Salvage enzymes in nucleotide biosynthesis

Balanced pools of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA, and ribonucleoside triphosphates (NTPs), the precursors of RNA, are crucial for a controlled cell proliferation. The dNTPs and NTPs are synthesized de novo via energy-consuming reactions involving low-weight molecules, and through a salvage pathway by recycling (deoxy)ribonucleosides originating from food and degraded DNA and RNA. The enzymes described in this thesis catalyze the first reaction in the salvage biosynthesis of dNTPs and NTPs. The crystal structures of three bacterial thymidine kinases (TKs) are described and the enzymes are investigated as potential targets for antibacterial therapies. TK is a deoxyribonucleoside kinase (dNK) with specificity for thymidine. In addition to the natural substrates, TK can also phosphorylate a number of nucleoside analogs used in antiviral and anticancer therapies. This thesis presents the structures of TKs from three pathogenic microorganisms: Ureaplasma urealyticum (parvum), Bacillus anthracis and Bacillus cereus, and compares them to the human thymidine kinase 1 (hTK1). The bacterial TKs and the hTK1 are structurally very similar and have a highly conserved active site architecture, which may complicate structure-based drug design. However, the different complex structures presented in this work provide information regarding the conformational changes of TK1-like enzymes during the time of reaction. The structure of human uridine-cytidine kinase 1 (UCK1) is also presented. Humans possess two uridine-cytidine kinases, UCK1 and UCK2. The expression pattern of these enzymes is tissue dependent, and despite high sequence as well as structural similarities they possess somewhat diverse substrate specificity. In addition to the natural substrates, uridine and cytidine, UCKs are able to phosphorylate a number of nucleoside analogs. The monomeric structure of UCK comprises four domains: a CORE domain, an NMP-binding domain, a LID domain and a β-hairpin domain, which upon substrate binding undergo dramatic conformational changes. In the structure described in this thesis the enzyme has been trapped in an intermediate conformation between a fully opened and fully closed form, which may represent a sequential mode of substrate binding

ANALYSIS OF THE RESULTS OF EXPERIMENTAL RESEARCH OF STAGE CULTURE FORMATION OF FUTURE TEACHERS OF MUSIC ART IN THE VOCAL TRAINING PROCESS

The article analyzes the organization of pedagogical experiment on the formation of the stage culture of future teachers of music in the vocal training process. The pedagogical experiment included three stages: ascertaining, forming, comparative. The stages of the formative expression are characterized – motivational, informative and creative-reflexive. Methods of experimental research are described. The effectiveness of experimental and experimental work, which is carried out on the basis of a comparative analysis of the stage and figurative culture of future teachers of musical art of experimental and control groups, is tested. The content and method of formation of stage-figurative culture of future teachers of music art in the process of vocal training during the forming experiment are characterized according to certain stages. At the first (motivational and motivational) stage, the formation of motivation of future teachers of music art to develop their own stage-figurative culture, motives and needs for the formation of stage, vocal and interpretative skills, value orientations for the improvement of this important quality was carried out. During realization of the second (informative) stage of realization of the method of formation of stage culture of the future teacher of musical art in the process of vocal training, attention was focused on enrichment of knowledge about stage and vocal skills, interpretation of musical image, formation of knowledge on stage-figurative culture (cultural studies, theatrical sciences, hermeneutics, art pedagogies, etc.); expanding thesaurus and identifying opportunities for practical application of knowledge in pedagogical activity. During the realization of the third (creative-reflexive) stage, attention was paid to the formation of vocal, stage, interpreting skills, development of vocal technique, artistic expressiveness, communicativeness, empathy, expression, etc., in order to influence the feelings, intellect of the students. The results of a formative experiment allow to confirm the effectiveness of sound pedagogical conditions and methods of forming the stage-figurative culture of future music teachers in the process of vocal training

Agriculture Income Assessment for the Purpose of Social Assistance: the Case of Ukraine

Ukraine belongs to the group of countries which are known for the widespread phenomenon of subsistence and semi-subsistence farming. Individual farmers are not obliged to produce financial reports and their incomes belong to the category of unobservable incomes. When checking the eligibility for social assistance the level of their incomes needs to be estimated. In a country, where poverty rate is quite high, the coverage of the poor with financial aid is relatively low and public finances under constant control, the importance of a fair and justified methodology for income imputation is particularly strong. In this situation, an outdated and unfair current system of agriculture income estimation in Ukraine calls for immediate changes. This paper presents recommendations for the Ukrainian government in the area of agriculture income imputation, where several methods of estimating farm income were proposed (including the one based on Household Budget Survey). The recommendations were preceded with the analysis of five countries' practices in this area: Kazakhstan, Kyrgyzstan, Moldova, Russia, and Poland. A review of different means testing methods, including direct means testing and proxy means testing, served as an introduction to the topic.subsistence and semi-subsistence farming, hard to verify income, farm household income, income (agro-income) imputation, means testing methods

Photothermal convection of a magnetic nanofluid in a direct absorption solar collector

Nanofluid-based direct absorption of solar heat results in thermal efficiencies superior to conventional solar thermal technology. In addition, convection of nanofluid can be sustained pump-free in the collector. In this article, we study an aqueous magnetic nanofluid capable to establish the photothermal convection in a lab-scale direct absorption solar collector equipped with a solenoid. The nanofluid consisted of 60-nm Fe2O3 particles dispersed in distilled water at concentration in the range 0.5% wt.-2.0% wt. An empirical model of the photothermal convection was developed based on the experiments. The model accounted for magnetic and thermophoretic forces acting within the nanofluid. The nanofluid with up to 2.0% wt. iron oxide nanoparticles obtained the velocity of ∼5 mm/s under the magnetic field of up to 28 mT. This resulted in the maximum thermal efficiency of the collector equal to 65%.publishedVersio

Photothermal conversion of biodegradable fluids and carbon black nanofluids

The paper is devoted to the topic of direct absorption solar collectors (DASCs). Various kinds of fluids can be used as heat transfer fluid in DASCs, and the main focus of our paper is on comparing nanofluids (water with carbon black nanoparticles, concentrations between 0.25 and 1.00% weight) and biodegradable coffee colloids. At first, these fluids were tested by exposing them to irradiation caused by artificial light in indoor experiments, and the corresponding temperature increase was recorded. The fluids were placed in a beaker with a relatively large size so that most of the fluid was not directly irradiated. In these experiments, the performance of the two studied fluids was similar: the resulting temperature increase varied between 46 and 50 °C. Our next experiments involved a smaller system subjected to irradiation obtained by using a solar collector. As a result, we detected an intense absorption on the nanoparticle surface so that the temperature rise in the nanofluid was higher than in the coffee colloids. Next, the process was analysed using a theoretical analysis that gave good correspondence with the experiments. Finally, we extended the theoretical analysis to a DASC with a flowing fluid. The model was validated against results from the literature, but it also supported our experimental findings.publishedVersio

Optimierung der therapeutischen Vakzination zur Behandlung chronischer Hepatitis B im präklinischen Model (Murmeltier)

More than 360 million people worldwide are persistently infected with the hepatitis B virus (HBV). The recommended treatment of chronic hepatitis B with interferon-α and/or nucleoside analogues does not lead to satisfactory results. It is well documented that the viral persistence is caused by an absence of the effective virus-specific T cell response. Therefore, the induction of HBV-specific T cells by therapeutic vaccination may be an innovative strategy to overcome virus persistence. Vaccination with commercially available HBV vaccines and DNA vaccines in patients did not result in an induction of the immune response, which would effectively control the HBV infection. Due to this, a more potent therapeutic vaccines are needed. The woodchuck (Marmota monax) is a useful preclinical model for developing new therapeutic approaches in chronic hepadnaviral infections. Several approaches using classical protein and DNA vaccines were tested previously in the woodchuck model without success. The high viral load observed in chronic hepadnaviral infections may have impaired the induction of an effective virus-specific T cell response. Therefore, the application of more potent vaccines, e.g. recombinant viral vectors in combination with antiviral treatment, may be required to achieve sustained antiviral response. In the presented study, an innovative therapeutic strategy, combining vaccination with optimized DNA and recombinant adenoviral vectors (AdV), as well as potent antiviral treatment with entecavir (ETV), was evaluated in the woodchuck model. It was hypothesized that this approach may be an effective strategy to improve WHV-specific immune responses and as a result lead to the resolution of chronic WHV infection. For that purpose, an improved DNA vaccine (pCGWHc), adenoviral vector serotype 5 (Ad5WHc) and chimeric Ad5 displaying Ad35 fiber (Ad35WHc) expressing high levels of woodchuck hepatitis core antigen (WHcAg) were constructed. The efficacy of the improved vaccines was tested first in mice and in naïve woodchucks. The improved WHcAg expression by pCGWHc caused a detection of significantly stronger CD8+ T cell response (interferon-γ production) and higher anti-WHc levels in C57BL/6 mice, compared to the previously used DNA plasmid vaccine. Immunization of mice by pCGWHc prime-AdV boost regimen enhanced CD8+ T cell response even more, compared to immunization with DNA alone. Moreover, the vigorous cytotoxic activity of these WHcAg-specific CD8+ T cells could be demonstrated in vivo. To further evaluate the efficacy of the new prime-boost regimen the vaccination of WHV transgenic mice (1217 WHV Tg) was performed. The immunizations elicited an unexpected anti-WHc and anti-WHs antibodies and WHcAg-specific CD8+ T cell response which led to a significant suppression of WHV replication in those mice. Next, the heterologous Ad5WHc/Ad35WHc immunization was performed in naïve woodchucks. The immunization resulted in the induction of a strong cellular immune response in woodchucks and protected them from WHV infection after the challenge. Altogether, these results clearly showed, that heterologous DNA-AdV immunization induces a more potent WHV-specific immune response than previously investigated strategies in mice and woodchucks. Therefore, this novel DNA prime-AdV boost regimen was used to treat chronically WHV-infected woodchucks in combination with entecavir (ETV) to induce a proper CTL response. Seven animals were treated with ETV for 23 weeks. Starting from week 8, five of them received additional 9 immunizations with DNA plasmids, expressing WHcAg and WHsAg, Ad5WHc and Ad35WHc. For the first time, the significant WHsAg- and WHcAg-specific proliferative responses (CD4) and degranulation responses (CD8) were detected in all chronic carriers that received immunizations in combination with ETV treatment. Moreover, 2 of 4 immunized woodchucks, which completed the ETV and vaccination treatment, demonstrated sustained antiviral response (undetectable viral load and development of anti-WHs). These findings indicate that the therapeutic approach evaluated in this study induces a potent T cell response and seems to be an effective strategy to achieve sustained control of chronic hepadnaviral infections. These results obtained in a preclinical model might be the base for the clinical trials of therapeutic vaccines which induce strong anti-core T cell response in combination with antivirals as a possible immunotherapy in chronically HBV-infected patients.Weltweit sind derzeit mehr als 360 Millionen Menschen mit dem Hepatitis B Virus (HBV) chronisch infiziert. Die empfohlenen Behandlungsstategien bei chronischer Hepatitis B, bestehend aus Interferon-α und/oder Nukleosid-Analoga, zeigen keine zufriedenstellenden Erfolge. In der Literatur ist beschrieben, dass die Viruspersistenz durch das Fehlen einer effektiven virusspezifischen T-Zellantwort mitunter verursacht wird. Daher stellt die Induktion einer HBV-spezifischen T-Zellantwort durch eine therapeutische Vakzinierung eine innovative Strategie zur Behandlung dar, um die Viruspersistenz zu überwinden. Die Schutzimpfung mit kommerziell erhältlichen HBV-Impfstoffen und DNA-Vakzinen in Patienten führt zu keiner ausreichenden Immunantwort, die HBV effektiv kontrolliert. Infolgedessen werden neue wirksame therapeutische Impfstoffe dringend benötigt. Das nordamerikanische Waldmurmeltier (Marmota monax; engl. woodchuck) ist ein nützliches, präklinisches Modell zur Entwicklung neuer therapeutischer Ansätze in chronischen hepadnaviralen Infektionen. Bisherige Therapieansätze im Murmeltier Model, unter Verwendung von klassischen Protein- und DNA-Vakzinen waren ohne Erfolg. Die hohe Viruslast, die in chronischen hepadnaviralen Infektionen beobachtet wird, kann die effektive, virusspezifische T-Zellantwort beeinträchtigen. Aus diesem Grund werden wirksamere Impfstoffe, wie zum Beispiel eine Kombination aus rekombinanten viralen Vektoren und antivirale Behandlung benötigt, um eine andauernde antivirale Antwort zu erzielen. In der vorliegenden Arbeit wurde eine innovative therapeutische Strategie im Murmeltier-Model verfolgt, die in einer kombinierten Vakzinierungsstrategie aus optimierten DNA- und rekombinanten Adenoviralen Vektoren (AdV) sowie aus einer wirksamen, antiviralen Behandlung mit Entecavir (ETV) bestand. Es wurde angenommen, dass dieser Ansatz eine effektive Immunisierungsstrategie darstellt, um eine spezifische Immunantwort gegen das Murmeltier Hepatitis Virus (WHV) zu verbessern und als Ergebnis zu einer Eliminierung der chronischen WHV-Infektion führt. Zu diesem Zweck wurden Vektoren konstruiert, die das Murmeltier Hepatitis Core Antigen (WHcAg) stark exprimieren: ein verbesserter DNA-Impfstoff (pCGWHc), ein Adenoviraler Vektor des Serotyps 5 (Ad5WHc) und einen chimären Ad5 mit präsentierenden Ad35 Fibern. Die Wirksamkeit dieser verbesserten Impfstoffe wurde zuerst in Mäusen und in naiven Murmeltieren getestet. Die durch pCGWHc verbesserte WHcAg Expression bewirkte eine signifikant stärkere CD8+ T-Zellantwort (Interferon γ Produktion) und höhere anti-WHc Level in C57BL/6 Mäusen als in zuvor verwendeten DNA Plasmid-Vakzinen. Im Vergleich zu DNA-Immunisierung konnte eine weitere Verbesserung der CD8+ T-Zellantwort erzielt werden, indem eine pCGWHc „prime-AdV boost“ Immunisierungsstrategie in Mäusen angewandt wurde. Darüber hinaus konnte gezeigt werden, dass diese WHcAg-spezifischen CD8+ T-Zellen eine starke zytotoxische Aktivität in vivo besaßen. Zur weiteren Beurteilung der neuen „prime-boost“ Strategie wurden WHV-transgene Mäuse (1217 WHV Tg) immunisiert. Die Immunisierung rief eine unerwartete anti-WHc und anti-WHs Antikörperproduktion sowei eine WHcAg-spezifische CD8+ T-Zellantwort aus, die zu einer signifikanten Verminderung der WHV Replikation in den Mäusen führte. Im Anschluss wurde die heterologe Ad5/Ad35WHc Immunisierung in naiven Murmeltieren durchgeführt, welche eine starke zelluläre Immunantwort ausgelöste. Des Weiteren bot diese Immunisierungsstrategie effektiven Schutz vor einer WHV-Infektion. Zusammenfassend zeigen diese Ergebnisse, dass eine heterologe DNA-AdV Immunisierung eine weitaus stärkere WHV-spezifische Immunantwort induzieren kann, als die zuvor in Mäusen und Murmeltieren angewandten Immunisierungsstrategien. Zu diesem Zweck wurde diese neuartige DNA „prime-AdV boost“ Strategie dazu verwendet, chronisch WHV-infizierte Murmeltiere in Kombination mit ETV für 23 Wochen zu behandeln. Ausgehend von Woche 8 erhielten 5 der chronisch WHV-infizierten Murmeltiere 9 Immunisierungen mit den DNA-Plasmiden, welche WHcAg und WHsAg sowie Ad5WHc und Ad35WHc exprimieren. Zum ersten Mal konnte eine signifikante WHsAg- und WHcAg-spezifische Proliferationsantwort (CD4) und Degranulationsantwort (CD8) in allen chronisch infizierten Murmeltieren detektiert werden. Des Weiteren konnte gezeigt werden, dass 2 aus 4 immunisierten Murmeltieren, die die Behandlung mit ETV und Vakzine abgeschlossen hatten, eine anhaltende antivirale Immunantwort aufwiesen. Diese Ergebnisse verdeutlichen, dass der gewählte therapeutische Ansatz eine wirksame T-Zellantwort induziert und eine effektive Behandlungsstrategie zu sein scheint, um eine anhaltende Kontrolle von chronischen hepadnaviralen Infektionen zu erzielen. Die in dieser Arbeit beschriebenen Befunde im prä-klinischen Model könnten die Basis für eine klinische Studie zur therapeutischen Vakzinierung zur Immuntherapie chronischer HBV-infizierter Patienten darstellen, die in Kombination mit antiviralen Agentien in der Lage ist eine starke anti-Core T-Zellantwort zu erzielen

Postnonclassical Practices іn the Content of Higher Art Education

The study of the phenomenon of transdisciplinarity in scientific discourse indicates the emergence of a new strategy for solving real practical problems. A new image of post-non-classical science is being formed. However, there are difficulties with the introduction of a transdisciplinary approach into the methodology of musical pedagogical research and with the practical implementation of the methods and techniques of transdisciplinarity. In order to solve this problem, the narrative “We grieved with one grief and rejoiced with one happiness”, based on the life and work of Boris Grinchenko, is presented and analyzed. Pedagogical observations prove that the form of narration created an open atmosphere for the transdisciplinary transfer of pedagogical skills, united the intellectual and creative potential of teachers and students, and proved the expediency of reorienting the musical and pedagogical processes in the context of postnonclassics. Due to a wide inter- and transdisciplinary synthesis, it has become possible to synergistically combine different ways of perceiving and comprehending the unstable and complex world of art