Increment cores were collected from 10 stands in mixed-conifer forest stands which had suffered varying levels of Douglas-fir tussock moth defoliation during 1946, 1964, and 1973 infestations in north central Idaho. Ring-width measurements, standardized to remove inherent growth trends, were compared between host (Douglas-fir and grand fir) and nonhost (western larch, ponderosa pine, western white pine) species for evidence of growth losses due to defoliation. Heavy defoliation caused growth of host species to decrease 75 %-90% in one year. Normal growth rates returned within 3-4 years after maximum defoliation, however. The effect of moderate defoliation could not be reliably identified in the data.

The basis for the selection of trees to be used in the production of dendrochronologies has long been an issue (Douglass 1946; Fritts 1976). In humid regions the common practice has been to use trees that appear to be in good health. As a part of a larger study involving the impact of ice storms on tree-ring increments (Travis 1989), we show that trees stressed as a result of ice damage produced a stronger climatic signal than nondamaged trees.

Telomeres are physical ends of chromosomes consisting of (TTAGGG)n DNA sequence and a specialized set of proteins that protect chromosomal ends from degradation and from eliciting DNA damage response. These specialized set of proteins, known as shelterin, directly bind to telomeric DNA. In addition, some DNA double-strand break (DSB) repair proteins such as, DNA-PKcs and KU70/80, play active roles in telomere maintenance. Mouse knock-out experiments have revealed that deletion of either DNA-PKcs or Ku70/80 resulted in elevated levels of telomeric fusion, indicative of dysfunctional telomeres. Artemis protein is involved in DNA DSB repair through non-homologous end joining (NHEJ) and it is phosphorylated by DNAPKcs. Human cells defective in Artemis have been identified and shown to be radiosensitive and patients with an Artemis defective gene suffer from radiosensitive severe-combined immune deficiency syndrome (RS-SCID). Mouse cells defective in Artemis have elevated levels of telomeric fusion. We have demonstrated in this thesis that Artemis defective human cell lines show a mild telomeric dysfunction phenotype detectable at the cytological level. The nature of telomere dysfunction phenotype appears to be similar to that observed in DNAPKcs defective cells as exemplified by the presence of IR induced chromatid telomeric fusions. We have also shown that (a) DNA damage occurring within the telomeric DNA is difficult to repair or irreparable in older cells and that (b) Artemis defective older cells show higher proportion of DNA damage at telomeres than their normal counterparts. Finally, we have demonstrated that inhibition of DNA-PKcs causes (a) an increase in telomeric fusions in Artemis defective cell lines relative to both normal cell lines after inhibition and Artemis cell lines before inhibition and (b)elevated levels of DNA damage at telomeres following exposure of cells to radiation relative to both irradiated normal cells exposed to a DNA-PKcs inhibitor and irradiated Artemis defective cells but not exposed to the DNA-PKcs inhibitor. These results suggest that the effects of Artemis and DNA-PKcs on telomeres are cumulative. We have also performed (a) experiments to examine telomere function in Artemis defective cell lines after knocking down DNA-PKcs levels by RNAi and b) preliminary experiments to knock-down Artemis in DNA-PKcs defective cells. Taken together, our results suggest that the Artemis defect causes mild telomere dysfunction phenotype in human cells.

Studies were undertaken to determine toxicological effects in a model species, Cyprinus carpio L. following hypoxic exposure either alone or in combination with representative heavy metal (i.e. copper; Cu) via a dietary route, at different levels of biological organisation (viz. biochemical, histological and individual level effects). Initially, the validation study of biological responses using a range of concentrations of dietary Cu as a relevant environmental contaminant was carried out (Chapter 3). The results showed a range of biological responses in exposed fish including significant genotoxic response as determined by induction of DNA strand breaks (i.e. the Comet assay) with bacterial enzymes Fpg and Endo-III (for detection of oxidative DNA damage) and reduction in growth rate suggesting the robustness of selected biomarkers. Subsequently, this approach was used initially to determine the biological responses following chronic hypoxic and hyperoxic exposure (Chapter 4). The results suggested that both hypoxic and hyperoxic conditions lead to a range of comparable biological responses. Following relative evaluation of chronic hypoxic and hyperoxic exposures, experiments were carried out to elucidate potential interactive effect of hypoxia in combination with dietary Cu (Chapter 5). The combined exposure of hypoxia and Cu induced a significantly higher level of DNA damage suggesting that DNA damage in fish can serve as a sensitive biomarker for changes in water quality as well as presence of genotoxic chemicals. The final sets of experiment were carried out to determine the biological responses in C. carpio following exposure to chronic hypoxic stress and subsequent recovery in normoxic condition for 7 days. Real-time PCR (qPCR) technology was used to examine the hypoxia inducible Factor-1 α (HIF-1α) gene expression pattern (Chapter 6). The results suggested that the expression levels of HIF-1α in response to hypoxia were significantly higher compared to normoxic controls, a high level of oxidative DNA damage under hypoxia and re-exposure to normoxic condition (i.e. recovery period). This will shed lights for development of adaptive response in higher vertebrates, which could also have significant clinical implications in human health.

A major concern affecting the efficient use of composite laminates in aerospace industry is the lack of understanding of the effect of low-velocity impact (LVI) damage on the structural integrity. This project aims to develop further knowledge of the response and damage mechanisms of composite laminates under LVI, and to explore the feasibility of assessing the internal impact damage with a visually inspectable parameter. The response and damage mechanisms of composite laminates under LVI have been investigated experimentally and numerically in this project. Various parameters including the laminates thickness, lay-up configuration, repeated impact, and curing temperature have been examined. The concept and the phenomena of delamination threshold load (DTL) have been assessed in details. It was found that DTL exists for composite laminates, but the determination of the DTL value is not straightforward. There is a suitable value of range between the impact energy and the laminates stiffness/thickness, if the sudden load drop phenomenon in the impact force history is used to detect the DTL value. It is suggested that the potential menace of the delamination initiation may be overestimated. The composite laminates tested in this project demonstrate good damage tolerance capacity due to the additional energy absorption mechanism following the delamination initiation. As a result, the current design philosophy for laminated composite structure might be too conservative and should be reassessed to improve the efficiency further. To explore the feasibility of linking the internal damage to a visually inspectable parameter, quasi-static indentation (QSI) tests have been carried out. The dent depth, as a visually inspectable parameter, has been carefully monitored and assessed in relation to the damage status of the composite laminates. It is proposed that the damage process of composite laminates can be divided into different phases based on the difference in the increasing rate of dent depth. Moreover, the internal damage has been examined under the optical microscope (OM) and the scanning electron microscope (SEM). Residual compressive strength of the damaged specimen has been measured using the compression-after-impact (CAI) test. The results further confirm the findings with regard to the overestimated potential menace of the delamination initiation and the proposed damage process assumption. The proposed damage process assumption has great potential to improve the efficiency and accuracy of both the analytical prediction and the structural health monitoring for damages in composite laminates under low-velocity impact.

A systematic investigation is presented into the microstructural and micromechanical influences on ductile damage nucleation with an emphasis on grain boundaries in polycrystals. Microstructures obtained from experiments on copper polycrystals are characterized using Electron Backscatter Diffraction (EBSD) and near-field High-Energy Diffraction Microscopy (nf-HEDM) and the occurrence of damage is compared with micromechanical values obtained using an elasto-viscoplastic model based on the Fast- Fourier Transform (EVPFFT). The model produces full-field solutions for the stress and strain in voxelized polycrystalline microstructures. In order to resolve the fields onto interfaces, local Cartesian moments of the polycrystalline grain structure are used to extract the normals of grain boundaries and the tangents of triple junctions directly from the voxelized microstructure. Thus projecting the stress yields a parameter with potential significance, i.e. the grain boundary surface tractions. We identify “traction hotspots”, i.e. regions with tractions that are significantly above the mean, for the case of uniaxial tension. These show correlations with the angle between the grain boundary normal and the loading axis, a trend that some experiments also show when boundaries that nucleated voids are analyzed using EBSD, though differences present between the simulation and experiment hint that further criteria are needed. Nf-HEDM was used to record microstructure images of a polycrystalline sample before and after it undergoes damage. The damage locations in the post-shocked image are mapped onto the pre-shocked image, allowing stress and strain values from the EVPFFT model in the regions that eventually nucleated damage to be correlated with the locations of the void. The unexpected result was that differences in plastic work across boundaries correlated with voids, whereas vi quantities such as triaxiality and normal forces across boundaries did not.

Ph.D (Industrial Psychology) / Brain injuries often occur suddenly and without warning, and from that instant, a normal life can be changed. Traumatic Brain Injury (TBI) may result in significant impairment of an individual’s physical, cognitive and psychosocial functioning. Although there is a fair amount of research on the concept of brain damage and the consequent effects on the family and return to work (RTW) prospects, there is far less information available on the actual assessment of workplace capability or workplace potential following the head injury incident. This is important, since the concept of work or job value has been well documented. Employment is far more than just a job; it not only provides for basic sustenance needs and decent living conditions, but also allows someone to fit into the world, create relationships, use talents and skills, learn, grow and build, and develop a sense of identity and belonging. While the value of work is well-documented, the statistics regarding unemployment following a head injury are concerning. It is estimated that approximately 1.5 million Americans sustain head injuries each year, with the majority of these people being under the age of 35 and in their prime years of vocational productivity. A baseline figure of 2% of the American population is currently living with disabilities as a result of head injuries. In South Africa, the rate of occurrence seems to be even more alarming, with published rates of 89,000 cases for 2009. In 2001, this was a baseline figure of 5% of the population living with disabilities resulting from head injuries. Various sources put the occurrence of head injuries in South Africa at 1.5 to 3.5 times higher than the estimated global rate. The negative impact of this on the individual, the family, community and economy is clear.

Hier werden einige Ansätze dargestellt, um das Schadenspotential von Stürmen abzuschätzen. Dabei sollen auch Aussagen zur Größenordnung des Schadens bei sehr unwahrscheinlichen Ereignissen gemacht werden. Die Naturgefahr Sturm wird getrennt nach außertropischen Zyklonen und Tornados betrachtet. Im ersten Fall werden empirische Verteilungsfunktion und mittels einer Markov Methode erzeugte synthetische Windreihen zur Abschätzung extremer Windgeschwindigkeiten genutzt. Eine Abschätzung der Böen wird durchgeführt und der Schaden mit Hilfe einer einfachen Beziehung zwischen Böengeschwindigkeit und Schaden ermittelt. Für die Abschätzung des Schadens im Falle eines Tornados werden von Dotzek angegebene Wahrscheinlichkeiten für Tornados in Deutschland und die Definition einer Tornadointensitätsskala als Schadensfunktion verwendet. / Some conceptions were explained, to estimate the risk of storm caused damages. The amount of damage by unlikely events is assessed. The natural hazard storm is considered separately for extratropical cyclones and tornadoes. Empirical distribution functions and synthetic series of wind speeds generated by a Markov chain model are used to derive the extreme wind speeds for cyclones. An estimation of gust speeds are performed and a simple relationship between gust speed and damage is applied. The likelihood of tornadoes to occur in Germany provided by Dotzek and the definition of an intensity scale are used to estimate the damage in the case of tornadoes.

The Saccharomyces cerevisiae Rmi1 protein is a component of the highly conserved Sgs1-Top3-Rmi1 complex, which is required for the maintenance of genome stability. The rmi1Δ deletion mutant has proven difficult to study because it exhibits very poor growth, and rapidly accumulates second site suppressor mutations. Furthermore, deletion of the putative HJ resolvase genes, MUS81-MUS81 or SLX1-SLX4 in rmi1Δ mutants causes synthetic lethality. In order to study phenotypes caused by loss of functional Rmi1, and to explore the genetic interactions between RMI1 and the MUS81, MUS81, SLX1 and SLX4 genes, a temperature sensitive mutant of RMI1 was isolated, named rmi1-1. Similar to rmi1Δ deletion mutants, rmi1-1 cells are highly sensitive to the DNA damaging agent, MMS and the replication inhibitor, HU. In addition, rmi1-1 mutants accumulate replication-associated branched DNA structures, and arrest in G₂/M after a transient exposure to MMS. These cells are proficient in DNA damage checkpoint activation. Deletion of SLX1, SLX4, MUS81 or MUS81 in the rmi1-1 strain causes synthetic lethality, which is associated with cell cycle defects. Following a transient exposure to MMS, rmi1-1 mutants accumulate homologous recombination intermediates. These intermediates are slowly resolved at the restrictive temperature, revealing a redundant resolution activity in the absence of functional Rmi1. This resolution depends upon Mus81-Mms4, but not on Slx1-Slx4 or Yen1. I propose that while the Sgs1-Top3-Rmi1 complex constitutes the main pathway for removal of homologous recombination intermediates following a perturbed S-phase, Mus81-Mms4 can act as a back up for resolution of these intermediates, which most likely represent double Holliday junctions. In this study, I also present screens for high copy suppressors of rmi1-1 phenotypes, and for novel Rmi1 interaction partners.

I have been interested in the molecular mechanisms of Haematopoietic malignant diseases such as leukaemia and lymphoma, especially those involving oncogenic tyrosine kinases. About 30 of the 90 tyrosine kinases in the human genome have been implicated in cancer (Blume-Jensen P, 2001). The oncogenic tyrosine kinases (OTKs), such as Bcr-Abl (product of chromosomal translocations of two genes bcr and abl) in Chronic Myelogenous Leukaemia, and Erythroblastic leukaemia viral oncogene homolog 2(Erb-B2) in mammary and other cancers, mediate their transforming effects via a diverse array of signalling pathways involved in DNA damage, cell survival and cell cycle regulation (Deutsch E, 2001; Skorski T, 2002; Kumar R, 1996). My work has been centred around the analysis of a mouse cancer model that is driven by an oncogenic tyrosine kinase – p56 Lck-F505 expressed on CD45 knock- out background (Baker M, 2000). The investigation of this mouse model has revealed that oncogenic inhibition of deamidation of the Bcl-xL survival protein plays a critical role in protecting thymocytes from DNA-damage induced apoptosis. Cells that would normally be eliminated due to accumulating DNA damage are instead preserved with an increasing load of double-stranded breaks, leading to genomic instability, chromosomal abnormalities and transformation. This work was published in Cancer Cell (An oncogenic tyrosine kinase inhibits DNA repair and DNA-damage-induced BclxL deamidation in T cell transformation. Zhao R, 2004). Following that I have tried to elucidate the different roles of the two deamidated species of Bcl-xL in apoptosis, and also the molecular mechanisms of DNA damage- induced Bcl-xL deamidation in order to understand the inhibition of Bcl-xL deamidation by oncogenic tyrosine kinases. Recently I have shown that Bcl-xL deamidation, whereby two critical Asn residues are converted to iso-Asp, cripples the ability of the protein to sequester pro-apoptotic BH3-only proteins such as Bim and p53- upregulated modulator of apoptosis (PUMA), thereby explaining its loss of pro-survival functionality. In vivo, DNA damage causes intracellular alkalinisation that is both necessary and sufficient to deamidate Bcl-xL, promoting apoptosis: no enzyme is necessary for this process. In pre-tumourigenic thymocytes alkalinisation is blocked, so preserving Bcl-xL in its pro-survival mode. Furthermore murine tumours are protected from genotoxic attack by native Bcl-xL, but enforced alkalinisation and consequent Bcl-xL deamidation promotes apoptosis. This part of work was published in Plos Biology (DNA damage-induced Bcl-xL deamidation is mediated by NHE-1 antiport regulated intracellular pH. Zhao R, 2007). Through collaboration with Prof AR Green’s research group at the Department of Haematology of the University of Cambridge, I have also analysed the Bcl-xL deamidation pathway in human myeloproliferative disorders, e.g. Polycythemia vera(PV) and Chronic Myelogenous Leukaemia (CML). We found that the oncogenic tyrosine kinases involved in these disorders, i.e. Jak2V617F and Bcr-Abl also inhibit the Bcl-xL deamidation pathway in DNA damage responses. These findings shed light on potential therapeutic application of the Bcl-xL deamidation pathway in human malignancies. This piece of work was recently published in the New England Journal of Medicine (Inhibition of the Bcl-xL deamidation pathway in myeloproliferative disorders. Zhao R, 2008). Overall the cited work has led to several important new insights into the molecular mechanisms involved in oncogenesis: first, that Bcl-xL deamidation is important in the cascade of events leading from DNA damage to apoptosis; second, that oncogenic tyrosine kinases inhibit these events in both the murine and human context; third, that up-regulation of the NHE-1 antiport and consequent intracellular alkalinisation are critical events in this DNA damage-induced cascade leading to apoptosis. In the process I have demonstrated the first in vivo mechanism for the deamidation of an internal protein Asn. Essentially, a completely new and unexpected signalling pathway has been uncovered that seems to pertain to all murine and human haematopoietic cell lineages that have been investigated so far.