Dissolved organic matter in water of Daugava river

Daugavpils UniversitāteDabaszinātņu un matemātikas fakultāteDaugavpils2012. gada 12.septembrīKONFERENCEEiropas Sociālā fonda projekts„Starpdisciplinārās zinātniskās grupas izveidošana jaunu fluorescentu materiālu un metožu izstrādei unieviešanai” Vienošanās Nr. 2009/0205/1DP/1.1.1.2.0/09/APIA/VIAA/152Ieguldījums tavā nākotnē!- 1 -

PROGRAMMA9.30 – 10.0010.00 – 10.1510.15 – 10.3010.30 – 10.4510.45 – 11.0011.00 – 11.1511.15 – 11.3011.30 – 11.4511.45 – 12.00Dalībnieku reģistrācija(Vienības iela 13, 214. auditorija)Pasākuma atklāšanaJuris Soms, Dr. geol., Daugavpils Universitātes Dabaszinātņu unmatemātikas fakultātes prodekānsSynthesis and Spectroscopic Study of Benzanthrone 3-N-Derivatives asNew Highly Fluorescent DyesBenzantrona 3-N-atvasinajumu kā jaunu fluorescentu krāsvielu sintēzeun spektroskopiskā izpēteJeļena Kirilova, Dr.chem., projekta zinātniskā vadītājaAminobenzanthrone Derivative ABM - Fluorescent Biomarker ofOrganism Immune StateAminobenzantrona atvasinājums ABM – organisma imūna statusafluorescentais biomārkerisInta Kalniņa, Dr. med., Daugavpils Universitātes Dabaszinātņu unmatemātikas fakultātes vadošā pētnieceAugu šūnu funkcionālā stāvokļa pētīšana, izmantojot jauniegūtusfluoroforusNatālija Škute, Dr.biol., Daugavpils Universitātes Dabaszinātņu unmatemātikas fakultātes vadošā pētnieceLuminescence and Structural Properties of Benzanthrone Dyes ThinFilmsBenzatrona krāsvielu plāno kārtiņu struktūra un luminiscenceIrēna Mihailova, MSc.ing., Daugavpils Universitātes Dabaszinātņu unmatemātikas fakultātes pētnieceČernobiļas katastrofas seku likvidācijas dalībnieku izpēte Latvijāizmantojot fluorescences zondesTija Zvagule, Dr. med., Rīgas Stradiiņa Universitāte, Darba Drošībasun Vides Veselības Institūta vadošā pētnieceFluorescentie benzantrona atvasinājumi: kvantu-ķīmiskie aprēķiniSvetlana Gonta, Dr. chem., Latvijas Universitātes Mikrobioloģijas unbiotehnoloģijas institūta vadošā pētnieceApplication of fluorescent technologies to identify viable pathogens indrinking waterTālis Juhna, Dr.sc.ing., Linda Mežule, Dr sc. ing., Rīgas Tehniskāuniversitāte, Ūdens Inženierijas un tehnoloģijas katedra12.00 – 12.30 Kafijas pauze (Vienības iela 13, 3.stāva foajē)12.30– 14.00 Stendu sesija (Vienības iela 13, 2.stāva foajē)- 3 -

14.00 – 14.1514.15 – 14.3014.30 – 14.4514.45 – 15.0015.00 – 15.1515.15 – 15.3015.30 – 15.4515.45 – 16.0016.00 – 16.1516.15 – 16.3016.30– 16.4516.45 – 17.30Application of fluorescent pigments and dyes in the research of plantstem cells and programmed cell deathMaija Selga, Dr.biol., Daugavpils Universitātes Dabaszinātņu un matemātikasfakultātes pētnieceApplication of new fluorescent dyes ABM, AM1, AM15 and P8 in plantcell biology – Problems and perspectivesAgrita Ozoliņa, MSc. biol., Daugavpils Universitātes Dabaszinātņu unmatemātikas fakultātepH izmaiņu ietekme uz fluorescento krāsvielu emisijas spektriemEvija Pīmane, MSc. biol, Latvijas Universitātes Mikrobioloģijas unbiotehnoloģijas institūtsJaunu organisko materiālu ar fotofizikālām īpašībām strukturāliepētījumiSergejs Beļakovs, Dr. phys., Latvijas Organiskās sintēzes institūtsMelanokortīna 1. receptora gēna polimorfismu funkcionālās ietekmesizvērtēšana, izmantojot konfokālo lāzerskenējošo mikroskopijuAija Ozola, MSc. biol., Latvijas Biomedicīnas pētījumu un studiju centrsJaunu - liposomas veidojošu katjonu karbazola atvasinājumuizmantošana šūnu transfekcijā in vitroAleksandra Vežāne, MSc. biol., Latvijas Biomedicīnas pētījumu un studijucentrsGlikogēna noteikšana raugos S. cerevisiae izmantojot jaunus uzbenzantrona bāzes sintezētos fluoroforusIrina Kralliša, Dr.biol., Latvijas Universitātes Mikrobioloģijas un biotehnoloģijasinstitūtsFluorescentās krāsvielas P8 izmantošana polihidroksibutirāta un polihidroksibutirāta-ko-hidroksivalerātalateksu kompozītplēvjuraksturošanaiLudmila Savenkova, Dr.biol., Latvijas Universitātes Mikrobioloģijas unbiotehnoloģijas institūtsBenzantrona atvasinājumu fizikāli-ķīmisko īpašību un ķīmiskāsstruktūras korelatīvā analīzeValerijs Savenkovs, Dr.biol., Latvijas Universitātes Mikrobioloģijas unbiotehnoloģijas institūtsSpektrofluorometrisko pētījumu metožu pielietojums smago metālu jonunoteikšanai ūdens vidēAleksandrs Pučkins, Msc. env. Daugavpils Universitātes Dabaszinātņu unmatemātikas fakultāteJaunās, uz 3-aminobenzantrona bāzes, sintezētās krāsvielasAnita Ruža, Msc. env. Daugavpils Universitātes Dabaszinātņu unmatemātikas fakultāteDiskusijaPasākuma noslēgumsEiropas Sociālā fonda projekts„Starpdisciplinārās zinātniskās grupas izveidošana jaunu fluorescentu materiālu un metožu izstrādei unieviešanai” Vienošanās Nr. 2009/0205/1DP/1.1.1.2.0/09/APIA/VIAA/152Ieguldījums tavā nākotnē!- 4 -

SATURS/CONTENTNosaukums/TitleAutors/AuthorSynthesis and Spectroscopic Study of Benzanthrone -N-Derivatives as NewHighly Fluorescent DyesE.M.Kirilova, A.I.Puchkin, I.D.Ivanova, A.Ruža, G.K.Kirilov, N.OrlovaSynthesis and Crystal Structure of Novel Amidino Derivatives ofBenzanthroneE.M.Kirilova, I.D.Ivanova, A.I.Puchkin, S.V.BelyakovSynthesis of New Solvatochromic Dyes by Reduction of BenzanthroneAzomethinesI.D.Ivanova, N.V.Orlova, E.M.KirilovaNovel Heterocyclic Imino and Amino Derivatives of Benzanthrone:Synthesis and PropertiesI.D Ivanova, N.V.Orlova, A.I .Puchkin, E.M.KirilovaStructural Investigation of Novel Organic Materials with PhotophysicalPropertiesS.Belyakov, A.Yanychev, M.FleisherThermal Properties of Chromium Complexes with N-benzanthronylamidinesA.I.Puchkin, I.D.Ivanova, E.M.KirilovaSpektrofluorometrisko pētījumu metožu pielietojums smago metālu jonunoteikšanai ūdens vidēA.Pučkins, E.KirilovaAmidines of Benzanthrone as Fluorescent Sensor for Metal IonsA.I.Puchkin, I.D.Ivanova, E.M.KirilovaJaunās, uz 3-aminobenzantrona bāzes, sintezētās krāsvielasA.Ruža, A.Volkova, E.M.KirilovaThin Films Fabricated from Benzanthrone Luminescent DyesG.K.Kirilov, A.S.Bulanov, M.Fleisher, E.M.Kirilova, I.Mihailova, S.GontaLuminescence and Structural Properties of Benzanthrone Dyes Thin FilmsI.Mihailova, G.Kirilov, A.Bulanovs, E.KirilovaApplication of Fluorescent Pigments and Dyes in the Research of Plant StemCells and Programmed Cell DeathM.Selga, T.Selga, A.OzolinaThe Influence Of High Temperature On Oxidative Processes and AcceleratedSenescence in Wheat Seedling (Triticum aestivum L.) ColeoptileM.Savicka, N.ŠkuteApplication of New Fluorescent Dyes ABM, AM, AM5 and P8 in Plant CellBiology. Problems and PerspectivesA.Ozolina, T.Selga, M.SelgaMycobacterium tuberculosis antibiotiku rezistenci izraisošo mutācijunoteikšana pēc DNS denaturācijas laikā novērotajām fluorescences līmeņaizmaiņāmA.Kalviša, I. Jansone, V. BaumanisLpp.Page8910111213141516171819202122- 5 -

Melanokortīna . receptora gēna polimorfismu funkcionālās lomasizvērtēšana, izmantojot konfokālo lāzerskenējošo mikroskopijuA.Ozola, R.Petrovska, I.Mandrika, O.Heisele, D.PjanovaResonance Energy Transfer Study of Lipid Bilayer Interactions of ApolipoproteinA-I VariantsG.Gorbenko, V.Trusova, E.Adachi, C.Mizuguchi, H.SaitoStyryl Derivatives as a New Probes For Yeast Cell Vitality DeterminationI.P.Goriacha, T.S.Dyubko, V.D.Zinchenko, I.V.Govor , L.D.Patsenke,T.Deligeorgiev, S.Kaloyanova, N.LesevMonitoring of Free Radical Processes in Protein-lipid Systems with a NewSquaraine DyeO.Kutsenko, V.Trusova, G.Gorbenko, T.Deligeorgiev, E.Slobozhanina,L.Lukyanenko, G.ZubritskayaStudying Zinc Biology with Fluorescent ProbesE.I.Slobozhanina,Y.M.Harmaza, N.M. Kozlova, A.V.TamashevskiApplication of Fluorescent Technologies to Identify Viable Pathogens inDrinking WaterM.Linda, T.JuhnaA New Aminobenzanthrone Dye for Identification of Amyloid FibrilsI. Maliyov , M.Romanova, K.Vus, A.Kastorna, G.Gorbenko, V.Trusova, E.Kirilova,G.Kirilov, I.KalninaInteraction of Fibrillar Insulin and Globin with Liposomes: Resonance EnergyTransfer StudyM.Romanova, I.Maliyov, A.Kastorna, K.Vus, V.Trusova, J.Molotkovsky,G.GorbenkoFluorescence Approaches to Detection of Protein AggregatesV.M.TrusovaFluorescence Study of Cytochrome c Binding to Model MembranesV.Trusova, G.Gorbenko, J.Molotkovsky, P.KinnunenAminobenzanthrone Dyes as Prospective Fluorophores for Detection andCharacterization of Amyloid FibrilsK.Vus, V.Trusova, G.Gorbenko, E.Kirilova, G.Kirilov, I.KalninaBinding of the Novel -tricyanovinylarylamine Dye to Fibrillar LysozymeK.Vus, V.Trusova, G.Gorbenko, T.Deligeorgiev, S.Kaloyanova, N.LesevAssociation of Novel Benzanthrone Probes with Model Lipid MembranesO.A.Zhitniakivska, V.M.Trusova, G.P.Gorbenko, E.M.Kirilova, G.K.Kirilov, I.KalninaNew ICT-dyes for Membrane StudiesO.Zhytniakivska, V.Trusova, G.Gorbenko, T.Deligeorgiev, S.Kaloyanova, N.LesevAlterations of Blood Plasma Albumin in Chernobyl Clean-up Workers withand without Type Diabetes MellitusT.Zvagule, N.Kurjane, A.Šķesters, A.Silova, N.Gabruseva, G.Gorbenko, G. Kirilov,I.Kalnina, E.Kirilova, S.GontaAlterations of Blood Plasma Albumin in Chernobyl Clean-up Workers withand without Concomitant DiseasesT.Zvagule, I.Kalnina, E.Kirilova, N.Kurjane, N.Gabruseva, J.Reste, G.Kirilov,A.Šķesters, A.Silova23242526272829303132333435363738- 6 -

Aminobenzanthrone Derivative ABM - Fluorescent Biomarker of ImmuneState of PatientsE.Kirilova, G.Gorbenko, I.Kalnina, T.Zvagule, G.Kirilov, N.KurjaneFunctional State of Beta Adrenoreactive System in Man and Animals:Fluorescent StudiesE. Kirilova, I. Kalnina,T. Zvagule, N. Kurjane, G. Kirilovs, N. GabrusevaFluorescent Studies of Human Blood Plasma Albumin Alterations andLymphocytes Subpopulations in Colorectal Cancer PatientsI. Kalnina, T. Zvagule, L. Klimkane, N. Kurjane, E. Kirilova, G. GorbenkoOksidatīvā stresa marķieru izmaiņas Černobiļas AES avārijas sekulikvidētājiem pēdējo 10 gadu laikāA. Šķesters, A. Silova , T. Zvagule, J. Reste, L. Lārmane, Ņ. Rusakova,N.GabruševaJaunu - liposomas veidojošu katjonu karbazola atvasinājumu izmantošanašūnu transfekcijā in vitroA. Vežāne, G. Apsīte, A. Baran, R. Brūvere, V. Ose, A. Plotniece, I. Timofējeva,T.KozlovskaGlikogēna noteikšana raugos S. cerevisiae izmantojot jaunus uz benzantronabāzes sintezētos fluoroforusI.KrallišaAlkilrezorcinolu izdalīšana un noteikšana baktērijās azotobacterchroococcumR.LindepH izmaiņu ietekme uz fluorescento krāsvielu emisijas spektriemE.PīmaneFluorescentās krāsvielas P8 izmantošana polihidroksibutirāta un polihidroksibutirāta-ko-hidroksivalerātalateksu kompozītplēvju raksturošanaiL. SavenkovaDzelzs jonu ietekme uz izmaiņām šūnā, alkilrezorcīnu un PHB veidošanosAzotobacter chroococcum šūnāsE.PīmaneFluorescentie benzantrona atvasinājumi: kvantu-ķīmiskie aprēķiniS. GontaBenzantrona atvasinājumu fizikāli-ķīmisko īpašību un ķīmiskās struktūraskorelatīvā analīzeV. Savenkovs, I.GotlibovičsAugu šūnu funkcionālā stāvokļa pētīšana, izmantojot jauniegūtus fluoroforusN. Škute39404142434445454646474849- 7 -

Synthesis and Spectroscopic Study of Benzanthrone 3-N-Derivatives asNew Highly Fluorescent DyesE.M.Kirilova 1 , A.I.Puchkin 1 , I.D.Ivanova 2 , A.Ruža 1 , G.K.Kirilov, N.Orlova 21 Chemistry Department, Daugavpils University, 13 Vienibas str., LV-5401, Daugavpils(Latvia);2 Faculty of Chemistry, University of Latvia, 48 Valdemara str., LV-1013, Riga (Latvia).e-mail: elena.kirilova@inbox.lvToday many techniques use fluorescent dyes for the labelling of biological objects. Newpractical uses call for the synthesis of new fluorescent probes with improved properties. Inpresent work, we have synthesized a number of 3-aminobenzanthrone N-derivatives –substituted amines, imines and amidines, obtaining highly fluorescent compounds [1].Synthesized dyes are the analogues of cell membrane hydrophobic probe – 3-methoxybenzanthrone,but new derivatives are long-wavelength light-emitting fluorescent dyes.YNR 1R 2YXOIIYNRNR 1R 2OIOIIIYNROIVThe spectral behaviour of the obtained dyes was investigated. We have studied thespectral properties of prepared derivatives – absorption and fluorescence spectra invarious solvents. For obtained dyes large Stokes shift values (about 100 nm) are observed,excitation maxima of the dyes are located near 500 nm and emission maxima near 650nm. In addition these compounds are showed strong fluorescent solvatochromism.It was found that many of synthesized compounds are quite sensitive to the surroundingenvironments and are potential fluorescent probes for screening structural and functionalalterations of cell membranes.- 8 -

Synthesis and Crystal Structure of Novel Amidino Derivatives ofBenzanthroneE.M.Kirilova 1 , I.D.Ivanova 2 , A.I.Puchkin 1 , S.V.Belyakov 31 Chemistry Department, Daugavpils University, 13 Vienibas str., LV-5401, Daugavpils(Latvia);2 Faculty of Chemistry, University of Latvia, 48 Valdemara str., LV-1013, Riga (Latvia);3 Latvian Institute of Organic Synthesis, Riga, Latvia.e-mail: elena.kirilova@inbox.lvToday many techniques use fluorescent dyes for the labeling of biological objects [1].Practical applications uses call for the synthesis of new fluorescent compounds withimproved properties for a specific requirement. In this connection the intensiveinvestigations for preparation of new fluorescent probes now have developed.In our previous investigations were synthesized a number of benzanthrone N-containingderivatives, obtaining highly fluorescent compounds [2, 3]. Synthesized dyes are sensitivelong-wavelength light-emitting fluorescent dyes, which have high photostability and lowcytotoxicity. It has been shown that some of obtained compounds are potential fluorescentprobes for screening structural and functional alterations of cell membranes and forestimation of the immune state [2].The aim of this work was the modification of some of benzanthrone derivatives (I, III) byincluding new substituents in chromophoric system in order to find potential fluorescentprobes with large luminescence intensity and high stability. Here we present the synthesisof several new benzanthrone derivatives (II, IV) with amidine group:NH 2NRNR 1R 2OIOIIBrBrN H 2OIIIRR 2R 1NNOIVSynthesized derivatives have bright from yellow to red fluorescence in organic solventsand solid state. The structure of obtained compounds was confirmed by NMR and IRspectroscopy. In addition thermal analysis and crystal structures of studied compoundshave been investigated.1. Kirilova, E. M; Kalnina, I; Kirilov, G. K; Meirovics, I. J. Fluoresc 2008, 18 (3-4), 645-648.2. Kirilova, E. M; Belyakov, S. V; Kalnina, I. Topics in Chemistry & Materials Science, Sofia:Heron Press (ed: G. Vayssilov, R. Nikolova) 2009, 3, 19-28.3. Gorbenko, G.; Trusova, V.; Kirilova, E.; Kirilov, G.; Kalnina, I.; Vasilev, A.; Kaloyanova, S.;Deligeorgiev, T. Chem. Phys. Lett, 2010, 495, 275–279.- 9 -

Synthesis of New Solvatochromic Dyes by Reduction of BenzanthroneAzomethinesI.D.Ivanova 1 , N.V.Orlova 1 , E.M.Kirilova 21 Faculty of Chemistry, University of Latvia, 48 Valdemara str., LV-1013, Riga (Latvia);2 Chemistry Department, Daugavpils University, 13 Vienibas str., LV-5401, Daugavpils(Latvia).Many derivatives of benzo[de]anthracene-7-one exhibit strong emission, which accountsfor their use in practice as active lasing media and fluorescent probes for investigation ofbiological objects. In present work a number of new derivatives of 3-aminobenzanthronewere synthesized. In summary, ten new dyes were synthesized in good yields (80-87%)via the reduction of corresponding azomethine derivative by sodium borohydride in DMFsolutions.The structure of obtained compounds was confirmed by NMR and FT-IR spectroscopyand mass spectrometry. Single-crystal structures of obtained dyes were determined by X-ray diffraction studies. In addition, thermal stability of the synthesized chromophores hasbeen undertaken using TG–DTA. The influence of solvents with various polarities uponabsorption and emission spectra was investigated. The absorption and luminescent spectraof the novel compounds in several solvents of different polarity were investigated. Thesynthesized dyes absorb at 520-580 nm with high extinction coefficients, have relativelylarge Stokes’ shifts (about 100 nm), and emit at 650-720 nm showing both absorption andfluorescence solvatochromism similarly to studied earlier 3-amino derivatives ofbenzanthrone [1,2]. The results indicated that these dyes were strongly dependent onsolvents and show generally bathochromic shifts as the polarity of solvents was increased.These characteristics of obtained dyes demonstrate their potential as biomedical probesfor proteins, lipids and cells.1. Kirilova, E. M; Kalnina, I; Kirilov, G. K; Meirovics, I. J. Fluoresc 2008, 18 (3-4), 645-648.2. Gorbenko, G.; Trusova, V.; Kirilova, E.; Kirilov, G.; Kalnina, I.; Vasilev, A.; Kaloyanova, S.;Deligeorgiev, T. Chem. Phys. Lett, 2010, 495, 275–279.- 10 -

Novel Heterocyclic Imino and Amino Derivatives of Benzanthrone:Synthesis and PropertiesI.D Ivanova 1 , N.V.Orlova 1 , A.I .Puchkin 2 , E.M.Kirilova 21 Faculty of Chemistry, University of Latvia, 48 Valdemara str., LV-1013, Riga (Latvia);2 Chemistry Department, Daugavpils University, 13 Vienibas str., LV-5401, Daugavpils(Latvia).e-mail: elena.kirilova@inbox.lvThe design of new fluorescent molecules is of continuing interest for many applications inresearch and industry. Especially donor–acceptor -conjugated organic materials haveattracted considerable interests owing to their potential wide applications for developmentof photoactive materials. Many derivatives of benzo[de]anthracene-7-one are known aseffective luminescent dyes with emission in the spectral region from green to red-purple,depending on the structure [1].Recently we reported the synthesis, molecular structures and spectral properties of a seriesof amino, amidino, and iminobenzanthrones, which appeared to be particularly interestingbecause they lead to perspective luminescent materials [1]. The aim of this work is tocreate a series of benzanthrone amines and azomethines by including new heterocyclicsubstituents in benzanthrone chromophoric system in order to find novel luminophoreswith large luminescence intensity and high stability.Target benzanthrone derivatives are synthesized by condensation reaction ofcorresponding aminobenzanthrone with appropriate heterocyclic aldehydes with followingreduction of obtained imines by NaBH 4 in DMF solution:R:NH 2ROHNRNaBH 4, DMFHNRNNNNOOO1 2a-g 3a-gSynthesized derivatives have bright from yellow to red fluorescence in organic solventsand solid state. The structure of obtained compounds was confirmed by NMR, IR andmass spectroscopy. In addition thermal properties and crystal structures of preparedcompounds have been investigated.1. B.M. Krasovitskii, B.M. Bolotin, Organic luminescent materials. - VCH Publishers, 19882. E. M Kirilova, I. Kalnina, G. K. Kirilov, S. V Belyakov, I. Meirovics. Spectroscopic Study ofBenzanthrone 3-N-Derivatives as New Hydrophobic Fluorescent Probes for Biomolecules//J.Fluoresc. – 2008 - Vol. 18 (3-4) – P.645-6483. V.M Trusova, E. Kirilova, I. Kalnina, G. Kirilov, O.A. Zhytniakivska, P.V. Fedorov, G.Gorbenko, Novel Benzanthrone Aminoderivatives for Membrane Studies.// J.Fluoresc.- 2012 -Vol. 22 (3) – P.953-959OSN- 11 -

Structural Investigation of Novel Organic Materials with PhotophysicalPropertiesS.Belyakov* 1 , A.Yanychev 2 , M.Fleisher 11 Latvian Institute of Organic Synthesis, Riga, Latvia;2 Riga Technical University, Riga, Latvia.e-mail: serg@osi.lvIn recent years a series of organic compounds with photophysical properties has beenstudied by means of X-ray diffraction and theoretical calculations. The molecular andcrystal structure of these compounds are intensively investigated, because there are muchunexpected results. In this presentation the results of the following investigations will bereported: molecular and crystal structures of 2-(4’-Dimethylamino)-benzylydene-1,3-indandione (DMABI) analogues, structure of 8-mercaptoquinoline and 8-selenoquinolinecomplexes containing metal atoms, structure of benzanthrone derivatives. Part of theseinvestigations has been published earlier as original articles [1-6]; the other part will bepresented for the first time.1. B Silina, E., Belyakov, S., Ashaks, J., Pecha, L., Zaruma, D. Tris(2-methylquinoline-8-selenolatok1 N,Se)antimony (III). Acta Crystallogr. 2007, vol.63.,Sec.C. p. m62-m64.2. Belyakov, S., Kampars, V., Pastors, P.J., Tokmakov, A. 2-(4,5,6,7,8,9-Hexahydro-6aazaphenylen-2-ylmethylene)indian-1,3-dione.Acta Crystallogr. 2008, vol.64.,Sec.E. p. o1200.3. Kirilova, E.M., Belyakov, S.V., Kirilov, G.K., Kalnina, I., Gerbreder, V. Luminescent propertiesand crystal structure of novel benzanthrone dyes. J. Luminescence 2009, vol.129, p. 1827-1830.4. Silina, E., Belyakov, S., Ashaks, J., Pecha, L., Zaruma, D. Crystal structure of catena-poly[bis (8-mercaptoquinolinato-N,S)disilver(I)], Ag 2 (C 9 H 6 NS) 2 . Zeitschrift für Kristallographie 2010,no.225. p. 211-212.- 12 -

Thermal Properties of Chromium Complexes with N-benzanthronylamidinesA.I.Puchkin 1 , I.D.Ivanova 2 , E.M.Kirilova 11 Chemistry Department, Daugavpils University, 13 Vienibas str., LV-5401, Daugavpils(Latvia);2 Faculty of Chemistry, University of Latvia, 48 Valdemara str., LV-1013, Riga (Latvia).e-mail: aleksandrs.puckins@du.lvThe chemistry of functional materials of metal complexes and their applications have beenof great interest in recent years. Compounds containing amidine group have playedimportant roles as ligands for various complexes of metals in organometallic chemistry.These systems have interesting chemical and spectral properties for practical applications.For example, the use of amidinate-ligated iridium complexes for fabrication of highefficiency phosphorescent organic light-emitting devices has been recently demonstrated[1]. Therefore it was of interest to prepare new complexes of transition metal ion andligand containing electron-donating amidine group.The object of the present research was to investigate the thermal behaviour of chromium(III) complexes incorporating earlier synthesized [2] N-benzanthronylamidine ligands andto characterize it by means of elemental analysis, infrared spectroscopy, and differentialthermal analysis (TG-DTA). It was investigated coordination of fifteen amidines withCr(NO 3 ) 3 , obtaining the formation of seven complex. Elemental analysis data with thehexacoordination tendency of Cr3+ complexes, let us to suggest an octahedral geometryfor obtained complexes with three molecules coordinated to the metal centre. The TG-DTA curves provided previously unreported information about the thermal stability andthermal decomposition of these compounds.1. Y. Liu, K. Ye, Y. Fan et al. Chem. Commun. 25 (2009) 3699-3701.2. E.M Kirilova, S. V Belyakov, I. Kalnina. in: G. Vayssilov, R. Nikolova (Eds), Topics inChemistry & Materials Science, Sofia: Heron Press, 3, 2009 pp.19-28.- 13 -

Fluorescence intensity, a. u.Spektrofluorometrisko pētījumu metožu pielietojums smago metālu jonunoteikšanai ūdens vidēA.Pučkins 1 , E.Kirilova 11 Daugavpils Universitāte, Daugavpils, Latvijae-mail: aleksandrs.puckins@du.lvVides piesārņošana ar smagajiem metāliem mūsdienās ir ļoti aktuāla problēma. Tāpēcpašlaik ir pievērsta liela uzmanība, lai izstrādātu jaunas jutīgas šo vielu noteikšanasmetodes.Ņemot vērā zināmo faktu, ka metālu joni var ietekmēt fluorescentā savienojuma,ar kuru saistās, fluorescences parametrus, tika veikts pētījums, kura laikā tika noskaidrotaspektrofluorometrisko metožu pielietojuma iespējamība smago metālu detektēšanai ūdensvidē.III600400200NiCoCu0400 500 600 700 800Wavelength, nmPētījumu laikā tika uzņemti vairāki krāsvielu fluorescences intensitātes spektri, pēckuriem spriežot tika secināts, ka dažādu smago metālu klātbūtne var izraisīt dažādusefektus: gan organisko fluoroforu fluorescences pavājināšanu, gan pastiprināšanu.Pētījuma gaitā tika noskaidrots, ka ar spektroflorometriskas metodes palīdzību var veiktgan kvalitatīvo, gan kvantitatīvo dažu smago metālu noteikšanu ūdens vidē.Spektrofluorometrisko pētījumu metožu grupai ir raksturīga vesela virkne priekšrocību,kas nepiemīt pārējām analīzes metodēm un lietderīgi atšķir to no citām metodēm: zemasizmantošanas izmaksas, eksperimentu vienkāršums, metodes straujums un jūtīgums.- 14 -

Amidines of Benzanthrone as Fluorescent Sensor for Metal IonsA.I.Puchkin 1 , I.D.Ivanova 2 , E.M.Kirilova 11 Chemistry Department, Daugavpils University, 13 Vienibas str., LV-5401, Daugavpils(Latvia);2 Faculty of Chemistry, University of Latvia, 48 Valdemara str., LV-1013, Riga (Latvia).e-mail: aleksandrs.puckins@du.lvPollution of the environment by toxic metals is a major problem in the last decays due totheir increasing utilization in industry and agriculture.. These pollutants are discharged ortransported into the atmosphere and aquatic and terrestrial environments and have thepotential to reach high concentrations. Large concentrations of some metal ions have beenshown to cause brain damage, and are potentially lethal. Due to potential health risksassociated with exposure to metals, legal constraints on the allowed abundance of metalsare becoming increasingly strict.As a result of the health threats posed by some metals, new methodologies that allow fortheir simple detection and quantitation are of interest. Today many fluorescent organicsensors were designed for recognition and detection of heavy and transition metal ions.These chemosensors are compounds that incorporate a binding site, a fluorophore, and ameans of communication between the two. The interaction of a metal ion with an organicligand may result in fluorescent enhancement or fluorescence quenching.Our recent efforts have been focused largely on synthesizing novel organic fluorophoressuch as N-containing benzanthrone derivatives [1,2]. The present work focuses on thespectral characterization of benzanthronylamidines and its complexes with transitionmetal ions. Obtained results testify that investigated dyes can be used as sensitivefluorescent chemosensors for transition metals: the one of them displays positive response(3-7-fold fluorescence increasing) to the presence of Ni 2+ or Co 2+ ions, but somesynthesized amidines show negative response (quenching) in fluorescence intensity to Crions. Further investigations can lead to development of new method for the detection ofsome cations.1. E.M Kirilova, S. V Belyakov, I. Kalnina. in: G. Vayssilov, R. Nikolova (Eds), Topics inChemistry & Materials Science, Sofia: Heron Press, 3, (2009) 19-28.2. V.M. Trusova, E. Kirilova, I. Kalnina, G. Kirilov, O.A. Zhytniakivska, P.V.Fedorov, G.Gorbenko. J.Fluoresc. 22 (2012) 953-959.- 15 -

Jaunās, uz 3-aminobenzantrona bāzes, sintezētās krāsvielasA.Ruža 1 , A. Volkova 1 , E.M.Kirilova 1 ,1 Ķīmijas fakultāte, Daugavpils Universitāte, Vienības iela 13., LV-5401, Daugavpils (Latvija)e-mail: elena.kirilova@inbox.lv; anita.ruza@gmail.comBenzantrons un tā atvasinājumi tiek plaši izmantoti dažādās zinātnes sfērās. Tā krāsvielasir labi zināmi luminofori, kam piemīt izteikta krāsa un fluorescences intensitāte, tās irstabilas un gaismas ietekmē nedegradējas. Ar benzantrona krāsvielām patreiz var iekrāsotgan ikdienā izmantojamas lietas (piem., audumus), gan dažādus polimērus un pat izmatottās LCD dienas gaismas spuldzītes un lāzeros.Krāsvielu sintēzei nepieciešamā 3-NH 2 -BA iegūšana notiek divos posmos: benzantronanitrēšana ar turpmāku reducēšanu Na 2 S klātbūtnē. Amidīnu sintēzes process ir sekojošs:3-NH 2 -BA kopā ar attiecīgo amīdu vāra kopā ar POCl 3 katalizatoru ~ 90 – 100 o C. Vēlākreakcijas maisījumu neitralizē ar piesātinātu amonjaka šķīdumu un izkritušās nogulsnesnofiltrē. Parasti pēc to izžūšanas ir nepieciešama nogulšņu pārkristalizācija, lai atbrīvotosno neorganikas piemaisījumiem un varētu izaudzēt vielu kristālus. Papildus tiek iegūti arīamidīni ar broma atomu, kas piesaistīts pie 2. vai 3. benzantrona oglekļa atoma. Bromaklātbūtnē mainās krāsvielu vizuālais izskats, luminescences krāsa un spektrālās īpašības.Spektrāliem pētījumiem tiek sagatavoti amidīnu šķīdumi dažādos organiskajosšķīdinātājos (DMF, CHCl 3 , u.c.) koncentrācijā 10 -4 M. Daļai no iegūtajām krāsvielām tiekpārbaudīta to spēja veidot kompleksos savienojumus ar smagajiem metāliem.Pašlaik jau ir sintezētas 36 krāsvielas un ir plānots iegūt vēl vismaz sešas. No esošajāmkrāsvielām, visizteiktākā luminescence sausā veidā ir savienojumiem AM16, AM20 unAM22.- 16 -

Thin Films Fabricated from Benzanthrone Luminescent DyesG.K.Kirilov 1 , A.S.Bulanov 1 , M.Fleisher 2 , E.M.Kirilova 1 , I.Mihailova 1 , S.Gonta 21 Daugavpils University, Vienibas 13, LV-5401, Daugavpils, Latvia2 Institute of Microbiology and Biotechnology, University of Latvia, Kronvalda blvd. 4, RigaLV-1586, Latviae-mail: georgk@inbox.lvOrganic thin films are used in a number of scientific and technical applications one ofwhich is producing cheap and large scale electronic and optical devices [1,2]. Thin filmssamples of novel organic luminophores - 3-N-(N’-methylacetamidino)benzanthrone and3-N-(N`,N`-dimethylbenzamidino)benzanthrone deposited on glass substrate wereprepared by thermal evaporation obtaining thin films of 2.5 to 3 µm thickness.The structural and spectroscopic properties of obtained films were investigated byconfocal microscope with input of femtosecond laser radiation, X-ray diffractometer andscanning electron microscope. In addition, quantum chemical calculations wereperformed, indicating electron properties of studied dye molecule in ground and excitedstate. Heats of formation, dipole moments and other parameters were extracted directlyfrom the data files following the geometry optimizations. The calculations show a planarconfiguration for the aromatic core of these compounds and two possible orientations ofamidine substituents.E= -4468.70 (kcal/mol)E= -4455.22 (kcal/mol)1. J. R. Sheats, H. Antoniadis, M. Hueschen, W. Leonard, J. Miller, R. Moon, D. Roitman, A.Stocking, Organic Electroluminescent Devices, Science, 273 (1996) 884-888.2. B.M. Krasovitskii, B.M. Bolotin, Organic luminescent materials. VCH Publishers, 1988.- 17 -

Luminescence and Structural Properties of Benzanthrone Dyes Thin FilmsI.Mihailova 1 , G.Kirilov 1 , A.Bulanovs 1 , E.Kirilova 21 Innovative Microscopy Center, Daugavpils University, Daugavpils, Latvia.2 Department of Chemistry, Daugavpils University, Daugavpils, Latvia.e-mail: irena.mihailova@du.lvWe report luminescence and structural properties of 3-N, N-diacetylaminobenzanthrone thin films deposited on glass substrate by thermalevaporation. The optical and structural properties of organic thin films were studied bymeans of the confocal microscope with an input of femtosecond laser radiation, X-raydiffractometer, and scanning electron microscope (SEM).Intense luminescence with the maximum at 530 nm was observed when excited bylaser radiation with the wavelengths 458-514 nm. In addition, the luminescence causedby two-photon absorption of infrared femtosecond (fs) laser radiation has beeninvestigated. The study of the structure of benzanthrone derivative thin films, createdby X-ray diffraction (XRD) methods, indicate the distance between molecular layersand ordered molecular fragments.It was found that prepared films of 3-N,N-diacetylaminobenzanthrone are highlyordered materials with molecular layers; X-ray diffraction analysis indicate that thedistance between these layers is ~6.5 Å. Similar ordering this compound shows inmonocrystal packing.- 18 -

Application of Fluorescent Pigments and Dyes in the Research of PlantStem Cells and Programmed Cell DeathM.Selga* 1 , T.Selga 2 , A.Ozolina 11 Daugavpils University, Daugavpils, Latvia;2 Faculty of Biology, University of Latvia, Riga, LatviaE-mail: turs.selga@lu.lvDifferent types of stem cells have been stated both in plants and animals [1]. The researchof stem cells particular abilities, regulation and fate becomes increasingly important dueto the fast development of regenerative medicine and new methods of plant propagation.However little is known about the flowering plant stem cell diversity, pools,developmental strategies and stemness of differentiated cells and regulation ofprogrammed cell death [2]. Cells of tobacco (Nicotiana tabacum L.), field beans (Viciafaba L.) and other plants cultivated in optimal conditions were researched by the light andconfocal laser-scanning microscopy. We have stated that: The asymmetric strategy of stem cell division and fate of daughter cells; Switching the structural mechanisms of mitosis that correlates with the reflectiveprocesses of cell differentiation and dedifferentiation; The lasting association of subordinated unequal daughter nuclei and cells viaplasmodesmata due to arrested asymmetric mitosis; Both mitosis and asymmetric programmed cell death is double regulated – byintrinsic and extrinsic signalling via evident structural pathways;Plastid-nucleus complex encircles mitosis in photosynthesising cells and existsduring programmed cell death [3].Peculiarities of staining with fluorescent dyes in different stages of cell development willbe discussed.1. Lin, H. Cell Biology of Stem Cells: an Enigma of Asymmetry and Self-renewal. J Cell Biol, 2008,vol.180, p 257-260.2. Krishnamurthy K.V., Krishnaraj R., Chozhavendan R. et al The programme of cell death in plantsand animals – A comparison. Current Sci. 2000. vol. 79, p. 1169-1181.3. Selga T., Selga M., Gobins V., et al. Plastid-nuclear complexes: permanent structures inphotosynthesizing tissues of vascular plants. Env and Exp Biol, 2010, vol. 8, p. 85–92.- 19 -

The Influence Of High Temperature On Oxidative Processes andAccelerated Senescence in Wheat Seedling (Triticum aestivum L.) ColeoptileM.Savicka* 1 , N.Škute 11 Institute of Ecology, Daugavpils University, Daugavpils, Latviae-mail: marina.savicka@du.lvMost environmental stresses are affecting on the production of active oxygen species inplants, causing oxidative stress. Temperature, along with light, humidity, etc., belongs tothe important factors in the environment of plants. It is also a tool for research, for inmany cases the processes of growth can be differentiated by their temperature responses.Heat shock is a form of the oxidative stress, resulting in the formation of many toxic ROSin plants (Tsang et al. 1991; Bhattacharjee, Mukherjee, 2003). High temperature inducedoxidative stress led to accelerated senescence of plants. Therefore, the objective of thiswork was to study the response of coleoptile of wheat seedlings to elevated temperatures.The present study investigated the effect of heat stress on different stages of growth inwheat (Triticum aestivum L.). The behaviour of wheat seedling coleoptile was studied,particularly at the physiological level. High temperature is known to induce oxidativeinjury in plants by inducing production of active oxygen species. Active oxygen species,cause lipid peroxidation (LP) and consequently membrane injury. High-temperature (15,30 and 60 min and 24h at 42°C) influence on senescent organ of wheat seedlings(Triticum aestivum L.) was analyzed taking into consideration the changes in somechanges on the molecular level, such as content of LP products (malodialdehyde (MDA)and conjugated dienes (CD)) and membrane permeability. The effect of high temperaturewas analyzed at the early stages (4 day-old) and at the late stages (7 day-old) of seedlingdevelopment. LP level was directly correlated with temperature, exposure time, and theirinteraction. Long-term high-temperature exposure (24h at 42°C) significantly increasedLP (MDA and CD content) in coleoptile of wheat seedlings at 4 and 7d of seedlingdevelopment. However, the increase in LP products was observed only at the late stagesof seedling development after short-term high temperature exposure. Heat shock causedan increase in the electric conductivity of cell membrane, which led to the loss ofmembrane selective permeability.1. Tsang, E.W.T., Bowler, C., Hérouart, D., Van Camp, W., Villarroel, R., Genetello, C. Differentialregulation of superoxide dismutases in plants exposed to environmental stress. Plant Cell. 1991, 3:783–792.2. Bhattacharjee, S., Mukherjee, A.K. Implications of reactive oxygen species in heat shock inducedgermination and early growth impairment in Amaranthus lividus L. Biologia Plantarum. 2003, 47(4): 517-522.- 20 -

Application of New Fluorescent Dyes ABM, AM1, AM15 and P8 in PlantCell Biology. Problems and Perspectives.A.Ozolina 1 , T.Selga 2 , M.Selga 11 Daugavpils University, Daugavpils, Latvia;2 Faculty of Biology, University of Latvia, Riga, Latviae-mail: turs.selga@lu.lvTo find out possible application of new stains ABM, AM1, Am15 and P8 in plant cellbiology we used different fixation and staining techniques in different combinations.Reliability of results were analysed with bright field and confocal laser-scanningmicroscopy.For bright field microscopy bulbs of onion Allium cepa L. were dissected and epidermisfrom different layers of leaf scales was removed. Epidermal peels from the middle oftobacco, pelargonium leaves and the outer, middle and inner layer of leaf scales of onionbulbs were stripped and transferred to droplets of aceto-ethanol on microscopic slides,washed and contrasted with aceto-orcein or methylene blue. Mesophyll was fixed with 2%glutaraldehyde in sodium cacodylate buffer or in aceto-ethanol, washed with water, frozenin a droplet of water, cut by razor under 50x magnification; and cuttings were contrastedon microscopic slides with aceto-orcein. Leaf pieces of mesophyll were excised in themiddle of the leaf blade among large veins, soaked in aceto-ethanol for, rinsed in dist.water, maturated in 1M HCl at 60 0 C, rinsed in dist. water, contrasted with aceto-orcein ormethylene blue, placed on microscopic slides, covered with cover slips, and gentlycrushed by pressure. Largely or partly separated palisade parenchyma cells due todestruction of cellulose were examined.Samples were examined under bright field illumination under a light microscope LEICADM 2000. Photographs were taken with a digital camera Leica EC3 and images processedwith LAS EZ and Paint Shop Pro 4.For confocal laser-scanning microscopy to observe plant tissue in vivo samples weremounted in tap water on a glass depression slide and placed under a cover slip. To limitwater evaporation and cover slip drift during long-term observations, cover slip wasattached to glass slide with an adhesive tape.Smples were fixed with 2% glutaraldehyde in sodium cacodylate buffer or in acetoethanoland washed with water. Samples were stained with propidium iodine, bodipy,mitotracker green, ABM, P8, AM1 or AM15 in different combinations.Laser scanning confocal microscopy was conducted with a Leica DM RA-2 microscopeequipped with a TCS-SL confocal scanning head (Leica Microsystems, Bannockburn,Ill.). Images were collected with a Leica 40x HCX PL FLUOTAR objective (NA=0.75)and 100× HCX PlAPO oil immersion objective (NA=1.40). Pictures were recorded bytaking z-stacks that captured multiple organelles. At least three such z-stacks were takenand analyzed for each sample. We used 0.3 μm thick optical sections and collected from 1to 10 μm thick samples.AM1,AM 15, P8 and ABM were excited with a 488-nm band of a four-line argon ionlaser. Fluorescence was detected between 530 nm and 580 nm. GFP was excited with a488-nm band of a four-line argon ion laser. Chlorophyll fluorescence was excited at633 nm. GFP fluorescence was detected between 500 nm and 560 nm. Chlorophyllfluorescence was detected between 680 nm and 700 nm.- 21 -

Mycobacterium tuberculosis antibiotiku rezistenci izraisošo mutācijunoteikšana pēc DNS denaturācijas laikā novērotajām fluorescences līmeņaizmaiņāmA.Kalviša 1 , I. Jansone 1 , V. Baumanis 11 Latvijas Biomedicīnas pētījumu un studiju centrs, Rīga, Latvijae-mail: adrija.kalvisa@gmail.comIevads: Ar rezistenci saistīto punktveida mutāciju spektrs un izvietojums M. tuberculosisgenomā ir plašs, tādēļ ir nepieciešama metode ātrai mutāciju klātbūtnes konstatēšanaivairākos genoma fragmentos vienlaicīgi. Augstas izšķirtspējas DNS kušanas līknesanalīze (high resolution melting jeb HRM) ir metode, kas pēc fluorescences nosakamutāciju izraisītas DNS fragmentu denaturācijas īpašību izmaiņas. HRM izmantošanaiepriekš veiksmīgi izmantota, nosakot mutācijas pēc DNS, kas izdalīta no M. tuberculosistīrkultūrām 1 , tomēr nav skaidras metodes pielietošanas iespējas atšķirīgas kvalitātesklīnisko paraugu analīzē.Mērķis: Noskaidrot HRM metodes pielietošanas iespēju robežas M. tuberculosisantibiotiku rezistences mutāciju molekulārģenētiskajai noteikšanai no klīniskā materiālaizdalītos DNS paraugos. Pārbaudīt metodes jutību genoma fragmentos ar atšķirīgiemmutāciju veidiem un mainīgu mutāciju daudzveidību.Metodes: Darbā tika izmantota M. tuberculosis DNS, izdalīta no slimnieku klīniskāmateriāla. Pētījuma objekti:a) ar streptomicīna rezistenci saistītās mutācijas 16S rRNS kodējošā rrs gēna fragmentā18 paraugos (2 dažādas mutācijas 8 paraugos, 10 savvaļas tipa paraugi).b) GyrA gēna hinolonu rezistenci determinējošā rajona (QRDR) mutācijas 32 paraugos (8dažādas mutācijas 13 paraugos, 8 savvaļas tipa paraugi un 3 jaukta tipa paraugi).DNS fragmenti pavairoti ar reālā laika PCR (polimerāzes ķēdes reakcijas) iekārtu Viia7(LifeSciences) DNS interkalējošas fluorescentās krāsas klātbūtnē. Katrs paraugsamplificēts divās paralēlās reakcijās. Mutāciju klātbūtne konstatēta, mērot interkalējošaskrāsas fluorescenci pakāpeniskā temperatūras pieaugumā un salīdzinot amplikonadenaturācijas temperatūru (T D ) ar atskaites DNS fragmenta T D . Visi iegūtie dati parmutāciju klātbūtni salīdzināti ar sekvenēšanas rezultātiem.Rezultāti: a) HRM analīze rrs gēna 8/8 mutantiem paraugiem un 7/10 savvaļas tipaparaugiem sakrita ar sekvenēšanas rezultātiem, savukārt atlikušie 3 savvaļas tipa paraugudati bija pretrunīgi.b) GyrA gēnam sakrita tikai 3/13 savvaļas tipa un 1/8 mutanto paraugu piederība, bet 3jaukta tipa paraugi deva pretrunīgus rezultātus.Secinājumi: HRM metode veiksmīgi izmantota streptomicīna rezistences mutācijukonstatēšanai un atšķiršanai no neitrālām mutācijām. Savukārt liela mutāciju daudzveidībaun mutācijas, kuru ietekme uz T D ir maza, apgrūtina mutāciju atšķiršanu un HRMizmantošanu hinolonu rezistences noteikšanai klīniskajos paraugos.1. Chen, X., Kong, F., Wang, Q., Li, C., Zhang, J., Gilbert, G.L. Rapid detection of isoniazid,rifampin, and ofloxacin resistance in Mycobacterium tuberculosis clinical isolates using highresolution melting analysis. J Clin Microbiol, 2011, vol. 49, p. 3450-7- 22 -

Melanokortīna 1. receptora gēna polimorfismu funkcionālās lomasizvērtēšana, izmantojot konfokālo lāzerskenējošo mikroskopijuA.Ozola* 1 , R.Petrovska 1 , I.Mandrika 1 , O.Heisele 1 , D.Pjanova 11 Latvijas Biomedicīnas pētījumu un studiju centrs, Rīga, Latvijae-mail: aija.ozola@biomed.lu.lvIevads. Melanoma ir ļaundabīgs ādas audzējs, kas veidojas no melanocītiem. Viens nozināmākajiem melanomas riska gēniem ir melanokortīna 1. receptora gēns (MC1R), kasiesaistīts ķermeņa pigmentācijas regulācijā. MC1R ir ļoti polimorfs. Asociāciju pētījumos irparādīta biežāk sastopamo MC1R polimorfismu saistība ar ķermeņa pigmentāciju un/vaipaaugstinātu melanomas risku, savukārt in vitro pētījumos ir pierādīta to ietekme uz receptorafunkcionālo aktivitāti. Pārējie MC1R polimorfismi ir reti sastopami, un tāpēc tiek uzskatīts, kato ietekme uz melanomas risku nav vērā ņemama. Tomēr daļa no šiem polimorfismiem varētumainīt receptora funkcionālo aktivitāti un līdz ar to arī ietekmēt melanoma risku.Mērķis. Izvērtēt Latvijas populācijā atrasto reto MC1R polimorfismu (g.133T>C Phe45Leu,g.248C>T Ser83Leu, g.265G>C Gly89Arg, g.284C>T Thr95Met, g.363C>G Asp121Glu,g.493G>A Val165Ile, g.550G>C Asp184His, g.562G>A Val188Ile, g.637C>T Arg213Trp)ietekmi uz receptora funkcionālo aktivitāti.Metodes. Izmantojot PCR mutaģenēzi, tika izveidoti MC1R konstrukti ar minētajiempolimorfismiem. Mikroskopijas vajadzībām tika izveidots otrs MC1R konstruktu komplekts,kurā polimorfismus saturošie MC1R varianti tika sajūgti ar GFP (green fluorescent protein).Visi konstrukti tika ekspresēti BHK (baby hamster kidney) šūnās. Konstrukti bez GFP tikaizmantoti cikliskā adenozīna monofosfāta (cAMP) aktivācijas līmeņa noteikšanai. Konstruktiar GFP tika analizēti, izmantojot konfokālo lāzerskenējošo mikroskopu Leica TCS SP2.Šūnām, kas tika analizētas mikroskopijā, membrānas tika iekrāsotas ar fluorescento krāsvieluAlexa Fluor 633 WGA (wheat germ agglutinin). MC1R konstruktu šūnas virsmas ekspresijaslīmeņa kvantitatīvai analīzei tika izmantots programma Leica Confocal Software (Las AFv2.6.0). Konfokālās mikroskopijas attēlos katrai no kopumā sešām šūnām (pa trim šūnām nodivām neatkarīgām transfekcijām) tika nejauši izvēlēti 10 lineāri ROI (regions of interest).ROI krustpunktā ar šūnas membrānu tika nolasīta GFP/WGA fluorescences intensitātesattiecība. Rezultātā katra konstrukta gadījumā tika iegūta datu kopa, kas sastāv no 120GFP/WGA vērtībām. Iegūtās GFP/WGA vērtības tika izmantotas kvantitatīvā receptora šūnasvirsmas ekspresijas līmeņa novērtēšanā, izmantojot neparametrisko Kruskal-Wallis testu unDunn’s daudzkārtējo salīdzinājumu testu.Rezultāti. Konfokālās lāzerskenējošās mikroskopijas attēlu analīze rāda, ka polimorfismuSer83Leu un Gly89Arg gadījumā MC1R ekspresija uz šūnas virsmas salīdzinājumā arsavvaļas tipa receptoru tiek pilnībā kavēta, Asp121Glu un Arg213T gadījumā virsmasekspresija ir pazemināta, bet pārējo polimorfismu gadījumā tā ir tādā pašā līmenī kā savvaļastipa receptoram. Matriksa tabula, kas tika izveidota, izmantojot ranga summas no Dunn’sdaudzkārtējo salīdzinājumu testa, rāda, ka ranga summu atšķirības starp polimorfajiem MC1Rvariantiem ar Ser83Leu, Gly89Arg, Asp121Glu vai Arg213T un savvaļas tipa receptoru irstatistiski ticamas (p

Relative quantum yieldRelative quantum yieldResonance Energy Transfer Study of Lipid Bilayer Interactions ofApolipoprotein A-I VariantsG.Gorbenko *1 , V.Trusova 1 , E.Adachi 2 , C.Mizuguchi 2 , H.Saito 21 V.N. Karazin Kharkov National University, Kharkov, Ukraine;2 Institute of Health Biosciences, The University of Tokushima, Tokushima, Japane-mail: galyagor@yahoo.comDue to its explicit distance dependence fluorescence resonance energy transfer (FRET)has long been used for obtaining structural information on a wide variety of biomolecularassemblies. In the present study this technique was employed to gain further insights intolipid interactions of apolipoprotein A-I (apoA-I) variants. ApoA-I is the main proteincomponent of high-density lipoproteins promoting cholesterol efflux from plasmamembrane and modulating phospholipid metabolism. Moreover, specific variants ofhuman apoA-I, particularly those having G26R substitution mutation, are capable offorming amyloid fibrils associated with renal or liver failure. The N-terminal fragment(amino acids 1-83) has been identified as the predominant form of apoA-I in amyloidfibril deposits. This fragment contains three tryptophan residues, Trp 8 , Trp 50 and Trp 72which have been recruited in this work as energy donors for membrane fluorescent probes4-dimethylaminochalcone (DMC) and 3-methoxybenzanthrone (MBA), located in theinterfacial bilayer region. Small unilamellar vesicles were prepared by extrusion techniquefrom egg phosphatidylcholine (PC) and its mixture with cholesterol(Chol) (30 mol%).Shown in Fig. 1 are typical FRET profiles obtained for 5 apoA-I 1-83 fragments, referredto here as apoA83 (without mutation), G26R, G26R/W8F_W50F, G26R/W8F_W72F andG26R/W50F_W72F.1.11.00.90.80.70.60.50.40.3G26R/W8F_W72F0.20 1 2 3 4 5 6Bound DMC, MG26R ApoA83G26R/W8F_W50FG26R/W50F_W72F0.40 1 2 3 4 5 6Bound DMC, MAFig. 1. FRET profiles for Trp-DMC donor-acceptor pair in PC (A) and PC/Chol (B)liposomes1.00.90.80.70.60.5G26R ApoA83G26R/W8F_W50FG26R/W50F_W72FG26R/W8F_W72FBFRET data have been quantitatively interpreted in terms of the model of energy transfer intwo-dimensional systems allowing for distance dependence of orientation factor. It wasfound that in PC membranes Trp residues adopt interfacial bilayer location, while inPC/Chol membranes they tend to reside in the aqueous phase. These findings support thehypothesis that Chol can affect the curvature of lipid-water interface in such a manner thatthermodynamically favorable location of apoA-I becomes less buried.This work was supported by the grants from European Social Fund (project number2009/0205/1DP/1.1.1.2.0/09/APIA/VIAA/152) and Fundamental Research State Fund ofUkraine (project number F.41.4/014).- 24 -

Styryl Derivatives as a New Probes For Yeast Cell Vitality DeterminationI.P.Goriacha 1 , T.S.Dyubko 1 , V.D.Zinchenko 1 , I.V.Govor 2 , L.D.Patsenke 2 , T.Deligeorgiev 3 ,S.Kaloyanova 3 , N.Lesev 31 Institute for Problems of Cryobiology and Cryomedicine of the National Academy ofSciences of Ukraine, Kharkiv, Ukraine;2 State Scientific Institution "Institute for Single Crystals", Kharkiv, Ukraine;3 University of Sofia, Faculty of Chemistry, Sofia, Bulgaria.e-mail: irynagor@gmail.comSolutions for a number of biological, biophysical and cryobiological tasks is necessary todetermine the viability of the cells in response to various factors cryobiological. In thiswork, we investigated the sensitivity of styryl fluorescent derivatives (Table) to a state ofyeast cells Saccharomyces cerevisiae in the native state, after boiling, after the action ofhigh doses of ozone (5 mg / l), and after the action of moderate doses of ozone (0,16, 0,48and 0,80 mg / L) to determine the sensitivity to the listed factors. Used by flow cytometryand fluorescence spectroscopy.tableNameFormulaλ maxexcitation, nmλ max fluor.,nmQuantumyield, [%]11-ST 410 - 8116-ST 510 - 6517-ST 450 618 7822-ST 37023-ST 445490600520610907524-ST 460 560 9526-ST 460 535 8130-ST 300 525 75Found that the probes 17-ST, 24-ST, 26-ST and 30-ST are sensitive to the action ofboiling and high doses of ozone. Probes 22-ST, 23-ST and 30-ST are sensitive to differentmoderate doses of ozone. Presumably, we attribute this to the sensitivity of dyes to studycell membrane potential, as well as to their ability to be integrated into the cell membraneand penetrate into the cells.- 25 -

Monitoring of Free Radical Processes in Protein-lipid Systems with a NewSquaraine DyeO.Kutsenko *1 , V.Trusova 1 , G.Gorbenko 1 , T.Deligeorgiev 2 , E.Slobozhanina 3 , L.Lukyanenko 3 ,G.Zubritskaya 31 V.N. Karazin Kharkov National University, Kharkov, Ukraine;2 Faculty of Pharmacy and Chemistry, University of Sofia, Sofia, Bulgaria;3 Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus,Minsk, Belaruse-mail: olzk@mail.ruAmyloids are highly ordered protein aggregates whose deposition in organs and tissues is oneof the characteristics of so called protein misfolding diseases. These disorders includeParkinson, Alzheimer, Huntington, prion diseases, etc. It was demonstrated that one of thereasons of amyloid cytotoxicity involves interaction of aggregated proteins with cellmembranes, particularly with red blood cell membrane. The mechanisms of erythrocyte injuryinclude modification of membrane fluidity, alterations in enzyme activity and oxidation state,apoptosis and oxidative stress.The present study was undertaken to explore amyloid effect on membrane lipid oxidation. Tothis end, we used model systems containing lysozyme amyloid fibrils (Lz), hemoglobin (Hb)and liposomes from phosphatidylcholine (PC) and its mixtures with cardiolipin (CL) andcholesterol (Chol). For identification of the presence of active oxygen species novel squarainedye SQ-1 was applied [1]. It has been previously found that Hb-induced formation of freeradicals leads to the drastic quenching of SQ-1 fluorescence, resulting presumably from thereaction between reactive oxygen species and SQ-1 squaric moiety [2]. Lz association withlipid bilayers was not followed by SQ-1 spectral changes indicating insensibility of this probeto Lz-induced membrane modification. Hb addition to liposomes without Lz was followed bysignificant decrease of SQ-1 emission indicating the formation of free radicals. SQ-1fluorescence quenching was found to depend on model membrane composition. In PC andPC/Chol bilayers the decrease of SQ-1 fluorescence was less pronounced than in CLcontainingmembranes. Interaction of the Hb with negatively charged membranes can befollowed by protein unfolding, heme displacement and dissociation of heme-globin complex.This facilitates heme-lipid interaction and consequent iron-induced formation of free radicalsleading to the increase of free radical formation in CL bilayers as compared to PCmembranes. Cholesterol prevents Hb penetration into the inner membrane regions, thusinhibiting oxidative processes. Interestingly, lysozyme fibrils suppressed SQ-1 fluorescencequenching in all types of lipid bilayers, with the magnitude of this effect being mostpronounced in CL-containing membranes. There are at least two explanations for Lzantioxidant effect. First, protein molecular groups can be the scavengers for free radicals. Onthe other hand, Hb and Lz can compete for the membrane binding sites, thereby reducing Hbpenetration into the membrane interior and formation of free radicals.This work was supported by the grant from Fundamental Research State Fund of Ukraine(project number F41.1/014) and Fundamental Research State Fund of Belarus Republic(project number B11K-152).1. Ioffe, V.M., Gorbenko, G.P., Deligeorgiev, T., Gadjev, N., Vasilev, A. Fluorescence study ofprotein-lipid complexes with a new symmetric squarylium probe. Biophys. Chem., 2007, vol. 128, p.75–86.2. Trusova, V.M., Gorbenko, G.P., Deligeorgiev, T., Gadjev, N., Vasilev, A. A Novel Squarylium Dyefor Monitoring Oxidative Processes in Lipid Membranes. J. Fluoresc., 2009, vol. 19, p. 1017–1023.- 26 -

Studying Zinc Biology with Fluorescent ProbesE.I.Slobozhanina *1 ,Y.M.Harmaza 1 , N.M. Kozlova 1 , A.V.Tamashevski 11 Institute of biophysics and cell engineering of NAS B, Minsk, Belaruse-mail: slobozhanina@ibp.org.byBackground: Zn 2+ is essential trace element that is required for a wide range of biologicalprocesses, including cell proliferation and differentiation. The physiological concentrationof Zn 2+ in serum is 2–15 μM, when free Zn 2+ level in most cells is extremely low (

Application of Fluorescent Technologies to Identify Viable Pathogens inDrinking WaterM.Linda 1 , T.Juhna* 11 Department of Water Engineering and Technology, Riga Technical University, Riga, Latvia.e-mail: Linda.mezule@rtu.lvWhen subjected to unfavourable environmental conditions or nutrient limitation manymicroorganisms (including pathogenic ones) can attain a state of non-cultivability (viablebut not cultivable, VBNC) where they are unable to form visible colonies on traditionalculture media 1 . In environmental studies Fluorescent in situ hybridization (FISH) withfluorescent oligonucleotide or peptide nucleic acid probes have become an important toolin monitoring and identification of certain bacterial species or groups. However, studieshave shown that FISH alone is not a direct marker of cell viability. To monitor cellmembrane integrity, metabolic activity, enzymatic activity or ability to divide as such,different molecular assays have been developed. Our studies have shown that many ofthese assays can be combined with specific identification of viable target species e.g. ableto divide Escherichia coli, in their natural environments, e.g. biofilm of drinking waterdistribution system. During the years analyses on occurrence of Legionella species in hotwater systems and enumeration of pathogenic protozoa in distribution systems have beenperformed with fluorescence microscopy. However, most studies have been performed onthe assessment of faecal indicator E. coli in drinking water distribution systems whichmeet current microbiological standards and potential inactivation techniques for theseorganisms.The results have shown that in low numbers able to divide E. coli can be detected even inthe biofilm of freshly treated water. On average around 53 % of all detected E. coli hadthe ability to divide and the concentration increased with water residence time in thenetwork. At the same time no colonies were isolated on culture media. Monitoring of E.coli concentration dynamics in drinking water distribution system and pilot scale reactorsshowed that after the application of disinfectant (chemical or mechanical) cells first losttheir cultivability (formation of colonies), then ability to divide (limited amount of time)and only then the metabolic activity (respiratory activity), indicating on possibleunderestimation of pathogen concentration when traditional techniques have been used,thus, posing the risk for the consumer.1. Oliver JD. Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMSMicrobiol Rev. 2010, vol. 34, p. 415–425.- 28 -

A New Aminobenzanthrone Dye for Identification of Amyloid FibrilsI. Maliyov *1 , M.Romanova 1 , K.Vus 1 , A.Kastorna 1 , G.Gorbenko 1 , V.Trusova 1 , E.Kirilova 2 ,G.Kirilov 2 , I.Kalnina 21 Department of Biological and Medical Physics, V. N. Karazin Kharkiv National University,Kharkiv, Ukraine;2 Department of Chemistry and Geography, Daugavpils University, Daugavpils, Latviae-mail: independentpart@mail.ruProtein polymerization into highly ordered fibrillar structures (amyloids) is currentlyregarded as determinant of a range of pathological conditions, particularly, endocrine andneuro-physiological diseases. Recent studies clearly indicate that amyloid cytotoxicity isprovoked by a continuum of cross-β-sheet aggregates including mature fibrils. To identifyamyloids, a wide variety of fluorescent dyes have been designed and evaluated withrespect to their sensitivity to different types of protein aggregates. Nevertheless, theproblem of amyloid detection by fluorescence tools is far from being fully resolved. Thepresent study addressed the question of whether amyloid fibrils can be effectively detectedusing aminobenzanthrone dyes. For this purpose, we examined the binding of onerepresentative of this class of dyes, ABM, to amyloid fibrils of two proteins - globin andinsulin (Fig. 1). The reaction of the protein fibrillization was conducted under stronglyacidic conditions (pH=2.2) at 60 o C during 8 days. The fluorimetric titration of the probeby proteins was performed with spectrofluorometer SM 2203, Solar, Belarus. The increaseof protein concentration was followed by the rise of fluorescence intensity and shortwavelengthshift of emission maximum. These spectral changes can be considered as theproof of dye-protein complexation. Quantitative characteristics of the dye binding toprotein aggregates ( Ka– association constant, n – binding stoichiometry, α – thedifference between molar fluorescences of the bound and free dye) were estimated byanalysing the isotherm of ABM-fibril binding in terms of Langmuir adsorption model(Table 1). The recovered parameters appeared to be the same order of magnitude as thoseknown for the other amyloid-specific dyes.Table 1.Parameters of ABM binding to amyloid fibrilsProtein K , µM -1 n, mol/mol α, µM -1aInsulin 0.14 0.16 4.7Globin 0.19 0.11 3.8Fig. 1. Transmission electron micrographsof negatively stained insulin fibrilsAs seen from Table 1, ABM binding characteristics insignificantly differ for insulin andglobin, suggesting that these proteins in fibrillar state have structurally similar ABMbinding sites.This work was supported by the grants from European Social Fund (project number2009/0205/1DP/1.1.1.2.0/09/APIA/VIAA/152) and Fundamental Research State Fund ofUkraine (project number F.41.4/014).- 29 -

Efficiency of energy transfer,%Efficiency of energy transfer,%Interaction of Fibrillar Insulin and Globin with Liposomes: ResonanceEnergy Transfer StudyM.Romanova *1 , I.Maliyov 1 , A.Kastorna 1 , K.Vus 1 , V.Trusova 1 , J.Molotkovsky 2 , G.Gorbenko 11 V.N. Karazin Kharkov National University, Kharkov, Ukraine;2 Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russiae-mail: romanova-mari@mail.ruA wide range of proteins are capable of assembling into cross β-sheet structures (amyloidfibrils) under pathological or nonphysiological conditions. The toxicity of amyloid fibrilsand their precursors is supposed to be associated with their action on structural andfunctional state of biological membranes. One way of approaching this problem is basedon the use of model systems consisting of lipid vesicles and protein aggregates formed invitro.The present study was directed towards clarifying the molecular details of interactionbetween globin or insulin fibrils and liposomes of different composition. Fibrillaraggregates were obtained by protein incubation in glycine buffer (pH 2.2) at 60 o C during8 days. To assess the extent of fibril binding to liposomes, we measured energy transferefficiency from tryptophan residues of globin or insulin to anthrylvinyl (AV) fluorophorecovalently attached to phosphatidylcholine (PC). Lipid vesicles were prepared from PCand its mixtures with anionic lipid phosphatidylserine (PS) (10, 20, 40 mol% PS).Increasing the concentration of globin fibrils resulted in the raise of AV fluorescenceintensity for all model systems under study (Fig.1). It was found that the most effectiveenergy transfer occurs when globin interacts with neutral PC vesicles. In the case ofinsulin, conversely, the energy transfer from Trp to AV-PC was negligible. Thisobservation can be explained by the fact that insulin has negative charge under theemployed experimental conditions (pH 7.4), which prevents protein binding to negativelycharged PC/PS liposomes.4.03.5PCPS10PS20PS404,03,53.03,02.52,52.02,01.51.00.50.00 2 4 6 8 10 12Concentration of Globin, M1,51,00,50,0InsulinGlobinFig.1. Efficiency of energy transfer as afunction of fibrillar globin concentrationFig.2. Comparison of energy transfer fromTrp to AV-PC for insulin and globinFig.2 illustrates the difference between fibrillar insulin and globin in the energy transferefficiency from Trp to AV-PC in PC bilayers. The results obtained allowed us to supposethat the character of interaction between globin fibrils and liposomes is mostlyhydrophobic, while in the case of insulin the role of electrostatics may be predominant.This work was supported by the grant from Fundamental Research State Fund of Ukraine(project number F.41.4/014).- 30 -

Fluorescence Approaches to Detection of Protein AggregatesV.M.TrusovaV.N. Karazin Kharkov National University, Kharkov, Ukrainevaltrusova@yahoo.comFluorescence spectroscopy is one of the most powerful tools for characterization of amultitude of biological processes 1 . Of these, the phenomenon of protein oligomerizationattracts especial interest due to its crucial role in the formation of fibrillar proteinaggregates (amyloid fibrils) involved in ethiology of so-called protein misfoldingdiseases. It is becoming increasingly substantiated that protein fibrillization in vivo can beinitiated and modulated at membrane-water interface 2 . All steps of membrane-assistedfibrillogenesis, viz., protein adsorption onto lipid bilayer, structural transition ofpolypeptide chain into a highly aggregation-prone partially folded conformation, assemblyof oligomeric nucleus from membrane-bound monomeric species and fiber elongation canbe monitored with a mighty family of fluorescence-based techniques 3-5 . Little or nodamage to the examined matter, requirement of material micromolar concentration, rathersimple methodology, involvement of relatively inexpensive instrumentation, highsensitivity and specificity make fluorometry a broadly used research tool for studying theprotein-protein interactions. Various kinds of fluorescence technique, namely steady-stateand time-resolved fluorescence, fluorescence polarization and fluctuation spectroscopy,stopped-flow and laser-induced fluorescence provide the information about the structure,microenvironment and distribution of protein complexes 1 . The immense range ofparameters measured for both intrinsic and extrinsic protein fluorophores includesfluorescence intensity, quantum yield, anisotropy and quenching, lifetime, Försterresonance energy transfer efficiency (FRET) efficiency, diffusion coefficients, to namejust a few. In the present contribution the applications of fluorescence spectroscopy tomonitoring protein oligomerization in a membrane environment are exemplified and someproblems encountered in such kinds of studies are highlighted. More specifically, suchaspects as fluorescence probing of protein-membrane binding, fluorescence monitoring ofprotein structural changes, detection of protein aggregates with intrinsic and extrinsicfluorophores are discussed. In addition, optimal strategies for fluorescence analysis ofprotein aggregation are suggested.References:1. Lakowicz, J.R. Principles of Fluorescence Spectroscopy. NY: Springer, 2006.2. Gorbenko, G.P., Kinnunen, P.K.J. The Role of Protein-Lipid Interactions in Amyloid-Type Proteinfibril Formation. Chem. Phys. Lipids, 2006, vol. 141, p. 72-82.3. Zerovnik, E. Amyloid-fibril Formation. Proposed Mechanisms and Relevance to ConformationalDisease, Eur. J. Biochem., 2002, vol. 269, p. 3362-3371.4. Domanov, Ye.A., Kinnunen, P.K.J. Islet Amyloid Polypeptide Forms Rigid Lipid–Protein AmyloidFibrils on Supported Phospholipid Bilayers. J. Mol. Biol., 2008, vol. 376, p. 42-54.5. Trusova, V.M., Gorbenko, G.P., Sarkar, P. et al. Forster Resonance Energy Transfer Evidence forLysozyme Oligomerization in Lipid Environment. J. Phys. Chem. B, 2010, vol. 114, p. 16773-16782.- 31 -

Fluorescence Study of Cytochrome c Binding to Model MembranesV.Trusova *1 , G.Gorbenko 1 , J.Molotkovsky 2 , P.Kinnunen 31 V.N. Karazin Kharkov National University, Kharkov, Ukraine;2 Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia;3 School of Science and Technology, Aalto University, Espoo, Finlande-mail: valtrusova@yahoo.comCytochrome с (cyt c) is a small highly basic hemoprotein abundant in the intermembranespace of mitochondria and possessing two at first glance unrelated functions. First, cyt с isan essential component in respiration, shuttling electrons from cyt c reductase (bc1complex) to cyt c oxidase. The second function of cyt c relates to its key role in theactivation of the apoptotic cascade, resulting from the release of this protein frommitochondria into the cytosol. It is generally established that control of the binding of cytc to the inner membrane of mitochondria (IMM) is involved in both of the abovefunctions. Despite the diversity of lipids constituting the mitochondrial membrane, centralto the cyt c – membrane interaction in IMM is cardiolipin (CL), which is indispensible forthe retention, stability and physiological functioning of cyt c. In this regard, the nature andspecificity of interactions between cyt c and CL attract particular interest. In the presentstudy fluorescence resonance energy transfer (FRET) between anthrylvinyl-labeledphosphatidylcholine as a donor and heme moiety of cytochrome c (cyt c) as an acceptorhas been employed to explore the protein binding to model membranes, composed ofphosphatidylcholine (PC) and CL. The existence of two types of protein-lipid complexeshas been hypothesized where either deprotonated or partially protonated CL molecules areresponsible for cyt c attachment to bilayer surface. To quantitatively describe cyt cmembrane binding, the adsorption model based on scaled particle and double layertheories has been employed, with potential-dependent association constants being treatedas a function of acidic phospholipid mole fraction, degree of CL protonation, ionicstrength and surface coverage. Multiple arrays of FRET data obtained under conditions ofvarying pH, ionic strength, CL content and protein-to-lipid molar ratio have been analyzedin terms of the model of energy transfer in two-dimensional systems combined with theadsorption model allowing for area exclusion and electrostatic effects. The set ofrecovered model parameters included effective protein charge, intrinsic associationconstants and heme distance from the bilayer midplane for both types of protein-lipidcomplexes. Upon increasing CL mole fraction from 10 to 20 mol % (the value close tothat characteristic of IMM) the binding equilibrium dramatically shifted towards cyt cassociation with partially protonated CL species. The estimates of heme distance frombilayer center suggest shallow bilayer location of cyt c at physiological pH, while at pHbelow 6.0 the protein tends to insert into membrane core. These findings support theviewpoint that hydrogen bonding of cyt c to protonated phosphate can be coupled with theformation of electrostatic contacts with deprotonated phosphate. Structural features of cytc do not exclude such a possibility. Cyt c molecule has two hydrophobic channelsconnecting heme crevice with the protein surface. The opening of one of these channels issurrounded by oppositely located Asn 52 and Lys 72 , Lys 73 . It has been hypothesized that cytc association with CL involves hydrogen bonding of one protonated phosphate moiety toAsn 52 with simultaneous electrostatic binding of deprotonated phosphate to lysine residuesand accommodation of one acyl chain within the hydrophobic channel.This work was supported by the grants from European Social Fund (project number2009/0205/1DP/1.1.1.2.0/09/APIA/VIAA/152) and Fundamental Research State Fund ofUkraine (project number F.41.4/014).- 32 -

Aminobenzanthrone Dyes as Prospective Fluorophores for Detection andCharacterization of Amyloid FibrilsK.Vus* 1 , V.Trusova 1 , G.Gorbenko 1 , E.Kirilova 2 , G.Kirilov 2 , I.Kalnina 21 Department of Biological and Medical Physics, V. N. Karazin Kharkiv National University,Kharkiv, Ukraine;2 Department of Chemistry and Geography, Daugavpils University, Daugavpils, Latviae-mail: katenka.vus@mail.ruIn view of dramatic role of pathological protein aggregates, amyloid fibrils, in thedevelopment of a number of severe diseases, detection and characterization of such entitiesrepresent the focus of current research efforts. Fluorescence spectroscopy has beenextensively employed in amyloid studies due to its inherent sensitivity and versatility, goodspatial and temporal resolution. The aim of this work was to test a series of novel fluorescentaminobenzanthrone dyes (BD) for their ability to identify and characterize fibrillar aggregatesof lysozyme prepared by protein denaturation in concentrated ethanol solution (FI) [1] or atstrongly acidic pH (FII) [2]. Analysis of the results of fluorimetric titration in terms of theLangmuir adsorption model yielded the parameters of BD binding to native and fibrillarprotein (association constant, binding stoichiometry and molar fluorescence). Furthermore,several additional quantities reflecting the preference of the probe to fibrillar proteinaggregates have been evaluated [3]. Based on the comprehensive analysis of the recoveredparameters, AM2 and A8 were selected as the most prospective tracers for FI and FII,respectively. Besides, A6 and A8 were also suitable for highly sensitive detection of both FIand FII types of amyloid fibrils, because of their high quantum yields in amyloid-bound state.Interestingly, comparison of quantum yields and binding parameters of the dyes in FI- andFII-bound states showed the differences in fibrillar morphologies. It was supposed that in FIIfibers protofilaments are packed more tightly than in FI, that complicates BD penetration intothe FII fibrils and is presumably responsible for the decrease of BD-protein binding extent.Finally, strong sensitivity of the novel probes to environmental viscosity was revealed byanalyzing their properties in ethanol (E) and in ethanol/glycerol mixture with glycerol contentof 67% (v/v) (EG). Substantially higher fluorescence anisotropy of BD in EG compared to Eis indicative of strong viscosity sensitivity of the novel dyes. These findings suggest thatfluorescence anisotropy of BD can be employed as an alternative parameter for studying thekinetics of amyloid fibril formation [4]. The present study demonstrated high potential ofaminobenzanthrone dyes in identification of fibrillar protein aggregates and uncovering theirstructural peculiarities.This work was supported by the grants from European Social Fund (project number2009/0205/1DP/1.1.1.2.0/09/APIA/VIAA/152) and Fundamental Research State Fund ofUkraine (project number F.41.4/014).References:1. Holley, M., Eginton, C., Schaefer, D., Brown, L.R. Characterization of Amyloidogenesis of HenEgg Lysozyme in Concentrated Ethanol Solution. Biochem Biophys Res Commun, 2008, vol. 373,p. 164-168.2. Morozova-Roche, L.A., Zurdo, J., Spencer, A., Noppe, W., Receveur, V., Archer, D.B., Joniau,M., Dobson, C.M. Amyloid Fibril Formation and Seeding by Wild-Type Human Lysozyme andIts Disease-Related Mutational Variants. J Struct Biol, 2000, vol. 130, p. 339-351.3. Vus, K., Trusova, V., Gorbenko, G., Kirilova, E., Kirilov, G., Kalnina, I., Kinnunen, P. NovelAminobenzanthrone Dyes for Amyloid Fibril Detection. Chem Phys Lett, 2010, vol. 532, p. 110-115.4. Sabate, R., Saupe, S.J. Thioflavin T Fluorescence Anisotropy: an Alternative Technique for theStudy of Amyloid Aggregation. Biochem Biophys Res Commun, 2007, vol. 360, p. 135-138.- 33 -

ABinding of the Novel 4-tricyanovinylarylamine Dye to Fibrillar LysozymeK.Vus* 1 , V.Trusova 1 , G.Gorbenko 1 , T.Deligeorgiev 2 , S.Kaloyanova 2 , N.Lesev 21 Faculty of Physics and Technology, V. N. Karazin Kharkiv National University, Kharkiv,Ukraine;2 Faculty of Pharmacy and Chemistry, University of Sofia “St. Kliment Ohridsky”, Sofia,Bulgariae-mail: katenka.vus@mail.ruAccumulation of fibrillar protein aggregates (amyloid fibrils) in different tissues is currentlyassociated with a variety of so called conformational diseases. The most widespreadtechniques employed in identification of amyloid fibrils are fluorescence and absorptionspectroscopy. Further progress in this research area is strongly coupled with the design andtesting of the novel chromo- and fluorophores. The present study was aimed at evaluation ofthe novel 4-tricyanovinylarylamine dye (referred here as 3q) as possible marker for fibrillarprotein aggregates. Amyloid fibrils of lysozyme were prepared at strongly acidic pH [1] .Tricyanovinylarylamine probes are characterized by high extinction coefficients and theability to form intramolecular charge transfer state upon excitation [2] . Fluorescent propertiesof these dyes are sensitive to their molecular structure and local environment. It appeared that3q has very low quantum yields in buffer solution and there was no fluorescence increase inthe presence of amyloid fibrils. For this reason, we tested this dye as possible analog of thetraditional amyloid marker Congo Red, non-fluorescing dye whose differential absorptionspectra are used for selective detection of amyloid fibrils. As seen in Fig. 1A, increasing theconcentration of fibrillar lysozyme leads to progressive decrease of 3q absorption, with clearisosbestic point. Quantitative characteristics of the dye binding to protein aggregates ( Ka–association constant, n – binding stoichiometry, α – the difference of extinction coefficients inthe bound and free states) were estimated by analysing the isotherm of 3q binding tolysozyme fibrils in terms of Langmuir adsorption model (Fig. 1B). The recovered parametersare close to those reported for existing amyloid-sensitive chromophores.0.10NC3q0.05NCCH 2CH 2N(CH 3) 3Lysozyme amyloidCNClO4concentration, M0.00244563-0.057892104-0.10116350 400 450 500 550 600 650H 2CNWavelength, nmABFig.1. Differential absorption spectra of 3q (A) and the isotherm of 3q binding to fibrillarlysozyme (B).To conclude, spectral behavior of 3q as one representative of tricyanovinylarylamine dyesallowed us to recommend these compounds for testing as possible amyloid markers similar toCongo Red.This work was supported by the grants from European Social Fund (project number2009/0205/1DP/1.1.1.2.0/09/APIA/VIAA/152) and Fundamental Research State Fund ofUkraine (project number F.41.4/014).- 34 -A 5020.100.080.060.040.020.00K a= 0.13 M -1n = 0.5 mol/mol = -0.0028 M -1 cm -10 20 40 60 80 100 120Protein concentration,

Association of Novel Benzanthrone Probes with Model Lipid MembranesO.A.Zhitniakivska* 1 , V.M.Trusova 1 , G.P.Gorbenko 1 , E.M.Kirilova 2 , G.K.Kirilov 2 , I.Kalnina 21 V.N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv, 61077, Ukraine;2 Department of Chemistry and Geography, Faculty of Natural Sciences and Mathematics,Daugavpils University, 13 Vienibas, Daugavpils LV5401, Latviae-mail: olya_zhitniakivska@yahoo.comAmong a wide variety of fluorescent dyes currently used in biomedical research and industrybenzanthrone derivatives are of particular interest due to their favorable spectral properties,viz. large extinction coefficient, marked Stokes shift; negligible fluorescence in an aqueousphase; high sensitivity of fluorescence parameters to environmental polarity, etc. Our recentstudy revealed high lipid-associating ability of a series of newly synthesized benzanthroneaminoderivatives. Moreover, spectral responses of the two examined dyes in different lipidsystems proved to correlate with increased bilayer hydration. These findings allowed us toassume that benzanthrone aminoderivatives may be effective fluorescent probes forexamining membrane-related processes, especially those coupled with the change in thedegree of lipid bilayer hydration. In view of this it seemed of interest to extend our previousinvestigation to a new series of benzanthrone dyes (referred to here as AM12, AM15, AM18,IAH, IBH and ISH), whose structures are given in Fig.1.NNHNNNNOAM12 OOAM15 OAM18OHNHNHNHO IAH OIBHFig. 1. Structures of aminobenzanthronesO ISHThe binding of aminobenzanthrones to the lipid membranes composed of zwitterionic lipidphosphatidylcholine (PC) and its mixtures with cholesterol (Chol) and anionic lipidcardiolipin (CL) was followed by significant increase in fluorescence intensity and dyequantum yields. To characterize benzanthrone-lipid binding quantitatively, we determined thedye partition coefficients for different lipid systems. CL and Chol addition to PC bilayerresulted in the increase of partition coefficients for IBH, ISH and IAH (relative to the neat PCmembrane), whereas AM12, AM15 and AM18 displayed the opposite behavior in cardiolipinandcholesterol-containing systems. As spectral properties of benzanthrone dyes stronglydepend on the nature of fluorophore environment (polarity, viscosity, the probability ofhydrogen bonding or other intermolecular interactions), it seems reasonable to interpret thedifferences between IBH, ISH, IAH and AM12, AM15, AM18 in terms of different bilayerlocation. It was assumed that ISH, IBH and IAH are located in the bilayer polar region, whileAM12, AM15 and AM18 penetrate more deeply, most probably in the membranehydrophobic part.This work was supported by the grants from European Social Fund (project number2009/0205/1DP/1.1.1.2.0/09/APIA/VIAA/152) and Fundamental Research State Fund ofUkraine (project number F.41.4/014).- 35 -

New ICT-dyes for Membrane StudiesO.Zhytniakivska* 1 , V.Trusova 1 , G.Gorbenko 1 , T.Deligeorgiev 2 , S.Kaloyanova 2 , N.Lesev 21 V.N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv, 61077, Ukraine;2 Faculty of Pharmacy and Chemistry, University of Sofia, Sofia, Bulgariae-mail: olya_zhitniakivska@yahoo.comDuring the past decades fluorophores which can undergo photoinduced intramolecular chargetransfer (ICT dyes) find increasing application in biological sciences. Fluorophores whichexhibit ICT state are widely used in different areas, including the development of solar cellsand chemosensing. High sensitivity of these compounds to the environmental polarityprovokes their use as fluorescent microenvironmental sensors, particularly, in the studies ofmembrane structure and protein-lipid interactions.The present study was undertaken to evaluate the sensitivity of newly synthesized ICT dyes,referred to here as ICT2, ICT4, ICT3 and ICT5, whose structures are given in Fig.1, to thechanges in physicochemical properties of lipid bilayer. At the first step of the study wecompared the lipophilic properties of the ICT-dyes and their sensitivity to the membraneenvironment. Fluorescence spectra of these dyes were recorded in buffer solution (5 mM Naphosphate,pH 7.4) and liposomal suspension. It appeared that only ICT2 and ICT4 arecapable of fluorescing, while ICT3 and ICT5 are non-fluorescent. ICT4 fluorescencedecreases, while emission maximum is shifted from 435 nm in buffer to about 420 nm on thedye transfer from the aqueous to lipid phase. On the contrary, the binding of ICT2 to themodel membranes was followed by the fluorescence increase without any shift of theemission maximum. Moreover, fluorescence quantum yield of ICT2 exhibited tenfoldincrease in the lipid bilayer compared to the buffer solution, while that of ICT4 decreasedabout twice.H 3 COCH 3 SO 4ISNCH 3SNCH 3NNCH 3CH 3OACHS3NNCH 3(CH 2 ) 3 NH 2 HBrCDFig. 1. Structure of the dyes ICT2 (A), ICT4 (B), ICT3(C) and ICT5 (D)To characterize ICT2-lipid binding quantitatively, at the next step of the study we determinedthe dye partition coefficient KPfor different lipid systems. The values of KPwere found tofall in the range (0.4 – 1.5)×10 4 indicating that the examined dyes possess high lipidassociatingability. Analysis of the partition coefficients shows that inclusion of anioniccardiolipin and cholesterol into phosphatidylcholine bilayer gives rise to the increase ofpartition coefficient relative to the neat phosphatidylcholine membrane.To summarize, a comparison of the fluorescence properties of the four newly synthesized ICTcompounds revealed that despite being structurally similar, these dyes display differentspectral and lipid-associating behavior. ICT2 was featured by the most effective partitioninginto lipid phase coupled with considerable increase of the fluorescence quantum yield. Therecovered properties of ICT2 allowed us to recommend this dye for further use in membranestudies. This work was supported by the grant from Fundamental Research State Fund ofUkraine (project number F.41.4/014).ISNCH 3 SO 4CH 3OCH 3B- 36 -

Alterations of Blood Plasma Albumin in Chernobyl Clean-up Workers withand without Type 2 Diabetes MellitusT.Zvagule 2 , N.Kurjane 2 , A. Šķesters 2 , A.Silova 2 , N.Gabruseva 2 , G.Gorbenko 3 , G. Kirilov 1 ,I.Kalnina 1 , E.Kirilova 1 , S.Gonta 41 Daugavpils University, Daugavpils,2 Riga Stradina University, Riga, Latvia,3 V.N. Karazin National University, Kharkov, Ukraine;4 Institute of Microbiology and Biotechnology, Latvia UniversityDiabetes mellitus has been one of the most crippling diseases that man has seen and itsprevalence has risen dramatically over the past two decades. Currently there are over the 150million diabetics word wide and this number is likely to increase to 300 million or moreby the year 2025. Diabetes mellitus increases the risk of many disorders includingcardio-vascular diseases. Understanding the molecular properties of diabetic progression is abig challenge in systems-biology era.ABM (3-aminobenzanthrone derivative) has been previously shown as a potential biomarkerfor determination of the immune state of patients with different pathologies. The aim ofthis study was to determine the several aspects of plasma albumin alterations in thegroup of Chernobyl clean-up workers with diabetes mellitus in relation with its commongroup without diabetes mellitus and humans having no professional contact withradioactivity. Patients: 1 Group type 2 diabetes mellitus. Complications associated withdiabetes- such as polyneuropa5thy, atherosclerosis, including chronic diseases withinflammation. Group 2 – prevalence of several chronic diseases (diabetes mellitus is asconcomitant disease. Cardiovascular disorders, progressive atherosclerosis with activeinflammation process, leucocytosis, metabolic syndrome, stroke. Group 3 – Chernobyl cleanupworkers without diabetes mellitus. ABM fluorescent characteristics are dependent onfunctional activity of CNS and indices of inflammatory processes. The most seriouscombination for patients-holisterinemy, progressive cerebral atherosclerosis, epilepsyparoxysms. The following parameters were examined: 1) the spectral characteristics ofABM in blood plasma; 2) “’effective” (EA) and total (TA) albumin concentration in bloodplasma; 3) quantitative parameters of albumin auto-fluorescence.Screening of the individuals with diabetes mellitus 25-26 years after the work inChernobyl revealed two groups of patients differing in structural/functional properties ofalbumin, first of all on conformations of plasma albumin and characteristics oftryptophanyl region of molecule. The revealed structural modifications of membranes aredependent on radiation- induced factors. Concomitant diseases (diabetes mellitus, cardiovasculardiseases) reinforce radiation induced effects in accordance with its manifestation.Results clarifies the heterogenous nature of albumin molecules and revealed quantitativedifferent its conformation in observed groups of patients. Albumin molecule in diabetics ismodified in the chronic hypoxia conditions provoked mainly by the hyperglycemia andoxidative stress.ABM is a sensitive probe of albumin alterations, and can be used to elucidate the changes inprotein systems. Significant differences in albumin dynamics exist between control (donors),and diabetics and non-diabetics groups of Chernobyl clean-up workers. The biomarker ofmodified albumin could also help to identify individuals with complex metabolic conditionswho have a higher risk of suffering from cardiovascular diseases.- 37 -

Alterations of Blood Plasma Albumin in Chernobyl Clean-up Workers withand without Concomitant DiseasesT.Zvagule 1 , I.Kalnina 2 , E.Kirilova 2 , N.Kurjane 1 , N.Gabruseva 1 , J.Reste 1 , G.Kirilov 2 ,A. Šķesters 1 , A.Silova 11 Riga Stradins University, Latvia2 Daugavpils University, Daugavpils, LatviaABM (3-aminobenzanthrone derivative) developed at Daugavpils University has beenpreviously shown as a potential probe for the determination of the immune state ofpatients with different pathologies. It is widely accepted that the dynamics of plasmaproteins (e.g. albumin) play a prime role immune characteristics of humans. Albumin is asingle source of ABM fluorescence in human blood plasma (650 nm). The aim was todetermine the several aspects of blood plasma albumin alterations of Chernobyl clean-upworkers, who had worked within the zone from 1986 till 1991. Most of them wereofficially documented as recipients of ionizing radiation exposure (1-50 cGy). For thestudy were selected the different groups of Chernobyl clean-up workers groups (common,after myocardial infarction, with epilepsy paroxysm, and 2 type diabetes mellitus), andcorresponding groups of patients having no professional contact with radioactivity. Thefollowing parameters were examined: 1) the spectral characteristics of ABM in bloodplasma, 2) plasma auto-fluorescence parameters; 3) “effective” and total albuminconcentrations in blood plasma. (“Effective „concentration –equivalent in plasma ofhealthy albumin and reflects its binding and carrier functions). The total albuminconcentration is more conservative. It has been noted that changes of pH in range from 3-12 strongly affect spectral characteristics of albumin bound ABM. The spectralcharacteristics of ABM in Chernobyl clean-up workers plasma results in splitting of thealbumin alterations onto two stages, acidic expansion stage (620-630 nm) and N-Ftransition stage (600-620 nm).These observations may be consistent with the decreasedbinding of ABM and/or conformational changes of albumin molecule, significantlydiffering in observed patients groups. The levels of pathological and pharmacologicalmetabolites (fatty acids, antioxidants, plasma levels of lipid peroxidation products etc.)balance differs in patients groups comparable to controls and hence their correlation toseizures pathophysiology and their degree. The metabolites caused conformationalchanges in albumin molecule and shifts in binding parameters are in agreement withresults of albumin auto-fluorescence data and ABM binding sites characteristics (bindingsites count, affinity for probe, polarity). A result clarifies the heterogeneous nature ofABM binding and revelled quantitative different conformation of albumin in observedgroups of patients. The spectral parameters of ABM in plasma (as in lymphocytes) arecoupled with cellular mechanisms of immune regulation in patients. The results showedstrong correlation with select immunological parameters (CD4+: CD8+ ratios,lymphocyte count etc.) and both clinical and pathological estimates. The morepronounced albumin structural/functional alterations were observed in clean-up workerswith concomitant diseases. One can with a certain degree of confidence that the revealedalterations of albumin are dependent on radiation induced factors. Concomitant diseasesreinforce radiation – induced effects in accordance of its manifestation. Therefore seemslikely that external radiation and incorporated radionuclides) predominate in alterations ofalbumin. Measures of ABM characteristics in blood plasma could potentially be a usefultool in clinical immunological screenings to estimate the immune state of patients.- 38 -

Aminobenzanthrone Derivative ABM - Fluorescent Biomarker of ImmuneState of PatientsE.Kirilova 1 , G.Gorbenko 3 , I.Kalnina 1 , T.Zvagule 2 , G.Kirilov 1 , N.Kurjane 21 Daugavpils University, Latvia;2 Riga Stradins University, Latvia;3 V.N.Karazin Kharkov National University, Kharkov, UkraineIn different pathologies membrane damage in immune cells and blood plasma albuminenvolves as a consequence of alterations in a immune state of patients. It is now widelyaccepted that the dynamics of these changes, along with the types of alterations instructures of the immune system cells and plasma proteins play a critical role in themaintenance of the immune status of any given organism. As a result of the importance ofchanges in the structural integrity of cells of the immune system, it is important forclinicians to receive information on the immunological status of organism via quick,reliable, reproducible methods. In this regard, fluorescent probes have shown to beexcellent tools for use in such protocols. Such an analysis has a great potential for notonly for helping to comprehed mechanisms of immunomodulation associated with theinduction/progression of pathologies, but might serve as a very important prognosticindicator of long- term survival among patients with pathologies. The advantages of probeABM:1. Spectral characteristics of aminobenzanthrones (e.g. ABM) satisfy all requirements foran ideal tracer (bright fluorescence, high extinction coefficient, photo-, thermo-andchemical stability). Specifically, it was found that spectral characteristics of this probecorrelate with a number of important parameters of artificial and cellular membranes suchas physicochemical state, microviscosity, proliferating and lipid metabolic activities ofcells, distribution of lymphoid subset, etc.2. The choise to examined albumin, among the myriad of constituents in plasma, is thatthis protein is practically the single source of ABM binding and subsequent fluorescencein plasma. Very important is that ABM can be used as a probe sensitive to conformationchanges in protein: the most prominent changes in fluorescence characteristics occured atpH values 3-12 known to cause conformation transitions of proteins. It is significant tonote that the probe emission in red region of spectra (650 nm) exceeds higher analyticalsensitivity of method (as compared with probes ANS, K-35, PR etc.). For exampleaccordingthe literature data probe ANS possess low sensitivity to conformative changesof albumin at pH 5.5-11.5. Only fluorescent probes allow to detect the „effective”concentration of albumin in blood plasma („effective” concentration- equivalent in bloodplasma of healthy albumin and reflects it’s binding and carrier functions). The totalalbumin concentration is more conservative. The detection of „effective ‘concentration ofalbumin using ABM is technically simple and not so time –consuming as in the cases ofother probes (don’t require additional steps of plasma and probe preparation). ABMspectral parameters in blood plasma are coupled with alterations in cellular mechanisms ofimmune regulation in the patients with different pathologies. Measures of ABMfluorescence intensity values for plasma albumin and/or for lymphocytes (total and amongdifferent subtypes) could potentially be a useful tool in clinical immunilogical screeningsto estimate the immune state of patients.- 39 -

Functional State of Beta Adrenoreactive System in Man and Animals:Fluorescent StudiesE.Kirilova 1 , I. Kalnina 1 , T.Zvagule 2 , N. Kurjane 2 , G. Kirilovs 1 , N. Gabruseva 21 Daugavpils University, Daugavpils, Latvia,2 Riga Stradins University, LatviaAnalysis of the structural /functional organization cell membranes, specifically, thestudies of the functional state of beta adrenoreceptors is a topical problem in medicine andbiology. The direct detection of functional activity of adrenoreceptors is methodologicallycomplete and therefore not suitable for systemic determination. Earlier works have shownthe unidirectivity of the changes in the adrenoreceptive properties of various tissues andorganisms (red blood cells including) during the pharmacological action on the adrenergicmechanisms of regulation in an organism. Thus, red blood cells are a convenient testobject which makes it possible to judge on the changes in the adrenoreactivity of anorganism on the whole.We investigated the binding of n-toluene sulfonate-4-(n-dimethylaminostyrene)-Imethylpyridinium(DSM) to human and animal erythrocyte membrane. Probe –cationDCM possess such properties as polychromaticity, sensitivity to the changes in polarity,viscosity of medium. The binding of probe to the erythrocyte membrane was studied inpathological states (myocardial infarction, instable stenocardia etc.; at high and lowadrenoreactivity of animals (quinea pigs, rats). Increased adrenoreactivity was due tointraperitoneal injection of quanethidine (30-50 mg/kg, 24 h); decreased adrenoreactivityby intraperitoneal injection of propranolol (5mg/kg, 20min).The emission spectra of DCMin erythrocytes consists of two bands with the maxima at 560-565 nm( exc. 470 nm) and610-620 nm (exc. 495 nm). The differences in the DCM binding parameters toerythrocytes were found at low and high adrenoreactivity of animals and observed patientsgroups as compared with control groups. Lower binding constants K and higher numberof binding sites N (an indication of less specific interactions) we found for the observedgroups of patients as compared with healthy subjects. At an increased adrenoreactivity ofthe organism it was found that the values of N do up and those of K down. At thedecreased adrenoreactivity there were lowering of N and rise of K. Changes offluorescence intensity (610-620 nm) correlates with dynamics of change in parameters Nand fluorescence intensity (560-565 nm). Similarity of the character of membraneresponse to the change in the adrenoreactivity of the organism during the effect of stressfactors and in diseases of the cardio-vascular system suggest that the along structuralrearrangement of erythrocytes membrane the structural and functional properties of theadrenoreceptors also change. Increased adrenoreactivity of animals led to enhancedaffinity of erythrocytes beta adrenoreceptors to propranolol and their binding tofluorescent robe were changed. It is concluded that probe DSM are promising in studies ofthe changes in membrane properties and in the beta-adrenoreceptive function both theerythrocytes and the organism as a whole.- 40 -

Fluorescent Studies of Human Blood Plasma Albumin Alterations andLymphocytes Subpopulations in Colorectal Cancer PatientsI. Kalnina 1 , T. Zvagule 2 , L. Klimkane 3 , N. Kurjane 3 , E. Kirilova 1 , G. Gorbenko 11 Daugavpils University, Latvia;2 Riga Stradins University, Latvia;3 Latvian Centre of Oncology, Riga, Latvia;The novel fluorescent probe ABM was used to characterize lymphocyte membranes andblood plasma albumin from colorectal cancer patients in context of the hostsimmunological parameters and state of cancer progression. For study patients withcolorectal cancer were examined: 10 one day before and 10 days after their surgicaltreatment ; 2) as disease worsened (Stages IIa, IIIb, IV); 3) advanced cancer patients. Theaim of studies was to evaluate the potential utility of measures of ABM fluorescenceparameters as a standart tool in the analysis of host immune status and for a clinicalinterpretation of alterations in albumin per se and lymphocytes functional activity inpatients. ABM spectral parameters in lymphocytes reflects interrelated properties of thecells: 1) outer membrane physicochemical state, 2 )membrane microviscosity, 3)proliferative activity, 4) lipid metabolism and/or 5) phenotypical profile. ABM binds withblood plasma albumin with high level of selectivity. Probe ABM is very sensitive to allknown conformational changes of albumin in the region pH 3-12. Spectral parameters ofABM binding with plasma albumin reflect albumin "effective" concentration (equivalentof "healthy" albumin in patients plasma), alterations of albumin globule, its physical andfunctional properties, characteristics of binding sites properties. The significant decreaseof ABM fluorescence intensity in plasma could be explained by bindingcapacity/conformational changes of the albumin in these patients. Specific relationshipwas obtained between ABM fluorescence in lymphocytes and characteristics of cells:anisotropy index, binding constant, binding sites number etc. The lymphocyte distributionamong the subsets also differed. Interestingly, the ABM fluorescence intensity in the cellsuspension and blood plasma correlate with select immunological parameters (CD4+,CD8+, ratio CD4+/CD8+, CD38+, CD16+ , level of immunoglobulines IgA, IgG, IgMetc.). Decrease in the CD4+/CD8+ ratio mainly depend on an increase in the T-suppressor cells in patients without metastases, whereas it is due to a decrease in the T-helper cell in most patients with metastatic disease. Surgical treatment affectsimmunological parameters and apperead to elevate lymphocytes functional activity. Thepreoperative immune state of patients is important for their survival. Immunosupressionincreased gradually with progress of cancer; capacity of albumin binding reserve and„effective” concentration decrease. These findings suggest likely physical (structural) andfunctional alterations in the patients cells and plasma were a function of cancer stage. Inadvanced cancer, in contrast to other groups, the absolute number of lymphocytes haddirect (not inverse) correlation with ABM fluorescence intensity.The higher level oflymphocytes number, T-cell proliferative activity, and albumin ‘’effective ‘’ concentrationhave a beneficial effect on overall survival. There was excellent agreement betweenchanges in spectral characteristics and both clinical and pathological estimates of diseaseseverity. Measures of ABM fluorescence intensity values in blood plasma andlymphocytes (as reflection of their functional activity) might be a useful tool in theevoluation of the immune status of patients in clinics, including prognosis, prediction oftherapeutic efficacy, treatment outcomes. The fluorescence-based method is lessexpensive, not very time-consuming, technically simple, 100 times more sensitive thanstandart ones.- 41 -

Oksidatīvā stresa marķieru izmaiņas Černobiļas AES avārijas sekulikvidētājiem pēdējo 10 gadu laikāA. Šķesters 1 , A. Silova 1 , T. Zvagule 2 , J. Reste 2 , L. Lārmane 1 , Ņ. Rusakova 1 , N. Gabruševa 2 .1RSU Bioķīmijas laboratorija, Rīga, Latvija;2Paula Stradiņa KUS Aroda un radiācijas medicīnas centrs, Rīga, Latvija1986. gada 26. aprīlis ir kļuvis par neatgriezenisku likteņa pavērsiena punktu vairāk kā 6000tūkstošiem Latvijas iedzīvotāju un viņu ģimeņu. Šī diena mainīja ne tikai cilvēku dzīves, betienesa ievērojamas izmaiņas visas Eiropas saimnieciskajā un finanšu sistēmā.Padomju „ kodolzinātnes eksperimenta” rezultātā, uz cilvēkiem iedarbojās ne tikai jonizējošāradiācija, bet atmosfērā nonāca radioaktīvie izotopi ( 137 Cs, 90 Sr, Ru, 131 J) un putekļi, cilvēkicieta no nekvalitatīvas pārtikas, ūdens, fiziskas pārpūles un psiho-emocionālās spriedzes. Šokaitīgo faktoru rezultātā, jau sākotnēji konstatēja akūtu oksidatīvo stresu, kurš vēlāk pārgājahroniskā formā. Pašreiz, attiecībā uz Černobiļas AES avārijas seku likvidētājiem, pieņemtslietot terminu – ilgstošs hronisks oksidatīvais stress, kura, pastāvīgas un ilgstošas iedarbībaslaukā, ir lipīdi un proteīni, kuri lokalizēti šūnu membrānas intra- un ekstra-celulārāsstruktūras, gan arī enzīmi un hormoni. Oksidatīvo stresu un tā pakāpi raksturojošos marķierusvar iedalīt pēc to veidošanās – no lipīdu oksidācijas procesa (LPO) nākušie, un tie marķieri,kuri veidojas proteīnu oksidācijas (PO) laikā. Bez tam, oksidatīvā stresa lielumu raksturoenzīmi-antioksidanti: Cu,Zn-superoksīddismutāze, katalāze, glutationa peroksidāze, tādi neenzimatiskieantioksidanti, kā vitamīns E, albumīni, selēns, arī varš, cinks, askorbāts, un citi.Mēs salīdzinājām dažus no minētajiem oksidatīvā stresa marķieriem, kurus analizējām Iposmā (1998/99. gadā) un II posmā (2010/11. Gadā).Nosakot lipīdu oksidācijas intensitāti I posma laikā, konstatējām ievērojamu oksidācijasprocesu intensificēšanos – lipīdu hidroperoksīdu (LOOH) saturs asins plazmā trīskārtīgipārsniedza LOOH saturu praktiski veselu atbilstoša vecuma vīriešu asinīs. Gandrīz dubultīgibija pārsniegta lipīdu (nepiesātināto taukskābju) oksidējamība. LPO procesa norises ātrumsČAES avārijas seku likvidētājiem, vairāk kā 3 reizes pārsniedza normu. Ievērojami palielinātsizrādījās arī malondialdehīds (MDA). Tai pašā laikā, avārijas seku likvidētāju asinīs,konstatējām ievērojamu selēna deficītu, kurš svārstījās robežās no neliela ( 68 ± 10 µg/L )līdz dziļam (46 ± 10 µg/L ) deficītam. Ievērojami samazināta bija glutationperoksidāzesaktivitāte, vitamīna E (α-tokoferola) saturs un kopējā antioksidatīvā organisma aizsardzība(AOA) asinīs. 10 gadus vēlāk, nosakot oksidatīvā stresa marķierus ČAES avārijas sekulikvidētājiem (pirms antioksidantu un nesteroīdo pretiekaisuma līdzekļu indicēšanas),konstatējām, ka, gandrīz visi iepriekšējie marķieri saglabājuši savas izmaiņas attiecībā pretnormu. Tiesa, daži no marķieriem ir ievērojami samazinājušies un tuvojas normas robežai(AOA, selēna un E vitamīna saturs asinīs), citi palikuši gandrīz nemainīgi (LOOH, MDA,lipīdu oksidējamība un LPO norises ātrums). Praktiski nemainīga saglabājusies kopējāorganisma antioksidatīvā aizsardzība. Tādējādi apstiprinājās pieņēmums, ka ČAES avārijasseku likvidētāju organismā joprojām saglabājas hronisks oksidatīvais stress.Pēc dabīgo antioksidantu (AO), ne-steroīdo pretiekaisuma (NSAID) līdzekļu vai AO/NSAIDkombinācijas lietošanas, ČAES avārijas seku likvidētājiem, ievērojami samazinājāsoksidatīvo stresu raksturojošie marķieri, daži – MDA, selēns, vitamīns E sasniedza zemākonormas robežu.Tādējādi esam ieguvuši apstiprinājumu, ka dabīgos antioksidantus vēlams iekļautkompleksajā terapijā gadījumos, kad oksidatīvā stresa marķieri uzrāda ievērojamas atšķirībasno normas. Paralēlajā klīniskajā pētījuma daļā atklātas labvēlīgas izmaiņas klīniskajā ainā pēcdabīgo antioksidantu lietošanas. Tai skaitā: samazinājās pieprasījums pēc recepšumedikamentiem, kritās holesterīna līmenis asinīs, mazinājās locītavu un citas lokalizācijassāpes, migrēna, depresija, kuņģa problēmas.- 42 -

Jaunu - liposomas veidojošu katjonu karbazola atvasinājumu izmantošanašūnu transfekcijā in vitroA. Vežāne 1 , G. Apsīte 1 , A. Baran 2 , R. Brūvere 1 , V. Ose 2 , A. Plotniece 2 , I. Timofējeva 1 , T.Kozlovska 11Latvijas Biomedicīnas pētījumu un studiju centrs (BMC), Rīga, Latvija2Latvijas Organiskās sintēzes institūts (OSI), Rīga, Latvijae-pasts: aleksandra.vezane@biomed.lu.lvPēdējā laikā strauji pieaug interese par fluorescento krāsvielu – t.s. zonžu pielietojumubioloģijā un medicīnā. Arī Latvijā, Latvijas Organiskas sintēzes institūtā (OSI), tieksintezēti jauni bioloģiski aktīvi savienojumi, kuru fluorescentās īpašības tiek plaši pētītasLatvijas Biomedicīnas pētījumu un studiju centrā (BMC).Konkrētā pētījuma mērķis bija izvērtēt 3 jauno - liposomas veidojošo katjonu karbazolaatvasinājumu izmantošanas potenciālu, pielietojot tos šūnu transfekcijā in vitro.Jauno - liposomas veidojošo katjonu karbazola atvasinājumu spēju veidot kompleksus arģenētisko materiālu (plazmīdas DNS), vērtēja, izmantojot gan elektronmikroskopiju, ganretardāciju agarozas gēlā. Jauno savienojumu spēja piegādāt ģenētisko materiālukultivējamām eikariotu šūnām jeb transfekcijas efektivitāte – tika noteikta izmantojotfluorescento mikroskopu.Ar tā palīdzību tika izvērtēts (GFP - zaļi fluorescējoša proteīna), ekspresijas līmenis, kuruieguvām, ievadot šūnās plazmīdu pEGFP. Pētāmās šūnās tika transficētas arī ar plazmīdupLac-Z. Šī fermenta β-galaktozidāzes aktivitātes pakāpe tika noteikta pielietojotspektrofotometriju. Lai noteiktu ekspresējošā GFP proteīna lokalizācijas vietu transficētāsšūnās, tika pielietota konfokālā lāzera skenējošā mikroskopija.Pētījumu rezultātā tika pierādīts, ka bioloģiski aktīvās fluorescentās zondes spēj ne tikaispecifiski saistīties ar šūnu membrānas struktūrām, tā iezīmējot šūnas un padarot tāsfluorescējošas, bet arī spēj saistīt ģenētisko materiālu, veidojot liposomas un tādējādi totransportēt mērķa šūnās. Šis darbs tika finansiāli atbalstīts no Valsts pētījumu programmaslīdzekļiem.1. Chang, C.-C. Chemistry and Biodiversity, 1, 1377 (2004).2. Saengkhae, C. Eur. J. Pharm., 559, 124 (2007).3. Tanious, F. Biochemistry, 39, 12091 (2000).4. Dias, N. Biochemistry, 43, 15169 (2004).5. Chang, C.-C. J. Chin. Chem. Soc. 50, 185 (2003).- 43 -

Glikogēna noteikšana raugos S. cerevisiae izmantojot jaunus uzbenzantrona bāzes sintezētos fluoroforusI.KrallišaLatvijas Universitātes Mikrobioloģijas un biotehnoloģijas institūts, Rīga, Latvijakrallish@latnet.lvFluorescentās metodes mūsdienās uzskata par progresīvu un efektīvu pieejumikroorganismu fizioloģijas pētījumiem, kā arī biotehnoloģijas jomā. Izmantojot uzbenzantrona bāzes sintezētās fluorescentās zondes, tika veikti Saccharomyces cerevisiaeiekš šūnu rezerves metabolīta glikogēna biosintēzes pētījumi oksidatīvā stresa - 10% (v/v)spirta barotnē un normālos augšanas apstākļos. S. cerevisiae ir vērtīgo bioloģiski aktīvovielu producents un plaši izmantots pētījumos kā eukariotisko šūnu modelis, tāpēc tas irļoti perspektīvs un piemērots modelis fluorescento metožu izstrādei ar mērķi pētīteikariotisko organismu šūnu noturības mehānismus dažādu stresa faktoru ietekmei uzšūnu fizioloģiju.Lai atlasītu efektīvas fluorescentās zondes tika pārbaudīta jauniegūto zonžu piemērotībaun aktivitāte attiecībā pret mērķa savienojumu glikogēnu, kurš tika izdalīts no S.cerevisiae šūnām. Fluorescento zonžu efektivitāte tika salīdzināta ar komerciāloglikogēnam specifisko akriflavīna efektivitāti. Kā efektivitātes rādītāji tika ņemti zonžuspektrālu parametru (emisijas maksimums un fluorescences intensitāte) atkarība noglikogēna koncentrācijas. Tika pārbaudītas vairākas fluorescentās zondes izmantojotglikogēna standarta šķīdumu , kā arī glikogēns tika izdalīts no S. cerevisiae šūnām augotsintētiskajā Ridera barotne vai YPG barotnē. Glikogēns tika izdalīts no raugu šūnāmizmantojot 0,25 M Na 2 CO 3 (sārmainā frakcija) un 0,5M HClO 4 (skābā frakcija), kā arīdots kopējais glikogēna saturs šūnās. Dati iegūtie glikogēna satura noteikšanā, izmantojotakriflavīnu un atlasītos uz benzantrona bāzes sintezētos fluorescentos zondus tikasalīdzināti ar glikogēna noteikšanu šūnu ekstraktos ar vispārpieņemto ķīmisko antronametodi.Pētījumu rezultātā tika parādīta pozitīva korelācija starp glikogēna saturu un fluorescencesintensitāti izmantotos paraugos, kas ļauj izmantot jaunos uz benzantrona bāzes sintezētosfluorescentos zondus glikogēna satura ātram skrīningam raugu šūnu ekstraktos. Raugušūnas oksidatīva stresa apstākļos palielina rezerves savienojuma glikogēna sintēzi, līdzīgikā agrāk novērotam trehalozes uzkrājumam palielināšanai līdzīgos oksidatīva stresaapstākļos.- 44 -

Alkilrezorcinolu izdalīšana un noteikšana baktērijās Azotobacterchroococcum.R.LindeLatvijas Universitātes Mikrobioloģijas un biotehnoloģijas institūts, Rīga, LatvijaRaimonds.linde@lu.lvVeikta alkilrezorcinolu izdalīšana no baktērijām Azotobacter chroococcum ar Fast Blue Breaģentu un to noteikšana ar dažādām metodēm. Tika pārbaudīts alkilrezorcinoludaudzums baktērijās Azotobacter chroococcum, kas fermentētas dažādos vides apstākļosizmantojot spektrofotometru Shimadzu UV-260, kā arī ar plānslāņa hromatogrāfiju.Alkilrezorcinolu absorbcija tika noteikta ar vairākām standartvielām.pH izmaiņu ietekme uz fluorescento krāsvielu emisijas spektriemE.PīmaneLatvijas Universitātes Mikrobioloģijas un biotehnoloģijas institūts, Rīga, Latvija;epimane@inbox.lvPētījumam tika izmantotas vairākas jaunas benzantronu atvasinājumu krāsvielas.Eksperimentā tika pārbaudīta krāsvielu fluorescences īpašību noturība, mainoties vides pHlīmenim. pH izmaiņas ietekmē krāsvielu fluorescences spektru emisijas maksimumus. pHlīmeņi destilētam ūdenim tika regulēti ar 1N un 4N HCl un 0,1N, 1N un 4N NaOH.Mērījumi tika veikti, izmantojot fluorescences spektrofotometru Fluoromax3.- 45 -

Fluorescentās krāsvielas P8 izmantošana polihidroksibutirāta unpolihidroksibutirāta-ko-hidroksivalerāta lateksu kompozītplēvjuraksturošanaiL. SavenkovaLatvijas Universitātes Mikrobioloģijas un biotehnoloģijas institūts, Rīga, Latvija;azot@latnet.lvNo PHB un PHB/HV lateksiem tika izveidotas kompozītplēves, atšķirīgas pēc PHAgranulu, polivinilspirta, glicerīna un fluorescentās krāsas P8 satura. Iegūtām PHA lateksapolimērplēvēm ir noteiktas mehāniskās īpašības. Ir secināts, ka kompozītplēvju sastāvsļoti lielā mērā ietekmē gan mehāniskās īpašības, gan stiprību, gan elastību.PHA lateksa kompozītplēvju virsmas struktūra tika vizualizēta ar konfokālā lāzeramikroskopijas metodi. Izveidoto kompozītplēvju izturību ūdens šķīdumos novērtēja pēcfluorescentās krāsvielas migrāciju inkubācijas vidē. Iegūtie dati par kompozītplēvjuvirsmas struktūras izmaiņām un fluorescentās krāsvielas migrāciju ļauj labāk izprast, kālateksu kompozītu sastāvs ietekmē plēvju mehāniskās un fizikālās īpašības.Dzelzs jonu ietekme uz izmaiņām šūnā, alkilrezorcīnu un PHB veidošanosAzotobacter chroococcum šūnāsE.PīmaneLatvijas Universitātes Mikrobioloģijas un biotehnoloģijas institūts, Rīga, Latvija;epimane@inbox.lvDzelzs jonu saturošiem savienojumiem ir ļoti liela nozīme atmosfēras slāpekli fiksējošaibaktērijai Azotobacter chroococcum. Dzelzs daudzums ietekmē A.chroococcum augšanuun poli-3-hidroksibutirāta (PHB) veidošanos, kas ir oglekļa un enerģijas rezervessavienojums. A.chroococcum šūnas novecojoties vai arī stresa apstākļos no aktīvi dalošāsveģetatīvās formas pāriet miera stāvoklī un pārvēršas par cistām. Cistu veidošanās ir lielanozīme alkilrezorcīniem, kas aizsargā šūnu no ārējās vides iedarbības. Vairāki pierādījumiliek domāt, ka metāli, tāpat kā alkilrezorcīni, var ietekmēt membrānu plūstamību.Fluorescentās metodes ir pietiekami jutīgas, ātras un tehniski vienkāršas un tādēļ var tiktizmantotas sākotnējai šūnu pārbaudei, lai noteiktu radušās izmaiņas.Ar fluorescentajām un spektrofotometriskajām metodēm tika pārbaudīta dzelzs ietekme uzalkilrezorcīnu un PHB saturu šūnā. Izmaiņas šūnu suspensijas fluorescences intensitātētika pārbaudītas ar fluorescento metodi, kur tika izmantota jauna, hidrofobā krāsviela P8(3-piperidinobenzantrons).- 46 -

Fluorescentie benzantrona atvasinājumi: kvantu-ķīmiskie aprēķiniS. GontaLatvijas Universitātes Mikrobioloģijas un biotehnoloģijas institūts, Rīga, Latvija;svetago@lu.lvLuminiscences krāsvielas ir atradušas plašu pielietojumu ķīmiskos un bioloģiskospētījumos. Bet interese par jaunām fluorescējošam krāsvielām turpina pieaugt. Ir zināms,ka aizvietoti N-aril amidīni parāda intensīvu luminiscenci šķīdumā. Lai izpētītuelektroniskās struktūras un īpašības sintezētiem N-aizvietotiem amidīnubenzo[de]antracen-7-ons atvasinājumiem, tika veikti kvantu-ķīmiskie aprēķini.Katram savienojumam veikta ģeometrijas optimizācija ar AM1 metodi. Aprēķini liecina,ka aromātiskam kodolam ir plakana konfigurācija, kā arī ir divi iespējamie virzieni noamidīna aizvietotāja. Aprēķina rezultāti korelē arī ar sarkano nobīdi savienojumuabsorbcijas un emisijas spektros.Amidīna aizvietotāji nerāda lielu ietekmi uz HOMO un LUMO enerģijām, kas aprēķinātasar jebkuru no izmantotajām metodēm. Aizvietotāji galvenokārt ietekmē oglekļa atomaamidīna grupas, no otras puses, pie amidīna fragmenta esošie aizvietotāji ievērojamiietekmē molekulas dipolu momentu. Tas aug, ja pie amidīna fragmentu molekulas irelektronu donoru grupas (Me, Et).Benzantrona atvasinājumu elektroniskie spektri tika aprēķināti izmantojot ZINDO/Smetodi. Aprēķini liecina par intensīvu (oscilators spēks ir ~ 0,6-0,9) absorbcijas joslu pie370-380 nm. Pamatā tas ir elektronu pāreja HOMO/LUMO.- 47 -

Benzantrona atvasinājumu fizikāli-ķīmisko īpašību un ķīmiskās struktūraskorelatīvā analīzeV. Savenkovs 1 , I.Gotlibovičs 11 Latvijas Universitātes Mikrobioloģijas un biotehnoloģijas institūts, Rīga, Latvija;e-pasts: newmikrob@latnet.lvDotā projekta ietvaros veidotā fluoroforu datu bāze atrodas pastāvīgā attīstībā. Ar katriemnākamajiem jaunajiem eksperimentālajiem datiem tiek uzstādīti jauni uzdevumi viņuapkopojumam. Lai atrisinātu tādus uzdevumus nepieciešams piesaistīt adekvātasmatemātiskās apstrādes metodes, kas pie pietiekoša eksperimentālo datu apjoma varpalīdzēt apkopot šo datu masīvu ar sekojošu to izmantošanu saistības meklēšanai starppētāmo savienojumu dažādiem parametriem.Atrastās saistības tālāk var kalpot par pamatu jaunsintezējamo fluoroforu to vai cituīpašību modelēšanai. Šajā datu bāzes attīstības etapā ir aprēķināti fluoroforu molekuluģeometriskie parametri, kā arī veikta korelatīvā analīze starp absorbcijas maksimumanobīdes lielumiem un emisiju rindai jaunsintezēto benzantrona atvasinājumu, kas parādāsatšķirīgos pēc polaritātes pakāpes šķīdinātājos ar šo savienojumu atsevišķu atomu lādiņulielumiem (AM1 un Zindo/S modeļi).- 48 -

Augu šūnu funkcionālā stāvokļa pētīšana, izmantojot jauniegūtusfluoroforusN. ŠkuteEcoloģijas Instituts, Daugavpils Universitāte, 13 Vienibas , LV-5401, Daugavpils (Latvia);e-mail: natalja.skute@inbox.lvŠūnu bioloģijā ļoti strauji attīstās gan tradicionālās (piemēram, izmantojot ELISE), ganmodernās (pielietojot daudzkrāsu proteīnus) fluorescentās analīzes metodes. Jaunufluorescentu zonžu atklāšana dod būtisko impulsu dažādos šūnu bioloģijas pētījumuvirzienos. Īpaša nozīme ir fluorescento zonžu atklāšanai, kuras būtu specifiskas noteiktušūnu sastāvdaļu iekrāsošanai un kvantitatīvai nukleīnskābju, olbaltumvielu u.c. bioloģiskisvarīgu vielu analīzei, kuras nebūtu toksiskas un izmantojamas pētījumos in vivo.Lai noskaidrotu jaunsintezēto fluoescento vielu toksiskumu tika analizēta šo vieluietekme uz antioksidantu fermentu izoformu veidošanos, pigmentu koncentrāciju,hlorofila absorbciju un fluorescenci dažādos augu orgānos [1, 2]. Tika analizēta projektāsintezēto vielu ietekme uz vesela auga orgāna augšanu, izmantojot tabakas lapu audukultūru un kviešu dīgstu modeli, kā arī ietekme uz augu šūnu dzīves ciklu, dažādu augušūnu organoīdu uzbūve un tās izmaiņas 3D [4]. Tika veikti pētījumi par augu šūnufizioloģiskā stāvokļa izmaiņām dažādos apstākļos (siltumšoks, sāļu stress) [2, 3]. Tikapētītas morfoloģisko parametru izmaiņas, augu šūnas membrānu caurlaidības izmaiņasfunkcionāli dažādos kviešu dīgstu orgānos siltuma šoka ietekmē. Tika veikti antioksidantuenzīmu elektroforetiskās aktivitātes pētījumi un lipīdu peroksidācijas produktu analīze [1].Lai noskaidrotu mitohondriju lomu oksidatīvajos bojāejas procesos tika veiktieksperimenti par ciklosporīna un antimicīna lomu šajos procesos.1. J.Orlova, N. Škute. Changes of fluorescence emission spectrum in first leaf and coleoptile cells ofwheat seedlings under light stress. 6 th International Conference ‘Research and Conservation ofBiological Diversity in Baltic Region’, Daugavpils, 2011, 28-29 April.2. A.Petjukevičs, N. Škute. Current approaches for bioindication, modeling and salt stress analysis inplants. 6 th International Conference ‘Research and Conservation of Biological Diversity in BalticRegion’, Daugavpils, 2011, 28-29 April3. M.Savicka, N.Škute. Effects of high temperature on malondialdehyde content, superoxideproduction and growth changes in wheat seedlings (Triticum aestivum L.) 2010. Ekologija. 56 (1–2),. 26–33.4. M. Selga, M. Selga, A. Ozoliņa. Kodola DNS 3D uzbūves un DNS novietojuma izmaiņas šūnasnovecošanas laikā. Daugavpils Universitātes 53. starptautiskā zinātniskā konference, Daugavpils2011. gada 13.-15. aprīlis.Eiropas Sociālā fonda projekts„Starpdisciplinārās zinātniskās grupas izveidošana jaunu fluorescentu materiālu un metožu izstrādei unieviešanai” Vienošanās Nr. 2009/0205/1DP/1.1.1.2.0/09/APIA/VIAA/152- 49 -