Radiation-induced damage to the genomic DNA of cells may lead to errors in transcription and replication and, if not repaired correctly, these may result in mutations, genomic instability and cell death. Laser microbeams have generally been used by many research groups to investigate the real-time dynamics of protein recruitment in response to DNA insults in mammalian cells; however, such irradiations induce a plethora of DNA damage (including UV base damage, base damage, SSBs and DSBs and complex damage). A novel experimental setup has been designed capable of following the dynamics of protein recruitment in response to DNA insults in mammalian cells shortly following submicrosecond- pulsed electron irradiation of living mammalian cells, not possible using conventional irradiation techniques. This arrangement was developed based on a 6 MeV electron pulse linear accelerator, to deliver sparsely ionising radiation, coupled to an automated, time-lapse inverted epifluorescence microscope imaging system. An integrated robotic system contained within a physiological environment of 37°C and 5% CO₂ was used to transfer remotely and repetitively custom-designed cell dishes containing the mammalian cells between irradiation and imaging locations. Following the development of the linear accelerator and associated imaging devices, preliminary ‘proof-of-principle’ investigations were carried out using living HT1080 mammalian cells containing YFP-tagged 53BP1, an established biomarker of DSB, to follow the recruitment and loss of 53BP1 to sites of radiation-induced DNA damage in real-time. This novel experimental setup has allowed for the first time observations of the appearance and disappearance of radiation-induced foci in the same cell population at very early times. These single-foci studies have provided evidence for the formation of not only promptly formed DSBs but also late appearing DNA damage signalled by 53BP1. These data highlight different classes of DSBs formed in response radiation damage. Additionally, the role of cell cycle on the repair kinetics was undertaken using HT1080- 53BP1-YFP cells which also express Geminin-mCherry under appropriate selection. Geminin is increasingly expressed from early S-phase onwards, but is degraded following mitosis. Geminin-associated fluorescence can be used as a marker of progression through the cell cycle. 53BP1 repair kinetics were characterised in response to radiation damage in combination with ATM and PARP inhibitors. These studies provided supporting evidence for the existence of different classes of DSBs, potentially assigned to radiation-induced replication breaks and DSBs formed by enzymatic conversion of clustered damage. These preliminary ‘proof-of-principle’ findings using DNA damage repair as an example, emphasize the use of this novel technology to explore the dynamics of numerous other biochemical processes in living cells in real-time with the knowledge of being able to quantify the range of damage induced by IR coupled with accurate dosimetry. The knowledge obtained may be used to identify potential biological targets coupled with drug discovery for translation into adjuncts for radiotherapy.

In recent years intensity modulated radiation therapy with photons (xIMRT) has gained attention due to its ability to reduce the dose in the tissues close to the tumour volume. However, this technique also results in a large low dose volume. Electron IMRT (eIMRT) has the potential to reduce the integral dose to the patient due to the dose fall off in the electron depth dose curves. This dose fall off makes it possible to modulate the dose distribution in the direction of the beam by selecting appropriate electron energies. The use of a computer based energy selection method was examined in combination with the IMRT technique to optimise the electron dose distribution. It is clearly illustrated that the energy optimisation procedure reduces the dose to lung and heart in a breast cancer treatment. To shape the multiple electron subfields (beamlets) that are used in eIMRT, an electron multi leaf collimator (eMLC) is needed. However, photons produced in a conventional electron treatment head could penetrate such an added eMLC, thus producing an undesirable dose contribution. The leakage levels normally achieved are acceptable for standard single electron field treatments but could become unacceptably high in eIMRT treatments where a lot of small subfields are combined. To limit this photon contribution, the photon MLC (xMLC) was used to shield off large parts of the photon leakage. The effect of this xMLC shielding on the reduction of photon leakage, the electron beam penumbras, and electron output (dose level), was studied using Monte Carlo methods for different electron treatment head designs. The use of helium as a mean to reduce the electron scatter in the treatment head, and thus the perturbating effect of the xMLC on electron beam penumbra and output, was also investigated. This thesis shows that the effect of the xMLC shielding on the electron beam penumbra and output can be made negligible while still obtaining a significantly reduced x-ray leakage dose contribution. The result is a large gain in radiation protection of the patient and a better dynamic range for the eIMRT dose optimisation. For this optimisation a computer based electron energy selection method was developed and tested on two clinical cases.

Radionuclide molecular imaging is an emerging multidisciplinary technique that is used in modern medicine to visualise diseases at cellular and molecular levels. This thesis is based on five papers (I-V) and focuses on the development of site-specific radiolabelled recombinant anti-HER2 Affibody molecules and preclinical evaluations in vitro and in vivo of the labelled conjugates. This work is part of a preclinical development of an Affibody molecule-based tracer for molecular imaging of HER2 expressing tumours. Papers I and II report the evaluation of the Affibody molecule ZHER2:2395-C, site-specifically labelled with the radiometals 111In (for SPECT) and 57Co (as a surrogate for 55Co, suitable for PET applications) using a thiol reactive DOTA derivative as a chelator. Both conjugates demonstrated very suitable biodistribution properties, enabling high contrast imaging just a few hours after injection. Papers III and IV report the development and optimization of a technique for site-specific labelling of ZHER2:2395-C with 99mTc using an N3S chelating peptide sequence. 99mTc-ZHER2:2395-C demonstrated high and specific tumour uptake and rapid clearance of non-bound tracer from the blood, resulting in high tumour-to-non-tumour ratios shortly after injection, enabling high contrast imaging. In addition, in the study described in paper IV, freeze-dried kits previously developed for 99mTc-labelling were optimised, resulting in the development of a kit in which all the reagents and protein needed for labelling of ZHER2:2395-C with 99mTc were contained in a single vial. Paper V reports the evaluation of an anti-HER2 Affibody molecule, ABY-025, with a fundamentally re-engineered scaffold. Despite the profound re-engineering, the biodistribution pattern of 111In-ABY-025 was very similar to that of two variants of the parental molecule. It seems reasonable to believe that these results will also be applicable to Affibody molecules towards other targets. Hopefully, this work will also be helpful in the development of other small proteinaceous tracers.

<p>Cerebral radiation necrosis as a consequence of radiation therapy is often observed in patients several months to years after treatment. Complications include painful headaches, seizures, and in the worst-case death. Radiation necrosis is an irreversible condition with the options available to manage it all having noticeable downsides. As such, there is a critical need for better ways of either preventing the onset of necrosis and/or managing its symptoms. As radiation necrosis cannot be induced in humans for ethical reasons, a mouse model that mirrors the features of radiation necrosis observed in patients would allow for new techniques to be tested before being used in human clinical trials. This thesis will explain how our lab designed a murine model of cerebral radiation necrosis that uses a 320 keV cabinet irradiator to produce radiation necrosis and MRI and histology to evaluate the development of radiation necrosis at multiple time points.</p><p><br></p> <p> </p> <p>Our model required the development of a mouse positioning apparatus that could be used in the cabinet irradiator used as well as the machining of lead shields so that focal semi-hemispheric irradiations could be conducted with other critical structures spared. The MRI scans used as well as the algorithm used to draw radiation necrosis lesions were based off what has been used in previous Gamma Knife models of radiation necrosis. Our initial work showed that since the cabinet irradiator has a relatively flat dose distribution unlike the Gamma Knife, the radiation lesion volumes produced in the former either plateaued or decreased, unlike in the case of the latter where lesion volumes tended to decrease over time. Further work analyzed the effects of fractionation and found minimal sparing using four different fractionation schemes. The effects of strain and sex on the development of radiation necrosis were also analyzed, with strain being found to be a statistically significant parameter while sex was not. Future research should focus on testing the effects of new drugs and techniques for better dealing with radiation necrosis.<b></b></p>

<p>The need to understand and accurately assess the health risks of low dose ionizing radiation is more important now than ever before. The global applications of ionizing radiation in medicine, mining, manufacturing, and the nuclear industry have increased exponentially in recent years. Parallel to this increase are the health concerns regarding occupational and medical exposures to radiation. The research presented here investigates the biological and health effects of ionizing radiation, specifically from medical diagnostic exposures.</p> <p>Medical diagnostic procedures such as x-rays and computed tomography (CT) scans account for a notable portion of the public's exposure to ionizing radiation. The health risk to humans associated with these low dose exposures is unknown. Often times they are correlated with risk estimates derived from much higher radiation doses. There is no doubt that very high dose ionizing radiation can be harmful; however, the same notion does not exist regarding exposures to low dose ionizing radiation such as that from medical diagnostic CT exposures.</p> <p>The objective of this research is to address the effects and risks associated with diagnostic CT scans. This research focuses on the biological outcome of cancer which remains a primary concern in health care and the development of radiation risk policies. The investigation utilized various mouse models that have differing sensitivities to radiation and susceptibilities to developing radiation-induced cancer.</p> <p>Results from this research found that low-dose diagnostic CT scans do not increase risk and can, in fact, induce protective effects. The hypothesis that harmful effects increase linearly with radiation dose is not supported by this research. With low doses of CT scans, protective biological effects such as reduced chromosomal aberrations, decreased radiation-induced oxidative DNA damage, and enhanced clearance of damaged cells have been observed. In cancer-prone mice, CT scans can increase longevity and reduce cancer risk by delaying the latency of specific cancers.</p> <p>This research advances the understanding of the biological effects and health risk associated with low-dose medical diagnostic procedures. This research is timely and important to allow medical practitioners, policy makers, and regulators to make informed decisions about using ionizing radiation in the clinic. Such knowledge is valuable as better, more complex, and perhaps more damaging modalities are being used to image and manage disease.</p> / Doctor of Philosophy (PhD)

Anxiety disorders are very common and the primary feature is abnormal or inappropriate anxiety. Fear and anxiety is often mediated by the amygdala, a brain structure rich in substance P (SP) and neurokinin 1 (NK1) receptors. To learn more about how the human amygdala is modulated by fear and anxiety in event-triggered anxiety disorders and to investigate if the SP/NK1 receptor system is affected, regional cerebral blood flow (rCBF) ([15O]-water; Study I and II) and the SP/NK1 receptor system ([11C]GR205171; Study III and IV) were studied with positron emission tomography (PET). In Study I we investigated the neural correlates of affective startle modulation in persons with specific phobia by measuring rCBF during exposure to fearful and non-fearful pictures, paired and unpaired with acoustic startle stimuli. Fear-potentiated startle was associated with activation of the affective part of the anterior cingulate cortex and the left amygdaloid–hippocampal area. In Study II short-term drug treatment effects on rCBF in patients diagnosed with social phobia was evaluated, comparing the NK1 receptor antagonist GR205171 to the selective serotonin reuptake inhibitor citalopram and placebo. Social anxiety and neural activity in the medial temporal lobe including the amygdala was significantly reduced by both drugs but not placebo. In Study III we investigated if activity in the SP/NK1 receptor system in the amygdala would be affected by fear provocation in individuals with specific snake or spider phobia. Fear provocation was associated with a decreased uptake of the NK1 antagonist [11C]GR205171 in the amygdala, possibly explained by an increase in endogenous SP release occupying the NK1 receptors. Study IV was conducted to explore the resting state NK1 receptor availability in PTSD patients as compared to healthy controls. Increased resting state binding of the tracer [11C]GR205171 in the amygdala of patients with PTSD suggested an increased amount of available receptors. In summary, fear and fear-potentiated startle modulates the human amygdala, possibly through the SP/NK1 receptor system.