Radiation induced bystander effects have changed our understanding of the biological effects of ionizing radiations. The original assumption was that biological effects require direct damage to DNA. The bystander effect eliminated that requirement and has become one main stream in radiation research ever since first reported over 20 years ago. Most bystander studies to date have been carried out by using conventional single cell in vitro systems , 2D cell array and 3D tissue samples, which are useful tools to characterize basic cellular and molecular responses. But to reveal the complexity of radiation responses and cellular communication, live animal models have many advantages. In recent years, models such as C. elegans and Zebrafish have been utilized in bystander effects research. In the Loma Linda/TAMU experiment, a L1 larva C. elegans model was devloped to study the radiation bystander effects by irradiating single intestine cell nuclei with a microbeam of protons. Due to the stochastic nature of particle interactions with matter and changing stopping power when protons slow down, precise dosimetry in the target nucleus is a difficult problem. This research was undertaken to provide a detailed description of the energy deposition in the targeted and surrounding non-targeted cell nuclei, and to evaluate the probabilities of the non-targeted cell nucleus being irradiated. A low probability is required to exclude the possibility of radiation biological an effect in non-targeted cells is caused by scattered particles. Mathematical models of the microbeam system and the worm body were constructed in this research. Performing Monte Carlo simulations with computer code, Geant 4, this research provided dosimetry data in cell nuclei in different positions and Geant 4, this research provided dosimetry data in cell nuclei in different positions and probabilities of scattering to non-targeted cell nuclei in various microbeam collima- tor configurations. The data provided will be useful for future collimated microbeam design.

Ionising radiation can cause damage to DNA that can result in gene mutations contributing to carcinogenesis. Radiation-protection policy currently estimates cancer risks from exposures to radiation in terms of excess risk per unit dose. At very low radiation dose-rates, where not all cells are absorbing radiation energy, this formula carries the inherent assumption that risk is limited to those cells receiving direct energy depositions. Numerous studies have now called this assumption into question. Such low dose-rates are in the relevant range that the public receives from natural background and man-made sources, and, if this fundamental assumption proves unfounded, current estimations of radiation-induced cancer risk at low doses will be incorrect. Accurate predictions of stochastic cancer risks from low-dose radiation exposures are crucial to evaluating the safety of radiation-based technologies for industry, power generation and the increasing use of radiation for medical diagnostic and screening purposes. This thesis explores phenomena known as radiation-induced bystander effects. The term bystander effects, as used here, describes biological responses to ionising radiation (hitherto observed in vitro) in cells not directly traversed by an ionising track, due to intercellular signals received from neighbouring cells that did receive energy depositions. This study aimed to determine whether radiation effects are communicated between irradiated and unirradiated cells in vivo, and if so, whether this effect alters current estimations of cancer risk following low-dose radiation exposures. In order to answer these questions, an in vivo experimental system for studying bystander effects in mice was developed. The method was based on the adoptive transfer of irradiated splenocytes into unirradiated hosts with simultaneous identification of irradiated donor cells, and biological endpoints in unirradiated bystander cells in situ using fluorescence microscopy and image analysis. Splenocytes from donor mice were radiolabelled with 3H-thymidine or received an acute X-ray dose. The irradiated donor cells, labelled with a fluorescent probe, were then adoptively transferred into unirradiated recipient mice via the tail vein, whilst control mice received sham-irradiated donor cells. A proportion of the cells lodged in the recipient mouse spleens where they remained for a period before the tissues were cryopreserved. The locations of donor cells were identified in frozen spleen sections by the fluorescent probe, and the levels of apoptosis and proliferation were simultaneously evaluated in situ in the surrounding unirradiated bystander cells using fluorescence-based assays. Transgenic pKZ1 recipient mice were also used to quantify chromosomal inversions in bystander cells. Since three-dimensional spatial relationships were preserved, responses could be measured in the local area surrounding irradiated cells as well as further afield. Following the development of the irradiated-cell adoptive transfer protocol and validation of the sensitivity and reproducibility of the biological assays in situ, a series of experiments was performed. In the initial experiments, 500,000 radiolabelled cells (0.33 mBq.cell-1) were injected into recipient mice and the spleen tissues were isolated 22 h later. No changes in apoptosis or proliferation were detected in local bystander spleen cells or throughout the spleen, compared to mice receiving sham-radiolabelled donor cells. In subsequent experiments, the effects of a number of experimental conditions were explored including the injection of tenfold more donor cells, analysis of spleen tissues after three days lodging in vivo, radiolabelling of donor cells with 100-fold higher 3H dose-rate and irradiation of donor cells ex vivo with 0.1 or 1 Gy X-rays. In each case, no changes in apoptosis or proliferation were observed. The in vivo method described here was designed to simulate the conditions of a bystander scenario from low dose-rate exposures relevant to public radiation protection. Contrary to the many reports of bystander effects in vitro, experiments using this sensitive method for examining the in vivo responses of unirradiated cells to neighbouring low-dose irradiated cells, have so far shown no changes in bystander cells in the spleen. This adoptive transfer method is the first in vivo method for examining the effects of known irradiated cells exposed to low radiation doses at low dose-rates, on neighbouring cells in situ that are truly unirradiated. Both the irradiated and bystander cells are normal, non-transformed primary spleen cells functioning in their natural environment. This in vivo experimental system allows the examination of tens of thousands of bystander cells and has shown a remarkable sensitivity, with statistical power to rule out changes in apoptosis &lt10% from the control. The relevance of in vitro bystander findings is unclear. Many reported bystander effects are more analogous to the systemic communication of abscopal effects from highly irradiated tissues. Disagreement between experimental systems and difficulty in reproducing key results between laboratories further complicate the translation of bystander data in vitro to human risk-estimation. The radiation protection community has expressed its need for in vivo validation of the bystander phenomenon before it can be included into the appraisal of carcinogenic risk. This adoptive transfer method is now available to study a range of bystander endpoints and potential signalling mechanisms in vivo, and provides a way to translate the wealth of data previously collected in vitro into findings directly relevant to human risk-estimation.

<p>Radiation induced bystander effects have given the cancer risk analysis a whole new paradigm. However the actual mechanism involved in producing the effects is still not clear. The basic bystander signal is assumed to be a biological signal. In this study we proposed and tried to quantify the presence of a physical signal in the form of electromagnetic radiation that can trigger a biological response in the bystander cells. In bystander effect studies where the cells are exposed to very low fluence of charged particles there could be several regions that can produce electromagnetic radiation due to the process of atomic/molecular excitations and relaxations. We focused on quantifying the number of ultraviolet photons emitted when charged particles pass through different media that have relevance to radiation biology experiments. The choice of UV photons was made due to the reason that its effects on living cells are very well documented. For this purpose we developed a system which employed the technique of single photon counting to measure the light emitted from samples irradiated under vacuum by a charged particle beam. Photon counting was done using a fast photomultiplier tube (Hamamatsu R7400p) with a peak cathode response at 420 nm wavelength.</p> <p>In the early set of “proof of principle experiments” we tested polystyrene targets for ion beam induced luminescence. Polystyrene is one of the materials that are used as a cell substrate for radiation biology experiments. The luminescence yield from polystyrene was measured in terms of absolute value i.e. number of photons per second per unit solid angle. The output appeared to have a non-linear behavior with the incident Ion fluence: it rose exponentially to an asymptotic value. We irradiated the samples with beam energies varying from 1 MeV to 2.0 MeV and showed saturation at or before an incident fluence rate of 3×10¹³ H⁺/cm²s. The average saturation value for the photon output was found to be 40 × 10⁶ cps. Some measurements were performed using filters to study the emission at specific wavelengths. In the case of filtered light measurements, the photon output was found to saturate at 28×10³, 10×10⁶, and 35×10⁶ cps for wavelengths of 280±5 nm, 320±5 nm and 340±5 nm respectively. Using the IBIL signal evolution characteristics with the ion fluence we determined the ions produce a damage having a cross section of the order of 10^-14 cm² in polystyrene. The average radiant intensity was found to increase at wavelengths of 280 nm and 320 nm when the proton energy was increased. Having found an evidence of a significant production of UV in ion irradiated, biologically relevant, material we extended this study further into the measurements from other relevant materials in radiation biology.</p> <p>Here charged particle irradiation was performed using positively charged protons (H⁺) ranging in energy from 1.2 MeV to 2.2 MeV at a fluence rate of 2.7×10¹⁰ protons mm^-2s^-1.The materials chosen for this study were polypropylene, Mylar, Teflon, and Cellophane as they are all materials commonly used in radiation biology experiments as cell substrates or containers. In addition, we performed measurements of two NIST standard materials derived from living cells: oyster tissue and citrus leaves. These materials were measured as a powder.</p> <p>All the container materials were found to emit UV frequency photons at emission levels that are significant enough to warrant further investigation of the potential biological consequences. In addition, the NIST standard reference materials oyster tissue and citrus leaves also emitted UV when irradiated. This suggested that biological materials may themselves emit UV at significant levels when irradiated with charged particles.</p> <p>We established this further by irradiated cells with β-particles. Cells were plated in Petri-dishes of two different sizes, having different thicknesses of polystyrene (PS) substrate. Exposure of the cell substrates (polystyrene) only resulted in the production of 1035 photons per unit activity in μCi of ⁹⁰Y which was equivalent to an exposure of 840 <em>β</em>-particles/cm² to the substrate. For a collimated electron beam exposure, we observed 158-167 photons per unit μCi (18 β-particles per cm² on the substrate) for different thicknessesof the substrate. Upon irradiating HPV-G cells plated on the PS dishes we determined that the luminescence gradually increased with the increasing exposure of β-particles; reaching up to 250 % of that of the luminescence without any cells for an activity of 180 μCi. For general irradiation conditions we found statistically significant difference in luminescence output for varying cellular densities with cells only and with the application of medium on top of the cells. The colourless medium increased the total luminescence yield while the coloured medium decreased it. When the cells were irradiated using a collimated beam of electrons it was found that the luminescence decreases with the increasing cellular density thus providing an evidence of re-absorption of photons within the surroundings.</p> <p>After establishing the fact that charged particles induce light emission from the materials that have a relevance to the radiation biology experiments. We extended our study further to find out other sources of radiation that could affect the dose distribution in radiation biology experiments. In radiation biology experiments the low doses of radiation are usually delivered usingamicrobeam charged particle accelerator. Microbeams delivers a highly localized and small dose to the biological medium by using a set of collimators that confine the charged particle beam to a very narrow (micron level) region. Since the collimation block a significant proportion of the beam therefore there is a chance of the production of low energy x-rays and secondary electrons. We used Monte Carlo simulations to investigate the production of particle induced x-rays and secondary electrons in the collimation system and its possible effects on the final dose delivery to the biological medium. We found no evidence of the escape of x-rays or secondary electrons from the collimation system for proton energies of up to 3 MeV. The thickness of the collimators was sufficient to reabsorb all the generated low energy x-rays and secondary electrons. However if the proton energy exceeds 3 MeV then a significant proportion of 10 keV and 59 keV (K-α) x-rays can be emitted by the collimator. Further it was established that due to the phase space distribution of particles in various orientations with the beam axis there are significant chances of hitting non-targetted cells in microbeams that employ a collimator to confine the beam.This may happen due to the beam particles travelling obliquely with the beam axis thus passing the collimator edge and hitting the non-targetted cells. Another reason could be the scatter of beam particles inside the collimator.</p> <p>The evidence of the production of UV in materials relevant to the radiation biology experiments suggest that the conclusions and hypotheses derived from some radiation bystander experiments need to be re-thought, as charged particle irradiation leads to some level of UV emission in experimental materials which may be the cause of some “non-targeted” effects.</p> / Doctor of Philosophy (PhD)

Workplace environments and the dynamics that exist within them affect everyone involved, especially coworkers. Although research has investigated how workplace bullying impacts its victims and the organization, little research has examined the effects of workplace bullying from the role of the bystander. Fewer have investigated how Perceived of Organizational Support (POS) may affect the employee's work engagement of those witnesses. The goal of this quantitative study was to determine the effect of POS on work engagement in the employees who witness workplace bullying. An online survey was used with the Negative Acts Questionnaire-Revised, (NAQ-R), Utrecht Work Engagement Scale (UWES) and POS instruments. It was launched on LinkedIn and 152 respondents participated and were asked to snowball the link. The study employed the affective events theory that presupposes that the occupational atmosphere influences those in proximity to negative behaviors. Regression results showed that only POS (t (150) = 5.14, p < 0.001) predicted employees' work engagement. On the other hand, witnessing workplace bullying (t (150) = -0.69, p = 0.49) did not affect employees' work engagement. This study provides a useful framework to illustrate how the environment of workplace bullying affects an organization's human and fiscal resources, contributing to the body of knowledge that can benefit organizations by helping to affect social change.

The historical narrative and our understanding about the low dose effects of radiation on the immune system has changed drastically from the beginning of the 20th century to now. A paradigm shift from the DNA target hit model to the one that also considers non-targeted effects (NTE) has attracted a lot of interest recently. Investigations to delineate mechanisms of NTE in the biological tissue have been carried out by various research groups where radiation induced genomic instability (RIGI), bystander effect (RIBE) and abscopal effect (AE) are the effects with most evidence available. This thesis addresses the question of whether low dose ionizing radiation (LDIR) stimulates or suppresses the immune system and how NTEs contribute to this immune modulation by adopting a two-pronged approach where first a narrative review constituting the introduction and literature review was performed followed by a systematic review using PRISMA guidelines to synthesize existing LDIR literature. This was prompted by our recent discovery that UVA photons are emitted by the irradiated cells and that these photons can trigger bystander effects in unirradiated recipients of these photons. Given the well-known association between UV radiation and the immune response, where these biphotons may pose as bystander signals potentiating processes in deep tissues as a consequence of ionising radiation, it is timely to revisit the field with a fresh lens. After reviewing various pathways and immune components that contribute to the beneficial and adverse effects induced by LDIR, it was found that these modulations can occur by way of NTE. However, the exact mechanistic underpinnings are still unclear and the literature examining low to medium dose effects of ionising radiation on the immune system is complex and controversial. Early work was compromised by lack of good dosimetry while later work mainly focuses on the involvement of immune responses in radiotherapy which typically uses high dose radiation. There is a lack of research in the LDIR/NTE field focussing on immune responses although bone marrow stem cells and lineages were critical in the identification and characterisation of NTE. This may be in part, a result of the difficulty of isolating NTE in whole organisms which are essential for good immune response studies. Models involving inter organism transmission of NTE are a promising route to overcome these issues. It is concluded that the simple question of whether LDIR stimulates or suppress the immune system is not as simple as initially hypothesized. An attempt was made to analyze if LDIR shifts the balance to immune suppression or enhancement via systematic review but, due to too many differences in the experimental methods in the current radiation and immune studies, a cookie-cutter answer was not possible. However, this thesis did point out the areas of concern such as lack of standardised tools in the field of radiobiological experimental research and quality of methods used which requires urgent attention. / Thesis / Master of Science (MSc)

This thesis demonstrates the relevance of bystander effect mechanisms after exposure to two Synchrotron modalities – Microbeam Radiation Therapy and Pencilbeam – that are currently at the preclinical stage but aim to treat brain tumours. We elucidate the relationship between the hyper-radiosensitivity phenomenon and radiation-induced bystander effects by studying the dose response of three glioma cell lines. The relevance of these low-dose effects for both Synchrotron modalities is because the tissue exposed to low valley-doses is predicted to be where hyper-radiosensitivity and bystander effects might be expected to predominate. In vivo experiments were conducted in the European Synchrotron radiation Facility in Grenoble, France and also in the University of Freiburg’s Hospital in Freiburg, Germany. Experiments conducted in vitro were performed at McMaster University. The most relevant results of this thesis revealed that the low-dose hyper-radiosensitivity phenomenon can coexist with radiation-induced bystander effects and evidence points towards bystander signalling mechanisms as the primary cause of cell killing during hyper-radiosensitivity. Bystander and abscopal effects can occur in rats and even in immune-compromised nude mice after exposure to Synchrotron Microbeam Radiation and Pencilbeam. Bystander effects can be communicated from irradiated rats to healthy unirradiated cage mate rats and the presence of a tumour modulates both the bystander and abscopal responses. The γ-H2AX biomarker can successfully be used for the detection of DNA damage in the brain of rodents after Synchrotron Radiation. In conclusion, this thesis considerably expands the understanding of the role of bystander effects in cells lines, tissues, and animals exposed to Synchrotron radiation. It is suggested that further exploration of the role of bystander effects and hyper-radiosensitivity during Synchrotron treatments could identify new targets leading to better tumour control. / Thesis / Doctor of Science (PhD)

abstract: Sexual harassment has emerged as a widespread problem facing women in public space in Egypt. Activism to combat sexual harassment began in 2005. However, just prior to and in the years following the January 25, 2011 Egyptian Revolution, which witnessed an increase in the collective sexual harassment, assault and rape of women, this activism has increased. Subsequently, scholarly attention to sexual harassment and public sexual violence has also expanded. Much of the attention in scholarly analyses has been directed toward politically motivated sexual violence, focused on understanding the state commissioning of sexual violence against female protestors to drive them from protest participation. There is an emerging critique of activist approaches that seems to ignore the politicalized nature of sexual harassment to focus instead on “cultural” targets. The early work of the Egyptian Center for Women’s Rights (ECWR) and current work of HarassMap have been criticized for depoliticizing sexual harassment by failing to include an analysis of state-commissioned sexual violence in their work. Similarly, both have been accused of expanding the scope of the security state by calling for increased policing of public space to protect women from “culturally-bad” men. With data collected through one year of participant observation with HarassMap, interviews with activists from eleven anti-sexual harassment initiatives and advocacy NGOs, and community-level surveys with non-activist individuals, this dissertation argues that “cultural” work undertaken through the community-based approaches by entities like ECWR and HarassMap is, in fact, an inherently political process, in which political engagement represents both an attempt to change political culture and state practice and a negotiative process involving changing patriarchal gender norms that underpin sexual harassment at a society-wide level. New conceptualizations of sexual harassment promoted by anti-sexual harassment initiatives and NGOs in Egypt frame it as a form of violence against women, and attempt to make sexual harassment an offense that may be criminalized. Yet, this dissertation contends there is a tension between activist and widespread public understandings of sexual harassment, predicated on the incomplete framing of sexual harassment as a form of violence. / Dissertation/Thesis / Doctoral Dissertation Anthropology 2016

L’efficacité d’une radioimmunothérapie (RIT) peut impliquer la coexistence des effets ciblés et des effets dit « non ciblés ». Les effets ciblés regroupent les effets biologiques observés dans les cellules ou tissus traversés par les particules ionisantes alors que les effets non-ciblés (ou bystander) sont observés dans des cellules qui n’ont pas été irradiées mais qui sont au proche voisinage des cellules exposées. Nous avons au cours de cette étude évalué in vitro et in vivo, la contribution des effets ciblés et non ciblés dans l’efficacité obtenue lors de la RIT alpha (212Pb, 213Bi) et de la RIT Auger (125I). Les effets ciblés ont été mesurés in vitro sur les cellules irradiées (cellules donneuses) alors que les effets bystander sont mesurés sur les cellules non irradiées (cellules receveuses) par une méthode de transfert de milieu. Elle consiste, à traiter les cellules receveuses dans un milieu de culture pré-incubé pendant 2h avec les cellules donneuses. Nos résultats montrent que la contribution des effets ciblés est nettement plus importante qu’en RIT alpha qu’Auger. En RIT alpha, on observe que les lésions de l’ADN (foci 53BP1et γ-H2AX) pourraient être différenciées en lésions complexes (sites multilésés = observation de gros foci) ou simples lésions (petit foci). Par contre en RIT Auger, ce sont les effets non ciblés qui prédominent sur les effets ciblés. L’utilisation d’inhibiteurs pharmacologiques des ROS montre l’implication du stress oxydatif dans ces effets non ciblés observés en RIT alpha et Auger. Ces effets non ciblés ont été observés également in vivo sur des souris athymiques porteuses de carcinoses péritonéales de petites tailles ; démontrant ainsi leur contribution dans l’efficacité thérapeutique finale observée après la RIT alpha et Auger. L’ensemble de ces résultats indiquent que même si des lésions de l’ADN sont produites après irradiation, que les effets non ciblés pourraient aussi contribuer à l’efficacité thérapeutique finale observée avec les anticorps couplés aux émetteurs de particules alpha ou d’électrons Auger. Ces résultats sont particulièrement intéressants pour la thérapie ciblée car les vecteurs utilisés n’ont pas souvent accès à l’ensemble des cellules constituant la tumeur. / We investigated in vitro and in vivo the relative contribution of targeted and non-targeted effects in the therapeutic efficacy against tumors of antibodies radiolabeled with alpha particle (212Pb, 213Bi) or Auger electron (125I) emitters. Targeted effects occurs in cells directly crossed by ionising particles while non-targeted effects are measured in cells neighbouring irradiated cells. Targeted effects were measured in vitro in cells exposed to antibodies radiolabeled with alpha or Auger emitters (donor cells) while non-targeted effects were investigated in recipient cells. Recipient cells consisted of cells not exposed to radiolabeled-mAbs, but grown in medium previously incubated for 2h with donor cells. We showed that the relative contribution of targeted effects versus non-targeted effects was higher during alpha RIT than Auger RIT. Alpha particles produced 53BP1 and gamma-H2AX foci in donor cells that could be differentiated in large, medium and small foci, while only small foci were observed in recipient cells. We assumed that large foci would correspond to locally multiply damage sites in DNA. Conversely, Auger RIT led predominantly to non-targeted effects compared with targeted effects. Use of radical scavengers showed that oxidative stress was involved in non-targeted effects. In vivo, we showed in athymic nude mice bearing tumor xenograft that non-targeted effects were also involved and participated to therapeutic efficacy of radiolabeled antibodies. These results indicate that although producing single DNA lesion, non-targeted effects can contribute to the therapeutic efficacy of mAbs radiolabeled with alpha particle or Auger electron emitters. These findings are particularly relevant for targeted therapy in which vectors cannot gain access to every tumor cell.

<p>Microbeam radiation therapy (MRT) is an experimental radiotherapy concept that has been primarily developed for the treatment of malignant brain tumours. MRT uses high flux synchrotron x-rays delivered as an array of parallel microbeams in high doses of irradiation in fractions of seconds. The aims of this study were to 1) investigate the induction of bystander effects after normal and tumour-bearing rat brains were exposed to MRT and homogenous radiation; 2) validate a brain bystander proteome by detecting protein expression throughout immunohistochemistry: and 3) to investigate whether communication of bystander signals can be produced between animals.</p> <p>Healthy and tumour-bearing Wistar rats were exposed to 17.5, 35, 70 or 350 Gy of MRT or homogenous field of synchrotron radiation to the right brain hemisphere. To study the communication of bystander effects between animals, irradiated rats shared the same cage with non-irradiated rats over a period of 48 hours. After euthanasia of the animals, brains and bladders were dissected, and samples for immunohistochemistry and bystander clonogenic assays were set up.</p> <p>Clonogenic survival of the reporter HPVG cells showed that bystander effects occurred in both the non-irradiated hemisphere and bladder of normal and tumour-bearing rats, while the irradiated hemisphere showed the direct effects of radiation. Moreover, communication of bystander signals was confirmed in the non-irradiated rats.</p> <p>In conclusion, the results suggest that the MRT and homogenous radiation of unilateral normal and tumour-bearing rat brains produce bystander signals that affect the whole organism and that those signals also can be transmitted to non-irradiated animals.</p> / Master of Science (MSc)

At low doses, relevant to nuclear incidents and accidental releases of radioactivity, the detriment of radiation extends beyond direct effects. This thesis investigates genomic instability, a subclass of non-targeted effects where damage and lethality is transmitted vertically and expressed in the progeny of cells many generations after initial radiation exposure. Through a series of experiments using clonogenic assay of human and fish cell culture, studies described in this thesis describe lethal mutations, hyper radiosensitivity and increased radioresistance – processes involving repair mechanisms that dictate survival in cells exposed to low doses. Further study investigates the difference in the relative biological effect of alpha particle radiation compared to what is expected at high doses. Results demonstrate increased radioresistance in a human cell line while also revealing increased lethality in a fish cell line confirming the need for consideration of dose-dependence as well as variance in behaviors of different cell lines and species. It is hoped the conclusions of this thesis will inspire the creation of protocols with greater attention to the indirect consequences of exposure to radiation at doses relevant to nuclear incidents and accidents. / Thesis / Master of Science (MSc)