Background: Glioblastomas (GBM) are characterised by genetic and epigenetic alterations in resistance-mediating genes and destructive infiltration of the surrounding normal brain. Cell adhesion molecules play an important role for intrinsic and acquired therapy resistances. Among the group of adhesion molecules, the collagen-binding discoidin domain receptor 1 (DDR1) is considered as potential, druggabale and promising cancer target owing to its own tyrosine kinase activity and its overexpression in various cancer types. This study evaluates the so far unknown role of DDR1 in GBM invasion and response to radiochemotherapy and investigates the underlying molecular mechanisms. Materials and Methods: Expression of DDR1 and GBM stem cell markers was evaluated in 13 biopsies of patients with primary GBM and correlated with overall survival. The effects of DDR1 inhibition by either DDR1-siRNA or DDR1 inhibitor (DDR1-IN-1), Temozolomide (TMZ) or a DDR1-IN-1/TMZ combination on GBM invasion capacity and cancer cell survival upon irradiation were evaluated in a panel of 7 GBM cell lines, 5 GBM stem-like cell cultures and an orthotopic GBM mouse model. Changes in underlying signal transduction mecha-nisms after DDR1 inhibition were assessed by Western blot and broad-spectrum phosphopro-teome analysis. Direct DDR1 binding partners were evaluated by immunoprecipitation, mass spectrometry, FRET analysis, truncated and mutated DDR1 variants and GST-pulldown. Results: DDR1 was found to be co-expressed with GBM stem cell markers in clinical GBM samples. High DDR1/Nestin co-localisation correlated with poor patient outcome. In GBM models a pharmacological DDR1 inhibition enhanced sensitivity and prolonged survival in response to radiochemotherapy with Temozolomide compared to conventional therapy. Moreover, a 50 % reduced invasion was determined in GBM cell cultures. Mechanistically, DDR1 inhibition impaired Akt/mTOR signalling and induced LC3b protein expression and formation of LC3b-positive autophagosomes suggesting autophagy for GBM radiochemosen-sitisation. Further, mass spectrometry on DDR1 immunoprecipitates identified a 14-3-3/Beclin-1/Akt1 protein complex as linker to the prosurvival Akt-mTOR axis. FRET analysis and GST-pulldown of truncated DDR1 variants identified complex assembly at DDR1 to be required for prosurvival Akt/mTOR signalling, regulation of autophagy and radichemosensiti-sation. Conclusion: These data suggest DDR1 targeting in GBM to be highly effective for radi-ochemosensitisation via autophagy induction, impairment of cell invasion and superior to the current conventional therapy. Based on its overexpression and key function in prosurvival mechanisms, targeting of the druggable DDR1 provides a rationale for combinatorial thera-pies with novel adjuvant autophagy-inducing agents administered with conventional radi-ochemotherapy.:Abbreviations V 1 Introduction 1 2 Background 3 2.1 Glioblastoma 3 2.1.1 Epidemiology 3 2.1.2 Molecular characteristics 5 2.1.3 GBM therapy 6 2.2 Physical and biological action of ionizing radiation 8 2.3 Therapy resistance 10 2.3.1 Tumour heterogeneity and cancer stem cells 12 2.3.2 Tumour microenvironment and cell-adhesion mediated resistance 13 2.3.3 Autophagy 16 2.4 Discoidin Domain Receptor 1 (DDR1) 17 2.4.1 Structure 17 2.4.2 Physiological role of DDR1 19 2.4.3 DDR1 in cancer 20 2.4.4 DDR1 signalling 22 3 Hypothesis and Aims 25 4 Materials and Methods 26 4.1 Materials 26 4.1.1 Devices 26 4.1.2 Further materials 28 4.1.3 Patient material 29 4.1.4 siRNA 29 4.1.5 Inhibitors 30 4.1.6 Chemotherapeutic agents 30 4.1.7 Plasmids 30 4.1.8 Primers 32 4.1.9 Polymerases, Restriction enzymes and ligases 32 4.1.10 Bacterial culture 33 4.1.11 Protein and DNA ladders 33 4.1.12 Method kits 34 4.1.13 Primary antibodies 34 4.1.14 Secondary antibodies 36 4.1.15 Solutions for cell biological applications 37 4.1.16 Solutions for protein-biochemical and molecular-biological applications 38 4.1.17 Solutions for immunofluorescence and immunohistological applications 40 4.1.18 Further solutions and chemicals 41 4.1.19 PC programs 41 4.2 Methods 42 4.2.1 Cell culture 42 4.2.2 Cell freezing and thawing 43 4.2.3 siRNA knockdown 43 4.2.4 Inhibitor treatment and chemotherapy 44 4.2.5 Radiation exposure 44 4.2.6 Colony formation assay 44 4.2.7 Sphere formation assay 46 4.2.8 Invasion Assay 46 4.2.9 Total protein extracts, SDS-PAGE and Western Blotting 47 4.2.10 Orthotopic mouse model 49 4.2.11 Histology and immunohistochemistry 51 4.2.12 Phosphoproteome analysis 52 4.2.13 Immunoprecipitation 52 4.2.14 Mass spectrometric analysis 53 4.2.15 Expression constructs, site-directed mutagenesis and transfection of plasmids 54 4.2.16 GST-Pulldown 57 4.2.17 Fluorescence microscopy 58 4.2.18 Fluorescence Resonance Energy Transfer (FRET) analysis 58 4.2.19 Autophagy analysis 59 4.2.20 Statistics 60 5 Results 61 5.1 DDR1 Expression in GBM 61 5.1.1 DDR1 expression analysis on publicly available data 61 5.1.2 DDR1 expression in GBM cell lines and GBM stem-like cells 63 5.1.3 The role of DDR1 in GBM stemness 64 5.2 The role of DDR1 in GBM radiochemoresistance and invasion 68 5.2.1 Effect of DDR1 silencing on GBM radiosensitivity 68 5.2.2 Effect of DDR1 silencing on GBM invasiveness 69 5.2.3 Pharmacological inhibition of DDR1 in vitro 69 5.2.4 DDR1 inhibition in an orthotopic GBM mouse model 73 5.3 DDR1 signalling in GBM 76 5.3.1 Akt/mTOR signalling axis 76 5.3.2 14-3-3/Beclin-1/Akt1 protein complex 79 5.4 The role of DDR1 inhibition in autophagy 85 6 Discussion 89 6.1 DDR1 expression correlates with GBM stemness and conveys radiochemoresistance and invasiveness 90 6.2 DDR1 inhibition impacts on Akt/mTOR signalling 92 6.3 A 14-3-3/Beclin-1/Akt1 complex physically connects DDR1 to the Akt/mTOR axis 93 6.4 DDR1 inhibition induces autophagy for radiochemosensitisation 94 7 Summary 97 8 Zusammenfassung 99 9 Figures 101 10 Tables 103 11 References 105 12 Acknowledgements 132 Appendix 133 Curriculum vitae 133 Veröffentlichungen 134 Anlage 1 136 Anlage 2 137 Darstellung des Eigenanteils 138

There is a large amount of interest and research currently going into studying the effects of low dose radiation on humans, and bridging the gap with the data from the effects of high dose radiation. Much work is to be done to understand low dose exposures such as from medical treatments and those who work with or around radiation. Two popular and widely known examples of low-dose phenomena are the radiation induced bystander effects and the radioadaptive response (RAR). This research involves the study of the impact of a low dose of radiation that is administered several hours after a high – even fatal – dose is given, which contrasts the traditional RAR where a low priming dose is given before a high dose and can lead to increased cell survival. Many different parameters were checked to see if cell survival can be enhanced or diminished depending on the stage of the cell cycle, cell growth conditions, and cell profiling differences in protein function (namely the TP53 gene). Additionally, the post-conditioning response was contrasted to see if it was possible to see any effects from the newly emerging area of bystander signalling, UV BioPhotons, would be present in cell lines that either did or did not exhibit a post-conditioning effect. It was shown that post-conditioning has a protective effect on survival of the cells in certain dose ranges and certain cell lines. The post-conditioning effect also appears to be stronger in magnitude than the classic RAR. No relationship between gamma-induced biophoton signalling and post-conditioning was observed, nor is it certain whether an acute gamma-field can induce significant UV biophoton damage. This thesis is aimed to explore the various parameters by which post-conditioning effects occur on various Human cancers. / Thesis / Master of Science (MSc) / This research looks at the effects of radiation on cells. More specifically, how do low doses of radiation affect cells after they have already been treated with higher doses of radiation. Moreover, can cells communicate through non-physical methods, such as through invisible light? The research focuses primarily on cancer cells and their responses to varying doses of radiation treatments.

Neste trabalho estudamos o efeito da exposição de células humanas à radiação ionizante e em associação a campos elétricos exógenos estáticos. A linhagem T47D de células de carcinoma ductal mamário foi irradiada com gamas no intervalo 0 8 Gy. A viabilidade celular da linhagem T47D exposta à radiação gama e campo elétrico estático (CEE) de 1.250 V/cm foi cerca de 12% inferior à viabilidade observada apenas com irradiação. Quando aplicado isoladamente por 24 e 72 horas o CEE não induziu toxicidade. A imunofluorescência realizada na linhagem normal MRC5 (fibroblasto de pulmão humano normal) quantificou a expressão da histona -H2AX. A quantidade de histonas fosforiladas foi cerca de 40% maior após irradiação com 2 Gy mais CEE aplicado por 1h, indicando que o campo elétrico interferiu negativamente no processo de reparo das quebras duplas de DNA. A análise de citometria de fluxo (FACS) mostrou que em células T47D tratadas com 1 e 2 Gy por 24 horas o CEE também interferiu negativamente no processo de reparo do DNA, notadamente pelo maior acúmulo de células na fase S. / Our goal is the study in human cells of the effect resulting from the association of irradiation with exposure to exogenous static electric fields. The T47D cell line of breast cancer cells was irradiated with gammas in the 0 8 Gy doses range. The viability of this T47D cells exposed to both gamma radiation and 1.250 V/cm static electric field (SEF) was about 12% lower than when only irradiated. The sole exposure of the cells to SEF by 24 and 72 hours didnt induce toxicity. Immunofluorescence runs carried out in irradiated normal MRC5 cell line of human lung fibroblast have quantified the expression of the g-H2AX histone. The amount of phosphorylated histones was approximately 40% higher after irradiation with 2 Gy plus exposure to a SEF by 1 hour, showing that the electric field negatively interfered in the repairing process of the DNA double strand breaks. The flow cytometry analysis with FACS showed that in T47D cells treated with 1 and 2 Gy by 24 hours the SEF also negatively interfered in the DNA repairing process, as evidenced by the higher accumulation of cells in the S phase.

Paracoccidioides brasiliensis é o agente da paracoccidioidomicose (PCM), uma doença sistêmica crônica prevalente na América Latina. Até o momento, não existe uma vacina eficaz para esta enfermidade. O potencial da radiação gama para a atenuação de patógenos e desenvolvimento de uma vacina foi explorado neste trabalho. Em nosso laboratório desenvolvemos leveduras de P. brasiliensis atenuadas por irradiação gama. Estas perderam a capacidade de reprodução, mas mantiveram a sua morfologia, a síntese e secreção de proteínas, o metabolismo oxidativo e o seu perfil antigênico. O objetivo deste trabalho foi avaliar a capacidade protetora induzida pela imunização com as leveduras radioatenuadas em modelo animal. A atenuação da virulência das leveduras radioatenuadas foi avaliada em camundongos BALB/c e Nude-Nude. O efeito protetor foi avaliado em grupos de camundongos BALB/c submetidos a uma ou duas imunizações. Cada grupo foi dividido em três subgrupos, que foram desafiados 30, 45 e 60 dias após a imunização. Os camundongos foram sacrificados 30 e 90 dias após o desafio. Os órgãos removidos foram usados para recuperação de unidades formadoras de colônias (UFCs), análise histológica e determinação de citocinas. Os soros foram coletados semanalmente para avaliar os títulos de IgG e o padrão de IgG1 e IgG2a produzido no curso da infecção. Para avaliar o tipo de resposta imune desencadeada, as citocinas IFN-g, TNF-a, IL-10 e IL-5 foram determinadas por PCR em tempo real. As leveduras radioatenuadas perderam a sua virulência, pois não foram capazes de provocar infecção em camundongos BALB/c e Nu/Nu. Nenhuma UFC foi recuperada nem foram observadas alterações histopatológicas nos camundongos infectados com o fungo radioatenuado. Os camundongos infectados com P. brasiliensis não irradiado mostraram altos níveis de produção do anticorpo IgG, enquanto a infecção com as leveduras radioatenuadas não estimulou alterações significativas destes níveis. Os camundongos infectados com a levedura radioatenuada apresentaram um aumento na produção de IFN-g e TNF-a, indicando uma estimulação da imunidade celular. O ensaio de proteção realizado com camundongos imunizados uma vez mostrou uma redução significativa na recuperação das UFCs avaliadas 30 dias após o desafio. Entretanto, foi verificado um aumento na recuperação das UFCs dos tecidos 90 dias após o desafio. Apesar de um grau significativo de proteção ter sido alcançado, estes resultados mostram que somente uma imunização não foi suficiente para conferir uma proteção a longo prazo, uma vez que alguns focos do fungo não foram eliminados. Uma proteção eficiente foi desenvolvida no grupo dos camundongos imunizados duas vezes. A imunização foi capaz de reduzir a infecção inicial e desencadear uma proteção a longo prazo. Uma proteção de 99,5% foi obtida nos camundongos analisados 90 dias após desafio. Neste mesmo período os níveis de IFN-g e TNF-a aumentaram significativamente, enquanto os de IL-10 e IL-5 diminuíram. Quando analisados 30 dias após o desafio os camundongos não desenvolveram um padrão totalmente polarizado de resposta Th1/Th2, mas uma tendência para um padrão Th1 foi evidente após 90 dias. Os níveis de IgG aumentaram em ambos os grupos após o desafio. Concluímos que a imunização com as leveduras radioatenuadas foi capaz de desencadear uma resposta imune protetora a longo prazo. Estas células são uma ferramenta valiosa em estudos da imunidade protetora e na pesquisa de vacina para a PCM. / Paracoccidioides brasiliensis is fungus agent of paracoccidioidomycosis (PCM), a chronic systemic disease prevalent in Latin American. To date, there is no effective vaccine. The potential of gamma radiation for pathogens attenuation and vaccine development was explored in this work. In our laboratory were developed yeast cells of P. brasiliensis attenuated by gamma radiation, which lose the reproductive ability, while retaining the morphology, the synthesis and secretion of proteins, the oxidative metabolism and the expression of the antigens present in the native yeast. The aim of the present work was to evaluate the protection elicited by the immunization with this cells in animal model. The virulence attenuated was evaluated in BALB/c and Nude-Nude mice. The protector effect was evaluated in BALB/c mice groups immunized once or twice. Each group was divided in three sub groups that were challenge 30, 45 or 60 days after the immunization. The mice were sacrificed 30 and 90 days after challenge. The removed organs were used for colonyforming units (CFUs) recover, histopathological analysis and cytokine determination. The sera were collected weekly to evaluate the IgG antibody titers and the IgG1 and IgG2a pattern in the course of infection. To evaluate the type of elicited immune response the cytokines IFN-g, TNF-a, IL-10 and IL-5 were determined by real time PCR. The radioattenuated yeast loses its virulence since fails in producing infection in BALB/c and Nude-Nude mice. No CFUs were recovered neither histological changes observed in the mice infected with the radioattenuated cells. The mice infected with the not irradiated P. brasiliensis showed a high level of antibody production while the infection with the radioattenuated yeast did not significantly change the antibody level. The mice infected with the radioattenuated yeast presented an increase in the IFN-g and TNF-a production and an inhibition of the IL-10 synthesis, indicating a stimulation of the cellular response. The protection assay performed with mice immunized once showed a significant reduction in the CFUs recovery evaluated 30 days after the challenge. However, when examined 90 days after the challenge the CFUs recovered from organs increased. Even so a significant degree of protection has been reached these results point that only one immunization was not enough to confer a long lasting protection, since some focus of the fungi were not eliminated. An efficient protection against highly infective forms of P. brasiliensis was developed in the mice group immunized twice. The immunization was able to reduce the initial infection and elicited a long lasting protection. A 99,5 % protection was obtained in the mice analyzed 90 days post challenge. At the same time the levels of IFN-gDQG71). increased significantly while the IL-10 and IL-5 decreased. When analyzed after 30 days, the mice had not developed a totally polarized pattern of Th1/Th2 response but, a trend to a Th1 response was evident after 90 days. The level of IgG increased in both immunized groups after the challenge. We concluded that the immunization with the radioattenuated yeast cells was able to elicit a long lasting protective immune response and these cells are valuable tool for protective immunity studies and vaccine research for PCM.

The chief aim in developing radiation treatment plans is to maximise tumour cell kill while minimising the killing of normal cells. The acceptance by a radiation oncologist of a radiation therapy treatment plan devised by the radiation therapist, at present is largely based on the oncologists' previous clinical experience with reference to established patterns of treatment and their clinical interpretation of the dose volume histogram. Some versions of radiotherapy planning computer software now incorporate a function that permits biologically based predictions about the probability of tumour control (TCP) and/or normal tissue complications (NTCP). The biological models used for these probabilities are founded upon statistical and mathematical principles as well as radiobiology concepts. TCP and NTCP potentially offer the capability of being able to better optimise treatments for an individual patient's tumour and normal anatomy. There have been few attempts in the past to correlate NTCPs to actual treatment complications, and the reported complications have generally not shown any significant correlation. Thus determining whether either or both NTCPs and TCPs could be correlated with the observed clinical outcomes of prostate radiotherapy is the central topic of this thesis. In this research, TCPs and NTCPs were prospectively calculated for prostate cancer patients receiving radiation therapy, and subsequently assessed against the clinical results of the delivered treatments. This research was conducted using two different types of NTCP models, which were correlated against observed treatment-induced complications in the rectum and bladder. The two NTCP models were also compared to determine their relative efficacy in predicting the recorded toxicities. As part of this research the refinement of some of the published bladder parameters required for NTCP calculations was undertaken to provide a better fit between predicted and observed complication rates for the bladder wall which was used in this research. TCPs were also calculated for each patient using the best available estimate of the radiosensitivity of the prostate gland from recent research. The TCP/NTCP data was analysed to determine if any correlations existed between the calculated probabilities and the observed clinical data. The results of the analyses showed that a correlation between the NTCP and a limited number of toxicities did occur. Additionally the NTCP predictions were compared to existing parameters and methods for radiotherapy plan evaluation - most notably DVHs. It is shown that NTCPs can provide superior discriminatory power when utilised for prospective plan evaluation. While the TCP could not be correlated with clinical outcomes due to insufficient follow-up data, it is shown that there was a correlation between the TCP and the treatment technique used.

No description available.

The purpose of this work is to make an attempt to quantify and model various types of cell death for a small cell lung cancer (SCLC) cell line (U1690) after exposure to a 137Cs source and as well as to compare cell survival models, the Linear-Quadratic (LQ) and Repairable Conditionally – Repairable model (RCR). This study is based on four different experiments that were taken place at Cancer Centrum Karolinska (CCK). A human small cell lung cancer (SCLC) cell line after the exposure to a 137Cs source was used for the extraction of the clonogenic cell survival curve. Additionally for the determination and quantification of various modes of cell death the method of fluorescence staining was implemented, where we categorized the cell death based on morphological characteristics. As next with the flow cytometry analysis we measured the properties of individual particles and more specifically the percentage of cells in each phase of the cell cycle. The quantification of senescent cells was performed by staining the samples with senescence associated-β-gal solution and then scoring as senescent cells those that had incorporated the substance. These data were introduced into a maximum likelihood fitting to calculate the best estimates of the parameters used by the model in section 2.8. In this model we sorted the modes of cell death into three categories: apoptotic, senescent and other types of cell death (nec/apop, necrotic, micronuclei, giant). In regards to the clonogenic cell survival assay the RCR model shows a ρ2 value that is equal to 6.10 whereas for the LQ model is 9.61. Moreover from the fluorescence microscopy and senescence assay we observed an initial increase of the probability of three different categories of cell death on day 2 and at higher doses there was saturation. On day 7 a significant induction of apoptosis in a dose and time dependent manner was evident whereas senescence was slightly increased in response to dose but not to time. As for the „other types of cell death‟ category on day 7 showed a higher probability that the one on day 2 and as well as a prominent dose dependence. A dose dependent accumulation of cells in the G2/M phase of the cell cycle was induced by photons on day 2. The accumulation in the G2/M phase on day 2 is released on day 7 and simultaneously an increase of the probability of apoptosis with time was observed. The RCR model is fitted better to the experimental data rather than the LQ model. On day 2 there is a slight increase of the apoptotic and senescent probability with dose. On the other hand on day 7 the shape of the curve of apoptosis differs and we observe a sigmoidal increase with dose. At both time points the mathematical model fit the data reasonable well. Due to the fact that the clonogenic survival doesn‟t coincide with the one extracted from the fluorescence microscopy, a more accurate way of quantification of cell death need to be used (e.g. CVTL). / Ο σκοπός αυτής της μελέτης είναι η ποσοτικοποίηση και μοντελοποίηση διαφόρων τύπων κυτταρικού θανάτου μικροκυτταρικού καρκίνου πνεύμονα μετά από ακτινοβόληση με πηγή Καισίου (137Cs) καθώς και η σύγκριση μοντέλων κυτταρικής επιβίωσης, Linear-Quadratic (LQ) και Repairable Conditionally-Repairable. Η μελέτη είναι βασισμένη σε τέσσερα ξεχωριστά πειράματα τα οποία πραγματοποιήθηκαν στο Cancer Centrum Karolinska (CCK). Ανθρώπινος μικροκυτταρικός καρκίνος πνεύμονα χρησιμοποιήθηκε για τον υπολογισμό της καμπύλης κυτταρικής επιβίωσης μετά από ακτινοβόληση με πηγή Καισίου (137Cs). Επιπρόσθετα για τον προσδιορισμό και την μοντελοποίηση των διαφόρων ειδών θανάτου εφαρμόστηκε η μέθοδος της φθορίζουσας μικροσκοπίας, με την βοήθεια της οποίας κατηγοριοποιήθηκε ο κυτταρικός θάνατος βάσει μορφολογικών χαρακτηριστικών. Στη συνέχεια μέσω της κυτταρομετρίας ροής υπολογίσαμε τις ιδιότητες μεμονομένων σωματιδίων (κυττάρων) και πιο συγκεκριμένα το ποσοστό των κυττάρων σε κάθε φάση του κυτταρικού κύκλου. Η ποσοτικοποίηση των κυττάρων γήρανσης πραγματοποιήθηκε μέσω της χρώσης των δειγμάτων με διάλυμα συσχετιζόμενο με την γήρανση και μετά καταγράφηκαν σαν κύτταρα γήρανσης αυτά τα οποία είχαν ενσωματώσει την ουσία. Τα δεδομένα χρησιμοποιήθηκαν σε μια διαδικασία προσαρμογής μέγιστης πιθανοφάνειας (maximum likelihood fitting) ώστε να υπολογιστούν οι βέλτιστες τιμές των παράμετρων που χρησιμοποιούνται από το μοντέλο στην ενότητα 2.8. Στο παρόν μοντέλο έχουμε ταξινομήσει τον κυτταρικό θάνατο σε τρεις κατηγορίες: απόπτωση, γήρανση και άλλοι τύποι κυτταρικού θανάτου (νεκ/αποπ, νέκρωση, μικροπυρήνες και γίγαντες). Όσον αφορά την κλωνογόνο κυτταρική επιβίωση το RCR μοντέλο παρουσιάζει τιμή χ2 ίση με 6.10 ενώ για το LQ μοντέλο ίση με 9.61. Επιπλέον μέσω της φθορίζουσας μικροσκοπίας και της χημικής δοκιμής για την κυτταρική γήρανση παρατηρήσαμε την 2η μέρα αρχική αύξηση της πιθανότητας και για τις τρεις κατηγορίες κυτταρικού θανάτου ενώ εμφανής ήταν ο κορεσμός στις υψηλότερες δόσεις. Την 7η μέρα παρουσιάστηκε επαγωγή της απόπτωσης με δοσο/χρονο-εξαρτώμενο τρόπο καθώς και το ότι η γήρανση των κυττάρων αυξήθηκε ελάχιστα με την δόση αλλά όχι με τον χρόνο. Σχετικά με την τρίτη κατηγορία ‘άλλοι τύποι κυτταρικού θανάτου’ την 7η μέρα ανέδειξε υψηλότερη πιθανότητα συγκριτικά με την 2η μέρα καθώς και μια έκδηλη εξάρτηση με την δόση. Κατά την ανάλυση του κυτταρικού κύκλου για την 2η μέρα αναδεικνύεται συσσώρευση των κυττάρων με δοσοεξαρτώμενο τρόπο στην φάση G2/M του κυτταρικού κύκλου. Η συσσώρευση των κυττάρων στην φάση G2/M την 2η μέρα απελευθερώθηκε την 7η μέρα με ταυτόχρονη αύξηση της πιθανότητας για απόπτωση συναρτήσει της δόσης. Βρέθηκε ότι το RCR μοντέλο προσαρμόζεται καλύτερα στα πειραματικά δεδομένα σε σχέση με το LQ μοντέλο. Την 2η μέρα παρατηρήθηκε πολύ μικρή αύξηση της πιθανότητας για απόπτωση και γήρανση συναρτήσει της δόσης. Ενώ την 7η μέρα η μορφή της καμπύλης της απόπτωσης διαφοροποιήθηκε και παρατηρήθηκε σιγμοειδής αύξηση με την δόση. Το μαθηματικό μοντέλο προσαρμόζεται αρκετά καλά στα δεδομένα για την 2η και 7η μέρα. Ένας πιο ακριβής τρόπος υπολογισμού της ποσοτικοποίησης του κυτταρικού θανάτου θα πρέπει να χρησιμοποιηθεί εξ’αιτίας του γεγονότος ότι η καμπύλη της κλωνογόνου επιβίωσης δεν συμπίπτει με αυτή που παράχθηκε από την μικροσκοπία φθορισμού.

Radiotherapy is the use of ionising radiation to induce localised DNA damage to cancerous tissues, leading to cell death and disease control. In order to maximise tumour growth control and to limit damage of the healthy surrounding tissues and the consequent side effects for the patient, molecular determinants of tumour radioresistance are investigated as potential clinical targets. A high-throughput siRNA colony formation assay screen in HeLa cervical carcinoma cells previously published by our laboratory identified modulators of radiosensitivity. From the list CSF1R, EPHB2, GAK and TOPK, were selected and validated. TOPK (T-LAK cell-originated protein kinase, also known as PDZ-binding kinase, PBK) was selected for further investigation because it is overexpressed in most malignancies but not in normal tissues, apart from testis and placenta. Knockdown of TOPK was shown to induce radiosensitisation in a panel of cancer cell lines with no significant effects on normal cells. A role for TOPK in the cell cycle response to ionising radiation (IR) was discovered in HCT116 colorectal cancer cells, with alterations in the G₁/S and G₂/M checkpoints. Furthermore, immunoprecipitation experiments identified a physical interaction between TOPK and CDKN1A (p21) at 8 hours after IR. Apoptosis and the number of multinucleated cells were significantly increased in TOPK depleted cells exposed to IR, suggesting the possibility of aberrant mitosis and mitotic catastrophe in these cells. High TOPK expression in early breast cancer patients was shown to be associated with poor recurrence-free survival. In addition, immunohistochemistry (IHC) analysis on samples from prostate cancer patients identified a strong correlation between high levels of TOPK and poor clinical response to radiotherapy. In order to facilitate future in vivo experiments, an HCT116 shRNA stable knockdown cell line was developed and two commercially available TOPK inhibitors were tested and optimised. Taken together, these data suggest that TOPK is a molecular determinant of radiosensitivity with a great potential for future clinical applications.

Radiation therapy (RT) plays a crucial role in the management of cancer, however, current standards of care have yet to account for patient specific radiation sensitivity. Raman spectroscopy (RS) is a promising technique for radiobiological studies as a way to measure radiation responses in biological samples and could provide a method for monitoring and predicting radiation response in patients. The work in this dissertation gives way to significant advances in the implementation of RS for applications in radiation oncology. Specifically, instrumentation improvements for clinical implementation of RS were achieved through the investigation and development of Raman microfluidic systems. Unique magnesium fluoride based microfluidic systems were engineered and evaluated for applications in radiobiological studies. These systems were found to yield superior spectral quality over traditional microfluidic designs. Furthermore, in order to assert RS as a key technique for clinical monitoring and prediction of radiation responses, human non-small cell lung cancer (NSCLC) and breast adenocarcinoma tumour xenograft models were investigated for Raman signatures of radiation response. These studies found that RS can identify unique and distinct signatures of radiation response in tumours, that can be tracked over time. In particular, NSCLC tumours were found to have key radiation induced modulations in cell cycle and metabolic linked spectral features- including glycogen. Breast adenocarcinoma tumours were found to exhibit distinct fluctuations in spectral features linked to cell cycle as well as protein content. In the case of NSCLC, radiation response signatures were found to be linked to tumour regression and hypoxic status of the tumour- a key factor that dictates radiation resistance in the disease. This work provides the first application of RS to measure radiation response signatures of tumours irradiated \textit{in vivo}. These results show that RS is a versatile technique that can offer insight into radiation induced molecular changes that are unique to the type of cancer and can be monitored over several days following radiation exposure. Together with improved instrumentation for radiobiological studies using microfluidics, the work presented in this dissertation further emphasizes the key role RS can have in radiation oncology and personalization of RT. / Graduate / 2019-08-21

Doctorat en Sciences / info:eu-repo/semantics/nonPublished