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<p>One of the hallmark features of cancer is altered metabolism, whereby rates of glucose and fatty acid turnover are constitutively elevated to support uncontrolled propagation. The key regulator of energy metabolism is the enzyme AMP-activated protein kinase (AMPK), which suppresses anabolic pathways that increase proliferation and enhanced catabolic pathways that liberate energy, all in an attempt to maintain energy homeostasis in the cell. In addition to regulating metabolism, AMPK has also been implicated as a tumour suppressor and we have suggested that it may be a modulator of radiation responses in cancer cells <em>in vitro</em>. Moreover, we investigated the molecular mechanisms that facilitate ionizing radiation (IR)-induced AMPK activation, as well as demonstrated that certain AMPK activating drugs can work as radiation sensitizers in a variety of cancer cell lines. Stemming from this framework, we also provided experimental evidence that suggests AMPK is centrally involved in pathways that regulate DNA damage and proliferation at the basal level, and in response to IR. One of the targets involved in these pathways that can also influence AMPK regulation is the stress-activated Sestrin 2 protein. We have provided evidence that Sestrin 2 mediates IR-induced activation and expression of AMPK. Taken together, this work has provided novel insight into the ability of IR to modulate the activity and expression of AMPK, which in turn is required to facilitate the appropriate stress-response in cancer cells. Given its emerging interest in the cancer field, AMPK may become an important biomarker for evaluating clinical outcomes in patients undergoing radiation therapy.</p> / Doctor of Philosophy (PhD)

<p><strong><em>Purpose</em></strong></p> <p>To examine the potential of the anti-diabetic agent Metformin (MET) to enhance responses of NSCLC to ionizing radiation (IR).</p> <p><strong><em>Experimental Design</em></strong></p> <p>Human NSCLC A549, H1299 and SK-MES cells were treated with IR, MET or the mTOR inhibitor rapamycin and subjected to proliferation, clonogenic, immunoblotting, cell cycle and apoptosis assays. A549 and H1299 cells were grafted into flanks of immunosuppressed mice and treated with MET and/or IR. Tumours were analyzed by immunoblotting and immunohistochemistry.</p> <p><strong><em>Results</em></strong></p> <p>MET(2.5uM-5mM) caused dose-dependent inhibition of proliferation (10-70%)in all lines, inibited clonogenic survival and sensitized cells to IR. In A549 cellsMET caused inhibition of proliferation comparable to rapamycin, stimulated expression and activation of the ATM and AMPK-p53-p21^cip1and inhibited the Akt-mTOR-4-EBP1 pathway.MET caused G1 arrest of cell cycle, enhanced apoptosis and induced sustained DNA repair foci of gH2AX. MET and IR alone inhibited xenograft growth and combined treatment enhanced that further. IR and MET induced sustained enhancement of expression and activity of ATM-AMPK-p53-p21^cip1and inhibitionof Akt-mTOR-4-EBP1 pathways in tumours also. MET reduced expression of angiogenesis and enhanced expression of apoptosis markers in both control and radiated tumours.</p> <p><strong><em>Conclusions</em></strong></p> <p>Clinically achievable(uM) doses ofMET inhibit human NSCLC cell and tumour growth and sensitize them to IR.This is accompanied by desirable modulation of molecular signals, inhibition of angiogenesis and induction of apoptosis. Our results suggest that MET could be a clinically useful adjunct to radiotherapy in NSCLC and support clinical investigation of MET in combination with radiotherapy.</p> / Master of Science (MSc)

Ionizing radiation exposure during pregnancy raises concerns of potentially harmful effects for both the mother and the unborn child. Fetal programming involves permanent changes in offspring phenotype due to stress experienced in-utero. This phenomenon has been well characterized in cardiometabolic disorders such as hypertension. The effects of prenatal ionizing radiation exposure on offspring cardiovascular endpoints following birth were studied in a mouse model. Pregnant wildtype C57Bl/6J mice were irradiated on day 15 of pregnancy with whole-body 137Cs gamma radiation at nominal doses of 5, 10, 50, 100, 300 or 1000 mGy. Post-natal measurements of offspring weight and blood pressure were completed. In female pups, blood pressure was significantly increased at 300 mGy and heart rate significantly decreased at 1000 mGy. Female pups were growth restricted over the study period at 50, 100 and 1000 mGy. Growth restriction in male pups was only observed at the highest dose of 1000 mGy. Unintended effects on the study measures caused by transportation of pregnant mothers to the irradiation facility were most evident in male offspring with increased blood pressure and heart rate and decreased body size. These unintended effects caused by transportation may have been attenuated with the 10 mGy in-utero exposure. Overall, these results suggest that prenatal radiation effects in mice are both dose- and gender-dependent, with even fairly low doses demonstrating (potentially adaptive) effects. There is a need for further study to better characterize the mechanism of this response. / Thesis / Master of Science (MSc)

Lake whitefish (Coregonus clupeaformis; LWF) are a cold-adapted freshwater species that are of both economic and cultural value. These fish spawn in lake areas where their embryos are exposed to thermal power plant effluents that may contain low levels of thermal, radiological and chemical stressors. Many studies on LWF embryonic development have looked at the individual effects of these stressors, but few have looked at the potential for combined effects. The combined effects of thermal and radiological stress were of interest due to growing evidence that mild thermal stress can produce an adaptive response, through the induction of the heat shock response (HSR), when followed with subsequent ionizing radiation stress. This thesis examined the combined impacts of thermal and radiological stress during LWF embryogenesis. LWF embryos were exposed to mild heat shocks (HS; Δ3 or 9°C) prior to a high dose of acute 137Cs gamma rays at 2, 6 and 24 hours post heat shock during the gastrulation or eyed stage. Heat shocked embryos were collected at each developmental stage and assessed for induction of heat shock protein (Hsp) genes. Following exposure, embryos were raised until hatch where mortality, morphometry, and embryo weight were measured. Mild HS induced Hsp70 mRNA expression at gastrulation, but not at the eyed stage. Embryos at hatch were not impacted by thermal or radiological exposure at the gastrulation stage. During the eyed stage, acute radiation treatment increased mortality and decreased body size at hatch. Mild HS prior to radiation did not provide protective effects and no adaptive response was observed. This thesis better defines the combined effects of thermal and radiological stress on the embryonic development of LWF. It also suggests that the ontogeny aspects of heat shock responses and radiosensitivity are important to consider for future adaptive response studies. / Thesis / Master of Science (MSc)

The communication of information between irradiated and non-irradiated bystander cell populations and the subsequent expression of radiation-like responses in the non-irradiated population, formally referred to as the radiation-induced bystander effect, is a very well established phenomenon in the study of radiobiology. Intercellular communication of bystander signals is known to occur via the exchange of soluble factors through biological fluids and via the transfer of molecules between adjacent cells via gap-junctions. Both of these communication methods require some degree of physical contact between biological entities. However, observations made in the literature demonstrating the induction of radiation effects in optically-coupled, yet chemically-separated organisms raises the hypothesis that alternative radiation bystander communication mechanisms may exist that have not yet been explored. Following the detection of significant photon emission from human keratinocyte cells exposed to ionizing beta-radiation by Ahmad in 2013, the involvement of an electromagnetic bystander signal was proposed. While not yet established in the field of radiobiology, intercellular communication via electromagnetic signalling is widely studied in the field of biophotonics. The emission of electromagnetic radiation from biological material, called biophoton emission, and the subsequent communication of effects using those signals has been characterized both spontaneously and as a result of perturbation by various stressors. This thesis therefore aimed to investigate intercellular communication via electromagnetic signalling stimulated by low-dose ionizing radiation to identify a possible convergence between the fields of biophoton communication and radiation-induced bystander effects. The characterization of biophoton emission from human cell cultures was accomplished using a single photon counting photomultiplier tube. The results revealed that biophoton emission is exacerbated by external stimulation (beta-radiation), it possesses a dependence upon the activity of radiation delivered, the density of the irradiated cell culture, and cell viability. These results suggest that biophoton emission is governed by physical transitions between excited and ground states and may further be modulated by metabolic processes. An effect of beta-radiation-induced biophoton emission upon non-irradiated bystander cells was identified and manifested as a reduction in cell survival. The modulatory effects observed following the application of photomodulating agents to the bystander cultures support ultraviolet electromagnetic radiation as a responsible factor in the communication of bystander signals. Observation of photon emission across the entire ultraviolet, visible and infrared spectra lead to the suggestion that ultraviolet wavelengths are only a portion of the signal responsible for eliciting bystander responses and that coherent interaction of multiple wavelengths is probable in the intercellular exchange of information. The possibility of a link between biophoton bystander signalling and soluble factor mediated bystander effects was investigated next by isolating exosomes from biophoton-exposed bystander cultures. Positive bystander responses were exhibited by secondary reporter cells incubated with the exosomes isolated from the biophoton-exposed bystander cultures, thereby suggesting that biophoton signalling is a possible form of biological redundancy where it acts as an intermediary to trigger soluble factor release and further reinforce intercellular communication. Finally, the effect of beta-radiation-induced biophoton signals upon mitochondrial activity was assessed and revealed the capacity for biophotons to downregulate Complex I and ATP synthase activity. The demonstrated effect of biophotons upon mitochondria elucidates a candidate mechanism worthy of further exploration to determine how biophotons may trigger responses in bystander cells. Overall, this thesis elucidates an additional mechanism for intercellular communication between biological systems perturbed by low doses of ionizing radiation, in the form of an electromagnetic signal. This work contributes to the current perspective on biophoton bystander signalling as a potential source of biological redundancy, facilitating a means of intercellular communication when optical coupling but not chemical contact is available in a given system. / Thesis / Doctor of Philosophy (PhD)

The commission on radiological protection through publication 118 decided to recommend a change to the eye dose limit in 2011. ICRP recommendations made in publications, especially ‘publication 60’ and its subsequent update ‘publication 103’ has served as standards for regulatory authorities worldwide in limiting ionizing radiation exposure both to workers and members of the public. For example in Canada, the Canadian Nuclear Safety Commission (CNSC) generally directly adopts recommendations from ICRP. The previous dose limit for the lens of the eye was 150 mSv year-1, based on Publication 60 and 103. Regulatory agencies worldwide have been using this value and subsequently nuclear facilities, hospitals and universities have designed their radiation protection program based on this dose limit for several decades. The new revised eye dose limit now being equivalent to the whole body dose limit will pose significant challenges for sectors where the eye exposure was not characterized as the limit was previously five times over the whole body exposure. A two-step approach was used in conducting this study, firstly a through literature search was conducted on the effects of ionizing radiation to the eye, its radiobiology, fundamentals in established both dose limits was analyzed. Secondly, the authors spent time researching institutions that use ionizing radiation and interviewed engineers, medical physicists, radiation safety officers and regulators from a wide array of fields and industries. Based on the ICRP publications, the review of the literature and the interviews conducted with the nuclear industry, there is consensus in Canada and among IAEA member states that the dose limit for the lens of the eye should be reduced from the original proposed limit of 150 mSv per year. However not to the recommendations suggested by ICRP 118, but, to a standard reasonable and an achievable limit that is 50 mSv per year. / Thesis / Master of Science (MSc) / The International Commission on Radiological Protection (“ICRP”), the independent governing body responsible for radiation protection, since the early 1950s has been issuing recommendations that are widely used as radiological protection standards by regulatory agencies worldwide, primarily UN member states. Since its inception in 1928, the ICRP has served as the basis for radiation protection and value based judgements in protecting both human and non-human biota. In 2011, the commission published (ICRP Pub. 118) its review of epidemiological studies and decided to recommend a change to the previously established eye dose limit. Based on the review of the literature and the research conducted within the academic, veterinary, nuclear and medical industry, there is general consensus in Canada and among IAEA members states that the dose limit for the lens of the eye should be reduced from the original proposed limit, but not to the recommendations suggested by ICRP 118.

Le traitement du cancer à l’aide d’une exposition aux radiations ionisantes (RI) peut mener au développement de plusieurs effets secondaires importants, dont un retard de réparation et de régénération des tissus. Les mécanismes responsables de ces effets demeurent largement inconnus encore aujourd’hui, ce qui a pour effet de limiter le développement d’approches thérapeutiques. À l’aide d’un modèle de guérison de plaie cutanée chez la souris, nous avons cherché à déterminer les mécanismes par lesquels l’exposition aux RI limite la régénération de la peau. Nos résultats démontrent que l’induction de la "stromal-derived growth factor 1α" (SDF-1α), une cytokine normalement surexprimée dans les tissus hypoxiques, est sévèrement diminuée dans les plaies de souris irradiées versus non-irradiées. Ce défaut corrèle avec un retard de guérison des plaies et est encore évident plusieurs mois suivant l’exposition aux RI, suggérant qu’il y a une altération permanente de la capacité de la peau à se réparer. Parce que SDF-1α est secrété principalement par les fibroblastes du derme, nous avons évalué le potentiel des cellules stromales multipotentes (MSCs), qui sont reconnues pour secréter des niveaux élevés de SDF-1α, à accélérer la régénération de la peau chez les souris irradiées. L’injection de MSCs en périphéries des plaies a mené à une accélération remarquable de la guérison de la peau chez les souris exposées aux RI. Les actions des MSCs étaient principalement paracrines, dû au fait que les cellules n’ont pas migré à l’extérieur de leur site d’injection et ne se sont pas différentiées en kératinocytes. L’inhibition spécifique de l’expression de SDF-1α a mené à une réduction drastique de l’efficacité des MSCs à accélérer la fermeture de plaie indiquant que la sécrétion de SDF-1α par les MSCs est largement responsable de leur effet bénéfique. Nous avons découvert aussi qu’un des mécanismes par lequel SDF-1α accélère la guérison de plaie implique l’augmentation de la vascularisation au niveau de la peau blessée. Les résultats présentés dans ce mémoire démontrent collectivement que SDF-1α est une importante cytokine dérégulée au niveau des plaies cutanées irradiées, et que le déclin du potentiel de régénération des tissus qui est observé suivant une exposition au RI peut être renversé, s’il est possible de restaurer le microenvironnement de la blessure avec un support stromal adéquat. / Cancer treatment using ionizing radiation (IR) may lead to significant side effects, like delayed tissue repair and regeneration. The mechanisms mediating these defects remain largely unknown at present, thus limiting the development of therapeutic approaches. Using a wound healing model, we investigate the mechanisms by which IR exposure limits skin regeneration. Our results show that induction of the stromal-derived growth factor 1α (SDF-1α), a cytokine normally overexpressed in hypoxic tissues, is severely impaired in the wounded skin of irradiated, compared to non-irradiated, mice. This defect is correlated with delayed healing, and is evident for several months following exposure to IR, suggesting permanent impairment of skin repair. Because SDF-1α is secreted mainly by dermal fibroblasts, we evaluated the potential of multipotent stromal cells (MSCs), which secrete high levels of SDF-1α, to improve skin regeneration in irradiated mice. Injection of MSCs into the wound margin led to remarkable enhancement of skin healing in mice exposed to IR. The MSC actions were mainly paracrine, as the cells did not migrate away from the injection site or differentiate into keratinocytes. Specific knockdown of SDF-1α expression led to drastically reduced efficiency of MSCs in improving wound closure, indicating that SDF-1α secretion by MSCs is largely responsible for their beneficial action. We also found that one mechanism by which SDF-1α enhances wound closure likely involves increased skin vascularization. Findings presented in this thesis collectively indicate that SDF-1α is an important deregulated cytokine in irradiated wounded skin, and that the decline in tissue regeneration potential following IR can be reversed, given adequate microenvironmental support.