The Henryk Niewodniczański INSTITUTE OF NUCLEAR PHYSICS Polish Academy of Sciences 152 Radzikowskiego str., Kraków, Poland

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1 The Henryk Niewodniczański INSTITUTE OF NUCLEAR PHYSICS Polish Academy of Sciences 152 Radzikowskiego str., Kraków, Poland Kraków, December 2014 Report No. 2077/AP XLV Polish Seminar on Nuclear Magnetic Resonance and Its Applications. Kraków, 1-2 December 2014 ABSTRACTS Organized by: The H. Niewodniczański Institute of Nuclear Physics PAN, Kraków and Committee of Physics, Polish Academy of Sciences Organizing Committee: Members: Ż. Bartel A. Osiak A. Birczyński A. Osip J. Blicharski Z. Olejniczak B. Błasiak W. Rutkowski K. Byk W. Piędzia M. Jabłońska T. Skórka K. Jasiński G. Stoch K. Kalita U. Tyrankiewicz Z.T. Lalowicz /Chairman/ W. P. Węglarz /V-Chairman/ M. Noga /Secretary/ Sponsors: BRUKER-POLSKA Sp. z o.o, Wydział III Nauk Ścisłych i Nauk o Ziemi POLSKIEJ AKADEMII NAUK

2 Addresses of the sponsors: BRUKER POLSKA SP. Z O.O mgr W. Leszczyński ul. Budziszyńska Poznań tel. (061) fax. (061) sekretariat@bruker.poznan.pl Wydział III Nauk Ścisłych I Nauk o Ziemi POLSKIEJ AKADEMII NAUK Pl. Defilad Warszawa

3 CONTENTS 1. FREEZING RESISTANCE OF CRYPTOBIOTIC POLYPEDILUM VANDERPLANKI LARVA BY 1H-NMR Ewelina Baran, Piotr Nowak, Hubert Harańczyk, Stanisław Knutelski, Kazimierz Strzałka, Takashi Okuda 2. MOLECULAR DYNAMICS OF METHANOL CONFINED IN NANOSCALE CAGES OF FAUJASITES. A DEUTERON NMR INVESTIGATION Z.T. Lalowicz, A. Birczyński, G. Stoch, K. Góra-Marek, J. Datka 3. 1H NMR BASED METABOLOMIC APPROACH TO MONITORING OF THE HEAD AND NECK CANCER TREATMENT RESPONSE Łukasz Boguszewicz, Agata Hajduk, Jolanta Mrochem-Kwarciak, Agnieszka Skorupa, Mateusz Ciszek, Krzysztof Składowski, Maria Sokół 4. LIVER CONDITION IN MICE MODEL OF ACUTE HEPATITIS BASED ON MAGNETIC - RESONANCE IMAGING Katarzyna Byk, Żaneta Bartel, Krzysztof Jasiński, Bogusław Tomanek, Tomasz Skórka 5. METABOLIC HALLMARKS OF VISUAL CORTEX DEGENERATION IN MOUSE MODEL OF GLAUCOMA (DBA/2J MICE) REVEALED BY IN VIVO PROTON MAGNETIC RESONANCE SPECTROSCOPY Michał Fiedorowicz, Jarosław Orzeł, Bartosz Kossowski, Marlena Wełniak-Kamińska, Piotr Bogorodzki, Paweł Grieb 6. IS HIGH-FAT HIGH-CARBOHYDRATE DIET (HFCD) NEUROPROTECTIVE? A MAGNETIC RESONANCE IMAGING STUDY IN WISTAR RATS Stefan Gaździński, Zuzanna Setkowicz, Joanna Osoba, Karolina Karwowska, Piotr Majka, Jarosław Orzeł, Bartosz Kossowski, Piotr Bogorodzki, Marlena Kamińska, Michał Fiedorowicz 7. VOXEL-WISE ANALYSES OF HIGH-FAT HIGH-CARBOHYDRATE DIET ON BRAIN STRUCTURE IN WISTAR RATS Piotr Majka, Zuzanna Setkowicz, Joanna Osoba, Karolina Karwowska, Jarosław Orzeł, Bartosz Kossowski, Piotr Bogorodzki, Marlena Kamińska, Michał Fiedorowicz, Stefan Gaździński 8. EFFECTS OF HIGH-FAT HIGH-CARBOHYDRATE DIET ON WHITE MATTER INTEGRITY: A DIFFUSION TENSOR IMAGING STUDY IN WISTAR RATS Stefan Gaździński, Andrzej Gaździński, Zuzanna Setkowicz, Joanna Osoba, Karolina Karwowska, You Zhang, Jarosław Orzeł, Bartosz Kossowski, Piotr Bogorodzki 9. HR MAS NMR METABOLIC PROFILES OF CARDIOMYOCYTES AFTER RADIATION EXPOSURE Michalina Gramatyka, Agnieszka Skorupa, Mateusz Ciszek, Łukasz i

4 Boguszewicz, Maria Sokół 10. THEORETICAL CALCULATIONS OF 13 C NMR CHEMICAL SHIFTS OF BROMINE-SUBSTITUTED CARBON ATOMS Adam Gryff-Keller, Dominika Kubica, Artur Wodyński 11. H-NMR IN LIVING ORGANISMS, TISSUES, AND OTHER BIOLOGICAL SYSTEM RESISTANT TO DRASTIC DEHYDRATION OR TO LOW TEMPERATURES Hubert Harańczyk 12. REMARKS ON THE FREE-APPROACH MODELS Łukasz Jaremko, Mariusz Jaremko, Michał Nowakowski, Andrzej Ejchart 13. COMPARISON OF MAGNETIC RESONANCE IMAGING OF THE MOUSE BRAIN IN VIVO USING DIFFERENT TYPES OF RF COILS AT 9.4 T J. Jasieniak, W. Piedzia, K. Jasinski, W.P. Weglarz 14. NMR STUDY OF P(MEO2MA) POLYMER NETWORKS J. Jenczyk, S. Kadłubowski, M. Olejniczak, M. Kozanecki, S. Jurga 15. QUANTITATIVE MRI IN STUDYING WHITE MATTER DAMAGE FOLLOWING SPINAL CORD INJURY Piotr Kozłowski 16. NEW HIGH DIMENSIONALITY NMR EXPERIMENTS FOR BIOMOLECULES Wiktor Koźmiński 17. INTRAMOLECULAR INTERACTION OF HYBRID OF URIDINE AND STILBENE DERIVATIVE Hanna Krawczyk, Przemysław Szczeciński 18. IMAGING METHODS IN RESEARCH AND DEVELOPMENT PROCESS OF GENERIC MODIFIED RELEASE MATRIX TABLETS Piotr Kulinowski, Krzysztof Woyna-Orlewicz, Gerd-Martin Rappen, Dorota Haznar-Garbacz, Władysław P. Węglarz, Przemysław P. Dorożyński 19. MOLECULAR MODELING OF SINGLE WALL CARBON NANOTUBE (SWCNT) CHEMICAL SHIFT DUE TO ADDITION OF DIATOMICS Teobald Kupka, Marzena Nieradka, Leszek Stobiński, Jakub Kaminský 20. MOLECULAR MODELING OF CHEMICAL SHIFT OF ATOMS, SMALL AND LARGE MOLECULES Teobald Kupka, Michał Stachów, Marzena Nieradka, Klaudia Radula-Janik, Roksana Wałęsa, Aneta Buczek, Małgorzata Broda 21. BADANIA SPEKTROSKOPOWE PT(II) Z 7,8-BENZOCHINOLINĄ ORAZ 2- FENOKSYPIRYDYNĄ Daria Niedzielska, Leszek Pazderski ii

5 22. FREEZING AND DRYING RESISTANCE OF ANTARCTIC TURGIDOSCULUM COMPLICATULUM THALLI AS OBSERVED BY 1 H- NMR METHODS Magdalena Bacior, Piotr Nowak, Paulina Kijak, Ewelina Baran, Hubert Harańczyk, Maria A. Olech 23. THE SOLUTION STRUCTURE OF THE MANEC-TYPE DOMAIN FROM HEPATOCYTE GROWTH FACTOR INHIBITOR 1 REVEALS AN UNEXPECTED PAN/APPLE DOMAIN-TYPE FOLD Michał Nowakowski, Zebin Hong, Chris Spronk, Steen V. Petersen, Jan S. Pedersen, Wiktor Koźmiński, Frans A.A. Mulder, Jan K. Jensen 24. ATLAS-BASED AUTOMATIC BRAIN MORPHOMETRY APPLIED TO DBA/2J MOUSE MODEL OF GLAUCOMA Jarosław Orzeł, Michał Fiedorowicz, Bartosz Kossowski, Marlena Wełniak-Kamińska, Piotr Bogorodzki, Paweł Grieb 25. MR TAGGING FOR EVALUATION OF MECHANICAL PROPERTIES OF FATTY LIVER TISSUE Anna Osiak, Krzysztof Jasiński, Paweł T. Jochym, Edyta Maślak, Tomasz Skórka 26. MRI-BASED METHOD FOR THE IN VIVO ASSESSMENT OF ENDOTHELIAL STATE IN MURINE MODELS Anna Osip, Krzysztof Jasiński, Żaneta Bartel, Tomasz Skórka, Stefan Chłopicki Na NMR STUDY OF Al- AND Ga- NANOPOROUS NATROLITES Mateusz Paczwa, Marcin Olszewski, Nikolaj Sergeev Aleksey.A. Sapiga, Aleksey.V. Sapiga 28. 2D AND 3D CP-VC AS TOOLS FOR DYNAMICS STUDY Piotr Paluch, Tomasz Pawlak, Julien Trébosc, Tatyana Polenova, Jean-Paul Amoureux, Marek J. Potrzebowski 29. EVALUATION OF THE RELAXATION AND THE IMAGING PROPERTIES OF SPIO LOADED NANOCAPSULES AT 9.4 T P. Piechota, K. Szczepanowicz, P. Warszyński, W. P. Węglarz 30. MR IMAGING OF THE MOUSE BRAIN USING CRYO-COIL AT 9.4 T - HISTOLOGY IN VIVO? W.Piedzia, N. Bock, K. Jasinski, K. Kalita, G. Stanisz, W.P. Weglarz 31. SUPERCONDUCTING DETECTION COIL FOR 0.2 T MRI SYSTEM Bartosz Proniewski, Henryk Figiel 32. PARA HYDROGEN INDUCED POLARIZATION OF PYRIDINE-LABELLED OLIGOPEPTIDES Tomasz Ratajczyk APPLICATION OF MICRO-MRI TECHNIQUES IN THE EVALUATION OF 41 iii

6 MUSCLE DEGENERATION AND REPAIR PROCESSES AFTER FEMORAL ARTERY OCCLUSION IN MICE Agnieszka Skorupa, Mateusz Ciszek, Łukasz Boguszewicz, Tomasz Cichoń, Ryszard Smolarczyk, Stanisław Szala, Maria Sokół 34. APPLICATION OF NMR RELAXATION MEASUREMENTS TO THE STUDY OF OXIDATION PROCESSES IN BIOLOGICAL SYSTEMS Dorota Wierzuchowska, Lech Skórski, Barbara Blicharska 35. CHEMICAL EXCHANGE SATURATION TRANSFER (CEST). FROM AN AGAR TO A MAN Greg J. Stanisz 36. NMR TOP SYGNALS OF THE 27 Al IN SOLID SOLUTIONS BASED ON THE YAG CRYSTAL Piotr Stępień, Marcin Olszewski, Nikolaj Sergeev, Bohdan Padlyak 37. SPI IMPLEMENTATION FOR 4.7T SYSTEM Grzegorz Stoch 38. DYNAMIC EFFECTS IN SINGLE CRYSTAL OF 9,10- DIMETHYLTRIPTYCENE-D12 ON BASIS OF PROTON NMR SPECTRA Piotr Bernatowicz, Tomasz Ratajczyk, Alexander Shkurenko, Bohdan Kamienski, Sławomir Szymański 39. DIVERSE DYNAMICS OF WATER MOLECULES CONFINED IN FAUJASITES. DEUTERON NMR INVESTIGATION A. Szymocha, Z.T. Lalowicz, Birczynski, K. Gora-Marek 40. CHLORINS - SYNTHESIS AND NMR SPECTROSCOPY STUDIES Justyna Śniechowska, Piotr Paluch, Marek J. Potrzebowski 41. NMR SPECTROSCOPY OF SERUM LIPID EXTRACTS OF SARCOIDOSIS PATIENTS Toczylowska Beata, Jastrzebski Dariusz, Zieminska Elzbieta, Zieleznik Karolina, Zebrowska Aleksandra, Ziora Dariusz, Mierzejewska Aneta, Kozielski Jerzy 42. EYE MORPHOLOGY QUANTITATED BY MAGNETIC RESONANCE IMAGING IN MICE Marlena Wełniak-Kamińska, Tomasz Chorągiewicz, Michał Fiedorowicz, Jarosław Orzeł, Piotr Bogorodzki, Paweł Grieb 43. THE CYTOSTATIC AGENT AS A CONTRAST AGENT FOR MRI Beata Wereszczyńska, Tomasz Zalewski, Magdalena Hałupka-Bryl, Marek Kempka, Stefan Jurga 44. USEFULNESS OF MR SEQUENCES: DTI AND 3D ASL IN RARE CHILD BRAIN TUMOR BASED ON A MR BRAIN CHILD EXAM BEFORE AND AFTER SURGERY Magdalena Wicher, Magdalena Machnikowska-Sokołowska, Anna Plechta, iv

7 Katarzyna Zymella, Dominika Wieja-Błach, Marcin Basiak, Marek Konopka 45. EXOGENOUS SILK PROTEIN AND SLES EFFECT ON PROPERTIES OF HYDRATED HAIR BY 1H-NMR AND SORPTION ISOTHERM D. Zalitacz, P. Nowak, A. Ciułkowska, K. Pieńkowska, H. Harańczyk 46. 4D NMR EXPERIMENT FOR PHOSPHORYLATION STUDIES OF IDPS Szymon Żerko, Gerald Platzer, Robert Konrat, Wiktor Koźmiński 47. THE ZN IONS AS IMPORTANT FACTOR REGULATED UBIQUITIN- ACTIVATING PROCESS. STRUCTURAL STUDIES OF THE PEPTIDE DERIVED FROM CYSTEINE CATALYTIC HALF-DOMAIN (SCCH) OF MOUSE E1 ENZYME Ilona Marszalek, Arkadiusz Bonna, Wojciech Bal, Igor Zhukov v

8 FREEZING RESISTANCE OF CRYPTOBIOTIC Polypedilum vanderplanki LARVA by 1H-NMR Ewelina Baran, Piotr Nowak, Hubert Harańczyk, Stanisław Knutelski, Kazimierz Strzałka, Takashi Okuda Jagiellonian University, Cracow, Poland 1 Institute of Physics, Cracow, Reymonta St. 4 2 Institute of Zoology, Jagiellonian University, Cracow, Poland 3 National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki , Japan ewelina.motyka@uj.edu.pl African chironomid Polypedilum vanderplanki is its larval form is the largest multicellular animal capable to survive anhydrobiosis process [1]. It populates temporary basins In Northern Nigeria and Uganda. Its maggot may survive deep dehydration during the prolonged dry season [2]. Dehydrated larvas of the chironomid Polypedilum vanderplanki were grown in a laboratory conditions [3]. We monitored the rehydration kinetics, the sorption isotherm, and 1H-NMR spectra. We analysed water content in dehydrated larva, a number and an arrangement of water binding sites on inner surfaces of its organism, and formation of tightly and of loosely bound water fractions at different temperatures. 1 H-NMR spectra were recorded on a Bruker Avance III spectrometer (Bruker Biospin), operating at the resonance frequency 300 MHz (B0 = 7 T). The pulse length was π/2 = 2.1 ms. For low values of the relative humidity, the gaseous phase hycdration courses revealed the anomalous form, which presumably may be caused by the growths of bacterias in the testins of specimen. For higher humidities the gaseous phase hydration courses show two phases of bound water, namely (i) very tightly bound water, and (ii) tightly bound water. The sorption isotherm is sigmoidal in form, much better fitted using Dent model than BET-approach. The relative mass of water bound to primary binding sites was ΔM/m0 = H-NMR spectra show superposition of the solid component well fitted by Gaussian function (ν 44 khz), coming from protons of dried tissues of P. vanderplanki; and Lorentzian line component (with ν 1.6 khz) coming from water tightly bound on inner and outer surfaces of solid tissue. This behaviour of bound water resembles that for Coleoptera alytron [4] or in DNA- CTMA complex [5]. 1H-NMR spectra temperature dependence show the gradual immobilization of bound water fraction without the formation of the ice crystallites, as it was detected in thalli of Antarctic lichenized fungi experienceed very low temperature, in vivo [6]. The detected by us contribution of liquid signal in dry form of P. vanderplanki maggot is higher as it was expected. References M. Sakurai, T. Furuki, K. Akao, D. Tanaka, Y. Nakahara, T. Kikawada, M. Watanabe, T. Okuda, vol. 105 no. 13, R. Cornette, Y. Kanamori, M. Watanabe, Y. Nakahara, O. Gusev, K. Mitsumasu, K. Kadono- Okuda, M. Shimomura, K. Mita,T. Kikawada, T. Okuda., J Biol Chem Nov 12;285(46): T. Okuda. O. Gusev, 2012, H. Harańczyk, P. Nowak,, M. Florek, S. Knutelski, APP, 2011 H. Harańczyk, J. Kobierski, J. Nizioł, E. Hebda, J. Pielichowski, D. Zalitacz, M. Marzec, and A. El-Ghayoury, Journal of Applied Physics,113, (2013) H. Harańczyk, P. Nowak, M. Bacior, Ł. Pater, M.A. Olech, APP, 2011 H. Harańczyk, P. Nowak, M. Bacior, M. Lisowska, M. Marzec, M. Florek, M.A. Olech, Antarctic Science, Volume 24, Issue 04, August 2012, pp The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (POIG /08). 1

9 MOLECULAR DYNAMICS OF METHANOL CONFINED IN NANOSCALE CAGES OF FAUJASITES. A DEUTERON NMR INVESTIGATION. Z.T. Lalowicz a, A. Birczyński a, G. Stoch a, K. Góra-Marek b, J. Datka b a H. Niewodniczański Institute of Nuclear Physics PAS, Radzikowskiego 152, Kraków, Poland b Faculty of Chemistry, Jagellonian University. Ingardena 3, Kraków, Poland Nuclear magnetic resonance (NMR) provides means to investigate molecular dynamics at every state of matter. Molecules confined in nanoscale cages of zeolites represent a particularly interesting system. Features of molecular dynamics characteristic for the gas phase, liquid-like layers and immobilized molecules were observed in the temperature range from 300K down to 20K. Narrow lines were observed at high temperature indicating basically isotropic reorientation. Spin-lattice relaxation rates provide evidence for a transition from translational diffusion to isotropic reorientation as the main mechanism of relaxation for molecules confined in nanoscale zeolite cages for D2, CD4 and CD3OD. Other molecules like D2O, ND3, and (CD3)2CO are strongly bonded, both mutually and to zeolite framework, and exhibit a much more restricted diffusion. Deuteron spin-lattice relaxation was measured for methanol-d4 molecules confined in zeolite NaX and NaY cages [1]. Experimental evidence was given for the formation of trimers, their existence was so far proposed only by theory. The conclusion was based on observation of different relaxation rates for methyl and hydroxyl deuterons undergoing a common dynamics. A change in the slope of the temperature dependence of both relaxation rates indicates a transition from the relaxation dominated by translational motion to prevailing contribution of reorientation at 222K. Trimers undergoing isotropic reorientation disintegrate and separate methanol molecules become localized on adsorption centers at 169.5K and 153.8K for NaX and NaY, respectively as indicated by extreme broadening of deuteron NMR spectra. The transition temperature, higher for NaX, indicates the dominating role of the hydrogen bonding to framework oxygen, contradicting common assumption of preferred adsorption on sodium cation. NMR spectra at low temperature are consistent with the model in which molecules are bonded at two positions: horizontal (methanol oxygen bonded to sodium cation) and vertical (hydrogen bonding of hydroxyl deuteron of methanol to zeolite framework oxygen). Molecules at vertical position remain localized up to high temperature. Mobility of single methanol molecules was observed for a lower loading (86 molecules/uc) in NaX. A direct transition from diffusion to localization was observed at 190K in this case. The magnetization recovery can be fitted quite accurately by three exponentials in the low temperature phase of localized molecules. Therefore a new method is introduced for analyzing deuteron spin-lattice relaxation in molecular systems with different mobility and a broad distribution of activation energies and correlation times [2]. A Gaussian distribution of the activation energy was assumed. Three parameters: the mean activation energy, the distribution width and the pre-exponential factor in the Arrhenius equation were calculated. The obtained parameters characterize the methyl and hydroxyl mobility of the methanol molecules at two different locations. [1] Z. T. Lalowicz, G. Stoch, A. Birczyński, M. Punkkinen, E. E. Ylinen, M. Krzystyniak, K. Góra-Marek, J. Datka, Solid State Nucl. Magn. Reson (2012) [2] G. Stoch, E. E. Ylinen, A. Birczyński, Z. T. Lalowicz, K. Góra-Marek, M. Punkkinen, Solid State Nucl. Magn. Reson (2013) The project was generously supported by the National Science Centre, Grant No. N N during

10 1H NMR BASED METABOLOMIC APPROACH TO MONITORING OF THE HEAD AND NECK CANCER TREATMENT RESPONSE Łukasz Boguszewicz 1, Agata Hajduk 2, Jolanta Mrochem-Kwarciak 3, Agnieszka Skorupa 1, Mateusz Ciszek 1, Krzysztof Składowski 2, Maria Sokół 1. 1 Department of Medical Physics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland. 2 I Radiotherapy Clinic, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland. 3 Analytics and Clinical Biochemistry Department, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland. Introduction and methodology The response of organisms to anticancer treatment is reflected in morphological and functional changes in treated volume as well as systemic and alterations. It may be also assessed by analyzing alterations in composition and concentrations of metabolites in body fluids, inter alia blood serum. The studied group consisted of 77 patients (median age 59) treated with radical radiotherapy or chemoradiotherapy in case of head and neck cancer (HNC). Serum samples were collected before or during the first days of treatment and after the treatment or within the last three days of treatment. During the treatment patients underwent weekly medical (laryngological and endoscopic, sonographic of neck lymphatic system) examinations, laboratory blood tests and acute radiation syndrome (ARS) evaluation. Serum samples were measured on MHz Bruker Avance III spectrometer using NOESY, CPMG and diffusion edited sequences with water presaturation. Acquired spectra were referenced to alanine signal and bucketed over the region ppm with a bucket size ppm. Multivariate projection techniques: principal component analysis, non-orthogonal and orthogonal partial least squares discriminant analysis and regression were exploited in order to find metabolic patterns reflecting response to treatment as well as to find correlations with available clinical and laboratory data. Results and discussion The directions of the highest variance in the NMR spectra are strongly influenced by the treatment itself but are independent of the treatment type (radiotherapy or chemoradiotherapy) and duration. Serum metabolic profiles show significant changes around 45 th day after the start of the treatment: increased 3-hydroxybutyrate (3HBT) levels became significantly lower while signals from lipids and fatty acids start to increase. 3HBT can be used as an energy source (when blood glucose is low) for high-energy demanding processes connected to healing acute radiation syndrome and tumor necrosis cleaning in irradiated place. 3

11 Liver condition in mice model of acute hepatitis based on magnetic resonance imaging Katarzyna Byk 1, Żaneta Bartel 1, Krzysztof Jasiński 1, Bogusław Tomanek 1,2, Tomasz Skórka 1 1 Department of Magnetic Resonance Imaging, Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland 2 Faculty of Medicine & Dentistry, Department of Oncology, Medical Physics Division, University of Alberta, 3-12 University Terrace, Street NW, Edmonton, AB T6G 2T4,Canada Purpose: The aim of this study was to prepare a mice model of acute hepatitis and assessing the influence of this disease on liver metabolism. Materials and methods: 15 BALB/c mice weighting (20.5 ± 1.5)g were divided into 2 groups based on concanavalin A (ConA, Sigma-Aldrich, USA) dose: 1 - control (8 animals) and 2-8mg/kg ConA dose administrated 24hrs before experiment (7 animals). ECG, temperature and respiratory were monitored (SA Inc., Stony Brook, NY). The experiment was conducted on Bruker 9.4T magnet (Ettlingen, Germany). Anatomical (TurboRARE sequence), perfusion (FAIR_Epi sequence) and contrast enhanced dynamic imaging (IntraGATE sequence) were performed. Gadolinium based contrast (Primovist, Bayer Schering Pharma AG, Germany) was injected into tail vain. Data were analyzed using empirical mathematical modelling [1], texture [2] and fractal [3] analyses. Analyses were performed with the use of ImageJ (NIH, USA), OriginPro (OriginLab Corporation, USA) and Statistica (StatSoft, USA) software with the p-value equal to Results: Animals from ConA group lost weight from g to g during 24 hrs while control animals weight remained stable. Peak of maximal enhancement after contrast injection appeared later in ConA group (9.7 ± 1.7) min than in control group (5.9 ± 1.1) min. The elimination half-life of enhancement increased from (32.3 ± 6.9) min in control group to (66 ± 11) min in ConA group. Perfusion in diseased group was equal to (61.9 ± 6.4) ml/min/100g and was reduced in comparison to healthy group (95.5 ± 2.6) ml/min/100g. Discussion: The parameters extracted from empirical mathematical modelling are useful for evaluating acute hepatitis in created mice model. The elimination half-life of enhancement was significantly increased in diseased group suggesting lesions and weakened metabolism of liver and the disruption of clearance function. Decrease in mice weight after ConA injection implied the digestive problems following the damaged livers. Blood perfusion was limited in diseased group pointing to liver tissue lesions. Acknowledgements: This work was supported by the European Regional Development Fund from European Union (grant coordinated by JCET-UJ, No WND-POIG /09-00). References: [1] - Fan, X., M. Medved, J. N. River, et al. New model for analysis of dynamic contrast enhanced MRI data distinguishes metastatic from nonmetastatic transplanted rodent prostate tumors. Magnetic Resonance in Medicine 2004:51(3): [2] - Cross, S. S. Fractals in pathology. The Journal of Pathology 1999:182(1):1-8. [3] - Jirak, D., M. Dezortova, P. Taimr and M. Hajek. Texture analysis of human liver. Journal of Magnetic Resonance Imaging 2002:15(1):

12 Metabolic hallmarks of visual cortex degeneration in mouse model of glaucoma (DBA/2J mice) revealed by in vivo proton magnetic resonance spectroscopy Michał Fiedorowicz (1), Jarosław Orzeł (1,2), Bartosz Kossowski (1,2), Marlena Wełniak - Kamińska (1), Piotr Bogorodzki (1,2), Paweł Grieb (1) (1) Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw (2) Faculty of Electronics and Information Technology, Warsaw University of Technology Objective: Glaucoma, a neurodegenerative disease of optic nerve and retinal ganglion cells extends beyond retina and affects extra-retinal vision-related brain structures, including visual cortex. The aim of this study was to indicate the hallmarks of degeneration within visual cortex in advanced stage mouse model of glaucoma (DBA/2J). Methods: Aged DBA/2J mice (14 months, n=10) and age matched C57Bl/6 mice (n=10) were anaesthetized with isoflurane (4% in oxygen induction, 1.5-2% - maintenance) and placed in 7T small animal-dedicated magnetic resonance tomograph (BioSpec 70/30USR; Bruker BioSpin, Ettlingen, Germany). T2-weighted images were obtained in order to precisely position two distinct voxels of interests (VOIs) corresponding to visual cortex (5x2x1.2 mm 3 ) and frontal cortex (4x2x1.5mm 3 ). Local shimming procedures (FASTMAP) were repeated for each voxel followed by manual water suppression procedure (VAPOR). The unsuppressed water line width was typically maintained at around Hz. Spectra were obtained with PRESS sequence (TR=2000ms, TE=20ms, NA=1024, Scan time=34min). The data were analyzed with LCModel software (Stephen Provencher Inc, Oakville, Ontario, Canada). Metabolite concentrations are reported as ratios to total creatine (sum of creatine and phosphocreatine, tcr). Spectra of poor quality (SNR ratio < 15) were excluded from further analysis. Statistical analysis was performed with U Mann-Whitney test. Results: Glutamate to tcr ratio was significantly higher (P<0.05) in the visual cortex of DBA/2J mice than in the control mice, while no significant difference was observed in the frontal cortex. Glutamine to tcr ratio was lower in DBA/2J than in C57Bl/6 mice in both VOIs (P<0.05). Taurine to tcr ratio was lower in DBA/2J mice than in controls in the visual cortex (P<0.001) but not in the frontal cortex. No significant differences were detected for N- acetylaspartate and total choline. Conclusion: Aged DBA/2J display complex picture within visual cortex related to the ongoing degenerative process. This neurodegeneration could be related to excitotoxicity that was indicated by increased glutamate levels. The study was supported by National Science Centre grant No. 2012/07/D/NZ4/

13 Is High-Fat High-Carbohydrate Diet (HFCD) Neuroprotective? A Magnetic Resonance Imaging Study in Wistar Rats. Stefan Gaździński 1, Zuzanna Setkowicz 2, Joanna Osoba 2, Karolina Karwowska 2, Piotr Majka 3, Jarosław Orzeł 4, Bartosz Kossowski 4, Piotr Bogorodzki 4, Marlena Kamińska 4, Michał Fiedorowicz 4 1 Military Institute of Aviation Medicine, Warsaw, 2 Jagiellonian University, Krakow, 3 Nencki Institute of Experimental Biology, 4 Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland Introduction: Obesity was associated with accelerated aging and elevated risk of neurodegenerative diseases. In animal models, high-fat high-carbohydrate diet (HFCD) is commonly used to induce obesity. We hypothesized that HFCD will lead to poorer memory, smaller hippocampi and lower concentrations of brain metabolites in hippocampi, which are predictors of neurodegenerative diseases both in humans and in laboratory animals. Methods: Twenty five male Wistar rats were put on HFCD (~35% fat, ~35% carbohydrates) on their 55th day of life, while 25 control male rats (CON) remained on chow. Both groups underwent memory tests in 8-arm radial maze at 3rd, 6th, and 9th month. At one year, all animals underwent MRI to evaluate hippocampal volumes and 1H magnetic resonance spectroscopy at 7T. Results: HFCD rats consumed slightly more calories than CON, but less proteins. However, their protein intake was within recommended amounts. Levels of sugar and ketone bodies were within healthy norms in both groups; however, numerically they were higher in the HFCD group. Contrary to our hypotheses, HFCD rats had better scores of memory than CON throughout the experiment. At one year, their hippocampi were by 3% larger than in CON (p=0.05), whereas concentration of N-acetylo-aspartate (NAA, marker of neuronal viability) was 8% higher (p=0.01). Conclusions: The results do not support the thesis that HFCD per se leads to degeneration of the nervous system. On the contrary, they consistently suggest that HFCD enhances memory and slows aging. More research is needed to pinpoint the mediating factors. Support: Polish National Science Centre (2011/03/B/NZ4/03771) to Stefan Gazdzinski. 6

14 Voxel-wise analyses of high-fat high-carbohydrate diet on brain structure in Wistar rats Piotr Majka 1, Zuzanna Setkowicz 2, Joanna Osoba 2, Karolina Karwowska 2, Jarosław Orzeł 3, Bartosz Kossowski 3, Piotr Bogorodzki 3, Marlena Kamińska 3, Michał Fiedorowicz 3, Stefan Gaździński 4 1 Nencki Institute for Experimental Biology, Polish Academy of Sciences, Warsaw, Poland, 2 Department of Neuroanatomy, Jagiellonian University, Krakow, Poland, 3 Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland, 4 Military Institute of Aviation Medicine, Warsaw, Poland Introduction: Smaller hippocampal volumes and extensive body fat accumulaton in midlife are risk factors for developing Alzheimer s disease. Our ROI analysis demonstrated that high fat high carbohydrate diet lead to larger hippocampal volumes in Wistar rats, without significant changes in cortical volume. Here, we applied voxel-wise analysis to determine focal changes in brain tissue structure. Furthermore, we compared the effects of template selection (Valdés-Hernández et. al. [1] template vs. study specific template) on the results. Methods: Twenty five male Wistar rats were put on HFCD (~40% fat, ~40% carbohydrates, ~7% proteins) on their 55th day of life, while 25 control male rats (CON) remained on chow. Structural T2-weighted TurboRARE (TR/TE=4700/30ms, RARE factor=4, resolution=125x125x500μm, no gap, NEX=7, TA=27 min) acquired on Bruker BioSpin working at 7T,, with a transmit cylindrical radiofrequency coil (15 cm inner diameter) and a receive-only coil array (2x2 elements) positioned over the animal s head. Eighteen datasets acquired for CON, 18 datasets for HFCD selected for analyses. Images resampled to isotropic resolution of 125μm/vox and processed with N4 algorithm to correct for intensity inhomogenity. Image of each specimen was registered into the Valdés-Hernández et. al. [1] template or study-specific template using SyN algorithm [2] resulting in a series of deformation field. Jacobian determinant of each deformation field was then computed and modulated with a gray matter probability, blurred with Gaussian filter of 250μm. Significance of differences between CON and HFCD was determined with two-sample unpaired T-test. Threshold-Free Cluster Enhancement permutation method [3] was used to threshold t-maps (FSL-randomise software). 10,000 permutations were used in tests and p=0.05 was chosen as a significance threshold. Results: Hippocampal volume are larger in HFCD-fed rats than in controls, especially in hippocampal CA1 field, but also in surrounding cortical areas, regardless of used template. Analysis with study specific template does not show regions of smaller volumes in HFCD fed group compared to CON. Conclusions: The results of voxel-wise comparisons not only confirm our ROI findings of larger hippocmpal volumes in HFCD fed rats, but also point to focal volume increases in temporal association cortex and ectorhinal cortex. Our results do not support the thesis that HFCD per se leads to degeneration of the nervous system. Moreover, as CA1 is selectively 7

15 affected by neurodegenerarative processes in Alzheimer s disease, our results seem to be of functional significance. References: 1. Valdes-Hernandez PA., Frontiers in Neuroinformatics, November2011 Volume5 Article26, doi: /fninf Avants, BB., et al., (2011) NeuroImage, 54(3), Smith, SM., & Nichols, TE. (2009), NeuroImage, 44(1), Support: Polish National Science Centre (2011/03/B/NZ4/03771) to Stefan Gazdzinski. 8

16 Effects of High-Fat High-Carbohydrate Diet on White Matter Integrity: A Diffusion Tensor Imaging Study in Wistar Rats Stefan Gaździński 1, Andrzej Gaździński 1, Zuzanna Setkowicz 2, Joanna Osoba 2, Karolina Karwowska 2, You Zhang 3, Jarosław Orzeł 4, Bartosz Kossowski 4, Piotr Bogorodzki 4 1 Military Institute of Aviation Medicine, Warsaw, Poland, 2 Department of Neuroanatomy, Jagiellonian University, Krakow, Poland, 3 Department of Radiology, Unoversity of California San Francisco, USA, 4 Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland. Introduction: Obesity worldwide has reached epidemic proportions, with more than 400 milion people affected. Currently, every third person in the world is overweight. Human DTI studies have demonstrated lower fractional anisotrophy (FA) and higher mean diffusivity (MD) in obese humans [1,2]. In animal models, high-fat high-carbohydrate diet (HFCD) is commonly used to induce obesity. We evaluate hypotheses that long-term HFCD use in male Wistar rats leads to Lower FA and higher MD than in control group. Methods: Twenty five male Wistar rats were put on HFCD (~40% fat, ~40% carbohydrates, ~7% proteins) on their 55th day of life, while 25 control male rats (CON) remained on chow; we obtained DTI data on 21 CON. MR measurements were performed on a 7T wide bore (30 cm) Bruker BioSpec at Mossakowski Medical Research Centre, Warsaw, Poland. Diffusion tensor was acquired with TE/TR=33/3750ms, along 72 directions, with resolution 0.156x0.172x0.7mm, no gap. Data were skull stripped and eddy-current corrected with FSL. Than, images were resized to 0.1x0.1x0.1 mm. FA images were than normalized to a study specific template using DARTEL in SPM8; Study-specific template was created by averaging all FA images. These transformations were applied to FA, MD, as well as perpendicular and parallel diffusivities. Images after normalization and smoothing with a smoothing kernel of 0.3mm at FWHM were compared between groups using two-sample t-tests (FWE<0.05, cluster size >27) with SPM8. Results: Right cerebral peduncle contains a region of lower MD in in rats fed with HFCD than in CON (p<0.05, FWE), accompanied by increased FA (p<0.001, uncorrected), contrary to our hypotheses. MD is elevated in corpus callosum and fimbria of HFCD-fed rats than in CON, as well as in trigeminal nerve (2b), consistent with our hypothesis. These changes were not accompanied by significant FA changes. No differences in parallel and radial diffusivities were noted. Conclusions: The results partially support the thesis that high fat high carbohydrate diet leads to worsening of WM integrity. The reason for such behavior is not known. References: 1. Stanek K. et al., Obesity, 500-4, Mueller K. et al., PLoS One, Apr 11;6(4):e18544, Support: Polish National Science Centre (2011/03/B/NZ4/03771) to Stefan Gazdzinski. 9

17 HR MAS NMR METABOLIC PROFILES OF CARDIOMYOCYTES AFTER RADIATION EXPOSURE Michalina Gramatyka, Agnieszka Skorupa, Mateusz Ciszek, Łukasz Boguszewicz, Maria Sokół Department of Medical Physics, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland. Clinical data from population of radiotherapy patients show that exposure of the heart to ionizing radiation during radiotherapy increases the subsequent rate of ischemic heart disease. Initially heart was considered as relatively radioresistant organ, but since the 1960s many studies have demonstrated that even low doses of radiation (<4 Gy) can have a negative impact on the cardiovascular system. The underlying mechanisms of this toxicity are not well understood. To address this topic we used human cardiomyocytes as a model system, and studied their metabolic response to radiation using high resolution magic angle spinning nuclear magnetic resonance techniques (HR MAS NMR). Cultured in vitro human cardiomyocytes were exposed to ionizing radiation and their survival was assessed by clonogenic assay. Changes in apoptosis intensity (TUNEL assay) after irradiation with a dose of 2 Gy was measured as well. Water-presaturated 1 H NOESY and CPMG MAS NMR spectra of cardiomyocytes were acquired at 293K using Bruker Avance 400 MHz spectrometer at a spinning rate of 3200 Hz. Survival of cardiomyocytes after NMR experiments was assessed by the Trypan blue exclusion assay. Exposure of cardiomyocytes to small doses of ionizing radiation (less than 4 Gy) had no effect on cell proliferation potential and intensity of cell death. However, analysis of HR MAS NMR metabolic profiles revealed changes in phospholipid and creatine metabolism. Trypan blue staining showed that after NMR experiments the cells remain viable. Results of this study show that ionizing radiation affects metabolic profiles of cardiomyocytes even at low doses, which potentially have no effect on cell viability. The results obtained from this experiment will be used in further in vivo studies on animal model. 10

18 THEORETICAL CALCULATIONS OF 13 C NMR CHEMICAL SHIFTS OF BROMINE-SUBSTITUTED CARBON ATOMS Adam Gryff-Keller a, Dominika Kubica a and Artur Wodyński b a Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, Warsaw b Faculty of Chemistry, University of Warsaw, Pasteura 1, Warsaw Reliable DFT-based methods of predicting 13 C NMR chemical shifts for carbon atoms bonded exclusively to the atoms of the first-row and even second-row of the periodic table of elements are now in common use. On the other hand, such predictions, in the case of carbons bonded to heavier atoms, are much more difficult, since theoretical calculations have to treat somehow the relativistic effects experienced by electrons in the vicinity of heavy nuclei. Such heavy atom on light atom (HALA) effects are well visible in the case of carbon atoms substituted by bromine or bromines. Presently, the DFT so-zora method accessible in ADF commercial program that allows introducing the scalar and spin-orbit relativistic corrections seems to be the most popular way of including relativity into quantum mechanical calculations of NMR parameters. In the case of halogens bearing three electron lone pairs, however, the electron correlation (EC) effects are also important. Actually, the EC and HALA effects cooperate in the case of bromine and a simple application of a relativistic analogue of the otherwise efficient non-relativistic method such as DFT/B3LYP/ G(2d,p) yields results which are not fully satisfying. We have found out that another, less popular functional, BHandH, collaborates well with ZORA and yields remarkably better results for brominated carbon atoms than B3LYP functional. Table 1. Comparison of the experimental and theoretically predicted 13 C NMR chemical shifts [ppm] calculated using the methods which neglect or include relativistic effects. Compound Method Exp a B3LYP b BHandH b ZORA/B3LYP c ZORA/BHandH c CBr Br-pyridine a CDCl3, TMS scale. b Basis: G(2d,p), solvent: PCM, program: Gaussian. c Basis: TZ2P, solvent: COSMO, program ADF. 11

19 1 H-NMR IN LIVING ORGANISMS, TISSUES, AND OTHER BIOLOGICAL SYSTEM RESISTANT TO DRASTIC DEHYDRATION OR TO LOW TEMPERATURES Hubert Harańczyk Institute of Physics, Jagiellonian University, Cracow, ul. Reymonta 4, Cracow For extremophilic living organisms, eg. Antarctic lichenized fungi [1-6] or insects [7]; for solid tissues, eg. Arthropode cuticle [8] or hair [9]; or for other biological systems, eg. DNA [10] or DNA/surfactant complexes [11-13], which may resist the drastic dehydration, proton NMR signal resembles the one observed for all microheterogeous solid samples at low hydration level, eg. for solidifying white synthetic cement [14]. Proton signal consists of a solid signal component, which may be well approximated using Gaussian function or in time domain by Abragam function [6]. For Antarctic fungi, the solid signal component reveals the contribution of Pake doublets [15], with the characteristic distance between relaxing protons as for protons of water molecule. Liquid signal component may be approximated by one or two Lorentzian lines which come from tightly or loosely bound water fraction. Both bound water fractions are defined by the proximity to the surfaces of solid matrix of specimen and sometimes averaged depending on size of pores in the structure. In extremophilic living organism at recovery after cryptobiotic form or in resistant tissue at rehydration, water soluble solid fraction dissolves with the increasing hydration level. It may ocurr on two ways, namely by simple dissolution mainly of sugars and/or polyols in case of some plant tissues [17], or by by enzyme-induced active bio-polymer decomposition, which is the way used by lichenized fungi, or at initial phases of seed imbibition [18]. This manifests in 1 H-NMR signal, either in time or in frequency domain, as a non-linear dependency of liquid signal component expressed in units of solid signal. If the rational function approximates this change, it enables to yield the saturation concentration of water soluble solid fraction. Acknowledgements: The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract no. POIG /08). References 1. R. Del-Prado, L.G. Sancho, Flora 195: (2000). 2. H. Harańczyk, On water in extremely dry biological systems, WUJ, Kraków (2003). 3. H. Harańczyk, M. Bacior, M.A. Olech, Antarctic Science 20, (2008). 4. H. Harańczyk, M. Bacior, P. Jastrzębska, M.A. Olech, Acta Phys. Polon. A115, (2009). 5. H. Harańczyk, Ł. Pater, P. Nowak, M. Bacior, M.A. Olech, Acta Phys. Polon. 121, , (2012). 6. H. Harańczyk, P. Nowak, M. Bacior, M. Lisowska, M. Marzec, M. Florek and M.A. Olech, Antarctic Science 24 (4), (2012); M. Watanabe, T. Sakashita, A. Fujita, T. Kikawada, D. D. Horikawa, Y. Nakahara, S. Wada, T. Funayama, N. Hamada, Y. Kobayashi, T. Okuda, Int. J. Radiat. Biol. 82, (2006). 12

20 8. H. Harańczyk, M. Florek, P. Nowak and S. Knutelski, Acta Phys. Polon. 121, , (2012). 9. D. Zalitacz, H. Harańczyk, P. Nowak, P. Delong, J. Investigative Dermatology 133, 1424 (2013). 10. H. Harańczyk, J. Czak, P. Nowak, J. Nizioł, Acta Phys. Polon. A117, (2010). 11. H.Harańczyk, J.Kobierski, D.Zalitacz, P.Nowak, A.Romanowicz, M.Marzec, J.Nizioł, Acta Phys. Polon. 121, , (2012). 12. H. Harańczyk, J. Kobierski, J. Nizioł, E. Hebda, J. Pielichowski, D. Zalitacz, M. Marzec, A. El-Ghayoury, J. Appl. Phys. 113, (2013) J. Nizioł, H. Harańczyk, J. Kobierski, E. Hebda, J. Pielichowski, B. Ostachowicz, J. Appl. Phys. 114, (2013). 14. R. Rumm, H. Harańczyk, H. Peemoeller, M. M. Pintar, Cement and Concrete Res. 21, (1991). 15. W. Derbyshire, M. Van den Bosch, D. Van Dusschoten, W. MacNaughten, I. A. Farhat, M. A. Hemminga, J.R. Mitchell, J. Magn. Res. 168, (2004). 16. J.Hetmańczyk, Ł.Hetmańczyk, A.Migdał-Mikuli, E.Mikuli, M.Florek-Wojciechowska, H.Harańczyk, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124, (2014). 17. H. Harańczyk, W. P. Węglarz, Z. Sojka, Holzforschung, 53, (1999). 18. H. Harańczyk, K. Strzałka, G. Jasiński, K. Mosna-Bojarska, Colloids &Surfaces, A115, (1996). 13

21 REMARKS ON THE FREE-APPROACH MODELS Łukasz Jaremko 1,2, Mariusz Jaremko 1, Michał Nowakowski 3, Andrzej Ejchart 4 1 Max Planck Institute for Biophysical Chemistry, Department for NMR-based Structural Biology, Am Fassberg 11, Göttingen, Germany, jaremko@gmail.com 2 Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Am Fassberg 11, Göttingen, Germany 3 Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, Warsaw, Poland,, lyam@chem.uw.edu.pl 4 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, Warsaw, Poland, aejchart@ibb.waw.pl One of the most successful and widely used approaches to the interpretation of nuclear magnetic relaxation data for biologically important polymers, mainly proteins, has become the model-free approach (MFA) requiring no particular physical model of motion(s) and a small number of parameters [1]. The model is based on two assumptions imposed on the form of the autocorrelation function: (1) internal motions and overall molecular tumbling are uncorrelated allowing to factorize the autocorrelation function into two components describing overall tumbling and internal motion, and (2) the internal correlation function can be approximated by a single exponential. In the frame of this model local mobility is described by two parameters, a generalized order parameter, S 2, which corresponds to the spatial freedom of the motion, and an internal correlation time, int, which corresponds to the rate of this motion in the pico- to nanosecond time scale faster than a single correlation time describing an isotropic overall molecular tumbling, R. Fourier transformation of autocorrelation function results in the expression for the spectral density function represented by two Lorentzian terms. By analysis of the backbone amide nitrogen relaxation, a global picture of the dynamics of a protein can be revealed. Soon it was found that model-free approach sometimes poorly reproduced experimental data in proteins, especially 15 N-{ 1 H} NOEs; experimental NOEs tended to be larger than their calculated values. This was attributed to the additional slow internal motion outside the extreme narrowing limit pointing at motions being faster than overall tumbling but slower than those obtained from the genuine MFA model. Therefore, the extended model-free approach (EMFA) taking into account more complex description of internal motions characterized by two different time scales (f - fast motion, s - slow motion) was introduced at the cost of larger number of model parameters [2]. One should bear in mind that NOE measurements are especially prone to systematic errors resulting from not fully relaxed spectra and/or saturation transfer due to exchange with water protons. Both these effects can increase apparent NOEs even beyond the theoretically expected maximum. An attempt to compensate such artifacts applying the extended model can result in false values of local parameters. Comparison of two free-approach models can be analyzed with the use of target function given by a widely accepted least-squares expression N χ= i=1 Γ i = i=1 N M j=1 [( P ij,exp P ij,calc ) 2 /σ ij 2 ] Simulations performed for MFA and EMFA revealed striking differences in target function dependences between these two models. The MFA displays well determined minima of quasi-parabolic target functions, ( int) and (S 2 ), in the least-squares procedure of the best motional parameter search as could be expected for well defined numerical problem (Fig. 1). On the other hand, the EMFA shows strongly diversified behavior of target function graphs for model parameters describing slow and fast internal motions (Fig. 2). 14

22 Fig. 1 (above). Dependence of target function values vs. local MFA model parameters, int and S 2, obtained from the fit to the synthetic R1, R2 and NOE data. Fig. 2 (right). Dependence of target function values vs. local EMFA model parameters obtained from the fit to the synthetic R1, R2 and NOE data. Plots ( f) and (Sf 2 ) are similar to those obtained for the MFA. On the contrary, the dependence ( s) is strongly asymmetric and nearly flat within a wide range of s values larger than its input value used in the simulations; opposite is true for the (Ss 2 ) graph. It usually results in a painfully long and unstable numerical search of global minimum of function and strongly unsymmetrical confidence boundaries of slow motion parameters s and Ss 2. One should focus an attention on the relation between a generalized order parameter and corresponding correlation time. As a rule, the increase of correlation time results in decrease of corresponding generalized order parameter. In real life, when experimental relaxation data bear unavoidable inaccuracies, the least squares procedure can deliver very large, non physically justified s values often exceeding overall correlation time. It is particularly important for the Ss 2 ( s) dependence since unrealistically large s value determined in the error sensitive EMFA-based minimization procedure can result in the assignment of unjustified internal mobility to given amino acid residues. Such behavior was observed for a number of residues in several proteins for which relaxation data were available in the literature and data bases. References [1] Lipari, G., Szabo, A., Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. J. Am. Chem. Soc. 1982, 104, [2] G.M. Clore, A. Szabo, A. Bax, L.E. Kay, P.C. Driscoll, A.M. Gronenborn, Deviations from the simple two-parameter model-free approach to the interpretation of nitrogen- 15 nuclear magnetic relaxation of proteins. J. Am. Chem. Soc. 1990, 112,

23 Comparison of magnetic resonance imaging of the mouse brain in vivo using different types of RF coils at 9.4 T J. Jasieniak, W. Piędzia 1, K. Jasiński 1, W.P. Węglarz 1 1 Department of Magnetic Resonance Imaging, Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland Purpose: The aim of this study was to compare quality of the mice`s brain MR images in vivo obtained with three different types of RF coils: cryo-coil and room temperature surface and volume coils. The radiofrequency field (B 1 ) mapping has also been made. Materials and methods: Three different RF coils: transmit/receive Bruker CryoProbe [1], mice brain receive coil (together with volume transmit birdcage coil) and 35 mm ID transmit/receive birdcage volume coil were used at 9.4T Bruker Biospec MR scanner for SNR assessment in doped water phantom and mice. ECG, temperature and respiratory were monitored during in vivo measurements. FLASH, MPRAGE and UTE pulse sequences were used for MR imaging. SNR was measured for all three coils. B 1 maps were obtained with double angle method. Images were analysed with the use of Fiji software. Results: The best quality of magnetic resonance imaging of the mouse brain in vivo has been obtained for cryo-coil. The SNR was ~3 to ~12-fold larger for the cryo-coil as compared to the birdcage [2] and ~2.3 to ~3-fold larger for the cryo-coil as compared to the mouse brain surface coil. Different B 1 maps have been obtained for the images of phantom of the 2 pulse position-3.5 mm and 5.5 mm. Discussion: MR imaging of the mouse brain requires good SNR due to the small size of the imaging object. This can be obtained reducing noise from the electronics of coil by lowering the temperature of the coil. Cryo-coil operates at a temperature of 20 K what makes it get the best SNR, which enable to get superior quality images of mouse brain in vivo in reasonable time. However, due to inhomogenous B 1 field (and thus spatially dependent flip angle) using of cryo-coil has also some limitations. Specifically, it often requires careful flip angle adjustment for chosen horizontal slice and/or flip angle (B 1 ) mapping. References: [1] Baltes, C., Radzwill, N. & Bosshard, S. at al. Micro MRI of the mouse brain using a novel 400 MHz cryogenic quadrature RF probe. NMR in Biomedicine, 2009;22(8),: [2]-W. Piędzia, K. Jasiński, K.Kalita, B.Tomanek, W.P. Węglarz. White and gray matter contrast enhancement in MR images of the mouse brain in vivo using IR UTE with a cryo-coil at 9.4 T

24 NMR STUDY OF P(MEO2MA) POLYMER NETWORKS J. Jenczyk, S. Kadłubowski 1, M. Olejniczak 2, M. Kozanecki 2, S. Jurga NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, Poznań, Poland 1 Institute of Applied Radiation Chemistry, Technical University of Lodz, Wroblewskiego 15, Lodz, Poland 2 Department of Molecular Physics, Technical University of Lodz, Zeromskiego 116, Lodz, Poland Hydrogels are three-dimensional networks, made of amphiphilic polymer chains, able to swell in water. Thermo-responsive gels have been extensively studied due to their potential applications as drug delivery systems, regenerative medicine, biosensors, responsive membranes, molecular machines, nanotemplates for nanoparticles formation, catalysis and photonic crystals The polymeric networks of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA), 2-hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA cross-linking agent) (molar ratios: 100:2:1) have been synthesized [1]. These cross-linked systems are characterized by lower critical solution temperature (LCST) which is directly related to volume phase transition (VPT). The VPT results in abrupt deswelling process (network collapse) and water release. The kinetics of this process can be effectively monitored by Nuclear Magnetic Resonance (NMR) spectroscopy. Moreover, the time-lapse NMR experiment enables one to assess the relative cross-linking level of studied samples [2]. Obtained results reveal how the irradiation dose and post-irradiation conditioning temperature influence on the degree of cross-linking. The analysis relies on the time evolution of the proton NMR spectra above LCST temperature. This evolution can be described by exponential relation and characterized by time constant τ. The τ value is directly proportional to cross-linking level due to the fact that water hydrogen bonding in a more densely crosslinked hydrogel is more resistant to disruption. Independently, wide line separation WISE experiments were performed for dried polymers in order to estimate mobile and static fraction of protons from MEO2MA side chains. 1 Time evolution of NMR spectrum (water signal) above LCST temperature. [1] S. Kadłubowski, M. Matusiak, J. Jenczyk, M. Olejniczak, M. Kozanecki, L. Okrasa Radiation Physics and Chemistry 100 (2014) [2] J. Yoon, Ch. Gayathri, R. R. Gil, T. Kowalewski, K. Matyjaszewski, Macromolecules 43 (2010)

25 QUANTITATIVE MRI IN STUDYING WHITE MATTER DAMAGE FOLLOWING SPINAL CORD INJURY Piotr Kozlowski University of British Columbia, UBC MRI Research Centre, and International Collaboration On Repair Discoveries (ICORD), Vancouver, BC, Canada Spinal cord injury (SCI) is a devastating event affecting mostly young, otherwise healthy population. It often results in severe physical impairment and disability that persists for the life of the affected individual. Apart from the obvious human tragedy and social costs, the economic burden on the health care system and society at large is enormous. It is widely recognized that the functional loss following SCI is largely caused by the damage to the white matter, thus most of the therapeutic procedures are oriented towards regeneration and functional restoration of the interrupted nerve fibre tracts. One of the critical aspects of a successful therapy is the ability to accurately assess the white matter damage prior to initiating the therapy, and to follow the efficacy of the treatment throughout the therapy. This requires a non-invasive imaging technique capable of quantitative measurements of white matter integrity in spinal cord. MRI is currently the most effective radiological method for assessing SCI. However, exact estimation of myelin content and axonal integrity in spinal cord is not possible with the standard MRI techniques used clinically. A number of quantitative MRI techniques have recently been developed with particular focus on white matter characterization. Among them, Myelin Water Imaging (MWI), Diffusion Tensor Imaging (DTI) and phase MRI have been particularly promising in SCI applications. This presentation will review physical bases of these techniques and show their applications in characterizing white matter damage in several pre-clinical models of SCI. Myelin Water Imaging: In a complex system, such as brain or spinal cord, multiple water compartments (e.g. myelin bilayers, intra-/extra-cellular space, cerebrospinal fluid) result in spin-spin relaxation becoming a multi-exponential process giving rise to multiple T2 values [1]. Quantitative analysis of T2 decay curves acquired with a multi-echo Carr-Purcell-Meiboom-Gill (CPMG) sequence produces continuous distributions of T2 values representing various T2 components present in the tissue (Figure 1). MWI was successfully applied to measuring myelin content in the normal and injured rat spinal cords ex vivo [2] and in vivo [3]. Figure 1. Continuous T2 distribution from a rat spinal cord. Multiple T2 components correspond to myelin water, intra-/extra-cellular water and CSF. Myelin Water Fraction (MWF) is a measure of myelin content in the tissue. Figure 2. MWI and DTI parametric maps obtained from rat spinal cords at 3 and 8 weeks following Dorsal Column transection injury. Luxol Fast Blue is a histological stain of myelin. Scatter plots show strong correlation between MWF and histological measure of myelin content. 18

26 Diffusion Tensor Imaging: DTI provides information about tissue fine structure and therefore is particularly useful for studying the structure and integrity of white matter in the control and injured spinal cord. It has been shown that diffusion anisotropy is affected both by the state of myelin and axonal structure [4]. Longitudinal diffusivity (diffusion coefficient along the axon) is sensitive to the axonal integrity, while transverse diffusivity (diffusion coefficient perpendicular to the axon) can provide some information on the myelin Figure 3. Fibre tracts reconstructed from a DTI data acquired ex vivo from a cervical spinal cord excised following contusion injury. The area of cavity (red arrows) and axonal damage to the ventral WM (yellow arrows) are clearly visible. content (Figure 2). Tractography is a technique that allows reconstruction of white matter tracts from DTI data. It can be used to identify tracts damaged as a result of SCI (Figure 3). Phase MRI: The MRI signal is complex in nature. Commonly, the magnitude of this complex number is used and the phase information is discarded. However, phase may reflect the relative resonance frequency of spins and can therefore be utilized to investigate the spins magnetic environment, such as the local tissue magnetic susceptibility. Theoretical models predict that the tissue microstructure has an influence on the phase of MRI signal [5], thus quantitative phase MRI can potentially be used to characterize white matter damage in an injury model in rat spinal cord. In a recent study [6], phase MRI has been shown to correlate with histological measures of myelin content and axonal damage in a Dorsal Column transection injury model in rat spinal cord (Figure 4). Figure 4. Frequency shift maps and histology sections of myelin (eriochrome, MBP), axonal (NF/Tub III), and degenerated myelin (degen-mbp) stains from rat spinal cords at baseline and 3 and 8 weeks after Dorsal Column (DC) transection injury. Images show sections 5 mm caudal (left) and 5 mm cranial (right) to injury site. The ascending DC tract (fasciculus gracilis) undergoes Wallerian degeneration on the cranial side (right) and the retrograde degeneration on the caudal side (left), while the descending DC tract (corticospinal tract) undergoes Wallerian degeneration on the caudal side (left) and the retrograde degeneration on the cranial side (right). References: 1. Whittall KP and MacKay AL, J Magn Reson, 84, (1989). 2. Kozlowski P, et al., J Neurotrauma, 25, (2008). 3. Kozlowski P, et al., Magn Reson Imaging, 32, (2014). 4. Beaulieu C, NMR Biomed, 15, (2002). 5. He et al., PNAS, 106, (2009). 6. Chen IE, et al., Proceedings of 21st Meeting of ISMRM, p. 347 (2013). 19

27 New high dimensionality NMR experiments for biomolecules Wiktor Koźmiński Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, Warsaw, Poland kozmin@chem.uw.edu.pl A variety of different methods was proposed to overcome the sampling limitation in multidimensional NMR spectroscopy. They could be utilized in two different ways, either to shorten the experiment duration without loss of resolution, or to perform experiments that are not obtainable conventionally, i.e. with significantly improved resolution and/or of high dimensionality. Most often first of these two, so called Fast NMR approach, is shown as the example of the utility of these methods, as it saves expensive spectrometer time. However, in many cases spectra featuring extraordinary resolution and high number of dimensions may be more interesting from scientific point of view as they reveal effects that are hidden, when spectral lines are broad, or enable resolving spectral ambiguities when peaks are overlapped. This second approach we refer to as Accurate NMR. Its full potential is manifested when the overall experiment time is less important than a new information available from spectra of high dimensionality (4-6D) or of high resolution approaching natural line-width. The new methods were applied for NMR studies of intrinsically disordered proteins, where the structural disorder in combination with highly repetitive amino-acid sequences causes severe peak overlap in the spectra. Recently, several novel 4-6D pulse sequences are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in indirectly detected dimensions. The experiments facilitate resonance assignment of intrinsically disordered proteins. [1] K. Kazimierczuk, J. Stanek, A. Zawadzka-Kazimierczuk, W. Koźmiński, Prog. Nucl. Mag. Res. Sp., 57, (2010). [2] K. Kazimierczuk, M, Misiak, J. Stanek, A. Zawadzka-Kazimierczuk, W. Koźmiński, Topics in Current Chemistry, 316, (2012). [3] K. Kazimierczuk, J. Stanek, A. Zawadzka-Kazimierczuk, W. Koźmiński, ChemPhysChem, 14, (2013) 20

28 Intramolecular interaction of hybrid of uridine and stilbene derivative Hanna Krawczyk and Przemysław Szczeciński Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, Warsaw, Poland, Hybrid molecules are defined as chemical entities with two (or more than two) structural domains having different biological functions. Their dual activity indicates that a hybrid molecule acts as two distinct pharmacophores. On the other hand chemical modification of nucleosides and their incorporation into nucleic acid oligomers represent one of the most successful drug design strategies when considering chemotherapeutic approaches, as is evidenced by the significant number of analogues in clinical trials for treatment of various diseases including cancer, inflammation, and viral infections.[1] Numerous modifications to the sugar ring as well as the heterocyclic nucleobase moieties have been utilized in recent years to increase chemotherapeutic activity.[2,3] A new class of modified nucleosides by stilbene derivative has recently been synthesised by us.[4] The purine nucleoside- uridine (first structural domain)- connected by a single C-C bond with stilbene derivatives (second structural domain) creates the hybrid molecules. This allows the modified nucleosides to more readily adopt to the spatial and other requirements of the binding site, while retaining the majority of the key structural features required for molecular recognition. The synthesized molecules have specific spectroscopic properties and could be biologically active, just like nucleosides-uridine- and stilbenes such as combretastatin- OXi4503, (Vascular Disrupting Agents ; VDAs), a phase I clinical trial for relapsed and refractory acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).[5] Our investigation concerns the determination of stereochemistry of new compounds focusing on various intramolecular interactions governing conformational equilibria (e.g. the interactions of the stilbene aromatic ring with nucleobase moieties and sugar scaffold). References [1] Papers presented at XV International Roundtable Nucleosides, Nucleotides and Nucleic Acids, Leuven, Belgium, September Nucleosides Nucleotides Nucl. Acids. 2003, 22, [2] Agrawal, S.; Zhao, Q. Y. Curr. Opin. Chem. Biol. 1998, 2, [3] De Clercq, E. Clin. Microbiol. Rev. 2003, 16, [4] K. Kordowska, H. Krawczyk, Nauka i Przemysł metody spektroskopowe w praktyce nowe wyzwania i możliwości- praca zbiorowa pod redakcją prof. dr hab. Zbigniewa Hubickiego (ISBN ) 2014, Lublin, [5] A service of the U.S. National Institutes of Healt, 21

29 Imaging Methods in Research and Development Process of Generic Modified Release Matrix Tablets Piotr Kulinowski a, Krzysztof Woyna-Orlewicz b, Gerd-Martin Rappen c, Dorota Haznar- Garbacz d, Władysław P. Węglarz e, Przemysław P. Dorożyński b a Institute of Technology, The Pedagogical University of Cracow, ul. Podchorążych 2, Kraków, Poland b Department of Pharmaceutical Technology and Biopharmaceutics, Pharmaceutical Faculty, Jagiellonian University, ul. Medyczna 9, Kraków, Poland c Physiolution GmbH, Walther-Rathenau-Strasse 49a, Greifswald, Germany d Center of Drug Absorption and Transport (C_DAT), Dept. of Biopharmaceutics and Pharmaceutical Technology, Felix-Hausdorff-Str. 3, Greifswald, Germany e Department of Magnetic Resonance Imaging, Institute of Nuclear Physics PAN, ul. Radzikowskiego 152, Kraków, Poland In vitro phase of R&D process of pharmaceutical product can be performed at relatively low cost compared to the biological (bioequivalence) studies, but there are no dedicated and effective methods for development of oral, generic, modified release formulations. The purpose of the study was to assess in vitro methodology for bioequivalence study risk minimization. Presented approach consisted of three independent steps: 1. Quality by Design / Design of Experiment (QbD/DoE) pharmaceutical strategy using compendial dissolution tests [1]; 2. Application of several, selected, imaging/analytical methods; 3. Biorelevant stress dissolution test [2]. At the first step, pharmaceutically equivalent quetiapine fumarate extended release dosage form of Seroquel XR was developed. The second step was performed using following methods: Magnetic Resonance Imaging in USP4 apparatus 4 performed using 4.7T research system with TMX (NRC, IBD, Canada) console with spin-echo sequence [3,4]. Multi-Echo Magnetic Resonance Imaging using 9.4T BioSpin research system (Bruker). Micro-CT imaging performed using Benchtop 160 CT 160 (Nikon Metrology Inc.) [5]. Texture analysis (penetrometry) with Compact Tabletop, Universal Tester EZ-SX (Shimadzu). The last three measurements were performed on samples removed from the dissolution apparatus at 2h of hydration. Despite pharmaceutical equivalence of the Seroquel XR and developed formulation developed and original dosage forms differed in micro/meso structure and consequently in mechanical properties. These differences were found to cause failure of biorelevant dissolution test using the stress dissolution apparatus performed at the third step of the study the test was used as a surrogate for bioequivalence study. The work was supported by the Polish Ministry of Science and Higher Education grant NN and German Federal Ministry of Education and Research grant BMBF FKZ 03IPT612C. [1] R.A. Lionberger, et al. AAPS J. 2008; 10(2):

30 [2] G. Garbacz et al. Exp. Opin. Drug Deliv. 7 (2010) [3] P. Kulinowski et al. Pharm. Res. 28 (2011) [4] P.P. Dorożyński at al. AAPS Pharmscitech 11 (2010) [5] P.R. Laity et al. Eur. J. Pharm. Biopharm. 74 (2010)

31 MOLECULAR MODELING OF SINGLE WALL CARBON NANOTUBE (SWCNT) CHEMICAL SHIFT DUE TO ADDITION OF DIATOMICS Teobald Kupka 1, Marzena Nieradka 1, Leszek Stobiński 2 i Jakub Kaminský 3 1 University of Opole, Faculty of Chemistry, Opole, Poland 2 Institute of Physical Chemistry, Polish Academy of Sciences, 44/52, Kasprzaka, Warsaw, Poland; 3 Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2., Prague, Czech Republic Density functional theory (DFT) studies on adsorption of several gaseous homo- and hetero-diatomic molecules (AB) including H2, O2, N2, NO and CO on external surface of H-capped pristine armchair (5, 5) single-walled carbon nanotube (SWCNT) were conducted. Structures of C70H10 and the corresponding C70H10 AB adducts were fully optimized at the B3LYP/6-311G* level of theory. Calculated 13 C NMR chemical shifts were analyzed and critically compared with available experimental data. Significant changes of carbon NMR atom chemical shifts (up to 100 ppm) and shielding anisotropies (up to -180 ppm) at sites of addition were observed. Fig. 1. The B3LYP/6-31G* optimized model of armchair (5, 5) SWCNT. A1 and A2 adsorption sites including C3, C4 and C22, C31 carbons are marked in blue. Two types of C-C bonds, tilted and perpendicular are marked off, too. [1] M. Nieradka, T. Kupka*, L. Stobiński and J. Kaminský*, DFT studies on armchair (5, 5) SWCNT functionalization. Modification of selected structural and spectroscopic parameters upon two-atom molecule attachment, J. Mol. Graphics Model., in press. 24

32 MOLECULAR MODELING OF CHEMICAL SHIFT OF ATOMS, SMALL AND LARGE MOLECULES Teobald Kupka*, Michał Stachów, Marzena Nieradka, Klaudia Radula-Janik, Roksana Wałęsa, Aneta Buczek i Małgorzata Broda 1 University of Opole, Faculty of Chemistry, Opole, Poland In this report we demonstrate the need of proper selection of method and basis set quality in calculations of NMR parameters. Hartree-Fock (HF), second-order Moller-Plesset (MP2), density functional theory (DFT) and coupled cluster (CC) studies on calculation of chemical shift of free noble gas atoms, their dimers and single atoms encapsulated in selected fullerene cages are reported. Several examples of DFT predicted chemical shifts and indirect spin-spin coupling constants (SSCC) in medium size molecules are also discussed. The importance of accounting for relativistic effects in case of typical 13 C NMR chemical shift in case of iodine attached to carbon atom is also demonstrated. Fig. 1. The optimized model of He@C28 [1] T. Kupka*, M. Nieradka, J. Kaminský and L. Stobiński, Modeling 21 Ne NMR parameters for carbon nanosystems, Magn. Reson. Chem., 51 (2013) [2] T. Kupka*, M. Stachów, E. Chełmecka, K. Pasterny, M. Stobińska, L. Stobiński and J. Kaminský, Efficient modeling of NMR parameters in carbon nanosystems, J. Chem. Theor. Comput., 9 (2013) [3] R. Wałęsa, T. Ptak, D. Siodłak, T. Kupka and M. A. Broda, Experimental and theoretical NMR studies of interaction between phenylalanine derivative and egg yolk lecithin, Magn. Reson. Chem., 52 (2014) [4] K. Radula-Janik, T. Kupka*, K. Ejsmont, Z. Daszkiewicz and S. P. A. Sauer, Halogen effect on structure and 13 C NMR chemical shift of 3,6-disubstituted-N-alkyl carbazoles, Magn. Reson. Chem. 51 (2013)

33 Badania spektroskopowe Pt(II) z 7,8-benzochinoliną oraz 2-fenoksypirydyną. Daria Niedzielska, Leszek Pazderski Zakład Chemii Analitycznej i Spektroskopii Stosowanej, Wydział Chemii, Uniwersytet Mikołaja Kopernika, Toruń Slowa kluczowe: kompleksów Pt (II/IV), związki kompleksowe Pt (II), 7,8-benzochinolina, 2- fenoksypirydyna, 1H NMR, 13C NMR, luminescencji. 7,8-benzochinolina (bzq) oraz 2-fenoksypirydyna są N-donorowymi ligandami heterocyklicznymi typu azynowego, które koordynują jony Pt(II/IV) w dwojaki sposób: jako ligand jednodonorowy za pomocą atomu azotu (związki kompleksowe) oraz ligand dwudonorowy za pomocą atomu azotu i zdeprotonowanego atomu węgla C(2 /10) (związki metaloorganiczne). Pierwszą formę kompleksowania obserwuje się w związku [Pt(L) 2Cl 2], natomiast drugą w [Pt(LL*)Cl] 2 (Schemat 2). Schemat 1 Schemat 2 Otrzymane związki te cieszą się dużym zainteresowaniem ze względu na ich właściwości katalityczne, luminescencyjne oraz cytotoksyczność. Związki Pt(II) z bzq oraz 2-popy są również stosowane jako prekursory podczas przygotowania innych związków mających właściwości przeciwnowotworowe, antybakteryjne, przeciwgrzybiczne oraz przeciwwirusowe. Celem niniejszych badań była synteza związków Pt(II/IV) z bzq i 2-popy oraz ich pełna charakterystyka spektroskopowa 1 H, 13 C i 15 N NMR, przy czym wszystkie sygnały zostały przypisane metodami 1 H- 13 C i 1 H- 15 N HMQC/HMBC. Zostały również wykonane widma fluorescencyjne w ciele stałym oraz w dichlorometanie. Wykonano również widma w średniej oraz dalekiej podczerwieni. Literatura: [1] Y.Fuchita, H. Leda, A. Kayama, J. Kinoshita-Nagaoka, H.Kawano, S. i M. Mikuriya Kamedac, J. Chem. Soc., Dalton Trans., 1998, [2] Jean-Ho Chu, Pi-Shan Lin Ming-Jung Wu, Organometallics, tom. 29, nr 18,

34 FREEZING AND DRYING RESISTANCE OF ANTARCTIC TURGIDOSCULUM COMPLICATULUM THALLI AS OBSERVED BY 1 H-NMR METHODS Magdalena Bacior 1, Piotr Nowak 2, Paulina Kijak 2, Ewelina Baran 2, Hubert Harańczyk 2, and Maria A. Olech 3 1 Department of Chemistry and Physics, Faculty of Agriculture and Economics, University of Agriculture, Cracow 2 Institute of Physics and 3 Institute of Botany, Jagiellonian University, Cracow Lichenized fungi can survive freezing and deep dehydration [1-3]. Numerous data suggest that freezing and dehydration resistance have a common origin and depend on lichen morphology and habitat [4, 5, 6]. Turgidosculum complicatulum (foliose thallus) samples were collected in the vicinity of Arctowski Polar Station, King George Island, Maritime Antarctic. 1 H-NMR spectra were collected on Bruker Avance III 300, Bruker Biospin, spectrometer (transmitter power 400 W; pulse length π/2 = 2.2 μs; bandwidths 300 khz). Proton Free Induction Decays (FIDs) were recorded at 30 MHz on a high power relaxometer WNS HB 65, Waterloo NMR Spectrometers (pulse lenghts /2 = 1.5 s, transmitter power 400 W). Proton FID consists of a solid signal component fitted well by Gaussian ( T 2 25 s) and two liquid components described by exponential functions coming from a tightly bound ( T s), and a loosely bound water fraction ( Gaussian [6, 7]. T s). Solid signal is fitted well by 1 H-NMR spectra are superpositions of a Gaussian component ( G 40 khz) coming from protons of solid matrix of thallus and one averaged Lorentzian component ( L 3000 Hz) coming from protons of all water fractions in different motional states. For thalli at low hydration level ( m/m 0<0.3) the amplitude (in time domain) and line area (in frequency domain) of liquid signal expressed in solid signal units, L/S, constantly decreases with decreasin temperature, what suggests non-cooperative immobilization of water molecules. For highly hydrated samples rapid decreasing of L/S with decreasing temperature suggests ice nucleation process. The hydration dependency of total liquid NMR signal component expressed in units of solid, L/S, both in time or in frequency domain is well described by the rational function suggesting the dissolving process of the thallus solid fraction at rehydratation. Acknowledgements: The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract no. POIG /08). We are thankful to colleagues from Maitri Station and participants of the 23 rd Indian Antarctic Expedition for their help during the field studies. [1] H. Harańczyk, On water in extremely dry biological systems, Wyd. UJ [2] H. Harańczyk, A. Pietrzyk, A. Leja, M. A. Olech, Acta Phys. Polon. 109, 411 (2006). [3] H. Harańczyk, M. Bacior, P. Jastrzębska, M.A. Olech Acta Phys. Polon. A115, (2009). [4] B. Schroeter, Ch. Scheidegger, New Phytol., 131, (1995) [5] H. Harańczyk, M. Bacior, M.A. Olech Antarctic Science 20, (2008). [6] Harańczyk H., Nowak P., Bacior M., Lisowska M., Marzec M., Florek M., Olech M.A., Antarctic Science, 24(4), (2012). 27

35 The solution structure of the MANEC-type domain from Hepatocyte Growth Factor Inhibitor 1 reveals an unexpected PAN/apple domain-type fold Michał Nowakowski 1, Zebin Hong 2, Chris, Spronk 4, Steen V. Petersen 3, Jan S. Pedersen 5, Wiktor Koźmiński 1, Frans A.A. Mulder 5 and Jan K. Jensen 2 1 Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Poland. 2 Department of Molecular Biology and Genetics, Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Denmark. 3 Department of Biomedicine, Aarhus University, Denmark. 4 Spronk NMR, Lithuania. 5 Interdisciplinary Nanoscience Center (inano), Aarhus University, Denmark. A decade ago, a Motif at N terminus with Eight-Cysteines or in short MANEC was defined as a new protein domain family. The domains were found exclusively in the N-terminus of > 400 multi-domain membrane proteins from multicellular animals. Despite the large number of MANEC-containing proteins, only one has been characterized: hepatocyte growth factor activator inhibitor-1 (HAI-1). HAI-1 is an essential protein shown to regulate the activity of matriptase, hepsin and hepatocyte growth factor activator, all serine proteases with crucial roles in epithelial development, cell growth and homeostasis. Misregulation of these systems has been implicated in severe pathological conditions such as skin diseases and cancer. Detailed functional understanding of HAI-1 and other MANEC-containing proteins is hampered by a lack of any structural information on MANEC. Here we present an NMR solution structure and biophysical characterization of the MANEC domain from HAI-1, as the first structure of a representative MANEC domain. Although no homologies were predicted based on sequence, the MANEC structure revealed it as a new subclass of the PAN/apple domain family. Intriguingly, in silico protein folding resulted in successful structure-based homology prediction, where sequence-based approaches fail. The MANEC structure represents a much needed tool for elucidation of function of MANEC-containing proteins as indicated by the homology to the PAN/apple domains as mediators of protein-protein and protein-glycan interactions. 28

36 Atlas-based automatic brain morphometry applied to DBA/2J mouse model of glaucoma Jarosław Orzeł (1,2), Michał Fiedorowicz (1), Bartosz Kossowski (1,2), Marlena Wełniak - Kamińska (1), Piotr Bogorodzki (1,2), Paweł Grieb (1) (1) Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw (2) Faculty of Electronics and Information Technology, Warsaw University of Technology Objective: Glaucoma is a common disease of sight that is associated with degeneration of retinal ganglion cells (and its axons forming an optic nerve). Recently, it was shown that neurodegeneration in glaucoma is not limited to retina but it is also present along whole visual pathway. However, dynamics and mechanisms underlying this process remain unclear. DBA/2J mice spontaneously develop glaucoma-like pathology. Magnetic Resonance Imaging (MRI) is a rapid, non-destructive and three-dimensional method that can be used to quantitate brain structures volumes. In vivo morphometric analysis of vision-related brain structures would provide a valuable tool for tracking the ongoing neurodegeneration. Methods: DBA/2J female mice with advanced glaucoma (aged 14 months, n=5) and age matched controls (C57BL/6J; n=5) were anaesthetized with isoflurane (4% in oxygen induction, 1.5-2% - maintenance) and placed in 7T small animal-dedicated magnetic resonance tomograph (BioSpec 70/30USR; Bruker BioSpin, Ettlingen, Germany). High resolution structural imaging with TURBORARE T2 sequence (TR=7000ms, TEeff=30ms, RARE factor=4, spatial resolution=0.86mm x 0.86mm, slice thickness=0.35mm, slices=42, scan time=23min). For the volumetric analysis we employed C57BL/6J atlas database published online by National University of Singapore ( and C57BL/6J tissue probability maps (TPM) from the SPMMouse toolbox ( Data were processed using SPM software ( and custom-made MATLAB scripts. Statistical analysis was performed with U Mann-Whitney test. Results: Calculated total brain volumes were significantly lower in DBA/2J mice (P<0.01). Most of the analyzed structures were also smaller in DBA/2J mice (including frontal cortex, visual cortex and auditory cortex). However, when normalisations were performed to the total brain volumes, significant differences were noted only for some of the segmented structures, namely visual cortex (P<0.01) and auditory cortex (P<0.01) but not for frontal cortex. Conclusion: Changes in relative brain structures volumes indicate an ongoing neurodegenerative process that particularly affects vision-related brain structures in DBA/2J model of glaucoma. The study was supported by National Science Centre grant No. 2012/07/D/NZ4/

37 MR TAGGING FOR EVALUATION OF MECHANICAL PROPERTIES OF FATTY LIVER TISSUE Anna Osiak 1, Krzysztof Jasiński 1, Paweł T. Jochym 2, Edyta Maślak 3, and Tomasz Skórka 1 1 Department of Magnetic Resonance Imaging, Institute of Nuclear Physics Polish Academy of Sciences, Kraków, Poland, 2 Department of Materials Research by Computers, Institute of Nuclear Physics Polish Academy of Sciences, Kraków, Poland, 3 Department of Endothelium Experimental Pharmacology, Jagiellonian Center for Experimental Therapeutics (JCET), Kraków, Poland Introduction: MR Tagging (MRt) has recently been proved useful for liver stiffness assessment. With the use of heart beating force deforming the adjacent liver instead of external pulsing device used in elastography, MRt can be alternative for MRE in studying liver elasticity. We propose MRt-based liver strain analysis, taking advantage from full strain tensor properties such as eigenvalues, eigenvectors, trace and absolute strain magnitude, enriched with parameterization of deformed area range and shape. We hypothesize that these features may potentially change in abnormal tissue conditions, thus we aimed to assess the accuracy of such a method for early alterations in non-alcoholic fatty liver disease (NAFLD). Methods: Two groups of 6-month old C57BL/6J mice: Control standard AIN-93G diet, NAFLD 45% + 60% kcal high-fat diet, were examined using 9.4T BioSpec scanner (Bruker, Germany): ECG-gated FLASH-cine sequence preceded with SPAMM tagging (TE 1.5ms, TR 8.5ms, data matrix, FOV 30 30mm 2, 1.0 mm slice thickness, tagging grid with 0.2 mm tag line, 0.6 mm tag span; LV short-axis heart projection). The liver deformation was computed from MRt images based on two-dimensional strain tensor ε (%) as the maps of: ε1 (stretching) and ε2 (compression) principal strains (%) with their spreading directions, fractional volume changes FVC=ε1+ε2 (%) and absolute strain magnitude AbsE=ε1 ε2 (%). The maps were quantified using two-dimensional central and invariant image moments for the deformation area and shape assessment. Results: In NAFLD, the deformed area within all maps was more irregular in comparison to healthy organ. The ε1 and ε2 directions showed symmetrical strain development about the central point of heart stimulation in Controls, while in NAFLD mice the strain penetration seemed to be limited. In FVC and AbsE maps of fatty livers the major amount of deformation was enclosed in narrow elliptical area. The central and invariant moments analysis showed decreased strain area and level within all ε1, ε2, FVC and AbsE maps in NAFLD group, with noticeably larger deformation shape eccentricity as compared to healthy Controls. Conclusions: The extended MRt-based analysis of cardiac-induced liver deformation, benefiting from all strain tensor properties showed restricted deformation area in NAFLD, what may suggest increased resistance and viscosity of FL tissue. The observations were compliant with significant differences between groups in quantitative analysis. The results proved the routine useful for in-vivo measurements of the structural changes within hepatic tissue in NAFLD. The method may be good alternative for classic MRE, which is often insensitive for liver elasticity alterations in early stage of the disease. Aknowledgements: Work supported by European Union (grant coordinated by JCET-UJ, No POIG /09) and by The Marian Smoluchowski Krakow Consortium Matter- Energy-Future from the resources of KNOW. 30

38 MRI-BASED METHOD FOR THE IN VIVO ASSESSMENT OF ENDOTHELIAL STATE IN MURINE MODELS Anna Osip 1,2, Krzysztof Jasiński 1, Żaneta Bartel 1, Tomasz Skórka 1, Stefan Chłopicki 2,3 1 Institute of Nuclear Physics, Polish Academy of Sciences, Department of MRI, Crakow 2 Jagiellonian University Medical College, Department of Experimental Pharmacology, Crakow, 3 Jagiellonian Centre for Experimental Therapeutics(JCET), Crakow Introduction Evaluation of endothelial dysfunction, in clinical conditions is currently based on the biochemical methods, involving detection of important endothelial markers or mediators, and physical direct methods, which allow for invasive or non-invasive assessment (1). On the other hand commonly used non-invasive methods, which are useful especially in clinical prevention, are limited to the study of peripheral arteries. Magnetic resonance imaging (MRI) seems to be promising method for assessing endothelial dysfunction in coronary arteries and also is adequate method to study this issue in mice models (2). The aim of the study was to implement a comprehensive method, for the reliable assessment of endothelial function in vivo in mice with the use of MR imaging techniques. Materials and methods MRI was performed on Bruker BioSpec 9.4T system (Ettlingen, Germany). Brachiocephalic artery (BCA) and left carotid artery (LCA) were imaged using 3D techniques based on the retrospectively gated IntraGate FLASH sequence in control mice (4-months old C57BL/6J mice, ACh: n=4, Galb/Pearm: n=4), in mice fed high-fat diet (HFD60-60 kcal%) (5-months old C57BL/6J mice fed for 4 months a HFD60, ACh: n=4, Galb/Pearm: n=8) and C57BL/6J mice treated with L-NAME (4-months old mice, ACh: 4). Assessment included the evaluation of endothelium-dependent vasomotion in response to acetylcholine (ACh - Sigma-Aldrich: 50 µl, 16,6 mg/kg i.p), or changes of endothelial permeability with the use of an albumin-binding gadolinium contrast agent (CA: Galbumin, BioPal, Worcester, MA - 25 mg/ml, 4.5 µl/g i.v.) and 3D IG-FLASH - based VFA - Variable Flip Angle technique. Blood vessel cross-sections area and volume after ACh administration were determined. Endothelial permeability was assessed by detection T1 relaxation time changes around vessel lumen and the number of pixels (Npx50), for which T1 has changed about 50%, 30 min after contrast agent administration. Data analysis was performed in ImageJ 1.46r program (NIH, USA) and scripts in Matlab (MathWorks, Natick, USA). Statistical analysis was performed in STATISTICA 10 (Stat Soft inc., USA). Results 25 min after ACh administration, the vasodilation of blood vessels in C57BL/6J mice and its paradoxical vasoconstriction in HFD60 mice was observed. Additionally, vasoconstriction response was higher in LCA. In mice treated with L-NAME ACh did not induced vasodilation. Group C57BL/6J C57/HFD60 C57/L-NAME BCA Area change ± 5.04 % ± 3.40 % 1.81±3.32 LCA Area change ± 2.57 % ± 4.02 % -3.91±4.99 BCA Volume change ± 3.19 % ± 2.28 % LCA Volume change ± 2.49 % ± 3.38 % 31

39 In HFD60 mice shortening of the T1 around BCA was observed as opposed to C57BL/6J mice, where shortening of T1 was not significant. Npx50 for HFD60 mice was significant different from Npx50 for C57BL/6J mice. Group T1 change Npx50 C57BL/6J ± 1.56 % 8 ± 2 C57/HFD ± 7.70 % 20 ± 4 Conclusion 3D MRI-based technique is suitable to detect systemic endothelial dysfunction in vivo in mice. Our results suggest that potentially, MRI-based assessment of endothelial permeability and endothelium-dependent vasomotion in response to acetylcholine may be useful for monitoring experimental, endothelial-targeted therapy. Acknowledgments This study was supported by European Union from the resources of the European Regional Development Fund under the Innovative Economy Pro- gramme (grant coordinated by JCET- UJ, No POIG /09). References 1. Nadar S, Blann AD, Lip GYH. Endothelial dysfunction: methods of assessment and application to hypertension. Curr. Pharm. 2004, Tom 10, Phinikaridou A, Andia ME, Protti A et al. Noninvasive MRI Evaluation of Endothelial Permeability in Murine Atherosclerosis Using an Albumin-Binding Contrast Agent. Circulation (Baltimore),. 2012, Tom 126,

40 23 Na NMR STUDY OF Al- AND Ga- NANOPOROUS NATROLITES Mateusz Paczwa, Marcin Olszewski, Nikolaj Sergeev Aleksey.A. Sapiga *), Aleksey.V. Sapiga *) Institute of Physics, University of Szczecin *) Faculty of Physics, V.I.Vernadskii Taurida National University, Simferopol, Crimea The Al-natrolite ( ) and Ga-natrolite ( ) are compounds with nanoporous structure. In the small nanochannels in the structure of these natrolites the water molecules and Na ions are located in the form of zig-zag chains [1,2]. In present report we represent the experimental results on the study of polycrystalline Al- and Ga-natrolites by 23 Na NMR methods. The sodium neighbours in Al- and Ganatrolites have a configuration of a distorted tetrahedron. In the tetrahedron corners there are two oxygen atoms belonging to a framework and two oxygen atoms of water molecules at an average distances of 2.37 Å. Furthermore there are two oxygen atoms of a framework at an average distances of 2.5 Å, four protons at an average distances of 2.8 Å and atoms of silicon and aluminum at an average distances of 3.0 Å [1]. The obtained 23 Na MAS NMR spectra of Al-natrolite are Fig shown in fig.1. The shapes of these spectra Na MAS NMR spectra of Al-natrolite at are determined only by the second-order rot = 10 khz. For Ga-natrolite we obtained the quadrupolar shift of the central transition and same spectra. (a) experimental spectrum at T = the obtained theoretical values of the 300 K. (b) - theoretical spectrum with NMR quadrupolar frequency Q and the asymmetry parameters: Q=879,6 khz; =0,64; CSA=8,19 parameter well coincide with experimental ppm; Gauss=73,11 Hz values obtained in [3]. Na2Al2Si3O10 2H2O From the comparison of the 23 Na NMR spectra, shown in fig.2, it appears that the 23 Na NMR spectra have the same shape at T = 300 K and T= 380 K. The NMR shape of 23 Na is determined by magnetic dipolar interactions and by electric quadrupolar interaction with the electric field gradient (EFG) on the 23 Na sites. The interaction with magnetic moment of 1 H nuclei give the main contribution to the magnetic dipolar interaction of 23 Na nuclei. However the 1 H decoupling, which was used to record the spectra of 23 Na NMR Na2Ga2Si3O10 2H2O Fig Na NMR spectra with 1 H- decoupling in Al-natrolite at T = 300 K and T= 380 K (fig.2), leads to averaging of the dipolar interactions between the magnetic moments of 1 H and 23 Na nuclei and so, from fig.2, it follows that in the temperature region T < 380 the electric field gradient (EFG) at the 23 Na sites does not depend on the temperature. From our theoretical calculation it follows that the contribution of electric dipolar moments of water molecules to the EFG at the 23 Na sites is compare to the contribution from other ions of lattice. However, according with 1 H NMR data of Al-natrolite water molecules at T < 380 K rotate about their quasi 2-fold axis [4]. The flipping of the water molecule does not change its 33

41 electric dipole moment and so the contribution of the dipoles of water molecules to the EFG of a 23 Na nuclei must be temperature independent. So our result do not conflict with the results obtained in [4]. Fig Na NMR spectra without 1 H- decoupling in Al-natrolite at T = 300 K and T= 380 K Fig.3. shows the 23 Na NMR spectra obtained without 1 H decoupling at T = 300 K and T= 380 K. The difference between the NMR spectra represented in fig.2 and fig.3 are related to the dipolar interactions between the magnetic moments of 1 H and 23 Na nuclei. From fig.3 it follows that increasing of the sample temperature leads to thermal averaging of dipolar interactions between the magnetic moments of 1 H and 23 Na nuclei. This averaging is connected with the rotations of water molecules about their quasi 2-fold axis [4]. These rotations do not change the contribution of the electric dipoles of water molecules to the EFG at the 23 Na positions but lead to averaging of the local magnetic fields from magnetic moments of the protons of water molecules at the 23 Na sites. The temperature dependences of the spin-lattice relaxation times T1 of 23 Na in Al- and Ganatrolite are shown in fig.4. An activation energy of about 25 kj/mol was obtained from data presented in fig.4 and this value agrees with the activation energies of the rotations of water molecules in other hydrates [4-6]. This result indicates that the rotation of the water molecules about their quasi 2-fold axis is responsible for the spin-lattice relaxation process of 23 Na nuclei in Al- and Ga-natrolites. These rotations do not change the contribution of the electric dipoles of water molecules to the EFG at the 23 Na positions but lead to the averaging of the local magnetic fields from magnetic moments of the protons of water molecules at the 23 Na sites. Fig.4. The temperature dependences of the T1 in Al natrolite (rhombus) and Ga-natrolite (circles) [1] A.V.Sapiga, N.A.Sergeev, NMR investigation of natrolite structure. Cryst. Res. Technol., 36 (2001) [2] A.A.Sapiga, M.Olszewski, M.Paczwa, A.V.Sapiga, N.A.Sergeev, NMR study of gallosilicate natrolite. Functional Materials, 21 (2014) [3] H.E.Petch, K.S.Pennington, Nuclear Quadrupole Coupling Tensors for 23 Na and 27 Al in natrolite, a Fibrous Zeolite. J.Chem.Phys., 36 (1962) [4] R.T.Thompson, R.R.Knispel, H.E.Petch, NMR study of the molecular motion of water in natrolite. Can. J. Phys., 52 (1974), [5] A.V.Sapiga, N.A.Sergeev, V.N.Shcherbakov, S.P.Gabuda, I.A.Belicky, Diffusion of water molecules in rhombic natrolite. J. Struct. Chem., 27 (1986) [6] J.Haase, K.D.Park, K.Guo, H.K.C.Timken, E.Oldfield, Nuclear Magnetic Resonance Spectroscopic study of spin-lattice relaxation of quadrupolar nuclei in zeolite. J. Phys. Chem., 95 (1991)

42 2D AND 3D CP-VC AS TOOLS FOR DYNAMICS STUDY Piotr Paluch a*, Tomasz Pawlak a, Julien Trébosc b, Tatyana Polenova c, Jean-Paul Amoureux b,d and Marek J. Potrzebowski a. a Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies, Sienkiewicza 112, PL Lodz, Poland b Unit of Catalysis and Chemistry of Solids (UCCS), CNRS-8181, University Lille North of France, Villeneuve d Ascq, France c Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA d Physics Department & Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai , China One of the biggest achievements of modern solid state NMR spectroscopy is its ability to determine accurate inter-nuclear distances, which can be afterward used as structural restraints for reconstruction of three-dimensional structures of the condensed matter. The most common strategy is based on the analysis of homo- and/or hetero-nuclear dipolar couplings, which are both inverse proportional to the cube of inter-nuclear distances. Among different interactions, C H and N H one-bond contacts are of great interest in the context of characterizing inter-molecular arrangement via hydrogen bonding as well as backbone and side-chain dynamics in biological molecules. Indeed, the partial averaging of C H and/or N H dipolar couplings gives information about the geometry and amplitude of the motional processes in the solid state. During last decades, different methodological approaches, both for static samples and samples under magic angle spinning (MAS), have been introduced in order to improve the quality and reliability of obtained data. The big achievement in the field of measurements of X 1 H dipolar couplings was the introduction of PISEMA technique, and its different variants which allowed determining dipolar interactions under MAS eg. PILGRIM. One years ago we demonstrated that a very simple experiment, Cross-Polarization with Variable Contact-time (CP-VC), is very efficient at ultra-fast MAS (vro=60 khz) to measure accurately the C H and N H distances, and to analyze the dynamics of bio-molecules. Very recently we developed new multidimensional solid-state NMR methodology which permits, in simple and accurate way, the analysis of the 1 H- 13 C dipolar splittings and further scrutiny of the molecular motions in side chains in nanocrystalline proteins. The power of the technique is demonstrated in 3D NMR CPVC-RFDR correlation experiments in two proteins, GB1 and DLC8. This presented methodology is general and can be extended to other systems. Fig. 2. F1/F2 planes extracted from 3D CPVC-RFDR spectrum of GB1 for the following residues: Tyr (a, b), Phe (c) and Trp (d). The F3 values for each plane are indicated in the figure. In my presentation I will briefly present some methodology and possibilities of advanced solid state NMR and latter I will discuss on probing dynamics of aromatic residues: phenylalanine, tyrosine and tryptophan using our new methodological approaches. 35

43 Evaluation of the relaxation and the imaging properties of SPIO loaded nanocapsules at 9.4 T P. Piechota 1, K. Szczepanowicz 2, P. Warszyński 2, W. P. Węglarz 1 1 Henryk Niewodniczański Institute of Nuclear Physics PAN, Krakow, Poland 2 Jerzy Haber Institute of Catalysis and Surface Chemistry PAN, Krakow, Poland Purpose: The aim of this work was to evaluate relaxation and visualization properties of nanocapsules with iron oxide nanoparticles (SPIO) incorporated into their shell, at 9.4 T. Materials and methods: Four different samples: SPIO, nanocapsules without SPIO, nanocapsules with single layer of SPIO in the shell and nanocapsules with double layer of SPIO in the shell were investigated using 9.4T Bruker Biospec MR scanner. CPMG and IR pulse sequences were used for T2 and T1 relaxation times measurements, respectively. RARE pulse sequence was used for MR imaging. Dependence of relaxivities on relative SPIO concentrations were obtained with the use of OriginLab software. Results: The best contrast effect was obtained for nanocapsules with double layer of iron oxides in the shell. The T1 relaxation time was 6 to 7 fold and the T2 relaxation time was 7.5 to 14 fold shorter as compared to three other samples. The best relaxivity was obtained for the highest concentration of iron oxides in the sample. Discussion: The higher the difference between relaxation times of two neighbouring tissues, the better contrast effect is obtained in MR images. Superparamagnetic iron oxides have ability to shorten relaxation times of tissues in which they are accumulated. Nanocapsules with double layer of iron oxides in the shell gave good contrast effect which indicate the possibility of imaging their distribution in the living organisms using MR imaging. 36

44 MR Imaging of the Mouse Brain using Cryo-coil at 9.4 T - Histology in vivo? W.Piędzia 1, N. Bock 2, K. Jasiński 1, K. Kalita 1, G. Stanisz 3, W.P. Węglarz 1 1 Institute of Nuclear Physics, Polish Academy of Sciences, Kraków, Poland 2 Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, Ontario, Canada 3 Sunnybrook Health Sciences Centre, Toronto, Ontario, Kanada Purpose White matter (WM) degeneration is caused by many disorders including Multiple Sclerosis. Among many constituents of WM, myelin damage is the most pronounced and affects brain function. Myelin changes can be evaluated by magnetic resonance imaging (MRI) through T2 and T1 relaxation times [1-5]. The inversion recovery ultra short echo time pulse sequence (IR-UTE) [3] has been shown to be well suited for T1 quantification measurements in vivo. However imaging of the whole brain is challenging using this techniques due to long total acquisition time. Recently, Bock et al [5] suggested MP-RAGE pulse sequence for high resolution and time efficient imaging of mouse brain that yields high quality myelin maps including cortical white matter. The purpose of our study was to assess feasibility of using segmented MP-RAGE pulse sequence and cryo-coil for contrasting WM/GM and quantification of T1 in mouse brain in vivo, as compared to room temperature brain surface coil. Methods A healthy C57BL/6J mice were scanned using a 9.4T/21cm horizontal bore Bruker Biospec MRI system. A dedicated mouse brain surface coil and a cryo-coil were used for obtaining a two sets of corresponding full 3D (multislice) images of the brain. Two mice were scanned with each coil using the same experimental parameters: TR/TE = 15/4.5 ms, Segment Repetition Time (SRT) = 6000 ms, FA = 12º, 22 horizontal slices with 0.3 mm thickness covering the whole brain volume, together with FOV = 1.5 x 2 cm and MTX = 256x256 were used resulting with in-plane resolution of 59x78 µm. Two averages and the following Inversion Times (TI): 500, 900, 1000, 1100, 1200, 1300, 1400, 1600 and 2500 ms were used. Pixel by pixel T1-maps were generated from the data using custom written Matlab based script. For 3 selected inversion times (1000, 1100 and 1200 ms for surface coil while 900, 1000 and 1100 ms for cryo-coil) images with NA = 6 were measured for another animal. Additionally, images without transmitting RF power (i.e. with pulse power attenuation of 150 db) were generated in order to properly assess noise level for SNR comparison between room temperature surface brain coil and cryo-coil. Results Calculations show times higher SNR for cryo-coil as compared to room temperature brain coil. The exact figure depends on which horizontal slice level the flip angle was adjusted for cryo-coil. Fig. 1 shows the comparison of selected horizontal slices obtained at two different TI s, illustrating switching of contrast between GM and WM, due to nulling of signal from WM at TI=900 ms, while GM signal is nulled at TI=1000ms. The corresponding TI values for room temperature coil were about 100 ms higher. The difference stem from the inhomogenous spatial distribution of flip angle in case of transmit/receive cryo-coil in contrary to room temperature setup where spatially homogenous flip angle is defined by volume transmit coil, while surface coil is used only for signal receiving. Fig. 2. shows calculated T1 maps from cryo-coil and for comparison from room temperature setup. Effect of FA inhomogeneity was not taken into account in calculation. Significantly more fine details is visible in cryo-coil based map, while already mentioned decrease in T1 values is observed. Fig.1 MP-RAGE images (TI = 1100 ms and 900 ms) and the T1 map of the mouse brain obtained from cryo-coil Conclusions We have shown that MP-RAGE sequence used with cryo-coil at 9.4T, due to significant 2-3 times larger SNR than available from the dedicated room temperature brain coil, allowed for obtaining high resolution images of the whole mouse brain in vivo, with easily adjustable WM/GM contrast trough choice of appropriate inversion time, within the experimental time very attractive for in vivo experiments. Proposed MP-RAGE/cryo-coil setup 37

45 is very promising for quantitative assessment of myelination in mouse models and potentially for in-vivo mouse brain histology. References [1] Wilhelm M.J. et al: PNAS; 109 (24): (2012) [2] Larson P.E.Z et al: Magn Reson Med; 56 (1): (2006) [3] Piędzia W. et al: Journal of Neuroscience Methods; 232: (2014) [4] Chavez S et al..: NMR Biomed.; 25(9): (2012) [5] Bock N. et al.: NeuroImage (2013) 38

46 SUPERCONDUCTING DETECTION COIL FOR 0.2 T MRI SYSTEM Bartosz Proniewski 1, Henryk Figiel 1 1 AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Al. Mickiewicza 30, Krakow, Poland In magnetic resonance imaging, the detection coil plays an important role, being responsible for both, the excitation of the resonance and the detection of the MR signal. The design of the detection coil is crucial in obtaining high quality images, as their sensitivity is directly translatable into signal to noise ratio (SNR), a standard approach in quantification of imaging quality. Quality factor Q of the radiofrequency (RF) is one electrical parameter that directly impacts the SNR and therefore influences the imaging quality of an MRI system. Its value can be measured on the bench and directly compared between coil designs. Using low electrical noise materials enables higher Q values of the coils, hence providing better image quality. In this work we have underlined key aspects of utilizing commercially available high temperature superconducting tapes for manufacturing a surface RF coil. Numerical calculations were carried out in order to determine the influence of external magnetic field on current flow in the superconductor with varying orientation of the tape. Based on the optimum orientation of the tape and the orientation of the external magnetic field in the system the coil was designed for, a coil type was selected to be in the form of a Figure of 8 and the tape layout was optimized using electromagnetic simulations. A cryostat built from multilayered wall was then designed to keep the room temperature for sufficient amount of time required to perform imaging experiments. Once all the key factors have been assessed, the final design was established and a prototype was built. Three identical coils were built one from a 1 st generation and one from the 2 nd generation HTS tapes supplied by American Superconductors Company and a third reference coil made out of copper wire. All cols have been tuned and matched inductively to the 8.86 MHz frequency and characterized on the bench, by measurements of their Q factors. Cryostat design was evaluated by measuring temperature on the outside, where the imaged object was to be placed. Performing imaging experiments using a simple phantom carried out experimental validation of the use of HTS tapes in RF coil design. Images were used to calculate the SNR in various planes for the three coils working in room temperature and in cryogenic conditions (77 K). Results show that HTS coils operating in cryogenic temperature can indeed provide higher SNR relative increase, compared to the copper coil. In fact the copper coil improved by 28-47% while the HTS coil by % when immersed in liquid nitrogen, compared to room temperature operation. 39

47 PARA HYDROGEN INDUCED POLARIZATION OF PYRIDINE-LABELLED OLIGOPEPTIDES Tomasz Ratajczyk Institute of Physical Chemistry Polish Academy of Sciences, Kasprzaka 44/52, Warszawa, Poland The application of Magnetic Resonance techniques is strongly limited by inherently low sensitivity. This problem can be solved by so-called hyperpolarization techniques. One of these techniques is the Para Hydrogen Induced Polarization (PHIP) [1]. In the PHIP variant called SABRE, the NMR signal is enhanced by the interaction of para-hydrogen with an appropriate molecular system via a catalyst in a complex arising in a reversible reaction [2]. When the labile complex is formed, high spin polarization is transferred from para-hydrogen to the substrate. Afterwards, the labile complex splits up into hydrogen molecule, the catalyst and the SABREhyperpolarized product. Biofunctional SABRE-hyperpolarized molecular systems would be of central importance for widening the scope of possible MRI applications. Thus, the design of appropriate SABRE-active molecular systems is crucial. Fig. 1 SABRE Fig. 2 SABRE activation Here this problem is addressed. We propose a guideline for designing of SABRE-active molecules. We show that SABRE-inactive molecular systems can by SABRE-activated using labelling with pyridine. In particularly, we report SABRE of simple pyridine-labelled oligopeptides. We reveal that these oligopeptides interact with catalyst and hydrogen molecule. When the 3-components labile complex is formed, high spin polarization from para-hydrogen is transferred only to the pyridine unit. For all studied compounds 1 H NMR signal enhancement factors were evaluated. [1] C.R. Bowers, D.P. Weitekamp, J. Am. Chem. Soc. 109 (1987) 5541; M.G. Pravica, D.P. Weitekamp, Chem. Phys. Lett. 145 (1988) 255; T.C. Eisenschmid, R.U. Kirss, P.P. Deutsch, S.I. Hommeltoft, R. Eisenberg, J. Bargon, R.G. Lawler, A.L. Balch, J. Am. Chem. Soc. 109 (1987) [2] R. W. Adams, J. A. Aguilar, K. D. Atkinson, M. J. Cowley, P. I. P. Elliott, S. B. Duckett, G G. Green, I. G. Khazal, J. Lopez-Serrano and D. C. Williamson, Science, 323 (2009), 1708; This work has been supported by the Polish National Science Centre (NCN) under Contract No.: SONATA-2011/03/D/ST4/

48 APPLICATION OF MICRO-MRI TECHNIQUES IN THE EVALUATION OF MUSCLE DEGENERATION AND REPAIR PROCESSES AFTER FEMORAL ARTERY OCCLUSION IN MICE Agnieszka Skorupa 1, Mateusz Ciszek 1, Łukasz Boguszewicz 1, Tomasz Cichoń 2, Ryszard Smolarczyk 2, Stanisław Szala 2, Maria Sokół 1 1 Department of Medical Physics, 2 Center for Translational Research and Molecular Biology of Cancer, Maria Skłodowska Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland. Introduction: Development of non-invasive techniques enabling evaluation of muscle damage and repair processes would provide a valuable insight into the effects of therapeutic interventions (for example injection mesenchymal stem cells into the injured muscles). The purpose of this work was to evaluate the kinetics of MRI-derived parameters characterizing muscle function after femoral artery ligation in mice. Materials and methods: MRI experiments were performed immediately, 3, 7, 14 and 21 days after femoral artery ligation on a 9.4 T vertical 89-mm-bore Bruker magnet equipped with a Bruker Micro2.5 gradient system of 1T/m and a transmit/receive birdcage radio frequency coil with an inner diameter of 30 mm. During data acquisition animals were anesthetized with sevoflurane. Body temperature and respiration rate were monitored using ECG/respiratory unit. 2D Time of flight (TOF) angiography was used to visualize collateral vessel formation after artery ligation. Multi-slice multi-echo (MSME) sequence and diffusion tensor imaging (DTI) with fat suppression were used to characterize muscle tissue in terms of transverse relaxation time (T2), fractional anisotropy (FA), apparent diffusion coefficient (ADC), radial (RD) and axial (AD) diffusities. Results and discussion: Longitudinal MRI monitoring of mice revealed decreased FA and increased T2, ADC and RD after experimental injury and progressive normalization of these parameters with time. The observed changes are presumably due to initial degeneration and subsequent rapid repair of the injured tissue. Though, the presence of intramuscular oedema (increased high T2 signal) on MRI carries an extremely broad differential, oedema-like changes are characteristic for acute phase of muscle injury, while abnormal DTI-derived parameters reflect disrupted muscle architecture. Conclusion: Multi-parametric MRI used in this study revealed dynamic response of T2, ADC, FA and RD parameters after ischemic muscle injury and provides information useful in monitoring of therapeutic effects of cellular therapies tested in animal models. 41

49 APPLICATION OF NMR RELAXATION MEASUREMENTS TO THE STUDY OF OXIDATION PROCESSES IN BIOLOGICAL SYSTEMS DOROTA WIERZUCHOWSKA*, LECH SKÓRSKI, BARBARA BLICHARSKA *Institute of Physics, Pedagogical University, ul. Podchorążych 2, Kraków, Poland Institute of Physics, Jagiellonian University, ul. Reymonta 4, Kraków, Poland So far only EPR and CI DNP (Chemically Induced Dynamic Nuclear Polarization) methods have been used to observe the action of free radicals in biological systems [1,2]. In this communication we present some new results obtained by NMR relaxation method, namely the time evolution measurements of relaxation time T1 in protein aqueous solutions such as: egg white and bovine serum albumin and rabbit blood serum, after initiation of oxidation process by addition of H2O2. Hydrogen peroxide is one of the strongest reactive oxygen spiecies and added to aqueous solutions causes changes of the water proton relaxation times [3,4]. Just after addition of H2O2/H2O (3%) to albumin solution samples the relaxation time T1 starts to short and after approximately 25 minutes stabilizes. The kinetics of this process depends on the structure and concentration of the protein and amount of added hydrogen peroxide. Measured time evolution curves may be well approximated by exponential decay. In rabbit serum, instead of stabilization, after reaching the minimum value the relaxation time T1 starts to regrow. Similar kinetics is observed in experiments with protein solutions containing a small amount of added antioxidants like ascorbic acid (vitamin C). Moreover, the supplementary addition of vitamin C to rabbit blood serum enhances the T1 behavior. It means that regrow of T1 might be a consequence of the action of antioxidants, which are known to be present in blood serum. We hope that in the future NMR relaxation measurements may be useful not only in investigation but also in diagnosis of some diseases, especially with free radical background. [1] D.A. Svistunenko, Biophys. Biochim. Acta 1546 (2001) [2] L.T. Kuhn, J. Bargon, Top.Curr.Chem. 276 (2007) [3] L.W. Skórski, B. Solnica, B. Blicharska, Journal of Laboratory Diagnostics 47(1) (2011) [4] H.C. Bertram, M. Kristensen, H. Osdal, C.P. Baron, J.F. Young, H.J. Andersen, J.Agric.Food Chem. 55 (2007)

50 CHEMICAL EXCHANGE SATURATION TRANSFER (CEST). FROM AN AGAR TO A MAN. Greg J Stanisz Physical Sciences, Sunnybrook Research Institute Departmental of Medical Biophysics, University of Toronto Toronto, Ontario, Canada This work describes the origins of the contrast mechanism chemical exchange saturation transfer (CEST) and methods for proton magnetic resonance imaging (MRI), with application in cancer. It focuses on endogenous techniques, which do not require the injection of a contrast agent, and the experimental and analytic techniques which allow quantitative metrics to be obtained. The theory behind CEST is presented, and it is modelled using a three pool system of Bloch equations which are a function of magnetization relaxation parameters, CEST pool concentration, and hydrogen exchange rate constant. A platform is created for optimization and parameter fitting, which is used to obtain parameter values and for pulse sequence experimental design. The contribution of semisolid macromolecules to the CEST spectrum is determined, and evidence given that the CEST parameters (in particular, the exchange rate constant) can be obtained independently of this additional pool. It is shown that there is a linear relationship between the log of the exchange rate constant and ph. CEST spectra are obtained in cancer xenografts in mice, exhibiting features from amide, amine and aliphatic protons as well as from magnetization transfer. Semi-quantitative CEST parameter maps are derived, showing the distribution of CEST features in tumours and in contrast with the surrounding normal-appearing muscle. Several exploratory experiments are performed in protein-containing phantoms and cell pellets in order to validate the origins of CEST spectra and their behaviour in conditions of changing ph and temperature. Cell pellet experiments support the hypothesis that CEST is sensitive to primarily intracellular conditions, and furthermore that the cell nucleus is a concentrated source of CEST-contributing proteins. Ultimately, the work contained herein supports the hypothesis that the endogenous CEST experiments provide new information not obtainable from other MRI experiments, leading to quantitative absolute intracellular ph mapping. This has potential for predicting the effectiveness of treatment regimens based upon the relationship between intracellular ph and drug uptake, establishing regions of tumour which are actively proliferating or which may be resistant to therapy. Figure 1: A visual comparison of the maps generated from each MRI contrast mechanism examined in this study, for a single scan time-point in a single Lewis Lung Carcinoma tumour xenograft in a mouse. Also shown, are the reference spin echo image and the TUNEL and H&E histology images for apoptosis. 43

51 NMR TOP SYGNALS OF THE 27 Al IN SOLID SOLUTIONS BASED ON THE YAG CRYSTAL Piotr Stępień, Marcin Olszewski, Nikolaj Sergeev, Bohdan Padlyak *) Division of Solis State Physics, Institute of Physics, University of Szczecin *) Spectroscopy Sector, Institute of Physical Optics, 23 Dragomanov Str., Lviv, Ukraine and Division of Spectroscopy of Functional Materials, Institute of Physics, University of Zielona Góra, 4a Szafrana Str., Zielona Góra The Two-dimensional One Pulse (TOP) experiment is the simplest 2D experiment [1-4]. In this experiment 2D signal s(t1,t2) of the sample, which rotates with frequency R is obtained from identical 1D signals - free induction decays (FID), separated by tr = 2 / R in both t1 and t2 dimensions. The main strength of TOP method lies in its rapid interpretation of MAS signals of half-integer quadrupolar nuclei [3]. In [1-3] affirms that TOP spectroscopy leads to a better resolution of information disguised in conventional 1D MAS spectra and it is an ideal method for study of satellite transition of quadrupolar nuclei. In this communication we represent the application of TOP method to study of 27 Al 2D spectra of nominally pure and Cr-doped yttrium-aluminium garnet YAG (Y3Al5O12 and Y3Al5O12:Cr) crystals. In Fig. 1 and Fig. 2 are presented the 27 Al MAS NMR spectra obtained for powdered crystalline samples Y3Al5O12 and Y3Al5O12:Cr. All 27 Al MAS NMR spectra, which are observed in YAG, contain two peaks corresponding to the tetragonal (AlO4) and octahedral (AlO6) structural atomic groups. The simulation of the experimental 27 Al MAS NMR spectra of the Y3Al5O12 and Y3Al5O12:Cr give the fractions of the AlO6 and AlO4 groups: N(AlVI) / N(AlIV) 0.67 for YAG and N(AlVI) / N(AlIV) 0.85 for YAG:Cr. So the doping by Cr of the Y3Al5O12 crystals leads to variation of the occupation by Al atoms both octahedrally- and tetrahedrallycoordinated sites of the garnet lattice. Fig. 1. The theoretical (a) and experimental (b) curves for 27 Al MAS NMR spectrum of the nominally-pure polycrystalline Y3Al5O12 sample. Fig. 2. The theoretical (a) and experimental (b) curves for 27 Al MAS NMR spectrum of the Y3Al5O12:Cr polycrystalline sample. The isotropic chemical shifts ( iso), quadrupolar coupling constants (CQ) of the 27 Al nuclei in the AlO4 and AlO6 structural groups obtained by Dmfit program [5] are presented in Table 1. 44

52 Table 1. Quadrupole coupling constants (CQ), isotropic chemical shifts ( iso), and the broadening parameters ( L) of the Lorentzian function, for AlVI and AlIV in the un-doped and Cr-doped YAG crystals. The asymmetry parameter = 0. The Al sites in Un-doped YAG Cr-doped YAG YAG crystal CQ,(MHz) iso,(ppm) L,(kHz) CQ,(MHz) iso,(ppm) L,(kHz) Tetrahedral Octahedral In Fig. 3 and Fig. 4 are presented the 27 Al TOP NMR spectra obtained for powdered crystalline samples Y3Al5O12 and Y3Al5O12:Cr. Fig. 3. The 27 Al TOP spectrum of YAG with different projections of 2D-spectrum. Fig. 4. The 27 Al TOP spectrum of YAG:Cr with different projections of 2D-spectrum. From comparison of Fig. 3 and Fig. 4 we conclude that the TOP MAS NMR spectroscopy is a sensitive and powerful method for investigating the local structure of main structural units in ordered and disordered solids and the redistribution of atoms between different sites caused by doping impurities. [1] B. Blumich, P. Blumler, J. Jansen, Presentation of sideband envelopes by two-dimensional one-pulse (TOP) spectroscopy, Solid State NMR, 1 (1992) [2] P. Blumler, B. Blumich, J. Jamsen, Two-dimensional one-pulse rotational echo spectra, Solid State NMR, 3 (1994) [3] D. Massiot, J. Hiet, N. Pellerin, F. Fayon, M. Deschamps, S. Sreuernagen, P.J. Grandinetti, Two dimensional one pulse MAS of half-integer quadrupolar nuclei, J. Magn. Res., 181 (2006) [4] Ph.J. Grandinetti, J.T. Ash, N.M. Trease, Symmetry pathways in solid-state NMR, Progress in NMR spectroscopy, 59 (2011) [5] D. Massiot, F. Fayon, M. Capron, I. King, S.Le Calve, B. Alonso, J.-O. Durand, B. Bujoli, Z. Gan, G. Hoatson, (2002) Modelling one- and two-dimentional solid-state NMR spectra. Magnetic Resonance in Chemistry, 40 (2002)

53 SPI Implementation for 4.7T System Grzegorz Stoch H. Niewodniczański Institute of Nuclear Physics of PAN, Kraków, Poland SPI is an imaging technique free from artifacts arising from B0 inhomogeneity, susceptibility variations, and chemical shift [1]. It is capable to deliver data for systems with very short T2 relaxation times. These capabilities are key points for extending MRI research on the field of solid state imaging - which has been not the case so far. Original approach to SPI implementation for our 4.7T system is presented (Oxford Instruments MARAN DRX console). References: [1]. B.J. Balcom, SPRITE imaging of short relaxation time nuclei, in: P. Bleumler, B. Bleumich, R. Botto, E. Fukushima (Eds.), Spatially Resolved Magnetic Resonance, Wiley-VCH, Toronto, 1998, pp

54 DYNAMIC EFFECTS IN SINGLE CRYSTAL OF 9,10- DIMETHYLTRIPTYCENE-D12 ON BASIS OF PROTON NMR SPECTRA Piotr Bernatowicz, 1 Tomasz Ratajczyk, 1 Alexander Shkurenko, 1 Bohdan Kamienski 2, Sławomir Szymański 2 1 Institute of Physical Chemistry, Polish Academy of Sciences, 2 Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, Warszawa, Poland Single-crystal and powder X-ray diffraction studies of 9,10-dimethyltriptycene (1) and its d12-isotopomer deuterated in the aromatic positions (1a), made lately by us and other authors, consistently delivered undistorted R-3c structure of the material. 1 In this structure, all methyl groups in the crystal occur in closely spaced pairs in a staggered conformation, with different pairs being both structurally and magnetically equivalent (see Scheme 1). On the other hand, the once obtained 1 H wide line NMR spectra of polycrystalline 1a were inconsistent with the above structure. 1 The presently 0.33 nm Scheme 1. reported 1 H spectra of a single crystal of 1a also reveal severe inconsistency with the R-3c structure determined for the same crystal from X-ray diffraction data. These NMR and X-ray data were both collected below 135 K where the methyl group dynamics are completely frozen on the NMR timescale. Apart from that shown in Scheme 1, the NMR spectra reveal another sharply defined environment of the methyl groups, involving as much as about 20 per cent of the latter. In this environment, the methyl groups apparently have the same orientations as in the pairs but suffer no dipole-dipole couplings to outer protons. The spectrum at 131 K is shown in Fig. 1, together with the theoretical fit. The theoretical component spectra corresponding to the two environments are also shown. The observed effects can tentatively be explained as follows: (i) in crystals of 1a point vacancies cause their nearest surroundings to form mesoscopic domains only slightly differing from the ordered R-3c phase; (ii) as such, these domains are practically invisible in the X-ray diffraction patterns; (iii) within the domains, the vacancies undergo rapid diffusion by purely translational jumps of the molecules of 1; (iv) in this way, the dipolar spin-spin couplings between the paired methyl groups are lost in a similar way as is the loss of J-couplings between rapidly dissociating and recombining fragments of molecules in solution. A precise definition of the domains mentioned under (i) is still to be elaborated. The dynamic disorder of this type is unusual because of its persistence down to relatively low temperatures. Fig H NMR spectrum of single crystal of 1a at 131 K (black), with the methyl groups' axes parallel to the magnetic field. The theoretical best fit spectrum and the component theoretical spectra of the ordered and dynamically disordered domains are depicted in red, blue, and green, respectively. The ratio of the intensities of the green and blue spectra is

55 Actually, below 90 K the spectra do start to broaden and change shape but in a way that cannot be unambiguously interpreted in terms of gradual freezing of the considered dynamic disorder. A distinctive feature of the proposed mechanism of the disorder is that the two types of methyl groups should experience essentially the same environment on the average. In one environment, the two given groups remain permanently in contact while in the other such close contacts are being constantly disrupted to be immediately restored, but with one partner changed. Above 160 K, effects of thermally activated dynamics of the methyl groups in 1a are evidenced in the spectra. A series of variable temperature spectra of 1a documenting these effects were measured for the orientation where the threefold axes of the methyl groups are directed at right angle to the external field. The spectra are shown in Fig. 2. They could be perfectly fitted with the damped quantum rotation (DQR) model 2 with the additional assumption that the dynamic properties of the isolated methyl groups and those coming in coupled pairs are identical. The conventional fits shown in the right panel of Fig. 2 are evidently defective in the region of slow and moderate exchange. Fig. 2. Variable temperature spectra of oriented single crystal of 1a (black lines) superposed with theoretical best fit spectra calculated with the DQR model (red lines, left panel) and the conventional random jump model (blue lines, right panel). The green line depicts the best fit spectrum to the experimental pattern measured at temperature where the methyl group dynamics are frozen. The threefold axes of the methyl groups are oriented at right angle to the external field. All calculations were performed under assumption that the ratio of the fractions of the isolated and pairwise coupled methyl groups is 0.220, i.e., the same as determined at 131 K. The perfect character of the DQR fits obtained for experimental spectra from an extended temperature range provides one more corroboration of the postulated mechanism of the dynamic disorder. If the methyl groups resided in two basically different environments, it would be unlikely for them to share the same dynamic parameters controlling their hindered rotation. 1 P. Bernatowicz, T. Ratajczyk, P. Kalicki, and S. Szymanski, Solid State NMR, 59-60, (2014). 2 S. Szymański, J. Chem. Phys. 111, (1999). 48

56 Diverse dynamics of water molecules confined in faujasites. Deuteron NMR investigation. A. Szymocha a, Z.T. Lalowicz b, Birczynski b, K. Gora-Marek c a H. Kollataj Academy of Agriculture, Mickiewicza 21, , Krakow, Poland b H. Niewodniczanski Institute of Nuclear Physics PAS, Radzikowskiego 152, Krakow, Poland c Faculty of Chemistry, Jagellonian University, ul. Ingardena 3, Krakow, Poland Study of dynamic behavior of water molecules in zeolites is a part of investigations aiming to elucidate also catalytic properties at molecular level. In a detailed microscopic model one expects features related to various interactions, such as electrostatic water-sodium cation, hydrogen bonding of water to framework oxygens, and water-water bonding, as well as their dependence on the loading and Si/Al ratio. There are two narrow components of different width in the spectra above 220K for all samples considered. Their relative weights change with temperature. We may conclude at this point that there are two phases and negligible exchange between them. Contribution of the narrow component undergoes thermally activated temperature dependence and can be attributed to water molecules forming clusters freely mowing in space, with O-D performing internal tetrahedral jumps. Other water molecules perform chaotic rotational jumps, and belong to bottom adsorption layers at sodium cations. Their contribution increases on decreasing temperature. All molecules become localized below 220K, as indicated by extreme broadening of the spectra, which in turn provide evidence for the symmetry of deuteron mobility. Three main components can be pointed out. Pake doublets, with the separation related to the quadrupole coupling constant, are attributed to immobile deuterons. The value of the quadrupole coupling constant, there are four similar values measured in all cases, allows to specify location of a deuteron on four chemically distinguishable oxygens in the zeolite framework. Twofold exchange of deuteron positions leads to the characteristic spectral shape. Gaussian spectral components, with the width decreasing on increasing temperature, represent chaotic reorientations leading to narrow spectra at high temperature. Contribution of these components depends on Si/Al ratio and loading. Pake doublets dominate at temperature below 70K, while twofold exchange was observed at the intermediate range. The project was generously supported by the National Science Centre, Grant No. N N during

57 CHLORINS SYNTHESIS AND NMR SPECTROSCOPY STUDIES Justyna Śniechowska, Piotr Paluch, Marek J. Potrzebowski Centre of Molecular and Macromolecular Studies Polish Academy of Science, Department of Structural Studies, Lodz, Poland Chlorins belong the group of heterocyclic compounds consisting, at the core, of three pyrroles and one pyrroline that are connected by bridging carbon atoms into a macrocyclic structure. Due to unique physical and chemical properties, they can be used as an excellent photosensitizers in photodynamic diagnosis and therapy as well as anticancer and antibacterial drugs. Knowledge about their chemistry and structural complexity is strongly desired. In this work we present method of the synthesis of several chlorins (Fig. 1) and their advanced structural study employing NMR spectroscopy in the liquid and solid state. Using high-resolution NMR techniques such as 1 H- 13 C HSQC/HMBC, 19 F- 13 C HSQC, 1 H- 15 N HSQC, 19 F- 19 F COSY and 19 F- 1 H HOESY the chemical shift assignment of signals in the spectra for the individual atoms of the molecule in the liquid state was done. The structure of major tautomer was established. Furthermore, the chlorins were found to be a very interesting and unique model for NMR studies, because of differentiation of hydrogen or fluorine atoms in side groups. It is probably caused by disturb of planarity system and restricted rotation of rings in meso-positions. In the solid state we employed both advanced two-dimensional (2D) NMR techniques and theoretical calculations that provide in-depth information about the structure and intermolecular interactions within host-guest (H-G) complexes. R R NH N N HN X R R R NH N N HN X R N R R Fig. 1. General structure of obtained chlorins 50

58 NMR SPECTROSCOPY OF SERUM LIPID EXTRACTS OF SARCOIDOSIS PATIENTS Toczylowska Beata 1,2, Jastrzebski Dariusz 3, Zieminska Elzbieta 4, Zieleznik Karolina 3, Zebrowska Aleksandra 5, Ziora Dariusz 3, Mierzejewska Aneta*, Kozielski Jerzy 3 1. Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Trojdena 4 2. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Pawinskiego 5A 3. School of Medicine with the Division of Dentistry, Chair and Department of Lung Diseases and Tuberculosis, Medical University of Silesia, Zabrze, Koziolka 1 4. Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Pawinskiego 5 5. Department of Physiological and Medical Sciences, Academy of Physical Education, Katowice, Mikołowska 72a Student of Warsaw University, work done in Institute of Biochemistry and Biophysics, Polish Acadamy of Sciences, Warsaw, Pawinskiego 5A The aim of this study was to determine the use of the lipid profile of patients with sarcoidosis and compare it with healthy subjects. We assume that lipid profile of serum in sarcoidosis differs from the lipid profile of control subjects. Serum was collected from 9 patients with II stage of sarcoidosis and 5 control subjects (healthy volunteers). Lipids were extracted from serum before analysis using modified Blight and Dyer method and dissolved in deuterated chloroform (Fig1). Proton NMR spectroscopy combined with discriminant analyses, PCA (principal component analysis) and PLS-DA (partial least squares projections to latent structures discriminant analysis), was used. NMR spectra were collected using 400MHz spectrometer and standard one pulse sequence. Thirty four NMR signals of lipid compounds were selected using home prepared software. Quantities of metabolites were expressed as relative intensities of spectral peak magnitudes as compared to the internal standard CHCl3 rest signal. Targeted profiling will be applied to each NMR spectrum as the data reduction technique. Partial least square discriminat analysis (PLS-DA) with Pareto scaling were used to analyze lipid profile. FA EC/FC FA/TC PL/TG EC PUFA TC FC/EC PC TG PC/SM TC Fig1. NMR spectrum of lipid extract ppm Univariate t-test analysis show significant differences in NMR signals of in 14 out of 34 of NMR signal levels from compounds: total cholesterol, both esterified and free cholesterol, phosphatidylcholine, triglycerides, fatty acids and unassigned compounds (4.94 ppm, 51

59 5.14 ppm, 4.05 ppm and 1.84 ppm) (p<0.05) and are presented on (Fig 2.) For analyzing lipid profile discriminant analysis was applied. Fig.2 Compound selected as significant different for control and sarcoidosis patients in PLS- DA analysis and t-test analysis. * - indicated significant differences in t-test (p<0.05) PLS-DA model consisted of three components and very good explain the data and also predict the data. Discriminant analysis correctly classified patients according to their groups for 100% of sarcoidose and 100% of control (Fig.3). Lipidomics indicated significant differences in phosphatidylcholine, triglycerides, fatty acids, sphigomyelin three unassigned compounds (4.05ppm, 4.94ppm, 5.14ppm) levels. Fig 3. Score The scores plot of the two-component PLS-DA model of the first two principal components t[1] and t[2]; t[1] represents the greatest amount of correlated variation in the data set, whereas t[2] represents the second greatest amount of correlated variation. From multivariate discriminant analysis we obtain a list of potential biomarkers which are statistically significant and which separate one class from another. These biomarkers are statistically significant, but not necessarily biochemically significant. They may have biochemical significance and they may be the biomarkers we are interested in, however, this must be established through extensive testing. Presented method allow to distinguish between healthy subject and sarcoidosis patients. 52

60 Eye morphology quantitated by magnetic resonance imaging in mice Marlena Wełniak - Kamińska (1), Tomasz Chorągiewicz (2) Michał Fiedorowicz (1), Jarosław Orzeł (1, 3), Piotr Bogorodzki (1, 3), Paweł Grieb (1) (1) Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw (2) 1st Eye Hospital, Medical University of Lublin, Lublin, Poland (3) Faculty of Electronics and Information Technology, Warsaw University of Technology Objective: Evaluating eye dimensions is crucial in studies of animal models of eye diseases. However, performing these measurements in alive mice is challenging. The aim of this study was to develop a method for quantitating eye morphology using planar surface MRI coil. Methods: Aged (18 months) C57Bl/6 female mice (n=5) were anaesthetized with isoflurane (4% in oxygen induction, 1.5-2% - maintenance) and placed in 7T small animal-dedicated magnetic resonance tomograph (BioSpec 70/30USR; Bruker BioSpin, Ettlingen, Germany). Small planar surface receive coil (internal diameter=10 mm, Bruker BioSpin, Ettlingen, Germany) was placed over left eye of the imaged animal. High resolution structural imaging with TURBORARE T2 sequence (TR=2700ms, TEeff=30ms, RARE factor=8, NA=12, spatial resolution=0.062mm x 0.062mm, slice thickness=0.3mm, slices=7, scan time=16min) was performed. Basic ocular dimensions and anterior chamber angle were measured manually using OsiriX software (Pixmeo, SARL, Bernex, Switzerland). Results: Mean anterior chamber depth in C57Bl/6 mice was mm (SD ±0.040) and anterior chamber angle (ACA) (SD ±5.599), vitreous chamber depth (VCD) mm (SD ±0.033), axial length (AL) mm (SD ±0.079) and horizontal length (HL) mm (SD ±0.061) of the eyeball, lens thickness (LT) mm (SD ±0.059) and optic nerve diameter (ON) mm (SD ±0.0520), retinal thickness (RT) mm (SD ±0.018). Conclusion: High resolution magnetic resonance imaging of the eye with use of small planar coil provide reproducible and consistent measures of key dimensions of eyeball. These results are consistent with results obtained with optical coherence tomography in C57Bl/6 mice [Chou et al. 2011, IOVS 52: ]. The study was supported by National Science Centre grant No. 2012/07/D/NZ4/

61 THE CYTOSTATIC AGENT AS A CONTRAST AGENT FOR MRI Beata Wereszczyńska a, Tomasz Zalewski b, Magdalena Hałupka-Bryl b, Marek Kempka a,b, Stefan Jurga a,b a Department of Macromolecular Physics, Adam Mickiewicz University, Umultowska 85, Poznań b NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, Poznań Superparamagnetic iron oxide nanoparticles (SPIO) are widely used in medicine especially as drug delivery systems and MRI contrast agents. The idea of combining those two applications resulted in verifying the possibility of use an existing medicine as MRI contrast agent. The object of the study were magnetic iron oxide nanoparticles loaded with doxorubicin (SPIO/DOX) synthesized for targeted drug delivery. Their magnetic properties and ability to accumulate in the tumor area makes them well suited to use in monitoring of tumor treatment by magnetic resonance imaging. In order to evaluate contrasting properties of SPIO/DOX studies of proton relaxation at three magnetic fields intensities (0.4T, 4.7T and 9.4T) have been performed. Relaxation times T1 and T2 have been measured (using Inversion-recovery and CPMG pulse sequences, respectively) for samples of different concentration (mm) of investigated nanoparticles in water and physiological saline and relaxivities r1 and r2 were calculated. Obtained results allow to conclude that investigated nanocomplex can be classified as effective T2 contrast agent in wide range of magnetic fields commonly used in human diagnosis. SPIO/DOX can also be used as T1 contrast agent up to magnetic fields of 1T. Series of T2-weighted MRI images of solution of SPIO/DOX injected into agarose gel phantom were obtained at 9.4T using Fast Spin Echo sequence. The successive images illustrate the expansion of the of contrast area in function of time after injection. In order to calculate contrast areas image profiles were performed. Threshold pixel value was determined and all the pixels, in regions of interest, below that value were counted. Results confirmed that investigated particles are efficient T2 contrast agent and have ability to easily spread by diffusion in environment with tissue-like density. Acknowledgements Financial support from the National Centre for Research and Development under research grant Nanomaterials and their application to biomedicine, contract number PBS1/A9/13/2012, is gratefully acknowledged. 54

62 USEFULNESS OF MR SEQUENCES: DTI AND 3D ASL IN RARE CHILD BRAIN TUMOR BASED ON A MR BRAIN CHILD EXAM BEFORE AND AFTER SURGERY Magdalena Wicher 1, Magdalena Machnikowska Sokołowska 1, Anna Plechta 1, Katarzyna Zymella 1, Dominika Wieja Błach 1, Marcin Basiak 1, Marek Konopka 1 1 SCANX Medical Imaging Sp. z o.o., ul. PCK 10, Katowice Purpose: To assess the usefulness of MR sequences: DTI (diffusion tensor imaging) and 3D ASL (3D Arterial Spin Labeling-non contrast, whole brain quantitative perfusion assessment) in MR imaging of rare child brain tumors. Materials and methods: Two MR brain examinations of a 9 years old boy before and after brain tumor surgery were analyzed. Exams were performed on GE MR Optima 450w 1,5T GEM with protocol: T1 and T2 SE (spin-echo), T2 FLAIR, 3D BRAVO. The protocol was supplemented with 3D ASL and DTI. 3D ASL uses magnetic properties of inflowing blood signed by energy with radio frequency (RF) pulses to invert the water spins of inflowing arterial blood. This technique uses water in arterial blood as an endogenous contrast media to help visualize tissue perfusion and provide quantitative assessment of cerebral blood flow (CBF) in ml/100g/min. 3D ASL image was acquired with following parameters: field of view 24 cm, echo time (TE) 10,7 ms, repetition time (TR) 4683 ms and 1 NEX (number of excitations). Arterial spin labeling was achieved on carotid arteries level (can be performed a few centimeters below FOV). Calculations of cerebral flow in ASL technique depends on time of arrival of arterial blood to imaging voxels, local tissue and blood time relaxation and magnetic balance of blood [1],[2],[3]. Pioneered pulsed-continuous arterial spin labeling method helps amplify signal and improve contrast. 3D ASL with spiral readout helps increase signal to noise (SNR), while minimizing motion and reducing susceptibility artifacts, compared to conventional gradient-echo based techniques. Rys.1. 3D ASL technique [4]. Another technique we used was diffusion tensor imaging (DTI). Diffusion of water molecules in the brain tissue is characterized as anisotropic, restricted by different barriers, such as myelin or cell membranes. Multidirectional diffusion differential requires more complex description with use of diffusion tensor. In case of diffusion tensor calculations it is necessary to make measurements of diffusion at least in six different directions. To show degree of anisotropy of examined structure fractional anisotropy (FA) (reaching values between 0 1) is used. 0 value corresponds with isotropic structure, whereas 1 value with structure, in which diffusion is possible only in one direction. There are many ways of diffusion tensor imaging (DTI). Most common are MD and FA images in grey scale, FA images coded by colour and tractography. In parametric FA maps coded by colour, tensor is defined by direction of maximum component of diffusion tensor (red: right left; green: front back; blue: up down), and intensity of colour depends on FA quantity [5]. 55

63 Results: Brain MR examination showed tumor originating in right lateral ventricle. Non-contrast perfusion (3D ASL) and diffusion tensor (DTI) in 6 directions were used. To minimize examination time instead of 25 directions we used only 6, which was enough for good fiber tracks visualization. Sequences were analyzed in Readyview (GE) software, with quantification of cerebral brain flow (CBF) for tumor and visualization of fiber tracks. Fiber tracks imaging helped spare neuronal fibres during surgery and was very useful in their observation in next patient MR exams. Based on 3D ASL, CBF value was calculated, which in first exam (before surgery) for a lesion deforming and thinning the corpus callosum CBF was: 86,44; in a second exam (after first surgery) despite bigger mass effect CBF was: 236,6. Conclusion: DTI (diffusion tensor imaging) and 3D ASL in MRI of brain children tumors are very useful during planning surgery and control examinations, help to spare neuronal fibres and plan neurosurgery (DTI) and specify total and residual tumor mass. 3D ASL is a noninvasive technique without a need for an exogenic injection, so it can be repeated as many times as necessary and enables to monitor perfusion in time. Both sequences have a vast usefulness in MRI of pediatric patients. References: [1] G. Jędrzejewski: Pol. J. Radiol. 2006, 71, 52. [2] J. Wang, D.C. Alsop, L. Lin: Magn. Reson. Med. 2002, 48, 242. [3] R. L. Wolf, D. C. Alsop, M. L. McGarvey: J Neuroimaging, 2003; 13, [4] A. Urbanik, K. Sprężak, Przegląd Lekarski 2013/70/5, strony:1-9 [5] R. Brecheisen, B. Platel, A. Vilanova, B. ter Haar Romeny: IEEE Trans.Vis. Comput. Graph 2009, 15 (6),

64 EXOGENOUS SILK PROTEIN AND SLES EFFECT ON PROPERTIES OF HYDRATED HAIR BY 1 H-NMR AND SORPTION ISOTHERM D. Zalitacz, P. Nowak, A. Ciułkowska, K. Pieńkowska, H. Harańczyk, Institute of Physics, Jagiellonian University, Cracow, Hydrolyzed silk protein is a main constituent of silk applied for improvement of hair properties [1], whereas sodium lauryl sulfate (SLES) is applied for hair purification. Hydration changes the properties of biological systems [2], eg. biopolymers [3]. The hydration process of human hair can be effectively approximated by the multilayer sorption of bond water saturating primary and secondary water binding sites [4]. Using hydration kinetics, sorption isotherm, 1 H-NMR spectroscopy we analysed the effect of silk proteins on a human terminal hair of the female individual. The kinetics and the saturation of the hydration process, and also the formation of tightly and loosely bound water fractions at different steps of hydration process were analyzed. We used terminal human hair of 35 years old Caucasian female. Prior to experiments, the sample was purified with a 25% water solution of SLES, and then rinsed with water [5]. Next on the hair surface the hydrolyzed silk protein was imposed. The results were compared with the control sample which was solely purified with SLES solution. The hydration kinetics shows three fractions of bound water, namely (i) a very tightly bound water fraction, (ii) a tightly bound water, and (iii) a loosely bound water. The sorption isotherm is sigmoidal in form reasonably well fitted using Dent model [6]. The relative mass of water saturating primary binding sites is average equal to ΔM/m0=0.050 for hair washed with a SLES and ΔM/m0 = for hair with hydrolyzed silk. The performed for air dry hair at room temperature 1 H-NMR spectra are the superpositions of a Gaussian component ( G 45kHz), coming from s solid matrix of hair, and one Lorentz component, coming from residual water bound in a hair structure. The halfwidth of the NMR liquid component was for SLES washed hair equal to L 2400 Hz, whereas L 2900 Hz) for hair doped with hydrolyzed silk. References [1]G.Secchi, Clinics in Dermatology, 26, (2008) [2]H.Harańczyk, On water in extremely dry biological systems, WUJ, Kraków (2003). [3]H.Harańczyk, J.Kobierski, D.Zalitacz, P.Nowak, A.Romanowicz, M.Marzec, J.Nizioł, Acta Phys. Polon. A121, (2012). [4]Zalitacz, D.; Haranczyk, H.; Nowak, P, Delong, P, 2013 "Mild hydration effect on boundwater dynamics in human hair monitored by H-1-NMR" Journal of investigative dermatology Vol. 133, , (2013) [5] G.Zhang, L.Senak. D. Moore, Journal of Biomedical Optics, 16(5), , 2011 [6] R.Dent, Textile Research J., 47, (1977). 57

65 4D NMR EXPERIMENT FOR PHOSPHORYLATION STUDIES OF IDPS Szymon Żerko* 1, Gerald Platzer 2, Robert Konrat 2, Wiktor Koźmiński 1 1 Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Żwirki I Wigury 101, , Warsaw, Poland 2 Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria The phosphorylation of a specific amino acid residue using NMR is usually detected basing on a chemical shift change between phosphorylated and not phosphorylated state of amino acid 1,2. Unfortunately, because of the severe signal crowding on spectra of disordered proteins of big sizes unambiguous identification of modified residue using 2D experiment such as 2D NHSQC, especially in case of existence of several phosphorylation sites, can be very difficult. Presented approach relay on a direct detection of the phosphorylation state. Thanks to the employment of a phosphorus filtering within the pulse sequence the direct detection of the phosphorylated residues is possible. To provide a good signal dispersion a three dimensional experiment was used. The result is a triple resonance experiment HNCO(P) with a phosphorus filter and a quadruple resonance experiment HNCOP. 3D HNCO(P) allows to an unambiguous identification of the phosphorylated and not phosphorylated amino acid residues. Moreover, 4D HNCOP allows to obtain an additional phosphorus chemical shift for each phosphorylated residue. Presented experiments were tested on a numerously phosphorylated human osteopontin, an intrinsically disordered protein containing 302 amino acid residues. Agilent 600MHz spectrometer equipped with a room temperature Penta probe was used. The study was carried out at the Biological and Chemical Research Centre, University of Warsaw, established within the project co-financed by European Union from the European Regional Development Fund under the Operational Programme Innovative Economy, [1]. Landrieu, L. Lacosse, A. Leroy, J. Wieruszeski, X. Trivelli, A. Sillen, N. Sibille, H. Schwalbe, K. Saxena, T. Langer, G. Lippens. JACS, 128(11): , [2] S. Liokatis, A. Dose, D. Schwarzer, P. Selenko. JACS, 132(42): ,

66 THE ZN IONS AS IMPORTANT FACTOR REGULATED UBIQUITIN- ACTIVATING PROCESS. STRUCTURAL STUDIES OF THE PEPTIDE DERIVED FROM CYSTEINE CATALYTIC HALF-DOMAIN (SCCH) OF MOUSE E1 ENZYME. Ilona Marszalek, 1 Arkadiusz Bonna, 1 Wojciech Bal, 1 and Igor Zhukov 1,2 1 Institute of Biochemistry and Biophysics, Polish Academy of Sciences ul. Pawińskiego 5a, Warsaw, Poland 2 NanoBioMedical Centre, Adam Mickiewicz University ul. Umultowska 85, Poznań, Poland Post-translational ubiquitination signal regulate the mechanism of protein degradation. It regulate a wide variety of cellular activities in eukaryotic organisms. The ubiquitilation process activated by an E1 enzyme by catalytic adenylation of the C-terminal -COOH group in ubiquitin (UBI) followed by linking via thioester bond UBI to the catalytic cysteine in catalytic domain of E1. The ubiquitin-activation signal triggering a cascade consisting of three classes of enzymes activation (E1), conjugation (E2), and ligation (E3). The ubiquitin-activating E1 enzyme presented as a one multidomain protein contained two adenylation (AAD active and IAD inactive) domains, four helix bundle domain (4HB), ubiquitin-like folded domain (UFD), and catalytic domain [1]. The later catalytic domain could be divided on two autonomously folded subunits FCCH (first catalytic cysteine half-domain) and SCCH (second catalytic cysteine halfdomain) [2]. The both sub-domains are located close in space, but sequentially interspersed with adenylation domain [2,3]. There are two other small proteins SUMO and NEDD8 which demonstrated similar activities, and referred as ubiquitin-like proteins (UBL). They are share the similar fold with UBI, but constitutes different set of E1, E2 and E3 enzymes. The structural and dynamic aspects of activation of ubiquitination and sumoilation activity were studied in details by several groups [4,5]. Nevertheless the structural studies were focused on yeasts (S. cerevisiae, S. pombe). From the other hand, the alignment performed for E1 enzymes reveal a specific sequence, which is highly conserved through all mammalian organisms, not observed in invertebrates and in plants (Figure 1). The selected sequence corresponded to 703 WGDCVTWACHHWHTEYC 719 segment comprise three cysteines, three histidines and three tryptophanes. This motif, which is very unusual for proteins, in crystal structure of SCCH it reveal an α-helical fold (pdb 1Z7L). We speculate that selected segment constitute the half of Zn(II)-binding interface, which is facilitated dimerization phenomena of Figu re 1. Sequence alignments of ubiquitin-activating E1 enzymes from different organisms. ubiquitin-activating E1 enzyme. Recently, the Zn(II) binding motif was detected in the SUMO E1 enzyme with unclear function [6]. In fact, only structures of catalytic domain from mouse is available in pdb [2,3], but consequences of Zn(II) binding on SCCH dimerization and protein degradation cascade were not studied before. The NMR experiments were performed on NMR sample obtained by solution of 3 mm nonlabeled peptide in 90%/10% H2O/D2O buffer contained 20 mm TRIS. ph was stabilized at 7.2. The measurements were conducted on Agilent DDR2 800 NMR spectrometer equipped with 59