Kororoit Creek and its adjacent area). The study uses data from a number of sources including Melbourne Water, NEXIS, VICMAP, a quick survey and literature. The developed procedure will help many practitioners in flood loss assessment and natural hazard risk management to face the challenges they have in establishing damage estimates with high accuracy.

Abstract not available

A novel method of predicting the multiaxial high-cycle fatigue strength of metallic components is proposed and verified for various steel, aluminium and cast iron alloys. The proposed Fatigue Damage Function shows superior multiaxial fatigue strength prediction compared to the established methods of Gough and Pollard, McDiarmid and Carpinteri and Spagnoli. A new material property, the Normal Stress Sensitivity Factor, is also introduced and its applicability is verified according to published test results of sixteen different structural alloys. To highlight the effectiveness of the proposed criterion, for industrial applications, a case study has been conducted on heat-treated and not heat-treated automatic transmission output shafts.