Chapt er8 ________________________________ Summary,concl usi ons andfutureperspectives
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8.1. Introduction This thesis deals with various aspects of intracellular behavior of antitumor-active dinuclear platinum complexes, i. e. their anticancer activity, cellular distribution and nephrotoxicity.This final chapter presents a summary of the most important results described in the thesis and gives an overview of the future perspectives in this research field. A maj or part of this thesis is focused on the investigation of cellular processing of dinuclear platinum anticancer drugs using fluorescence microscopy.Besides, design of new dinuclear platinum anticancer compounds is described, the relationships between the structure and nephrotoxicity of dinuclear complexes are discussed, and application of dinuclear platinum complexes in drugtargetingis evaluated. 8.2. Summary Chapter 1 presents an introduction to the research topics described in this thesis and gives an overview of the relevant literature.First, the basic concepts of platinum antitumor chemistry and biochemistry are introduced, and then the relevant research areas are reviewed in detail. New antitumor-active azine-bridged dinuclear platinum complexes are described in Chapter 2.These complexes contain 2,5-dimethylpyrazine, quinazoline and phthalazine as bridgingligands.They are structurally similar to the recently described cytotoxic complexes with pyrazine, pyrimidine and pyridazine,1 and thus, extend this new class of dinuclear platinum anticancer drugs.The complexes described in Chapter 2 show only moderate cytotoxicity in various cancer cell lines. However, they induce apoptosis and partly overcome cisplatin resistance in mouse leukemia cells.The structure-activity relationship for azine-bridged dinuclear platinum complexes is established.The complexes that possess additional groups in the azine ring, which induce steric hindrance and hamper bindingof the complex to nuclear DNA, are less cytotoxic than the complexes with unsubstituted azines. The substituents on the ligand, which can provide additional interaction with DNA such as intercalation, significantly improve the antitumor properties of the complex. Chapters 3–5 deal with investigation of intracellular distribution of dinuclear platinum anticancer drugs using fluorescence microscopy.Fluorescence microscopy is a powerful method, which allows to study drug processing in living cells.However, most of platinum drugs are not fluorescent. Two different approaches have been considered to enable fluorescence microscopy studies of platinum complexes.One approach has been the design of new platinum antitumor drugs with fluorescent ligands.This method combines the development of new drugs and investigation of their intracellular behavior.Another approach has been the labeling of promising platinum anticancer complexes with a fluorescent reporter.The first approach has been applied in Chapters 3 and 4.These chapters report cellular
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processing of
new
dinuclear
platinum
complexes
with
fluorescent
diaminoanthraquinones and the respective free ligands in different cancer cell lines. Both the anthraquinones and their dinuclear complexes were earlier shown to exhibit high cytotoxicity in A2780human ovarian carcinoma cells.2 Their cellular processing in A2780and cisplatinresistant A2780cisR cell lines is described in Chapter 3. The ligands are processed similarly by sensitive and resistant cells, which is in agreement with the cytotoxicity data showing that the compounds overcome cisplatin resistance in A2780cisR cells. In contrast, the intracellular distribution of the dinuclear platinum complexes with diaminoanthraquinones in the resistant and sensitive cell lines is very different. In A2780cisR cells, the complexes are sequestered in acidic vesicles in the cytosol, which prevents them from binding to nuclear DNA. This phenomenon was not observed in the sensitive A2780cell line. The cytotoxicity tests showed that the dinuclear complexes with anthraquinones are cross-resistant with cisplatin in A2780cisR. Thus, sequestration of the complexes in lysosomal vesicles explains their decreased activity in this cell line. However, this mechanism was found to be not related to the deactivation of platinum complexes by glutathione, which plays an important role in the resistance profile of A2780cisR cell line. Therefore, encapsulation of the complexes and their inactivation by intracellular GSH are two different resistance mechanisms, which appear to operate independently. Chapter 3 presents the first example of sequestration in cellular organelles as a mechanism of cisplatin resistance. Chapter 4 is closely related to Chapter 3. It deals with cellular processing of the abovementioned dinuclear platinum complexes with diaminoanthraquinones and the respective free ligands in U2-OS human osteosarcoma cell line and its cisplatin-resistant derivative, U2-OS/ Pt subline. Cellular distribution of the compounds in U2-OS sensitive/ resistant pair of cell lines is different from their distribution in A2780 sensitive/ resistant cell line pair. Cellular processing of the platinum complexes, as well as the anthraquinones, is similar in U2-OS and U2-OS/ Pt cells. This finding is consistent with the results of the cytotoxicity tests, as all the compounds overcome resistance in the U2-OS/ Pt cell line. Furthermore, no sequestration of the platinum complexes in this cell line has been observed. Higher activity and different intracellular distribution of the dinuclear complexes in U2-OS/ Pt cells compared to A2780cisR cells results from different resistant profiles of these cell lines. Intracellular GSH content in A2780cisR is much higher than in U2-OS/ Pt cells,3,4 which partly accounts for cross-resistance of the dinuclear complexes with cisplatin. In contrast to U2-OS/ Pt cells, A2780cisR cells sequester the complexes in lysosomes, thereby preventing them form binding to nuclear DNA. Sequestration of the platinum compounds in A2780cisR cell line has been found to result from alkalinization of lysosomes. Failure to maintain normal lysosomal pH leads to a general defect in endocytosis, which appears to facilitate sequestration. Chapter 5 describes new fluorescent-labeled dinuclear platinum complexes designed for investigation of cellular processing of promising dinuclear platinum antitumor drugs. The
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cis- and trans-configured complexes have been modified with a fluorogenic tag. The modified compounds have been shown to be good models for the parent dinuclear platinum anticancer complexes. They interact with a guanine model base similarly to the label-free complexes. The labeled complexes also mimic some of the pharmacokinetic properties of the unlabelled compounds. Cellular processing of the new complexes with a fluorescent label in U2-OS and cisplatin-resistant U2-OS/Pt human osteosarcoma cells has been investigated. A platinum-free compound with the same label has been used in the control experiment. Both cis and t rans dinuclear platinum complexes have been found to accumulate in the nucleus one hour after internalization, and to be excreted out of the cell via the Golgi complex. Platinum-free control compounds showed no specific localization, which confirms that the observed results are induced by the dinuclear platinum moiety, and not just by the label. Application of dinuclear platinum complexes in drug targeting is described in Chapter 6. The dinuclear platinum moiety has been found to be suitable for coupling small organic drugs to carrier proteins. However, the synthesis of drug-carrier conjugates based on dinuclear platinum unit is rather complicated, and the overall yield is quite low. For these reasons, dinuclear platinum complexes are not very likely to find a broad application in drug targeting. Chapter 7 discusses the relationships between structure of dinuclear platinum complexes and their nephrotoxicity. In the case of the dinuclear platinum complexes with rigid ligands, sterically hindered complexes are less toxic because of their poor uptake and lower reactivity. Toxicity of the dinuclear complexes with flexible ligands depends on the geometry of the ligands around platinum:cis-configured compounds are more toxic than their trans-counterparts. 8.3. Conclusions and future perspectives Nowadays, cancer is one of the most widespread diseases in the world. It can start growing in any organ of the body and may later have serious consequences for the whole organism. A number of cancer types exist, and they are all very different from one another. Each cancer has its specific features, which makes treatment a challenging and complicated task. Chapter 4 clearly shows that drug behavior in different tumor types is not the same. Therefore, investigation of cellular processing of platinum and organic anticancer drugs in various cancer cell lines is of great importance. Fluorescence microscopy appears a very useful tool in these studies. As shown in this thesis, platinum complexes with fluorescent ligands or fluorescent-labeled analogues of platinum drugs can be used for investigation of cellular distribution of platinum compounds. Resistant cell lines with different resistant profiles may be studied. It would be also very interesting to compare cellular processing of platinum drugs in cell lines with intrinsic and acquired resistance to cisplatin. Identification
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of proteins involved in intracellular transport of platinum complexes is of great interest. It has been recently shown that copper homeostasis proteins are involved in uptake and efflux of platinum anticancer drugs.5-8 Therefore, the role of copper transporters in intracellular distribution of platinum complexes deserves detailed investigation. Platinum drugs are now widely used in cancer chemotherapy. However, toxicity and tumor resistance significantly limit their clinical use. Therefore, development of new anticancer agents remains of great importance. Targeting drugs to a tumor appears a very promising approach, as it may increase the therapeutic efficacy of drugs, decreasing at the same time undesirable side effects. Drug targeting may also help to overcome resistance. As shown in this thesis, drugs can be coupled to carrier proteins via a platinum moiety. This approach presents some advantages over the widely used covalent coupling. It allows coupling of drugs, which lack chemically reactive groups for covalent binding. Furthermore, the rate of drug release in the targeting conjugates based on platinum complexes can be controlled by choosing an appropriate coordination surrounding of platinum. W orldwide an increasing number of new drug candidates is being designed. M any of them show high anticancer activity and overcome resistance. However, it is very important to perform preliminary toxicity tests on new compounds synthesized as potential antitumor drugs. Defining structure-activity and structure-toxicity relationships for new classes of compounds is of great interest. It will help in the design of new drugs, which exhibit high antitumor activity in combination with low toxicity. References 1. Komeda, S.;Kalayda, G. V.;Lutz, M .;Spek, A. L.;Yamanaka, Y.;Sato, T.;Chikuma, M .;Reedijk, J. Journal of Medicinal Chemistry 2003, 46, 1210. 2. Jansen, B. A. J.;W ielaard, P.;Kalayda, G. V.;Ferrari, M .;M olenaar, C.;Tanke, H. J.; Brouwer, J.;Reedijk, J. J.Biol.Inorg.Chem.2004, 9, 403. 3. Perego, P.;Caserini, C.;Gatti, L.;Carenini, N.;Romanelli, S.;Supino, R.;Colangelo, D.; Viano, I.;Leone, R.;Spinelli, S.;Pezzoni, G.;M anzotti, C.;Farrell, N.;Zunino, F. Mol. Pharmacol.1999, 55, 528. 4. Perez, J. M .;M ontero, E. I.;Quiroga, A. G.;Fuertes, M . A.;Alonso, C.;NavarroRanninger, C. Metal-Based Drugs 2001, 8, 29. 5. Lin, X. J.;Okuda, T.;Holzer, A.;Howell, S. B. Mol.Pharmacol.2002, 62, 1154. 6. Samimi, G.;Safaei, R.;Katano, K.;Holzer, A. K.;Rochdi, M .;Tomioka, M .;Goodman, M .;Howell, S. B. Clin.Cancer Res.2004, 10, 4661. 7. Katano, K.;Safaei, R.;Samimi, G.;Holzer, A.;Tomioka, M .;Goodman, M .;Howell, S. B. Clin.Cancer Res.2004, 10, 4578. 8. Safaei, R.;Howell, S. B. Critical Reviews in Oncology Hematology 2005, 53, 13.
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Samenvatting (Summary in Dutch) Vandaag de dag is kanker een van de meest wijdverbreide ziektes ter wereld. Volgens de Wereldgezondheidsorganisatie leden tien miljoen mensen aan kanker in 2000, waarvan zes miljoen gestorven zijn. Kanker ontstaat vaak onopvallend en groeit heel snel. De groei ervan kan beginnen in welk orgaan dan ook en heeft ernstige gevolgen voor het hele lichaam. Er zijn een aantal verschillende kankers, en ze lijken vaak niet op elkaar. Elke kanker heeft zijn specifieke eigenschappen wat de behandeling heel ingewikkeld en uitdagend maakt. Cisplatina is een van de vaakst gebruikte antikankermiddelen. Dit medicijn is met name heel effectief in de behandeling van testikel- en ovariumkanker. Cisplatina heeft echter een aantal nadelen. Het is slecht oplosbaar in water waardoor het klinische gebruik van het medicijn onhandig wordt. Daarnaast veroorzaakt cisplatina-chemotherapie ernstige bijwerkingen, zoals misselijkheid, haaruitval en nierbeschadiging. Bovendien kunnen tumors tijdens de behandeling resistentie voor cisplatina ontwikkelen, waardoor ze niet meer reageren op het medicijn. De nadelen van cisplatina stimuleren de zoektocht naar nieuwe antikankermiddelen. In de afgelopen jaren is er een groot aantal nieuwe platinacomplexen gemaakt, waarvan slechts 28 verbindingen in aanmerking kwamen voor klinische proeven. Drie platinacomplexen (zogenaamde carboplatina, oxaliplatina en nedaplatina) zijn goedgekeurd voor routine toepassing, en ze worden nu regelmatig gebruikt bij de behandeling van kanker. Dinucleaire (en polynucleaire) platinacomplexen behoren tot een groep van zeer veelbelovende antikankermiddelen. Ze bevatten twee (of meer)platina-atomen die door een zogenaamd brugmolecuul verbonden worden. De meeste dinucleaire platinaverbindingen zijn positief geladen waardoor ze veel beter oplosbaar zijn in water dan het neutrale cisplatina. Bovendien vergroot de positieve lading de affiniteit van de complexen voor DNA, het veronderstelde ultieme doelwit van platina-antikanker-medicijnen binnen de cel. De meeste di- en polynucleaire complexen tonen hoge activiteit tegen verschillende soorten van kanker. Veel van deze complexen behouden hun activiteit in resistente cellen. Het vermogen van polynucleaire platinacomplexen om de resistentie tegen cisplatina te overwinnen is waarschijnlijk de meest belangrijke eigenschap van deze verbindingen. In dit proefschrift worden verschillende aspecten van de invloed van dinucleaire antikanker-platinacomplexen op kankercellen en gezonde cellen behandeld. Er wordt onder andere naar de antikankeractiviteit, toxische effecten en het gedrag van deze veelbelovende antikankermedicijnen in de cellen gekeken. Het onderzoek naar het transport van dinucleaire platinaverbindingen binnen kankercellen krijgt veel aandacht in dit proefschrift. Daarnaast wordt een verhouding tussen structuur en toxiciteit van dinucleaire complexen besproken en
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wordt het gebruik van dinucleaire platinaverbindingen in het ontwerpen van nieuwe geneesmiddelafgiftesystemen geëvalueerd. De inleiding tot de onderzoeksonderwerpen van dit proefschrift en een overzicht van de relevante literatuur worden gegeven in Hoofdstuk 1. Eerst worden de belangrijkste begrippen uitgelegd en vervolgens zijn de belangrijkste ontwikkelingen in de betrokken onderzoeksgebieden samengevat. Nieuwe dinucleaire antikanker-platina complexen met azine-liganden worden beschreven in Hoofdstuk 2. Deze complexen bevatten 2,5-dimetylpyrazine, chinazoline en ftalazin als bruggen tussen twee platina-atomen. De complexen die in dit hoofdstuk gepresenteerd worden, vertonen matige activiteit in diverse kankercellijnen. Ze leiden echter tot geprogrammeerde celdood en overwinnen bovendien deels de resistentie tegen cisplatina. Deze verbindingen worden vergeleken met de complexen die andere azines (pyrazine, pyrimidine en pyridazine) bevatten. De structuur-activiteitsrelatie voor de groep van dinucleaire complexen met azines is vastgesteld. De azinebrug, die het binden van het dinucleaire complex aan DNA verhindert, leidt tot de verlaagde activiteit van de stof. Het azine-ligand, dat zelf aan DNA kan binden (zoals een intercalator - een aromatisch molecuul dat tussen de nucleobasen van het DNA kan schuiven), verhoogt de activiteit van het complex. In Hoofdstukken 3 tot en met 5 wordt het transport van dinucleaire antikankerplatinacomplexen binnen de cel onderzocht met behulp van fluorescentiemicroscopie. Fluorescentiemicroscopie is een nuttige methode om het gedrag van fluorescerende stoffen in de cellen te bestuderen. De meeste platinamedicijnen fluoresceren echter helaas niet. Twee verschillende aanpakken zijn gebruikt om het onderzoek van platina-antikankermiddelen met fluorescentiemicroscopie mogelijk te maken. De eerste aanpak is het ontwerpen van nieuwe dinucleaire antikanker-platinacomplexen met fluorescerende bruggen. In dit geval gaat het ontwikkelen van de nieuwe medicijnen samen met het bestuderen van hun gedrag binnen de kankercel. De andere aanpak is het vastmaken van een fluorescerende label aan potentiële antikankermiddelen. De eerstgenoemde aanpak wordt in Hoofdstukken 3 en 4 toegepast. In deze hoofdstukken wordt het gedrag van de dinucleaire platinacomplexen met diaminoantrachinonen als bruggen tussen twee platina-atomen en diaminoantraquinonen zelf binnen ovariumkanker en osteosarcoma (botkanker) cellen onderzocht. De activiteit en het transport van de bovengenoemde antraquinonen en hun dinucleaire platinacomplexen in A2780 (cisplatina-gevoelige) en A2780cisR (cisplatina-resistente) ovarium-kankercellijnen wordt beschreven in Hoofdstuk 3. De vrije liganden (antrachinonen)
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worden op eenzelfde manier verwerkt door gevoelige en resistente cellen. Dit komt overeen met de resultaten van de activiteitstesten die laten zien dat de antrachinonen even actief zijn in de A2780- en A2780cisR-cellijnen. Het gedrag van de dinucleaire platinacomplexen met antrachinonen is verschillend in gevoelige en resistente ovarium-kankercellen. In de resistente A2780cisR-cellijn worden de complexen afgezonderd in lysosomen (organellen waarbinnen lage pH heerst, bestemd voor het verwerken en vervoer van de stoffen binnen de cel). In de gevoelige A2780-cellen is dat niet het geval. Het afzonderen van de platinacomplexen in A2780cisR-cellen heeft tot gevolg dat deze stoffen niet meer aan DNA kunnen binden, waardoor ze minder actief zijn tegen deze resistente cellen vergeleken met de A2780 (gevoelige) cellen. Het afzonderingsverschijnsel zou verklaard kunnen worden door een verhoogde concentratie van het zwavel-tripeptide glutathion in A2780cisR (resistente) cellen. Glutathion heeft namelijk een grote affiniteit voor platinaverbindingen en zou het afzonderen van de bestudeerde complexen in lysosomes kunnen bevorderen. Het onderzoek heeft echter laten zien dat het glutathion geen rol speelt in het afzonderingsproces. Hoofdstuk 4 dat verwant is aan Hoofdstuk 3, beschrijft de activiteit en het transport van de bovengenoemde diaminoantrachinonen en hun dinucleaire platinacomplexen in U2-OS (cisplatina-gevoelige) en U2-OS/Pt (cisplatina-resistente) osteosarcoma-cellijnen. De verbindingen worden heel anders verwerkt door osteosarcoma-cellen dan door ovariumkankercellen. In tegenstelling tot ovarium kankercellen verplaatsen de platinacomplexen zich op dezelfde manier binnen gevoelige en resistente osteosarcoma-cellen. Daarnaast is er geen afzondering in lysosomes waargenomen in de resistente cellijn. Het blijkt ook dat de complexen ongeveer dezelfde activiteit hebben in de gevoelige en de resistente osteosarcoma cellijn. Het verschil tussen U2-OS/Pt resistente cellen en A2780cisR resistente cellen, die behandeld worden in het voorafgaande hoofdstuk, wordt verklaard door bijzonder hoge pH in de lysosomen in de A2780cisR-cellijn vergeleken met de lysosomen in de U2-OS/Pt-cellijn. Lage pH in lysosomes is noodzakelijk voor de cel om het vervoer van stoffen in stand te houden. Verhoogde pH leidt tot een algemene storing in het invoer/afvoer-systeem van de cel en vervolgens tot het afzonderen van de platinaverbindingen in lysosomes. Het ontwerpen van nieuwe fluorescerend-gelabelde dinucleaire platinacomplexen wordt beschreven in Hoofdstuk 5. De complexen met fluorescerende labels maken het onderzoek van het transport van potentiële dinucleaire platina-antikankermedicijnen binnen kankercellen mogelijk. De dinucleaire platina-antikankermiddelen van de cis- en transconfiguratie zijn succesvol gemodificeerd met twee verschillende fluorescente labels. De gelabelde complexen blijken goede modellen te zijn voor de originele medicijnen. Ze kunnen dus worden gebruikt in het onderzoek van het gedrag van dinucleaire platinaantikankermiddelen binnen de cel.
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In Hoofdstuk 6 wordt er naar een mogelijke toepassing van dinucleaire platina complexen in het ontwerpen van nieuwe geneesmiddelafgiftesystemen gekeken. Het blijkt dat een dinucleaire platina-unit geschikt is voor de koppeling van kleine organische medicijn-moleculen aan het dragende eiwit. De synthese van een geneesmiddelafgiftesysteem gebaseerd op dinucleaire platinaverbindingen is echter ingewikkeld, en de opbrengst is bovendien laag. In Hoofdstuk 7 worden de structuur-toxiciteitsrelaties voor verschillende groepen van dinucleaire platina-antikankermiddelen behandeld. Binnen de groep van dinucleaire complexen met rigide liganden zijn flexibele complexen meer toxisch dan complexen met een stijve structuur. Dat komt door de betere opname van flexibele complexen en hun hogere reactiviteit met glutathion, een belangrijke antioxidant binnen de cel. Van de complexen met flexibele liganden zijn trans-isomeren minder toxisch dan cis-isomeren. De belangrijkste resultaten die beschreven worden in voorafgaande hoofdstukken, worden samengevat in Hoofdstuk 8. In het laatste hoofdstuk van dit proefschrift worden ook een vooruitzicht op en suggesties voor verder onderzoek gegeven.
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Ʉɨɪɨɬɤɢɣ ɡɦɿɫɬ (Summary in Ukrainian) ɇɚ ɫɶɨɝɨɞɧɿɲɧɿɣ ɞɟɧɶ ɪɚɤ ɽ ɨɞɧɿɽɸ ɡ ɧɚɣɛɿɥɶɲ ɩɨɲɢɪɟɧɢɯ ɯɜɨɪɨɛ ɭ ɫɜɿɬɿ. Ɂɚ ɞɚɧɢɦɢ ȼɫɟɫɜɿɬɧɶɨʀ Ɉɪɝɚɧɿɡɚɰɿʀ Ɉɯɨɪɨɧɢ Ɂɞɨɪɨɜ’ɹ, ɡ ɞɟɫɹɬɢ ɦɿɥɶɣɨɧɿɜ ɥɸɞɟɣ, ɫɬɪɚɠɞɚɸɱɢɯ ɜɿɞ ɪɚɤɭ ɭ 2000 ɪɨɰɿ, ɲɿɫɬɶ ɦɿɥɶɣɨɧɿɜ ɩɨɦɟɪɥɨ ɜɿɞ ɰɿɽʀ ɯɜɨɪɨɛɢ. ȼ ɛɿɥɶɲɨɫɬɿ ɜɢɩɚɞɤɿɜ ɪɚɤ ɡ’ɹɜɥɹɽɬɶɫɹ ɧɟɩɨɦɿɬɧɨ ɿ ɪɨɡɜɢɜɚɽɬɶɫɹ ɞɭɠɟ ɲɜɢɞɤɨ. Ɋɚɤ ɦɨɠɟ ɜɪɚɡɢɬɢ ɛɭɞɶ-ɹɤɢɣ ɨɪɝɚɧ ɥɸɞɢɧɢ ɿ ɡ ɱɚɫɨɦ ɜɢɤɥɢɤɚɬɢ ɩɨɪɭɲɟɧɧɹ ɨɫɧɨɜɧɢɯ ɮɭɤɧɰɿɣ ɜɫɶɨɝɨ ɨɪɝɚɧɿɡɦɭ. ȱɫɧɭɽ ɜɟɥɢɤɚ ɤɿɥɶɤɿɫɬɶ ɪɿɡɧɢɯ ɜɢɞɿɜ ɪɚɤɭ, ɿ ɜɨɧɢ ɚɛɫɨɥɸɬɧɨ ɧɟ ɫɯɨɠɿ ɨɞɢɧ ɧɚ ɨɞɧɨɝɨ ɡɚ ɫɜɨɽɸ ɩɨɜɟɞɿɧɤɨɸ. Ʉɨɠɧɢɣ ɪɿɡɧɨɜɢɞ ɪɚɤɭ ɦɚɽ ɫɜɨʀ ɨɫɨɛɥɢɜɨɫɬɿ, ɳɨ ɞɭɠɟ ɭɫɤɥɚɞɧɸɽ ɥɿɤɭɜɚɧɧɹ. ɐɢɫɩɥɚɬɢɧ ɽ ɨɞɧɢɦ ɡ ɧɚɣɛɿɥɶɲ ɲɢɪɨɤɨ ɡɚɫɬɨɫɨɜɭɜɚɧɢɯ ɩɪɟɩɚɪɚɬɿɜ ɩɪɨɬɢ ɪɚɤɭ. ȼɿɧ ɽ ɞɭɠɟ ɟɮɟɤɬɢɜɧɢɦ ɩɪɢ ɥɿɤɭɜɚɧɧɿ ɪɚɤɭ ɠɿɧɨɱɢɯ ɬɚ ɱɨɥɨɜɿɱɢɯ ɫɬɚɬɟɜɢɯ ɨɪɝɚɧɿɜ. Ɉɞɧɚɤ ɰɢɫɩɥɚɬɿɧ ɦɚɽ ɪɹɞ ɧɟɞɨɥɿɤɿɜ. Ƀɨɝɨ ɪɨɡɱɢɧɧɿɫɬɶ ɭ ɜɨɞɿ ɧɟɜɢɫɨɤɚ, ɳɨ ɧɟɡɪɭɱɧɨ ɩɪɢ ɤɥɿɧɿɱɧɨɦɭ ɡɚɫɬɨɫɭɜɚɧɧɿ. ɐɢɫɩɥɚɬɢɧ ɦɚɽ ɩɨɛɿɱɧɿ ɞɿʀ, ɡɨɤɪɟɦɚ, ɜɢɤɥɢɤɚɽ ɧɭɞɨɬɭ, ɜɢɩɚɞɚɧɧɹ ɜɨɥɨɫɫɹ ɿ ɡɚɲɤɨɞɠɚɽ ɧɨɪɦɚɥɶɧɿɣ ɪɨɛɨɬɿ ɧɢɪɨɤ. Ʉɪɿɦ ɰɶɨɝɨ, ɪɚɤɨɜɿ ɭɬɜɨɪɟɧɧɹ ɦɨɠɭɬɶ ɪɨɡɜɢɧɭɬɢ ɫɬɿɣɤɿɫɬɶ ɞɨ ɰɢɫɩɥɚɬɢɧɭ ɜ ɩɪɨɰɟɫɿ ɥɿɤɭɜɚɧɧɹ. ȼ ɬɚɤɨɦɭ ɜɢɩɚɞɤɭ ɩɨɞɚɥɶɲɟ ɡɚɫɬɨɫɭɜɚɧɧɹ ɰɶɨɝɨ ɩɪɟɩɚɪɚɬɭ ɜɬɪɚɱɚɽ ɫɟɧɫ. ɇɟɞɨɥɿɤɢ ɰɢɫɩɥɚɬɢɧɭ ɫɬɢɦɭɥɸɸɬɶ ɩɨɲɭɤ ɧɨɜɢɯ ɩɪɨɬɢɪɚɤɨɜɢɯ ɩɪɟɩɚɪɚɬɿɜ. ȼ ɨɫɬɚɧɧɿ ɞɟɫɹɬɢɪɿɱɱɹ ɛɭɥɚ ɫɢɧɬɟɡɨɜɚɧɚ ɜɟɥɢɤɚ ɤɿɥɶɤɿɫɬɶ ɧɨɜɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ, ɚɥɟ ɥɢɲɟ 28 ɡ ɧɢɯ ɜɢɹɜɢɥɢɫɹ ɜɚɪɬɢɦɢ ɩɨɞɚɥɶɲɢɯ ɤɥɿɧɿɱɧɢɯ ɞɨɫɥɿɞɠɟɧɶ. Ɍɪɢ ɧɨɜɿ ɤɨɦɩɥɟɤɫɢ ɩɥɚɬɿɧɢ (ɬɚɤ ɡɜɚɧɿ ɤɚɪɛɨɩɥɚɬɢɧ, ɨɤɫɚɥɿɩɥɚɬɢɧ ɿ ɧɟɞɚɩɥɚɬɿɧ) ɛɭɥɢ ɜɢɡɧɚɧɿ ɩɪɢɞɚɬɧɢɦɢ ɞɥɹ ɥɿɤɭɜɚɧɧɹ ɪɚɤɭ. Ɂɚɪɚɡ ɰɿ ɫɩɨɥɭɤɢ ɲɢɪɨɤɨ ɡɚɫɬɨɫɨɜɭɸɬɶɫɹ ɜ ɯɟɦɨɬɟɪɚɩɿʀ. Ⱦɜɨɹɞɟɪɧɿ ɿ ɩɨɥɿɹɞɟɪɧɿ ɤɚɬɿɨɧɧɿ ɤɨɦɩɥɟɤɫɢ ɩɥɚɬɢɧɢ ɧɚɥɟɠɚɬɶ ɞɨ ɧɚɣɛɿɥɶɲ ɩɟɪɫɩɟɤɬɢɜɧɢɯ ɩɪɨɬɢɪɚɤɨɜɢɯ ɩɪɟɩɚɪɚɬɿɜ. Ɂɚ ɪɚɯɭɧɨɤ ɫɜɨɽʀ ɿɨɧɧɨʀ ɛɭɞɨɜɢ ɰɿ ɫɩɨɥɭɤɢ ɧɚɛɚɝɚɬɨ ɤɪɚɳɟ ɪɨɡɱɢɧɹɸɬɶɫɹ ɭ ɜɨɞɿ, ɧɿɠ ɰɢɫɩɥɚɬɢɧ ɿ ɣɨɝɨ ɚɧɚɥɨɝɢ. Ʉɪɿɦ ɬɨɝɨ, ɩɨɡɢɬɢɜɧɢɣ ɡɚɪɹɞ ɤɨɦɩɥɟɤɫɧɨɝɨ ɤɚɬɿɨɧɭ ɡɛɿɥɶɲɭɽ ɫɩɨɪɿɞɧɟɧɿɫɬɶ ɞɨ ȾɇɄ. ɐɟ ɽ ɜɚɠɥɢɜɢɦ, ɨɫɤɿɥɶɤɢ ɫɚɦɟ ɜɡɚɽɦɨɞɿɹ ɩɥɚɬɿɧɨɜɢɯ ɤɨɦɩɥɟɤɫɿɜ ɡ ȾɇɄ ɪɚɤɨɜɨʀ ɤɥɿɬɢɧɢ ɜɪɟɲɬɿ ɪɟɲɬ ɩɪɢɡɜɨɞɢɬɶ ɞɨ ʀʀ ɡɚɝɢɛɟɥɿ. Ȼɿɥɶɲɿɫɬɶ ɞɜɨ- ɿ ɩɨɥɿɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɩɪɨɹɜɥɹɸɬɶ ɜɢɫɨɤɭ ɚɤɬɢɜɧɿɫɬɶ ɩɪɨɬɢ ɪɿɡɧɨɦɚɧɿɬɧɢɯ ɜɢɞɿɜ ɪɚɤɨɜɢɯ ɭɬɜɨɪɟɧɶ. Ȼɚɝɚɬɨ ɡ ɰɢɯ ɫɩɨɥɭɤ ɡɛɟɪɿɝɚɸɬɶ ɫɜɨɸ ɚɤɬɢɜɧɿɫɬɶ ɭ ɪɚɤɨɜɢɯ ɤɥɿɬɢɧɚɯ, ɫɬɿɣɤɢɯ ɞɨ ɰɢɫɩɥɚɬɢɧɭ. ɉɨɞɨɥɚɧɧɹ ɰɿɽʀ ɫɬɿɣɤɨɫɬɿ ɽ ɧɚɣɛɿɥɶɲ ɜɚɠɥɢɜɨɸ ɜɥɚɫɬɢɜɿɫɬɸ ɩɨɥɿɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ. ɍ ɞɚɧɿɣ ɞɢɫɟɪɬɚɰɿʀ ɜɢɜɱɟɧɿ ɪɿɡɧɿ ɚɫɩɟɤɬɢ ɞɿʀ ɞɜɨɹɞɟɪɧɢɯ ɩɪɨɬɢɪɚɤɨɜɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɧɚ ɪɚɤɨɜɿ ɤɥɿɬɢɧɢ ɿ ɡɞɨɪɨɜɿ ɤɥɿɬɢɧɢ, ɚ ɫɚɦɟ ɩɪɨɬɢɪɚɤɨɜɚ ɚɤɬɢɜɧɿɫɬɶ, ɬɨɤɫɢɱɧɿɫɬɶ ɿ ɩɨɜɟɞɿɧɤɚ ɰɢɯ ɩɟɪɫɩɟɤɬɢɜɧɢɯ ɩɪɨɬɢɪɚɤɨɜɢɯ ɩɪɟɩɚɪɚɬɿɜ ɭ ɤɥɿɬɢɧɿ. ȼɟɥɢɤɚ ɭɜɚɝɚ ɩɪɢɞɿɥɟɧɚ ɜɧɭɬɪɿɲɧɶɨɤɥɿɬɢɧɧɨɦɭ ɬɪɚɧɫɩɨɪɬɭ ɞɜɨɹɞɟɪɧɢɯ ɫɩɨɥɭɤ ɩɥɚɬɢɧɢ. Ʉɪɿɦ
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ɰɶɨɝɨ, ɪɨɡɝɥɹɧɭɬɢɣ ɡɜ'ɹɡɨɤ ɦɿɠ ɫɬɪɭɤɬɭɪɧɢɦɢ ɨɫɨɛɥɢɜɨɫɬɹɦɢ ɿ ɬɨɤɫɢɱɧɿɫɬɸ ɞɜɨɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ, ɿ ɨɩɢɫɚɧɟ ɜɢɤɨɪɢɫɬɚɧɧɹ ɞɜɨɹɞɟɪɧɢɯ ɫɩɨɥɭɤ ɩɥɚɬɢɧɢ ɜ ɫɢɫɬɟɦɚɯ ɞɨɫɬɚɜɤɢ ɥɿɤɭɜɚɥɶɧɨɝɨ ɡɚɫɨɛɭ ɞɨ ɞɿɥɹɧɤɢ ɞɿʀ. Ɂɚɝɚɥɶɧɢɣ ɜɫɬɭɩ ɿ ɨɝɥɹɞ ɥɿɬɟɪɚɬɭɪɢ ɧɚɜɟɞɟɧɿ ɭ ɝɥɚɜɿ 1. ɍ ɰɿɣ ɝɥɚɜɿ ɩɨɹɫɧɟɧɿ ɨɫɧɨɜɧɿ ɩɨɧɹɬɬɹ ɿ ɨɩɢɫɚɧɿ ɧɚɣɜɚɠɥɢɜɿɲɿ ɞɨɫɹɝɧɟɧɧɹ ɭ ɧɚɭɤɨɜɢɯ ɨɛɥɚɫɬɹɯ, ɳɨ ɫɬɨɫɭɸɬɶɫɹ ɬɟɦɢ ɞɢɫɟɪɬɚɰɿʀ. ɇɨɜɿ ɞɜɨɹɞɟɪɧɿ ɩɪɨɬɢɪɚɤɨɜɿ ɤɨɦɩɥɟɤɫɢ ɩɥɚɬɢɧɢ ɡ ɚɡɢɧɚɦɢ ɨɩɢɫɚɧɿ ɭ ɝɥɚɜɿ 2. ɐɿ ɤɨɦɩɥɟɤɫɢ ɦɿɫɬɹɬɶ 2,5-ɞɢɦɟɬɢɥɩɿɪɚɡɢɧ, ɯɿɧɚɡɨɥɿɧ ɿ ɮɬɚɥɚɡɢɧ ɜ ɹɤɨɫɬɿ ɦɿɫɬɤɨɜɢɯ ɥɿɝɚɧɞɿɜ. Ʉɨɦɩɥɟɤɫɢ, ɩɪɟɞɫɬɚɜɥɟɧɿ ɜ ɰɿɣ ɝɥɚɜɿ, ɩɪɨɹɜɥɹɸɬɶ ɩɨɦɿɪɧɭ ɚɤɬɢɜɧɿɫɬɶ ɩɪɨɬɢ ɪɿɡɧɢɯ ɬɢɩɿɜ ɪɚɤɨɜɢɯ ɤɥɿɬɢɧ. Ɉɞɧɚɤ ɜɨɧɢ ɡɞɚɬɧɿ ɩɨɞɨɥɚɬɢ ɫɬɿɣɤɿɫɬɶ ɞɨ ɰɢɫɩɥɚɬɢɧɭ. Ɂɪɨɛɥɟɧɨ ɩɨɪɿɜɧɹɧɧɹ ɰɢɯ ɤɨɦɩɥɟɤɫɿɜ ɡ ɤɨɦɩɥɟɤɫɚɦɢ, ɳɨ ɦɿɫɬɹɬɶ ɿɧɲɿ ɚɡɢɧɢ (ɩɿɪɚɡɢɧ, ɩɿɪɢɦɿɞɢɧ ɿ ɩɿɪɢɞɚɡɢɧ) ɜ ɹɤɨɫɬɿ ɦɿɫɬɤɨɜɢɯ ɥɿɝɚɧɞɿɜ. ȼɫɬɚɧɨɜɥɟɧɨ ɜɡɚɽɦɨɡɜ'ɹɡɨɤ ɦɿɠ ɫɬɪɭɤɬɭɪɨɸ ɤɨɦɩɥɟɤɫɿɜ ɿ ʀɯ ɩɪɨɬɢɪɚɤɨɜɨɸ ɚɤɬɢɜɧɿɫɬɸ. Ɇɿɫɬɤɨɜɢɣ ɥɿɝɚɧɞ, ɳɨ ɩɟɪɟɲɤɨɞɠɚɽ ɚɛɨ ɫɩɨɜɿɥɶɧɸɽ ɜɡɚɽɦɨɞɿɸ ɞɜɨɹɞɟɪɧɨɝɨ ɤɨɦɩɥɟɤɫɭ ɡ ȾɇɄ, ɡɧɢɠɭɽ ɚɤɬɢɜɧɿɫɬɶ ɤɨɦɩɥɟɤɫɭ, ɚ ɦɿɫɬɤɨɜɢɣ ɚɡɢɧ, ɹɤɢɣ ɫɚɦ ɡɞɚɬɧɢɣ ɡɜ'ɹɡɭɜɚɬɢɫɹ ɡ ȾɇɄ, ɩɿɞɜɢɳɭɽ ɚɤɬɢɜɧɿɫɬɶ ɤɨɦɩɥɟɤɫɭ. ɍ ɝɥɚɜɚɯ 3 – 5 ɨɩɢɫɚɧɨ ɞɨɫɥɿɞɠɟɧɧɹ ɜɧɭɬɪɿɲɧɶɨɤɥɿɬɢɧɧɨɝɨ ɬɪɚɧɫɩɨɪɬɭ ɞɜɨɹɞɟɪɧɢɯ ɩɪɨɬɢɪɚɤɨɜɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɡɚ ɞɨɩɨɦɨɝɨɸ ɮɥɸɨɪɟɫɰɟɧɬɧɨʀ ɦɿɤɪɨɫɤɨɩɿʀ. Ɏɥɸɨɪɟɫɰɟɧɬɧɚ ɦɿɤɪɨɫɤɨɩɿɹ ɽ ɞɭɠɟ ɤɨɪɢɫɧɢɦ ɦɟɬɨɞɨɦ ɞɥɹ ɜɢɜɱɟɧɧɹ ɩɨɜɟɞɿɧɤɢ ɮɥɸɨɪɟɫɰɟɧɬɧɢɯ ɫɩɨɥɭɤ ɭ ɤɥɿɬɢɧɚɯ. ɇɚ ɠɚɥɶ, ɛɿɥɶɲɿɫɬɶ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɧɟ ɽ ɮɥɸɨɪɟɫɰɟɧɬɧɢɦɢ. Ⱦɥɹ ɬɨɝɨ, ɳɨɛ ɡɪɨɛɢɬɢ ɦɨɠɥɢɜɢɦ ɞɨɫɥɿɞɠɟɧɧɹ ɩɥɚɬɢɧɨɜɢɯ ɩɪɨɬɢɪɚɤɨɜɢɯ ɩɪɟɩɚɪɚɬɿɜ ɡɚ ɞɨɩɨɦɨɝɨɸ ɮɥɸɨɪɟɫɰɟɧɬɧɨʀ ɦɿɤɪɨɫɤɨɩɿʀ, ɛɭɥɨ ɜɢɤɨɪɢɫɬɚɧɨ ɞɜɚ ɪɿɡɧɿ ɩɿɞɯɨɞɢ. ɉɟɪɲɢɦ ɫɩɨɫɨɛɨɦ ɛɭɜ ɞɢɡɚɣɧ ɿ ɫɢɧɬɟɡ ɧɨɜɢɯ ɞɜɨɹɞɟɪɧɢɯ ɩɪɨɬɢɪɚɤɨɜɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɡ ɮɥɸɨɪɟɫɰɟɧɬɧɢɦɢ ɦɿɫɬɤɨɜɢɦɢ ɥɿɝɚɧɞɚɦɢ. ȱɧɲɢɦ ɫɩɨɫɨɛɨɦ ɛɭɥɨ ɜɜɟɞɟɧɧɹ ɮɥɸɨɪɟɫɰɟɧɬɧɢɯ ɦɿɬɨɤ ɜ ɫɬɪɭɤɬɭɪɭ ɩɟɪɫɩɟɤɬɢɜɧɢɯ ɩɪɨɬɢɪɚɤɨɜɢɯ ɩɪɟɩɚɪɚɬɿɜ. ɉɟɪɲɢɣ ɩɿɞɯɿɞ ɨɩɢɫɚɧɢɣ ɭ ɝɥɚɜɚɯ 3 ɿ 4, ɚ ɞɪɭɝɢɣ – ɭ ɝɥɚɜɿ 5. ɍ ɝɥɚɜɚɯ 3 ɿ 4 ɪɨɡɥɹɧɭɬɨ ɩɨɜɟɞɿɧɤɭ ɞɜɨɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɡ ɞɢɚɦɿɧɨɚɧɬɪɚɯɿɧɨɧɚɦɢ ɿ ɫɚɦɢɯ ɞɢɚɦɿɧɨɚɧɬɪɚɯɿɧɨɧɿɜ ɭ ɤɥɿɬɢɧɚɯ ɪɚɤɭ ɹɽɱɧɢɤɿɜ ɿ ɭ ɤɥɿɬɢɧɚɯ ɪɚɤɭ ɤɿɫɬɨɤ. ɍ ɝɥɚɜɿ 3 ɨɩɢɫɚɧɨ ɩɪɨɬɢɪɚɤɨɜɭ ɚɤɬɢɜɧɿɫɬɶ ɿ ɜɧɭɬɪɿɲɧɶɨɤɥɿɬɢɧɧɢɣ ɬɪɚɧɫɩɨɪɬ ɜɢɳɟɡɝɚɞɚɧɢɯ ɚɧɬɪɚɯɿɧɨɧɿɜ ɬɚ ɞɜɨɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɡ ɰɢɦɢ ɥɿɝɚɧɞɚɦɢ ɭ Ⱥ2780 ɤɥɿɬɢɧɧɿɣ ɥɿɧɿʀ ɿ ɭ ɫɩɨɪɿɞɧɟɧɿɣ, ɫɬɿɣɤɿɣ ɞɨ ɰɢɫɩɥɚɬɢɧɭ Ⱥ2780cisR ɤɥɿɬɢɧɧɿɣ ɥɿɧɿʀ ɪɚɤɭ ɹɽɱɧɢɤɿɜ. Ɍɪɚɧɫɩɨɪɬ ɜɿɥɶɧɢɯ ɚɧɬɪɚɯɿɧɨɧɿɜ ɭ ɤɥɿɬɢɧɚɯ, ɱɭɬɥɢɜɢɯ ɞɨ ɰɢɫɩɥɚɬɢɧɭ, ɧɟ ɜɿɞɪɿɡɧɹɽɬɫɹ ɜɿɞ ɬɪɚɧɫɩɨɪɬɭ ɭ ɤɥɿɬɢɧɚɯ, ɫɬɿɣɤɢɯ ɞɨ ɰɢɫɩɥɚɬɢɧɭ. Ⱥɤɬɢɜɧɿɫɬɶ
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ɚɧɬɪɚɯɿɧɨɧɿɜ ɭ Ⱥ2780 ɿ Ⱥ2780cisR ɤɥɿɬɢɧɧɢɯ ɥɿɧɿɹɯ ɦɚɣɠɟ ɨɞɧɚɤɨɜɚ, ɳɨ ɞɨɛɪɟ ɭɡɝɨɞɠɭɽɬɶɫɹ ɡ ɪɟɡɭɥɶɬɚɬɚɦɢ ɞɨɫɥɿɞɠɟɧɧɹ ɜɧɭɬɪɿɲɧɶɨɤɥɿɬɢɧɧɨɝɨ ɬɪɚɧɫɩɨɪɬɭ ɰɢɯ ɫɩɨɥɭɤ. ɉɨɜɟɞɿɧɤɚ ɞɜɨɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ ɡ ɞɢɚɦɿɧɨɚɧɬɪɚɯɿɧɨɧɚɦɢ ɭ ɱɭɬɥɢɜɢɯ ɿ ɫɬɿɣɤɢɯ ɞɨ ɰɢɫɩɥɚɬɢɧɭ ɤɥɿɬɢɧɚɯ ɞɭɠɟ ɪɿɡɧɚ. ɇɚ ɜɿɞɦɿɧɭ ɜɿɞ Ⱥ2780 ɰɢɫɩɥɚɬɢɧ-ɱɭɬɥɢɜɢɯ ɤɥɿɬɢɧ, ɭ Ⱥ2780cisR ɰɢɫɩɥɚɬɢɧ-ɫɬɿɣɤɿɣ ɤɥɿɬɢɧɧɿɣ ɥɿɧɿʀ ɩɥɚɬɢɧɨɜɿ ɤɨɦɩɥɟɤɫɢ ɩɨɝɥɢɧɚɸɬɶɫɹ ɥɿɡɨɫɨɦɚɦɢ ɤɥɿɬɢɧ ɿ ɧɚɤɨɩɢɱɭɸɬɶɫɹ ɬɚɦ. ɇɚɤɨɩɢɱɟɧɧɹ ɤɨɦɩɥɟɤɫɿɜ ɭ ɥɿɡɨɫɨɦɚɯ ɩɨɡɛɚɜɥɹɽ ʀɯ ɦɨɠɥɢɜɨɫɬɿ ɪɟɚɝɭɜɚɬɢ ɡ ȾɇɄ ɪɚɤɨɜɨʀ ɤɥɿɬɢɧɢ ɿ ɬɚɤɢɦ ɱɢɧɨɦ ɩɪɢɡɜɨɞɢɬɶ ɞɨ ɧɚɛɚɝɚɬɨ ɧɢɠɱɨʀ ɩɪɨɬɢɪɚɤɨɜɨʀ ɚɤɬɢɜɧɨɫɬɿ ɭ Ⱥ2780cisR ɤɥɿɬɢɧɚɯ ɩɨɪɿɜɧɹɧɨ ɡ Ⱥ2780 ɤɥɿɬɢɧɚɦɢ. Ȼɭɥɨ ɩɪɢɩɭɳɟɧɨ, ɳɨ ɩɨɝɥɢɧɚɧɧɹ ɫɩɨɥɭɤ ɩɥɚɬɢɧɢ ɥɿɡɨɫɨɦɚɦɢ ɜɢɤɥɢɤɚɧɨ ɫɿɪɤɨɜɦɿɫɧɢɦ ɬɪɢɩɟɩɬɢɞɨɦ ɝɥɸɬɚɬɿɨɧɨɦ. Ⱥ2780cisR ɤɥɿɬɢɧɢ ɦɿɫɬɹɬɶ ɞɭɠɟ ɜɟɥɢɤɭ ɤɿɥɶɤɿɫɬɶ ɝɥɸɬɚɬɿɨɧɭ, ɹɤɢɣ ɜ ɫɜɨɸ ɱɟɪɝɭ ɦɚɽ ɜɟɥɢɤɭ ɫɩɨɪɿɞɧɟɧɿɫɬɶ ɞɨ ɩɥɚɬɢɧɢ. Ɉɞɧɚɤ ɞɨɫɥɿɞɠɟɧɧɹ ɩɨɤɚɡɚɥɢ, ɳɨ ɝɥɸɬɚɬɿɨɧ ɧɟ ɩɪɢɣɦɚɽ ɭɱɚɫɬɿ ɭ ɩɪɨɰɟɫɿ ɧɚɤɨɩɢɱɟɧɧɹ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɭ ɥɿɡɨɫɨɦɚɯ. Ƚɥɚɜɚ 4 ɬɿɫɧɨ ɩɨɜ'ɹɡɚɧɚ ɡ ɝɥɚɜɨɸ 3. ɍ ɧɿɣ ɪɨɡɝɥɹɧɭɬɨ ɩɪɨɬɢɪɚɤɨɜɭ ɚɤɬɢɜɧɿɫɬɶ ɿ ɜɧɭɬɪɿɲɧɶɨɤɥɿɬɢɧɧɢɣ ɬɪɚɧɫɩɨɪɬ ɜɢɳɟɡɝɚɞɚɧɢɯ ɚɧɬɪɚɯɿɧɨɧɿɜ ɬɚ ɞɜɨɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɡ ɰɢɦɢ ɥɿɝɚɧɞɚɦɢ ɭ U2-OS ɰɢɫɩɥɚɬɢɧ-ɱɭɬɥɢɜɿɣ ɤɥɿɬɢɧɧɿɣ ɥɿɧɿʀ ɿ ɫɩɨɪɿɞɧɟɧɿɣ U2-OS/Pt ɰɢɫɩɥɚɬɢɧ-ɫɬɿɣɤɿɣ ɤɥɿɬɢɧɧɿɣ ɥɿɧɿʀ ɪɚɤɭ ɤɿɫɬɨɤ. ɉɨɜɟɞɿɧɤɚ ɰɢɯ ɫɩɨɥɭɤ ɭ ɤɥɿɬɢɧɚɯ ɪɚɤɭ ɤɿɫɬɨɤ ɞɭɠɟ ɜɿɞɪɿɡɧɹɽɬɶɫɹ ɜɿɞ ʀɯ ɩɨɜɟɞɿɧɤɢ ɭ ɤɥɿɬɢɧɚɯ ɪɚɤɭ ɹɽɱɧɢɤɿɜ, ɳɨ ɨɩɢɫɚɧɚ ɭ ɩɨɩɟɪɟɞɧɿɣ ɝɥɚɜɿ. ɇɚ ɜɿɞɦɿɧɭ ɜɿɞ ɤɥɿɬɢɧ ɪɚɤɭ ɹɽɱɧɢɤɿɜ, ɬɪɚɧɫɩɨɪɬ ɞɜɨɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ ɭ ɱɭɬɥɢɜɢɯ ɿ ɫɬɿɣɤɢɯ ɞɨ ɰɢɫɥɚɬɢɧɭ ɤɥɿɬɢɧɚɯ ɪɚɤɭ ɤɿɫɬɨɤ ɿɞɟɧɬɢɱɧɢɣ. Ʉɪɿɦ ɬɨɝɨ, ɩɨɝɥɢɧɚɧɧɹ ɤɨɦɩɥɟɤɫɿɜ ɥɿɡɨɫɨɦɚɦɢ ɫɬɿɣɤɢɯ ɤɥɿɬɢɧ ɧɟ ɜɿɞɛɭɜɚɽɬɶɫɹ. ɉɪɨɬɢɪɚɤɨɜɚ ɚɤɬɢɜɧɿɫɬɶ ɞɜɨɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɭ U2-OS ɿ U2-OS/Pt ɤɥɿɬɢɧɧɿɣ ɥɿɧɿʀ ɦɚɣɠɟ ɨɞɧɚɤɨɜɚ. Ɋɿɡɧɢɰɹ ɦɿɠ U2-OS/Pt ɬɚ Ⱥ2780cisR ɰɿɫɩɥɚɬɢɧ-ɫɬɿɣɤɢɦɢ ɤɥɿɬɢɧɚɦɢ (ɝɥɚɜɚ 3) ɩɨɥɹɝɚɽ ɭ ɧɚɞɡɜɢɱɚɣɧɨ ɧɢɡɶɤɿɣ ɤɢɫɥɨɬɧɨɫɬɿ ɥɿɡɨɫɨɦ Ⱥ2780cisR ɤɥɿɬɢɧ ɩɨɪɿɜɧɹɧɨ ɡ ɥɿɡɨɫɨɦɚɦɢ U2-OS/Pt ɤɥɿɬɢɧ. ȼɢɫɨɤɚ ɤɢɫɥɨɬɧɿɫɬɶ ɥɿɡɨɫɨɦ ɽ ɧɟɨɛɯɿɞɧɨɸ ɞɥɹ ɩɿɞɬɪɢɦɚɧɧɹ ɧɨɪɦɚɥɶɧɨɝɨ ɨɛɦɿɧɭ ɪɟɱɨɜɢɧ ɭ ɤɥɿɬɢɧɿ. Ɉɬɠɟ, ɩɨɧɢɠɟɧɚ ɤɢɫɥɨɬɧɿɫɬɶ ɩɪɢɡɜɨɞɢɬɶ ɞɨ ɩɨɪɭɲɧɧɹ ɨɛɦɿɧɭ ɪɟɱɨɜɢɧ ɿ ɫɤɨɩɢɱɟɧɧɹ ɩɥɚɬɢɧɨɜɢɯ ɤɨɦɩɥɟɤɫɿɜ ɜ ɥɿɡɨɫɨɦɚɯ Ⱥ2780cisR ɤɥɿɬɢɧ. Ⱦɢɡɚɣɧ ɿ ɫɢɧɬɟɡ ɧɨɜɢɯ ɮɥɸɨɪɟɫɰɟɧɬɧɨ-ɦɿɱɟɧɢɯ ɞɜɨɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɨɩɢɫɚɧɨ ɭ ɝɥɚɜɿ 5. Ʉɨɦɩɥɟɤɫɢ ɡ ɮɥɸɨɪɟɫɰɟɧɬɧɢɦɢ ɦɿɬɤɚɦɢ ɧɚɞɚɸɬɶ ɦɨɠɥɢɜɿɫɬɶ ɞɨɫɥɿɞɠɟɧɧɹ ɜɧɭɬɪɿɲɧɶɨɤɥɿɬɢɧɧɨɝɨ ɬɪɚɧɫɩɨɪɬɭ ɩɨɬɟɧɰɿɣɧɢɯ ɩɪɨɬɢɪɚɤɨɜɢɯ ɩɪɟɩɚɪɚɬɿɜ ɧɚ ɨɫɧɨɜɿ ɩɥɚɬɢɧɢ. ɉɪɨɬɢɪɚɤɨɜɿ ɞɜɨɹɞɟɪɧɿ ɤɨɦɩɥɟɤɫɢ ɩɥɚɬɢɧɢ ɰɢɫ- ɿ ɬɪɚɧɫ-ɝɟɨɦɟɬɪɿʀ ɛɭɥɢ ɭɫɩɿɲɧɨ ɦɨɞɢɮɿɤɨɜɚɧɿ ɞɜɨɦɚ ɪɿɡɧɢɦɢ ɮɥɸɨɪɟɫɰɟɧɬɧɢɦɢ ɦɿɬɤɚɦɢ. Ⱦɨɫɥɿɞɠɟɧɧɹ ɩɨɤɚɡɚɥɢ, ɳɨ ɦɿɱɟɧɿ ɤɨɦɩɥɟɤɫɢ ɽ ɯɨɪɨɲɢɦɢ ɦɨɞɟɥɹɦɢ ɜɢɯɿɞɧɢɯ ɞɜɨɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ. Ɉɬɠɟ, ɧɨɜɿ ɞɜɨɹɞɟɪɧɿ ɤɨɦɩɥɟɤɫɢ ɡ ɮɥɸɨɪɟɫɰɟɧɬɧɢɦɢ ɦɿɬɤɚɦɢ ɦɨɠɭɬɶ ɛɭɬɢ ɜɢɤɨɪɢɫɬɚɧɿ ɩɪɢ ɜɢɜɱɟɧɧɿ ɩɨɜɟɞɿɧɤɢ ɰɢɯ ɩɟɪɫɩɟɤɬɢɜɧɢɯ ɩɪɨɬɢɪɚɤɨɜɢɯ ɩɪɟɩɚɪɚɬɿɜ ɭ ɤɥɿɬɢɧɚɯ.
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ɍ ɝɥɚɜɿ 6 ɪɨɡɝɥɹɧɭɬɚ ɦɨɠɥɢɜɿɫɬɶ ɡɚɫɬɨɫɭɜɚɧɧɹ ɞɜɨɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɭ ɞɢɡɚɣɧɿ ɧɨɜɢɯ ɫɢɫɬɟɦ ɞɨɫɬɚɜɤɢ ɥɿɤɭɜɚɥɶɧɨɝɨ ɡɚɫɨɛɭ ɞɨ ɞɿɥɹɧɤɢ ɞɿʀ. Ⱦɨɫɥɿɞɠɟɧɧɹ ɩɨɤɚɡɚɥɢ, ɳɨ ɞɜɨɹɞɟɪɧɿ ɫɩɨɥɭɤɢ ɩɥɚɬɢɧɢ ɡɞɚɬɧɿ ɫɥɭɠɢɬɢ ɦɿɫɬɤɨɦ ɦɿɠ ɜɿɞɧɨɫɧɨ ɦɚɥɟɧɶɤɨɸ ɨɪɝɚɧɿɱɧɨɸ ɦɨɥɟɤɭɥɨɸ ɦɟɞɢɱɧɨɝɨ ɡɚɫɨɛɭ ɿ ɛɿɥɤɭ, ɳɨ ɧɚɩɪɚɜɥɹɽ ɚɤɬɢɜɧɭ ɪɟɱɨɜɢɧɭ ɞɨ ɞɿɥɹɧɤɢ ɞɿʀ. Ɉɞɧɚɤ ɫɢɧɬɟɡ ɬɚɤɢɯ ɫɢɫɬɟɦ ɞɨɫɬɚɜɤɢ ɽ ɞɭɠɟ ɫɤɥɚɞɧɢɦ ɿ ɩɪɨɦɢɫɥɨɜɨ ɧɟɜɢɝɿɞɧɢɦ. ɍ ɝɥɚɜɿ 7 ɪɨɡɝɥɹɞɚɸɬɶɫɹ ɜɡɚɽɦɨɡɜ’ɹɡɤɢ ɦɿɠ ɫɬɪɭɤɬɭɪɨɸ ɞɜɨɹɞɟɪɧɢɯ ɩɪɨɬɢɪɚɤɨɜɢɯ ɤɨɦɩɥɟɤɫɿɜ ɩɥɚɬɢɧɢ ɬɚ ʀɯɧɶɨɸ ɬɨɤɫɢɱɧɿɫɬɸ. ɋɟɪɟɞ ɞɜɨɹɞɟɪɧɢɯ ɤɨɦɩɥɟɤɫɿɜ ɡ ɠɨɪɫɬɤɢɦɢ ɥɿɝɚɧɞɚɦɢ ɛɿɥɶɲ ɫɬɟɪɢɱɧɨ ɭɫɤɥɚɞɧɟɧɿ ɫɩɨɥɭɤɢ ɽ ɦɟɧɲ ɬɨɤɫɢɱɧɢɦɢ. ɋɟɪɟɞ ɤɨɦɩɥɟɤɫɿɜ ɡ ɝɧɭɱɤɢɦɢ ɥɿɝɚɧɞɚɦɢ ɰɢɫ-ɿɡɨɦɟɪɢ ɽ ɛɿɥɶɲ ɬɨɤɫɢɱɧɢɦɢ, ɧɿɠ ɬɪɚɧɫ-ɿɡɨɦɟɪɢ. ɇɚɣɜɚɠɥɢɜɿɲɿ ɪɟɡɭɥɶɬɚɬɢ, ɨɩɢɫɚɧɿ ɭ ɞɚɧɿɣ ɞɢɫɟɪɬɚɰɿʀ, ɨɝɥɹɧɭɬɿ ɭ ɝɥɚɜɿ 8. ɍ ɰɿɣ ɝɥɚɜɿ ɬɚɤɨɠ ɪɨɡɝɥɹɧɭɬɿ ɦɨɠɥɢɜɿ ɧɚɩɪɹɦɤɢ ɿ ɩɟɪɫɩɟɤɬɢɜɢ ɩɨɞɚɥɶɲɢɯ ɞɨɫɥɿɞɠɟɧɶ.
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Curriculum Vitae Ganna Kalayda was born on August 2, 1979 in Nikopol, a town in the south of Ukraine. From 1986 until 1992, she attended secondary school in Kiev. In 1992 she entered the Technical Gymnasium of Ukrainian National Technical University in Kiev and graduated in 1996 with honour. She started to study chemistry at Kiev Taras Shevchenko University (Ukraine) in September 1996 and obtained her B.Sc. degree with honour in 2000. In 1999 Ganna spent two months in Tufts University (Medford, Massachusetts, USA) as a research scholar under supervision of Prof. Elena Rybak-Akimova. She continued her study at Leiden University (The Netherlands) and obtained a M.Sc. degree in chemistry in August 2001. She performed a research project in the field of bioinorganic and medicinal chemistry in the group of Prof. Dr. Jan Reedijk. In February 2002, Ganna Kalayda started to work on her PhD project in this group. During the following four years, she carried out platinum antitumor drug research, which mainly dealt with various aspects of intracellular behavior of dinuclear platinum anticancer drugs. She intensively collaborated with the group of Prof. Dr. Hans Tanke (Leiden University Medical Center). In 2004 she spent two months at University of Münster working on a research project in the group of Prof. Dr. Bernt Krebs. Some of the results described in this thesis were presented at several international conferences and meetings: “Metal Compounds in the treatment of cancer and viral diseases” COST D20 mid-term evaluation meeting (Trieste, Italy, September 2003); XXXVIth International Conference on Inorganic Chemistry (Merida, Yucatan, Mexico, July 2004); “Metal Compound in the Treatment of Cancer”COST D20 meeting (Jerusalem, Israel, April 2005); 12th International Conference on Biological Inorganic Chemistry (Ann Arbor, Michigan, USA, August 2005).
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List of publications 1. Pavlishchuk, V. V.; Kalaida, A. V.; Goreshnik, E. A. A new method of synthesizing [Cu(Bipy)3](ClO4)2. Crystal structure revisited and spectral and magnetic characterization. Russian Journal of Coordination Chemistry, 1999, 25, 7, 507 - 510. 2. Rybak-Akimova, E. V.; Nazarenko, A. Y.; Silchenko, S. S.; Kalayda, G. V. Double-bond migration in macrocyclic complexes dictated by coordination requirements of the metal ion. Abstracts of Papers of the American Chemical Society, 2000, 219, 70. 3. Komeda, S.; Kalayda, G. V.; Lutz., M.; Spek, A. L; Yamanaka, Y.; Sato, T.; Chikuma, M.; Reedijk, J. New isomeric azine-bridged dinuclear platinum(II) complexes circumvent cross-resistance to cisplatin. Journal of Medicinal Chemistry, 2003, 46, 7, 1210-1219. 4. Herrera, A. M.; Kalayda, G. V.; Disch, J. S.; Wikstrom, R. P.; Korendovych, I. V.; Staples, R. J.; Campana, C. F.; Nazarenko, A. Y.; Haas, T. E.; Rybak-Akimova, E. V. Reactions at the azomethine C=N bonds in the nickel(II) and copper(II) complexes of pyridine-containing Schiff-base macrocyclic ligands. Journal of Chemical Society, Dalton Transactions, 2003, 23, 4482-4492. 5. Kalayda, G. V.; Komeda, S.; Ikeda, K.; Sato, T.; Chikuma, M.; Reedijk, J. Synthesis, structure and biological activity of the new azine-bridged dinuclear platinum(II) complexes - a new class of anticancer compounds. European Journal of Inorganic Chemistry, 2003, 24, 4347-4355. 6. Meistermann, I.; Kalayda, G. V.; Hotze, A. C. G.; Reedijk, J. Preparation of a new Ruthenium(II) building block for the synthesis of mixed-metal complexes. Tetrahedron Letters, 2004, 45, 2593-2596. 7. Jansen, B. A. J.; Wielaard, P.; Kalayda, G. V.; Ferrari, M.; Molenaar, C.; Tanke, H. J.; Brouwer, J.; Reedijk, J. Dinuclear platinum complexes with N,N’-bis(aminoalkyl)-1,4diaminoanthraquinones as linking ligands. Part I. Synthesis, cytotoxicity, and cellular studies in A2780 human ovarian carcinoma cells. Journal of Biological Inorganic Chemistry, 2004, 9, 4, 403-413. 8. Kalayda, G. V.; Jansen, B. A. J.; Molenaar, C.; Wielaard, P.; Tanke, H. J.; Reedijk, J. Dinuclear platinum complexes with N,N’-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part II. Cellular processing in A2780 cisplatin-resistant human ovarian
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carcinoma cells. New insights in the mechanism of resistance. Journal of Biological Inorganic Chemistry, 2004, 9, 4, 414-422. 9. Kalayda G.V.; Jansen, B.A.J.; Wielaard, P.; Tanke, H. J.; Reedijk, J. Dinuclear platinum anticancer complexes with fluorescent N,N’-bis(aminoalkyl)-1,4-diaminoanthraquinones: cellular processing in two cisplatin resistant cell lines reflects the differences in their resistance profiles. Journal of Biological Inorganic Chemistry, 2005, 10, 305-315. 10. Kalayda G.V.; Zhang, G.; Abraham, T.; Tanke, H. J.; Reedijk, J. Application of fluorescence microscopy for investigation of cellular distribution of dinuclear platinum anticancer drugs. Journal of Medicinal Chemistry, 2005, 48, 5191. 11. Kalayda G. V.; Fakih, S.; Bertram, H.; Ludwig, T.; Oberleithner, H.; Krebs, B.; Reedijk, J. Structure-toxicity relationships for different types of dinuclear platinum complexes. Journal of Inorganic Biochemistry, 2005, submitted.
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Acknowledgements Four years ago, on the 1st of February 2002, I started my PhD study in Leiden. These years have now passed by. My thesis is ready, and I will leave Leiden shortly. This makes me look back and remember the people who in one or another way made a contribution to this thesis, who gave me help and support in the past years. I first joined CBAC group in 2000 when I came to Leiden for my M.Sc. study. I did my M. Sc. project (as well as my PhD research) in the platinum subgroup. The members of this subgroup at that time, Seiji, Bart and Marc, introduced me to the chemistry of platinum anticancer drugs. They helped me to learn new techniques and to acquire new skills, which were also very useful during my PhD time. Steven and Elena started their PhD research just a little bit earlier than I did, and we have been working in the same lab and in the same office for most of the past four years. I would also like to mention the people who joined the platinum subgroup later, Eva, Patricia and Paul. I had a good time within CBAC, for which I am grateful to the group members, in particular Stefania, Karlijn, Anna, Giannnis, Christophe, Meenal, Patrick and others. My only M.Sc. student was Guofang Zhang (Ella) who spent one year with me working on her research project. Being smart and hardworking, Guofang was very helpful in my research. In fact, she performed a great deal of synthetic work described in Chapter 5. Ingrid Bekooy, Yvonne Snellenberg and Marianne Kooistra were always ready to help in organizing things and solving administrative problems. I am very grateful to John van Dijk for his patience and persistence during HPLC purifications and during LC-MS and MS measurements, which were not always as successful as we wanted. I would like to thank Jos van Brussel for a number of elemental and ICP analyses. NMR experiments would be a lot more difficult without the help of Fons Lefeber and Cees Erkelens. I also appreciated that I could always come to Hans Den Dulk for an advice concerning cell culture. Some of the cytotoxicity tests described in Chapter 2 were carried out at Osaka University of Pharmaceutical Sciences. I am grateful to Seiji Komeda, Takaji Sato and Prof. Chikuma for performing these experiments. I spent a lot of time at the Sylvius laboratory doing fluorescence microscopy experiments, which formed the basis of Chapters 3 – 5. I am grateful to all the people from the Cytochemistry & Cytometry group, in particular Chris Molenaar, Tsion Abraham and Ilke Krouwels, for their help. I would like to give special thanks to Chris who taught me to use the technique. Working on the Drug Delivery project together with the people from Kreatech Biotechnology and Groningen University was very interesting and pleasant for me. Chapter 6 presents some results of this collaboration.
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At the end of 2004, I spent two months at the University of Münster investigating epithelial toxicity of platinum drugs, which is described in Chapter 7. I would like to thank the people from the Inorganic Chemistry group there. I am especially grateful to Sarah Fakih who arranged my stay in Münster and to Helga Bertram, a co-worker from the Institute of Physiology, for her assistance with the cell culture and with the measurements. I would like to thank Dr. Vitaly Pavlishchuk for opening the world of chemistry for me and being my first teacher in science. But life consists (fortunately!) not of science alone. I always had a great time with my friends, no matter where we were – in Holland, in Ukraine, in Germany or in other places in the world. I thank them all for their support, for the wonderful moments we had together. The so-called ‘paranymphs’ are the two people who may give me a hand during the thesis defence, at least, according to the original idea. I am very grateful to my ‘paranymphs’, who have helped a lot in the past years: to Ira for our friendship, and to Klaas for always being at my side. My last words are for my family. In the first place, I thank my parents; they brought me up to who I am. The support of my parents-in-law in the past two years was valuable for me. I thank my brother Andrey for being my friend. And of course Klaas for our past, present and future.
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