Tsüklilise adenosiinmonofosfaadi biosensori arendamine ja rakendamine G valguga seotud retseptorite signaaliülekande uurimiseks

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Maailma rahvastik on pidevas kasvus ning vananemises ning sellest on tingitud mõnd haigust või sündroomi põdevate patsientide üha suurenev hulk. Enamik haigustest on põhjustatud häiretest organismi metabolismi või rakkude signaaliülekande regulatsioonis. G valguga seotud retseptorid (GPCR) moodustavad suure rakkudevahelist suhtlust reguleeriva valkude perekonna. Keemilise signaaliülekande moduleerimine on levinud ravistrateegia, mida kinnitab ka tõsiasi, et ligi kolmandik kõigist tänapäeva retseptiravimitest on suunatud just GPCR-tele. Ravimid ehk bioaktiivsed ühendid on seega tänapäeva maailma asendamatu osa. Uute sünteetiliste ühendite otsinguil on väga olulisteks uute molekulide testimine ning saadud katsetulemuste võrdlus struktuurselt sarnaste või kehaomaste signaalimolekulide omadustega. See aitab iseloomustada ühendite erinevaid omadusi ning annab väärtuslikku informatsiooni uute ühendite disainiks. Käesolevas doktoritöös arendati välja katsesüsteem erinevate bioloogiliselt aktiivsete molekulide iseloomustamiseks elusates rakkudes. Selleks kasutati fluorestsentsil põhinevat biosensorit. Biosensori kasutamiseks on vajalik selle olemasolu uuritavas objektis (rakud toodavad sensorvalku ise, lähtudes neisse eelnevalt viidud sensori geenijärjestusest). Saavutamaks biosensori ühtlast taset erinevates uuritavates rakkudes kasutati käesolevas töös putukaviiruseid. Inimesele ohutud viirused transpordivad sensorit kodeeriva geenijärjestuse uuritavasse rakku, kus sellest sünteesitakse uus valk, tundlik biosensor. Süsteem võimaldas kasutatava viiruse kogusega reguleerida biosensori taset, mille tulemuseks oli ühtlane ja hea korratavusega valkude tootmine ning usaldusväärne katsesüsteem. Töö käigus demonstreeriti biosensori rakendatavust mitmetes erinevates rakkudes väga erinevate retseptorite aktivatsiooni uurimiseks. Tänu katsesüsteemi tundlikkusele sobib see näiteks ka kehaomaste hormoonide kontsentratsiooni määramiseks erinevates proovides, mis on olulise tähtsusega nii meditsiini kui ka uute tehnoloogiate arendamise seisukohalt. Töö tulemusena on loodud tööriist, mille abil on võimalik määrata erinevate struktuursete omadustega ühendite bioloogilist aktiivsust vastavate retseptorite aktivatsiooni kaudu.World’s population is constantly growing and aging. As a result, the number of patients suffering from some sort of illness or disease is rising as well. Most of the illnesses are caused by disturbances in cellular signaling or metabolism of the organism. G protein-coupled receptors (GPCR) form a large family of cell surface proteins responsible for the regulation of signal transduction. Over one third of all todays’ prescription drugs are targeted to GPCRs. In search for new synthetic compounds targeting these receptors functional testing is of great importance. Obtained experimental data need to be evaluated and compared with data from structurally similar or endogenous ligands. This yields information about the properties of the new ligand and for molecular design of future compounds. In the current thesis an assay system for characterization of different biologically active molecules on living cells was developed based on the use of fluorescent biosensor proteins. To use a biosensor, it needs to be present inside the cells of interest. To achieve a reliable and uniform cellular level of the biosensor protein, a gene delivery system based on the use of insect viruses was employed. These viruses, harmless to humans, transport the biosensor gene sequence in to the cells, where a new functional protein is synthesized and is ready to be used in functional experiments. The level of protein expression can be regulated by the amount of the applied virus, which results in homogeneous expression and a reliable assay with high reproducibility. During the studies the applicability of the new biosensor assay was demonstrated by monitoring the activation of very different GPCRs in various cells. Due to high sensitivity of the assay it is also suitable for quantification of endogenous hormones in different probes and preparations. This is of importance for the field of medicine as well as for the development of novel technologies. The result of this work is the developed biological tool that enables us to monitor GPCR activation for measurement of biological activity of compounds with various structural properties

Контрафакція як дії, що порушують авторські права

Мазіна О. О. Контрафакція як дії, що порушують авторські права : автореф. дис. ... канд. юрид. наук : 12.00.03 / О. О. Мазіна; кер. роботи О. І. Харитонова; Нац. ун.-т "Одеська юридична академія". – Одеса, 2014. – 19 с.Дисертація на здобуття наукового ступеня кандидата юридичних наук за спеціальністю 12.00.03 – цивільне право і цивільний процес; сімейне право; міжнародне приватне право. – Національний університет «Одеська юридична академія», Одеса, 2014. Дисертація є спеціальним комплексним дослідженням контрафактних дій, які порушують авторські права, та відповідальності за їх здійснення. Досліджено наукову концепцію щодо причин виникнення, умов існування та наслідків контрафакції як дій, що порушують авторські права. Надане авторське визначення понять «контрафакція» як дій, що порушують авторські права, та інших порушень авторських прав – «піратство» і «плагіат». Визначено відмінності від дій з вільного правомірного використання творів. Охарактеризовано окремі види контрафакції, піратства і плагіату та запропоновано підстави для їх класифікації, а також для розмежування контрафакції від інших дій, що порушують авторські права. Досліджено порядок здійснення цивільно-правового захисту та застосування мір відповідальності за порушення авторських прав внаслідок контрафакції. Сформульовано конкретні пропозиції щодо вдосконалення законодавства у сфері захисту авторських прав від контрафакції.Диссертация на соискание научной степени кандидата юридических наук по специальности 12.00.03 – гражданское право и гражданский процесс, семейное право, международное частное право. – Национальный университет «Одесская юридическая академия», Одесса, 2014. Диссертация является специальным комплексным исследованием контрафактных действий, нарушающих авторские права, и ответственности за их осуществление. Исследовано понятие контрафакции, его признаки и содержание отдельных действий, являющихся контрафактными. Сформулировано понятие контрафакции как механического воспроизведения произведения: в полном объеме или в его части, с коммерческой целью и соблюдением авторства, но без согласия правообладателя. Установлено, что в зависимости от вида контрафактных действий, различают контрафактные произведения, превышающие по количеству тираж, предусмотренный условиями договора; распространяющиеся вне территории, определенной договором; изготовленные на ином материальном носителе, чем предусмотрено в договоре. Установлено, что признаками экземпляров контрафактных произведений является идентичность оригиналу по внешнему виду, качеству и способу механического воспроизведения. Обобщены основания для классификации действий, составляющих контрафакцию, плагиат и пиратство. Предложено авторское определение пиратства и плагиата как действий, нарушающих авторские права. Разграничены контрафакция и иные нарушения авторских прав, в частности, пиратство и плагиат. Пиратство и контрафакция различаются в зависимости от качества механического воспроизведения, внешнего вида экземпляра произведения, попытки контрафакторов ввести в заблуждение при распространении контрафактных экземпляров. Контрафакцию от плагиата отличает факт соблюдения личных неимущественных прав. Обосновано, что контрафакторами могут являться не только физические или юридические лица, незаконно использующие произведения, но и непосредственно обладатель авторских прав. Изучена судебная практика по применению мер защиты и мер ответственности за контрафакцию и иные нарушения авторских прав. Рассмотрены общие способы защиты гражданских прав и специальные способы защиты авторских прав. Проанализированы нормы уголовного законодательства и законодательства об административных правонарушениях в части привлечения к ответственности за контрафакцию и иные действия, нарушающие авторские права. Проведено сравнение с нормами зарубежного законодательства, предусматривающими ответственность за нарушение авторских прав. Сформулированы конкретные предложения по усовершенствованию законодательства в сфере защиты авторских прав от контрафакции.Dissertation for the degree of candidate of legal sciences, specialty 12.00.03 – civil law and civil procedure, family law, private international law. – National University «Odessa Law Academy», Odessa, 2014. The thesis represents a special comprehensive analysis of counterfeiting actions that violate copyrights and responsibilities for them. The thesis is examined the scientific conception counterfeiting origin causes as actions that violate copyrights and their consequences. The author suggested definition of «counterfeiting» as actions that violate copyrights and other violations such as «piracy» and «plagiarism». In the research the distinctions of named actions and legal usage of a work were determined. Separate kinds of counterfeiting, plagiarism and piracy were characterized; the criteria of its classifications and differentiations were suggested. The author also set up the demarcation counterfeiting with other actions that violate copyrights. The procedure of prosecution for copyright violation as a result of counterfeiting and liability for such actions was analyzed. According to the results of the research the author makes conclusions and suggests practical recommendations for improving the current civil legislation and protecting copyright from counterfeiting

Determination of biological activity of gonadotropins hCG and FSH by Forster resonance energy transfer based biosensors

Determination of biological activity of gonadotropin hormones is essential in reproductive medicine and pharmaceutical manufacturing of the hormonal preparations. The aim of the study was to adopt a G-protein coupled receptor (GPCR)-mediated signal transduction pathway based assay for quantification of biological activity of gonadotropins. We focussed on studying human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH), as these hormones are widely used in clinical practice. Receptor-specific changes in cellular cyclic adenosine monophosphate (cAMP, second messenger in GPCR signalling) were monitored by a Forster resonance energy transfer (FRET) biosensor protein (T)Epac(VV) in living cells upon activation of the relevant gonadotropin receptor. The BacMam gene delivery system was used for biosensor protein expression in target cells. In the developed assay only biologically active hormones initiated GPCR-mediated cellular signalling. High assay sensitivities were achieved for detection of hCG (limit of detection, LOD: 5 pM) and FSH (LOD: 100 pM). Even the smallscale conformational changes caused by thermal inactivation and reducing the biological activity of the hormones were registered. In conclusion, the proposed assay is suitable for quantification of biological activity of gonadotropins and is a good alternative to antibody- and animal-testing-based assays used in pharmaceutical industry and clinical research.Peer reviewe

Ultrasonic Nanomedicine in the Therapy of Oncological Diseases

In this paper the authors develop the concept of using solid-phase nanoscale inclusions in biological structures, as the concentrators of acoustic energy for ultrasound therapy of oncological diseases. Particu-lar attention is paid to possibility of synthesis of these inclusions directly in the tumor tissue. The validity of the hypothesis of solid-phase sonosensitization has been confirmed in vitro on bacterial cultures. Super-additive effect of combined action of ultrasound and solid-phase sonosensitizer on bacterial cultures was shown. Experimental studies on animals showed that the ultrasound exposure of malignant tumors con-taining nanoparticles of gold and some complex compounds (e.g. theraphthal) results in a significant ther-apeutic effect, which is expressed in a considerable inhibition of tumor growth. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/3541

Quantitation and Identification of Organic N-Chloramines Formed in Stomach Fluid on Ingestion of Aqueous Hypochlorite

The chemical reactions that hypochlorite undergoes in the body when chlorinated water is ingested have received very little attention. Because amino nitrogen compounds are important components of the average diet, the reactions of hypochlorite with amino compounds in the stomach were investigated. Stomach fluid was recovered from Sprague-Dawley rats that had been fasted for 48 hr and administered 4 mL deionized water. The chlorine demand of the stomach fluid was determined. An average volume-independent demand of 2.7 mg chlorine was measured. At doses below 40 mg/L chlorine reducing reactions appeared to account for reduction of all oxidizing species within 15 min as measured by the FAS-DPD titrimetric method. At least part of the chlorine demand is associated with amino acids present in the stomach fluid. Amino acids were identified and quantified in the stomach fluid by precolumn derivatization with o-phthalaldehyde and high-pressure liquid chromatography (HPLC). When stomach fluid is chlorinated to concentrations of chlorine between 200 and 1000 mg/L, organic N-chloramines are formed. After derivatization of chlorinated stomach fluid with dansyl sulfinic acid, fluorescent derivatives of chloramines were separated by HPLC. Three chloramino acid derivatives, N-chloroalanine, N-chloroglycine, and N-chlorophenylalanine, were identified by cochromatography with known standards using two chromatographic methods. The yield of a chloramine that would form in stomach fluid on administration of hypochlorite to animals was determined using tritiated piperidine and doses of 200 and 1000 mg/L chlorine. Yields of tritiated N-chloropiperidine in recovered stomach fluid were 70% and 42%, respectively, of the theoretical amount expected

Mechanisms of transcriptional regulation of ecdysone response

The mechanisms of ecdysone-dependent expression have been studied for many decades. Initially, the activation of individual genes under the influence of ecdysone was studied on the model of polythene chromosomes from salivary glands of Drosophila melanogaster. These works helped to investigate the many aspects of the Drosophila development. They also revealed plenty of valuable information regarding the fundamental mechanisms controlling the genes’ work. Many years ago, a model describing the process of gene activation by ecdysone, named after the author – Ashburner model – was proposed. This model is still considered an excellent description of the ecdysone cascade, which is implemented in the salivary glands during the formation of the Drosophila pupa. However, these days there is an opinion that the response of cells to the hormone ecdysone can develop with significant differences, depending on the type of cells. The same genes can be activated or repressed under the influence of ecdysone in different tissues. Likely, certain DNA-binding transcription factors that are involved in the ecdysonedependent response together with the EcR/Usp heterodimer are responsible for cell-type specificity. A number of transcriptional regulators involved in the ecdysone response have been described. Among them are several complexes responsible for chromatin remodeling and modification. It has been shown by various methods that ecdysone-dependent activation/repression of gene transcription develops with significant structural changes of chromatin on regulatory elements. The description of the molecular mechanism of this process, in particular, the role of individual proteins in it, as well as structural interactions between various regulatory elements is a matter of the future. This review is aimed to discuss the available information regarding the main regulators that interact with the ecdysone receptor. We provide a brief description of the regulator’s participation in the ecdysone response and links to the corresponding study. We also discuss general aspects of the mechanism of ecdysone-dependent regulation and highlight the most promising points for further research

THE UNIQUE OPPORTUNITIES OF ACCOUNTING TO PROMOTE TRANSPARENCY OF ECONOMIC PROCESSES

The article is devoted an important role the Institute of Accounting in the modern economy. This role is supported by unique institutional and informational opportunities determined by its specific characteristics. On the one hand, accounting is the legislative and normative institution through which the controlling and regulating institutions influence the economic processes occurring at each enterprise. On the other hand, the accounting system creates an information database on the actual economic activity of each enterprise required by users. Such a database is formed due to the fact that accounting is an exclusive registrar of business activities: documentation, inventory, assessment, calculation, accounts, double-entry bookkeeping, balance, reporting. These methods have such a mathematical feature that has made it possible to fully them with the latest information technologies. All of these factors allow accounting to provide transparent accounting information to any users and institutions