INSTITUTE OF BIOLOGY, MEDICINAL CHEMISTRY & BIOTECHNOLOGY
 
  Drug Discovery
  Molecular Analysis
  Organic and Organometallic Chemistry
  Medicinal Chemistry
  Structural Biology & Chemistry
  Molecular Endocrinology
  Signal Mediated Gene Expression
  Molecular & Cellular Ageing
  Biomedical Applications
  Holistic Approaches in Health
  Chemical Carcinogenesis and Genetic Toxicology
  Metabolic Engineering-Bioinformatics
  Biotechnology
  Enzyme and Synthetic Biotechnology
  Biomimetics & Nanobiotechnology

 

Biomimetics & Nanobiotechnology

Biomimetics & Nanobiotechnology

 

The Biomimetics and Nanobiotechnology group deals with studies on the behavior of bioactives, such as peptides, enzymes, drugs, vitamins and antioxidants in model systems. The studies are focused on structural and functional aspects of the biomolecules when hosted in nano-scale environments such as nanodispersions (microemulsion or nanoemulsions) and related systems.

In such systems the coexistence of water nano-droplets in a continuous non-polar organic phase (oil) and vice versa, allows the solubilization of substances with various polarities. The lipophilic ones are localized in the oil phase and the hydrophilic ones (such as antioxidants) are encapsulated in the aqueous phase where they react through a huge interface.

 

Research Team

Research Staff
Dr. Aris Xenakis, Researcher Α'
Dr. Vassiliki Papadimitriou, Researcher B'
Dr. Maria Zoumpanioti, Research Engineer Β’

Scientific Technical Personnel
Mr. G. Sotiroudis, MSc. Pharmacist

Researcher Emeritus
Dr. Theodoros G. Sotiroudis, Chemist

Post Doctoral Fellows
Dr. Spyridon Avramiotis, Chemist
Dr. Maria Chatzidaki, Chemical Engineer
Dr. Ilias Matis, Chemist

PhD Students
Ioanna Theochari, Msc. Chemist, University of Thessaly, Greece
Evgenia Mitsou, Msc. Biologist, University of Ioannina, Greece
Evdokia Vassiliadi, Msc. Biologist, University of Ioannina, Greece
Sotiria Demisli, Msc. Chemist, University of Thessaly, Greece

Msc. Students
Eirini Karvouni, Msc. Agriculturist-Ichthyologist, Agricultural University of Athens

Bsc. students
Anastasios Aridas, Department of Biological Applications, University of Ioannina, Greece
Evdoxia Karpenisioti, Department of Biological Applications, University of Ioannina, Greece
Xrysa Kourtesi,
Technical University of Athens, Greece
Maria Kyriakaki, Department of Biological Applications, University of Ioannina, Greece
Maria Vlachou
, Department of Biological Applications, University of Ioannina, Greec

[Full List of students]

Visiting Members/ Recent collaborators (since 2012)

  • Ines Nicolic, Department of Pharmaceutical Technology and Cosmetology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia
  • Alexis Leroy, University Grenoble Alpes, France
  • Paulina Kulpa, University of Opole, Poland
  • Marija N. Todosijević, Department of Pharmaceutical Technology and Cosmetology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia
  • Marwa Charri, Department of Biological Engineering, National Engineering Scholl in Sfax, Tunisia
  • Ioanna Chazapi, University of Cyprus, Nicosia, Cyprus
  • Nehir Arik, Izmir Katip Celbi Universites, Menemen. Turkey
  • Maissa Khemakhem, Ecole Supérieure des Industries Alimentaires de Tunis, Tunis, Tunisia
  • Milan Nikolic, Faculty of Pharmacy, University of Belgrade, Serbia
  • Karen Mendeiros Gonçalves, Faculty of Pharmacy, Federal University of Rio de Janeiro, Brazil
  • Ivaldo Itabaiana Junior, Faculty of Chemistry, Federal University of Rio de Janeiro, Brazil
  • Andreia Fatima Zanette, Department of Chemical Engineering, State University of Maringa, Brazil

 

Overview of research activities

The activities developed within the Biomimetics and Nanobiotechnology group comprise:

  • Development and structural characterization of nanodispersions - nanoformulation
  • Structure and function studies of immobilised enzymes - biocatalysis.

Our lab has the expertise in formulation of nanodispersions such as microemulsions and nanoemulsions, especially on biocompatible ones, as well as enzyme immobilization and use for the synthesis of high added value products. Main aim of our group is the broadening of their applications in different aspects of life from food products to cosmetics and pharmaceuticals.

 

Formulation of water-in-oil (W/O) microemulsion [Video]

 

Current projects

Isolation of nutrients from microalgae cultivation

Colloidal systems could be applied in the food sector as products with enhanced nutritional value. An interesting type of bioactive molecules is naturally occurring antioxidants. Antioxidants of different polarities could be encapsulated in emulsions or nanoemulsions for potential use as functional foods.

The scavenging effect of antioxidants found in Extra Virgin Olive Oil (ORAC, DPPH, EPR) using different in vitro techniques in homogeneous and micro-heterogeneous media are examined. Moreover, a two-step digestion model gives an insight on the relationship between colloidal dispersions and digestive enzymes.

 

 

Biocatalysis in surfactant-based and surfactant-free microemulsions

Microemulsions are regarded as "the ultimate enzyme microreactors" for liquid catalysis. Their structure, composed by water nanodroplets dispersed in a non-polar medium, provides several benefits for their use as media for enzymatic transformations. They have the ability to overcome the solubility limitations of hydrophobic substrates, enhance the enzymatic activity (superactivity phenomenon) and stability, while providing an interface for surface-active enzymes. Of particular interest is the use of such systems to study biotransformations catalyzed by lipases and other enzymes towards the production of high added value products. Weak self-assembly systems consisted by a nonpolar hydrocarbon, an alcohol and water can form macroscopically homogeneous liquid media with distinct microenvironments of varying polarity. These ternary systems are very similar to classic water in oil microemulsions although they don’t have any amphiphilic surfactant. They were thus, initially called “surfactantless” or “detergentless” microemulsions. One of the main application fields that these systems cover is the one of biocatalysis in non-conventional media, a pillar of industrial biotechnology.

 

Enzyme immobilization on gels based on natural polymers

Gels, either based on water-in-oil microemulsions (organogels) or on water (hydrogels) can be formed using various natural polymers such as gelatin, agar, chitosan or cellulose derivatives. Enzymes entrapped in such systems can keep their activity and enhance their stability within the gel matrix. The importance of these catalysts leans on their numerous potential biotechnological applications. An important example is the use of various lipase-containing systems for hydrolytic or synthetic reactions.

 

Microemulsions as carriers of potential anticancer drugs for targeted therapies.

Nanodispersions based on biocompatible, safe materials are formulated as delivery vehicles of drugs with potential anticancer activity.
Most of the chemotherapeutic drugs currently available are poorly water-soluble and need to have their bioavailability improved through the development of enabled formulations and effective delivery mechanisms. One of the most important fields of modern "translational research" is the design and development of delivery systems as carriers for chemotherapeutic drugs. Their targeted-controlled release in tumor tissues is an attempt to avoid side effects and face major clinical problems such as tumor resistance.

In context of STHENOS project, (O/W) micro- and nanoemulsions were formulated as delivery vehicles of lipophilic antitumor drugs against various cancer types (e.g melanoma) correlated with the BRAFV600E mutation. Structural characterization of the proposed systems was obtained using viscosity, dynamic light scattering (DLS), electron paramagnetic resonance (EPR) spectroscopy and cryogenic-transmission electron microscopy (Cryo-TEM). This project was followed by STHENOS-β which aims to the formulation of biocompatible gels and (O/W) micro-  and nanoemulsions as delivery vehicles of lead compounds against cancer types correlated with the BRAFV600E mutation but also lead compounds as inhibitors of glucocorticoid receptor (GR).

 

Nanodispersions for intranasal delivery of bioactive compounds

The drug delivery through nasal route offers advantages including rapid absorption, avoidance of first-pass hepatic metabolism etc. In this respect, nanodispersions based on Extra Virgin Olive Oil will be used as carriers for a variety of bioactive compounds through nasal route. Intranasal administration of nanodispersions offers a practical, non-invasive, convenient and cost-effective route of administration. The nanodispersions will be tested for their possible toxic in nasal cells. It has to be underlined that preliminary studies have not shown any toxic effect in human colon cells of the above systems even in high concentrations. The construction of a nasal epithelium that is mimicking the physiological will be constructed with the cultivation of specific cell lines in Transwell device. The artificial epithelium will be used in order to prove if the proposed systems enhance the permeability of different encapsulated compounds in nasal mucosa and if cause morphological changes in the epithelium.

 

 

Current Funded projects:

  1. “QFytoTera: Nanoemulsions of plant oils with moisturizing and insect repellent properties”. NHRF, Operational Programme Competitiveness, Entrepreneurship and Innovation 2014- 2020, Industrial Partner: Qualia Pharma (Coordinator Dr S. Zographos, IBMCB/NHRF) (Budget: 630,005.37 €, Budget IBMCB/NHRF: 289.900) WP1 leader
  2. “Bioconversion of CO2 into high added value bioproducts through sustainable microalgae cultivation processes” Budget 1.000.000 € (Biomimetics & Nanobiotechnology 150.000,10 €), NHRF, Operational Programme Competitiveness, Entrepreneurship and Innovation 2014- 2020 Coordinator NHRF: Dr. Maria Zoumpanioti
  3. Program ΕΣΠΑ - “RIS3-AGROFOOD”. “Study and exploitation of olive oil as innovative raw material for special industrial applications to baby food products”. Budget 250.000 € (Biomimetics & Nanobiotechnology 65.000 €), Coordinator NHRF: Dr. Maria Zoumpanioti
  4. “Nanobiotechnology”, National Hellenic Research Foundation Project 2402, 2015-2018, Budget 162.635 €. Coordinator NHRF: Dr. A. Xenakis
  5. Program Agence Nationale de Recherche (ANR), Institut Carnot, Projet Lipides pour l’industrie et la santé (LISA), France. 2013-14. “Micro/nano-émulsions de type e/h biocompatibles et riches en micronutriments : préparation et caractérisation” Budget: 43.000 €. Coordinator: F.Calderon, INP Bordeaux.
  6. EU INCO FP7: “Mediterranean Science, Policy, Research & Innovation Gateway (MEDSPRING)”, 2013-6 Budget: 289.650 €. Coordinator NHRF: Dr A. Xenakis
  7. EU INCO FP7: “EURO-MEDITERRANEAN” Cooperation through ERANET joint activities and beyond (ERANETMED, 2013-6, Budget: 107.649,45 €. Coordinator NHRF: Dr A. Xenakis
  8. Program National Strategic Reference Framework, Action “Developmental Projects of Research Organisations- Kripis”2013-2015: “STHENOS”, Targeted therapeutic approaches against degenerative diseases, with special focus on cancer and ageing. Budget: 1.500.000,00 € (Biomimetics 174.773€). Coordinator Dr A. Pintzas
  9. Program ΕΣΠΑ PAVET “Application of Nanoemulsion Technology to food products based on Oil in Water Emulsions such as vegetable cream and fudge icings” Budget: 300.000,00 € Contractor: JOTIS SA Budget :61.250€ Coordinator NHRF, Dr. V. Papadimitriou.

Complete list of projects

 

Collaboration with industries (since 2012)

The collaboration of our team with industries has two main axes:

  • Provision of services in order to determine properties of different types of products (use of EPR, DLS etc.)
  • Product development through common projects (food products, cosmetics etc.)

JOTIS SA

  • “RIS3-AGROFOOD” “Study and exploitation of olive oil as innovative raw material for special industrial applications to baby food products” ΠΑΒΕΤ 2013.
  • “Application of Nanoemulsion Technology to food products based on Oil in Water Emulsions such as vegetable cream and fudge icings” (BeNeFIC)
  • Submission ΠΑΒΕΤ 2013. “Exploitation of Greek vinery waste for the development of high added value traditional foods”.
  • Submission Greek-Germany Cooperation «Food grade surfactant-free microemulsions for healthy and safe food preparations».

ALGAE S.A.

  • Joint program ΕΣΠΑ “Bioconversion of CO2 into high added value bioproducts through sustainable microalgae cultivation processes”

Public Power Corporation (ΔΕΗ)

  • Joint program ΕΣΠΑ “Bioconversion of CO2 into high added value bioproducts through sustainable microalgae cultivation processes”

APIVITA S.A.

QUALIA PHARMA

  • QFytoTera: Nanoemulsions of plant oils with moisturizing and insect repellent properties”. NSRF, Operational Programme Competitiveness, Entrepreneurship and Innovation 2014- 2020.

PAGONIS-DIAIRY

VIORYL S.A.

  • Common Programme ΕΣΠΑ “Cooperation” 2010-2. “Development of nanoemulsions as new materials for managing phytoprotective products to reduce the environmental charge”

 

Group activities – Honors

  • Scholarship from Hellenic Foundation and Research Foundation (HFRI) and the General Secretariat for Research and Technology (GSRT)
    PhD student E. Mitsou was selected by the Hellenic Foundation and Research Foundation for a 18month PhD scholarship.

  • Scholarship from Institut Français Gréce
    Dr. M. Chatzidaki was selected by the Institut Français Gréce for a three-month Post-Doc scholarship «Séjours scientifiques de haut niveau» 2016.

  • Innovathens 2016 – Olea team
    Our team has been selected by scientists specialized in food science, economists and entrepreneurs to participate in the Business Accelerator called “Food for Business” organized by the Innovathens team- Hub of Innovation and Entrepreneurship, Technopolis city of Athens. (For more information visit: http://www.innovathens.gr/). “Food for Business” is a three-month program of accelerated advisory that provides high quality information services, education and personalized mentoring/coaching in groups and startups who aspire to try the fields of foods, gastronomy and tourism.

  • Ecotrophelia competition 2015 – (µeRÅki)
  • Ekkinisi Lab 2014

 

Recent patents

  • Papadimitriou K., Chatzidaki M. D., Alexandraki S., Papadimitriou V., Tsakalidou E., Xenakis A. 2015. “Water-in-oil (W/O) microemulsions as carriers of bacteriocins for the antimicrobial protection of foods” (OBI – 1008858).
  • Chatzidaki M. D., Mitsou E., Theochari I., Papadimitriou V., Xenakis A., 2015. “Edible microemulsions with encapsulated plant extracts as dressing type products” (OBI – 1008863).
  • Filippou K., Zoumpanioti M., Xenakis A. 2014. “Immobilized catalyst in a continuous flow system for the synthesis of high added value products” (OBI – 1008307).
  • Kalaitzaki A., Xenakis A., Papadimitriou V. (2013) “Biocompatible nanodispersions as media for encapsulating bioactive substances with phytoprotective activity” (OBI- 20130100305).

 

Selected Recent Publications

  1. Ioannou, P. C., Arbez-Gindre, C., Zoumpanioti, M., Raptopoulou, C.P., Psycharis, V., Kostas, I.D., Kyritsis, P. Catalytic reactivity of the complexes [Pd{(Ph2P)2N(tBu)-P,P´}X2], X = Cl, Br, I, in the Suzuki-Miyaura C−C coupling reaction: Probing effects of the halogeno ligand X− and the ligand's tBu group. J. Organomet. Chem. (2019) 879, 40-46. (IF 1.946)
  2. Mitsou, E., Kalogianni, E.P., Georgiou, D., Stamatis, H., Xenakis, A., Zoumpanioti, M. Formulation and structural study of a biocompatible Water-in-Oil microemulsion as an appropriate enzyme carrier: The model case of Horseradish peroxidase. Langmuir (2019) 35(1) 150-160 (IF 3.789)
  3. Vassiliadi E., Xenakis, A.. Zoumpanioti, M.. Chitosan hydrogels: A new and simple matrix for lipase catalysed biosynthesis. Mol Catal, (2018) 45, 206-212 
  4. Golfomitsou, I., Mitsou, E., Xenakis, A., Papadimitriou, V. Development of O/W nanoemulsions as carriers of vitamin D: A structural study, J. Mol. Liq. (2018) 268, 734–742. (IF 4.513)
  5. Chatzidaki, M.D, Balkiza, F., Gad, E., Alexandraki, V., Avramiotis, S., Georgalaki, M., Papadimitriou, V., Tsakalidou, E., Papadimitriou, K., Xenakis, A. Reverse micelles as nano-carriers of nisin against foodborne pathogens. Part II: The case of essential oils, Food Chem., (2018) 278, 415-423. (IF 4.946)
  6. Chatzidaki, M.D.; Papadimitriou, K.; Alexandraki, V.; Balkiza, F.; Georgalaki, M.; Papadimitriou, V.; Tsakalidou, E.; Xenakis, A. Reverse Micelles as Nanocarriers of Nisin against Foodborne Pathogens. Food Chem. (2018), 255, 97–103. (IF 4.946)
  7. Theochari, I.; Papadimitriou, V.; Papahatjis, D.; Assimomytis, N.; Pappou, E.; Pratsinis, H.; Xenakis, A.; Vasiliki Pletsa, V. Oil-in-water microemulsions as hosts for benzothiophene cytotoxic compounds. An effective combination. Biomimetics (2018) 3, 13.
  8. Chaari, M., Theochari, I., Papadimitriou, V., Xenakis, A., Ammar, E. Encapsulation of carotenoids extracted from halophilic Archaea in oil-in-water (O/W) micro- and nano-emulsions. Colloids and Surfaces B (2018) 161, 219-227 (IF 3.902)
  9. Bubic Pajić, N.; Nikolic, I.; Mitsou, E.; Papadimitriou, V.; Xenakis, A.; Randjelovic, D.; Dobric, V.; Smitran, A.; Cekic, N.; Calija, B.; Savic, S. Biocompatible microemulsions for improved dermal delivery of sertaconazole nitrate: phase behavior study and microstructure influence on drug biopharamaceutical properties. Journal of Molecular Liquids (2018) 272, 746-758 (IF 4.513)
  10. Theochari, I., Goulielmaki, M., Danino, D., Papadimitriou, V., Pintzas, A., Xenakis, A. Drug nanocarriers for cancer chemotherapy based on microemulsions: the case of Vemurafenib analog PLX4720. Colloids and Surfaces Β: Biointerfaces (2017) 154, 350-356 (Corresponding author) (IF2015 3.902)


Complete list of publications & patents

Collaborations with Industries

 

Collaborations (since 2012)

  • Cirad, Argicultural Research for Development, Montpellier, France
  • Institut de Chimie & Biologie des Membranes & des Nano-objets (CBMN), Pessac, France
  • CNRS- Centre National de la Recherche Scientifique, Marseille, France
  • University of Örebro, Örebro, Sweeden
  • Technion- Israel Institue of Technology, Haifa, Israel
  • University of Copenhagen, Copenhagen, Denmark
  • Ecole Supérieure des Industries Alimentaires de Tunis, Tunis, Tunisia
  • Izmir Katip Celbi Universites, Menemen. Turkey
  • National Engineering School in Sfax, Sfax, Tunisia
  • Department of Pharmaceutical Technology and Cosmetology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia
  • Department of Chemistry, Univercity of Nicosia, Cyprus
  • Faculty of Pharmacy, Federal University of Rio de Janeiro, Brazil
  • Biocatalysis and Organic Synthesis Group, Chemistry Institute, Federal University of Rio de Janeiro, Brazil
  • State University of Maringa, Department of Chemical Engineering, Maringa, Brazil
  • Argicultural University of Athens, Athens, Greece
  • Department of Biological Applications & Biotechnology, University of Ioannina, Ioannina, Greece
  • Faculty of Biology, National & Kapodistrian University of Athens, Athens, Greece
  • University of Thessaly, Larissa, Greece
  • Department of Food Technology, Technological Educational Institute of Athens, Athens, Greece
  • Department of Food Technology, School of Argicultural Technology, Thessaloniki, Greece
  • Benaki Phytopathological Institute, Kifissia, Greece
  • Material Synthesis and Physical Chemistry, Theoretical & Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece

 

 

Upcoming conferences

34th ECIS 2020 Athens, Greece

The conference of the European Colloid and Interface Society will be held in Athens organized by our team and a group of prestigious scientists in the field of colloids. The annual ECIS Conference brings together participants from the broad, interdisciplinary field of colloid and interface science, including chemists, physicists, biologists and engineers.

 

 

8th Greek Lipid Forum / National Hellenic Research Foundation, Athens, Greece

The aim of the conference, with the participation of our team in the organizing committee, is to provide a platform for the exchange of scientific and technological knowledge in the field of lipids and oils. The aim of GLF (member of the European Federation for the Science and Technology of Lipids) is to inform the public about current lipid developments by bringing together scientists from the academic and research community, who deal with olive oil, edible fats, their technology and applications, non-food lipid applications etc.

 

Participation in Science Communication Projects

Our team has an active participation in Science Communication Projects such as Researcher’s Night and Athens Science Festival. Our aim is to give the opportunity to children to explore scientific and technological advancments in an entertaining way.

 

Scientific equipment

Dynamic Light Scattering (DLS)

DLS is a non-invasive, well-established technique that can be used to determine the size and the size distribution of particles in the sub-micron region. Some of the DLS applications which are used in our lab are the determination of particle size, polydispersity index and z-potential of different types of emulsions such as microemulsions and nanoemulsions.

 

 

Electron Paramagnetic Resonance (EPR)/ Bruker EMX

EPR is a powerful, versatile, nondestructive and nonintrusive analytical method yielding structural and dynamical information, even from ongoing chemical or physical processes.

The Bruker EMX EPR spectometer is a research grade scientific instrument. It is capable of routine measurements, as well as sophisticated and advanced experiments.

Some of the EPR applications which are commonly used in our laboratory are listed below:


  • Study of interfacial properties and dynamic structures in different nanodispersions, using spin labels such as 5-doxyl stearic acid (5-DSA)
  • Investigation of proteins dynamics and conformation in a variety of environments by bounding an enzyme’s active site with appropriate spin-label reagents
  • Sensitive and rapid method (spin trapping) for evaluating the antioxidant activity of potential antioxidant compounds by using free radicals (Galvinoxyl, DPPH etc.)
  • Detection of paramagnetic species

Our group has expertise in EPR studies with decades of experience and plentiful bibliography (See the complete list publication archive) in a wide range of applications from biotechnology and pharmaceuticals to food applications.

 

Gas Chromatograph

A useful tool for monitoring enzymatic bioconversions

 

High-performance liquid chromatography

Used for qualitative and quantitative analysis of reaction samples

 

High Pressure Homogenizer (PANTA PLUS – GEA)

PandaPlus 1000 is a table-top laboratory unit, designed for continuous operation at high pressure, for high pressure processing of nanoparticles, nanodispersions, nanoemulsions and for cell disruption. In addition, it can be used for processing dairy products, fruit juices, liquid food, food additives and ingredients as well as cosmetic and chemical products. Tests can be run even with small sample volumes, and the results are comparable with those achieves on bigger size machines.


Probe homogenizer (UNIDRIVE X1000 homogenizer)

It is a homogenizer for general laboratory purpose with electronically stabilized speed control. The stirring medium is subjected to a combination of mechanical shearing and sonic energy. It is suitable for the formulation of emulsions, nanoemulsions and Pickering emulsions

 

Franz Diffusion Cell

Franz Diffusion Cell is generally regarded as the most common in vitro release testing method. It is often used to monitor release of active compounds from semi-solid drug products across a membrane (synthetic membrane, a tissue construct or biological sample such as cadaver skin). It is suitable for testing the release of active substances encapsulated in microemulsions or gels.

 

 

High pressure apparatus

Our high pressure apparatus is suitable for performing enzymatically catalyzed reactions under supercritical conditions. Since supercritical carbon dioxide (scCO2) has emerged as perhaps the most promising “green” solvent (apart from water) due to its being toxicologically harmless, not inflammable, readily available and inexpensive, enzymatic reactions in scCO2 are increasingly attracting interest. Using this apparatus, we aim in replacement of any organic solvent used in the conventional biocatalytic process by harmless CO2.

 

Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) System

The hybrid quadrupole / linear ion trap technology of the 3200 QTRAP System offers the ability to identify and quantitate components from complex samples in a single run. LC-MS/MS can be used for qualitative and quantitative analysis of lipid complex mixtures but also for the monitoring of chemical reactions (esterification, epoxidation).
The mass spectrometer has a wide range of applications and among others, it has been used in our lab for the identification and determination of smoking biomarkers in meconium.

 

 

 

News

Dr. A Xenakis interview in ''Ελευθεροτυπία'' Newspaper (26-5-2013)

Collaboration with Örebro, Sweden

 

 

 

 
 

 

   
       

 

 

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