This thesis investigates the tissue sparing effect of FLASH (>40 Gy/s) radiation, as opposed to CONV (conventional, dose rates typically between 0.01­0.1 Gy/s) radiation. We irradiated zebrafish embryos (4 days past fertilisation) with 116 MeV protons. The aim was (1) to measure the effect, and (2) if the effect were significant, see whether it depended on the oxygen concentration in the tissue, as the oxygen depletion hypothesis (a popular theory on the underlying mechanics of the FLASH effect) predicts. We irradiated embryos with either FLASH or CONV, where a possible FLASH effect would reduce toxicity of the FLASH radiation. We did the same for zebrafish which were deliberately put in a hypoxic condition prior to irradiation. In that case, the depletion hypothesis would predict that the difference between FLASH and CONV disappears. Our biomarkers for radiobiological damage were the survival rate and γ­H2AX foci formation. In our experimental conditions, the radiation effect on the survival rate was eclipsed by other factors which could not be isolated. We did confirm the possibility of using γ­H2AX foci formation as a marker for radiobiological damage in full­body irradiated zebrafish embryos. There were individual samples that showed clear and localised specific γ­H2AX signal, but these were too scarce and the signal was too inconsistent across samples to gather meaningful statistics. This was most often caused by limited antibody penetration in the embryo. We were therefore unable to draw conclusions about the FLASH effect. Better and more consistent antibody penetration, e.g. by longer digestion in collagenase before antibody staining, could change this in the future. We further custom­built and validated a hypoxic aquarium to produce hypoxic zebrafish tissue, as well as a computational model of the irradiation setup to simulate the dose distribution in the zebrafish container. We found the dose distribution to be sufficiently homogeneous for our experiment, at least 91.47% uniform for CONV and 90.72% uniform for FLASH.

The main challenge of radiotherapy, used for treating cancer, is to deposit the prescribed dose in the tumor volume while sparing the surrounding tissue.The depth-dose distribution of protons makes proton therapy an alternative to conventional radiotherapy for some tumor sites. A better knowledge of the proton radiobiological mechanisms can improve the effectiveness of radiotherapy treatments. Holland Proton Therapy Center (HPTC) is one of the Dutch proton therapy centers. One of the purposes of its experimental beamline is to perform radiobiological experiments. To conduct different types of pre-clinical experiments, the beamline must be equipped to provide large field irradiation with precise dose characterization. Moreover, having a reliable Monte Carlo(MC) model of the system allows to perform in silico verification of the beamline design and contribute to its optimization. In this context, the goals of this project were: implementing a dual-ring scattering system in the experimental room of HPTC to produce homogeneous fields of different sizes; creating a MC model of the HPTC passive beamline able to reproduce the experimental setup.A dual-ring double scattering system was implemented in the HPTC horizontal beamline, starting from a single 150MeV pencil beam. The resulting passive irradiation fields were characterized by measuring and analyzing the lateral beam profiles, the depth-dose distributions and the relative dose at target position. Moreover, the beam characteristics and setup were implemented in theTOPAS MC code. The beam source parameters, input of the MC model, were found by comparing the experimental and simulated beam envelope and depth-dose distributions of the pencil beam. Then, the passive system model was benchmarked with experimental data by evaluating the lateral profiles, Braggcurves and dose distributions.The results show that the implemented passive system can achieve dose uniformity between 96% and 99% for field sizes between 4x4cm2 and 20x20cm2for a 150 MeV proton beam. Moreover, using a collimator with a 5x5cm2aperture, uniformity of at least 97% in the different Bragg peak regions is achieved. Good uniformity is also obtained for beam energies in the range 115MeV-150 MeV, showing robustness of the setup. Furthermore, the range of the proton beams traversing the beam-shaping elements, as well as the energy arriving to target, were studied. Moreover, with a ridge modulator, Spread-OutBragg peaks were obtained with a width up to 3,4cm and of uniformity 98,5%.The MC model produced in TOPAS was first benchmarked against a 150MeVproton beam in air. Secondly, the experimental data of the large fields were compared with the simulated ones. The simulation of depth-dose distributions and lateral beam profiles agreed with the experimental data. Furthermore, the model was used to estimate the number of initial protons required to achieve an experimental dose and to estimate the location of the Bragg peaks.All in all, the dual-ring passive scattering setup has been successfully implemented and is ready to be used to perform radiobiological experiments. Also, the TOPAS MC model can be used to assist in the preparation of these experiments and to further optimize the beamline design.

As protons have a localized depth-dose distribution, proton therapy nowadays is a form of radiotherapy that is being implemented for part of the cancer patients. Compared to photon therapy it allows more precise targeting of tumours and sparing of surrounding healthy tissue. Besides patient treatment, research on proton therapy topics is being done to gain more knowledge on and keep improving the technique. Since the 1950s almost 100 proton therapy facilities are operating clinically worldwide. One of those facilities is HollandPTC, a proton therapy center located in Delft, The Netherlands. The ProBeam Varian cyclotron serves two treatment gantries, an eye treatment room, and a research experimental room. The experimental room is dedicated to research topics in the proton therapy field and is equipped with a fixed horizontal beam line that can be used for physics experiments and also for radiobiological experiments. In order to perform those types of research the beam needs to be fully characterized in terms of dose, shape, size and energy, this is the purpose of this work. The second goal was to commence with a setup for creating homogeneous fields by means of passive scattering and determine the optimal distances required between the elements of the setup. The single pencil beam characterization has been done by performing a large variety of experiments, making use of setups with different types of detectors. The experiments include beam spot, beam envelope, beam current, and depth-dose distribution measurements, and have been performed for nominal beam energies between 70 and 240MeV. For the passively scattered field a dual ring setup has been implemented. Spread-out Bragg peaks have been created with a ridge filter. An energy of 150 MeV was used, as the scattering elements have been designed for this energy specifically. The single pencil beam characterization resulted in beam spot sizes at the isocenter varying from 3.54 mm for 240 MeV, up to 5.47 mm for 70 MeV, with an asymmetry of 1.7% at most. The beam envelope measurements showed that the beam spot size just after the exit window is 2-3 mm and diverges up to 12 mm at 2 m from the exit window. The beam current measurements gave the transmission efficiency of the system, ranging from 0.04% for a 70 MeV beam up to 5.6% for 240 MeV, increasing exponentially. The depth-dose measurements provided the difference between the nominal beam energy and the beam energy at the isocenter. The difference becomes smaller as the nominal beam energy increases and is 2.4% for a 70 MeV nominal beam and 0.3% for a 230 MeV nominal beam. With the dual ring setup fields of up to 25 cm in diameter have been formed with a uniformity of at least 97%. The ridge filter created a spread-out Bragg peak of 2.8 cm with a uniformity of 98% and also gave good results for other beam energies (100-200MeV). It can be concluded that the goals of the project have been reached as the beam has been characterized in terms of shape, current, and energy. Also some preliminary work for the passive field has been accomplished. The work on the dual ring setup can be further expanded by placing the ridge filter and a collimator in the setup, after which the field can be characterized in terms of dose. The results obtained from the single pencil beam characterization allowed for the first physics experiments to start at the experimental room of HollandPTC during the last year.