Overview of DNA Damage Laboratory and Group - PowerPoint Presentation

1. Overview of DNA Damage Laboratory and GroupAlexandros G. Georgakilas1,* (h-index = 52, >13000 citations)1DNA Damage laboratory, Physics Department, School of Applied Mathematical and Physical Sciences, National Technical University of Athens (NTUA), Zografou 15780, Athens, Greece *Correspondence to A.G. Georgakilas: e-mail: alexg@mail.ntua.grhttps://scholar.google.gr/citations?user=MAD6KTcAAAAJ&hl=elhttps://www.linkedin.com/in/alexgeorgakilasntua/Twitter: @GeorgakilasAlexhttp://dielectricsgroup.physics.ntua.gr/index.php/about-medical-physics-group/

2. Innate immunityAdaptive immunitytime Immunological memoryOur laboratory is working on the following projects:-Development of state-of-the-art methodologies for the detection in situ in cell and tissues of complex DNA damage.Role of DNA damage and repair gene biomarkers in radiation response from plants to humans.Use of bioinformatics, metanalysis and omics for the better understanding of relationship between Radiation Response and the immune system. Apply Machine Learning for radiation effectsDevelopment of radiosensitivity DNA damage-based patient biomarkers.-Use of simulations for evaluating the response of biological systems to electromagnetic radiations, metallic nanoparticles.DNA Damage Laboratory (Dr. Georgakilas)http://dielectricsgroup.physics.ntua.gr/index.php/about-medical-physics-group/

3. DNA damage detection facilityLaboratory Equipment General cell culture capability, flow chamber, inverted microscope, centrifuge, pH meter, cytometer, ultrasonic baths, Orbital shakers.Dry sterilization furnace, pure water distiller, hot plates, precision balance, Vortex stirrers, refrigerators Electrophoresis electrophoresis devices, water bath, UV-transilluminator, micro-centrifugeImmunofluorescence (in situ immunofluorescence)- Fluorescence microscope (wide field) Cytogenetics (Chromosomal aberrations) optical microscope, camera Ability to grow cell colonies (colony assay) stereoscope, camera DNA sequencing using MinIon reader, PC cluster (need upgrade)Atomic Force Microscopy (AFM) – Scanning Electron Microscopy FacilityPhysics Department equipmentDielectric Spectroscopy - Equipment of the Dielectric TeamBroadband Dielectric Spectroscopy (BDS), Thermally Stimulated Depolarization Currents (TSDC), Differential Scanning Calorimetry (DSC), Thermal Conductivity - Laser Flash Analysis (LFA), Water and Organic Vapor Sorption / Diffussion, High Frequency Spectrscopy.12. In house Low dose alpha particle apparatus. The system includes a 234U alpha (α)- source of total activity 0.77 ± 0.03 MBq. Irradiation of cells with doses 0.01 – 1 Gy. 13. Associated facility : Use of Transmission Electron Microscopy (TEM) in Medical School Lab

4. Using fluorescence microscopy for detection of complex DNA damage Through foci localization patternsHigh Colocalization of APE1 (green) and 53ΒP1 (red) foci on A549 cells exposed to Micro-Léman collimated X-rays, 10 min after irradiation .Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET). Nikitaki Z, Nikolov V, Mavragani IV, Mladenov E, Mangelis A, Laskaratou DA, Fragkoulis GI, Hellweg CE, Martin OA, Emfietzoglou D, Hatzi VI, Terzoudi GI, Iliakis G, Georgakilas AG. Free Radic Res. 2016 Nov;50(sup1):S64-S78.Nikitaki, Z., Pariset, E., Sudar, D., Costes, S.V. and Georgakilas, A.G. (2020) In Situ Detection of Complex DNA Damage Using Microscopy: A Rough Road Ahead. Cancers, 12, 3288.

5. Experimental Design at the Imaging Medical Beam Line (IMBL), Synchrotron MRT & BB Radiation, Melbourne, Australia 0, 10 or 40 Gy local irradiation 6-week old female C57BL/6J mice (5 mice per group) 5 cm Systemic effects of radiation in vivo (with Dr. Olga Martin)1. Lobachevsky, P.N., Ventura, J., Giannakandropoulou, L., Forrester, H., Palazzolo, J.S., Haynes, N.M., Stevenson, A.W., Hall, C.J., Mason, J., Pollakis, G. et al. (2019) A Functional Immune System Is Required For The Systemic Genotoxic Effects Of Localised Irradiation. International Journal of Radiation Oncology*Biology*Physics, 103 1184-1193.2. Ventura, J., Lobachevsky, P.N., Palazzolo, J.S., Forrester, H., Haynes, N.M., Ivashkevich, A., Stevenson, A.W., Hall, C.J., Ntargaras, A., Kotsaris, V. et al. (2017) Localized Synchrotron Irradiation of Mouse Skin Induces Persistent Systemic Genotoxic and Immune Responses. Cancer Res, 77, 6389-6399.

6. DIELECTRIC SPECTROSCOPY FACILITYDIELECTRIC MATERIALSSTUDY OF POLYMERS/BIOPOLYMERS

7. GRAPHICAL ABSTRACTAn overview of the use of Broadband Dielectric Spectroscopy (BDS) towards the measurement of biophysical and structural changes in mammalian tissues after exposure to radiations (Ionizing radiation-IR or non-IR) or growing tumors-induced stress. M.P. Souli et al. Applying Broadband Dielectric Spectroscopy (BDS) for the Biophysical Characterization of Mammalian Tissues under a Variety of Cellular Stresses. Int J Mol Sci 18 (2017).

8. THE USE OF TRANSMISSION ELECTRON MICROSCOPY (TEM) ON STUDYING AuNPs RADIOSENSITIZATIONTEM is a basic assay in order to study the cellular uptake of metallic NPs which play key role in their radiosensitization effect (see Figure 1)TEM can be used along with other exp. Procedures for identifying and quantifying DNA damage markers (single or double immunolocalization/immunogold labelling) (see Figure 2)Figure 1: Cellular uptake of 15 nm PEG‐capped GNPs in PC3 cells. Double thick arrows indicate vesicles and single thin arrows indicate the cytoplasm.N: nucleus, G: Golgi apparatus, RER: rough endoplasmic reticulum, m: mitochondriaFigure 2: PC3 cells after immunogold labelling for DNA damage detection. Double thick arrows indicate AuNPs uptake. 10 nm immunogold particles and 25 nm immunogold particles are used for labelling γH2AX (single thin arrows) and OGG1 (single thick arrows) respectively. n: nucleolusTremi, I.; Havaki, S.; Georgitsopoulou, S.; Lagopati, N.; Georgakilas, V.; Gorgoulis, V.G.; Georgakilas, A.G. A Guide for Using Transmission Electron Microscopy for Studying the Radiosensitizing Effects of Gold Nanoparticles In Vitro. Nanomaterials 2021, 11, 859. https://doi.org/10.3390/nano11040859

9. Initiatives/researches/projects in progress and Past/in progress Collaborations with private industries, research centers and government:Member of the scientific committee of InSyBio, Ltd with the important role to guide the research activities of the company in the fields of personalized cancer treatment, DNA damage response and immune response mechanisms. Ongoing-Past Research Projects (NTUA): (more than 10 young scientists PhD-Postdocs trained over the years in NTUA )Various National and International Projects1. Title: “Processing of oxidatively induced clustered DNA lesions under a double strand break repair deficiency in human tumor cells”. Source: Marie Curie Career Integration Grants (CIG) —EU (Proposal no: 303514). Budget: 150,000 euros; Role: Principal Investigator (PI: 20%); Dates: 1/6/2012-30/5/2016.2. Title: “Research and development of novel multifunctional polymer nanocomposites”. Source: THALIS-Greek Secretariat Research and Technology (Proposal no: 356 MPN). Budget: 510,000 euros; Role: co-Principal Investigator (co-PI: 2.5%); Dates: 1/8/2012-30/7/2016.3. Title: “Mechanistic insights on the etiology of space radiation-induced chromosomal aberrations and risk for carcinogenesis using conventional and interphase cytogenetics”. Source: European Space Agency (ESA-AO-10-IBER-24). Budget: 375,000 euros. Role: co-Principal Investigator (co-PI: 2.5%). Dates: 1/1/2013-30/12/2014. Pending final funding decision from the Greek Secretariat of Research and Technology.4. Title: "Biomimetic radical chemistry". Source COST ACTION NUMBER: CM1201. Budget: 740,000 euros. Role: co-PI and regional coordinator for Greece (PI: Dr. C. Chatgilialoglu-Italy). Dates: 1/12/2012-30/12/2016.5. Title: “DNA Damage and Repair and Their Relevance to Carcinogenesis”. Source German Academic Exchange Service (DAAD) (No 57339330). Budget: 400.000 euros. Role co-PI (PI: Professor G.Iliakis, University of Essen). Dates: 1/5/2017-30/4/2022.6. Title: “Effects of ionizing radiation on humans, complex DNA damage and systemic effects”. Source Greek National Funds for Research and Development ESPA (MIS 5004875). Budget: 150.000 euros PI: A. Georgakilas. Dates: 1/5/2018-1/9/2019.  Completed Research Projects USA (ECU 2004-2011): A total funding level of $600k has been received from different organizations: NIH, NC Biotechnology Center Grant, ECU Excellence Award Program, International Union Against Cancer (UICC). .

10. The Georgakilas Group at NTUAStella Logotheti, Tremi Ioanna, Spyros Kalospyros, Antonio Pantelias, Zacharenia Nikitaki. Post doc collaboratorVasiliki Zanni, Maria Souli, Christine Vasileiou, Angeliki Gkikoudi, Hector DeLastic : Phd studentsCollaboratorsS. Costes , GeneLab Project Manager and Principal Investigator, NASAG. Iliakis, University of Essen, GermanyO. Martin, Peter MacCallum Cancer Center, Australia C. Hellweg, Institute of Aerospace Medicine, Koln, Germany; R. Stewart, University of Washington, USAGreeceV. Gorgoulis, School of Medicine, University of Athens, GreeceG. Pantelias, G. Terzoudi , NCSR ‘Demokritos’, GreeceD. Emfietzoglou, University of Ioannina, GreeceC. Vorgias, Biology Department, University of Athens, GreeceThis work partially funded International Union Against Cancer (UICC), EU Marie Curie IRG Grant, Thalis’ Grant GSRT Greece. COST Action CM1201. DAAD Grant ‘DNA Repair’