Radiation Type- and Dose-Specific Transcriptional Responses across Healthy and Diseased Mammalian Tissues

Ionizing radiation (IR) is a genuine genotoxic agent and a major modality in cancer treatment. IR disrupts DNA sequences and exerts mutagenic and/or cytotoxic properties that not only alter critical cellular functions but also impact tissues proximal and distal to the irradiated site. Unveiling the molecular events governing the diverse effects of IR at the cellular and organismal levels is relevant for both radiotherapy and radiation protection. Herein, we address changes in the expression of mammalian genes induced after the exposure of a wide range of tissues to various radiation types with distinct biophysical characteristics. First, we constructed a publicly available database, termed RadBioBase, which will be updated at regular intervals. RadBioBase includes comprehensive transcriptomes of mammalian cells across healthy and diseased tissues that respond to a range of radiation types and doses. Pertinent information was derived from a hybrid analysis based on stringent literature mining and transcriptomic studies. An integrative bioinformatics methodology, including functional enrichment analysis and machine learning techniques, was employed to unveil the characteristic biological pathways related to specific radiation types and their association with various diseases. We found that the effects of high linear energy transfer (LET) radiation on cell transcriptomes significantly differ from those caused by low LET and are consistent with immunomodulation, inflammation, oxidative stress responses and cell death. The transcriptome changes also depend on the dose since low doses up to 0.5 Gy are related with cytokine cascades, while higher doses with ROS metabolism. We additionally identified distinct gene signatures for different types of radiation. Overall, our data suggest that different radiation types and doses can trigger distinct trajectories of cell-intrinsic and cell-extrinsic pathways that hold promise to be manipulated toward improving radiotherapy efficiency and reducing systemic radiotoxicities

Bridging Plant and Human Radiation Response and DNA Repair through an In Silico Approach

The mechanisms of response to radiation exposure are conserved in plants and animals. The DNA damage response (DDR) pathways are the predominant molecular pathways activated upon exposure to radiation, both in plants and animals. The conserved features of DDR in plants and animals might facilitate interdisciplinary studies that cross traditional boundaries between animal and plant biology in order to expand the collection of biomarkers currently used for radiation exposure monitoring (REM) in environmental and biomedical settings. Genes implicated in trans-kingdom conserved DDR networks often triggered by ionizing radiation (IR) and UV light are deposited into biological databases. In this study, we have applied an innovative approach utilizing data pertinent to plant and human genes from publicly available databases towards the design of a ‘plant radiation biodosimeter’, that is, a plant and DDR gene-based platform that could serve as a REM reliable biomarker for assessing environmental radiation exposure and associated risk. From our analysis, in addition to REM biomarkers, a significant number of genes, both in human and Arabidopsis thaliana, not yet characterized as DDR, are suggested as possible DNA repair players. Last but not least, we provide an example on the applicability of an Arabidopsis thaliana—based plant system monitoring the role of cancer-related DNA repair genes BRCA1, BARD1 and PARP1 in processing DNA lesions

Repair of oxidatively-induced clustered DNA lesions, after exposure of human cancer cells to ionizing radiation

In the present thesis, the induction and repair of complex DNA lesions, double strand breaks (DSBs) of high complexity accompanied by oxidatively-induced base lesions in their close vicinity were studied after exposure to ionizing radiation (IR) of varying quality i.e. linear energy transfer (LET). These lesions are considered the hallmark of ionizing radiation as well as of the high levels of oxidative stress. Complex DNA damage may be consisted by DSBs, single strand breaks (SSBs) and base lesions within a very short DNA fragment up to 10-15 base pairs. Clustered DNA damage challenges the cell DNA repair machinery and therefore the eventual misrepair could lead to mutations, genomic instability or even carcinogenesis. The in situ immunofluorescence-based detection of this kind of damage is generally accepted as a very challenging goal and can be performed currently only at the chromosomal level and within DNA regions much larger than the above mentioned above. The main experimental method followed in this thesis, it is the in situ indirect immunofluorescence, using specific antibodies against several human DNA repair proteins i.e. for DSBs (γ-Η2ΑΧ), for oxidative base lesions (OGG1) and for abasic DNA sites (hAPE1). Moreover complementary meta-analysis of experimental results was performed, achieving the systematical review of underlying mechanisms. The scientific interest for DNA damage as well as for the consequential cellular response can be attributed to the dual action of ionizing radiation. On one hand, coincidental exposure to ionizing radiation could disrupt DNA integrity, causing mutations, sometimes beneficial, promoting evolution or detrimental causing genomic instability and sometimes cancer. On the other hand, one of the most important therapies against cancer, radiotherapy utilizes ionizing radiation to target the DNA of tumor cells and thus to induce programmed cell death (apoptosis). In the framework of the present thesis, several human cancer cell lines originating from breast cancer (MCF7), hepatocellular carcinoma (HepG2), lung carcinoma (A549), glioblastoma (MO59K and their isogenic MO59J) but also normal immortalized keratinocytes (FEP18-11-T1), as well as lymphocytes from healthy donors were studied. Different types of ionizing radiation and LET, electromagnetic (Low LET: γ- and X- rays) and charged particles (high-LET: a- particles and Argon ions) were used. Increasing complexity of the induced damage was detected with the increase of LET of the incident radiation. Regarding the repair of this damage, a significant delay in the process of complex lesions was also observed. Chemical inhibition of the DNA repair protein DNA-PK was induced in cell cultures, using two different and highly specific inhibitors, namely the NU7026 and the NU7441. NU7441 was found to induce stronger inhibition in double strand break repair process than NU7026. DNA-PK was also tested by using a pair of cell lines (MO59K and MO59J), MO59J are DNA-PKcs deficient. DNA repair was delayed but finally was achieved. In order to detect complex DNA lesions that also include oxidative base lesions in the vicinity of a DSB, we defined the proper protocol which allows their visualization as colocalized fluorescence signal coming from fluorescent antibodies that target the DSB repair proteins (γ-Η2ΑΧ or 53BP1) and the base lesion repair proteins (OGG1 or APE1).In order to evaluate the colocalization levels based on “fluorescent” images, as a result of the present work, we introduced a criterion-Parameter called Pclc (colocalization parameter). Our criterion conserves the focus topology for the DSBs, which are considered the ‘centers’ of repair, and regarding the oxidative base lesions, it uses their mean luminosity. Pclc compares the surface (or spatial in the case of 3D images) luminosity of the signal coming from the labeled antibody that targets the base repair enzymes, considered over the DSB foci area with the same value considered over the rest of the cell nucleus area. Using our suggested parameter Pclc, normalization of any fluctuation among images has been achieved, making possible the comparison among images resulting by different experiments. The main advantage of this consideration is that it allows unbiased the decision whether or not the occurring colocalization is random or of biological importance. Significant Pclc increases were detected with increasing LET and this trend was verified with some minor variations for all radiations used. In addition, specific correlations were made with repair time and focus size synonymous with damage complexity. Approaching the DNA repair of complex DNA lesions on organism level (systems biology), data analysis coming from universal data bases was performed, utilizing bioinformatics. For this purpose several open source tools were utilized, and multilevel systematic proofing of the results was performed. Proper gene nomenclatures were case –specifically used. Gene ontology terms hold the key role in the gene screening that was performed, making the results reliable. This analysis has been based exclusively on experimentally evaluated data. Specific correlations were done between DNA damage response (DDR) and repair genes with the immune system and inflammatory response especially in the case of radiation-induced systemic effects which consisted a significant part of our bioinformatical work. The idea of radiation non-targeted effects holds currently a prominent position in radiation biology and our main hypothesis has been that complex DNA lesions and their repair can be regarded as the first ‘danger’ signal due to the difficulty the cell encounters in processing and amending these genome problems. Bioinformatics analysis revealed numerous (human and plant) genes that we believe that are also participating in DNA repair machinery, following the exposure to IR and especially to the repair of clustered DNA lesions. Several other genes where identified and are proposed as potential biomarkers of exposure to endogenous (replication, oxidative) and to exogenous stress (ionizing radiation), being also applicable to biological dosimetry. Regarding cancer therapy, the role of DNA repair genes was explored and several genes were suggested as potential targets for chemical/pharmaceutical inhibition in order to enhance radiotherapy in the frame of Synthetic Lethality, moreover the idea of Multi-pathway Lethality was also proposed. Summarizing, the results of this study are centered on the biophysical mechanisms induction and repair of complex DNA lesions. They led to the development of protocols for the detection of these lesions on the cellular level, by using in situ immunofluorescence. The scientific importance of these findings relies primarily on two aspects: 1. the technical improvements and advancement of the field of in situ detection of complex damage as well as 2. the biophysical/biological importance of the findings on the processing of complex DNA damage consisting of a DSB and neighboring oxidative lesions challenging the repair machinery of the cell. More importantly, the biophysical experimental and complementary bioinformatics work has been published in the following papers: [1]Z. Nikitaki, V. Nikolov, I. V. Mavragani, I. Plante, D. Emfietzoglou, G. Iliakis, and Alexandros G. Georgakilas, "Non-DSB clustered DNA lesions. Does theory colocalize with the experiment?," Radiation Physics and Chemistry, 2016. 128: p. 26-35.AbstractIonizing radiation results in various kinds of DNA lesions such as double strand breaks (DSBs) and other non-DSB base lesions. These lesions may be formed in close proximity (ie, within a few nanometers) resulting in clustered types of DNA lesions. These damage clusters are considered the signature of ionizing radiation, notably charged particles of high linear energy transfer (LET). Accumulating theoretical and experimental evidence suggest the induction of these clustered lesions appears under various irradiation conditions but also as a result of high levels of oxidative stress. The biological significance of these clustered DNA lesions pertains to the inability of cells to process them efficiently as isolated lesions. The results in the case of unsuccessful or erroneous repair can vary from mutations up to chromosomal instability. In this mini review, we discuss of several Monte Carlo simulations codes and experimental evidence regarding the induction and repair of radiation-induced non-DSB complex DNA lesions. We also critically discuss the most widely used methodologies (ie, gel electrophoresis and fluorescence microscopy [in situ colocalization assays]). Based on the comparison of different approaches, we present examples and suggestions for the improved detection of these lesions in situ. Based on the current status of knowledge, we conclude that there is a great need for improvement of the detection techniques at the cellular or tissue level, which will provide valuable information for understanding the mechanisms used by the cell to process clustered DNA lesions.[2]Z. Nikitaki, V. Nikolov, I. V. Mavragani, E. Mladenov, A. Mangelis, D. A. Laskaratou, Georgios I. Fragkoulis, Christine E. Hellweg, Olga A. Martin, Dimitris Emfietzoglou, Vasiliki I. Hatzi, Georgia I. Terzoudi, George Iliakis and Alexandros G. Georgakilas, "Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)," Free Radic Res, pp. 1-45, Sep 5 2016.AbstractDetrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of double strand breaks (DSBs), single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase of the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing linear energy transfer (LET) i.e., γ-, X-rays 0.3-1 keV/μm, α-particles 116 keV/μm and 36Ar ions 270 keV/μm. Using ɣ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5-16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1) i.e. damage foci as probes for oxidized purines or abasic sites respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also made correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage. [3]Z. Nikitaki, I. V. Mavragani, D. A. Laskaratou, V. Gika, V. P. Moskvin, K. Theofilatos Konstantinos Vougas, Robert D. Stewart and Alexandros G. Georgakilas., "Systemic mechanisms and effects of ionizing radiation: A new 'old' paradigm of how the bystanders and distant can become the players," Semin Cancer Biol, vol. 37-38, pp. 77-95, Jun 2016.AbstractExposure of cells to any form of ionizing radiation (IR) is expected to induce a variety of DNA lesions, including double strand breaks (DSBs), single strand breaks (SSBs) and oxidation, as well as loss of bases, i.e. abasic sites. The damaging potential of IR is primarily related to the generation of electrons, which through their interaction with water produce free radicals. In their turn, free radicals attack DNA, proteins and lipids. Damage is induced also through direct deposition of energy. These types of IR interactions with biological materials are collectively called ‘targeted effects’, since they refer only to the irradiated cells. Earlier and sometimes ‘anecdotal’ findings were pointing to the possibility of IR actions unrelated to the irradiated cells or area, i.e. a type of systemic response with unknown mechanistic basis. Over the last years, significant experimental evidence has accumulated, showing a variety of radiation effects for ‘out-of-field’ areas (non-targeted effects-NTE). The NTE involve the release of chemical and biological mediators from the ‘in-field’ area and thus the communication of the radiation insult via the so called ‘danger’ signals. The NTE can be separated in two major groups: bystander and distant (systemic). In this review, we have collected a detailed list of proteins implicated in either bystander or systemic effects, including the clinically relevant abscopal phenomenon, using improved text-mining and bioinformatics tools from the literature. We have identified which of these genes belong to the DNA damage response and repair pathway (DDR/R) and made protein-protein interaction (PPi) networks. Our analysis supports that the apoptosis, TLR-like or NOD-like receptor signaling pathways are the main pathways participating in NTE. Based on this analysis, we formulate a biophysical hypothesis for the regulation of NTE, based on DNA damage and apoptosis gradients between the irradiation point and various distances corresponding to bystander (5 mm) or distant effects (5 cm). Last but not least, in order to provide a more realistic support for our model, we calculate the expected DSB and non-DSB clusters along the central axis of a representative 200.6 MeV pencil beam calculated using Monte Carlo DNA damage simulation software (MCDS) based on the actual beam energy-to-depth curves used in therapy.  [4]A. G. Georgakilas, A. Pavlopoulou, M. Louka, Z. Nikitaki, C. E. Vorgias, P. G. Bagos, Ioannis Michalopoulos,"Emerging molecular networks common in ionizing radiation, immune and inflammatory responses by employing bioinformatics approaches," Cancer Lett, vol. 368, pp. 164-72, Nov 28 2015.AbstractEfficient radiation therapy is characterized by enhanced tumor cell killing involving the activation of the immune system (tumor immunogenicity) but at the same time minimizing chronic inflammation and radiation adverse effects in healthy tissue. The aim of this study was to identify gene products involved in immune and inflammatory responses upon exposure to ionizing radiation by using various bioinformatic tools. Ionizing radiation is known to elicit different effects at the level of cells and organism i.e. DNA Damage Response (DDR), DNA repair, apoptosis and, most importantly, systemic effects through the instigation of inflammatory ‘danger’ signals and innate immune response activation. Genes implicated both in radiation and immune/inflammatory responses were collected manually from the scientific literature with a combination of relevant keywords. The experimentally validated and literature-based results were inspected, and genes involved in radiation, immune and inflammatory response were pooled. This kind of analysis was performed for the first time, for both healthy and tumor tissues. In this way, a set of 24 genes common in all three different phenomena was identified. These genes were found to form a highly connected network. Useful conclusions are drawn regarding the potential application of these genes as markers of response to radiation for both healthy and tumor tissues through the modulation of immune and/or inflammatory mechanisms.[5]N. Hekim, Z. Cetin, Z. Nikitaki, A. Cort, and E. I. Saygili, "Radiation triggering immune response and inflammation," Cancer Lett, vol. 368, pp. 156-63, Nov 28 2015.AbstractRadiation therapy (RT) is a well-established but still under optimization branch of Cancer Therapy (CT). RT uses electromagnetic waves or charged particles in order to kill malignant cells, by accumulating the energy onto these cells. The issue at stake for RT, as well as for any other Cancer Therapy technique, is always to kill only cancer cells, without affecting the surrounding healthy ones. This perspective of CT is usually described under the terms “specificity” and “selectivity”. Specificity and selectivity are the ideal goal, but the ideal is never entirely achieved. Thus, in addition to killing healthy cells, changes and effects are observed in the immune system after irradiation. In this review, we mainly focus on the effects of ionizing radiation on the immune system and its components like bone marrow. Additionally, we are interested in the effects and benefits of low-dose ionizing radiation on the hematopoiesis and immune response. Low dose radiation has been shown to induce biological responses like inflammatory responses, innate immune system activation and DNA repair (adaptive response). This review reveals the fact that there are many unanswered questions regarding the role of radiation as either an immuneactivating (low dose) or immunosuppressive (high dose) agent.[6]Z. Nikitaki, I. Michalopoulos, and A. G. Georgakilas, "Molecular inhibitors of DNA repair: searching for the ultimate tumor killing weapon," Future Med Chem, vol. 7, pp. 1543-58, Aug 2015.AbstractDNA repair (DR) inhibitors are small molecules that interact with DR proteins in order to disrupt their function and induce a ‘strike’ to the high fidelity of the mammalian DNA repair systems. Many anticancer therapies aim to harm the DNA of the usually highly proliferative cancer cell, causing it to undergo apoptosis. In response to this, cancer cells attempt to fix the induced lesion and reconstitute its genomic integrity, in turn reducing the efficacy of treatment. To overcome this, DR inhibitors suppress DNA repair proteins’ function, increasing the potency and tumor killing effect of chemotherapy or radiotherapy. In this review, we discuss clinically applied novel inhibitors under translational investigation and we apply bioinformatic tools in order to identify repair proteins implicated in more than two phenomenically distinct DNA repair pathways (e.g., base excision repair and nonhomologous end joining), that is, the concept of ‘synthetic lethality’. Our study can aid towards the optimization of this therapeutic strategy and, therefore, maximizing treatment effectiveness like in the case of radiation therapy.[7]Z. Nikitaki, C. E. Hellweg, A. G. Georgakilas, and J. L. Ravanat, "Stress-induced DNA damage biomarkers: applications and limitations," Front Chem, vol. 3, p. 35, 2015.Abstract A variety of environmental stresses like chemicals, UV and ionizing radiation and organism’s endogenous processes such as replication stress and metabolism can lead to the generation of reactive oxygen and nitrogen species (ROS/RNS) that can attack cellular vital components like DNA, proteins and lipid membranes. Among them, much attention has been focused on DNA since DNA damage plays a role in several biological disorders and aging processes. Thus, DNA damage can be used as a biomarker in a reliable and accurate way to quantify for example radiation exposure and can indicate its possible long term effects and cancer risk. Based on the type of DNA lesions detected one can hypothesize on the most probable mechanisms involved in the formation of these lesions for example in the case of UV and ionizing radiation (e.g., X- or α-,γ-rays, energetic ions, neutrons). In this review we describe the most accepted chemical pathways for DNA damage induction and the different types of DNA lesions, i.e., single, complex DNA lesions etc. that can be used as DNA damage biomarkers. We critically compare DNA damage detection methods and their limitations. In addition, we suggest the use of DNA repair gene products as biomarkes for identification of different types of stresses i.e., radiation, oxidative, or replication stress, based on bioinformatic approaches and meta-analysis of literature data.[8]V. I. Hatzi, D. A. Laskaratou, I. V. Mavragani, Z. Nikitaki, A. Mangelis, M. I. Panayiotidis, et al., "Non-targeted radiation effects in vivo: a critical glance of the future in radiobiology," Cancer Lett, vol. 356, pp. 34-42, Jan 1 2015.AbstractRadiation-induced bystander effects (RIBE), demonstrate the induction of biological non-targeted effects in cells which have not directly hit by radiation or by free radicals produced by ionization events. Although RIBE have been demonstrated using a variety of biological endpoints the mechanism(s) of this phenomenon still remain unclear. The controversial results of the in vitro RIBE and the evidence of nontargeted effects in various in vivo systems are discussed. The experimental evidence on RIBE, indicate that a more analytical and mechanistic in depth approach is needed to secure an answer to one of the most intriguing questions in radiobiology.Στη διατριβή αυτή μελετήθηκαν οι σύνθετες οξειδωτικές βλάβες DNA, δίκλωνες θραύσεις αυξημένης πολυπλοκότητας που συνοδεύονται και από βλάβες βάσεων στην εγγύς τους περιοχή. Οι βλάβες αυτές θεωρούνται η υπογραφή των ιοντιζουσών ακτινοβολιών αλλά και του ισχυρού οξειδωτικού στρες στο κύτταρο. Αποτελούνται από διάφορες βλάβες, όπως δίκλωνες θραύσεις, μονόκλωνες και αλλοιώσεις βάσεων πολύ κοντά η μία στην άλλη εντός 10-15 ζευγών βάσεων DNA. Λόγω της μεγάλης δυσκολίας επιδιόρθωσής τους μπορούν να οδηγήσουν πολύ εύ

Study of Photodynamic therapy combined with antioxidants in prostate cancer cells

135 σ.Εθνικό Μετσόβιο Πολυτεχνείο--Μεταπτυχιακή Εργασία. Διεπιστημονικό-Διατμηματικό Πρόγραμμα Μεταπτυχιακών Σπουδών (Δ.Π.Μ.Σ.) “Μικροσυστήματα και Νανοδιατάξεις”Η Φωτοδυναμική Θεραπεία(ΦΔΘ) είναι μια πολλά υποσχόμενη μέθοδος αντιμετώπισης καλοήθων και μη ασθενειών, οι οποίες εμφανίζουν υπερανάπτυξη ανεπιθύμητων ή μη φυσιολογικών κυττάρων. Η βασική ιδέα στηρίζεται στη χορήγηση μιας μη τοξικής ουσίας, του φωτοευαισθητοποιητή (ΦΕ), ο οποίος συσσωρεύεται επιλεκτικά στα κύτταρα του όγκου, καθιστώντας τα ευάλωτα στην οπτική ακτινοβολία. Όταν ολοκληρωθεί η επωαστική περίοδος με τον ΦΕ, ακολουθεί ακτινοβόληση του όγκου με ορατό φως. Παρουσία οξυγόνου, η ενεργοποίηση του ΦΕ από την ακτινοβολία προκαλεί την παραγωγή ελευθέρων ριζών, οι οποίες είναι κυτταροτοξικές. Αποτέλεσμα της επαγόμενης κυτταροτοξικότητας είναι ο κυτταρικός θάνατος και επομένως η αποδόμηση του πάσχοντος ιστού. Η χρήση της ΦΔΘ στην αντιμετώπιση του καρκίνου θεωρείται ιδιαιτέρως ελκυστική, δεδομένης της θεμελιώδους εξειδίκευσης και επιλεκτικότητας που παρέχει. Στην παρούσα εργασία ερευνάται η συνδυαστική δράση Φωτοδυναμικής Θεραπείας και αντιοξειδωτικών σε ανθρώπινα καρκινικά κύτταρα προστάτη. Ειδικότερα μελετήθηκε η Φωτοδυναμική δράση με δύο σκευάσματα του ΦΕ mTHPC (απλή μορφή-Foscan& λιποσωμική μορφή-Fospeg) και εκχύλισμα προερχόμενο από τον φλοιό του πεύκουPinushalepensis,πλούσιο σε πολυφαινόλες. Η αντιοξειδωτική ή/και προοξειδωτική δράση του εκχυλίσματος μελετήθηκαν συστηματικά. Καθώς παρόμοιες μελέτες οι οποίες συνδυάζουν τη ΦΔΘ με αντιοξειδωτικά σε προοξειδωτικές δόσεις έχουν δείξει συνεργειακή δράση, μελετάται εδώ το συγκριμένο εκχύλισμα. Το κείμενο είναι χωρισμένο σε δύο μέρη, στο θεωρητικό και το πειραματικό.Το θεωρητικό μέρος ξεκινά με την εισαγωγή στη Φωτοδυναμική Θεραπεία όπου παρουσιάζονται αναλυτικά οι δύο κύριες συνιστώσες της, οι φωτοευαισθητοποιητές και τα lasers. Στη συνέχεια εξηγούνται οι μηχανισμοί επίδρασης σε ατομικό, μοριακό, χημικό, υποκυττάριο και κυτταρικό επίπεδο. Ακολούθως αναφέρονται οι μηχανισμοί καταστροφής των όγκων από κυτταρικό επίπεδο μέχρι την ενεργοποίηση του ανοσοποιητικού συστήματος. Τα τελευταία δύο κεφάλαια του πρώτου μέρους αφιερώνονται στη συμβολή της Νανοτεχνολογίας στη ΦΔΘ και τους τρόπους ενίσχυσης της Φωτοδυναμικής δράσης. Στο πειραματικό μέρος περιγράφονται τα πρωτόκολλα που αναπτύχθηκαν, αναφέρονται τα υλικά, οι διατάξεις και οι μέθοδοι που χρησιμοποιήθηκαν και παρουσιάζονται τα αποτελέσματα που εξήχθησαν. Στο τέλος γίνεται συζήτηση για τα συμπεράσματα και τις μελλοντικές προοπτικές. H μελέτη της επίδρασης του εκχυλίσματος του φλοιού του πεύκου Pinus halepensis, πραγματοποιήθηκε με πειράματα βιωσιμότητας, στα ακόλουθα στάδια: Έλεγχος Ι: τοξικότητα σκότους των δύο σκευασμάτων του ΦΕ mTHPC, Foscan και Fospeg. Έλεγχος ΙΙ: επίδραση μόνο της ακτινοβόλησης. Έλεγχος ΙΙΙ: κλασική ΦΔΘ με τα δύο προαναφερθέντα σκευάσματα. Έλεγχος ΙV: προσδιορισμός συγκέντρωσης αντιοξειδωτικού στην οποία ξεκινά η προοξειδωτική δράση. Έλεγχος V: τοξικότητα σκότους αντιοξειδωτικού και Foscan, τοξικότητα σκότους αντιοξειδωτικού και Fospeg. Έλεγχος VI: τοξικότητα αντιοξειδωτικού και ακτινοβόλησης. Έλεγχος VII: πιθανή συνεργειακή δράση ΦΔΘ και αντιοξειδωτικού.Photodynamic therapy is considered to be a promising method for the treatmentof malignant and non-malignant diseases that are generally characterized by overgrowth of unwanted or abnormal cells. The basic concept for this method consists of administering a non-toxic drug, known as a photosensitizer, which selectively accumulates by the tumor cells. After the incubation time, irradiation of the tumor follows, with visible light. In presence of oxygen, the activation of the photosensitizer through irradiation, leads to the generation of cytotoxic species and consequently to cell death and tissue devastation. The use of Photodynamic Therapy as a cancer therapy is considered particularly attractive, due to its fundamental specificity and selectivity. In the present study is investigated the combination of Photodynamic Therapy and the use of antioxidants on human prostate cancer cells. Speciffically the photodynamic action of two formulations of PS mTHPC (simple form – Foscan & liposomal form – Fospeg) and the extract from the pine Pinus halepensis bark, rich in polyphenols, is investigated. The antioxidant and/or prooxidant properties of this extract were studied in depth. Since similar studies compining PDT with antioxidants in prooxidant concentrations have demonstrated synergetic action, this particular extract is investigated. This thesis is divided in two parts, the theoretical and the experimental. The theoretical part includes firstly an introduction to Photodynamic Therapy which details its two main components, the photosensitizers and lasers. Secondly, themechanisms of the PD effect inatomic, molecular, chemical, subcellular and cellular level are explainedfollowed by the mechanisms of tumor destruction at a cellular level leading to the activation of the immune system. The last two chapters of the first part are devoted to the contribution of Nanotechnology in PDT and other ways of enhancing the photodynamic action. The experimental part details the protocols developed during the experiments, the materials, devices and methods that were used. Finally the results are presented followed by the conclusions and a discussion about future prospects. Control I: Dark toxicity of the two formulations of PS mTHPC, Foscαn and Fospeg. Control II: The effects of illumination Control III: Classic PDT using the two previously mentioned formulatios Control IV: Determination of the antioxidants concentration where its prooxidant action begins Control V: Dark toxicity of antioxidant and Foscan, dark toxicity of antioxidant ant Fospeg. Control VI: Toxicity of antioxidant under illumination Control VII: Possible synergetic action of PDT with the antioxidant.Ζαχαρένια Γ. Νικητάκ

In Situ Detection of Complex DNA Damage Using Microscopy: A Rough Road Ahead

Complexity of DNA damage is considered currently one if not the primary instigator of biological responses and determinant of short and long-term effects in organisms and their offspring. In this review, we focus on the detection of complex (clustered) DNA damage (CDD) induced for example by ionizing radiation (IR) and in some cases by high oxidative stress. We perform a short historical perspective in the field, emphasizing the microscopy-based techniques and methodologies for the detection of CDD at the cellular level. We extend this analysis on the pertaining methodology of surrogate protein markers of CDD (foci) colocalization and provide a unique synthesis of imaging parameters, software, and different types of microscopy used. Last but not least, we critically discuss the main advances and necessary future direction for the better detection of CDD, with important outcomes in biological and clinical setups

Using Machine Learning Techniques for Asserting Cellular Damage Induced by High-LET Particle Radiation

This is a study concerning the use of Machine Learning (ML) techniques to ascertain the impacts of particle ionizing radiation (IR) on cell survival and DNA damage. Current empirical models do not always take into account intrinsic complexities and stochastic effects of the interactions of IR and cell populations. Furthermore, these models often lack in biophysical interpretations of the irradiation outcomes. The linear quadratic (LQ) model is a common way to associate the biological response of a cell population with the radiation dose. The parameters of the LQ model are used to extrapolate the relation between the dosage and the survival fraction of a cell population. The goal was to create a ML-based model that predicts the α and β parameters of the well known and established LQ model, along with the key metrics of DNA damage induction. The main target of this effort was, on the one hand, the development of a computational framework that will be able to assess key radiobiophysical quantities, and on the other hand, to provide meaningful interpretations of the outputs. Based on our results, as some metrics of the adaptability and training efficiency, our ML models exhibited 0.18 median error (relative root mean squared error (RRMSE)) in the prediction of the α parameter and errors of less than 0.01 for various DNA damage quantities; the prediction for β exhibited a rather large error of 0.75. Our study is based on experimental data from a publicly available dataset of irradiation studies. All types of complex DNA damage (all clusters), and the number of double-stranded breaks (DSBs), which are widely accepted to be closely related to cell survival and the detrimental biological effects of IR, were calculated using the fast Monte Carlo Damage Simulation software (MCDS). We critically discussed the varying importance of physical parameters such as charge and linear energy transfer (LET); we also discussed the uncertainties of our predictions and future directions, and the dynamics of our approach

A Mathematical Radiobiological Model (MRM) to Predict Complex DNA Damage and Cell Survival for Ionizing Particle Radiations of Varying Quality

Predicting radiobiological effects is important in different areas of basic or clinical applications using ionizing radiation (IR); for example, towards optimizing radiation protection or radiation therapy protocols. In this case, we utilized as a basis the ‘MultiScale Approach (MSA)’ model and developed an integrated mathematical radiobiological model (MRM) with several modifications and improvements. Based on this new adaptation of the MSA model, we have predicted cell-specific levels of initial complex DNA damage and cell survival for irradiation with 11Β, 12C, 14Ν, 16Ο, 20Νe, 40Αr, 28Si and 56Fe ions by using only three input parameters (particle’s LET and two cell-specific parameters: the cross sectional area of each cell nucleus and its genome size). The model-predicted survival curves are in good agreement with the experimental ones. The particle Relative Biological Effectiveness (RBE) and Oxygen Enhancement Ratio (OER) are also calculated in a very satisfactory way. The proposed integrated MRM model (within current limitations) can be a useful tool for the assessment of radiation biological damage for ions used in hadron-beam radiation therapy or radiation protection purposes

GENE EXPRESSION COLLECTIVE DATA ANALYSIS FOR STUDYING THE EFFECTS OF HIGH-LET IONIZING RADIATION: A BIOINFORMATICS APPROACH

The use of high linear energy (LET) ionizing radiation (IR) is progressively being incorporated in radiation therapy (RT) due to its precise dose localization and high relative biological effectiveness. At the same time, these benefits of particle radiation become a high risk for astronauts in the case of inevitable long-term cosmic radiation exposure. Nonetheless, DNA Damage Response (DDR) activated via complex DNA damage on healthy tissue, occurring from such types of radiation, may be instrumental in the induction of various chronic and late effects. A method of approach in understanding the possible underlying mechanisms, is studying alterations in gene expression. To this end we identified Differentially Expressed Genes (DEGs) in IR-exposed healthy human tissue, utilizing microarray data available in public repositories. DEG analysis was conducted using R programming language. Consequently, through functional enrichment and biological network analysis, we identified biological pathways and processes implicated in DDR. By comparing low and high-LET radiation effects, our primary results indicate the induction of a differential biological response for high-LET, like an enhanced inflammatory response.In addition, patterns of DNA repair are substantially distinct compared to low-LET. Finally, we expanded our study in search of possible comorbidities for HZE particle exposure. Pathway enrichment analysis suggests the involvement of mechanisms, tightly correlated with neurodegenerative disorders like amyloid fibrils formation. Regarding blood tissue, platelet activation signaling was found, upholding the connection to cardiovascular disease. This holistic bioinformatics approach revealed cellular trends towards inflammation and degeneration which might be central to the development of late effects of high-LET radiation exposure. It can contribute to the identification of molecular targets for effective countermeasures

Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis

Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs), single strand breaks (SSBs) and base lesions within a short DNA region of up to 15–20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1) repair resistant, increasing genomic instability (GI) and malignant transformation and (2) can be considered as persistent “danger” signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity). Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair