Low-energy X-rays in the range 10 - 50 keV have a wide application. One important application in radiological diagnostics is mammography, whereas, in radiotherapy, they are used for irradiation of superficial tumours, in brachytherapy and photon activation therapy. The importance of soft X-rays for fundamental radiobiological research is based on the fact, that all species of ionizing radiation produce a wide spectrum of secondary electrons, mainly responsible for the primary damage to be transformed into an observable radiobiological effect. By variation of the primary soft X-ray energy, a variation in the secondary electron spectra and therefore in the local energy deposition is provided. However, up to now no definitive conclusions about the relative biological effectiveness (RBE) of soft X-rays can be made due to its dependence on the photon energy, biological endpoint and dose range and the consequent large spread of the published data. The superconducting electron linear accelerator of high brilliance and low emittance (ELBE) at the Forschungszentrum Rossendorf with an electron energy up to about 40 MeV is used, among all, to generate X-rays in a wide energy range. One method for production of intensive, quasi-monochromatic X-rays in the energy range 10 - 100 keV tunable in energy, is by channeling of electrons in a perfect crystal. This X-ray source has many advantages over the most widespread X-ray source, which is the X-ray tube. Although the physical basis of the channeling effect has been previously investigated, the feasibility of an X-ray source based on channeling radiation (CR) for radiobiological studies has been for the first time theoretically and experimentally studied and a dedicated CR source built and optimised in the frame of this thesis. CR has been theoretically characterised in order to estimate its applicability for radiobiological studies. A good agreement between the theoretical predictions and the measured data has been found. The intrinsic properties of the CR source have led to the conclusion that monochromatisation is necessary. A monochromator based on HOPG mosaic crystals, was designed and manufactured. The dosimetrical methods have been investigated at the CR source as well as at an X-ray tube. Absolute dose measurements using an ionisation chamber and spectral dose distribution determination using semiconductor detectors have been performed. In addition, an unconventional system based on thermally stimulated exoelectron emission (TSEE) detectors, allowing to measure dose in a small volume and in the real cell environment has been tested and has proven itself appropriate in a variable dose range and in a liquid environment, in cases where reproducible laboratory conditions are provided. The biological endpoints clonogenic cell survival and micronucleus induction have been optimised for two established cell lines. The human mammary epithelial cells MCF-12A have been chosen due to the importance of RBE of soft X-rays for determination of risk from mammography. On the other hand, the use of the widespread mouse fibroblast cell line NIH/3T3 allows to compare the results with previously published data. The influence of the specific irradiation procedure at ELBE on the control level of cell survival and micronucleus induction has been tested and an irradiation system was developed and constructed. In addition, the RBE for soft X-rays was determined by X-ray tube irradiation at the Medical Department of Technische Universität Dresden. The RBE of 10 kV and 25 kV X-rays relative to 200 kV X-rays was determined. The RBE was found to be in the range from 1.0 to 1.4, depending on the used radiation quality, cell line and the biological endpoint, in agreement with previously published data for the same radiation qualities. These results confirm that systematical studies of RBE dependence on photon energy at the ELBE CR source are necessary and feasible.

Recently a powerful electron accelerator, 50 MeV race-track microtron, has been taken into clinical use. This gives the opportunity to treat patients with higher x-ray and electron energies than before. Furthermore, treatments can be performed were the entire fractional dose can be delivered in parts of a second. The relative biological effectiveness (RBE) of high energy photons (up to 50 MV) was studied in vitro and in vivo. Oxygen enhancement ratio (OER) of 50 MV photons and RBE of 50 MeV electrons were investigated in vitro. Single-fraction experiments, in vitro, using V-79 Chinese hamster fibroblasts showed an RBE for 50 MV x-rays of approximately 1.1 at surviving fraction 0.01, with reference to the response to 4 MV x- rays. No significant difference in OER could be demonstrated. Fractionation experiments were carried out to establish the RBE at the clinically relevant dose level, 2 Gy. The RBE calculated for the 2 Gy/fraction experiments was 1.17. The RBEs for 20 MV x-rays and 50 MeV electrons were equal to one. In order to investigate the validity of these results, the jejunal crypt microcolony assay in mice was used to determine the RBE of 50 MV x-rays. The RBE for 50 MV x-rays in this case was estimated to be 1.06 at crypt surviving fraction 0.1. Photonuclear processes are proposed as one possible explanation to the higher RBE for 50 MV x-rays. Several studies of biological response to ionizing radiation of high absorbed dose rates have been performed, often with conflicting results. With the aim of investigating whether a difference in effect between irradiation at high dose rates and at conventional dose rates could be verified, pulsed 50 MeV electrons from a clinical accelerator were used for experiments with ultra high dose rates (mean dose rate: 3.8 x 10^ Gy/s) in comparison to conventional (mean dose rate: 9.6 x 10"^ Gy/s). V-79 cells were irradiated in vitro under both oxic and anoxic conditions. No significant difference in relative biological effectiveness (RBE) or oxygen enhancement ratio (OER) was observed for ultra high dose rates compared to conventional dose rates. A central issue in clinical radiobiological research is the prediction of responses to different radiation qualities. The choice of cell survival and dose response model greatly influences the results. In this context the relationship between theory and model is emphasized. Generally, the interpretations of experimental data are dependent on the model. Cell survival models are systematized with respect to their relations to radiobiological theories of cell kill. The growing knowledge of biological, physical, and chemical mechanisms is reflected in the formulation of new models. This study shows that recent modelling has been more oriented towards the stochastic fluctuations connected to radiation energy deposition. This implies that the traditional cell survival models ought to be complemented by models of stochastic energy deposition processes at the intracellular level. / <p>S. 1-44: sammanfattning, s. 47-130: 5 uppsatser</p> / digitalisering@umu

[EN] The hormones Gibberellins (GAs) are growing regulators that control fruit set and fruit growth. GAs are perceived by its nuclear receptors GID1 (GA INSENSITIVE DWARF1) (GID1A, GID1B y GID1C), which then trigger degradation of downstream repressors DELLA which are negative regulators of GA response. Our general goal is to know the molecular mechanisms by which GA-mediated fruit set. To understand which of the three GA receptor genes and four DELLA proteins are involved during fruit initiation in Arabidopsis, we have examined their temporal and spatial localization and expression, respectively and identify tissue-specific interactions between GID1 and DELLA. Our data suggest that GID1A can interact with RGA and GAI in all tissues, whereas GID1C-RGL1 and GID1B-RGL2 interactions only occur in valves and ovules, respectively. Functional study of gid1 mutant combinations confirms that GID1A plays a major role during fruit-set and growth, whereas GID1B and GID1C have specific roles in seed development and pod elongation, respectively. Therefore, in ovules, GA perception is mediated by GID1A and GID1B, while GID1A and GID1C are involved in GA perception in valves. On the other hand, to identify which are the genes that may participate in fruit set mediated by GAs, we have used transcriptomic analysis to detect genes regulated in the 4xdella mutant, fruits induced by GAs and pollinated fruits. As many as 10,000 genes appear to be differentially regulated, which suggest the complexity of this process. Among the differential genes, many of them encode for transcription factors that may regulated the GA response in fruit and some of them were tested as early targets of DELLA. The implication in the GA pathway of early target genes, RBE and PIL2, was studied by mutant phenotype analysis and in vitro assays for protein-protein interaction. / [ES] Las giberelinas (GAs) son reguladores del crecimiento que controlan la formación y desarrollo del fruto. Las GAs son percibidas por los receptores nucleares GID1 (GA INSENSITIVE DWARF1) (GID1A, GID1B y GID1C), mediando así la degradación de las proteínas DELLA que actúan como reguladores negativos de la respuesta a GAs. Nuestro objetivo es conocer cuáles son los mecanismos moleculares por los cuales las GAs median la formación y desarrollo del fruto. Para comprender qué receptores GID1 y qué proteínas DELLA (GAI, RGA, RGL1 y RGL2) participan en la formación del fruto hemos analizado su expresión espacial y temporal, identificando las posibles interacciones específicas de tejido entre los GID1 y DELLA. GID1A puede interactuar con RGA y GAI en todos los tejidos del pistilo, mientras que las interacciones entre GID1C-RGL1 y GID1B-RGL2 solamente ocurren en valvas y óvulos, respectivamente. El análisis de alelos mutantes gid1 indica que GID1A tiene una función principal en el crecimiento del fruto, mientras que GID1B y GID1C tienen funciones específicas en desarrollo de la semilla y elongación de la vaina, respectivamente. Por tanto la percepción de GAs en óvulos es mediada por GID1A y GID1B, mientras que en la valva lo es por GID1A y GID1C. Por otro lado, para identificar cuáles son los genes regulados por GAs que participan en la formación del fruto, hemos realizado un análisis transcriptómico en el mutante 4xdella y en frutos partenocárpicos inducidos por GAs y polinizados, identificando más de 10,000 genes diferenciales, lo que sugiere la complejidad del proceso. De éstos se han reconocido varios factores transcripcionales que potencialmente pueden mediar la respuesta a GAs en el fruto, y hemos determinado cuáles de ellos son dianas directas de DELLA. Hemos analizado con más detalle la función de dos de ellos, RBE y PIL2, mediante el análisis de los fenotipos mutantes e interacciones in vitro proteína-proteína. / [CAT] Les gibberel·lines (GAs) són reguladors del creixement que controlen la formació i desenvolupament del fruit. Les GAs són percebudes pels receptors nuclears GID1 (GA INSENSITIVE DWARF1) (GID1A, GID1B y GID1C), intervenint així la degradació de les proteïnes DELLA que actuen com reguladors negatius de la resposta a GAs. El nostre objectiu és conéixer quins són els mecanismes moleculars pels quals les GAs mitjancen la formació i desenvolupament del fruit. Per a comprendre que receptors GID1 i que proteïnes DELLA participen en la formació del fruit hem analitzat la seua expressió espacial i temporal, identificant les possibles interaccions específiques de teixit entre els GID1s i DELLAs. GID1A pot interactuar amb RGA i GAI en tots els teixits, mentre que les interaccions entre GID1C-RGL1 i GID1B-RGL2 solament ocorren en valves i òvuls, respectivament. L'anàlisi d'al·lels mutants gid1 indica que GID1A té una funció principal en el creixement del fruit, mentre que GID1B i GID1C té funcions específiques en desenvolupament de la llavor i elongació de la beina, respectivament. Per tant la percepció de GAs en òvuls és mitjançada per GID1A i GID1B, mentre que en la valva ho és per GID1A i GID1C. Per altra banda, per a identificar quins són els gens regulats per GAs que participen en la formació del fruit, hem realitzat una anàlisi transcriptòmatic en el mutant 4xdella i en fruits induïts per GAs i pol·linitzats, identificant més de 10,000 gens diferencials, la qual cosa sugereix la complexitat del procés. D' aquests s'han identificat diversos factors transcripcionals que potencialment poden intervenir en la resposta a GAs en el fruit, i hem determinat quins d'ells són dianes directes de DELLAs. Hem analitzat amb més detall la funció de dos d'ells, RBE i PIL2, mitjançant l'anàlisi dels fenotips mutants i interaccions in vitro proteïna-proteïna. / Gallego Giraldo, C. (2015). PERCEPCIÓN Y SEÑALIZACIÓN DE LAS GIBERELINAS DURANTE LA FRUCTIFICACIÓN EN ARABIDOPSIS THALIANA [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/50429 / TESIS

Protonentherapie ist eine wichtige Behandlungsmodalität in der Radioonkologie. Aufgrund einer vorteilhaften Dosisverteilung im bestrahlten Volumen kann diese Bestrahlungsmethode das tumorumgebende Normalgewebe schützen. Dadurch können Nebenwirkungen in bestimmten Patientenpopulationen, zum Beispiel Kindern oder Patienten mit Gehirntumoren, verringert werden. Trotzdem können nach Protonenbestrahlung von Gehirntumorpatienten Normalgewebsschäden auftreten. Gründe dafür können der notwendige klinische Sicherheitssaum im Normalgewebe, der Einfluss der relativen biologischen Wirksamkeit RBE sowie eine erhöhte Strahlensensitivität bestimmter Gehirnregionen sein. Um diese Aspekte zu beleuchten, werden geeignete präklinische Modelle für die Normalgewebsreaktion im Gehirn nach Protonenbestrahlung benötigt. Darüber hinaus kann eine Risikostratifizierung der Patienten durch die Vorhersage von Nebenwirkungswahrscheinlichkeiten oder der Tumorantwort den Behandlungserfolg erhöhen. Auch hier können präklinische Modelle helfen, um neue prädiktive Biomarker zu finden und um die zugrunde liegenden Mechanismen strahleninduzierter Gehirnschäden besser zu verstehen. Das Ziel dieser Dissertation war die Etablierung und Charakterisierung von adäquaten präklinischen Modellen für die Untersuchung von strahleninduzierten Normalgewebsschäden im Gehirn. Diese Modelle bilden die Grundlage für zukünftige Studien zur Untersuchung von RBE Effekten, der spezifische Strahlensensitivität einzelner Gehirnregionen und neuer Biomarker. Die getesteten Modellsysteme waren in vitro Kulturen von adulten organotypischen Gehirnschnitten, Tumorschnittkultur sowie in vivo Bestrahlung von Gehirnsubvolumina, jeweils mit dem Modellorganismus Maus. Die Etablierung eines Bestrahlungssetups in der experimentellen Protonenanlage und dessen dosimetrische Charakterisierung waren von großer Bedeutung für die Durchführung der biologischen Experimente. Ein weiteres Hauptziel war die Definition klinisch relevanter Endpunkte für frühe und späte Nebenwirkungen. Die Gewebsschnitte wurden durch Messungen des Zellüberlebens und der Entzündungsreaktion, sowie mittels in situ Analyse von Zellmorphologie und DNA Schäden untersucht. Als ergänzendes Modell wurde die Tumorschnittkultur etabliert und ähnliche Endpunkte analysiert. Adulte Gehirnschnitte stellten sich als ungeeignet für präklinische Experimente in der Radioonkologie heraus. Die Messungen von Zelltod und Entzündungswerten zeigten eine starke Zellreaktion auf die Inkulturnahme, aber keine auf die Protonenbestrahlung. In der Histologie wurden gestörte Zellmorphologie, reduzierte Vitalität und eingeschränkte Reparaturfähigkeit von DNA Schäden beobachtet. Daher sollten für strahlenbiologische Experimente andere 3D Zellkulturmodelle in Betracht gezogen werden, wie zum Beispiel Organoide oder durch Tissue Engineering hergestellte Kulturen. Durch die Publikation der Daten leistet diese Dissertation einen wichtigen Beitrag zur aktuellen Forschung, da so künftig die limitierten Ressourcen, die für strahlenbiologische Experimente mit Protonen zur Verfügung stehen, auf relevantere Modelle verwendet werden können. Die Bestrahlung von Gehirnsubvolumina in Mäusen wurde mit dem Ziel etabliert, klinisch vergleichbare Felder zu erreichen. Das gewählte Zielvolumen war der rechte Hippocampus; der Protonenstrahl sollte in der Mitte des Gehirns stoppen. Im Rahmen des Projekts wurde ein Arbeitsablauf für präzise und reproduzierbare Bestrahlung entwickelt. Zur Verifizierung wurde der induzierte DNA Schaden ausgewertet und anschließend mit Monte-Carlos Dosissimulationen korreliert. Die Maushirnbestrahlung lieferte wertvolle Ergebnisse für frühe Zeitpunkte (d.h. innerhalb 24 h nach Bestrahlung). Im Verlauf des Projekts wurde ein Algorithmus erstellt, der schnell und zuverlässig die räumliche Verteilung des DNA Schadens in Relation zur Gesamtzellzahl analysiert. Diese Auswertung zeigte, wie bei der Bestrahlungsplanung vorgesehen, ein Stoppen des Protonenstrahls im Gehirn. Eine anschließende Korrelation der Schadensverteilung mit der applizierten Dosis weist nach, dass das Modell einen wichtigen Beitrag zur Untersuchung des RBE leisten kann. In einer darauf folgenden Studie wurde der Dosis-Zeitverlauf der beobachteten Strahlenreaktion des Normalgewebes genauer beleuchtet. Dafür wurden Untersuchungen des Allgemeinzustands der Versuchstiere, regelmäßige Magnetresonanztomografie (MRI) Messungen über einen Zeitraum von sechs Monaten, sowie abschließende Histologie korreliert. Die Volumenzunahme des Kontrastmittelaustritts, die den Zusammenbruch der Blut-Hirn-Schranke anzeigt, wurde konturiert; aus diesen Daten entstand ein prädiktives Dosis-Volumen Modell. Die Pilotstudie konnte eine dosisabhängige Strahlenreaktion nachweisen, die sich im Zusammenbruch der Blut-Hirn-Schranke, einer Hautreaktion mit vorrübergehender Alopezie, Gewichtsabnahme und zelluläre Veränderung äußerte. Das von den MRI Messungen abgeleitete Modell konnte zuverlässig das Eintreten der Nebenwirkungen, den Krankheitsverlauf, sowie die geschätzte Überlebensdauer der Mäuse vorhersagen. Zusätzlich konnte ein Zusammenhang zwischen den MRI Bildänderungen und den pathologischen Gewebsveränderungen beobachtet werden. Durch die außerordentlich homogene Strahlenreaktion der Tiere können aus den vorliegenden Daten künftig zuverlässig geeignete Dosen für spezifische experimentelle Endpunkte bestimmt werden. Zusammenfassend wurden in dieser Arbeit zwei präklinische Modelle für die Protonengehirnbestrahlung etabliert, nämlich organotypische Gewebsschnitte als 3D Zellkulturmodell sowie in vivo Bestrahlung von Gehirnsubvolumina in Mäusen. Während Zellkulturexperimente die Erwartungen nicht erfüllen konnten, stellen sich die Tierexperimente als hervorragendes Modell für translationale Radioonkologie heraus, welches zusätzlich für andere Strahlenqualitäten eingesetzt werden kann. Darauf basierend können aktuelle und zukünftige Studien die Ursachen von strahleninduzierten Normalgewebsschäden im Gehirn beleuchten, RBE Effekte untersuchen und neue prädiktive Biomarker erforschen.:Contents Abstract i Zusammenfassung v Publications ix List of Figures xiii List of Acronyms and Abbreviations xiv 1 Introduction 3 2 Background 5 2.1 Proton therapy for brain cancer treatment 5 2.1.1 Fundamentals of radiobiology 5 2.1.2 Proton therapy 6 2.1.3 Tumors of the central nervous system 8 2.2 Radiation effects on brain cells 8 2.2.1 Neurons and myelin 9 2.2.2 Blood-brain barrier 9 2.2.3 Astrocytes 10 2.2.4 Microglia 10 2.3 Principles of histology 11 2.3.1 Hematoxylin & eosin staining 12 2.3.2 Immunohistochemistry 13 2.3.3 Bioimage analysis 13 2.4 Techniques in medical imaging 14 2.4.1 Projectional radiography 14 2.4.2 Computed tomography 14 2.4.3 Magnetic resonance imaging 15 2.5 Preclinical models for radiation injury 17 2.5.1 Technical requirements 17 2.5.2 In vitro models 17 2.5.3 Small animal models 18 3 Applying Tissue Slice Culture in Cancer Research – Insights from Preclinical Proton Radiotherapy 19 3.1 Aim of the study 19 3.2 Conclusion 19 3.3 Author’s contribution 19 3.4 Publication 21 4 High-precision image-guided proton irradiation of mouse brain sub-volumes 41 4.1 Aim of the study 41 4.2 Conclusion 41 4.3 Author’s contribution 41 4.4 Publication 43 5 Late side effects in normal mouse brain tissue after proton irradiation 51 5.1 Aim of the study 51 5.2 Conclusion 51 5.3 Author’s contribution 52 5.4 Publication 53 6 Discussion 71 6.1 Establishment of preclinical models for radiooncology 71 6.1.1 3D cell culture 71 6.1.2 In vivo irradiation of brain subvolumes 73 6.2 Current applications of the mouse model 75 6.2.1 Ongoing data analysis 75 6.2.2 Innovating on-site imaging 76 6.2.3 RBE investigations 77 6.3 Future studies of radiation-induced brain tissue toxicities 79 Acknowledgement XV Supplementary Material XVII 1 Applying Tissue Slice Culture in Cancer Research – Insights from Preclinical Proton Radiotherapy XVII 2 High-precision image-guided proton irradiation of mouse brain sub-volumes XXVI 3 Late side effects in normal mouse brain tissue after proton irradiation XXXI / Proton therapy is an important modality in radiation oncology. Due to a favorable dose distribution in the irradiated volume, this treatment allows to spare tumor-surrounding normal tissue. Although this protection can lead to reduced side effects in certain patient populations, such as brain tumor or pediatric patients, normal tissue toxicities can occur to some extend. This could be due to clinical safety margins around the tumor that lead to dose deposition in the normal tissue. The underlying causes might also be related to relative biological effectiveness (RBE) variations or elevated radiosensitivity of certain brain regions. To address these issues, suitable preclinical models for normal brain tissue reaction after proton therapy are needed. In addition, patient stratification to predict the tumor response or the probability of side effects will contribute to increased treatment effectiveness. Preclinical models can improve the process of finding new predictive biomarkers and help to understand underlying mechanisms of radiation-induced brain injury. The aim of this thesis was to establish and characterize suitable preclinical models of brain tissue irradiation effects and set the base for future studies designed to reveal RBE effects, brain region specific radiation sensitivities, and novel biomarkers. The tested model systems were in vitro organotypic brain slice culture (OBSC) and in vivo irradiation of brain subvolumes, both on mouse brain tissue. Setup establishment at the experimental proton beam line and subsequent dosimetry built the foundation for conducting the biological experiments. Additionally, one main goal was defining clinically relevant endpoints for both short- and long-term effects. For OBSC, assays for cell death and inflammation, as well as in situ analysis of cell morphology and DNA damage induction were tested. As comparative model to OBSC, tumor slice culture was established and the results were also used for proton investigation. Adult OBSC turned out as inadequate model for preclinical experiments in radiation oncology. The assays measuring cell death and inflammation indicated a severe reaction during the first days in culture, but no response to irradiation. Histology revealed deficient cell morphology, reduced vitality and impaired DNA damage repair. In conclusion, other 3D cell culture models, such as organoids or tissue engineered constructs, should be considered for radiobiological experiments with protons. By publishing the observations, this thesis contributes to conserving the limited resources of proton radiobiology for more meaningful models. A methodology for irradiation of mouse brain subvolumes was established with a focus on creating fields comparable to clinical practice. The chosen target was the right hippocampus and the goal was to stop the proton beam in the middle of the brain. The project included a workflow for this precise irradiation in a robust and reproducible manner. Evaluation of the induced DNA damage and its correlation to Monte Carlo dose simulations were used for verification. Irradiation of mouse brain subvolumes yielded valuable results for early (i.e. within 24 h after irradiation) time points. An evaluation algorithm was designed for fast and robust analysis of spatial DNA damage distribution in relation to the total cell count. This ratio showed that the beam stopped in the brain tissue, in accordance to the treatment planning. Furthermore, the DNA damage could be reliably correlated with the dose simulation, which proves the value of the presented model for future RBE studies. In a follow-up experiment, the dose-time relationship of induced normal tissue reactions was analysed. For this, scoring of the animals' health status was combined with regular MRI measurements over the course of up to 6 months, and final histopathology. The volume increase of contrast agent leakage - representing breakdown of the blood brain barrier (BBB) - was contoured and the data was used to create a dose-volume response model. This pilot study on long-term radiation effects revealed dose-dependent normal tissue toxicities, including breakdown of the BBB, a skin reaction with temporary alopecia, weight reduction and changes on the cellular level. The model derived from MRI data reliably predicts onset of side effects, volume of brain damage as well as the expected animal survival. In addition, MRI image changes could be correlated to underlying tissue alterations by histopathology. Due to the uniform radiation response of the animals this data set enables to determine endpoint-specific dose values in future experiments. In conclusion, two preclinical models for proton brain irradiation were established, namely OBSC as 3D cell culture model and in vivo irradiation of mouse brain subvolumes. While the former could not yield the anticipated results, the latter emerged as excellent model for translational radiooncology, which can also be applied for experiments with other radiation types. Ongoing and future studies will focus on revealing the causes of normal brain tissue toxicities, studying RBE effects, and investigating new predictive biomarkers.:Contents Abstract i Zusammenfassung v Publications ix List of Figures xiii List of Acronyms and Abbreviations xiv 1 Introduction 3 2 Background 5 2.1 Proton therapy for brain cancer treatment 5 2.1.1 Fundamentals of radiobiology 5 2.1.2 Proton therapy 6 2.1.3 Tumors of the central nervous system 8 2.2 Radiation effects on brain cells 8 2.2.1 Neurons and myelin 9 2.2.2 Blood-brain barrier 9 2.2.3 Astrocytes 10 2.2.4 Microglia 10 2.3 Principles of histology 11 2.3.1 Hematoxylin & eosin staining 12 2.3.2 Immunohistochemistry 13 2.3.3 Bioimage analysis 13 2.4 Techniques in medical imaging 14 2.4.1 Projectional radiography 14 2.4.2 Computed tomography 14 2.4.3 Magnetic resonance imaging 15 2.5 Preclinical models for radiation injury 17 2.5.1 Technical requirements 17 2.5.2 In vitro models 17 2.5.3 Small animal models 18 3 Applying Tissue Slice Culture in Cancer Research – Insights from Preclinical Proton Radiotherapy 19 3.1 Aim of the study 19 3.2 Conclusion 19 3.3 Author’s contribution 19 3.4 Publication 21 4 High-precision image-guided proton irradiation of mouse brain sub-volumes 41 4.1 Aim of the study 41 4.2 Conclusion 41 4.3 Author’s contribution 41 4.4 Publication 43 5 Late side effects in normal mouse brain tissue after proton irradiation 51 5.1 Aim of the study 51 5.2 Conclusion 51 5.3 Author’s contribution 52 5.4 Publication 53 6 Discussion 71 6.1 Establishment of preclinical models for radiooncology 71 6.1.1 3D cell culture 71 6.1.2 In vivo irradiation of brain subvolumes 73 6.2 Current applications of the mouse model 75 6.2.1 Ongoing data analysis 75 6.2.2 Innovating on-site imaging 76 6.2.3 RBE investigations 77 6.3 Future studies of radiation-induced brain tissue toxicities 79 Acknowledgement XV Supplementary Material XVII 1 Applying Tissue Slice Culture in Cancer Research – Insights from Preclinical Proton Radiotherapy XVII 2 High-precision image-guided proton irradiation of mouse brain sub-volumes XXVI 3 Late side effects in normal mouse brain tissue after proton irradiation XXXI

Thesis (MSc (Radiography))--Cape Peninsula University of Technology, 2018 / Background: Breast cancer is one of the most commonly diagnosed among woman in South Africa, and a more resilient effort should be focused on treatment improvements. Worldwide, proton therapy is increasingly used as a radiation treatment alternative to photon therapy for breast cancer, mostly to decrease the risk for radiation-induced cardiovascular toxicity. This in vitro study aims to determine a better understanding of the radiosensitivity of both tumour and normal breast cell lines to clinical proton irradiation. In addition, we propose to investigate whether the increase in linear energy transfer (LET) towards the distal part of the proton beam results in an increase in relative biological effectiveness (RBE) for both cell lines. Methods: Malignant (MCF-7) and non-malignant (MCF-10A) breast cells were irradiated at different water equivalent depths in a 200 MeV proton beam at NRF iThemba LABS using a custom-made Perspex phantom: the entrance plateau, 3 points on the Bragg peak, the D80% and the D40%. A cytokinesis-block Micronucleus (CBMN) assay was performed and Micronuclei (MNi) were manually counted in binucleated cells (BNCs) using fluorescent microscopy. Reference dosimetry was carried out with a Markus chamber and irradiations were performed with a clinical proton beam generated at NRF iThemba LABS that was degraded to a R50 (half-value depths) range of 120 mm, with a field size of 10 cm x 10 cm and a 50 mm SOBP. The phantom could be adjusted to accommodate different perspex plates depending on the depth required within the proton beam. Cells were then exposed to 0.5, 1.0, 2.0, 3.0 and 4.0 Gy doses for each cell line independently and for each dose point. Results and Discussion: For the CBMN results, a program was developed on Matlab platform to calculate the 95% confidence ellipse on the co-variance parameters α and β. These values were determined by fitting the linear quadratic dose response curve to the average number of radiation induced MNi per 1000 BN cells. The ellipse region around a coordinate (the average MN frequency) for both MCF-7 and MCF-10A cells at the plateau region was defined by the mean estimate of the α-value and the β-value that were plotted on the X-axis and Y-axis respectively. The ratio of the two parameters, α/β, is a measure of the impact of fractionation to determine the biological effective dose. In fractionated proton therapy, the MCF10A cells will repair less between two fractions compared to the MCF7 cells. This is not an indication of therapeutic gain from a fractioned treatment protocol. For this reason, the hypofractionated stereotactic treatment protocols that can be applied with protons could be to the befit of the breast cancer patient. The above argument is based only on the radiosensitivity of the two cell lines exposed in the plateau region. Further analysis of the 95% confidence ellipse of both cell lines also showed a clear increase of the alpha value toward the distal portion of the beam and indicates an increase in energy transfer in this region. The gradual increase in α and β parameters with depth for protons for both cells is of clinical importance, since it implicates a non-homogeneous dose within the targeted area and an unwanted high dose behind the targeted area. Distal energy modulation could be investigated especially with larger breast tumours. RBE was calculated as the ratio of the dose at the different positions to the dose at the entrance plateau position (reference) to obtain an equal level of biological effect. A statistically significant difference in radiosensitivity could be observed between malignant and non-malignant cells at all positions (p<0.05). The variation in RBE was between 0.99 to 1.99 and 0.92 to 1.6 for the MCF-7 and MCF10A cell respectively. Conclusions: There is a variation in RBE along the depth-dose profile of a clinical proton beam. In addition, there is difference in radiosensitivity between the cancerous cells and the normal breast cells. While this study highlights a variation in sensitivity between cells it could be used by the modelling community to further develop biologically motivated treatment planning for proton therapy.

Low-energy X-rays in the range 10 - 50 keV have a wide application. One important application in radiological diagnostics is mammography, whereas, in radiotherapy, they are used for irradiation of superficial tumours, in brachytherapy and photon activation therapy. The importance of soft X-rays for fundamental radiobiological research is based on the fact, that all species of ionizing radiation produce a wide spectrum of secondary electrons, mainly responsible for the primary damage to be transformed into an observable radiobiological effect. By variation of the primary soft X-ray energy, a variation in the secondary electron spectra and therefore in the local energy deposition is provided. However, up to now no definitive conclusions about the relative biological effectiveness (RBE) of soft X-rays can be made due to its dependence on the photon energy, biological endpoint and dose range and the consequent large spread of the published data. The superconducting electron linear accelerator of high brilliance and low emittance (ELBE) at the Forschungszentrum Rossendorf with an electron energy up to about 40 MeV is used, among all, to generate X-rays in a wide energy range. One method for production of intensive, quasi-monochromatic X-rays in the energy range 10 - 100 keV tunable in energy, is by channeling of electrons in a perfect crystal. This X-ray source has many advantages over the most widespread X-ray source, which is the X-ray tube. Although the physical basis of the channeling effect has been previously investigated, the feasibility of an X-ray source based on channeling radiation (CR) for radiobiological studies has been for the first time theoretically and experimentally studied and a dedicated CR source built and optimised in the frame of this thesis. CR has been theoretically characterised in order to estimate its applicability for radiobiological studies. A good agreement between the theoretical predictions and the measured data has been found. The intrinsic properties of the CR source have led to the conclusion that monochromatisation is necessary. A monochromator based on HOPG mosaic crystals, was designed and manufactured. The dosimetrical methods have been investigated at the CR source as well as at an X-ray tube. Absolute dose measurements using an ionisation chamber and spectral dose distribution determination using semiconductor detectors have been performed. In addition, an unconventional system based on thermally stimulated exoelectron emission (TSEE) detectors, allowing to measure dose in a small volume and in the real cell environment has been tested and has proven itself appropriate in a variable dose range and in a liquid environment, in cases where reproducible laboratory conditions are provided. The biological endpoints clonogenic cell survival and micronucleus induction have been optimised for two established cell lines. The human mammary epithelial cells MCF-12A have been chosen due to the importance of RBE of soft X-rays for determination of risk from mammography. On the other hand, the use of the widespread mouse fibroblast cell line NIH/3T3 allows to compare the results with previously published data. The influence of the specific irradiation procedure at ELBE on the control level of cell survival and micronucleus induction has been tested and an irradiation system was developed and constructed. In addition, the RBE for soft X-rays was determined by X-ray tube irradiation at the Medical Department of Technische Universität Dresden. The RBE of 10 kV and 25 kV X-rays relative to 200 kV X-rays was determined. The RBE was found to be in the range from 1.0 to 1.4, depending on the used radiation quality, cell line and the biological endpoint, in agreement with previously published data for the same radiation qualities. These results confirm that systematical studies of RBE dependence on photon energy at the ELBE CR source are necessary and feasible.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-29T13:21:36Z No. of bitstreams: 2 Dissertação - Weslaine Alline da Silva Faria Santos - 2017.pdf: 2097732 bytes, checksum: 70179731d7ebabb4b4c354dab5ac03d9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-29T13:24:48Z (GMT) No. of bitstreams: 2 Dissertação - Weslaine Alline da Silva Faria Santos - 2017.pdf: 2097732 bytes, checksum: 70179731d7ebabb4b4c354dab5ac03d9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-11-29T13:24:48Z (GMT). No. of bitstreams: 2 Dissertação - Weslaine Alline da Silva Faria Santos - 2017.pdf: 2097732 bytes, checksum: 70179731d7ebabb4b4c354dab5ac03d9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-09-29 / This research, of a descriptive and analytical nature, was made in the master’s course program of Post-Graduation in Education (PPGE) at Federal University of Goiás (UFG), the following analyses is focus on a specific problem: Which are the teorical incursions about education of the body that has been marking the Scientific production researches published on the journals Revista Brasileira de Educação, Revista Brasileira de Ciências do Esporte and Revista Brasileira de História da Educação in the last twenty years (1997-2017)? These Journal's articles are the primer source of this research. The seek of the analysis corpus occurred over the last twenty years of these journals (1997- 2017), with the exception of Revista Brasileira de História da Educação (RBHE) which was published for the first time in 2001 using the descriptors bodyeducation of the body-history of the education of the body. The purposes of this research were: 1) To map the journals publications which had body education as the object of main study, investigation and debate; 2) To perform a description of the constitutive elements of the scientific production published; and 3) To analyse the chosen elements description searching how body education has been inserted on physical education, history of education and education. For lecture and interpretation of the research sources was used Pierre de Bourdieu’s sociology and his categories analyses of recurrence and singularity, inspired on Cristina Borges de Oliveira (2009) working paper. The description and analyses of these academic sources allowed us to notice, beside others, that body-education knowledge has been developed by a few physical-education professors binded to Colleges and Post-Graduation Programs of Education from south and southeast Brazil and that belonging feeling of the writers restrain the tematics clipping choices and the periodization. Also, was verified teorical and epistemologycal fragility showed on dispersion and generalization concepts. / A pesquisa, de caráter descritivo-analítico, foi realizada no curso de mestrado do Programa de Pós-Graduação em Educação (PPGE) da Universidade Federal de Goiás (UFG), nosso interesse está delimitado ao seguinte problema: quais incursões teóricas sobre a educação do corpo têm marcado a produção científica das pesquisas publicadas na Revista Brasileira de Educação, na Revista Brasileira de Ciências do Esporte e na Revista Brasileira de História da Educação nos últimos vinte anos (1997-2017)? Os escritos publicados nestas Revistas constituem as fontes de pesquisa. A busca do corpus de análise ocorreu nas edições destas revistas nos últimos vinte anos, (1997- 2017), com exceção apenas da Revista Brasileira de História da Educação (RBHE) que teve sua primeira publicação apenas no ano de 2001 com o uso dos descritores corpo-educação do corpo-história da educação do corpo. Os objetivos da pesquisa foram: 1) – mapear as publicações nas revistas que tenham o a educação do corpo como objeto de estudo e tema de pesquisa; 2) – realizar uma descrição de elementos constitutivos da produção cientifica divulgada; e 3 – analisar a descrição dos elementos escolhidos tendo em vista compreender como a educação do corpo tem se inserido nos campos acadêmicos da educação física, educação e história da educação. Para a leitura e interpretação das fontes de pesquisa buscou-se a inspiração da sociologia de Pierre de Bourdieu sendo utilizada também as categorias de análise recorrência e singularidade, inspirada no trabalho de Cristina Borges de Oliveira (2009). A descrição e análise das fontes permitiu perceber, entre outros, que o conhecimento sobre educação do corpo tem sido desenvolvido por (poucos) professores de educação física vinculados a faculdades e programas de pós-graduação em educação das regiões sul-sudeste brasileira e que este pertencimento dos autores condiciona as escolhas de recortes temáticos e a periodização. Também foi constatada fragilidade teórica e epistemologia expressas na dispersão e generalização conceitual.

L'Efficacité Biologique Relative (EBR) des faisceaux de protons énergétiques (70-250 MeV) utilisés dans les différents centres de protonthérapie est classiquement estimée à 1,10 par rapport aux photons du Cobalt-60. Bien qu'en accord avec la mesure de la régénération des cryptes intestinales chez la souris après exposition à une dose unique de 10 à 15 Gy, cette valeur moyenne a été contestée par la microdosimétrie. Ces incertitudes nous ont conduits à analyser l'effet des protons mis en œuvre dans les deux faisceaux médicaux (76 et 201 MeV) du Centre de Protonthérapie de l’Institut Curie à Orsay (ICPO) sur deux lignées cellulaires humaines tumorales, et en partie sur une lignée fibroblastique. Les résultats font apparaître une différence de la valeur de l'EBR pour la survie au rayonnement dans la partie distale du SOBP (Spread-Out Bragg Peak) en fonction de l'énergie incidente, ainsi qu'une absence de corrélation entre la réponse en survie et l'incidence des cassures double-brin de l'ADN dans le faisceau de 76 MeV. Nous montrons cependant, grâce à l'utilisation de lignées défectives dans les voies de signalisation et de réparation des cassures double-brin de l'ADN par le D-NHEJ, que ces voies déterminent la valeur de l'EBR dans la partie distale du SOBP de 76 MeV. La réponse aux dommages de l'ADN dans cette région suggère que les dommages létaux appartiennent à la classe des “lésions complexes” (LMDS) de l'ADN. D'autre part, il apparaît que la fluence des particules constitue un paramètre majeur qui doit être pris en compte dans la partie distale des faisceaux. / Treatment planning in proton therapy uses a generic value for the Relative Biological Efficiency (RBE) of 1.1 relative to 60Co gamma-rays throughout the Spread Out Bragg Peak (SOBP). We have studied the variation of the RBE at three positions in the SOBP of the 76 and 201 MeV proton beams used for cancer treatment at the Institut Curie Proton Therapy in Orsay (ICPO) in two human tumor cell lines using clonogenic cell death and the incidence of DNA double-strand breaks (DSB) as measured by pulse-field gel electrophoresis without and with endonuclease treatment to reveal clustered lesions as endpoints.The RBE for induced cell killing by the 76 MeV beam increased with depth in the SOBP. However for the 201 MeV protons it was close to that for 137Cs gamma-rays and did not vary significantly. The incidence of DSBs and clustered lesions was higher for protons than for 137Cs g-rays, but did not depend on the proton energy or the position in the SOBP.In the second part of our work, we have shown using cell clones made deficient for known repair genes by stable or transient shRNA transfection, that the D-NHEJ pathway determine the response to protons. The response of DNA damages created in the distal part of the 76 MeV SOBP suggests that those damages belong to the class of DNA "complex lesions" (LMDS). It also appears that the particle fluence is a major determinant of the outcome of treatment in the distal part of the SOBP.

Protonen töten Zellen wirksamer ab als Photonen. Die klinisch verwendete konstante relative biologische Wirksamkeit (RBW) für Protonen vernachlässigt jedoch erste klinische Evidenz einer RBW-Variabilität, die vom linearen Energietransfer (LET) abhängt. Diese Arbeit trägt dazu bei, die RBW-Variabilität in Protonen-Bestrahlungsplänen zu berücksichtigen, um potenzielle Nebenwirkungen zu vermindern. Zuerst wurde ein erhöhtes Risiko für RBW-induzierte Nebenwirkungen bei Hirntumorpatienten festgestellt. Dies konnte jedoch nicht systematisch durch klinische Planungsstrategien reduziert werden. Zweitens ergab eine multizentrische europäische Studie, dass die zentrums-spezifischen, nicht standardisierten LET-Berechnungen erheblich voneinander abweichen. Eine harmonisierte LET-Definition wurde vorgeschlagen und reduzierte die Variabilität zwischen den Zentren auf ein klinisch akzeptables Niveau, was künftig eine einheitliche Dokumentation des Therapieergebnisses ermöglicht. Abschließend wurden vier Strategien zur RBW-Reduktion in der Planoptimierung bei Hirntumorpatienten angewandt, die das Risiko für Nekrose und Erblindung erheblich reduzierten. LET-Optimierung in Hochdosisregionen erscheint besonders geeignet, um die Sicherheit der Patientenbehandlung künftig weiter zu verbessern.:List of Figures vii List of Tables viii List of Acronyms and Abbreviations ix 1 Introduction 1 2 Theoretical background 3 2.1 Proton interactions with matter 4 2.2 Biological effect of radiation 8 2.2.1 Linear-quadratic model 8 2.2.2 Relative biological effectiveness 9 2.3 Proton beam delivery and field formation 13 2.4 Treatment planning 14 2.4.1 Patient modelling and structure definition 15 2.4.2 Treatment plan optimisation 16 2.4.3 Treatment plan evaluation 19 2.5 Proton therapy uncertainties and mitigation strategies 22 2.5.1 Clinical mitigation strategies 23 2.5.2 Optimisation approaches beyond absorbed dose 26 3 Variable biological effectiveness in PBS treatment plans 29 3.1 LET and RBE recalculations of proton treatment plans with RayStation 30 3.1.1 Monte Carlo dose engine 30 3.1.2 Monte Carlo scoring extensions 32 3.1.3 Graphical user interface 33 3.2 LET assessment and the role of range uncertainties 36 3.2.1 Patient cohort and treatment plan creation 37 3.2.2 Simulation of range deviations 38 3.2.3 Treatment plan recalculation settings 39 3.2.4 Resulting impact of range deviations 40 3.3 Patient recalculations in case of side effects 46 3.3.1 Image registration and range prediction 48 3.3.2 Retrospective treatment plan assessment 49 3.4 Benefit of an additional treatment field 50 3.4.1 Patient and treatment plan information 50 3.4.2 Results of variable RBE recalculations 51 3.5 Discussion 51 3.6 Summary 59 4 Status of LET and RBE calculations in European proton therapy 61 4.1 Study design 62 4.1.1 Treatment planning information 64 4.1.2 Data processing and treatment plan evaluation 67 4.2 Treatment plan comparisons in the water phantom 68 4.2.1 Absorbed dose evaluation 69 4.2.2 Centre-specific LET calculations 69 4.2.3 Harmonised LET calculations 71 4.3 Treatment plan comparisons in patient cases 72 4.3.1 Dose-averaged linear energy transfer for protons 73 4.3.2 Centre-specific RBE models and parameters 76 4.4 Discussion 77 4.5 Summary 82 5 Biological treatment plan optimisation 83 5.1 Treatment plan design 84 5.1.1 Clinical goals 86 5.1.2 Novel treatment plan optimisation approaches 87 5.2 Treatment plan quality assessment with a constant RBE 90 5.3 Assessment of NTCP reductions with a variable RBE 90 5.4 Discussion 95 5.5 Conclusion 100 6 Summary 103 7 Zusammenfassung 107 Bibliography 111 Danksagung 137 / Protons are more effective in cell killing than photons. However, the clinically applied constant proton relative biological effectiveness (RBE) neglects emerging clinical evidence for RBE variability driven by the linear energy transfer (LET). This thesis aims to safely account for RBE variability in proton treatment plans to mitigate potential side effects. First, an elevated risk for RBE induced overdosage was found in brain tumour patients. However, this could not be mitigated systematically by clinical planning strategies. Second, a multicentric European study revealed that centre-specific non-standardised LET calculations differed substantially. A harmonised LET definition was proposed which reduced the inter-centre variability to a clinically acceptable level and allows for future consistent outcome reporting. Finally, four strategies to include RBE variability in treatment plan optimisation were applied to brain tumour patients, which considerably reduced the estimated risk for necrosis and blindness. Of these, LET optimisation in high dose regions may be suited for clinical practice to further enhance patient safety in view of a variable RBE.:List of Figures vii List of Tables viii List of Acronyms and Abbreviations ix 1 Introduction 1 2 Theoretical background 3 2.1 Proton interactions with matter 4 2.2 Biological effect of radiation 8 2.2.1 Linear-quadratic model 8 2.2.2 Relative biological effectiveness 9 2.3 Proton beam delivery and field formation 13 2.4 Treatment planning 14 2.4.1 Patient modelling and structure definition 15 2.4.2 Treatment plan optimisation 16 2.4.3 Treatment plan evaluation 19 2.5 Proton therapy uncertainties and mitigation strategies 22 2.5.1 Clinical mitigation strategies 23 2.5.2 Optimisation approaches beyond absorbed dose 26 3 Variable biological effectiveness in PBS treatment plans 29 3.1 LET and RBE recalculations of proton treatment plans with RayStation 30 3.1.1 Monte Carlo dose engine 30 3.1.2 Monte Carlo scoring extensions 32 3.1.3 Graphical user interface 33 3.2 LET assessment and the role of range uncertainties 36 3.2.1 Patient cohort and treatment plan creation 37 3.2.2 Simulation of range deviations 38 3.2.3 Treatment plan recalculation settings 39 3.2.4 Resulting impact of range deviations 40 3.3 Patient recalculations in case of side effects 46 3.3.1 Image registration and range prediction 48 3.3.2 Retrospective treatment plan assessment 49 3.4 Benefit of an additional treatment field 50 3.4.1 Patient and treatment plan information 50 3.4.2 Results of variable RBE recalculations 51 3.5 Discussion 51 3.6 Summary 59 4 Status of LET and RBE calculations in European proton therapy 61 4.1 Study design 62 4.1.1 Treatment planning information 64 4.1.2 Data processing and treatment plan evaluation 67 4.2 Treatment plan comparisons in the water phantom 68 4.2.1 Absorbed dose evaluation 69 4.2.2 Centre-specific LET calculations 69 4.2.3 Harmonised LET calculations 71 4.3 Treatment plan comparisons in patient cases 72 4.3.1 Dose-averaged linear energy transfer for protons 73 4.3.2 Centre-specific RBE models and parameters 76 4.4 Discussion 77 4.5 Summary 82 5 Biological treatment plan optimisation 83 5.1 Treatment plan design 84 5.1.1 Clinical goals 86 5.1.2 Novel treatment plan optimisation approaches 87 5.2 Treatment plan quality assessment with a constant RBE 90 5.3 Assessment of NTCP reductions with a variable RBE 90 5.4 Discussion 95 5.5 Conclusion 100 6 Summary 103 7 Zusammenfassung 107 Bibliography 111 Danksagung 137

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