Interferon regulatory factor 3 (IRF3) plays an important role in activating the innate immune response in a variety of conditions, including viral infection. As well as regulating the immune response to viruses, IRF3 is involved in regulating cellular functions including apoptosis. Apoptosis and the inflammatory response to viral infection are very different; therefore, it is obvious that IRF3 plays dramatically different roles in the cell depending on the conditions. We previously identified a non-activating phosphorylation of IRF3 in response to adenovirus (Ad) in which Serine-173 is phosphorylated. In addition to Ad infection, IRF3- S173 is phosphorylated in response to genotoxic stresses including ultraviolet (UV) irradiation and etoposide. In this study, I show that this phosphorylation event is involved in a variety of processes including protein stability, cell survival and IRF3 regulation. Thus, phosphorylation of IRF3-S173 is a novel and important event in a complex regulatory pathway of an integral protein.

Myc is a transcription factor frequently found deregulated in human cancer. Cells with deregulated expression of Myc carry a selective advantage against its neighbours due to the fact that Myc-mediated transcription governs crucial cellular events such as proliferation and growth. In addition, Myc has been implicated in several other aspects of tumour biology like cellular immortality, the formation of new blood vessels and the colonization of distant tissues through the process of metastasis. Therapy aimed at disrupting essential pathways regulated by Myc is important because of the many different types of cancers that depend on continued signalling along these pathways.  This thesis describes new treatment opportunities for cancers with a high Myc signature. In Paper Ι, we describe a new role for the DNA methyltransferase inhibitor Decitabine in the treatment of Myc transformed tumours cells. We show that the therapeutic potential of Decitabine in the treatment of Burkitt Lymphoma relies not only on its ability to cause reactivation of silenced genes such as pro-apoptotic PUMA, but also on the DNA damage that this drug induces. In vivo, Decitabine delays disease progression of transplanted lymphoma cells. In Paper ΙΙ, we identify the DNA damage checkpoint kinase Chk1 as a therapeutic target in Myc overexpressing cancers. We show that targeting Chk1 with shRNA or with a novel small molecule inhibitor cause a delay in disease progression of transplanted lymphoma cells in vivo. In Paper ΙΙΙ, the Chk1-related kinase Chk2 is evaluated as a therapeutic target in Myc overexpressing cancers. Myc overexpressing cells are not dependent on Chk2 but we show that Chk2 abrogation using shRNA causes polyploidization and protection against DNA damage. However, Chk2-targeted therapy elicits a synergistic lethal response in combination with inhibition of the DNA repair associated protein PARP. In conclusion, this thesis shows the potential of targeting the DNA damage machinery and the functional hubs important for maintenance of genomic stability in tumours with a deregulated expression of Myc.

Ultraviolet radiation reaching the Earth’s surface (UVR, 280-400 nm) may penetrate deep into the clear oligotrophic waters influencing a large part of the euphotic layer. Marine heterotrophic bacteria at the surface of the oceans are especially sensitive to the damaging solar radiation due to their haploid genome with little or no functional redundancy and lack of protective pigmentation. In a context of climate change and ozone depletion, it is clearly important to understand the physiology and underlying molecular UVR responses of abundant marine bacteria species. We chose the marine ultramicrobacterium Sphingopyxis alaskensis as a reference species to study the impact of solar radiation due to its numerical abundance in oligotrophic waters and its photoresistance, previously reported. For this purpose, we focused on the formation of the two major UVB-induced DNA photoproducts (CPDs and 6-4PPs) as well as the differential protein expression under solar radiation. We first demonstrated that the GC content of prokaryotic genome had a major effect on the formation of UVB-induced photoproducts, quantified by HPLC-MS/MS. Due to its high GC content, S. alaskensis presented a favoured formation of highly mutagenic cytosine-containing photoproducts and therefore would be more susceptible to UVinduced mutagenesis. By comparing S. alaskensis to another marine bacterium Photobacterium angustum, we observed for the latter strain a remarkable resistance to high UVB doses associated with a decrease in the rate of formation of CPDs explained by a non-conventional activity of photolyase. We also demonstrated that DNA damage in S. alaskensis was markedly modulated by growth temperature and time spent in stationary phase. In order to assess the effects that environmental UV-R had on regulatory networks and pathways of S. alaskensis, and determine how the cell’s physiology was affected, a quantitative proteomics investigation was performed. Changes in proteome were analyzed, with the recent and powerful mass spectrometry based approach using iTRAQ methodology. Approximately, one third of the proteome of S. alaskensis was identified, with 119 statistically and significantly differentially abundant proteins. Cellular processes, pathways and interaction networks were determined and gave us unique insight into the biology of UV response and adaptation of S. alaskensis.

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Selenium (Se) is an essential micronutrient, being a component of more than twenty seleno-proteins in humans. Previous studies suggested that increased intake of Se may reduce the risk of degenerative diseases including cancer; however, excessive intake can be toxic. Wheat is one of the major dietary sources of Se in humans, mainly in the form of L-selenomethionine (Se-met) but the impact of this source of Se on human health at the genome level was previously unexplored. This PhD project aimed to (a) determine the safe dose-range and bio-efficacy of Se-met in vitro; (b) identify the optimal concentration of Se-met for reduction of genome damage in vitro; (c) investigate the optimal concentration of Se-met for improving resistance to gamma radiation or hydrogen peroxide induced genome damage in vitro; d) determine the bioavailability and bioefficacy of Se in vivo, in the form of either Semet or wheat biofortified with Se; e) identify the nutrients and food groups that are correlated with Se intake/status and f) identify the nutrients, food groups and plasma mineral concentrations that are correlated to baseline lymphocyte DNA damage. The in vitro study was performed on the peripheral blood lymphocytes isolated from six males and cultured with media supplemented with Se-met in a series of Se concentrations from 3 to 3850 μg Se/l while keeping the total methionine (i.e. Se-met + L-methionine) concentration constant. Baseline genome stability of lymphocytes and the extent of DNA damage induced by 1.5 Gy γ-ray or 7.5 μM hydrogen peroxide (H₂O₂) were investigated using the Cytokinesis-block Micronucleus Cytome (CBMNCyt) assay and the alkaline Comet assay with and without glycosylase (Fpg or Endo III) treatment after 9 days of culture. Results showed that high Se concentrations (≥1880 μg Se/l) caused strong inhibition of cell division, extensive DNA damage and increased cell death indicating cytotoxicity and genotoxicity. Baseline frequency of nucleoplasmic bridges (NPBs) and nuclear buds (NBud) declined significantly as Se concentration increased from 3 μg Se/l to 430 μg Se/l (P trend = 0.03 and 0.008, respectively); however, a significant trend of increase in Comet DNA damage was also observed (P trend <0.05) in lymphocytes. Selenium concentration (≤ 430 μg Se/l) had no significant effect on baseline frequency of micronuclei (MN) or DNA oxidation and had no protective effect against γ-ray-induced or H₂O₂-induced genome damage in lymphocytes. A randomised double-blind placebo-controlled intervention trial was conducted on healthy South Australian males (n = 62, age (mean ± SD) 56 ± 7.0 years) with Se dosage increased every 8 weeks for a total duration of 24 weeks. This study compared the bioavailability, by using plasma Se concentration as the biomarker, and bioefficacy of Se, by using platelet glutathione peroxidase (GPx) activity and lymphocyte DNA damage as biomarkers, from wheat process-fortified with Se-met (PROFORT) and high-Se wheat biofortified with Se (BIOFORT) compared to non-fortified normal (CONTROL) wheat. It was found that increased Se intake from BIOFORT wheat increased plasma Se concentration effectively in a dose-response manner from a baseline of 122 μg/l up to 190 μg/l (P<0.001). Increased Se intake from PROFORT wheat also increased plasma Se with a plateau at 140 μg/l, being therefore less effective than BIOFORT wheat (P<0.001). There was no significant change in Se status in the CONTROL group. Improved plasma Se concentrations had no effect on platelet GPx activity or lymphocyte DNA damage in either of the intervention groups. Results from the food frequency questionnaire (FFQ) survey (n = 173) and plasma Se concentration survey (n = 179) suggested that the study population screened for participation in the in vivo trial described above had a mean plasma Se concentration (± SD) of 102 (± 12) μg/l and a mean (± SD) estimated Se intake of 165 (± 68) μg/d. This is a higher estimated Se intake than found in previous Australian studies. The major dietary sources of Se were found to be bread/cereals, fish/seafood and meat. However, increased intake of nuts/seeds, which are rich in Se, may have undesirable effects on lymphocyte DNA oxidation in this Se-replete population. In conclusion, the in vitro studies suggest that (1) Se-met at higher concentrations at greater or equal to 1880 μg Se/l is cytotoxic; (2) Se-met may improve specific genome stability biomarkers such as nucleoplasmic bridge and nuclear bud at concentrations up to 430 μg Se/l, but further studies are needed to verify this effect. The in vivo studies in older men showed that Se from BIOFORT wheat is more effective in raising plasma Se concentration than Se from wheat process-fortified by the addition of Se-met, when both wheat products were subjected to strong heat. However, the platelet GPx activity and lymphocyte DNA damage appeared not to be modified by improved Se status. This work contains two publications: 1) "The effect of selenium, as selenomethionine, on genome stability and cytotoxicity in human lymphocytes as measured by the cytokinesis-block micronucleus cytome assay". Mutagenesis 2009 May;24(3):225-32. 2) "Increased consumption of wheat biofortified with selenium does not modify biomarkers of cancer risk, oxidative stress or immune function in Australian males" Environmental Molecular Mutagenesis. 2009 July; 50 (6):489-501 The latter one was not able to be published in a journal of higher impact factor due to part of the data had been published elsewhere. Both articles are attached in Appendix. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1523459 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2010

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Ultraviolet radiation reaching the Earth’s surface (UVR, 280-400 nm) may penetrate deep into the clear oligotrophic waters influencing a large part of the euphotic layer. Marine heterotrophic bacteria at the surface of the oceans are especially sensitive to the damaging solar radiation due to their haploid genome with little or no functional redundancy and lack of protective pigmentation. In a context of climate change and ozone depletion, it is clearly important to understand the physiology and underlying molecular UVR responses of abundant marine bacteria species. We chose the marine ultramicrobacterium Sphingopyxis alaskensis as a reference species to study the impact of solar radiation due to its numerical abundance in oligotrophic waters and its photoresistance, previously reported. For this purpose, we focused on the formation of the two major UVB-induced DNA photoproducts (CPDs and 6-4PPs) as well as the differential protein expression under solar radiation. We first demonstrated that the GC content of prokaryotic genome had a major effect on the formation of UVB-induced photoproducts, quantified by HPLC-MS/MS. Due to its high GC content, S. alaskensis presented a favoured formation of highly mutagenic cytosine-containing photoproducts and therefore would be more susceptible to UVinduced mutagenesis. By comparing S. alaskensis to another marine bacterium Photobacterium angustum, we observed for the latter strain a remarkable resistance to high UVB doses associated with a decrease in the rate of formation of CPDs explained by a non-conventional activity of photolyase. We also demonstrated that DNA damage in S. alaskensis was markedly modulated by growth temperature and time spent in stationary phase. In order to assess the effects that environmental UV-R had on regulatory networks and pathways of S. alaskensis, and determine how the cell’s physiology was affected, a quantitative proteomics investigation was performed. Changes in proteome were analyzed, with the recent and powerful mass spectrometry based approach using iTRAQ methodology. Approximately, one third of the proteome of S. alaskensis was identified, with 119 statistically and significantly differentially abundant proteins. Cellular processes, pathways and interaction networks were determined and gave us unique insight into the biology of UV response and adaptation of S. alaskensis.

Thesis (Ph.D.) -- McMaster University, 1998. / Includes bibliographical references (leaves 167-182). Also available via World Wide Web.

Thesis (Ph. D.)--McMaster University (Canada), 1990. / Source: Dissertation Abstracts International, Volume: 52-10, Section: B, page: 5244. Supervisor: D.R. McCalla.